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Biomedical subjects

F Müller

Publications and source records attributed to F Müller.

At least 127 records · Page 7Linked to original sources
PubMed1998

Phosphorylation of mammalian olfactory cyclic nucleotide-gated channels increases ligand sensitivity.

In vertebrate olfactory sensory neurons, odorant receptors couple the sensory signal to the synthesis of the second messenger cAMP. Cyclic nucleotide-gated (CNG) channels are activated by binding of cAMP and conduct a depolarizing receptor current that leads to electrical excitation of the neuron. The sensitivity of olfactory CNG channels for cAMP can be significantly reduced by binding of calmodulin to a regulatory domain that resides within the N-terminus of the alpha-subunit of the channel. This regulatory domain also contains a consensus phosphorylation sequence for protein kinase C (PKC). We have investigated the effect of channel phosphorylation by PKC and found that phosphorylation increases ligand sensitivity without counteracting modulation of the channel by calmodulin. We have identified the amino acid residue that is phosphorylated by PKC and have localized three isoforms of PKC in olfactory sensory cilia. The results of this study provide information about the control of ligand sensitivity in olfactory CNG channels by an intrinsic regulatory domain, representing both a calmodulin-binding site and a substrate for PKC.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Radiometric quantification of Mycobacterium avium complex.

The purpose of this study was to establish a system that would allow rapid and reliable quantification of Mycobacterium avium complex infection with a method that was as sensitive as counting of colony-forming units but less time-consuming and safer in the laboratory. The radiometric system for quantification of mycobacteria (Bactec, Becton Dickinson, USA) which calculates growth curves, was found to be faster, safer, and as sensitive as the established method of counting colony-forming units, with a low intra-assay variation and a wide assay range. Furthermore, the calculated growth curves provided important additional information about replication characteristics of the mycobacteria.

Carbon Dioxide↗
PubMed1998

[Signal transduction in photoreceptor cells].

Vertebrate photoreceptors respond to light with a brief hyperpolarization of their membrane potential. In the dark, photoreceptors are depolarized by cation influx through channels in the plasma membrane which are kept open by the second messenger cGMP. Light absorption activates an enzyme cascade that hydrolytically destroys cGMP, resulting in channel closure and hyperpolarization of the membrane. In addition, processes are initiated that allow photoreceptors to adapt their sensitivity to the ambient illumination. Although these adaptational mechanisms are less well understood, it is clear that they are strongly controlled by the intracellular Ca2+ concentration. This review describes our present knowledge about the signal transduction and its fine tuning by a complex network of Ca(2+)-mediated processes in vertebrate photoreceptors.

Animals↗
PubMed1998

Different effect of 1,25-dihydroxyvitamin D3 on replication of Mycobacterium avium in monocyte-derived macrophages from human immunodeficiency virus-infected subjects and healthy controls.

Mycobacterium avium complex (MAC) is the most common cause of disseminated bacterial infection in patients with acquired immune deficiency syndrome (AIDS) and macrophage dysfunction is important both in the pathogenesis of AIDS- and MAC-infection. 1,25-Dihydroxyvitamin D3 (1,25D), the active metabolite of vitamin D, has a number of effects on cell types of the immune system including monocytes/macrophages. The present study was designed to investigate whether 1,25D supplementation in vitro could modulate MAC replication in macrophages from HIV-infected patients. It was therefore of particular interest to examine whether the effect of 1,25D differs between cells from HIV-infected patients and healthy control subjects. After 3 and 7 days of infection, 1,25D supplementation increased numbers of bacteria in cells from control subjects. In contrast, there was no change or even a decrease in numbers of bacteria in cells from HIV-infected patients. These findings suggest that HIV infection may significantly modulate the macrophage response to 1,25D stimulation, and that 1,25D may have inhibitory effects on MAC replication in macrophages from HIV-infected patients.

Adult↗
PubMed1998

Adhesion molecules in common variable immunodeficiency (CVID)--a decrease in L-selectin-positive T lymphocytes.

