Determination of murein precursors during the cell cycle of Escherichia coli

A convenient and reliable method has been established that allows a quantitative determination of m-diamino[3H]pimelic acid-labelled murein precursors in 1 ml culture samples of Escherichia coli. Prior to separation by reversed-phase high-pressure liquid chromatography the lipid-linked intermediates were hydrolysed to release the muropeptides. The accuracy for the measurement of UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide) was +/- 1.9% (SD), for undecaprenyl-P-P-MurNAc-pentapeptide (lipid I) +/- 10% (SD) and for undecaprenyl-P-P-(GlcNAc-beta 1----4)MurNAc-pentapeptide (lipid II) +/- 5% (SD). The ratio of UDP-MurNAc-pentapeptide:lipid I:lipid II was about 300:1:3 for E. coli MC4100. The relative cellular concentrations of all three precursor molecules were found not to vary throughout the cell cycle. It is concluded that elongation and division of the murein sacculus is not controlled by oscillations in the concentrations of these late murein precursors.     

Grading follicular lymphoma on fine needle aspiration specimens. Comparison with proliferative index by DNA image analysis and Ki-67 labeling index

OBJECTIVE: To determine whether follicular lymphoma (FL) can be graded on fine needle aspiration (FNA) biopsies by determining the percentage of centroblasts in the neoplastic follicles on the smears. STUDY DESIGN: Eighty-nine cases of histologically confirmed cases of FL, including 31 grade 1, 46 grade 2 and 12 grade 3, were evaluated. Proliferative index (PI) by DNA image analysis (DIA) and Ki-67 labeling index (LI) were obtained on all cases. A minimum of 200 cells were counted per case (range, 200-800 cells) at 40x magnification, and the number of large cells (centroblasts) was expressed as a percentage of the total number of cells counted within the follicles. RESULTS: The percentage of centroblasts in the follicular aggregates was 9.7 +/- 2.9% in grade 1 FLs, 24.7 +/- 5.6% in grade 2 and 48.4 +/- 7.5% in grade 3. These differences were significant (P < .05). DNA image analysis of PI and Ki-67 LI differed significantly between grade 1 FLs and grade 2 and 3 FLs (P < .05), but there were no significant differences between grade 2 and 3 FLs. CONCLUSION: Determining the percentage of centroblasts in the follicular aggregates on FNA specimens is a good method of grading FLs. Using the percentage of centroblasts per follicular structure, FL grades 1, 2 and 3 were adequately distinguished. PI by DIA and Ki-67 LI clearly distinguished FL grade 1 from FL grades 2 and 3; however, it did not clearly distinguish between grades 2 and 3.     

The effect of measurement time when evaluating static muscle endurance during sustained static maximal gripping

The purpose of this study was to examine the useful measurement time when evaluating static muscle endurance by comparing various parameters during sustained static gripping for 1, 3 and 6 min. Fifteen males (mean +/- SD age 20.8 +/- 1.3 yr, height 172.9 +/- 4.6 cm, body mass 67.7 +/- 5.7 kg) and fifteen females [mean +/- SD age 20.2 +/- 0.9 yr, height 158.5 +/- 3.2 cm, body mass 55.9 +/- 4.6 kg] volunteered to participate in this study. The subjects performed the sustained static maximal grip test with a sagittal and horizontal arm position for 1, 3 and 6 min on different days. Eleven force-time parameters were selected to evaluate static muscle endurance. The trial-to-trial reliability of each measurement time of sustained static maximal gripping was very high (rxy = 0.887-0.981 (1 min), 0.912-0.993 (3 min), 0.901-0.965 (6 min)). The errors of exertion values between trials were very small (below 10%). A significant correlation was found in the following parameters: the final strength and the exponential function between 1 min and 3 min, all parameters except for the time required to reach 80% of maximal grip, the regression coefficient at post-inflection between 3 min and 6 min, and the decreasing rate between all measurement times (1 min, 3 min, and 6 min). Significant differences between the measurement times were found in all parameters except for the time to 60, 70, and 80% force decreases, and the regression coefficient of pre-inflection. There was a tendency that the longer the measurement time, the larger the decreasing force. It is suggested that for the 6 min measurement, the subjects unconsciously restrained the maximal gripping force, influenced by a psychological factor as the pain became greater. The 1 min measurement may evaluate only the remarkable decreasing phase of the decreasing force, and not evaluate the phase of an almost steady state.     

