GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells

The Haemophilus ducreyi homolog of GroEL, a 58.5-kDa heat shock protein (Hsp), is a dominant protein produced not only in response to heat stress but also under in vitro growth conditions. Extracellular localization of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supernatants of washed bacteria by immunoblotting with a Haemophilus ducreyi GroEL-specific mouse monoclonal antibody (BB11). To investigate binding of the Hsp to eukaryotic cells, the 58.5-kDa Hsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 cells, HeLa cells, and human fibroblasts; and then analyzed by immunoblotting. Direct involvement of the 58.5-kDa Hsp in the adherence of H. ducreyi to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results. In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to the eukaryotic cells tested but exerted only limited (about 20%) inhibition of H. ducreyi adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of H. ducreyi is associated with the bacterial surface, binds to eukaryotic cells, and partially influences H. ducreyi adherence to HEp-2 cells, indicating possible involvement of the 58.5-kDa Hsp in the attachment of bacteria to host cells and to each other.     

Electrostatic contributions to heat capacity changes of DNA-ligand binding

Significant heat capacity changes (DeltaCp) often accompany protein unfolding, protein binding, and specific DNA-ligand binding reactions. Such changes are widely used to analyze contributions arising from hydrophobic and polar hydration. Current models relate the magnitude of DeltaCp to the solvent accessible surface area (ASA) of the molecule. However, for many binding systems-particularly those involving non-peptide ligands-these models predict a DeltaCp that is significantly different from the experimentally measured value. Electrostatic interactions provide a potential source of heat capacity changes and do not scale with ASA. Using finite-difference Poisson-Boltzmann methods (FDPB), we have determined the contribution of electrostatics to the DeltaCp associated with binding for DNA binding reactions involving the ligands DAPI, netropsin, lexitropsin, and the lambda repressor binding domain.     

The haemolytic activity of some fibrous amphiboles and its relation to their specific surface areas

Determination and quantitation by mass spectrometry can be difficult for compounds in complex biological mixtures where chromatographic interferences are frequently encountered. A gas chromatographic-mass spectrometric system is described which utilizes reverse spectral search and retention time screening to provide a high degree of compound specificity. Computer control of instrument operation, and of data acquisition, analysis and printout allows technologist operators to obtain highly reliable, precise quantitative results using relatively crude sample preparation procedures and short chromatographic times.     

Physicochemical parameters of the surface of bacteria and the penicillin-binding capacity in relation to the sensitivity to penicillins and streptomycin

Development of resistance to penicillins and streptomycin in bacteria results in an increase in the electrokinetic potential changes in the hydratation rate and isoelectric levels of pH. Changes in the physico-chemical properties of the surface of penicillin-resistant Escherichia depends mainly on the accompanying dissociation. The ability of Escherichia to bind labeled benzylpenicillin is shown. Binding of 35S-benzylpenicillin by Escherichia in S-form correlates with sensitivity of the culture to the antibiotic. The penicillin-resistant mutants in R-form bound the antibiotic to a greater extent than sensitive R-cultures. The role of penicillinase in binding 35S-benzylpenicillin by Escherichia was not shown experimentally. Only increased synthesis of the enzyme by the penicillin-resistant mutants was observed. The ability of Escherichia to bind 35S-benzylpenicillin does not depend on the physico-chemical properties of the cell surface and dissociation.     

Effect of ischemia--reperfusion on heat shock protein 70 and 90 gene expression in rat liver: relation to nutritional status

p53 gene mutations occur in most human cancers and result in an altered protein product that accumulates within the cell. Although the observed endogenous human CTL response to p53 is weak, high-affinity, human p53-specific CTLs have been generated from HLA A2.1 transgenic mice immunized with human CTL epitope peptides. In this study, we examine the ability of HLA A2.1-restricted and human p53-specific CTLs from HLA A2.1 transgenic mice to suppress the growth of p53-overexpressing human tumors in severe combined immunodeficient (SCID) mice. In vitro, murine p53(149-157)-specific CTLs selectively lysed the p53-overexpressing pancreatic carcinoma cell line Panc-1 but did not recognize HLA A2.1- tumor cells or HLA A2.1+ normal human fibroblasts. Furthermore, in vivo, the growth of established human tumor xenografts in SCID mice was significantly reduced and survival was prolonged after the administration of p53-specific CTLs but not after the administration of control CTLs or PBS alone. Following treatment with p53(149-157)-specific CTLs, regressing Panc-1 tumors were infiltrated by the CD8+ CTLs, as demonstrated by immunohistochemistry. These findings suggest that p53(149-157)-specific and HLA A2.1-restricted murine CTLs suppress the growth of established Panc-1 tumors following adoptive transfer into SCID hosts and prolong their survival.     

