Comparison in sensitized and unsensitized guinea-pigs of tuberculin PPDs RT 23 and PPD-M by skin tests and lymphocyte stimulation tests. Effect of immunization time

The biological activities of tuberculin PPD RT 23 and the International Standard for Purified Protein Derivative of Mammalian Tuberculin (PPD-M) were compared in sensitized and unsensitized guinea-pigs by skin tests and lymphocyte stimulation (LS) tests. Estimates of relative potency (RP) from skin test results were dependent on the dose level, on the immunogen used, and, in guinea-pigs immunized with killed tubercle bacilli in oil, also on the immunization time. Relative potency estimates from LS results were dependent on the source of the lymphocytes and were different from estimates obtained from skin tests. Lymphocyte stimulation dose-response curves for the tuberculins were qualitatively different. In contrast to RT 23, PPD-M gave rise to non-specific skin reaction in unsensitized guinea-pigs. Both tuberculins were mitogenic to lymph node lymphocytes isolated from unsensitized guinea-pigs, PPD-M being the more mitogenic of the two tuberculins. The present results confirm that qualitatively different tuberculins cannot be unambiguously calibrated in identical terms and thus emphasize that the uncritical use of (international) standards should be avoided in tuberculin calibration.     

The utility of the macrophage migration inhibition test for in vitro titration of tuberculins

The purpose of this study was to explore the fundamental principle of the potency estimation of tuberculins, as applied in vitro by the macrophage migration inhibition test (MIT) under agarose. The MIT was performed using specifically sensitized mouse and guinea-pig peritoneal macrophages and serial dilutions of the analogous PPD (purified protein derivatives of tuberculin) tuberculins as antigen. Statistical studies performed included (a) the standard deviation of the mean migration areas, (b) the analysis of variance, (c) the regression analysis, (d) its corresponding linearity test and (e) the determination of the related correlation coefficient. It was shown for the first time and in both animal species under study that there is correspondence between the log dose-response relationship of the tuberculin PPD in MIT under agarose and the well known in tuberculin cutaneous reaction. The MIT may therefore successfully replace the in vivo titration of tuberculins.     

Characterisation of the external features ofSchistocephalus solidus (Müller, 1776) (Cestoda) from different geographical regions and an assessment of the status of the Baltic ringed sealPhoca hispida botnica (Gmelin) as a definitive host

A comparative study of some morphological (segment number, scolex morphology and biometry, length and weight) and biological (maturation in different hosts) features ofSchistocephalus solidus plerocercoids and adults from different geographical regions (Baltic Sea and the British Isles) was carried out. The length of the plerocercoids fromGasterosteus aculeatus was shown to be the variable that best correlated with segment number. A very clear bimodal distribution of segment numbers separated the majority of British and Baltic plerocercoids (British n=21, mean length 25.48, SD 5.63, range 14–34 mm; mean segment number 66.33, SD 8.68, range 51–86. Baltic n=30, mean length 33.23, SD 4.64, range 23–48 mm; mean segment number 117.27, SD 10.30, range 99–138). AdultS. solidus from the intestines of Baltic ringed sealsPhoca hispida botnica and from a Welsh cormorantPhalacrocorax carbo carbo were also compared, and a similar bimodal distribution of segment numbers was found (Baltic n=70, mean segment number 106.16, SD 10.60, range 77–136; Welsh n=98, mean segment number 73.13, SD 8.78, range 54–97). Neither the morphology nor measurements of the scolex from apical-view scanning electron microphotographs provided distinguishing features for taxonomic purposes. Of 580 adult worms from Baltic ringed seals only 2.9% were gravid, 2.1% from spring and 10.5% from autumn samples. By contrast, of 98 adults from the Welsh cormorant 46.7% were gravid. The proportion of gravid worms did not increase with increasing worm numbers in seals. Reasons for poor maturation are discussed. Plerocercoids of BritishS. solidus were fromleiurus (gymnurus) forms ofG. aculeatus, which were relatively small, whereas in the northern Baltic plerocercoids were fromsemiarmatus ortrachurus forms, which were larger. As segment number was definitively established during the growth of the plerocercoid in the stickleback, the hypothesis is proposed that segment number is a phenotypic variable related to stickleback length (size).     