Excretion of trimethylselenonium ion in human urine

A method to permit isolation and measurement of trimethylselenonium ion [TMSe, (CH3)3Se+] from 1 liter of human urine was developed. The method was based on precipitation of TMSe with ammonium reineckate, preseparation with anion-exchange resin, and final thermal decomposition and collection of the product in HNO3. It was tested for recovery and separation from other selenium moieties present in urine using both in vivo-labeled rat urine and human urine spiked with unlabeled TMSe. Recoveries from the former were in the range 76.8-87.0% (mean +/- SD: 81.8 +/- 3.7%, n = 5), while for the latter they were in the range 72.0-93.0% (mean +/- SD for three occasions (%): 80.9 +/- 5.5, 81.4 +/- 7.8, and 78.9 +/- 1.0). The reliability of the method was tested against an HPLC procedure using in vivo-labeled rat's urine. The mean (+/- SD) percentage of urine radioactivity appearing as TMSe was 36.0 +/- 5.7% for the present and 36.2 +/- 6.6% for the HPLC method. The mean of deviations, as percentage of the HPLC method, was -0.03 +/- 8.8%. The linear regression equation for the two methods was y = -0.805 + 1.029x (r2 = 0.81). Excretion of TMSe was measured in urine samples from several persons (range: 0.18-0.37 micrograms Se/liter; mean +/- SD: 0.26 +/- 0.07, n = 9). One subject consumed three separate doses of unlabeled selenite on alternate days (Day 1, 197 micrograms Se; Day 3, 395; and Day 5, 592). For the first 24 h of each period, TMSe excretions (micrograms Se/24 h) were 0.24, 0.53, and 0.97, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous determination of oxprenolol and 2H6-labelled oxprenolol in human plasma by gas chromatography/mass spectrometry

A gas chromatographic/mass spectrometric method for the specific determination of oxprenolol and 2H6-labelled oxprenolol when both are present in the same sample is described. After addition of 13C3-labelled oxprenolol as internal standard, plasma is alkalized and extracted by a mixture of dichloromethane and diethyl ether. The residue following evaporation of the organic phase is derivatized with heptafluorobutyric anhydride. Negative ion detection with N2O as reagent gas is used for the measurements at m/z 488, 491 and 494 for oxprenolol, the 13C3-labelled internal standard and 2H6-labelled oxprenolol, respectively. The precision and accuracy of the analytical method were investigated using samples containing both unlabelled and 2H6-labelled oxprenolol. The overall mean recovery (% +/- SD, n = 70) in the concentration range 20-1500 nmol l-1 (around 6-450 ng ml-1 of the hydrochloride salts) was 100.6 +/- 3.3 and 101.0 +/- 3.5 for oxprenolol and 2H6-labelled oxprenolol, respectively. The limit of quantification was around 20 nmol l-1 for both compounds.     

Methodological and physiological variability within the ventilatory response to hypoxia in humans.

Measurement of the acute hypoxic ventilatory response (AHVR) requires careful choice of the hypoxic stimulus. If the stimulus is too brief, the response may be incomplete; if the stimulus is too long, hypoxic ventilatory depression may ensue. The purpose of this study was to compare three different techniques for assessing AHVR, using different hypoxic stimuli, and also to examine the between-day variability in AHVR. Ten subjects were studied, each on six different occasions, which were >/=1 wk apart. On each occasion, AHVR was assessed using three different protocols: 1) protocol SW, which uses square waves of hypoxia; 2) protocol IS, which uses incremental steps of hypoxia; and 3) protocol RB, which simulates an isocapnic rebreathing test. Mean values for hypoxic sensitivity were 1.02 +/- 0.48, 1.15 +/- 0.55, and 0.93 +/- 0.60 (SD) l. min(-1). %(-1) for protocols SW, IS, and RB, respectively. These differed significantly (P < 0.01). The coefficients of variation for measurement of AHVR were 20, 23, and 36% for the three protocols, respectively. These were not significantly different. There was a significant physiological variation in AHVR (F (50,100) = 3.9, P < 0. 001), with a coefficient of variation of 26%. We conclude that there was relatively little systematic variation between the three protocols but that AHVR varies physiologically over time.     