Coronary intercapillary distance during growth: relation to PtO2 and aerobic capacity

Intercapillary distance (ICD) was measured in left ventricles of rats beating in situ. Between 40 and 400 days of age, left ventricular weight increased threefold and ICD increased from 12.5-19.5 mum. ICD could be decreased by at least 2 mum at all ages studied. The number of capillaries which must be recruited to reduce ICD by 2 mum fell from 1,200/mm2 at 40 days to 280/mm2 at 400 days. Ventricular growth did not affect the O2 sensitivity of precapillary sphincters or the uniformity of capillary spacing. Calculations indicate that under basal conditions tissue PO2 (Pto2) in subepicardium is about the same at 40 and 400 days, even though VO2 per gram, capillary density, and ICD change twofold, twofold, and 7 mum, respectively. Nevertheless, as the ventricle grows, capillary recruitment becomes progressively less effective in defending Pto2 under conditions of stress. Diminished coronary capillary compensation for stress may, in part, account for the effect of age on the maximum aerobic capacity of the whole animal.     

Erythrocyte sodium lithium countertransport in normal and hypertensive pregnancy: relation to haemodynamic changes

OBJECTIVE: To establish the changes in erythrocyte sodium lithium countertransport (SLC) with advancing normal pregnancy and to determine if these changes were different in pregnancy induced hypertension (PIH). The changes in both groups were assessed in relation to haemodynamic changes. DESIGN: SLC, mean arterial pressure (MAP), cardiac output (CO) and total peripheral vascular resistance (TPVR) were determined serially during normal pregnancy and cross-sectionally in PIH. Women were studied again 20 weeks after delivery where possible. SETTING: Routine antenatal clinic and antenatal ward of a regional reference centre. SUBJECTS: Fifty-one normal primigravid women were studied serially and 41 primigravid women with PIH were studied at time of diagnosis. RESULTS: During normal pregnancy SLC (mmol Li/h/l cells) increased from a nonpregnant value of 0.24 +/- 0.02 (mean +/- SEM) to 0.32 +/- 0.02 at 14 weeks, and 0.37 +/- 0.02 at 20 weeks gestation. This was maintained until 38 weeks (0.40 +/- 0.02). The increase until 20 weeks occurred at the time of greatest change in CO (5.10 +/- 0.18 to 6.79 +/- 0.20 l/min) and TPVR (1327 +/- 58 to 969 +/- 33 dyn/s/cm-5). The decrease in TPVR with a rise in SLC is opposite to the relation reported in essential hypertension so that a functional relation is unlikely. However, the changes within pregnancy were positively correlated (r = 0.43, P < 0.01). In hypertensive pregnancies TPVR was elevated compared with normotensive pregnancies (1543 +/- 100 vs 1090 +/- 37) but the SLC was not different from that found in normotensive pregnancies (0.43 +/- 0.02 vs 0.40 +/- 0.02). CONCLUSIONS: The changes in SLC activity suggest dynamic effects on erythrocyte membrane function during pregnancy. However, no differences could be found between normal and hypertensive pregnancy and SLC is unlikely to be of value as a marker of hypertensive risk during pregnancy.     

Exercise capacity in patients with beta-thalassemia major: relation to left ventricular and atrial size and function

