Biomedical subjects

M Müller

Publications and source records attributed to M Müller.

At least 19 recordsLinked to original sources

PubMed1992

Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function.

The membrane insertion of the mannitol permease (MtlA protein) of Escherichia coli, a polytopic cytoplasmic membrane protein possessing an uncleaved amphiphilic signal sequence, was studied using a cell-free protein synthesis system. The MtlA protein synthesized in the presence of inside-out cytoplasmic membrane vesicles was shown to insert into the membranes based on the following criteria: (a) co-sedimentation of the majority of the MtlA protein with the vesicles; (b) selective extraction of the membrane-associated MtlA by doxycholate but not by urea treatment; and (c) protease resistance of a defined MtlA fragment observed exclusively in the presence of membranes. Post-translational addition of membrane vesicles allowed membrane association of MtlA but did not allow efficient integration. In cell-free systems having reduced levels of the export factors SecA and SecB and exhibiting defective translocation of preOmpA and preLamB, insertion of the in vitro synthesized MtlA apparently occurred normally. In contrast, when membranes from the secY24ts mutant or trypsin-treated membranes were used, insertion of MtlA was reduced. In vivo experiments monitoring the permease activity of MtlA in the secA and secY mutants supported the conclusions of the in vitro results. Thus, the insertion of MtlA is essentially SecA- and SecB-independent but may be dependent on SecY and/or an as yet unidentified membrane protein. The requirements for the insertion of the mannitol permease are therefore clearly different from those for the translocation of most proteins having a cleavable hydrophobic signal sequence.

Adenosine Triphosphatases↗

PubMed1992

Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection.

An important aspect of gene transfer into farm animals is the improvement of disease resistance. The mouse Mx1 protein is known to be sufficient to confer resistance to influenza viruses. Gene constructs containing the mouse Mx1 cDNA controlled by the human metallothionein IIA promoter (hMTIIA::Mx), the SV40 early enhancer/promoter region (SV40::Mx) and the mouse Mx1 promoter (mMx::Mx) were transferred into pigs. The results of the gene transfer experiments with the hMTIIA::Mx and the SV40::Mx constructs indicate that the permanent high-level synthesis of Mx1 might be deleterious to the organism: the gene transfer efficiency was surprisingly low, and all transgenic piglets born had rearrangements in their transgene copies that abolished protein synthesis. The use of the interferon (IFN)- and virus-inducible mMx::Mx construct resulted in normal gene transfer efficiency. Two transgenic pig lines could be established which expressed IFN-inducible mouse Mx1 mRNA. Extensive protein analysis did not detect mouse Mx1 in IFN-treated transgenic animals.

Animals↗

PubMed1992

Low molecular weight, non-peptide fibrinogen receptor antagonists.

The tetrapeptide H-Arg-Gly-Asp-Ser-OH (1) (RGDS), representing a recognition sequence of fibrinogen for its platelet receptor GP IIb-IIIa (integrin alpha IIb beta 3), served as lead compound for the development of highly potent and selective fibrinogen receptor antagonists. Replacement of the N-terminal arginine by p-amidinophenylalanine or the Gly moiety by m-aminobenzoic acid led to compounds which are superior to the lead peptide with regard to activity and selectivity for GP IIb-IIIa vs the closely related vitronectin receptor alpha v beta 3. By random screening [(p-amidinobenzenesulfonamido)ethyl]-p-phenoxyacetic acid derivatives have been identified as fibrinogen receptor antagonists. Further structure-activity relationship studies culminated in the preparation of N-[N-[N-(p-amidinobenzoyl)-beta-alanyl]-L-alpha-aspartyl]-3-phenyl-L- alanine (29h, Ro 43-5054) and [[1-[N-(p-amidinobenzoyl)-L-tyrosyl]-4-piperidinyl]oxy]acetic acid (37f, Ro 44-9883), which exhibit very high activity as platelet aggregation inhibitors (IC50s 0.06 and 0.03 microM, respectively, human PRP/ADP) as well as marked selectivity for GP IIb-IIIa vs alpha v beta 3. Since the activity of 37f in dogs declines according to a two-compartment model with an initial phase having a t1/2 of 8 min and a second phase with a t1/2 of 110 min, this compound is a suitable candidate for the development as iv platelet inhibitor.