Timing of cortical excitability changes during the reaction time of movements superimposed on tonic motor activity.

Seated subjects were instructed to react to an auditory cue by simultaneously contracting the tibialis anterior (TA) muscle of each ankle isometrically. Focal transcranial magnetic stimulation of the leg area of the motor cortex (MCx) was used to determine the time course of changes in motor-evoked potential amplitude (MEP) during the reaction time (RT). In one condition the voluntary contraction was superimposed on tonic EMG activity maintained at 10% of maximal voluntary contraction. In the other condition the voluntary contraction was made starting from rest. MEPs in the TA contralateral to the stimulation coil were evoked at various times during the RT in each condition. These were compared to the control MEPs evoked during tonic voluntary activity or with the subject at rest. The RT was measured trial by trial from the EMG activity of the TA ipsilateral to the magnetic stimulus, taking into account the nearly constant time difference between the two sides. The MEPs became far greater than control MEPs during the RT (mean = 332%, SD = 44 %, of control MEPs, P < 0.001) without any measurable change in the background level of EMG activity. The onset of this facilitation occurred on average 12.80 ms (SD = 7.55 ms) before the RT. There was no difference in the onset of facilitation between the two conditions. Because MEPs were facilitated without a change in the background EMG activity, it is concluded that this facilitation is specifically due to an increase of MCx excitability just before voluntary muscle activation. This conclusion is further reinforced by the observation that MEPs evoked by near-threshold anodal stimuli to the MCx were not facilitated during the RT, in contrast to those evoked by near-threshold transcranial magnetic stimulation. However, several observations in the present and previous studies indicate that MEP amplitude may be more sensitive to alpha-motoneuron activity than to motor cortical neuron activity, an idea that has important methodological implications.     

Volumetric dynamic computed tomography angiography in the topical diagnosis of sporadic insulinomas

The authors analyzed the data of contrast-enhanced multislice spiral computed tomography (MSCT) angiography versus those of volumetric dynamic computed tomography, percutaneous and endoscopic ultrasonography, selective angiography, and arterial stimulation blood sampling in the diagnosis of sporadic insulinomas depending on the size and site of the tumor. Forty-five patients (25 women and 20 men) aged 20 to 55 years (mean age, 43.4 +/- 2.8 years) with characteristic clinical symptoms and Whipple's triad, were examined. These were found to have 45 sporadic insulinomas that were located in the head and isthmus (n = 13 (28.9%)), body (n = 19 (42.2%)), and tail (n = 13 (28.9%)). The insulinomas measured 4.0 to 10.0 mm (mean size, 6.5 +/- 2.1 mm) (n = 9), 11.0 to 20.0 mm (mean size, 13.2 +/- 2.5 mm) (n = 23), and 21.0 to 25.0 mm (mean size, 22.6 +/- 2.8 mm) (n = 13). Volumetric dynamic CT angiography increases detection rates for tumors less than 1.0 cm in size to 77.8%. It enabled insulinomas to be localized in 82.2% of cases.     

Quantification of apolipoprotein D in human urine by zone immunoelectrophoresis assay: a methodological and clinical study.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in unconcentrated native human urine. A standard curve, linear between 1 and 8 mg apo D/l was obtained with ZIA. The relative coefficients of variation for this method were 5-9% (n = 15 x 6) with a mean +/- SD of 7 +/- 1.4% and below 11% (n = 6 x 15) for within-run and between-run reproducibility, respectively. Equal amounts of apo D in unconcentrated and diluted urines, in serum and of the purified protein produced the same zone migration distances indicating parallelism between the immunologic reactions of apo D in different sample matrixes. Storage experiments with normal urines demonstrated good stability of apo D in both acidic and alkalinized urine over at least 2 days at +5 degrees C and during several days at -20 degrees C to -40 degrees C. Using ZIA, urine samples from 50 normal healthy men aged 23-65 years were analyzed for apo D. Mean and SD were: 2.8 +/- 2.1 mg/l, 2.6 +/- 1.8 micrograms/min and 0.24 +/- 0.13 mg/mmol for concentration, rate of excretion and mass/creatinine concentration, respectively.     