CVID is immunologically characterized by defective antibody production. Various additional immunological abnormalities have been reported, but little is known of the role of adhesion molecules in CVID. In 31 CVID patients serum levels of L-selectin (CD62L), vascular cell adhesion molecule-1 (VCAM-1) (CD106) and intercellular adhesion molecule-1 (ICAM-1) (CD54) were significantly elevated compared with controls. In 15 CVID patients investigated, the number of L-selectin-positive cells was significantly reduced in both CD4+ and CD8+ lymphocytes compared with controls, and these changes were observed in both CD45RA+ and CD45RO+ subsets. In CD19+ lymphocytes the percentage of ICAM-1+ cells was significantly increased compared with controls. Fifty percent of the patients had splenomegaly. These patients demonstrated even higher serum levels of adhesion molecules, a lower percentage of L-selectin-positive and a higher percentage of CD38+ cells in many T lymphocyte subsets compared with both other CVID patients and controls. Finally, in this patient group the percentage of L-selectin-positive CD19+ lymphocytes was significantly reduced compared with both other patients and controls. These findings indicate a state of ongoing T lymphocyte activation in CVID, especially in the subgroup of patients with splenomegaly, which may contribute to the impaired antimicrobial defence observed in these patients.

ADP-ribosyl Cyclase↗
PubMed1998

Enhanced interleukin-10 production in response to Mycobacterium avium products in mononuclear cells from patients with human immunodeficiency virus infection.

Patients with advanced human immunodeficiency virus (HIV) infection are susceptible to infections with Mycobacterium avium complex (MAC). Interleukin (IL)-10 may impair immunity to MAC; therefore, the effect of different MAC preparations on IL-10 production was examined in mononuclear cell cultures from HIV-infected patients. IL-10 levels in cultures for 26 patients were higher than those in 20 control cultures. The highest IL-10 levels were found in cultures from patients with the most advanced HIV disease. Monocytes were the major IL-10 producers, while little IL-10 could be attributed to Th2 lymphocytes. Cultures for patients produced reduced levels of tumor necrosis factor-alpha and normal levels of IL-12; the production of these cytokines increased after neutralization of IL-10. Circulating IL-10 was higher in HIV-infected patients than in controls, with the highest levels in the AIDS group. Elevated monocyte/macrophage-derived IL-10 production may contribute to the high susceptibility to MAC infection seen in patients with advanced HIV disease.

AIDS-Related Opportunistic Infections↗
PubMed1998

Circulating levels of RANTES in human immunodeficiency virus type 1 infection: effect of potent antiretroviral therapy.

RANTES has been found to suppress human immunodeficiency virus type 1 (HIV-1) replication. To further elucidate the role of this chemokine in HIV-1 infection, RANTES levels were analyzed in serum and platelet-free plasma (PFP) in 53 HIV-1-infected patients and 20 controls. RANTES levels were significantly elevated in both serum and PFP in all clinical stages of HIV-1 infection, with the highest levels in CDC groups A and B. In longitudinal testing, the progressors were characterized by a pronounced decline in serum levels over time; the nonprogressors, however, had only a slight reduction or an increase in RANTES levels. During 16 weeks of indinavir therapy, there was an increase in circulating RANTES levels and enhanced release of RANTES from stimulated CD8+ lymphocytes. The decline in RANTES levels along with disease progression is compatible with RANTES having a beneficial role in HIV-1-infected patients. The increase in RANTES levels during protease inhibitor-containing regimens may represent a previously unrecognized immunologic effect of such therapy.

Adult↗
PubMed1998

Mapping a telomere using the translocation eT1(III;V) in Caenorhabditis elegans.

In Caenorhabditis elegans, individuals heterozygous for a reciprocal translocation produce reduced numbers of viable progeny. The proposed explanation is that the segregational pattern generates aneuploid progeny. In this article, we have examined the genotype of arrested embryonic classes. Using appropriate primers in PCR amplifications, we identified one class of arrested embryo, which could be readily recognized by its distinctive spot phenotype. The corresponding aneuploid genotype was expected to be lacking the left portion of chromosome V, from the eT1 breakpoint to the left (unc-60) end. The phenotype of the homozygotes lacking this DNA was a stage 2 embryonic arrest with a dark spot coinciding with the location in wild-type embryos of birefringent gut granules. Unlike induced events, this deletion results from meiotic segregation patterns, eliminating complexity associated with unknown material that may have been added to the end of a broken chromosome. We have used the arrested embryos, lacking chromosome V left sequences, to map a telomere probe. Unique sequences adjacent to the telomeric repeats in the clone cTel3 were missing in the arrested spot embryo. The result was confirmed by examining aneuploid segregants from a second translocation, hT1(I;V). Thus, we concluded that the telomere represented by clone cTel3 maps to the left end of chromosome V. In this analysis, we have shown that reciprocal translocations can be used to generate segregational aneuploids. These aneuploids are deleted for terminal sequences at the noncrossover ends of the C. elegans autosomes.

Animals↗
PubMed1998

Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection.

