When Kim Clijsters thinks about changing her nationality to thatof Australian fiance Lleyton Hewitt once they marry, there is noBelgian waffle.
The No. 2-ranked women's tennis player will continue to be fromBelgium, no matter how many folks Down Under call her Aussie Kim."
Sidelined a month with a wrist injury, Clijsters was back on thecourt Saturday in Bree, Belgium, for a love match" against Hewitt. Herallied from a 6-4 deficit to win the best-of-17 game match 9-8.
I'll never say, …
Abstract
To determine whether older Quebecers are eating adequately and whether summary scores represent diet quality, a representative subset of participants aged 55 to 74 (weighted n=460, 47% male) was studied from the 1990 Enquete quebecoise sur la nutrition dataset. Participants' diet quality was scored from adjusted 24-hour recalls. Foods were coded into Canada's Food Guide to Healthy Eating food groups. Usual Dietary Adequacy Score (maximum=18) and Dietary Diversity Score (maximum=4) were calculated from adjusted food guide portions and validated internally in relation to achievement of nutrient recommendations using correlation analysis. Average usual Dietary Adequacy Score …
Asian stocks showed signs of life Tuesday after a global market rout the previous day, with several markets climbed into positive territory or paring losses after opening sharply lower.
Australian stocks jumped after its central bank cut interest rates by a bigger-than-expected 1 percentage point to 6 percent in response to the unfolding global financial crisis. The S&P/ASX-200 index rose 2 percent to 4,634 after opening down 3.7 percent.
Japan's benchmark Nikkei 225 index erased some losses after briefly falling over 5 percent to below 10,000 for the first time in almost five years. By midday, it was trading about 1.7 percent lower at 10,292.
…
A chain of hotels based near Bath experienced its best April foryears - despite going into administration last month.
Peasedown St John-based von Essen, which runs the Royal CrescentHotel and Homewood Park, said the double four-day bank holidayweekends had produced a surge in late bookings as couples opted fora taste of royal wedding-style class.
The parent company which runs the hotels has gone intoadministration, after a turbulent couple of years in the high-endhotel and spa business.
The venues themselves are operating as normal whileadministrators at Ernst and …
WASHINGTON - A raid on suspected terrorists in northern Iraq failed after Iraqi soldiers ran out of gas and couldn't send half their men to the mission, a U.S. commander says.
The incident illustrates "a critical weakness" in the Iraqi security forces, which still have "a way to go" in learning logistics and other soldiering skills, said U.S. Army Col. Stephen M. Twitty.
Twitty commands the 4th Brigade, 1st Cavalry Division in northern Iraq, where the main job is doing counterinsurgency operations and training local forces in intelligence, engineering, medical evacuation and other skills they need to take over the country's security themselves.
"We lost valuable …
SAO PAULO (AP) — Zico has stepped down as Flamengo's executive director, saying he was unable to do his job because many people were unhappy with his presence in the club.
"It is not possible to do what I would like to do at Flamengo," Zico said in a statement posted on Friday on his website. "I realize that my presence has caused the discontent of many people. The conditions are not right …
LONDON (AP) — American hurdler Duane Ross has been disqualified retroactively from the 2004 Athens Olympics for doping.
The IOC stripped Ross of his Athens results following the U.S. Anti-Doping Agency's decision to suspend him for two years for using banned drugs.
Ross finished fifth in his semifinal heat of the 100-meter hurdles in Athens and did …
Rescue Mission ponders fate of its neon
It's not quite the clock tower, but some people are wondering: Just what's going to happen to the Boise Rescue Mission's iconic sign once the building gets torn down? Boise developers Clay Carley and Gary Christiansen recently received design review approval for a 23-story building that will take the place of the Boise Rescue Mission on the corner of 6th and Front streets. The building is the first of a series of developments of two square blocks that, once complete, will also replace the breakfast eatery Addie's Diner and other businesses.
Although Boise's newest skyscraper proposal has been discussed for several months, neither the …
| First Round |
| Tom Purtzer 30-32_62 |
| Allen Doyle 33-30_63 |
| Jim Thorpe 30-33_63 |
| D.A. Weibring 31-33_64 |
| John Harris 32-33_65 |
| Mark McNulty 34-31_65 |
| Eduardo Romero 33-32_65 |
| Jay Haas 31-34_65 |
| Mark Wiebe 32-33_65 |
| Jerry Pate 37-29_66 |
| Bob Gilder 33-33_66 |
Stepping off my bus to encounter the gut-wrenching smell of thenearby breweries, I just knew it would be a stinker of a game.
As soon as I reached the Tynecastle gate, I was practically strip-searched by the police before being allowed in.
I bought a pie and went to my seat, which was so far up the steepstand that I felt like I had climbed Everest to reach it.
My pie did look burnt when I bought it, but even clarted in sauceI could only manage one bite.
It was so disgusting I'm sure even the Gorgie rats would haveturned their noses up at it!
The real disaster of the day was the …
Andre Liebich. From the Other Shore: Russian Social Democracy after 1921. Cambridge, MA: Harvard University Press, 1997. xii, 476 pp. Illustrations. Notes. Sources. Index. $48.00, cloth.
Andre Liebich begins and ends this meticulous biography of Menshevism by insisting, not without some warnings, on the utility of studying exile movements. His introduction deplores the lack of historical imagination which has brought scholars overwhelmingly to concentrate upon the winners, ignoring those who, by the fact of their exile, embody lost causes, defeat, rejection. Liebich's interest in exile movements generally prompts him to address this phenomenon. A second interest, equally …
Russia has accused the British Embassy's top trade official in Moscow of espionage, the British Foreign Office confirmed Friday.
The accusation appears likely to worsen Russian-British relations, already strained in part by the continuing fight for control at the TNK-BP oil producer, which is jointly owned by the British company and a group of Russian billionaires.
The British Foreign Office said the accused diplomat was acting head of U.K. Trade and Investment at the embassy and confirmed his name was Chris Bowers.
The espionage accusation was first made Thursday in a report carried by the Interfax news agency, which cited a source in Russia's …
NEW YORK (AP) — Brett Gardner hit a bases-loaded triple and the New York Yankees beat the Baltimore Orioles 4-2 Sunday.
Freddy Garcia (10-7) struck out six in six innings and allowed two runs on five hits to send the Yankees 22 games over .500, their highest point of the season.
Derek Jeter left after getting hit by a pitch, but X-rays only showed a bruised right middle finger.
New York, which hadn't announced any deals by the time the non-waiver trade deadline passed at 4 p.m., improved its major league best record in day games to 31-7 and won for the 20th time in its last 26 home games.
Yankees reliever David Robertson struck out the side in the eighth and pitched 1 1-3 innings of perfect relief overall before Mariano Rivera pitched the ninth for his 27th save in 31 chances.
Baltimore's Jake Arrieta (10-8) walked six batters in five innings, giving up five hits along with four runs. Only two of the runs were earned.
The Orioles have lost six of their last seven and 18 times in 24 games.
Adam Jones put the Orioles up early with an RBI single on a hit-and-run in the third inning. Jeter went far to his left at shortstop to reach Jones' hopper up the middle, but his throw to second was off and couldn't get Nick Markakis in time.
Gardner came to bat after Eric Chavez walked to lead off the fourth, Russell Martin reached on shortstop J.J. Hardy's fielding error and Eduardo Nunez singled. Gardner slowed imperceptibly to glance into the right-field corner as he rounded second, then motored straight into third to give the Yankees the lead with his sixth triple this season.
