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Biomedical subjects

B Brenner

Publications and source records attributed to B Brenner.

At least 109 records · Page 6Linked to original sources
PubMed1998

Structural features of force-generating cross-bridges. A 2D-X-ray diffraction study.

In several concepts of muscle contraction it was proposed that during force generation cross-bridges assume a rigor-like conformation. However, so far 2D-X-ray diffraction patterns recorded during isometric contraction did not reveal structural features of rigor-like cross-bridges. It was therefore supposed that the number of force generating cross-bridges in isometric steady state contraction is too low to be detected in 2D-X-ray diffraction patterns. To test this concept we studied the features of 2D-X-ray diffraction patterns of different fractions of rigor-like cross-bridges by varying the concentration of the nucleotide analog ATP gamma S at high [Ca++]. Although we had a fully activated system, i.e., that weak binding cross-bridges were attached to the activated thin filament, none of the patterns was like the active pattern. Analysis of the intensity change of the actin layer lines at 1/370 A-1 and at 1/59 A-1 in diffraction patterns recorded during isometric contraction vs. relaxed and rigor conditions as well as mechanical experiments revealed that the fraction of force generating cross-bridges apparently is as high as 55-70%. Yet, in agreement with previous studies, in spite of this large fraction of force generating cross-bridges, no rigor-like features were detected in diffraction patterns recorded during isometric contraction. Moreover, the intensity distribution along the actin layer lines was clearly different in rigor compared to isometric contraction. Taken together these results provide evidence that in contrast to the above mentioned models the majority of the force generating cross-bridges seems to be in a conformation which is different from rigor.

Adenosine Triphosphate↗
PubMed1998

Cellular stimulation via CD95 involves activation of phospho-inositide-3-kinase.

Several distinct intracellular pathways have been recently shown to be activated during CD95/Fas/APO-1-mediated apoptosis. Here, we demonstrate that CD95 ligation induces a rapid and transient tyrosine phosphorylation and activation of phosphoinositide-3-kinase (PI-3-K) in Jurkat T lymphocytes or CD95-sensitive glioma cells. Experiments using p56lck-deficient or p56lck-reconstituted Jurkat clones and the tyrosine kinase inhibitor herbimycin A revealed that tyrosine phosphorylation and activation of PI-3-K by CD95 depends on expression of Src-like tyrosine kinases, in particular p56lck. PI-3-K stimulation seems to be critical for CD95 receptor signalling since, first, inhibition of PI-3-K prevents CD95-mediated apoptosis and, second, CD95 receptor ligation fails to induce tyrosine phosphorylation or activation of PI-3-K in CD95-resistant glioma cells. Thus, PI-3-K activation may be an early signalling event during CD95-induced apoptosis, and failure to stimulate PI-3-K may predict tumor cell resistance to CD95-triggered apoptosis.

Apoptosis↗
PubMed1998

Cone and plate(let) analyzer: monitoring glycoprotein IIb/IIIa antagonists and von Willebrand disease replacement therapy by testing platelet deposition under flow conditions.

Exposure of citrated whole blood samples to polystyrene plates under flow conditions with the cone and plate(let) analyzer (CPA) results in surface immobilization of plasma von Willebrand factor (vWF) followed by platelet deposition. Staining the plates allows measurement of the percentage of surface covered (SC) by the adhered particles and their average size (AS). Both SC and AS parameters depend on platelet count and hematocrit level and reach maximal values after 2 minutes; only AS is shear rate dependent. Under optimal assay conditions (2 minutes at 1800 sec(-1)) normal blood samples yielded an SC of 14.9% +/- 2.5% and an AS of 39.4 +/- 5.2 microm2. Severe von Willebrand disease (eight patients) yielded low SC (5.2% +/- 2.1%), which was restored to normal by testing with vWF precoated surfaces. Glanzmann's thrombasthenia (six patients) samples demonstrated no adhesion of platelet at all. Blocking of the glycoprotein (GP) IIb/IIIa receptor by the chimeric antibody abciximab, and of the GPIb by a recombinant vWF fragment, have yielded a dose-response inhibition demonstrating the crucial role of these receptors in platelet deposition on polystyrene plates in this system. Evaluation of a patient with severe von Willebrand disease receiving replacement vWF factor VIII therapy revealed a comparable response as tested by both the CPA and the Ricof methods. In vitro testing of the GPIIb/IIIa blocking by a nonpeptidic analogue tirofiban and abciximab revealed a good correlation between the CPA test and the routine aggregometry. Eleven patients treated by abciximab after coronary angioplasty were studied by the CPA during the first 24 hours, demonstrating a marked decrease in SC and AS, with some diversity in the responses. We conclude that the CPA test is suitable for evaluation of primary hemostasis and for monitoring of anti-platelet drugs.

