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Biomedical subjects

B Brenner

Publications and source records attributed to B Brenner.

At least 73 records · Page 4Linked to original sources
PubMed1999

Sarcomeric binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment in skinned fibers of skeletal muscle.

Intact caldesmon and particularly the actin-binding C-terminal fragment (20-kDa) of caldesmon have been shown in skeletal muscle fibers to selectively displace low affinity, weakly bound cross-bridges from actin without significantly altering the actin attachment of force producing, strong binding cross-bridges (Brenner et al., 1991; Kraft et al., 1995a). However, the sarcomeric distribution and the specific binding of externally added caldesmon to the myofilaments of skeletal muscle fibers was not known. It was e.g., unclear whether caldesmon binds along actin in a manner similar to tropomyosin or whether it also binds to myosin. In this study, we determined the binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment, respectively, in MgATP-relaxed rabbit skeletal muscle fibers using electron (EM) and confocal fluorescence microscopy (CFM). EM showed that similar to what has been demonstrated earlier for smooth muscle thin filaments (Lehman et al., 1989), intact caldesmon binds periodically every 38 nm along the thin filaments. CFM revealed that rhodamine-labeled intact caldesmon and the 20-kDa caldesmon fragment bind along nearly the entire length of the thin filaments. A portion of the I-band near the Z-line appears unlabeled, both when equilibrated at normal and long sarcomere lengths. The width of the unlabeled region seems to depend on ionic strength. The 20-kDa C-terminal caldesmon fragment binds in essentially the same pattern as intact caldesmon. This indicates that the high fluorescence intensity in the overlap region seen with intact caldesmon does not depend on caldesmon binding to myosin. X-ray diffraction was used to monitor the effects of filament lattice. Intact caldesmon at > 0.3 mg/ml induced disorder in the myofilament lattice. No such disordering was observed, however, when fibers were equilibrated with up to 0.8 mg/ml of the 20-kDa caldesmon fragment.

Actins↗
PubMed1999

A single-fiber in vitro motility assay. In vitro sliding velocity of F-actin vs. unloaded shortening velocity in skinned muscle fibers.

We describe an approach that allows us to form a micro in vitro motility assay with as little myosin as can be retrieved from a short (approximately 10 mm) segment of a single skinned skeletal muscle fiber (diameter some 100 microm). Myosin is directly extracted from the single fiber segment by a high ionic strength solution in the presence of MgATP, and the extracted myosin is immediately applied to a miniaturized flow cell that has been pretreated with BSA. The observed sliding velocities of fluorescently labeled F-actin are essentially identical with those reported in the literature. Since at the single fiber level most muscle fibers contain only a single myosin heavy chain isoform this approach allows us to determine without additional purification steps, the sliding velocity driven by myosins with different heavy chain isoforms. In addition, this approach can be used to directly correlate under identical experimental conditions unloaded shortening velocity measured in segments of skinned muscle fibers with the in vitro sliding velocity of fluorescently labeled F-actin by extraction of myosin from the same skinned fibers. Such direct correlation was performed with different myosin heavy chain isoforms as well as at different temperatures and ionic strengths. Under all conditions studied, unloaded shortening velocity was 4- to 8-fold faster than sliding velocity in the motility assay even at high temperature (22 degrees C) and ionic strengths >50 mM. This suggests that sliding velocity in the motility assay is limited by additional factors beyond those thought to limit velocity of unloaded shortening in muscle fibers. One such factor might be unspecific ionic interactions between F-actin and the substrate in the motility assay resulting in somewhat higher sensitivity for ionic strength of sliding velocity in the motility assay. This might become of special relevance when using in vitro sliding velocity in assessing functional consequences of mutations involving charged residues of actin or myosin.

Actins↗
PubMed1999

A force transducer for measuring mechanical properties of single cardiac myocytes.

We have described a transducer design capable of recording forces generated by single cardiac myocytes with sufficient temporal resolution to detect force responses to rapid length changes. Our force sensors were made from thin steel foils that act as cantilevers whose bending is monitored by reflection off a laser beam. Deflection of the laser beam is measured by a differential photodiode detector. A small, 50-micron-thick tungsten needle attached to the free end of the steel foil allowed us to glue single cardiac cells to the force transducer. The transducers have compliances of approximately 0.02 m/N and resonance frequencies between 2 and 3 kHz. The resolution is approximately 18 nN rms at a detector bandwidth of 16 kHz, so we were able to resolve 0.2% of the maximum isometric force ( approximately 12 microN) developed by a single cardiac myocyte. We have demonstrated that the transducer is well suited to analysis of mechanical properties of single ventricular myocytes, for example, the recording of isometric forces and rate constants of force redevelopment after rapid release-restretch maneuvers.