Baltimore's Chris Davis, acquired the day before in a trade for Koji Uehara, arrived Sunday morning and played first base. He went 0 for 4, striking out in his first at-bat. The Orioles, who struck out 10 times overall, also acquired right-hander Tommy Hunter in the deal, and he will join the team in Kansas City.
Jeter was hit by a pitch from Arrieta on his right knuckles in the third inning and lifted for pinch-hitter Francisco Cervelli in the fourth.
Vladimir Guerrero hit an RBI single in the sixth inning and Robert Andino stole three bases for the Orioles.
Notes: Baltimore, which was swept in a day-night doubleheader on Saturday, hasn't won a series since June 24-26 against the Cincinnati Reds. The O's last AL series win was a three-game sweep of Oakland from June 6-8. ... Cervelli, normally the Yankees' backup catcher, had to take over at second in the fifth inning after batting for Jeter. The Yankees' usual 2B, Robinson Cano, was the DH Sunday. Cano later moved to second and reliever Hector Noesi had to go into the lineup in the second spot. When the No. 2 place came up again in the order, Robertson was lifted for pinch-hitter Andruw Jones with two outs in the eighth. ... When Baltimore LF Felix Pie batted in the fourth inning, the scoreboard showed the Greek letter pi, along with 15 decimal places of the constant: 3.141592653589793.
BUENOS AIRES, Argentina -- Thousands of Argentines traveled to areligious shrine Monday, some tearfully raising stalks of wheatsymbolizing jobs gained, to give thanks for an improving economy thathas cut the unemployment rate to close to single digits for the firsttime in 13 years.
Four years after the worst economic crisis on record shattered thejob market in South America's second-largest economy, Argentines madetheir annual pilgrimage to a Roman Catholic Church of San Cayetano --a saint revered as the patron of bread, jobs and prosperity.
Many of the worshippers said they were thankful for an economy onthe mend but still worried about the future.
President Nestor Kirchner inherited an economy in tatters when hetook power three years ago. A 2002 financial meltdown brought hunger,despair and an unemployment rate that shot above 21 percent.
Kirchner said the rate for the second quarter of 2006 was 10.4percent, down from 11.4 in the first quarter.
ST. CAJETAN STARTED BANK
"I've come to give thanks that things are getting better and thatour economy seems to be slowly returning to normal," said 66-year-old retired schoolteacher Nelida Burdazco.
Known in English as St. Cajetan, San Cayetano died in Naples in1547. Among his works was the founding of a bank to help the poor andoffer an alternative to loan sharks.
Pharmaceutical and chemical maker Bayer AG has filed a federal lawsuit alleging that drugmaker Abbott Laboratories' best-selling drug, Humira, infringes on a decade-old Bayer patent.
The German company's Bayer HealthCare LLC unit sued Abbott and two of its subsidiaries, claiming the popular biotech drug used to treat severe types of arthritis and other immune disorders infringes on the U.S. patent that Bayer was awarded in 1997.
Bayer's lawsuit was filed six years after Humira was approved for U.S. sales _ and in the plaintiff-friendly U.S. District Court for the Eastern District of Texas. The lawsuit seeks triple damages for past and future patent infringement, attorney fees and other relief, but does not seek to halt sales of Humira.
Humira, which is injected, is Abbott's top revenue driver, with sales jumping 50 percent to $1.2 billion in the third quarter. In October, the company boosted its forecast for full-year sales to $4.4 billion.
Bayer does not have any products that compete with Humira, according to spokeswoman Marcy Funk.
North Chicago-based Abbott, which was sent a summons by the court on Tuesday, will "vigorously defend against this lawsuit," spokesman Scott Stoffel said.
"Humira does not infringe Bayer's patent and Abbott believes Bayer's patent is invalid," Stoffel said.
Humira is approved to treat several serious immune system disorders, including rheumatoid and other kinds of arthritis as well as Crohn's disease. The drug carries the risk of serious, possibly fatal, infections, including tuberculosis.
The Bayer patent in question, No. 5,654,407, covers a genetically engineered antibody that binds to and blocks the action of a type of immune system cell called tumor necrosis factor, or TNF. TNF is one of the cytokines, the "messengers" of the immune system, and stimulates inflammation, a key problem in the conditions that Humira treats.
According to the lawsuit, filed on Dec. 24 in Tyler, Texas, Bayer chose to file in the Eastern District of Texas because Abbott and subsidiaries Abbott Bioresearch Center Inc. and Abbott Biotechnology Ltd. sell and advertise Humira there.
Bayer's lead lawyer in the case, T. John Ward Jr., is the son of a federal judge in Marshall, Texas, who helped build the Eastern District into a patent lawsuit hotbed.
Humira competes with the drug Remicade, made by Johnson & Johnson's Centocor unit. That company and New York University filed a patent infringement suit against Abbott in April 2007, also in the Eastern District of Texas. The case is ongoing, according to Stoffel.
Abbott has a number of patents that cover Humira, including a "composition of matter" patent that doesn't expire until 2016, he said.
Bayer said its patent runs until August 2014.
Bayer's top drugs include the erectile dysfunction treatment Levitra, the antibiotics Cipro and Avelox, contraceptives Mirena and Yasmin, and Glucobay for diabetes. The company also makes agricultural products, coatings and plastics.
___
AP Business Writer David Koenig in Dallas contributed to this report.
ABSTRACT
The temperature dependencies of the infrared absorption CO bands of carboxy complexes of horseradish peroxidase (HRP(CO)) in glycerol/water mixture at pH 6.0 and 9.3 are interpreted using the theory of optical absorption bandshape. The bands' anharmonic behavior is explained assuming that there is a higher-energy set of conformational substates (CSS^sub h^), which are populated upon heating and correspond to the protein substates with disordered water molecules in the heme pocket. Analysis of the second moments of the CO bands of the carboxy complexes of myoglobin (Mb(CO)) and hemoglobin (Hb(CO)), and of HRP(CO) with benzohydroxamic acid (HRP(CO)+BHA), shows that the low energy CSS^sub h^ exists also in the open conformation of Mb(CO), where the heme pocket is spacious enough to accommodate a water molecule. In the HRP(CO)+BHA and closed conformations of Mb(CO) and Hb(CO), the heme pocket is packed with BHA and different amino acids, the CSS^sub h^ has much higher energy and is hardly populated even at the highest temperatures. Therefore only motions of these amino acids contribute to the band broadening. These motions are linked to the protein surface and frozen in the glassy matrix, whereas in the liquid solvent they are harmonic. Thus the second moment of the CO band is temperature-independent in glass and is proportional to the temperature in liquid. The temperature dependence of the second moment of the CO peak of HRP(CO) in the trehalose glass exhibits linear coupling to an oscillator. This oscillator can be a moving water molecule locked in the heme pocket in the whole interval of temperatures or a trehalose molecule located in the heme pocket.