OBJECTIVES: The objective of this study was to examine the association between exercise capacity and echocardiographic indexes of atrial and ventricular function and size in patients with beta-thalassemia major. BACKGROUND: In patients with beta-thalassemia major, the assessment of cardiac function with echocardiography alone does not always correspond to their functional status. Peak oxygen uptake and anaerobic threshold, on the other hand, constitute 2 objective and reproducible determinants of exercise capacity in patients with heart failure. METHODS AND RESULTS: Forty consecutive patients (22 women and 18 men, 18 to 30 years old) who were in stable condition while receiving regular transfusions and 30 age- and sex-matched control subjects were studied. At 2 to 3 days after the last transfusion, each subject underwent complete echocardiographic study followed by cardiopulmonary exercise testing. Left atrial volumes (maximal [Vmax], at onset of atrial systolic [Vp], and minimal [Vmin]) and left ventricular volumes were measured with the biplane area-length method, and left atrial active emptying fraction (ACTEF) and left ventricular ejection (LVEF) fraction were calculated. Peak oxygen uptake (Vo 2 max) and anaerobic threshold (AT) were also estimated. After transfusion, patients with beta-thalassemia major had reduced Vo 2 max and AT and greater left atrial volume in comparison with control subjects. Also, ACTEF and LVEF were significantly lower in the patient group. Moreover, Vo2 max and AT were inversely related to Vmax (r = -0.74 and r = -0.80, respectively) and directly related to ACTEF (r = 0.85 and r = 0.82, respectively) in beta-thalassemia major, whereas they were poorly related to LVEF (r = 0.50 and r = 0. 53, respectively). In the control group, Vo 2 max and AT parameters were related to Vmax and ACTEF in a similar way to that in the beta-thalassemia group. CONCLUSIONS: In patients with beta-thalassemia major, exercise capacity does not correlate with left ventricular dimensions and function. On the contrary, left atrial size and systolic dysfunction are probably predictors of decreased exercise capacity.     

Cytogenetic changes in 67 cranial and spinal meningiomas: relation to histopathological and clinical pattern

The cytogenetic analysis of 67 meningiomas (58 intracranial and 9 spinal tumors) identified chromosomal abnormalities in 63% of cases. When chromosomes involved in numerical and structural changes with a frequency of more than one standard deviation above the mean were considered, distinct cytogenetic patterns could be identified according to sex, anatomical location and histology. The chromosomes more frequently affected were 1, 2, 3, 4, 8, 14, 15, 19, 22, Y. No conclusion could be drawn regarding the prognostic significance of these karyotypic alterations.     

Cytological changes and conjunctival hyperemia in relation to sensory eye irritation

Expression of certain variants of dihydrofolate reductase (DHFR) in mammalian cells protects them from methotrexate. Retroviral transfer of the gene for such a variant DHFR into hematopoietic cells might permit selection of modified cells in vivo by antifolate administration or alleviate antifolate-induced myelosuppression in patients receiving antifolate therapy. We examined protection of cells of the human lymphoblastoid line, CCRF-CEM, transduced with variants of mouse DHFR. In transduced cells selected with G418 but not with antifolate, the variant that had arginine substituted for leucine 22 did not protect against either methotrexate or trimetrexate; however, four other variants did offer protection, with the best having leucine 22 changed to tyrosine. Polyclonal cultures transduced with the different variants express DHFR at about the same level, but clones within each polyclonal population differ in DHFR expression levels and in resistance. These differences in expression were shown to reflect different integration sites for proviral DNA. Exposure to trimetrexate selects highly resistant clones, with high expression due to both high copy number and integration sites that are favorable for expression. Differences in the resistance of cultures expressing different variants at the same level are due to differences in the catalytic activity of the expressed DHFR, its affinity for antifolates, and its stability.     

Histomorphometric changes of the skin in rats in relation to age

Histomorphometric evolution of skin was studied using 74 Wistar rats aged from 2 days to 34 months. Epidermal and dermal thickness, as well as surface density of collagen bundles in the superficial dermis was investigated by image analysis. Average epidermal thickness decreases progressively up to the 4th week, than it remains almost constant. Dermal thickness has a biphasic evolution. It increases rapidly during the first 3 weeks (+166%). This increase is followed by an important decrease (-55%), than dermal thickness increases again and reaches at the age of 1 year 5 times its value at birth. That thickness persists up to the end of life. Surface density of collagen bundles follows the rate of increase of dermal thickness, but variations are less important. Total increase of surface density of dermal collagen bundles between birth and the end of life corresponds to 56% of the initial value.     

Cold denaturation of yeast phosphoglycerate kinase: kinetics of changes in secondary structure and compactness on unfolding and refolding