Animals↗
PubMed1999

Tailoring treatment for classical Kaposi's sarcoma: comprehensive clinical guidelines.

Classical Kaposi's sarcoma (CKS) is a rare indolent proliferative disease which is particularly prevalent among Jews of Ashkenazi and Mediterranean origin. To define guidelines for its comprehensive management, we conducted a retrospective analysis of 123 patients, focusing mainly on treatment modalities. The CKS-related mortality was 4% (5 patients). Of the 39 patients for whom observation only was the primary approach, 15 (38%) remained progression-free for 1-83 months (median, 4 months). Twenty-nine of the 52 (56%) patients who underwent surgery as the primary approach remained recurrence-free for 1-162 months (median, 15 months). Radiotherapy achieved an objective response in 74 courses (85%), including 50 (58%) complete responses. Symptomatic relief was reported in 95% of the patients. Vinblastine (27 series) achieved an objective response in 73% of series, including 22% complete responses. Multivariate analysis of time to progression with observation alone identified immunosuppression as the only significant independent factor that predicted disease progression. Our study suggests that observation alone may be sufficient for immunocompetent asymptomatic patients; symptomatic resectable lesions are suitable for simple excision; and more advanced disease or unresectable lesions require radiotherapy. If disease is extensive or the other approaches fail, chemotherapy is appropriate. Tailoring the treatment for CKS is an integrative process, requiring good understanding of the role of each available modality in the different clinical disease settings.

Adult↗
PubMed1999

Safety of low-molecular-weight heparin in pregnancy: a systematic review.

Unfractionated heparin (UFH) remains the anticoagulant of choice during pregnancy. Low-molecular-weight heparins (LMWH) are an attractive alternative to UFH due to their logistic advantages and their association with a lower incidence of osteoporosis and HIT. We reviewed all published clinical reports concerning the use of LMWH during pregnancy. In addition, participants of an international interest group contributed a cohort of pregnant women treated with LMWH. Pregnancies were divided into two groups; those with and those without maternal comorbid conditions. The number of adverse fetal outcomes and the occurrence of maternal complications were evaluated in the two groups. In the group of women with comorbid conditions (n = 290), 13.4% of the pregnancies were associated with an adverse fetal outcome. In contrast, in the group of women without comorbid conditions (n = 196), 3.1% were associated with an adverse outcome, which is comparable to that seen in the normal population. We conclude that LMWH appear to be a safe alternative to unfractionated heparin as an anticoagulant during pregnancy.

Anticoagulants↗
PubMed1999

Thrombophilic polymorphisms are common in women with fetal loss without apparent cause.

An association between fetal loss and thrombophilia has recently been described but has not been yet fully elucidated. We have evaluated prospectively the prevalence of the three common thrombophilic polymorphisms (TP) factor V G1691A (Leiden), thermolabile-methylenetetrahydrofolate reductase (TL-MTHFR) C677T and factor II G20210A mutations, in 76 women with fetal loss (> or =3 in first, > or =2 in second, > or =1 in third trimester) without apparent cause and 106 controls without fetal loss. Thirty seven out of 76 (49%) of the women in the fetal loss group had at least one TP compared to only 23/106 (22%) in the control group (p = 0.0001 ). Factor V-Leiden was more common in the fetal loss group 24/76 (32%) compared to the control group 11/106 (10%) (OR = 4.0, 95% CI: 1.8-8.8, p <0.001). Five of the 76 patients (7%) were homozygous for factor V-Leiden compared to none of the controls (p = 0.012). A trend, albeit no statistically significant difference was found between women with fetal loss and control groups regarding factor II G20210A (8% vs. 4% respectively, OR = 2.2, 95% CI: 0.6-8.0, p = 0.23) and MTHFR C677T (18% vs. 10% respectively, OR = 1.95, 95% CI: 0.83-4.6, p = 0.12). Combined TP were documented in 6/76 (8%) patients compared to 1/106 (1%) in controls (OR = 9.0, 95% CI: 1.1-76, p = 0.02). Second or third trimester fetal loss were more common cause of pregnancy termination in 37 patients with TP compared to 39 patients without TP (57/158 (36%) vs. 23/135 (17%) respectively, (p = 0.0004). Thrombophilic polymorphisms are common in women with fetal loss without apparent cause and are associated with late pregnancy wastage. Combinations of TP increase the risk for fetal loss.

Abortion, Spontaneous↗
PubMed1999

Prospective evaluation of hemostatic system activation and thrombin potential in healthy pregnant women with and without factor V Leiden.