Circadian variation and age dependence of human atrial natriuretic peptide levels in hospitalized patients

Circadian variation of plasma levels of human atrial natriuretic peptide (hANP) was studied in 8 patients less than or equal to 65 a of age (mean +/- SD = 43.8 +/- 13 a; 5 females, 3 males) and in 15 patients greater than 65 years of age (mean +/- SD = 81.4 +/- 5.7 a; 9 females, 6 males). Intraindividual variation was up to 40% relative to the day's mean level in both groups. A significant elevation of hormone levels in the evening was observed in patients greater than 65 years of age (P less than 0.002), no circadian rhythm could be detected in patients less than or equal to 65 years of age. All patients except the two eldest had average plasma levels of hANP within our normal range of 3-75 ng/l (N = 106; mean +/- SD = 29.9 +/- 15.3), found in healthy persons up to 65 years of age. We propose, that in the elderly hANP levels rise during the day by edema because of latent renal and/or cardial insufficiency. At rest, in the evening and during the night edema is eliminated by the well known nycturia, which might well be facilitated or at least partially caused by elevated levels of hANP.     

Maintenance of myoglobin concentration in human skeletal muscle after heavy resistance training

The aim of this study was to determine the effects of 8 weeks of resistance training (RT) on the myoglobin concentration ([Mb]) in human skeletal muscle, and to compare the change in the [Mb] in two different RT protocols. The two types of protocol used were interval RT (IRT) of moderate to low intensity with a high number of repetitions and a short recovery time, and repetition RT (RRT) of high intensity with a low number of repetitions and a long recovery time. A group of 11 healthy male adults voluntarily participated in this study and were divided into IRT (n = 6) and RRT (n = 5) groups. Both training protocols were carried out twice a week for 8 weeks. At the completion of the training period, the one-repetition maximal force values and isometric force were increased significantly in all the subjects, by about 38.8% and 26.0%, respectively (P < 0.01). The muscle fibre composition was unchanged by the 8 weeks of training. The muscle fibre cross-sectional areas were increased significantly by both types of training in all fibre types (I, IIa and IIb, mean + 16.1 %, P < 0.05). The [Mb] showed no significant changes at the completion of the training [IRT from 4.63 (SD 0.63) to 4.48 (SD 0.72), RRT from 4.47 (SD 0.75) to 4.24 (SD 0.80) mg x g(-1) wet tissue] despite a significant decrease in citrate synthase activity [IRT from 5.27 (SD 1.45) to 4.49 (SD 1.48), RRT from 5.33 (SD 2.09) to 4.85 (SD 1.87) micromol x min(-1) x g(-1) wet tissue; P < 0.05] observed after both protocols. These results suggested that myoglobin and mitochondria enzymes were regulated by different mechanisms in response to either type of RT. Moreover, the maintained [Mb] in hypertrophied muscle should preserve oxygen transport from capillaries to mitochondria even when diffusion distance is increased.     

Body temperature and oxygen uptake in the kinkajou (Potos flavus, Schreber), a nocturnal tropical carnivore.

Two kinkajous (Potos flavus, Procyonidae) showed marked nycthemeral variations in their rectal temperature. The mean Tr at night was 38.1 +/- 0.4 degrees C SD and 36.0 +/- 0.6 degrees C SD while resting during the day. Body temperature and O2-consumption were measured at ambient temperatures from 5-35 degrees C. With one exception at 35 degrees C, hypo- or hyperthermia was never observed. At air temperatures above 30 degrees C the bears reacted with behavioural responses. O2-consumption was minimal at Ta's from 23-30 degrees C. The mean basal metabolic rate was 0.316 ml O2 g-1 h-1 which is only 65% of the expected value according to the Kleiber formula. Below 23 degrees C heat production followed the equation : y (ml O2 g-1 h-1) = 0.727--0.018 Ta. The minimal thermal conductance was 90% of the predicted value according to the formula : C (ml O2 g-1 h-1 degrees C-1) = 1.02 W-0.505 (HERREID & KESSEL, 1967). Kinkajous are another distinct exception to the mouse to elephant curve.     