Under mildly destabilizing conditions (0.7 M GuHCl), phosphoglycerate kinase from yeast undergoes a reversible two-step equilibrium unfolding transition when the temperature is lowered from 30 to 1 degree C (Griko, Y. V., Venyaminov, S. Y., & Privalov, P. L. (1989) FEBS Lett. 244, 276-278). The kinetics of the changes in compactness and secondary structure have been studied by means of dynamic light scattering and far-UV circular dichroism, respectively. It turned out that unfolding and refolding after an appropriate temperature jump (T-jump) was performed proceeded in substantially different ways. After a T-jump from 30 to 1 degree C, a multiphasic unfolding behavior was observed, reflecting the independent unfolding of the N-terminal and C-terminal domains with time constants of about 7 and 45 min, respectively. A remarkable feature of the unfolding process is the simultaneous change of compactness and secondary structure. Refolding after a T-jump from 1 degree C to higher temperatures occurs in two stages. At the first stage an appreciable amount of secondary structure is formed rapidly within the dead time of the T-jump, while the overall dimensions of the polypeptide chain remain essentially unchanged. Thus, an extended folding intermediate is formed at an early stage of folding. Further information of secondary structure proceeds slowly within a time range of minutes in parallel with the increase of compactness. At 30 degrees C, both domains refold simultaneously, while at 15 degrees C, independent folding can be observed. These findings are discussed with respect to predictions of existing models of folding.     

Prenatal and postnatal lead exposure induces 70 kDa heat shock protein in young rat brain prior to changes in astrocyte cytoskeleton

In previous studies we found that chronic postnatal (PN) lead exposure [1 g% (w/v)] induced astroglial hypertrophy in rat hippocampus. Since astrocytic responses change upon the stage during which exposure occurs, astroglial reactions in cerebral cortex and hippocampus of young animals were studied and compared when exposure began during development. Lead-treatment started 90 days prior to mating, and was maintained during gestation and after birth up to PN160. Alterations observed from PN21 to PN140 were assessed by antibodies to the 70kDa heat shock proteins (hsp), glial fibrillary acidic protein (GFAP) and vimentin (VIM) using immunohistochemistry, transmission electron microscopy (TEM), and computer assisted image analysis. The induction of hsp was seen from PN21 to PN45 in non-pyramidal neurons and astrocytes, and at the same time, astroglial swelling was noticed. After PN45 the resolution of this edema coincided with an increase of gliofilaments and GFAP and VIM immunoreaction (PN60-PN90). Recovery of VIM expression persisted after PN120 in the hilus; meanwhile, lipofuscin-like bodies appeared in neurons and astrocytes. Lead exposure during rapid brain growth induced hsp after weaning in neurons and astrocytes prior to astrocyte cytoskeletal changes. Astroglial and neuronal alterations could modify complex neuron-glia interactions, disturbing brain function in consequence.     

The use of Fluoresceincadaverine for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells

The use of Fluoresceincadaverine as a primary amine donor for detecting the endogenous substrates for active transglutaminase in living cells was studied. Fluoresceincadaverine was found to be suitable for labelling cells in culture as it did not induce cytotoxicity when used at 0.5 mM in culture media and diffused throughout the cell. After appropriate fixation using methanol, Fluoresceincadaverine-labelled cells were observed by direct fluorescence microscopy, allowing visualization of the substrates for active transglutaminase. Simultaneous detection of transglutaminase and of Fluoresceincadaverine incorporated into proteins strongly suggested that cytosolic transglutaminase was inactive in these living cells. However, transglutaminase co-distributed with Fluoresceincadaverine-labelled structures, which resembled a lattice. Fluoresceincadaverine-labelled proteins detected by Western blotting using an anti-Fluorescein antibody showed that, in living cells, the major transglutaminase substrate migrated at an apparent molecular weight of 220 kDa, as does fibronectin. Fibronectin was found to co-distribute with Fluoresceincadaverine-labelled lattice. This confirmed that these lattice structures were extracellular and, therefore, that transglutaminase is in an active form in this compartment. This opportunity to perform morphological and biochemical analyses in the search for transglutaminase substrates in living cells should help in determining the specific function of transglutaminases in a particular cell type as well as in universal cellular events, such as apoptosis or cell growth.     

联络巷对采空区氧化升温带影响的多场耦合模拟研究

在U+L型通风条件下,联络巷的存在对采空区遗煤自燃有重要的影响.为保证矿井安全生产,并为预防遗煤自燃提供依据,根据煤体低温氧化的反应机理,使用UDF将煤氧反应的机理编入FLUENT,对联络巷存在时采空区氧化升温带的分布规律进行多场耦合数值模拟研究.结果表明:联络巷的存在使采空区内风流场、氧浓度场及温度场都发生变化,氧化升温带不仅向回风侧偏移,而且向采空区深部移动且变宽;联络巷与工作面的距离影响氧化升温带的宽度,联络巷距工作面20 m时氧化升温带宽度最大约为25 m;反应进行10 d后,U+L型通风下采空区高温点的升温速率可达1.24 K·d^-1,是U型通风的1.5倍,但联络巷相对工作面的位置对高温点几乎没有影响;与U型通风时相比,U+L通风时回风侧的温度场中联络巷口温度最高,而且比U型通风时相同坐标位置的温度平均每天高出4 K,随着联络巷与工作面距离的不断增加,联络巷口升温速率由0.1 K·d^-1可升至0.9 K·d^-1,这在整体温度场中虽然不属于高温区域,但具有很好的升温潜质.     