INTRODUCTION
Heme proteins (e.g., myoglobin, Mb, and hemoglobin, Hb) significantly change configuration upon coordination of different ligands and can be studied using virtually all spectroscopic techniques covering wide intervals of times and temperatures. Therefore they are intensively used to address the problem of protein dynamics and its role in the protein functioning (1,2). The role of protein dynamics in influencing the protein function was shown by studies of CO recombination in myoglobin (Mb) after photolysis. These studies have led to the view of conformational substates (CSS) and energy barriers between them (3,4). The authors explained nonexponentiaJ kinetics of the ligand rebinding at low temperatures, postulating that, in the glassy matrix, the protein molecules are frozen in slightly different conformations, corresponding to various CSSs. Existence of CSSs was supported by the observation that proteins containing heme derivatives show inhomogeneously broadened optical spectra (5-8). Consequently, one can describe protein dynamics as a superposition of two types of motions (9). The first type is the non-protein-specific motion, corresponding to harmonic vibrations of relatively small protein parts (e.g., amino-acid internal vibrations). The second is a large-amplitude protein-specific motion, which corresponds to the protein molecule transition from one CSS to another; these transitions are affected by the protein surroundings and are strongly hindered in a glassy matrix (10-23).
The CSSs are organized hierarchically, being grouped in tiers of different energy; their population controls the protein dynamics (4). Studies of the temperature dependence of the population of these CSSs were performed using infrared (IR) (24,25). Experimental data on the temperature dependence of the intensities of the M�ssbauer and neutron scattering spectra (9,26-28) also were used to study the effect of the temperature and the solvent glass-liquid transition on the mean-square displacements of protein atoms. However, it was suggested recently (29) that the temperature dependence of these intensities stems mainly from the motion of the protein as a whole and do not reflect the internal protein dynamics.
The position of a band corresponding to the IR absorption by the heme-coordinated carbon monoxide is affected by the static protein electric field (30-40), whereas the temperature dependence of the shape of this band is sensitive to the dynamics of the heme environment (41-43). The CO band width of horseradish peroxidase (HRP(CO)) manifests very specific temperature-dependence: it weakly changes at temperatures lower than the temperature of the glass-liquid transition (T^sub c^ = 170-180 K) of the solvent (glycerol/water, pH 6.0 and pH 9.3) and increases dramatically upon heating in the liquid solvent. Earlier (41,42) we analyzed the temperature dependence of the second moment (M^sub 2^) of this band and showed that this broadening is caused by the transition of the protein between the CSSs of the lower- and higher-energy sets (CSS^sub l^ and CSS^sub h^, respectively). However, in the procedure of the evaluation of the second moments the additional uncertainty originates, which, in particular, strongly depends on the correct subtraction of the background. Therefore, the precision of the obtained parameters did not allow us to make conclusions about the nature of this state.
In this article we use the theory of optical band shape (44-46) to interpret the experimental data on the temperature dependence of the shape (not only of M^sub 2^) of the CO band of HRP(CO) at pH 6.0 and 9.3 using simultaneous fitting procedures of the spectra obtained at different temperatures. This allows us to obtain the parameters of CSS^sub h^ and, as a result, suggest the nature of this state. Using this knowledge we also discuss the effect of the trehalose glass and substrate coordination on the dynamics and function of HRP, as well as the relationship between soivent-dependent and -independent protein motions and the resultant influence of the solvent on the heme center.
Theoretical background
Our previous theoretical studies of the effect of electric field on the CO vibrational frequency (ohm) showed that 1 [Angstrom] motion of a unit point charge changes ohm by the order of 10 cm^sup -1^ (32,34). This implies that there must be weak coupling between the C-O stretching coordinate and motion of the charged or polar parts of the heme environment. This is pure dephasing, caused by this interaction, that mainly contributes to the thermal broadening of the CO band; the contribution of the relaxation processes hardly affects this dependence (47).
RESULTS
We consider different simplest models of the heme environment dynamics, which can be used to interpret the experimental data. Two of them are based on an assumption that the heme environment explores only one low-energy set of CSSs, whereas other models take into account a possibility of thermal population also of higher-energy CSSs. Note that, below, the term "heme environment" will be used to describe only a part of the whole heme environment that notably affects the CO bandshape.
Models of the heme environment dynamics
1. The energy of CSS^sub h^ is too high to be populated, and the heme environment motion is not affected by the glassliquid transition. In this case the motion of the environment is harmonic and M^sub 2^ is expected to have the characteristic temperature-dependence of Eq. 3 or, if the classic limit (Eq. 5) is fulfilled, Eq. 6.
3. The prolein including the heme environment can exist in CSS^sub 1^ and CSS^sub h^, whereas the transition between them is linked to the protein surface. However, in each of CSSs, the heme environment motion is disconnected from the protein surface and is harmonic in the whole interval of temperatures.
4. The glassy matrix arrests the motion of the heme environment in each of CSSs and does not affect the transition between them. The band has shape, described by Eq. 13, where T has to be substituted for T^sub eff^ and vice versa, T [Lef-right arrow] T^sub eff^. Note that from the physical point of view this model does not look reasonable; the fitting procedure presented below supported this conclusion.
5. Both the transition between CSS^sub 1^ and CSS^sub h^ and the motion in each of these CSSs are connected to the protein surface and, consequently, are frozen in the glassy matrix. In this case the bandshape depends on the temperature only in the liquid sample; this point can be described mathematically substituting T [arrow right] T^sub eff^ in Eq. 13.
6. Neither the transition between CSS^sub 1^ and CSS^sub h^, nor the motions in each of the states are connected to the protein surface. In this case, the bandshape is described by Eq. 13 with T^sub eff^ [arrow right] T.
The HRP(CO) spectra
It was shown earlier (41,42) that heating of the liquid samples of HRP(CO) in the liquid 60% glycerol/water (v/v) solvent at pH 6.0 and 9.3 causes very strong increase of M^sub 2^ of the CO band, which is much steeper than proportional to T. In Eq. 7, this unambiguously shows that, in the liquid solution, the heme environment moves anharmonically. At the same time, thermal broadening of the CO band at T < T^sub c^ could be well described in the framework of the harmonic model (Eq. 3).
Models 1 and 2 cannot explain such behavior of the band and should be ruled out. Models 3-6 differ only by their behavior at T < T^sub c^ and include thermal population of CSS^sub h^, which can lead to the experimentally observed broadening of the CO band.
To make the fitting procedure faster, only nine representative spectra, which correspond to the glassy (five spectra) and liquid (four spectra) samples (T^sub c^ = 180 K (41)) and span the whole experimental interval of temperatures, were considered. The fitting was performed using the LevenbergMarquardt procedure. To accommodate the experimental uncertainty, the rigid shift of the band and up to 5% variation of the intensity (I^sub 0^) upon the temperature change was allowed. Taking into account the fact that all the spectra are relatively narrow, we simulated the background by a baseline.
The result of the fitting procedure showed that only Model 3 gives a reasonable fit. The fitting procedure automatically led to the conclusion that B^sub 1^ = 0 (see Eq. 4)-implying that CSS^sub 1^ hardly contributes to the thermal broadening in both the samples. It was also obtained that criterion 5 is fulfilled in both the samples and the spectra were fitted in the classical limit (see Fig. 1 and Table 1 for the fitting parameters). Note, that the fitting procedure leads to slightly different ohm^sub h^ and ohm^sub 1^ values for the spectra measured at different temperatures, the difference between them being constant. Their mean values are presented in Table 1, and the standard deviations of the ohm^sub 1^ and ohm^sub h^ distributions in the pH 6.0 and 9.3 samples are 0.5 and 0.6 cm^sup -1^, respectively. These deviations are in the range of the experimental uncertainty.