Blood Circulation↗
PubMed1998

Reactivity patterns of antiphospholipid antibodies and endothelial cells: effect of antiendothelial antibodies on cell migration.

Antiphospholipid syndrome (APS) is characterized by the presence of a heterogeneous class of antibodies directed against phospholipids and associated with high occurrence of thrombotic complications. Antiendothelial cell antibodies (AECAs) have been identified in various autoimmune disorders including APS, but their reactivity patterns remain unclear. We used eluted endothelial membrane-bound antibodies (EC eluates) to investigate possible cross-reactivity of AECAs and their pathogenic effects on endothelial cell integrity. The heterogeneous and nonspecific nature of AECAs was confirmed by our finding that they cross-react with fibroblasts and platelets and bind to cardiolipin. In addition, platelet-bound antibodies from sera of patients with APS reacted with endothelial cells. A dose-dependent binding of human monoclonal anticardiolipin antibody was demonstrated, but this antibody did not compete with AECAs in EC eluates, indicating that only small portion of AECAs are directed against cardiolipin. Although sera from APS patients prolonged coagulation tests, EC eluates did not affect coagulation, suggesting that AECAs may belong to antiphospholipid antibodies subsets that does not interfere with coagulation. Vascular damage is a common feature of autoimmune disorders associated with AECAs. Possible effects of AECAs on vascular perturbance were investigated by cytotoxicity, attachment, and migration assays. Although AECAs were not shown to be cytotoxic or to affect cell attachment, sera from APS patients caused reduced cellular migration (by 30%), and EC eluates caused even more significant inhibition (by 50%). These findings suggest possible interference of AECAs in vascular repair mechanisms and provide an explanation for the thrombotic complications frequently seen in APS patients.

Antibodies, Antiphospholipid↗
PubMed1998

The acute asthmatic patient in the ED: to admit or discharge.

Treating asthma in the emergency department (ED) always involves the potentially difficult decision as to whether to discharge the patient, to continue treatment, or to admit to the hospital. The following are useful guidelines. (1) The duration of the bronchospasm, frequency of visits, history of previous endotracheal intubation, pulse rate, and accessory muscle use are findings affecting successful discharge from the ED. (2) Patients with peak expiratory flow rate (PEFR) of < 20% and who do not respond to inhalant therapy, with PEFR values persisting at < 40% of predicted, will require 4 or more days to resolve and should be admitted to the hospital. (3) Patients with a PEFR between 40% and 70% of predicted after initial inhalant therapy may well be responsive to steroids in the ED, but an ED will adequately need to care for the patient for 5 to 12 hours while waiting for the onset of action of glucocorticoids. Discharged with glucocorticoids, this group has a 6% relapse rate within 10 days of the ED visit. (4) Patients with a PEFR of > or = 70% have a 14% relapse rate after discharge without glucocorticoids. Other reasons to consider admission are pneumonia, barotrauma, lability, prominent psychiatric difficulties, poor access to medications, poor educability, fear of steroids, patients on glucocorticoids or those who have recently stopped glucocorticoids, and evening discharges of patients from the ED, which all predispose to relapses of acute asthma. To decrease the relapse rate, provocative factors should be reviewed with the patient, as well as access to medication and use of spacers, inhaler techniques, PEFR meters, self-management plans, and referral to a defined appointment at 24 to 48 hours of the ED visit.

Acute Disease↗
PubMed1998

Sugar transport and glut transporter expression in a variety of human immunodeficiency virus-1 (HIV-1) chronically infected target cell lines.

In this study, sugar transport and the cellular content of the human Glut 1 and 3 glucose transporters were ascertained in uninfected and chronically HIV-infected Jurkat and H9 cell lines (T-cell lines) and U937 cells (a promonocytic cell line). Sugar transport was determined by monitoring 2-deoxy glucose uptake (2DG) and glut transporter content was determined by Western analysis. Although 'acute' HIV infection of H9 cells led to increased cellular transport activity and Glut 3 transporter content, chronic HIV infection exhibited no significant differences in sugar transport in any of the cell types investigated whether log or stationary phase cultures were employed. When uninfected and chronically HIV-infected cell lines were compared, all cell lines expressed the Glut 1 transporter, however, significant differences in Glut 1 transporter content were not observed. The Glut 3 transporter which could only be detected in the H9 cell line exhibited no differences in Glut 3 content in uninfected or chronically HIV-infected cells (2.1 +/- 0.6 versus 3.8 +/- 2.1 x 10(-3) arbitrary units/microgram protein). A trend towards lower amino acid uptake was seen in the chronically HIV-infected cells but this was not significantly different from uninfected cell cultures. The data indicate that: (1) glucose transport and the Glut 1 and 3 transporters are not increased in cells chronically infected with HIV-1 and (2) the expression of the Glut 3 sugar transporters is not the same in all target cells.

