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Biomedical subjects

P Löw

Publications and source records attributed to P Löw.

At least 19 recordsLinked to original sources
PubMed2002

Initiation of HAART in drug-naive HIV type 1 patients prevents viral breakthrough for a median period of 35.5 months in 60% of the patients.

The introduction of potent combinations of antiviral drugs is a major breakthrough in the treatment of HIV. We investigated the long-term virologic outcome and the development of resistance after initiating highly active antiretroviral therapy (HAART) in drug-naive patients in daily clinical practice. Twenty-five treatment-naive HIV-1 patients were started on HAART. Fifteen patients responded with a drop in viral load below the limit of detection during 35.5 (interquartile range: 7) months of therapy. In 6 of 10 patients with virologic failure, virus with resistance-related mutations against the received drugs emerged. Compared with responders (R), nonresponding (NR) patients were in a later disease stage at therapy start (p = 0.0089) with lower CD4 cell counts at baseline (p = 0.040), and a lower proportion of nonresponders showed protease inhibitor (PI) levels above C(min) (p = 0.049). More NR patients showed secondary PI mutations at baseline (p = 0.079), and the CCR2-64I coreceptor polymorphism was absent among NR patients, compared with 38.5% of R patients displaying CCR2-64I (p = 0.053), although the differences were not significant. In conclusion, starting HAART in antiretroviral drug-naive HIV-infected patients followed in daily clinical practice prevented viral breakthrough for up to 44 months in 60% of the patients. Virologic failure was associated with the development of resistance-related mutations, a later stage of disease at start of therapy and lower PI drug levels.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Tubulin and microtubule are potential targets for brain hexokinase binding.

The metabolite-modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non-mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC(50)=4.0+/-1.4 microM). This value virtually was not affected by specific ligands such as ATP or glucose 6-phosphate. Microtubule-bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell-free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.

Adenosine Triphosphate↗
PubMed2001

Proteolytic activity of 26s proteasomes isolated from muscles of the tobacco hornworm, Manduca sexta: differences between surviving muscles and those undergoing developmentally programmed cell death.

The intersegmental muscles (ISMs) of tobacco hornworm,Manduca sexta are a well-characterised model system for examining the biochemical changes that accompany programmed cell death during development. When the ISMs become committed to die, there are dramatic increases in both the ubiquitin-expression, and ubiquitin-dependent proteolysis. Since the 26S proteasome is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its enzymatic properties. Specific chymotrypsin-like proteolytic activity of 26S proteasomes isolated from ISM is four times higher than that of surviving flight muscle (FM). However, specific activity does not change between developmental stages within ISM or FM. The difference between proteolytic capacity of the two kinds of muscles is even higher when the ISM become committed to die because 26S proteasome content of ISM increases just before cell death. These observations underline the role of 26S proteasome in programmed cell death.

Animals↗
PubMed2000

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin.

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Sequential steps in clathrin-mediated synaptic vesicle endocytosis.

Synaptic vesicles are recycled with remarkable speed and precision in nerve terminals. A major recycling pathway involves clathrin-mediated endocytosis at endocytic zones located around sites of release. Different 'accessory' proteins linked to this pathway have been shown to alter the shape and composition of lipid membranes, to modify membrane-coat protein interactions, and to influence actin polymerization. These include the GTPase dynamin, the lysophosphatidic acid acyl transferase endophilin, and the phosphoinositide phosphatase synaptojanin. Protein perturbation studies in living nerve terminals are now beginning to link the actions of these proteins with morphologically defined steps of endocytosis.

Animals↗
PubMed2000

Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6' (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD). Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres. S6' is present in degraded material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and also in degraded material. S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes. These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD. Cell Death and Differentiation (2000) 7, 1210 - 1217.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Biased TCR repertoire in HIV-1-infected patients due to clonal expansion of HIV-1-reverse transcriptase-specific CTL clones.