Note that postulating of the intensity independence of temperature and simultaneous fitting of all of the spectra ailowed taking into account the contribution of broad and low-intense subbands (see, for example, spectra at 170 and 290 K in Fig. 2). If the fitting had been done for each temperature separately, these contributions would easily escape into erroneously determined background. This could lead to an incorrect conclusion about the reduction of the CO band intensity upon heating.
M^sub 2^ of A^sub 0^ band of Mb(CO)
M^sub 2^ of this band also increases very steeply upon heating in the liquid solvent (see Fig. 4 a) and cannot be interpreted in the framework of the harmonic approximation (24), It is successfully fitted to Eq. 14, neglecting the shift of the CO band upon the CSS^sub 1^ [arrow right] CSS^sub h^ transition (ohm^sub h^ = ohm^sub 1^) (fitting parameters are presented in Table 1). Note that, as remarked in the Introduction, the uncertainty of the obtained parameters is larger than in cases of fitting of spectra by themselves.
M^sub 2^ of A^sub 1^ bands of Mb(CO) and Hb(CO), and of the CO band of HRP(CO) with aromatic substrate analog, benzohydroxamlc acid (BHA)
Temperature dependences of M^sub 2^ of the A^sub 1^ band of Mb(CO) and Hb(CO), and of the CO infrared absorption band of HRP(CO)+BHA. are presented on Fig. 4 b. In this case, the M^sub 2^ increase upon heating is much weaker, than in the case of HRP(CO) and can be fitted to Model 2. The fitting parameters are presented in Table 1.
M^sub 2^ of the CO band of HRP(CO) in trehalose
The temperature dependence of M^sub 2^ of the central peak of the HRP(CO) spectrum in trehalose can be fitted in the framework of the harmonic approximation to Model 1, Eq. 3, the fitting parameters being A = 1.4 � 0.4 cm^sup -2^, B =0.17 � 0.01 cm^sup -1^, and ω = 223 � 18 cm^sup -1^.
DISCUSSION
Modeling the heme environment dynamics in HRP(CO)
As it was noted in Results, Models 1 and 2 cannot explain the anharmonic temperature dependence of the infrared absorption CO band of HRP(CO) at pH 6.0 and 9.3.
Models 3-6 include the anharmonicity (thermal population of CSS^sub h^) and in principle can cause the very strong thermal broadening. However, it is very difficult to imagine how the glassy matrix can arrest the motion inside each of CSSs and does not arrest the transition between CSSs, the latter being expected to cause much stronger change in the prolein geometry. This explains why Model 4 fails to fit to the experimental data.
Model 5 assumes that the heme environment dynamics, and, consequently, the CO bandshape are temperatureindependent in the glassy matrix. To understand why it does not work, one should carefully inspect the temperature dependence of the experimental spectra (see Fig. 2). It follows from Fig. 2 that the 12( 15) K spectra notably differ from the corresponding 170 K spectra, the 170 K spectra having much longer tails. This shows that the heme environment motion depends on temperature even al T < T^sub c^ = 180 K.
Model 6, which assumes that the populations of CSS^sub h^ and CSS^sub 1^ are changing even in the glassy matrix, leads to the toostrong temperature dependence of the bandshape al T < T^sub c^.
Model 3 fits well to the experimental data. Fig. 2 clearly shows how the increasing amplitude of heme environment motion in CSS^sub h^ upon heating leads to the appearance of the band tails in the glassy matrix. It is also clearly seen how the heating in the liquid solvent increases population of CSS^sub h^, causing essentially non-Gaussian bandshape at 230 K, where CSS^sub l^ and CSS^sub h^ are nearly equally populated. Further heating strongly reduces the contribution of CSS^sub l^ into the spectrum, signaling an almost complete transition of the system in CSS^sub h^.
The temperature dependence of the CSS^sub h^ population can be calculated using Eq. 11 and the corresponding parameters of Table 1. Fig. 3 shows that the population strongly depends on the temperature, and at 290 K ~95% of the protein molecules in the pH 6.0 sample exist in CSS^sub h^, and ~90% in the pH 9.3 sample. It follows from Fig. 3 that, in the samples with a solvent with T^sub c^ < 180 K, the band is expected to manifest stronger temperature dependence at T < 180 K than in the glycerol/water sample. This conclusion can be tested experimentally.
Note that, in principle. Models 3-6 could be generalized by invoking a larger number of the higher-energy sets of the CSSs. However, this generalization would increase a number of parameters. Since the temperature dependence under consideration is described in the framework of Model 3, this generalization would be superfluous.
Nature of the excited conformatlonal substate
The simplest explanation for the nature of CSS^sub h^ would be to suggest that it corresponds to a different protein conformation, in which the amino acids forming the HRP heme pocket move with much larger amplitude than in CSS^sub l^. The cause of this larger dynamics could be a cleavage of some hydrogen bonds that exist in CSS^sub l^ and anchor some specific amino acid. However, the close inspection of the magnitudes of parameters presented in Table 1 shows that this suggestion is most probably wrong.
The pKα of the HRP(CO) distal histidine (His^sup 42^) was reported to be 8.3 (52-55). Increasing pH above this value causes the deprotonation of His^sup 42^, transforming its positively charged imidazolinium to neutral imidazole, which has a moderate dipole moment. This change alters the structure of the distal part of the heme pocket (43). The changes in the pocket structure and the His^sup 42^ charge essentially affect the electrostatic interaction between the C-O dipole moment and the heme pocket amino acids. As a result, the CO band (40,43) notably shifts upon the change in pH from 6.0 to 9.3 (see Fig. 1).
Consequently, if the thermal broadening of the CO band was caused by the motion of the heme pocket amino acids, then the parameter of the electrostatic interaction of this motion with the C-O dipole moment (B in Eq. 4) would be essentially different at pH 6.0 and 9.3. However, it follows from Table 1 that B^sub h^ is weakly affected by the pH change, decreasing only by <10% upon the His^sup 42^ deprotonation (this decrease is of the order of the uncertainty of the fitting procedure; see Table 1), whereas B^sub l^ is the same at both pH values. Moreover, the CO band position hardly shifts upon the transition of the protein from CSS^sub l^ to CSS^sub h^ in both the pH 6.0 and 9.3 samples; this fact points to the weak change in the heme pocket structure upon the transition. Therefore, one should conclude that, most probably, the CO band broadening is caused not by the increase in the heme pocket dynamics in CSS^sub h^, but by some other factor.
From our point of view, the best candidate for this role is a disordered water molecule, which appears in the heme pocket upon heating, and CSS^sub l^ and CSS^sub h^ correspond to the protein conformation without and with this molecule in the heme pocket. Indeed, HRP has a big pocket, which can accommodate not only water, but also much bigger substrate molecules and their analogs (56,57), and the analysis of the crystal structure of ferric HRP suggests the presence of a disordered water molecule in the heme pocket (56). The pocket is less polar than the solvent, and therefore CSS^sub l^ corresponds to the protein conformation with the water molecule outside of the pocket. H2O has a strong dipole moment; it can weakly bind in different places of the heme pocket and move at these places and between them, affecting the CO band width without notably changing its position. H2O entry to the pocket from the solvent increases the entropy of the entire system; this qualitative conclusion coincides with the results of the fitting procedure (see Table I ). Moreover, at pH 6.0 (His^sup 42^ is protonated) the heme pocket is more polar and has more places to bind H2O, than at pH 9.3. Therefore, both the increase in entropy upon the water entrance (ΔS) and the inhomogeneous broadening in CSS^sub h^ (A^sub h^) are expected to be stronger at pH 6.0 than at pH 9.3. This qualitative conclusion also coincides with the results of the fitting procedure (see Table 1; note, however, the uncertainty of the ΔS evaluation). Water motion inside the heme pocket is hardly connected to the protein surface and is expected to take place in CSS^sub h^ even in the glassy environment. On contrary, the water entrance into the pocket (CSS^sub l^ [arrow right] CSS^sub h^ transition) is coupled to the large amplitude motions of the heme pocket, their arrest by the glassy matrix making water entrance impossible. The latter two features explain the ability of Model 3 (and only of Model 3) to fit the experimental data.