Biological Transport, Active↗
PubMed1998

Differential effects of mucosal pH on human (Caco-2) intestinal epithelial cell motility, proliferation, and differentiation.

Mucosal pH abnormalities are associated with anastomotic dehiscence, ischemia, and malignancy. We postulated that intraluminal pH influences intestinal epithelial motility, proliferation, and differentiation and studied extracellular pHo (7.0-8.5) effects on human (Caco-2) intestinal epithelial motility, proliferation, and differentiation. Mucosal healing was modeled by sheet migration and differentiation by alkaline phosphatase and dipeptidyl dipeptidase specific activity. In parallel differentiation and motility studies, we inhibited proliferation with mitomycin to dissociate indirect mitogenic effects. Intracellular pHi was quantitated using BCECF/AM at varying extracellular pHo and in migrating cells. Motility was maximal at pHo 7.6 and proliferation at 7.2. Each decreased with acidity and alkalinity. By contrast, brush border enzyme activity was lowest at pHo 7.0 and highest at pHo 8.5. pHi was highest at pHo 8.5. Migrating cell pHi was higher than static cell pHi. Thus, extracellular pHo deviations perturb Caco-2 pHi homeostasis and motility. Alkalinity promotes differentiation while acidity induces proliferation and limits differentiation.

Caco-2 Cells↗
PubMed1998

Fas/CD95/Apo-I activates the acidic sphingomyelinase via caspases.

Fas/CD95/Apo-I has been shown to stimulate a variety of molecules including several members of the caspase family and the acidic sphingomyelinase (Martin and Green 1995; Gulbins et al, 1995). Here, we demonstrate that Fas receptor-triggered activation of the acidic sphingomyelinase, consumption of sphingomyelin, release of ceramide, and subsequent activation of JNK and p38-K are regulated by caspases. Inhibition of caspases by Ac-YVAD-chloromethylketone or transient CrmA transfection prevented stimulation of acidic sphingomyelinase, release of ceramide and activation of JNK and p38-K upon Fas-receptor crosslinking. Likewise, Fas triggered apoptosis was almost completely blocked by Ac-YVAD-chloromethylketone or CrmA mediated inhibition of caspases. The results suggest a new signalling cascade from the Fas receptor via caspases to acidic sphingomyelinase, ceramide and JNK/p38-K.

Acids↗
PubMed1998

Coexistence of acquired protein S and protein C deficiency and the Arg506Gln mutation in factor Va in a child with severe thromboembolic disease.

An 11-y-old girl who presented with cellulitis and clinical signs of deep vein thrombosis (DVT) is reported here. She developed staphylococcal sepsis, recurrent septic emboli and a large vegetation on the tricuspid valve. The patient was found to be heterozygous for the Arg506Gln mutation in factor Va and had low levels of protein C and protein S during the sepsis. The coexistence of the two thrombophilic states may explain the severe thromboembolic manifestations.

Alleles↗
PubMed1998

Classic Kaposi sarcoma: experience at Rabin Medical Center in Israel.

Classic Kaposi sarcoma is an indolent cutaneous proliferative disease affecting mainly elderly people of Mediterranean and Jewish origin. The authors review the epidemiologic and clinical findings in Israeli patients with classic Kaposi sarcoma treated at the Institute of Oncology, Rabin Medical Center. A total of 123 patients were identified. The average age at diagnosis was 68 years (range, 20-90 years) and, as expected, there was a strong predominance of men (2.4:1). All but two patients were Jewish. The distribution of Ashkenazic Jews and Sephardic Jews was almost equal. Twenty-three patients (19%) had secondary malignancies that were mostly solid tumors. The clinical course was indolent and rarely fatal (4% disease-related mortality). Multivariate analysis revealed that non-Ashkenazic origin, age over 70 years, multiple lesions (>10), and immunocompromised conditions adversely affected survival. Radiotherapy for localized skin lesions yielded an 88% objective response, with symptomatic relief in 95% of patients. Chemotherapy was similarly effective (76% response rate) in patients with disseminated disease. These data demonstrate the indolent nature of classic Kaposi sarcoma which usually requires a less aggressive therapeutic approach than in the African and AIDS-related types of Kaposi sarcoma.