Normal pregnancy is associated with alterations of the hemostatic system towards a hypercoagulable state and an increased risk of venous thromboembolism. The risk of venous thrombosis is higher in pregnant women with factor V Leiden (FVL) than in those with wildtype factor V. Routine laboratory assays are not useful to detect hypercoagulable conditions. A prospective and systematic evaluation of hemostatic system activation in women with and without FVL during an uncomplicated pregnancy employing more sensitive markers of hypercoagulability, such as prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), D-Dimer, or the endogenous thrombin potential (ETP), an indicator of the plasma's potential to generate thrombin, has not been performed. We prospectively followed 113 pregnant women with (n = 11) and without (n = 102) FVL and measured F1+2. TAT, D-Dimer and the ETP at the 12th, 22nd and 34th gestational week as well as 3 months after delivery (baseline) in each subject. None of the women developed clinical signs of venous thromboembolism during pregnancy or postpartum. Pregnant women with and without FVL exhibited substantial activation of the coagulation and fibrinolytic system as indicated by a gradual increase of F1+2, TAT and D-Dimer throughout uncomplicated pregnancy up to levels similar to those found in acute thromboembolic events (p < 0.0001 by analysis of variance for each parameters). Levels of F1+2 and TAT were comparable between women with and without FVL, but levels of D-Dimer were significantly higher in women with FVL than in those without the mutation (p = 0.0005). The ETP remained unchanged in both women with and without FVL at all timepoints. Our data demonstrate a substantial coagulation and fibrinolytic system activation in healthy women with and without FVL during uncomplicated pregnancy. An elevated F1+2, TAT or D-Dimer level during pregnancy is not necessarily indicative for an acute thromboembolic event. The normal ETP in both women with and without FVL suggests that the capacity of the plasma to generate thrombin after in vitro activation of the clotting system is not affected by pregnancy. Higher levels of D-Dimer in women with FVL than in women with wildtype factor V at baseline as well as during pregnancy indicate increased fibrinolytic system activation in carriers of the mutation.

Adult↗
PubMed1998

A missense mutation in gamma-glutamyl carboxylase gene causes combined deficiency of all vitamin K-dependent blood coagulation factors.

To identify potential mutations in the gamma-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined in 4 patients from a consanguineous kindred with combined deficiency of all vitamin K-dependent procoagulants and anticoagulants and results were compared with normal genomic sequence. All 4 patients were homozygous for a point mutation in exon 9 that resulted in the conversion of an arginine codon (CTG) to leucine codon (CGG) at residue 394. Screening of this mutation based on introduction of Alu I site in amplified fragment from normal allele but not from the mutated allele showed that 13 asymptomatic members of the kindred were heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation which is the first reported in the gamma-glutamyl carboxylase gene fits perfectly with phenotype of the disorder and confirms the suggested autosomal recessive pattern of inheritance of combined deficiency of all vitamin K-dependent procoagulants and anticoagulants in this kindred. The mutated carboxylase protein expressed in Drosophila cells was stable but demonstrated threefold reduced activity compared with WT carboxylase, confirming that the L394R mutation results in a defective carboxylase.

Animals↗
PubMed1998

von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.

BACKGROUND: Thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome are severe microvascular disorders of platelet clumping with similar signs and symptoms. Unusually large multimers of von Willebrand factor, capable of agglutinating circulating platelets under high shear stress, occur in the two conditions. We investigated the prevalence of von Willebrand factor-cleaving protease deficiency in patients with familial and nonfamilial forms of these disorders. METHODS: Plasma samples were obtained from 53 patients with thrombotic thrombocytopenic purpura or hemolytic-uremic syndrome. Von Willebrand factor-cleaving protease was assayed in diluted plasma samples with purified normal von Willebrand factor as the substrate. The extent of the degradation of von Willebrand factor was assessed by electrophoresis in sodium dodecyl sulfate-agarose gels and immunoblotting. To determine whether an inhibitor of von Willebrand factor-cleaving protease was present, we measured the protease activity in normal plasma after incubation with plasma from the patients. RESULTS: We examined 30 patients with thrombotic thrombocytopenic purpura and 23 patients with the hemolytic-uremic syndrome. Of 24 patients with nonfamilial thrombotic thrombocytopenic purpura, 20 had severe and 4 had moderate protease deficiency during an acute event. An inhibitor found in 20 of these patients was shown to be IgG in five of five tested plasma samples. Of 13 patients with nonfamilial hemolytic-uremic syndrome, 11 had normal levels of activity of von Willebrand factor-cleaving protease during the acute episode, whereas in 2 patients, the activity was slightly decreased. All 6 patients with familial thrombotic thrombocytopenic purpura lacked von Willebrand factor-cleaving protease activity but had no inhibitor, whereas all 10 patients with familial hemolytic-uremic syndrome had normal protease activity. In vitro proteolytic degradation of von Willebrand factor by the protease was studied in 5 patients with familial and 7 patients with nonfamilial hemolytic-uremic syndrome and was normal in all 12 patients. CONCLUSIONS: Nonfamilial thrombotic thrombocytopenic purpura is due to an inhibitor of von Willebrand factor-cleaving protease, whereas the familial form seems to be caused by a constitutional deficiency of the protease. Patients with the hemolyticuremic syndrome do not have a deficiency of von Willebrand factor-cleaving protease or a defect in von Willebrand factor that leads to its resistance to protease.