The appearance of the disordered water molecule in the heme pocket can be also caused by a cleavage upon heating of the hydrogen bond between one of the ordered heme pocket water molecules and the corresponding amino acid (56). We cannot exclude this possibility, but note that the presence of a hydrogen-bonded ordered water molecule in the pocket produces a static electric field. This field is expected to notably shift the CO band position. Al the same time the disordered water molecule only broadens the band. Consequently, one should expect not only broadening, but also a notable shift of the CO band upon heating when the hydrogen bonded is cleaved and the water moves in the pocket. This conclusion is in contradiction to the very close values of Ω^sub l^ and Ω^sub h^, and makes this mechanism less likely.
Additional x-ray diffraction experiments at low temperatures can be suggested to distinguish between these two mechanisms. If a water molecule enters the heme pocket at higher temperatures (in liquid solvent), the number of ordered water molecules at low and room temperatures is expected to be the same. On the contrary, if heating of the sample liberates an ordered hydrogen-bonded water molecule, the experiment must clearly show this.
It follows from the consideration presented above that position of the CO band in HRP(CO) is controlled by the electrostatic interaction with heme pocket amino acids (43), whereas main contribution to the band broadening most probably stems from the interaction with the disordered water molecule in the pocket.
Other heme proteins
On Fig. 4 a, the temperature dependences of M^sub 2^ of the CO bands of HRP(CO) at pH 6.0 and 9.3 (calculated using Eq. 14 and parameters from Table 1) and of the A^sub 0^ band of Mb(CO) at pH 5.0 (24) are presented. The A^sub 0^ CO infrared absorption band was shown (34,58-61) to correspond to the open protein conformation with the distal histidine located outside of the heme pocket. In this conformation the distal part of the heme pocket is big enough to accommodate nitrite (62) as well as the water molecule. The myoglobin heme pocket is hydrophobic; consequently the CSS^sub 1^ must correspond to the state with the water molecule out of the pocket. Upon heating, the water molecule can enter the pocket, forming CSS^sub h^. This naturally explains why Model 3, which involves an assumption about the presence of CSS^sub h^, fits in the experimental data. Our interpretation of CSS^sub h^ as CSS with a disordered water in the pocket is also supported by the fact that the band position hardly depends on the temperature (24), suggesting a small difference between Ω^sub h^ and Ω^sub 1^. Note that this result reinstates an earlier proposition (25,63) that, in the open conformation, there is CSS^sub h^ (called A'^sub 0^ by the authors). Population of this CSS^sub h^ strongly increases upon heating (see Fig. 3), being lower than for HRP in nearly the entire interval of the temperatures studied. This result is easily understandable, because the Mb heme pocket is much smaller and less polar than that of HRP.
In the closed conformation, which is mostly populated at pH 6.8, the distal histidine is located inside the heme pocket, leaving much less free space. Consequently, the CSS^sub h^ energy is much higher and its population is expected to be much less. As a result, it hardly contributes to the CO band, and the thermal broadening temperature dependence of the related A^sub 1^ CO band is expected to behave harmonically in the liquid solvent. In the glassy environment, the band is expected to depend on temperature harmonically if the heme environment is not linked to the protein surface, and be temperature independent if the heme environment is strongly linked to the protein surface. Fitting shows (see Fig. 4 b) that, in the closed conformation of Mb(CO), the latter situation takes place (Model 2). The same is true for HRP(CO) + BHA and the closed conformation of Hb(CO), showing that the BHA binding in the HRP heme pocket displaces the disordered water even at room temperature (57). Most probably in all these cases the CO broadening is caused by the electrostatic coupling to the motions of the heme pocket amino acids, which are linked to the protein surface. This cause of the broadening also presumably exists in the CSS^sub h^ of the HRP(CO) and Mb(CO) open conformation, but is masked by a much stronger contribution of the disordered water.
It was obtained recently (64) that the distal histidine relaxation after the Mb(CO) photolysis is mostly disengaged from the solvent. However, as the authors (64) note, this disengagement most probably arises from the inward direction of the histidine relaxation movement. Moreover, this displacement is relatively small. On the contrary, in the open protein conformation the distal histidine is displaced significantly outward of the pocket; this displacement is expected to be strongly linked to solvent.
Trehalose (T^sub c^ = 331 K) exists in a glassy state in the entire interval of temperatures studied in this article. The infrared absorption spectra of the pH 6.0 sample of HRP(CO) in trehalose consist of three clearly distinct peaks, at least at low temperatures (42). This is very different from other HRP(CO) spectra (Fig. 1), which manifest only one absorption peak in the interval 1900-2000 cm^sup -1^. In Fig. 4 b, the temperature dependence of M^sub 2^ of the most intense central peak is presented; despite the glassy environment, it significantly depends on temperature. This dependence was fitted to the harmonic model 1, Eq. 3. which suggests that there is no CSS^sub 1^ [arrow right] CSS^sub h^ transition. This dependence is much stronger than that of the closed conformations of Mb(CO), Hb(CO), and HRP(CO)+BHA, where there is also no CSS^sub 1^ [arrow right] CSS^sub h^ transition. Moreover, this is the only case where the quantum effects are clearly seen and the effective frequency of the active vibration is found out: ω = 223 cm^sup -1^. All these facts imply that, in this case, the heme pocket structure significantly differs from that in the glycerol/water mixture.
Three hypotheses can be invoked to explain the experimentally observed temperature dependence under consideration. First, the HRP(CO) trehalose sample contains disordered water. This is possible because the solid sample was prepared from the trehalose-water solution (42), and the probability for a water molecule to enter the pocket at T > 300 K in liquid solvent is close to 1 (see Fig. 3). Formation of the glassy matrix upon the sample drying can lock the water in the pocket keeping the system in CSS^sub h^ at all the temperatures studied. This assumption is supported by the close magnitudes of the coupling of the CO band to the heme environment: B^sub h^ = 0.24 � 0.04 (glycerol-water solvent) and 0.17 � 0.01 (trehalose glass) cm^sup -1^. The quantum effects in the temperature dependence can stem from the change in the water molecule motion, because in the glass under osmotic stress the protein in general and the heme environment in particular can become more compact.
Another possibility is that the reduction of the distances between the CO and the pocket amino acids upon the heme pocket contraction in the glassy matrix increases the coupling of the CO band to the internal vibrations of these amino acids. If this interpretation is correct, the 223 cm^sup -1^ is an effective frequency, which corresponds to a group of internal vibrations of the heme pocket amino acids.