Besides their role in hemostasis, platelets are involved in inflammatory and immunological processes, and we hypothesize that platelet activation may play an immunopathogenetic role in HIV-1 infection. Blood was drawn from 15 controls and 20 HIV-1-infected patients with normal platelet counts, classified into groups of non-AIDS and AIDS. Platelet activation was detected using flow cytometry with mAbs against the release markers P-selectin and CD63, mAb against GPIb, and the probe annexin V detecting surface exposure of aminophospholipids. The amount of microvesicles was measured using mAb against GPIIIa. Compared to controls, blood samples from HIV-1-infected patients showed significantly enhanced levels of microvesicles and activated platelets as detected by their exposure of P-selectin, CD63, and aminophospholipids, as well as reduction in GPIb expression. Increased expression of P-selectin and amounts of microvesicles were most pronounced in advanced clinical and immunological disease. When studying the effect of HIV-1 protease inhibitor therapy (indinavir) on platelet activation, we found that concomitant with a profound decrease in plasma viral load, there was a near normalization of several of the parameters reflecting enhanced platelet activation. Finally, we demonstrated that platelets may be an important source of the chemokine RANTES in HIV-1-infected patients. Although both unstimulated and SFLLRN-stimulated platelets from asymptomatic patients had enhanced release of RANTES, platelets from AIDS patients were characterized by markedly enhanced spontaneous, but decreased SFLLRN-stimulated release of this chemokine. Taken together, these results, which demonstrate for the first time increased platelet activation in HIV-1-infected patients with normal platelet counts, may represent a previously unrecognized immunopathogenic factor in HIV-1 infection.

Anti-HIV Agents↗
PubMed1998

Protein kinase A type I antagonist restores immune responses of T cells from HIV-infected patients.

Cyclic AMP-dependent protein kinase A (PKA) type I has been established as an acute inhibitor of T cell activation. For this reason, we investigated the possible role of PKA type I in HIV-induced T cell dysfunction. T cells from HIV-infected patients have increased levels of cAMP and are more sensitive to inhibition by cAMP analog than are normal T cells. A PKA type I-selective antagonist increases the impaired proliferation of T cells from HIV-infected patients to normal or subnormal levels (up to 2.8-fold). Follow-up of patients after initiation of highly active antiretroviral treatment revealed that a majority of patients have a persistent T cell dysfunction that is normalized by incubation of T cells with Rp-8-Br-cAMPS. These observations imply that increased activation of PKA type I may contribute to the progressive T cell dysfunction in HIV infection and that PKA type I may be a potential target for immunomodulating therapy.

Adult↗
PubMed1998

Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger PH 2.5 acid phosphatase.

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90 degrees C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70 degrees C. After heat denaturation at temperatures up to 90 degrees C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90 degrees C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of >/=80 degrees C. In feed pelleting experiments performed at 75 degrees C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85 degrees C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80 degrees C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.

6-Phytase↗
PubMed1998

Calcium-binding proteins and nitric oxide in retinal function and disease.

Regulation of phototransduction in photoreceptor cells occurs via several feedback loops. Some of these circuits involve the neuron-specific Ca2+-binding proteins recoverin and guanylyl cyclase-activating protein. Recent findings suggest that these proteins are also involved in retinal diseases. Another Ca2+-regulated process in the retina is the synthesis of nitric oxide, a substance of potential neurotoxicity. This review discusses several Ca2+-mediated processes in the retina.

Animals↗
PubMed1998

Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis.