Genetic changes in colorectal adenoma and cancer in relation to various morphologic aspects

Diffuse hypoxic pneumonia was found to be caused by angiotensin converting enzyme (ACE) inhibitors in two patients given enalapril and fosinopril for hypertension. Both patients developed sub-acute respiratory failure and lost weight. Imaging explorations showed multiple areas of alveolar consolidation, moderate pleural effusion and in one case linear opacities. In both cases, peripheral eosinophila was found and the bronchoalveolar lavage fluid contained lymphocytes. Progressive improvement was achieved after withdrawal of the ACE and corticosteroid therapy for three months. Subsequent x-rays and respiratory function tests returned to normal apart from persistently low CO diffusion in one patient. In view of other cases reported in the literature, ACE inhibitors should probably be included in the list of drugs capable of inducing pneumonia, notably eosinophilic pneumonia.     

Seasonal changes in heat balance of dogs acclimatized to outdoor climate

Heat production (M), dry heat loss (R+C), evaporative heat loss (E) and rectal temperature (Tre) were measured in a direct calorimeter in female mongrel dogs acclimatized to outdoor climate at Kanazawa (latitude; 36 degrees 35" N), Japan. M and total dry and evaporative heat losses (HL) were minimum at calorimeter wall temperatures (TW) of 26-29 degrees C in summer and 22-26 degrees C in winter (thermoneutral temperature; TNT). The seasonal shift of the lower critical temperature was confirmed. At TW below TNT, the values of M and HL were significantly higher in summer. At TW above TNT, these values increased. A TNT and above, M and HL were significantly higher in winter. (R+C) decreased linearly with increasing TW in both seasons. AT TW below 26 degrees C, (R+C) were significantly higher in summer. At TW above 26 degrees C, E increased greatly. The values of E were significantly higher in winter at TW 29-32 degrees C. Tre remained nearly constant at TNT and below, and increased at TW above TNT in both seasons. Mean body surface temperature (Tsf) decreased with decreasing TW. Body thermal conductance (K) was minimum at TW below 26 degrees C in summer and at TW below 22 degrees C in winter. At TW above these temperatures, K increased significantly. Whole body insulation (I) was significantly higher in winter, particularly at TW 18 degrees C. These results suggest that the dogs reared outdoors in winter acclimatized to cold in two ways; by increasing the insulating effect of the fur coat and by elevating resting heat production.     

Microbiological quality of filled pasta in relation to the nature of heat treatment

BACKGROUND: The role of nitric oxide in the ischemia/reperfusion injury of the pancreas is still unclear. In other organs, protective as well as aggravating effects have been described. We have, therefore, investigated the effect of the nitric oxide donor sodium nitroprusside on pancreatic ischemia/reperfusion injury. METHODS: In Landrace pigs, after transsection of the pancreas, complete vascular isolation of the pancreatic tail was performed. The tail was subjected to 3 hr of warm ischemia and thereafter reperfusion (6 hr). The animals were divided into a control group (n=7) and a treatment group (n=7) that received 15 mg of sodium nitroprusside after reperfusion intra-arterially into the splenic artery. RESULTS: The morphological tissue damage and lipase activity in the venous effluent of the pancreas were significantly lower in the treatment group. Partial oxygen tension in the tissue after reperfusion was markedly reduced in the control group, indicating an impairment of microcirculation. In the treatment group, however, partial oxygen tension in the tissue was significantly higher (43 vs. 20 mmHg; P<0.014). Furthermore, total blood flow through the pancreatic tail in the treatment group was found to be significantly higher in the late reperfusion period (14 vs. 9.5 ml/min at 5 hr after reperfusion; P<0.05). CONCLUSION: There is a marked impairment of pancreatic microcirculation after reperfusion. Sodium nitroprusside counteracts this impairment and has a protective effect on ischemia/reperfusion injury of the pancreas.