ADAM Proteins↗
PubMed1998

L-selectin stimulates the neutral sphingomyelinase and induces release of ceramide.

Selectins have been shown to be crucial in the rolling process of leukocytes during lymphocyte homing and in the early phase of inflammatory processes. Recently, we and others have shown that binding of L-selectin to its ligands correlates with a rapid induction of several intracellular signaling molecules, in particular, Src-like tyrosine kinases, MAP-kinases, Jun NH2-terminal kinase, the small G-proteins Ras and Rac, and a release of Ca2+ in leukocytes. Here, we demonstrate the activation of a novel signaling pathway by L-selectin. Stimulation of Jurkat T-lymphocytes via L-selectin results in an increase of neutral sphingomyelinase activity. This activity correlates with a consumption of cellular sphingomyelin and a release of ceramide. The activation of the neutral sphingomyelinase by L-selectin does not depend on tyrosine kinase activity and, therefore, represents an alternative and novel pathway to stimulate lymphocytes via L-selectin.

Ceramides↗
PubMed1998

Fas or ceramide induce apoptosis by Ras-regulated phosphoinositide-3-kinase activation.

We demonstrate a rapid and transient activation of phosphoinositide-3-kinase (PI-3-K) by Fas receptor triggering or cellular treatment with synthetic C6-ceramide. The stimulation of PI-3-K is critical for Fas or C6-ceramide-induced programmed cell death because transfection with a transdominant inhibitory PI-3-K construct or pre-treatment with the PI-3-K inhibitor wortmannin almost completely prevented Fas or C6-ceramide-mediated apoptosis. Treatment with the caspase inhibitor Ac-YVAD-cmk or cellular transfection with transdominant inhibitory N17Ras prevented PI-3-K stimulation by Fas, suggesting that Fas activates PI-3-K via caspases and Ras. N17Ras expression also prevented C6-ceramide-initiated PI-3-K stimulation. The notion of a PI-3-K regulation by Ras upon Fas receptor ligation or ceramide treatment is supported by co-immunoprecipitation experiments revealing an activation-dependent association of PI-3-K and Ras.

Androstadienes↗
PubMed1998

Fluorescence of NBD-labelled troponin-I as a probe for the kinetics of thin filament activation in skeletal muscle fibers.

Using fluorescence of NBD-labelled troponin I incorporated into skinned fibers of the rabbit psoas muscle by chasing native troponin by troponin with the NBD-labelled TnI subunit we attempted to study kinetics of thin filament activation at different Ca(++)-concentrations. Since fluorescence of NBD-labelled TnI is sensitive to both, changes in Ca++, and strong cross-bridge attachment, we were able to induce changes thin filament activation by rapidly dropping the fraction of strongly attached cross-bridges to very low levels, e.g. by switching from isometric to isotonic contraction conditions. At any [Ca++], the time course of the resulting changes in fluorescence of NBD-labelled TnI was found at least an order of magnitude faster than the time course of force redevelopment subsequent to the period of isotonic contraction. Modelling shows that with the kinetics of thin filament activation derived from these studies, common kinetic schemes of the actomyosin ATPase predict regulation to act via changes in cross-bridge cycling kinetics, as we had previously proposed.

Animals↗
PubMed1998

X-ray diffraction studies of the cross-bridge intermediate states.

Two dimensional x-ray diffraction was obtained from skinned rabbit psoas muscle fibers. The goal is to correlate structures of the cross-bridge population with various intermediate states in the cross-bridge cycle by using nucleotides and their analogs. It was found that in a relaxed muscle in ATP containing solutions, cross-bridges are distributed in three populations in equilibrium: those detached from actin and ordered on the myosin helix, those that are detached and disordered, and those weakly attached to actin in random orientations. The distribution among the three populations is highly dependent on temperature. Those that are detached and yet ordered in a helical structure surrounding the myosin backbone are very likely in the M.ADP.Pi state, supporting an earlier suggestion by Wray (1987). It was also found that the attached cross-bridges with bound MgADP are structurally distinct from those without nucleotide, in agreement with one of our earlier findings by osmotic compression (Xu et al., 1993). Another finding of interest is that the analog AMP-PNP was found to be an ATP analog, rather than an ADP analog as it has been reported previously by many research groups.

Adenosine Triphosphate↗
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↗