Trichogramma releases in North Carolina cotton: why releases fail to suppress heliothine pests

Field studies were conducted in 1996 and 1997 to determine the fate of naturally oviposited F3 heliothine eggs in cotton plots treated with augmentative releases of Trichogramma exiguum Pinto & Platner and nontreated plots. Four cohorts of newly oviposited eggs (< 24 h old) were followed in 1996 and two cohorts in 1997. In 1996, mean +/- SD percent parasitism, estimated by in-field studies following the fate of naturally oviposited eggs, ranged from 7 +/- 7 to 61 +/- 8% in T. exiguum release plots and 0 +/- 0 to 35 +/- 13% in control plots. The mean +/- SD percent of eggs hatched in T. exiguum release plots ranged from 1 +/- 2 to 11 +/- 4% and 7 +/- 4 to 28 +/- 10% in control plots. In 1997, mean +/- SD percent egg parasitism ranged from 27 +/- 4 to 40 +/- 3% in T. exiguum release plots and 15 +/- 18 to 25 +/- 8% in control plots. The mean +/- SD percent of eggs hatched in T. exiguum release plots ranged from 7 +/- 3 to 12 +/- 2% and 18 +/- 6 to 28 +/- 8% in control plots. Despite increased parasitism and reduced egg hatch in T. exiguum release plots, overall, there was no significant difference in larval density (all instars combined) between T. exiguum release and control plots. Combined analysis of the heliothine larval populations and egg fate data revealed that the additional egg mortality produced by released T. exiguum was offset by lower larval mortality in release plots. Because of the occurrence of compensatory mortality, the egg stage of heliothines is not an appropriate target for biological control using Trichogramma wasp releases.     

1,3-dihydroxy-5-(tridec-4′,7′-dienyl)benzene: a new cytotoxic compound from Lithraea molleoides

A dichloromethane extract from the leaves of Lithraea molleoides (Anacardiaceae), an argentine medicinal plant, showed cytotoxicity on human hepatocellular carcinoma cell line. Bioassay guided fractionation of this extract led to the isolation of a new active 5-alkyl resorcinol: 1,3-dihydroxy-5-(tridec-4',7'-dienyl)benzene. Chemical structure was established based on spectroscopic data (UV, IR, MS, 1H-NMR, 13C-NMR, COSY). This compound presented cytotoxic activity on 3 human tumoral cell lines: hepatocellular carcinoma cell line-Hep G2 (IC50 +/- SD of 68 +/- 2 microM), mucoepidermoid pulmonary carcinoma cell line-H292 (IC50 +/- SD of 63 +/- 5 microM) and mammary gland adenocarcinoma cell line -MCF7 (IC50 +/- SD of 147 +/- 5).     

The accuracy of the American College of Sports Medicine metabolic equation for walking at altitude and higher-grade conditions

The purpose of this study was to examine the accuracy of the American College of Sports Medicine (ACSM) walking equation at low walking speeds, altitude (1,550 m), and higher grades. Twenty men and women (mean +/- SD, age, 28 +/- 6 years; height, 171 +/- 13 cm; weight, 67.8 +/- 18.1 kg) completed 2 randomized testing sessions under altitude (AL) (P(I)o(2) = 123.1 mm Hg [20.93%]) and sea level control (SLC) (P(I)o(2) = 147.3 mm Hg [25.00%]) conditions. Steady-state oxygen uptake (Vo(2)) was measured while subjects walked at 50 m.min(-1) at 8 separate grades (0, 5, 10, 15, 18, 21, 24, and 27%). Steady-state Vo(2) measurements from the last 2 minutes of each grade in AL and SLC were compared to the predicted Vo(2) of each grade according to the ACSM walking equation. Mean Vo(2) differences between predicted and AL values ranged from -0.5 to 1.4 ml.kg(-1).min(-1), averaged -0.1 ml.kg(-1).min(-1) across all grades, and were significant (p < 0.05) at 0 percent grade. Mean Vo(2) differences between predicted and SLC values ranged from 0.6 to 3.0 ml.kg(-1).min(-1), averaged 1.4 ml.kg(-1).min(-1) across all grades, and were statistically significant (p < 0.05) at 0 and 5 percent. The standard error of the estimate (SEE) for the prediction of Vo(2) under AL and SLC were 2.2 and 2.0 ml.kg(-1).min(-1), respectively. Total errors for the prediction of Vo(2)max under AL and SLC were 2.3 and 2.6 ml.kg(-1).min(-1), respectively. Overall, the findings indicate that the current ACSM prediction equation for walking is appropriate for application at low speeds, moderate altitude, and higher grades.     