To study whether an expansion of HIV-1-specific CTL is contributing to the skewed TCR repertoire in HIV-1-infection, we characterized the TCR usage of CTL clones specific for a conserved epitope in HIV-1 reverse transcriptase (RT/476-484). CTL clones from three HIV-1-infected patients displayed highly similar TCR usage and used the identical Vbeta6.1 and Valpha2.5 gene segments. CTL clones from two patients showed a very high degree of similarity within the TCR complementarity-determining region-3 (CDR-3). In accordance with the similar molecular structure, all three CTL clones also exhibited a similar functional activity with regard to recognition of variant peptides and cytokine secretion pattern. In one subject clonal expansion of a single CTL specificity could be shown over a 10-mo period. TCR spectratyping of PBMC from two patients revealed a marked expansion of CDR-3 segments of a certain length within the Vbeta6-family. Sequence analysis of these CDR-3 yielded sequences identical to the RT/476-484-specific CTL previously isolated from the same patients. This analysis demonstrates that clonal expansion of HIV-1-specific CTL is contributing to the skewed TCR repertoire in HIV-1-infected patients.

Amino Acid Sequence↗
PubMed1999

A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins.

The large clostridial cytotoxins (LCTs) constitute a group of high molecular weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins. We demonstrate that a novel LCT (TcdB-1470) from Clostridium difficile strain 1470 is a functional hybrid between "reference" TcdB-10463 and Clostridium sordellii TcsL-1522. It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522. All three toxins had equal enzymatic potencies but were equally cytotoxic only when microinjected. When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522. The small GTP-binding protein R-Ras was identified as a target for TcdB-1470 and also for TcsL-1522 but not for TcdB-10463. R-Ras is known to control integrin-extracellular matrix interactions from inside the cell. Its glucosylation may be a major determinant for the cell rounding and detachment induced by the two R-Ras-attacking toxins. In contrast, fibroblasts treated with TcdB-10463 were arborized and remained attached, with phosphotyrosine containing structures located at the cell-to-cell contacts and beta3-integrin remaining at the tips of cellular protrusions. These components were absent from cells treated with the R-Ras-inactivating toxins. The novel hybrid toxin will broaden the utility of the LCTs for clarifying the functions of several small GTPases, now including also R-Ras.

3T3 Cells↗
PubMed1999

Pyruvate kinase as a microtubule destabilizing factor in vitro.

Endogenous control of microtubule dynamism is essential in many cell types. Numerous microtubule-adhering proteins stabilize the polymer status, while very few protein factors are described with opposite effects. The brain- and muscle-specific M1 isoform of the enzyme pyruvate kinase is investigated here in this respect. Three pieces of evidence indicate antimicrotubular effects of this protein. (1) Pyruvate kinase inhibits taxol-induced tubulin polymerization into microtubules as revealed by turbidimetry. (2) Pelleting experiments show that pyruvate kinase partially disassembles taxol-stabilized microtubules into less sedimentable oligomers leading to the appearance of tubulin in the supernatant fractions. (3) Electron microscopy reveals the kinase-induced formation of great amounts of thread-like tubulin oligomers which tend to accumulate in a light/less sedimentable fraction. Immunoelectron micrographs using labeled antibody against pyruvate kinase provide evidence for the binding of pyruvate kinase to the thread-like oligomeric forms. The present data allow the assumption that pyruvate kinase may display multiple regulatory functions as a glycolytic control enzyme and as a modulator of microtubule dynamism.

Animals↗
PubMed1999

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function.

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.

Animals↗
PubMed1999

Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis.

Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Perturbation of the synaptic release machinery in hippocampal neurons by overexpression of SNAP-25 with the Semliki Forest virus vector.

We have examined whether the Semliki Forest virus (SFV) expression vector can be used to manipulate the exocytotic machinery in cultured hippocampal neurons. Autaptic responses were recorded in individually identified neurons which overexpressed either a non-synaptic protein, the transferrin receptor, or the synaptic SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDA). In neurons overexpressing the transferrin receptor, autaptic responses occurred in a similar proportion and had similar amplitudes (12-18 h postinfection) as in uninfected control neurons. With increasing time after the infection, an increasing proportion of the transferrin receptor-overexpressing neurons showed changes in the shape of the cell body, but the autaptic responses appeared normal as long as recordings could be performed (up to 30 h postinfection). In contrast, in SNAP-25-overexpressing neurons, the proportion of responding cells was reduced 12-18 h after the infection, and the amplitude of the autaptic current in responding neurons was also reduced. The sensitivity to exogenously applied glutamate was, however, unchanged. Biochemical analysis showed that 50% of the overexpressed SNAP-25 was palmitoylated. The levels of two other SNAREs, syntaxin and synaptobrevin (also called vesicle-associated membrane protein), were not affected. Our results indicate that the SFV vector can provide an effective tool to study the function of proteins participating in neurotransmitter release.