Angulate and leopard tortoises in the Thicket Biome, Eastern Cape, South Africa: populations and biomass estimates

The population parameters (morphometrics, population size and biomass) were determined for leopard and angulate tortoises in the Thicket Biome, Eastern Cape, South Africa. Leopard tortoises had a mean total length of 306.47mm (SD = 94.65mm, range 59–496mm, n = 89). Males were smaller (mean total length 277.31mm, SD = 71.85mm, range 73–377mm, n = 36) than females (mean total length = 332.33mm, SD = 97.64mm, range 59–496mm, n = 53). Angulate tortoises had a mean total length of 155.17mm (SD = 33.89mm, range 59–194mm, n = 18). Males were larger (mean total length = 186mm, SD = 8.40mm, range 174–194mm, n = 5) than females (mean total length = 143.31mm, SD = 32.48mm, range 59–178mm, n = 13). The estimated population density for leopard tortoises was 0.85 tortoises ha⁻¹ with an estimated biomass of 6.02kg ⁻¹. Angulate tortoise population density was estimated at 0.12 tortoises ha⁻¹ with a mean biomass of 0.06kg ha⁻¹. The possible consequences of this high density and biomass are discussed.     

Intraaortic administration of protamine: Method for heparin neutralization after cardiopulmonary bypass

In neutralizing heparin with intravenous protamine sulfate, hypotension may be prevented by administering the drug intraarterially. Forty patients underwent cardiac surgery with extracorporeal circulation in our hospital; each received a rapid injection of nondiluted protamine sulfate in the aortic root to reverse the effects of heparin. To maintain the blood volume at a constant level, volume expanders and inotropic drugs were avoided. The intraaortic injections ranged in duration from 0.2 min to 2.8 min, with a mean of 1.1 min. The mean systolic pressure only dropped from 92 mm Hg (SD +/- 21) before protamine injection to 85 mm Hg (SD +/- 23) after injection (p < 0.0001). In seven patients (18%), no hypotension was evident; in the remaining patients, the systolic pressure returned to preinjection values within a mean of 2.2 min. Coagulation was observed within 3 to 4 min (mean = 2.2 min) after the initiation of injection. This study indicates that intraaortic administration of protamine is a rapid and safe technique for heparin reversal after cardiopulmonary bypass.     

On the relationship between body dimensions and appendix length

In the last decades a lot of significant correlations between clinical and anthropometric parameters were found, e.g. the relationships between somatotypical and endocrine parameters or between body dimensions and echocardiographic heart size. During operation, surgeons often find a wide range of appendix size of patients with varying body dimensions. A goal of this work is the detection of the relationships between anthropometric characteristics (height, weight, body mass index (= BMI)), inflammation parameters (histological inflammation degree, leukocyte count and C-reactive protein (= CRP)) and the appendix length with n=167 consecutive patients (time period 1.1.2004 - 31.12.2006; range of age 7-95 years, median age 20 years; average age of men 27.7, women 26.6 years), which had to be operated because of acute appendicitis. Appendectomy was laparoscopic in 95 % of the patients and open in 5 %. The appendix length of the male patients was high-significantly longer with a mean of 7.5 cm (SD = 2.1) than those of the female patients with 6.3 cm (SD = 1.6). The following mean anthropometric values were registered: height 169.8 (SD = 15.6) cm for the males and 165.2 (SD = 9.6) cm for the females, weight 70.4 (SD = 23.4) kg for the males and 61.9 (SD = 16.1) kg for the females, BMI 24.1 (SD = 5.4) kg/m2 for the males and 22.9 (SD = 4.8) kg/m2 for the females. The mean leukocyte count was 12,700 (SD = 5500)/ml for the males and 11,600 (SD = 4900)/ml for the females, CRP was 4.3 (SD = 5.4) mg/l for the males and 4.5 (SD = 8.9) mg/l for the females. Concerning the histopathological diagnosis, n=76 patients (45.5 %) suffered from a submucous fibrosis bliteration, n 1 (12.6 ) from an acute purulent inflammation degree, n = 65 (38.9 ) from an ulcerative hlegmonous inflammation degree and n (3 ) from a perforation. While no significant Spearman's correlation coefficient between appendix length and CRP was found, the appendix length correlated highly significantly to the body weight (r = 0.25) and significantly to leukocyte count (r = 0.16), body height (r = 0.16), BMI (r = 0.17) and the histological inflammation degree (r = 0.18). Thus, the correlations found were lower than the relationships between anthropometric and endocrine or echocardiographic parameters described in literature.     