Abrogation of oxidative stress improves insulin sensitivity in the Ren-2 rat model of tissue angiotensin II overexpression

To evaluate the role of renin-angiotensin system (RAS)-mediated oxidative stress in insulin resistance (IR), we compared the effects of the angiotensin II (ANG II) receptor blocker (ARB) valsartan and a superoxide dismutase (SOD) mimetic, tempol, on whole body glucose tolerance and soleus muscle insulin-stimulated glucose uptake in transgenic hypertensive TG(mREN-2)27 (Ren-2) rats. Ren-2 rats and Sprague-Dawley (SD) controls were given valsartan (30 mg/kg) or tempol (1 mmol/l) in their drinking water for 21 days. IR was measured by glucose tolerance testing (1 g/kg glucose ip). IR index (AUC(glucose) x AUC(insulin)) was significantly higher in the Ren-2 animals compared with SD controls (30.5 +/- 7.0 x 10(6) arbitrary units in Ren-2 vs. 10.2 +/- 2.4 x 10(6) in SD, P < 0.01). Both valsartan and tempol treatment normalized Ren-2 IR index. Compared with SD controls (100%), there was a significant increase in superoxide anion production (measured by lucigenin-enhanced chemiluminescence) in soleus muscles of Ren-2 rats (133 +/- 15%). However, superoxide production was reduced in both valsartan- and tempol-treated (85 +/- 22% and 59 +/- 12%, respectively) Ren-2 rats. Insulin (INS)-mediated 2-deoxyglucose (2-DG) uptake (%SD basal levels) was substantially lower in Ren-2 rat soleus muscle compared with SD (Ren-2 + INS = 110 +/- 3% vs. SD + INS = 206 +/- 12%, P < 0.05). However, Ren-2 rats treated with valsartan or tempol exhibited a significant increase in insulin-mediated 2-DG uptake compared with untreated transgenic animals. Improvements in skeletal muscle insulin-dependent glucose uptake and whole body IR in rats overexpressing ANG II by ARB or SOD mimetic indicate that oxidative stress plays an important role in ANG II-mediated insulin resistance.     

Accumulated oxygen deficit increases with inclination of uphill running.

This study examined whether accumulated oxygen deficit depends on treadmill grade during uphill running. Oxygen uptake was measured during steady-state submaximal running. By linear extrapolation at each grade, energy demand was estimated for short exhaustive runs. Oxygen deficit was the difference between this estimate and accumulated oxygen uptake. Six subjects ran at grades of 1, 15, and 20% (study I), and five males trained for anaerobic metabolism ran at 1, 10.5, and 15% (study II). Accumulated oxygen deficit was 40 +/- 11 (SD), 72 +/- 20, and 69 +/- 8 ml O2/kg, respectively (study I), and 57 +/- 8, 78 +/- 10, and 100 +/- 7 ml O2/kg (study II). The finding that accumulated oxygen deficit became larger with treadmill inclination could reflect involvement of an increasing muscle mass. However, variation in accumulated oxygen deficit was too large to make this possibility the only explanation. More likely at small treadmill inclinations energy demand for high-intensity running is underestimated by extrapolation from oxygen uptake during submaximal exercise. At high grades of uphill running, accumulated oxygen deficit reached a maximum that may reflect the subjects' anaerobic capacity for running. This hypothesis was substantiated by an enhanced accumulated oxygen deficit in the anaerobically trained subjects during 15%, but not during 1%, uphill running.     

Mevalonate kinase in lysates of cultured human fibroblasts and lymphoblasts: kinetic properties, assay conditions, carrier detection and measurement of residual activity in a patient with mevalonic aciduria