Adult↗
PubMed1998

Glanzmann thrombasthenia in two Iraqi-Jewish siblings is caused by a novel splice junction mutation in the glycoprotein IIb.

An A-->G transition in the acceptor splice site at the intron 19/exon 20 junction of the glycoprotein IIb gene was defined as a novel mutation causing Glanzmann thrombasthenia in two Iraqi-Jewish siblings. This mutant DNA was transcribed into four distinct species of mRNA, one of which resulted in a premature termination codon and the other three predicting deletions of 50, 61 or 72 amino acids, respectively.

Aged↗
PubMed1998

Platelet activation in patients with antiphospholipid syndrome.

The binding of antiphospholipid antibodies to circulating platelets and the potential association with thrombocytopenia and platelet activation was investigated in 25 patients with primary antiphospholipid syndrome (APS). Fourteen patients had a platelet count above 150 x 10(9)/l, and 11 patients had mild to moderate thrombocytopenia of 50-150 x 10(9)/l. The presence of platelet autoantibodies was investigated by immunofluorescent binding. No correlation between the presence of autoantibodies on platelets and thrombocytopenia was found. The binding of antibodies in patients' serum and platelet eluates was investigated by performing enzyme-linked immunosorbent assays with phospholipids as antigens. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. Platelet activation was measured by flow cytometry using a fluorescein isothiocyanate (FITC) labeled monoclonal antibody to P-selectin (CD62). The binding of anti-P-selectin to patients' platelet surface P-selectin was not increased, compared with the binding to platelets obtained from normal donors. Platelet serotonin concentration in APS patients was significantly lower than that found in the platelets of normal controls. More studies are necessary to determine the exact role of antiphospholipid antibodies in the pathogenesis of thrombocytopenia, and to elucidate the cause of low serotonin levels in platelets of APS patients.

Adolescent↗
PubMed1998

Clinical studies and thrombin generation in patients homozygous or heterozygous for the G20210A mutation in the prothrombin gene.

A genetic variation in the prothrombin gene, the G-->A transition at nucleotide 20210, is a risk factor for venous thrombosis in heterozygotes and is associated with increased prothrombin activity. The homozygous phenotype and the extent of thrombin generation in heterozygous and homozygous subjects are unknown. We investigated a family that included 2 homozygous and 5 heterozygous carriers of the 20210 A allele. The homozygous propositus and his presumably heterozygous father suffered from deep-vein thrombosis. His presumably heterozygous mother and his homozygous sister had recurrent phlebitis at a young age. The remaining 5 affected family members are still asymptomatic. We studied thrombin generation in the family and in 22 unrelated carriers of the 20210 A allele by measuring (1) prothrombin fragment F1+2 (F1+2) as an index of ongoing thrombin generation and (2) the endogenous thrombin potential (ETP) as an index of the possible thrombin-forming capacity. Their F1+2 levels were not different from those of age-matched controls, and thus, ongoing hemostatic system activation was not detectable. A significantly increased ETP was found in the heterozygous carriers of the 20210A allele compared with the controls (527.8+/-114.9 versus 387+/-50.1 nmol/L x min, P<0.0001). In the 2 homozygotes, the ETP was almost twice (639 and 751 nmol/L x min, respectively) as high as in the controls. We conclude that homozygosity for the G20210A mutation in the prothrombin gene is associated with a severe, albeit more benign, thrombotic diathesis compared with homozygosity for deficiencies of antithrombin, protein C, or protein S. In carriers of the 20210 A allele, the pathomechanisms leading to thrombosis should be sought in the higher amounts of thrombin that may be formed once thrombin generation is triggered, rather than in ongoing thrombin generation in vivo.

Adult↗
PubMed1998

Natural porcine surfactant (Curosurf) down-regulates mRNA of tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha type II receptor in lipopolysaccharide-stimulated monocytes.