The serum level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D], the biologically most potent metabolite of vitamin D, is tightly regulated within narrow limits in human healthy adults. 1,25-(OH)2D deficiency is rare and is associated with disturbances in calcium and bone metabolism. We have previously reported a marked decrease in serum levels of 1,25-(OH)2D in human immunodeficiency virus (HIV)-infected patients. The present study was designed to further examine the causes and consequences of severe 1,25-(OH)2D deficiency in these patients. The design was a prospective cohort study. Fifty-four HIV-infected patients clinically classified according to the revised criteria from Centers for Disease Control and Prevention and healthy controls were studied. Parameters related to vitamin D and calcium metabolism as well as immunological and nutritional status were determined. Twenty-nine of the patients (54%) had serum levels of 1,25-(OH)2D below the lower reference limit, and 18 of these had undetectable levels. In contrast, HIV-infected patients had normal serum levels of 25-hydroxyvitamin D and vitamin D-binding protein. HIV-infected patients as a group had modestly depressed serum calcium and PTH levels. There were, however, no correlations between these parameters and serum levels of 1,25-(OH)2D. There were no differences in serum calcium or PTH levels or nutritional status when patients with severe 1,25-(OH)2D deficiency were compared to other patients, but patients with undetectable 1,25-(OH)2D had significantly elevated serum phosphate levels. Furthermore, patients with undetectable 1,25-(OH)2D levels were characterized by advanced clinical HIV infection, low CD4+ lymphocyte counts, and high serum levels of tumor necrosis factor-alpha (TNFalpha). We conclude that inadequate 1alpha-hydroxylation of 25-hydroxyvitamin D seems to be the most likely cause of 1,25-(OH)2D deficiency in HIV-infected patients, possibly induced by an inhibitory effect of TNFalpha. The low 1,25-(OH)2D and high TNFalpha levels observed may impair the immune response in HIV-infected patients both independently and in combination and may represent an important feature of the pathogenesis of HIV-related immunodeficiency. Markedly depressed 1,25-(OH)2D serum levels are also present in certain other disorders characterized by immunological hyperactivity. Thus, the findings in the present study may not only represent a previously unrecognized immune-mediated mechanism for induction of 1,25-(OH)2D deficiency in human disease, but may also reflect the importance of adequate serum levels of 1,25-(OH)2D for satisfactory performance of the immune system in man.

Adolescent↗
PubMed1997

Dysregulation of membrane-bound tumor necrosis factor-alpha and tumor necrosis factor receptors on mononuclear cells in human immunodeficiency virus type 1 infection: low percentage of p75-tumor necrosis factor receptor positive cells in patients with advanced disease and high viral load.

The correlation of persistent tumor necrosis factor-alpha (TNF-alpha) activation with disease progression in patients infected with human immunodeficiency virus type 1 (HIV-1), suggests a role for TNF-alpha in the pathogenesis of HIV-1 infection. In the present study, we examined by flow cytometry the expression of membrane-bound (m) components of the TNF system in 33 HIV-1-infected patients and 12 healthy controls. While peripheral blood mononuclear cells (PBMC) from asymptomatic and symptomatic non-acquired immune deficiency syndrome (AIDS) patients showed a significantly increased percentage of mTNF-alpha+ and mTNF receptor (TNFR)+ cells compared with controls, this was not found in the AIDS group. Compared with healthy controls, AIDS patients had a significantly decreased percentage of both monocytes and lymphocytes expressing p75-TNFR. PBMC from AIDS patients showed a higher p75-TNFR mRNA level and a higher spontaneous release of soluble p75-TNFR than healthy individuals, suggesting enhanced cell surface turnover of this TNFR. The low expression of TNFRs on both lymphocytes and monocytes in the AIDS group was associated with high numbers of HIV-1 RNA copies in plasma, low numbers of CD4+ lymphocytes, and high serum levels of soluble TNFRs. AIDS patients had a decreased percentage of CD8+ lymphocytes expressing TNFRs compared with healthy controls. In contrast, these patients, as well as symptomatic non-AIDS patients, had an increased percentage of TNF-alpha+ and TNFRs+ cells among remaining CD4+ lymphocytes. The pattern of abnormalities seen in AIDS patients suggests a role for persistent activation of the TNF system in the accelerated CD4+ lymphocyte destruction, the enhanced HIV-1 replication, and the markedly impaired antimicrobial defense in advanced HIV-1-related disease.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Termination of mammalian rDNA replication: polar arrest of replication fork movement by transcription termination factor TTF-I.

A replication fork barrier (RFB) at the 3' end of eukaryotic ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. We have reproduced the RFB in vitro in HeLa cell extracts using 3' terminal murine rDNA fused to an SV40 origin-based vector. The RFB is polar and modularly organized, requiring both the Sal box transcription terminator and specific flanking sequences. Mutations within the terminator element, depletion of the RNA polymerase I-specific transcription termination factor TTF-I, or deletion of the termination domain of TTF-I abolishes RFB activity. Thus, the same factor that blocks elongating RNA polymerase I prevents head-on collision between the DNA replication apparatus and the transcription machinery.

3T3 Cells↗
PubMed1997

Visualization of a covalent intermediate between microsomal epoxide hydrolase, but not cholesterol epoxide hydrolase, and their substrates.

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of alpha/beta-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-(14)C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1,1,1-trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalcone-oxide-sensitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-(14)C]cholesterol-5alpha,6alpha-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.

Amino Acid Sequence↗
PubMed1997

Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos.

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the beta-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.

Actins↗