Quantitative determination of juvenile hormone III and 20-hydroxyecdysone in queen larvae and drone pupae of Apis mellifera by ultrasonic-assisted extraction and liquid chromatography with electrospray ionization tandem mass spectrometry

In this paper, a method for the rapid and sensitive analysis of juvenile hormone III (JH III) and 20-hydroxyecdysone (20E) in queen larvae and drone pupae samples was presented. Ultrasound-assisted extraction provided a significant shortening of the leaching time for the extraction of JH III and 20E and satisfactory sensitivity as compared to the conventional shake extraction procedure. After extraction, determination was carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in electrospray ionization positive ion mode via multiple reaction monitoring (MRM) without any clean-up step prior to analysis. A linear gradient consisting of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid, and a ZORBAX SB-Aq column (100 mm × 2.1 mm, 3.5 μm) were employed to obtain the best resolution of the target analytes. The method was validated for linearity, limit of quantification, recovery, matrix effects, precision and stability. Drone pupae samples were found to contain 20E at concentrations of 18.0 ± 0.1 ng/g (mean ± SD) and JH III was detected at concentrations of 0.20 ± 0.06 ng/g (mean ± SD) in queen larvae samples. This validated method provided some practical information for the actual content of JH III and 20E in queen larvae and drone pupae samples.     

The Effect of Radiotherapy on Ultrasound-Guided Fine Needle Aspiration Biopsy and the Ultrasound Characteristics of Neck Lymph Nodes in Oral Cancer Patients after Primary Treatment

Objectives

To investigate the effect of radiotherapy (RT) on ultrasound-guided fine needle aspiration (USgFNA) and sonographic characteristics in the assessment of cervical lymph nodes (LNs) in oral squamous cell carcinoma (OSCC) patients after primary treatment.

Materials and Methods

88 treated OSCC patients underwent 111 USgFNAs of the neck LNs after US evaluation. Among them, 48 USgFNAs were performed on 40 patients following RT and 63 USgFNAs on 48 patients without previous RT. The results of USgFNA and the US characteristics were compared between these two groups.

Results

USgFNA had a sensitivity of 88.0%, specificity of 91.4%, positive predictive value (PPV) of 88.0%, negative predictive value (NPV) of 91.4% and accuracy of 90.0% in patients without previous RT, and a sensitivity of 97.1%, specificity of 83.3%, PPV of 94.3%, NPV of 90.9% and accuracy of 93.5% in those with previous neck RT. The ranges of the short-axis and long-axis length were 13.3 ± 8.0 mm (mean ± SD) versus 17.8 ± 9.1 mm, and 18.6 ± 9.0 mm versus 24.4 ± 10.9 mm for recurrent LNs from patients with, versus without, previous RT (both ps < 0.05), respectively. 76.5% (26/34) of the recurrent nodes after RT and 48% (12/25) of the recurrent nodes without previous RT exhibited an irregular margin (p < 0.05). Additionally, irradiated recurrent LNs had a significantly decreased percentage of discernable calcification compared with non-irradiated recurrent nodes (48% versus 20.6%, p < 0.05).

Conclusions

RT had influence on sonographic characteristics but no influence on USgFNA in diagnosing recurrent LNs in treated OSCC patients.     

Thermolabile 5,10-methylenetetrahydrofolate reductase as a cause of mild hyperhomocysteinemia.

Thermolability of 5,10-methylenetetrahydrofolate reductase (MTHFR) was examined as a possible cause of mild hyperhomocysteinemia in patients with premature vascular disease. Control subjects and vascular patients with mild hyperhomocysteinemia and with normohomocysteinemia were studied. The mean (+/- SD) specific MTHFR activity in lymphocytes of 22 control subjects was 15.6 (+/- 4.7) nmol CH2O/mg protein/h (range: 9.1-26.6), and the residual activity (+/- SD) after heat inactivation for 5 min at 46 degrees C was 55.3 (+/- 12.0)% (range: 35.9-78.3). By measurement of MTHFR activity, two distinct subgroups of hyperhomocysteinemic patients became evident. One group (n = 11) had thermolabile MTHFR with a mean (+/- SD) specific activity of 8.7 (+/- 2.1) nmol CH2O/mg protein/h (range: 5.5-12.7) and a residual activity, after heat inactivation, ranging from 0% to 33%. The other group (n = 28) had normal specific activity (+/- SD) of 21.5 (+/- 7.2) nmol CH2O/mg protein/h (range: 10.0-39.0) and a normal residual activity (+/- SD) of 53.8 (+/- 9.2)% (range: 33.1-71.5) after heat inactivation. The mean (+/- SD) specific activity of 29 normohomocysteinemic patients was 20.7 (+/- 6.5) nmol CH2O/mg protein/h (range: 9.4-33.8), and the mean (+/- SD) residual activity after heat inactivation was 58.2 (+/- 10.2)% (range: 43.0-82.0). Thus, in 28% of the hyperhomocysteinemic patients with premature vascular disease, abnormal homocysteine metabolism could be attributed to thermolabile MTHFR.     