And finally, the central peak of HRP(CO) in trehalose at pH 6.0 can correspond to the conformation with a trehalose molecule located in the pocket (despite its big size, trehalose is flexible and can enter the pocket). In this case, the stronger thermal broadening than in the cases of HRP(CO)+BHA, and closed conformations of Mb(CO) and Hb(CO) and the quantum effects, can be interpreted as a manifestation of the coupling of the CO band to internal vibration of the trehalose molecule. This assumption is supported by the facts that trehalose has vibrations in this region of 223 cm^sup -1^ (65) and another peroxidase accommodates a molecule of co-solvent, glycerol, in its pocket (66).
Note that in this article we constrained ourselves to the simplest model of the protein dynamics, which invokes only one CSS^sub h^. It is clear that it can be (and probably are) several higher energy conformational states. However, the fact that the simplest model allows interpreting the experimental data successfully shows that no conclusions about the larger number of CSSh and their nature can be done on the basis of the experimental data under consideration.
CONCLUSION
The temperature dependences of the infrared absorption CO bands of HRP(CO) at different pH values and of second moments of the CO bands of Mb(CO) and Hb(CO) were interpreted using the theory of optical absorption bandshape, The interpretation revealed very different dynamics of the heme environment in these proteins: anharmonic, caused by the presence of the high-energy CSS^sub h^; harmonic in the liquid solvent and frozen-in by the glassy matrix; and harmonic in the whole interval of temperatures studied.
In HRP(CO) and the open conformation of Mb(CO) a high-energy set of CSS^sub h^ exists. Its population strongly increases upon heating and becomes close to 1 at room temperature. Most probably this CSS^sub h^ corresponds to the protein substate with a disordered water molecule in the heme pocket. It is clear that the disordered water is also expected to affect the structure and dynamics of the heme pocket itself. Note that, to our best knowledge, this is the first example of an almost complete transition of a native protein from a lowenergy set of conformational substates to a high-energy one, caused by heating. It is the population of this CSS^sub h^, which was shown to cause the anharmonic behavior of the CO bands under discussion.
In the HRP(CO)+ BHA and closed conformations of Mb(CO) and Hb(CO) the disordered water is forced out of the pocket by BHA or the distal histidine. This strongly increases the CSS^sub h^ energy and causes a very small population of CSS^sub h^, even at the highest studied temperatures. Thus, the only contribution to the thermal broadening of the CO band stems from the electrostatic coupling of the CO vibration to the amino acids of the heme pocket. Their motions are linked to the motion of the protein surface. Therefore these motions are frozen in the glassy matrix, whereas in the liquid solvent they are well described in the harmonic approximation. As the result, the CO band second-moment is temperature independent in the glassy matrix and is proportional to the temperature in a liquid solvent.
Finally, the harmonic behavior of the central CO peak in the infrared absorption spectra of HRP(CO) in trehalose manifests electrostatic coupling of the CO vibration to an oscillator which is disengaged from the protein surface motion.
Usually enzymes have big pockets near their active site, which can accommodate the substrates. These pockets are more hydrophobic than the protein environment. Therefore the water entrance in this pocket upon heating and its presence at room temperature can be a general property of different enzymes. It is obvious that change in the number of the disordered water molecules in the pocket should affect the mechanism and dynamics of the enzyme functioning and must be taken into account when interpreting the experimental data. For example, the enthalpy and entropy of the enzymatic reactions is usually obtained from the temperature dependence of their equilibrium and rate constants. Doing so, one must remember that most probably the temperature change affects the population of different sets of the enzyme's conformational substates, which differ by the number of disordered water molecules in the active site pocket.
We thank Dr. A. Cupane for providing us with the Hb(CO) experimental data and Dr. N. Agmon for very useful discussions.
This work was supported by National Insulines of Health grant No. GM 48130.
[Reference]
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[Author Affiliation]
Andras D. Kaposi,* Jane M. Vanderkooi,[dagger] and Solomon S. Stavrov[double dagger]
* Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary; [dagger] Johnson Research Foundation,
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and
[double dagger] Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine,
Tel Aviv University, Tel Aviv, Israel
[Author Affiliation]
Submitted June 8.2005. and accepted for publication August 23, 2006.
Address reprint requests to S, S. Stavrov, Tel.: 972-3-640-9859; E-mail: stavrov@post.tau.ac.il.
I must acknowledge the letter in last week's Chronicle from Steveand Sue Skinner referring to the withdrawing of the MobilityComponent of the Disabled Living Allowance from all those disabledyoung adults in residential care.
There is absolutely no doubt that this category of person is byfar the most vulnerable, very often having no mobility, and in thecase of our son, who is quadriplegic, very reduced skills in copingwith the normal facets of life, like washing and, dressing andtoiletting.
So what has this Government decided to do but remove the oneallowance that can make their life more bearable and that gives thema tiny bit of independence? As people who know us will agree, wehave cared for our son throughout his young life and it was only aswe got older that residential care became necessary. There are 17people who live in Trowbridge with Richard, all with very differentneeds but equally disabled, and who face a life that the Governmentdeems should revolve round hospital, doctors and dentalappointments, and little else, because they are removing the fundsfor any other journeys that the residents would normally pay for.
What really concerns me is the support our MPs should be givingtheir more vulnerable constituents.
So if your readers care about our disabled young adults I suggestthey plan their future voting patterns accordingly.
I wonder what Mr Foster feels about the vindictiveness of thisCoalition Government? ANNE KEAT Rudloe, Corsham
Abstract: Two techniques (surface photographic analysis [SPA] and microscopic tumour analysis [MTA]) are described for assessing the stage of tumour development induced by intraperitoneal 4-(methyl nitrosamino)- 1 -(3 pyridyl)-l-butanone in syngeneic A/J mice. Parameters used to assess the number and size of tumours were surface tumour count (STC), surface tumour area (STA), and relative tumour surface area (RTSA) - all using SPA; and mean tumour count (MTC), mean tumour area, and relative tumour area (RTA) - all using MTA. In a study examining the possible effects of Millipore chamber implantation on lung tumours, the stage of tumour development was assessed in 239 mice using both SPA and MTA. Statistically significant correlations (Spearman-Rank) were apparent between the derived parameters: STC vs. MTC (r = 0.6, P <0.0001); STA vs. mean tumour area (r = 0.5, P <0.0001); RTSA vs. RTA (r = 0.4, P <0.0001). Therefore, it is concluded that SPA - the simpler technique - is an appropriate method for assessing the stage of tumour development in this model.
Key words: Lung neoplasms. Models, biological. Mice. Microscopy.
Introduction
Carcinoma of the lung remains the most common cause of death in the UK. Although surgery offers the only potential cure, the five-year recurrence-free survival rate, even with early stages of the disease, is only 40-70%.1 Recurrences are caused by malignant cells that remain after surgery. If few in number, the immune system, which is known to contribute to tumour defence,2 should be able to eliminate them. Unfortunately, surgery has an adverse effect on many components of the immune response,3,4 and can be regarded as a `two-edged sword' - on the one hand being the only effective treatment, and on the other impairing the ability of the immune system to eliminate residual tumour cells.
Ideally, there should be a way of performing a surgical resection without depressing the immune response, and results of a study that looked at the effect of lung implanted subcutaneously in rats suggested a possible way to achieve this.5 It was shown that lung contained within a Millipore chamber (MPC) and implanted into an allogeneic (rather than syngeneic) host produced a prolific immune response around the implanted chamber.
The inference from this work is that necrosing lung within the chamber releases antigens that pass through the pores of the MPC and interact with local tissues. It is hypothesised that lung tumour implanted in a similar manner would result in the release of tumour antigen into the local tissues, causing an immune reaction, and that the MPC would prevent local spread.