Amino Acid Sequence↗

PubMed1992

Leukotriene uptake by hepatocytes and hepatoma cells.

The uptake of tritiated cysteinyl leukotrienes (LTC4, LTD4, LTE4) and LTB4 was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial-rate conditions. Leukotriene uptake by hepatocytes was independent of an Na+ gradient and a K+ diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low-Km system and a non-saturable system for the uptake of cysteinyl leukotrienes as well as LTB4 under the conditions used. AS-30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB4. Moreover, only LTB4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB4 (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S-decyl glutathione, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, probenecid, docosanedioate, and hexadecanedioate (100 microM each), but not by taurocholate, the amphiphilic cations verapamil and N-propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB4 uptake by Hep G2 hepatoma cells under the same experimental conditions. Leukotriene-binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [3H]LTE4 and [3H]LTB4 as the photolabile ligands. Predominant leukotriene-binding proteins with apparent molecular masses in the ranges of 48-58 kDa and 38-40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [3H]LTE4 in Hep G2 cells. With [3H]LTB4 a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected. Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB4, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.

Affinity Labels↗

PubMed1992

New S-adenosylmethionine decarboxylase inhibitors with potent antitumor activity.

Methylglyoxal bis(guanylhydrazone) (MGBG) has been studied clinically as an antitumor and antileukemic agent and is recognized as a potent but nonspecific inhibitor of the key polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (SAMDC). A series of four SAMDC inhibitors with structural features similar to MGBG have been found to have improved potency and specificity toward the target enzyme, SAMDC. Relative to MGBG, the new derivatives were much more effective in inhibiting partially purified preparations of SAMDC (50% inhibitory concentration, 10 to 100 nM), much less effective at inhibiting diamine oxidase, and inactive toward ornithine decarboxylase. The inhibitors varied relative to MGBG in their ability to compete with spermidine for uptake, with two being similar and two being less effective. Against L1210 leukemic cells and T24 bladder carcinoma cells, the compounds were slightly less effective than MGBG at inhibiting cell growth, with 50% inhibitory concentration values of 1 to 10 microM as compared with 0.5 and 1.1 microM, respectively, for MGBG. Under 50% growth-inhibitory conditions, the inhibitors decreased SAMDC activity, increased ornithine decarboxylase activity and putrescine pools, and markedly depleted spermidine and spermine pools of L1210 cells. At the same time, mitochondrial integrity as assessed by whole-cell pyruvate oxidation and mitochondrial DNA content was not affected as it was with MGBG. At doses less than one tenth that of the maximally tolerated dose, all of the new inhibitors strongly suppressed the growth of B16 melanoma in vivo with minimal weight loss or toxicity. At doses less than one sixth the maximally tolerated dose, they effectively inhibited the growth of T24 human bladder carcinoma xenografts. In these same systems, MGBG showed only marginal antitumor activity. These studies identify two potent and efficacious inhibitors of SAMDC as potential antitumor agents and reaffirm the importance of SAMDC as a target in anticancer drug discovery.

Adenosylmethionine Decarboxylase↗

PubMed1992

Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY.

SecY is an integral plasma-membrane protein of Escherichia coli which is essential for the export of periplasmic and outer-membrane proteins containing cleavable signal sequences. We have synthesized SecY in vitro using an E. coli transcription/translation system. In the absence of membranes, SecY remained largely soluble but cosedimented on sucrose gradients with the membrane fraction when inside-out plasma-membrane vesicles (INV) had been added cotranslationally. Membrane association of SecY was unaffected if the endogenous SecY of the INV had been inactivated by either antibodies, a mutation or trypsin treatment. In contrast, inactivation of the INV SecY interfered with membrane targeting and, consequently, the processing of precursors to beta-lactamase and lambda receptor. When SecY-deprived INV were, however, first functionally reconstituted with in-vitro-synthesized SecY, targeting and translocation of the lambda receptor were partially restored. Thus, the assembly of SecY into INV in vitro leads to an active enzyme. In addition, we show that the prlA4 allele of the secY gene suppresses signal-sequence mutations of the lambda receptor in vitro. Collectively, our results demonstrate that SecY, while functioning as a membrane-located receptor for precursors of exported proteins, appears to be virtually independent of pre-existing SecY for its own membrane integration.