An assay has been developed for the measurement of mevalonate kinase activity in extracts of cultured human fibroblasts and lymphoblasts. Individual elements of the assay were investigated in order to achieve optimum conditions. Apparent Michaelis constants (KMapp) for the substrates mevalonic acid and adenosine-5'-triphosphate were 22 +/- 10 mumol/l and 0.42-0.53 mmol/l, respectively, in lysates of control fibroblast lines. The same values in lysates of a control lymphoblast line were 17 mumol/l and 0.23 mmol/l, respectively. Mevalonate kinase activity in extracts of cultured fibroblasts derived from 6 control individuals was 3.24 +/- (SD) 0.91 nmol/min/mg protein. The activity in extracts of fibroblasts derived from a patient with mevalonic aciduria was 0.15 +/- 0.10 nmol/min/mg protein, approximately 5% of the control mean. The parents and brother of the patient displayed mevalonate kinase activities in fibroblast extracts approximating 38-42% of the control mean. Substantially higher mevalonate kinase activity was documented in extracts of cultured lymphoblasts. When assayed on various occasions, the mean activity of mevalonate kinase in extracts of lymphoblasts derived from the parents, brother and maternal grandmother of the patient ranged from 27 to 32% of the mean activity of 9.8 +/- (SD) 3.4 nmol/min/mg protein measured in a parallel control lymphoblast line, while the mean activity in a maternal and paternal uncle approximated 65-89% of the same control mean. The mean activity in extracts of lymphoblasts derived from the patient approximated 2% of the control mean. The data suggest that the parents, brother and maternal grandmother are carriers of the defective gene responsible for mevalonate kinase deficiency, consistent with an autosomal recessive mode of inheritance.     

The lamina cribrosa in the eyes of rats, hamsters, gerbils and guinea pigs

The scleral lamina cribrosa in the eyes of adult rats, hamsters, gerbils and guinea pigs was examined by ordinary histology and by scanning electron microscopy after soft tissue digestion. The complexity of the lamina, when mounted for scanning electron microscopy, was graded on a scale of 0 to 4.5 by three independent observers under X 60 magnification in a stereo microscope. The observers were unaware of the species and were offered the 44 specimens twice in random order. The average variance attributable to an observer was 22 +/- 3% (SE) of the total variance of the gradings. The rat eyes had the least developed lamina cribrosa, with only 1-2 layers of sparse connective tissue. The mean complexity grading of 12 rat eyes was 1.6 +/- 0.15. The lamina cribrosa of the eyes of gerbils and guinea pigs was much more developed with at least 3 layers of abundant connective tissue, the mean grades of complexity being 3.4 +/- 0.09 and 3.5 +/- 0.15, respectively, in 12 eyes of each species. The lamina cribrosa in the hamster eyes was somewhat more developed than that of the rat, but much less than that of the gerbil and guinea pig. The mean grade of complexity was 2.4 +/- 0.14 in 8 eyes. In 6 pairs of rat eyes there was no correlation in grade of laminar complexity between the two eyes of the same animal. The present study makes the rat eye a candidate for experiments where a possible influence of the lamina cribrosa as such is undesired.     

Anti-tumor efficacy of interleukin-2-activated killer cells in human neuroblastoma ex vivo

We studied the ex vivo sensitivity of continuously cultured neuroblastoma cells from 3 different patients towards interleukin-2-induced cell-mediated cytotoxicity. A mean (+/- SD) target cell lysis (4 h 51Cr release) of 49 +/- 11, 46 +/- 8, and 32 +/- 11% in SMS-SAN, LA-N-1, and SK-N-BE2 cell lines, respectively, was achieved when neuroblastoma cells were co-cultured at an effector-to-target (E:T) ratio of 50:1 with peripheral blood mononuclear cells (PBMC) that had been preincubated for 4 days in the presence of recombinant interleukin-2 (rIL-2; 100 U/ml). Under identical conditions, 93 +/- 9% of Daudi cells (a standard target for rIL-2-activated killer cells) were lysed. Preincubation of rIL-2-induced PBMC cultures in the presence of irradiated neuroblastoma targets (LA-N-1, SK-N-BE2) resulted in a significant cytolytic augmentation. At E:T ratios of 50:1 and 10:1, day-4 rIL-2/LA-N-1-stimulated PBMC produced 69 +/- 7 and 41 +/- 11% lysis of LA-N-1 cells, as compared to 46 +/- 8 and 22 +/- (mean +/- SD) 7% lysis by untargeted PBMC that were preincubated with rIL-2 (100 U/ml) in the absence of LA-N-1 target cells (p less than 0.05). Co-incubation of rIL-2-induced PBMC preparations with irradiated LA-N-1 and SK-N-BE2 cells, respectively, did not significantly enhance the cytolytic activity against other neuroblastoma targets and the standard Daudi cell line (p greater than or equal to 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in regional cerebral blood flow distribution during postexercise hypotension in humans.