Rearing of Procypris rabaudi during early life-history stages

The early life‐history of Chinese rock carp Procypris rabaudi was investigated during a 56‐day rearing period: 318 artificially propagated P. rabaudi larvae were reared throughout metamorphosis in a small‐scale recirculation system (345 L water volume, 10 × 18 L rearing tanks, 150 L storage and filter compartment with bioballs, 20–30 larvae L^?1) at the Institute of Hydrobiology, Wuhan, Hubei Province, China. The newly hatched larvae had an initial total length of 8.93 ± 0.35 mm SD (n = 10) at 3 days post‐hatch and reached an average total length of 33.29 mm (±1.88 mm SD, n = 10) 56 days after hatching. Length increment averaged 0.45 mm day^?1, resulting in a mean growth of 24.4 mm within the 56‐day period. High mortality rates of up to 92% derived from an introduced fungus infection and subsequent treatment stress with malachite green. Our results indicate that Chinese rock carp can be raised successfully from artificially fertilized eggs. We therefore assume this species to be a candidate for commercial aquaculture.     

Reproducibility of the multiple inert gas elimination technique

Although measurement errors in the multiple inert gas elimination technique have a coefficient of variation of approximately 3%, small biological fluctuations in ventilation, blood flow, or other variables must contribute additional variance to this method of assessing ventilation-perfusion (VA/Q) mismatch. To determine overall variance of computed indices of VA/Q mismatch, an analysis of variance was carried out using a total of 400 duplicate pairs of inert gas samples obtained from canine (N = 118) and human (N = 282) studies in the past 2 years. In both sets VA/Q mismatch ranged from minimal (2nd moment of ventilation and blood flow distributions, log SDV and log SDQ, respectively approximately equal to 0.3 each) to severe (log SDV and log SDQ approximately equal to 2.0). Differences between duplicate log SD values were computed and found to be a constant fraction of the mean log SD of each duplicate pair, averaging 13% for both canine and human ventilation and blood flow data. The resultant coefficient of variation for a single measurement of log SD about its mean averaged 8.6% for all data combined. This analysis demonstrates excellent reproducibility of these dispersion indices over a wide range of conditions, and if the mean of duplicate values is used, thus reducing variability by square root 2 to 6.1%, log SD can be estimated with an approximately 95% confidence limit of +/- 12%.     

Improved and simplified LC-ESI-MS/MS method for homocysteine determination in human plasma: application to the study of cardiovascular diseases

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the determination of human plasma homocysteine (Hcy), an important independent risk factor for cardiovascular disease, with a simplified sample pretreatment procedure and a zero blank free of endogenous Hcy for calibrator/QC preparation. Following protein precipitation, chromatographic separation was performed on Hypersil Aquasil C18 column (50 mm x 2.1mm, 5 microm, Thermo) using mobile phase of aqueous 10% methanol containing 0.02% formic acid at 0.25 mL/min. Hcy and deuterated internal standard were detected in the multiple reaction monitoring mode with precursor to product ion transitions of m/z 136.1/90.0 and 140.1/94.0, respectively. The retention time was 1.2 min, and the total run time was 2 min per injection. A streamlined three-point calibration curve and one-point QC was used. Excellent linearity was observed with correlation coefficient (r)>0.99. The intra- and inter-batch were < or =3.24% and < or =4.04%, and accuracy was within +/-10%. Method comparison between the proposed method (y) and FPIA assay (x) demonstrated a correlation equation of y=1.003x + 0.4318 (r=0.9589). The developed method, improved for automation with cost-effective reagents, was proven to be suitable for high-throughput quantitative determination of Hcy in clinical practice by successfully applying it to the cardiovascular disease study.     