Bacterial Outer Membrane Proteins↗

PubMed1992

[HIV infection: survey among practicing physicians in Bern].

With increasing numbers of HIV-infected patients, practitioners will play a crucial role in the prevention of HIV infection and in the care of infected persons. To assess the need for accurate education programs we investigated by questionnaire the current practice, attitudes and knowledge of 1057 practitioners (general, internists, surgeons, dermatologists, gynecologists), on care, prevention and treatment of HIV infection and HIV-related problems in the Canton of Berne (population 1 million), Switzerland. 65% of the practitioners provided completely evaluable responses. More than 90% had already prescribed HIV tests by the end of 1989. A majority rejected routinely performed tests (e.g. for all pregnant women, for all patients before surgery). Only about 50% considered a patient's informed consent mandatory before each test. Primary prevention by sexual history-taking was only rarely done in current practice. All recommendations from the Swiss "Stop Aids" campaign were supported, and even experimental measures such as allowing i.v. drug use in street corner agencies or controlled distribution of heroin were supported by the majority. Mandatory testing of risk groups was favored by about 50% but almost nobody wanted to establish name records of persons with risk behaviour. By the end of 1989 about one third of practitioners regularly cared for HIV-infected persons. Most practitioners would prefer to care independently for asymptomatic HIV-infected persons, but would favor a specialist support when caring for symptomatic patients. Lack of medical skills and knowledge was the main reason for their reluctance in caring for patients. Skilled and experienced practitioners were more prone to care for additional patients and were more active in prevention. Knowledge about HIV transmission, HIV-related complications and treatment was good among general practitioners and internists. However, there was some confusion about the indications for active vaccination in Aids patients. The readiness of Bern practitioners to care for HIV-infected persons is already large and could be increased by improving their medical knowledge. However, it is unlikely that more medical information alone will improve their prevention activities. It appears that education programs aimed at improving the prevention activities of practitioners are best designed and performed by their skilled and experienced colleagues.

AIDS Serodiagnosis↗

PubMed1992

Investigations on the hepatic uptake systems for organic cations with a photoaffinity probe of procainamide ethobromide.

Azido procainamide methoiodide (APM), a photolabile derivative of the transport model compound procainamide ethobromide (PAEB), shows a close resemblance to PAEB from a physicochemical point of view. Like PAEB it is effectively taken up by the liver and excreted into bile. Kinetics of the uptake of APM in isolated hepatocytes revealed that in addition to a non-saturable process, two saturable uptake systems are involved (Km1 = 3 microM, Vmax1) = 80 pmol/min/10(6) cells, Km2 = 100 microM, Vmax2 = 130 pmol/min x 10(6) cells). The uptake rate of APM was inhibited markedly in the presence of other organic cations. Organic anions and uncharged compounds generally had no inhibitory effect on the APM uptake. These results support the theory that there is a separate hepatic uptake system for organic cations like APM. Photoaffinity labeling of intact hepatocytes as well as plasma membrane sub-fractions enriched with sinusoidal domains disclosed two major binding polypeptides with apparent M(r) of 48,000 and 72,000. Such labeling patterns were not observed in membranes from hepatoma cells that are deficient in organic solute uptake. Differential photoaffinity labeling with other cationic compounds such as tributylmethyl ammonium and d-tubocurarine reduced the incorporation of APM in these polypeptides. The 48- and 72-kDa proteins might be involved in carrier-mediated transport of type I organic cations at the hepatic uptake level.

Affinity Labels↗

PubMed1992

ATP-sensitive K+ channels in insulinoma cells are activated by nonesterified fatty acids.