This investigation compared patterns of regional cerebral blood flow (rCBF) during exercise recovery both with and without postexercise hypotension (PEH). Eight subjects were studied on 3 days with randomly assigned conditions: 1) after 30 min of rest; 2) after 30 min of moderate exercise (M-Ex) at 60-70% heart rate (HR) reserve during PEH; and 3) after 30 min of light exercise (L-Ex) at 20% HR reserve with no PEH. Data were collected for HR, mean blood pressure (MBP), and ratings of perceived exertion and relaxation, and rCBF was assessed by use of single-photon-emission computed tomography. With the use of ANOVA across conditions, there were differences (P < 0.05; mean +/- SD) from rest during exercise recovery from M-Ex (HR = +12 +/- 3 beats/min; MBP = -9 +/- 2 mmHg), but not from L-Ex (HR = +2 +/- 2 beats/min; MBP = -2 +/- 2 mmHg). After M-Ex, there were decreases (P < 0.05) for the anterior cingulate (-6.7 +/- 2%), right and left inferior thalamus (-10 +/- 3%), right inferior insula (-13 +/- 3%), and left inferior anterior insula (-8 +/- 3%), not observed after L-Ex. There were rCBF decreases for leg sensorimotor regions after both M-Ex (-15 +/- 4%) and L-Ex (-12 +/- 3%) and for the left superior anterior insula (-7 +/- 3% and -6 +/- 3%), respectively. Data show that there are rCBF reductions within specific regions of the insular cortex and anterior cingulate cortex coupled with a postexercise hypotensive response after M-Ex. Findings suggest that these cerebral cortical regions, previously implicated in cardiovascular regulation during exercise, may also be involved in PEH.     

Effect of increased hydrostatic pressure on lymphatic elimination of hyaluronan from sheep lung

The effects of increased hydrostatic pressure on the concentrations of hyaluronan (hyaluronic acid) in lung lymph and serum were investigated in awake sheep with a cannula in the efferent vessel from the caudal mediastinal lymph node. Lung lymph was sampled at base line [left atrial pressure (LAP) 6.5 +/- 1.7 mmHg] and after two increases of LAP to 25.7 +/- 2.2 mmHg (level 1) and 37.0 +/- 5.1 mmHg (level 2). The lung lymph flow increased from 1.9 +/- 0.5 at base line to 9.3 +/- 2.2 and 15.9 +/- 0.7 ml/30 min, and the lymph-to-plasma concentration ratio of total protein decreased from 0.63 +/- 0.02 to 0.32 +/- 0.04 and 0.32 +/- 0.05 at the two elevated levels of LAP, respectively. The hyaluronan concentration in lung lymph was unchanged, and there was a flow-dependent elimination of hyaluronan from the lung that increased from 23 +/- 8 to 87 +/- 19 and 137 +/- 37 micrograms/30 min, respectively. The lung concentration of hyaluronan was 167 +/- 28 micrograms/g fresh lung, and at base line it was calculated that slightly less than 2% of the lung hyaluronan was eliminated by the lymphatic route in 24 h. If extrapolated to 24 h, the elimination rate of hyaluronan seen during elevated LAP would result in lymphatic elimination of 18% of the lung hyaluronan over this time period. Since hyaluronan is responsible for part of the protein exclusion in the extracellular matrix, it is plausible that washout of interstitial hyaluronan contributes to the decrease in albumin exclusion from the interstitium that occurs after an elevation of LAP.     

A short acute octreotide test for response prediction of long-term treatment with somatostatin analogues in acromegalic patients.

The usefulness of the acute octreotide test in the selection of acromegalic patients for chronic somatostatin depot analogues treatment is controversial. The aim of the present study was to determine its accuracy for chronic response prediction, and the reliability of a short version of the classic 6-hour test. The data from 26 acromegalics (19 women, 7 men, mean age 52.6+/-13.1 years) studied with an acute octreotide test (6 hours sampling for GH measurement after octreotide 100 microg s. c.) were retrospectively analyzed. Eighteen of them followed chronic somatostatin depot analogues treatment for 12 months. GH nadir was always detected at 2 hours (mean decrease 75.9+/-24%). GH levels at 2 hours positively correlated with the other time-points (r(s) 0.97, 0.98, 0.97, 0.96 at 3, 4, 5 and 6 h, respectively; p<0.0001). During chronic treatment with maximal effective dose for 12 months, 61% of the patients achieved IGF1 <3 SD and 22% reached IGF1 <2 SD. GH nadir correlated with IGF1 decrease at 12 months (r(s) 0.76, p<0001). GH nadir of 9.2 ng/ml predicts IGF1 <3 SD with 82% sensitivity and 58% specificity (75% PPV, 67% NPV); for IGF1<2 SD, 75% sensitivity and 58% specificity are obtained for GH nadir 3.6 ng/ml, with 33% PPV and 89% NPV. Acute octreotide test reliably predicts response to long-term treatment; the short, 2-hour version is fully informative for therapeutic decisions in acromegalic patients.     