A proposed clinical application is that, following surgical resection of a lung carcinoma, a segment of tumour contained within an MPC be re-implanted subcutaneously into the patient to augment the antitumour immune response, thereby reducing the likelihood of recurrent disease.
The mouse lung adenoma model6 is a long-established method for assessing potential treatments for lung carcinoma. 4-(methyl nitrosamino)-1-(3 pyridyl)1-butanone (NNK) is the most carcinogenic of the tobacco-specific nitrosamines.7 Lung tumours are induced effectively in syngeneic A/J mice by a single intraperitoneal dose of NNK, and multiple lung tumours develop within 16 weeks.8
Tumours appear as white, spherical lesions that are obvious to the naked eye, and counting these lesions has become a standard method for analysis.9,10 However, this procedure assumes that the tumour development apparent on the surface of the lung accurately reflects the situation within.
In order to assess tumour development within the lung, it is necessary to take multiple histological sections; and it has been demonstrated that sections (5 (mu)m thick) taken at 50 (mu)m intervals through the lung will identify all tumours (+/-2%) present.11 However, this process is considerably more laborious than surface assessment, and would be a major disadvantage in large-scale trials.
A study to examine the effect of subcutaneous implantation of MPCs containing lung tumour into mice with lung tumours, in order to assess a proposed method to reduce recurrence following surgical resection for lung carcinoma, is underway. An important part of this study is the analysis of the stage of tumour development in experimental subjects. Two methods are used: one is based on an examination of the lung surface (surface photographic analysis [SPA]); the other on examination of multiple histological sections of the lung (microscopic tumour analysis [MTA]).
Equivalent parameters are derived using these two techniques to assess tumour frequency and size, and a comparison made between them to determine whether the more simple surface analysis is a valid approximation of tumour development or if it is necessary to perform the more time-consuming microscopic analysis.
Here, the two techniques used to compare lung tumour development in the murine lung adenoma model are described and compared.
Materials and methods
Tumour induction
Lung tumours were induced with a single intraperitoneal injection of 10 pmol/L NNK (Lancaster Synthesis Ltd, Morecambe, Lancs, UK) in 295 syngeneic A/J mice (female, six to nine weeks old). Sixteen weeks later, MPCs containing either normal lung or lung tumour harvested from appropriate donor mice were implanted in the induced tumour group. Between one and eight weeks after MPC implantation, subgroups of mice were sacrificed and their left lungs harvested for analysis of the stage of tumour development (211 subjects in 19 experimental groups). The right lungs were used for analysis of cellular content, which formed part of the overall study but not reported here. Uniform tumour distribution across both lungs has been reported previously in this model.12
Analysis of stage of tumour development
The left lung was harvested from each sacrificed experimental subject and placed in Bouin's solution for 4 h, then placed in 70% ethanol overnight. Each lung was photographed and histological processing completed to paraffin wax for subsequent sectioning.
Surface photographic analysis: Standardised black and white photographs were taken of the medial and lateral surfaces of each lung (Figure 1). Each photograph was analysed to derive the following parameters: surface tumour count (STC) - the total number of visible tumours identified on the medial and lateral surfaces of the lung; surface tumour area (STA) - the total surface area of the visible tumours (each tumour appeared as a circular lesion, and by taking the measured radius [r] of each visible tumour and summing all these to obtain R, the STA was derived from pi[R]^sup 2^); and relative tumour surface area (RTSA) - the ratio (%) of the STA to the lung surface area (LSA), which was derived by approximating the shape of the analysed lung to a right-angled triangle, and the product of the maximum length and width approximated to the LSA (medial and lateral surfaces).
Microscopic tumour analysis: Following embedding in paraffin wax, each lung was sectioned (5 (mu)m thick) every 50 (mu)m, two sections were picked up onto each microscope slide, and stained with haematoxylin and eosin (H&E). In the course of the study, over 4000 sections were produced and mounted on over 2000 slides. In order to make analysis feasible, a computerised image analysis system was used (Alpha Imager(TM) 2000 Documentation and Analysis System, Alpha Innotech Corporation, USA). Using a camera linked to the computer screen, the outer borders of each lung section and tumour were 'marked' using the computer mouse, and the relevant areas calculated automatically (Figure 2). From these measurements, the following parameters were derived: mean tumour count (MTC) - the total number of tumours identified for each lung, divided by the number of sections; mean tumour area - mean area of each section occupied by tumour; and relative tumour area (RTA) -the ratio (%) of the mean tumour area to the mean lung area.
Statistical analysis
The parameters derived by the two techniques assessed equivalent measurements. Both STC and MTC were measures of the number of lung tumours present. STA and mean tumour area were both measures of the absolute size of the tumours. RTSA and RTA were measures of the size of the tumours, relative to the lung.
These equivalent parameters derived from each experimental subject were compared using the Spearman-Rank correlation test to assess their similarity in assessing the degree of tumour development. A correlation coefficient based upon ranked rather than the original observed values was used so that the outcome would not be affected severely by extreme or influential observations.13
Results
The data obtained by the two methods (mean +/- SEM) are shown in Table 1. This was compared for each assessment of tumour development (tumour frequency, tumour area and RTA) between the values obtained by SPA and MTA. The resulting correlation coefficients and the associated statistical significance were as follows:
Assessment of tumour frequency, STC vs. MTC: r = 0.6, P < 0.0001
Assessment of tumour area, STA vs. mean tumour area: r = 0.5, P <0.0001
Assessment of relative tumour area, RTSA vs. RTA: r = 0.4, P <0.0001
Overall, no differences were apparent between the results of either SPA or NITA to assess the stage of tumour development. At one week post-implantation, there was significantly less tumour development in group 2, compared with groups I and 3. At eight weeks post-implantation, there was significantly greater tumour development in group 19, compared with groups 15, 16, 17 and 18. These differences between groups were demonstrated by both SPA and MTA.
Discussion
Prerequisites for use of an animal tumour model to assess different manipulations on tumour development are that measurements of the stage of tumour development are accurate, robust and reproducible. Moreover, it should be relatively simple to ensure that the study is viable. However, if simplicity results in a less accurate study, it would have little value.
Two techniques for analysis of stage of tumour development were used: SPA and MTA. SPA is based upon the surface appearance of the lungs, and attempts to increase the accuracy of the standard simple tumour count by using a permanent photographic record, and includes parameters to assess the size of the tumours (both absolute and relative to the lungs).
Whilst this approach provides more information than a simple tumour count, it assumes that the external appearance reflects tumour development within the lung parenchyma. An analysis based upon examination of histological sections does not rely upon such an assumption; however, the number of sections required to reflect tumour development accurately is unclear.
Previously, it has been reported that sections of lung taken every 50 ltm would identify all (+/-2%) tumours,11 and this protocol (MTA) was used as the second technique for assessing the stage of tumour development. On average, 20 sections were cut from each lung, and, for the whole study, this equated to over 4000 sections on over 2000 slides.
Whilst a computerised image analysis system was used, the time commitment required for analysis of the sections was considerable (over 200 man-hours); in addition to which there was the time required to produce the slides. It is likely that MTA would be unfeasible in a significantly larger study.
The question that this report seeks to answer is whether or not a less time-consuming technique, based upon the surface appearance of the lung, would provide sufficiently comparable results from which to derive valid conclusions. The results presented here show statistically significant correlations (P <0.0001) between equivalent parameters measured by the two techniques (SPA and MTA); however, the correlation coefficients varied between the two techniques (0.4-0.6), indicating that 'similar' estimates are not identical measurements.