Photoaffinity labeling of tubulin with (2-nitro-4-azidophenyl)deacetylcolchicine: direct evidence for two colchicine binding sites

A new photoaffinity analogue of colchicine, (2-nitro-4-azidophenyl)deacetylcolchicine (NAPDAC), bound to two classes of sites on bovine renal tubulin and photolabeled both the alpha- and beta-subunits. The apparent Ki for the photoaffinity analogue was 1.40 +/- 0.17 microM (mean +/- SD, n = 3) as measured by competition with [3H] colchicine. Values of the apparent KdS for the two sites, as measured by the direct binding of the [3H]NAPDAC to tubulin, were 0.48 +/- 0.11 microM and 11.6 +/- 3.5 microM (mean +/- SD, n = 6), and the corresponding stoichiometries of binding of the two sites were 0.25 +/- 0.06 and 1.3 +/- 0.4 mol/mol of tubulin (mean +/- SD, n = 6). NAPDAC was a potent inhibitor of microtubule formation as detected by electron microscopy. When tubulin was photolabeled with NAPDAC at 25 degrees C, 15 +/- 3 mol % (mean +/- SD, n = 6) of the [3H]NAPDAC was covalently bound to the alpha-subunit, and 67 +/- 9 mol % (mean +/- SD, n = 6) was covalently bound to the beta-subunit. Since NAPDAC is a mixture of two interconvertible diastereomers, the photoincorporation of each was also examined. One diastereomer photolabeled both alpha- and beta-tubulin; however, the other did not significantly photolabel either subunit. Tubulin photolabeled with NAPDAC (1:1 mole ratio) exhibited a 23% decrease in colchicine binding. Preblocking and prephotolysis experiments with colchicine, NAPDAC, or ANPAH-CLC [Williams et al. (1985) J. Biol. Chem. 260, 13794-13802] provided evidence for conformational changes in tubulin upon colchicine binding. Peptide maps of [3H]NAPDAC-labeled alpha- and beta-tubulin, using Staphylococcus aureus V8 protease, demonstrated the presence of NAPDAC in one peptide of the alpha-subunit and in five peptides of the beta-subunit as detected by autoradiography. NAPDAC provides the first direct evidence for two colchicine binding sites on tubulin.     

Effect of exercise on cerebral perfusion in humans at high altitude.

The effects of submaximal and maximal exercise on cerebral perfusion were assessed using a portable, recumbent cycle ergometer in nine unacclimatized subjects ascending to 5,260 m. At 150 m, mean (SD) cerebral oxygenation (rSO2%) increased during submaximal exercise from 68.4 (SD 2.1) to 70.9 (SD 3.8) (P < 0.0001) and at maximal oxygen uptake (.VO2(max)) to 69.8 (SD 3.1) (P < 0.02). In contrast, at each of the high altitudes studied, rSO2 was reduced during submaximal exercise from 66.2 (SD 2.5) to 62.6 (SD 2.1) at 3,610 m (P < 0.0001), 63.0 (SD 2.1) to 58.9 (SD 2.1) at 4,750 m (P < 0.0001), and 62.4 (SD 3.6) to 61.2 (SD 3.9) at 5,260 m (P < 0.01), and at .VO2(max) to 61.2 (SD 3.3) at 3,610 m (P < 0.0001), to 59.4 (SD 2.6) at 4,750 m (P < 0.0001), and to 58.0 (SD 3.0) at 5,260 m (P < 0.0001). Cerebrovascular resistance tended to fall during submaximal exercise (P = not significant) and rise at .VO2(max), following the changes in arterial oxygen saturation and end-tidal CO(2). Cerebral oxygen delivery was maintained during submaximal exercise at 150 m with a nonsignificant fall at .VO2(max), but at high altitude peaked at 30% of .VO2(max) and then fell progressively at higher levels of exercise. The fall in rSO2 and oxygen delivery during exercise may limit exercise at altitude and is likely to contribute to the problems of acute mountain sickness and high-altitude cerebral edema.