System for dynamic measurements of membrane capacitance in intact epithelial monolayers.

Dynamic measurements of exocytosis have been difficult to perform in intact epithelial monolayers. We have designed a system that estimates with +/-1% accuracy (99% confidence) the total membrane capacitance of monolayers represented by a lumped model. This impedance measurement and analysis system operates through a conventional transepithelial electrophysiology clamp, performing all signal measurements as frequently as every 5 s. Total membrane capacitance (the series combination of apical and basolateral membranes) is the inverse of one of three unique coefficients that describe the monolayer impedance. These coefficients are estimated using a weighted, nonlinear, least-squares algorithm. Using the estimated coefficients, solution ranges for individual membrane parameters are calculated, frequently providing results within +/-20% of true values without additional electrophysiological measurements. We determined the measurement system specifications and statistical significance of estimated parameters using 1) analytical testing with circuit simulation software and equation-generated data; 2) a system noise analysis combined with Monte Carlo simulations; and 3) analog model circuits for calibration of the electronic system and to check equation-generated results. Finally, the time course of capacitance changes associated with purinergically stimulated mucin exocytosis are quantified in monolayers of the colonic goblet cell-like cell line HT29-CI.16E.     

Breast carcinoma, intratumour heterogeneity and histological grading, using geostatistics.

Tumour progression is currently believed to result from genetic instability. Chromosomal patterns specific of a type of cancer are frequent even though phenotypic spatial heterogeneity is omnipresent. The latter is the usual cause of histological grading imprecision, a well documented problem, without any fully satisfactory solution up to now. The present article addresses this problem in breast carcinoma. The assessment of a genetic marker for human tumours requires quantifiable measures of intratumoral heterogeneity. If any invariance paradigm representing a stochastic or geostatistic function could be discovered, this might help in solving the grading problem. A novel methodological approach using geostatistics to measure heterogeneity is used. Twenty tumours from the three usual (Scarff-Bloom and Richardson) grades were obtained and paraffin sections stained by MIB-1 (Ki-67) and peroxidase staining. Whole two-dimensional sections were sampled. Morphometric grids of variable sizes allowed a simple and fast recording of positions of epithelial nuclei, marked or not by MIB-1. The geostatistical method is based here upon the asymptotic behaviour of dispersion variance. Measure of asymptotic exponent of dispersion variance shows an increase from grade 1 to grade 3. Preliminary results are encouraging: grades 1 and 3 on one hand and 2 and 3 on the other hand are totally separated. The final proof of an improved grading using this measure will of course require a confrontation with the results of survival studies.     

Skeletal muscle collagen content in humans after high-force eccentric contractions.

The purpose of this study was to investigate the effects of high-force eccentric muscle contractions on collagen remodeling and on circulating levels of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in humans. Nine volunteers [5 men and 4 women, mean age 23 (SD 4) yr] each performed a bout of 100 maximum voluntary eccentric contractions of the knee extensors. Muscle biopsies were taken before exercise and on days 4 and 22 afterward. Image analysis of stained tissue sections was used to quantify endomysial collagen staining intensity. Maximum voluntary contractile isometric force was recorded preexercise and on days 1, 2, 3, 4, 8, 11, and 14 postexercise. Venipuncture blood samples were also drawn on these days for measurement of serum creatine kinase activity and concentrations of MMP-9, TIMP-1, TIMP-2, and the MMP-2/TIMP-2 complex. Maximum voluntary contractile force declined by 39 +/- 23% (mean +/- SD) on day 2 postexercise and recovered thereafter. Serum creatine kinase activity peaked on day 4 postexercise (P < 0.01). Collagen type IV staining intensity increased significantly on day 22 postexercise to 126 +/- 29% (mean +/- SD) of preexercise values (P < 0.05). Serum MMP-9 levels increased on day 8 postexercise (P < 0.01), and serum TIMP-1 was also significantly elevated on days 1, 2, 3, 4, and 14 postexercise (P < 0.05). These results suggest that a single bout of eccentric muscle contractions results in remodeling of endomysial type IV collagen, possibly via the MMP pathway.