We have previously shown that Curosurf, a natural porcine surfactant, and its phospholipids effectively suppressed secretion of tumor necrosis factor (TNF-alpha) by resting and through lipopolysaccharide (LPS)-stimulated human monocytes. In this study the effect of Curosurf on monocyte mRNA for TNF-alpha and TNF-alpha type II-receptor (TNF-alpha-RII) were analyzed to evaluate the cellular mechanisms involved in the modulation of TNF-alpha expression. LPS-stimulated monocytes simultaneously exposed to Curosurf (500 microg/mL for 24 h) expressed approximately 70% less TNF-alpha mRNA when compared with control subjects (p < 0.05). In addition, 86% less TNF-alpha RII mRNA was found in monocytes exposed to Curosurf (p < 0.001). Decreased mRNA expression was clearly associated with significantly reduced secretion of TNF-alpha protein (Curosurf-exposed LPS-stimulated monocytes 3628 +/- 1873 pg/mL TNF, LPS-stimulated monocytes 31,376 +/- 2524 pg/mL TNF; mean +/- SEM, p < 0.001). The activation of the transcription factor nuclear factor-kappaB upon LPS stimulation is not affected by Curosurf incubation. This excludes that the decrease in mRNA and protein levels of TNF-alpha and TNF-alpha-RII is due to an inhibition of nuclear factor-kappaB activation by Curosurf. We conclude that Curosurf affects TNF-alpha release of LPS-stimulated monocytes at a pretranslational site by down-regulating both mRNA for TNF-alpha and TNF-alpha-RII, therefore acting as an anti-inflammatory agent within alveolar space.

Adult↗
PubMed1998

Passive deformability of mature, immature, and active neutrophils in healthy and septicemic neonates.

Obstruction of narrow vessels by rigid neutrophils may contribute to ischemic organ injury. In septicemia, a substantial portion of the neutrophils may become activated and the number of circulating immature neutrophils may rise sharply. Volume and deformability of mature (PMN) and immature neutrophils in healthy preterm and full-term infants and in septicemic neonates were studied by means of a micropipette system. Membrane cytoplasm tongues were aspirated into 2.5-microm (diameter) pipettes over a period of 60 s. Volume and tongue growth of mature resting PMN were similar in healthy preterm and full-term neonates and adults. Compared with mature PMN (about 360 fl), the volumes of band cells (415 fl), metamyelocytes (470 fl), and less mature cells (myeloblasts, promyelocytes, and myelocytes; 490 fl) were significantly increased (p < 0.005). Final tongue lengths of band cells, metamyelocytes, and less mature cells were decreased by about 50, 60, and 70%, respectively, when compared with passive mature cells. In septic neonates, the percentage of immature neutrophils was increased, but the deformability and volume of the cell subpopulations were not affected by septicemia. Active PMN were characterized by pseudopod formation. More active PMN were found in group B streptococcal (14% of total PMN), gram-negative (12%), and Staphylococcus epidermidis septicemia (8%) than in healthy neonates and adults (4%). The main bodies of active PMN were less deformable than passive PMN, and the pseudopods showed very little membrane deformation. The increased number of rigid active and immature neutrophils may contribute to impaired microcirculation and the high risk for organ injury in septic patients.

Adult↗
PubMed1997

Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells.

Invasion of human mucosal cells by N. gonorrhoeae via the binding to heparansulfate proteoglycan receptors is considered a crucial event of the infection. Using different human epithelial cells and primary fibroblasts, we show here an activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM) by N. gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacological blockade of ASM and PC-PLC cause inhibition of cellular invasion by N. gonorrhoeae. Complementation of ASM-deficient fibroblasts from Niemann-Pick disease patients restored N. gonorrhoeae-induced signaling and entry processes. The activation of PC-PLC and ASM, therefore, is an essential requirement for the entry of N. gonorrhoeae into distinct nonphagocytic human cell types including several epithelial cells and primary fibroblasts.

Bacterial Proteins↗
PubMed1997

Evidence for a novel function of the CD40 ligand as a signalling molecule in T-lymphocytes.

The interaction of the CD40 receptor with its ligand has been shown to be crucial for the activation of B-lymphocytes. Here, we provide evidence that the pg39 molecule/CD40 ligand (gp39/CD40L) also functions as a stimulatory molecule for T-lymphocytes. Activation of T-lymphocytes via gp39/CD40L induced a strong activation of Jun-N-terminal kinase (JNK) and p38-K. Activation of these kinases correlates with a stimulation of Rac1 and inhibition of Rac1 prevents gp39/CD40L triggered JNK/p38-K activation. Further, cellular stimulation via the CD40 ligand results in tyrosine phosphorylation of cellular proteins and the activation of p56(lck). Inhibition of src-like kinases inhibits Rac1 as well as JNK/p38-K stimulation suggesting a signalling cascade from the gp39/CD40L via p56(lck) and Rac1 to JNK/p38-K.

Animals↗