Animals↗
PubMed1998

Suppression of HHV-8 viremia by foscarnet in an HIV-infected patient with Kaposi's sarcoma and HHV-8 associated hemophagocytic syndrome.

Human herpes virus 8 (HHV-8) seems to be involved in the pathogenesis of Kaposi s sarcoma. In vitro, antiviral drugs with activity against herpes viruses also can suppress HHV-8, however, little is known about the antiviral activity against HHV-8 in vivo. In this report we describe the effects of foscarnet on HHV-8 viremia in an HIV-infected patient with disseminated Kaposi s sarcoma and a presumably HHV-8 associated hemophagocytic syndrome. HHV-8 DNA could be detected in this patient by PCR in peripheral blood mononuclear cells (PBMC), in bronchoalveolar fluid and tumor biopsies. After initiation of foscarnet because of a severe hemophagocytic syndrome HHV-8 PCR turned negative in PBMC, but stayed positive in pleural effusions and in a tumor biopsy. After termination of foscarnet therapy HHV-8 DNA in PBMC persistently reappeared. Under treatment with foscarnet the hemophagocytic syndrome dramatically improved, suggesting that HHV-8 had a pathogenetic role in this syndrome.

AIDS-Related Opportunistic Infections↗
PubMed1998

Dissociation between Ca2+-triggered synaptic vesicle exocytosis and clathrin-mediated endocytosis at a central synapse.

We have tested whether action potential-evoked Ca2+ influx is required to initiate clathrin-mediated synaptic vesicle endocytosis in the lamprey reticulospinal synapse. Exo- and endocytosis were temporally separated by a procedure involving tonic action potential stimulation and subsequent removal of extracellular Ca2+ (Ca2+e). A low concentration of Ca2+ ([Ca2+]e of 11 microM) was found to be required for the induction of early stages of endocytosis. However, the entire endocytic process, from the formation of clathrin-coated membrane invaginations to the generation of synaptic vesicles, proceeded in the absence of action potential-mediated Ca2+ entry. Our results indicate that the membrane of synaptic vesicles newly incorporated in the plasma membrane is a sufficient trigger of clathrin-mediated synaptic vesicle endocytosis.

Animals↗
PubMed1997

CD80 expression on monocytes in HIV-infected patients.

A prominent feature of HIV infection is a progressive anergy of T cells and an increase of activation-dependent T-cell death. CD80 (B7.1), the ligand of CD28, is an important co-stimulatory molecule on antigen-presenting cells that delivers an essential second signal for T-cell activation. To test whether immunologic dysfunction in HIV disease involves CD80, we studied CD80 expression on circulating monocytes in heparinized whole blood of 33 HIV-infected patients and 13 controls. Most monocytes in patients and controls expressed significant amounts of CD80. There was no statistical difference of mean fluorescence intensity (MFI) of CD80 expression when all HIV-infected patients were compared with healthy controls. However, asymptomatic patients in clinical Centers for Disease Control and Prevention (CDC) stage A showed a significantly stronger CD80 expression than did healthy controls. Additionally, patients receiving antiretroviral therapy exhibited significantly higher CD80 expression than did patients not receiving therapy and healthy controls. We did not find a correlation with the presence of HIV p24 antigenemia and counts of CD4+ and CD8+ T cells. Although we studied CD80 expression only on circulating monocytes and not in HIV-infected monocytes or in activated macrophages, our data do not support a role for a general impairment of CD80 expression in induction of anergy in HIV disease.

B7-1 Antigen↗
PubMed1997

Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions.

The proline-rich COOH-terminal region of dynamin binds various Src homology 3 (SH3) domain-containing proteins, but the physiological role of these interactions is unknown. In living nerve terminals, the function of the interaction with SH3 domains was examined. Amphiphysin contains an SH3 domain and is a major dynamin binding partner at the synapse. Microinjection of amphiphysin's SH3 domain or of a dynamin peptide containing the SH3 binding site inhibited synaptic vesicle endocytosis at the stage of invaginated clathrin-coated pits, which resulted in an activity-dependent distortion of the synaptic architecture and a depression of transmitter release. These findings demonstrate that SH3-mediated interactions are required for dynamin function and support an essential role of clathrin-mediated endocytosis in synaptic vesicle recycling.

Amino Acid Sequence↗