To a certain extent, the apparent highly significant P value is a reflection of the large overall size of the study group. The differences in measurements obtained by the two techniques may be due either to one of them providing a less accurate estimate, or to the possibility that they are measuring different aspects of that parameter. This study does not differentiate between these two possibilities. It is likely that MTA provides a more accurate measurement, and SPA is an approximation to this measurement. However, in any experimental study, the relevance of the accuracy of a measurement is whether or not it is sufficient to determine true differences if they exist between experimental groups.
Both MTA and SPA identified differences between the same experimental groups; therefore, whilst it has not been possible to demonstrate whether or not SPA is as accurate as MTA, both provided 'similar' measures and identical conclusions in the overall study.
Conclusions
Surface photographic analysis provides a valid assessment of tumour development in the mouse tumour model used, and is particularly appropriate for largescale studies. Microscopic tumour analysis, whilst potentially the more accurate assessment, should be restricted to small-scale studies because it requires a considerable amount of time to perform.
[Reference]
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[Author Affiliation]
LINDSAY C. H. JOHN
Department of Cardiothoracic Surgery, Kings College Hospital, Denmark Hill, London SE5 9RS, UK
(Accepted 10 April 2001)
Pollution prevention is an increasingly important mission in the chemical process industries (CPI). Some firms are even adopting it as a way of doing business, not only for the environmental benefits it brings, but also because it makes sense economically.
An earlier CEP article, "Follow this Path to Pollution Prevention," by James A. Dyer and Kenneth L. Mulholland, Jan. 1998, pp. 34-42, presented a methodology for identifying and implementing pollution-prevention technologies and practices. A follow-up article, "Prevent Pollution via Better Reactor Design and Operation," Feb. 1998, pp. 6166, discussed one area in more detail and outlined strategies to minimize wastes by improving reactor selectivity.
In this issue, the authors look at two additional areas of technology and practice:
* "Prevent Pollution in Batch Processes," pp. 24-29 (coauthored by Robert A. Keller)
* "Prevent Pollution in Equipment and Parts Cleaning Operations," pp. 30-34.
These authors have addressed in detail other aspects of pollution prevention, including program development, economics, and numerous other technologies and practices applicable throughout the CPI, in a new book published by AIChE, "Pollution Prevention: Methodology, Technologies, and Practices." Information about the book and how to order it is available on the AIChE Web site at http://www.aiche.org/ publication/pollutionprevention.htm, or from the AIChExpress Call Center at 1-800-242-4363 (U.S.) or 1-212-591-8100.
Because Zyvox or its derivatives have not been used for treatinginfectious diseases, it may have an advantage over other antibiotics,because bacteria might be slower to develop resistance to it.
Drug-resistant bacteria have become a growing concern in recentyears, in part because of the indiscriminate use of antibiotics.Overexposure to drugs allows bacteria to develop resistant genes.Because bacteria evolve quickly, a few resistant microbes can becomemillions literally overnight.
Because of this, the FDA has recommended that use of the drug belimited_at least at first_to patients in hospitals or otherinstitutions, to avoid overuse, which could lead to resistance.
A top Iraqi official said Sunday there was no "conclusive" evidence that Shiite extremists have been directly supplied with some Iranian arms as alleged by the United States.
Government spokesman Ali al-Dabbagh said Iraq does not want trouble with any country, "especially Iran."
Al-Dabbagh was commenting on talks this week in Tehran between an Iraqi delegation and Iranian authorities aimed at halting suspected Iranian aid to some Shiite militias.
Asked about reports that some rockets made in 2007 or 2008 and seized in raids against militias were directly supplied by Iran, al-Dabbagh replied: "There is no conclusive evidence."
The U.S. accuses Iran of financing and training Shiite militants in Iraq and of funneling lethal weapons into the country. Iranian officials have denied the allegations.
Al-Dabbagh said Iraq wants friendly ties with Iran and stressed both countries share common interests.
"We can't ignore or deny we are neighbors. We do not want to be pushed in a struggle with any country, especially Iran," he told a news conference.
"We are fed up with past tensions that we have paid a costly price for because some parties have pushed Iraq (in the past) to take an aggressive attitude to Iran."
But he also said a crackdown on Shiite militants will not stop, despite word that Iran will not restart security talks with the United States until the fighting is halted.
Al-Dabbagh told reporters that the Iraqi government is "seizing every opportunity to establish good relations with Iran" but that it also has a responsibility to "implement the rule of law."
"I think that the ongoing military operations in Iraq are an internal Iraqi affair and concern the Iraqi government and the coalition forces in Iraq," al-Dabbagh said. "No other party, except the Iraqis, has anything to do with this issue."
A five-member Iraqi delegation returned Saturday from Tehran where they held meetings aimed at halting the suspected Iranian aid to militiamen.
One of the meetings was with Gen. Ghassem Soleimani, commander of the Quds Force, an elite unit of Iran's Revolutionary Guard Corps that has been accused of training and funneling weapons to Shiite extremists in Iraq.
The Iraqi delegation was said to have carried documents and other material implicating the Quds Force in supplying weapons and training Shiite fighters.
U.S. military officials have said the evidence includes caches of weapons that have date stamps showing they were produced in Iran this year _ including mortars, rockets and armor-piercing roadside bombs known as explosively formed penetrators, or EFPs.
U.S.-backed Iraqi forces are in the midst of a crackdown on the Mahdi Army militia of radical Shiite cleric Muqtada al-Sadr, who is believed to be living in Iran.
According to officials familiar with the meeting, the delegation received a frosty reception from Soleimani, who questioned the origin of the documents. The officials asked not to be named for security reasons.
Iran's Fars news agency reported that Iranian negotiators told their Iraqi counterparts that as long as the U.S. carried out attacks against the Mahdi Army in Sadr City, Iran would not restart talks with the Americans.
U.S. military spokesman Rear Adm. Patrick Driscoll told reporters that the "multi-national force endorses all dialogue."
But he said Iranian involvement in destabilizing Iraq was mostly an "issue between the government of Iraq _ a sovereign nation _ and Iran to discuss and seek resolution."
____
Associated Press reporter Qassim Abdul-Zahra contributed to this story.
Japan's Honda says it plans to expand production at several factories in China to keep up with strong demand, raising its total capacity by more than a quarter to 830,000 vehicles a year by late 2012.
Guangqi Honda Automobile Co., Honda's joint venture with Guangzhou Auto Group, will spend 930 million yuan ($136 million) to raise its capacity to 480,000 vehicles a year by the second half of 2011, from the current 360,000 vehicles, the company said in a statement Tuesday.
Guangqi Honda makes Accord sedans, compact City and Fit models, and Odyssey minivans. Its sales rose 19 percent in 2009 over the year before to 365,000 vehicles, it said. Sales in January-April were up 36 percent from a year earlier at 134,000 vehicles.
Honda Motor Co. earlier announced plans to add a second production plant to its joint venture in central China's Wuhan city with Dongfeng Motor Group.
Overall, the expansions will raise Honda's capacity in China to 830,000 vehicles from the current 650,000, the statement said.
Sales of all vehicles in China in January-April climbed 61 percent from the same period of 2009 to 6.2 million. Sales growth for the year is forecast at about 15 percent.