Both 86Rb+ efflux experiments and electrophysiological studies have shown that arachidonic acid and other nonesterified fatty acids activate ATP-sensitive K+ channels in insulinoma cells (HIT-T15). Activation was observed with arachidonic, oleic, linoleic, and docosahexaenoic acid but not with myristic, stearic, and elaidic acids. Fatty acid activation of ATP-sensitive K+ channels was blocked by antidiabetic sulfonylureas such as glibenclamide. The activating effect of arachidonic acid was unaltered by indomethacin and by nordihydroguaiaretic acid, indicating that it is not due to metabolites of arachidonic acid via cyclooxygenase or lipoxygenase pathways. Moreover, the nonmetabolizable analogue of arachidonic acid, eicosatetraynoic acid, was an equally potent activator. Activation of ATP-sensitive K+ channels by fatty acids was potentiated by diacylglycerol and was inhibited by calphostin C, an inhibitor of protein kinase C. These findings indicate that fatty acid activation of ATP-sensitive K+ channels is most likely due to the participation of arachidonic acid (and other fatty acid)-activated protein kinase C isoenzymes. Activation of ATP-sensitive K+ channels by nonesterified fatty acids is not involved in the control of insulin secretion since arachidonic acid stimulates insulin secretion from insulinoma cells instead of inhibiting it.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1992

Activation and inhibition of ATP-sensitive K+ channels by fluorescein derivatives.

Fluorescein derivatives are known to bind to nucleotide-binding sites on transport ATPases. In this study, they have been used as ligands to nucleotide-binding sites on ATP-sensitive K+ channels in insulinoma cells. Their effect on channel activity has been studied using 86Rb+ efflux and patch-clamp techniques. Fluorescein derivatives have two opposite effects. First, like ATP, they can inhibit active ATP-sensitive K+ channels. Second, they are able to reactivate ATP-sensitive K+ channels subjected to inactivation or "run-down" in the absence of cytoplasmic ATP. Therefore reactivation of the inactivated ATP-sensitive K+ channel clearly does not require channel phosphorylation as is commonly believed. The results indicate the existence of two binding sites for nucleotides, one activator site and one inhibitor site. Irreversible binding at either the inhibitor or the activator site on the channel was obtained with eosin-5-maleimide, resulting in irreversible inhibition or activation of the ATP-sensitive K+ channel respectively. The irreversibly activated channel could still be inhibited by 2 mM ATP. After activation by fluorescein derivatives, ATP-sensitive K+ channels become resistant to the classical blocker of this channel, the sulfonylurea glibenclamide. Negative allosteric interactions between fluorescein/nucleotide receptors and sulfonylurea-binding sites were suggested by results obtained in [3H]glibenclamide-binding experiments.

Adenosine Triphosphate↗

PubMed1992

Identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus.

In Gram-negative bacteria, exported proteins are synthesized with an amino-terminal signal sequence which is cleaved off by the signal peptidase during, or shortly after the translocation process. Here, we report the identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus which cleaves homologous and heterologous precursor proteins at the authentic cleavage site. This signal peptidase is the first identified component of the R. capsulatus protein export machinery.

Amino Acid Sequence↗

PubMed1992

Proteolysis in protein import and export: signal peptide processing in eu- and prokaryotes.

Numerous proteins in pro- and eukaryotes must cross cellular membranes in order to reach their site of function. Many of these proteins carry signal sequences that are removed by specific signal peptidases during, or shortly after, membrane transport. Signal peptidases have been identified in the rough endoplasmic reticulum, the matrix and inner membrane of mitochondria, the stroma and thylakoid membrane of chloroplasts, the bacterial plasma membrane and the thylakoid membrane of cyanobacteria. The composition of these peptidases varies between one and several subunits. No site-specific inhibitors are known for the majority of these enzymes. Accordingly, signal peptidases recognize structural motifs rather than linear amino acid sequences. Such motifs have become evident by employing extensive site-directed mutagenesis to investigate the anatomy of signal sequences. Analysis of the reaction specificities and the primary sequences of several signal peptidases suggests that the enzymes of the endoplasmic reticulum, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts all have evolved from bacterial progenitors.

Animals↗

PubMed1992

Distamycin-induced inhibition of homeodomain-DNA complexes.

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.

Animals↗

PubMed1992

Structure and function of the cochlea in the African mole rat (Cryptomys hottentotus): evidence for a low frequency acoustic fovea.

The cochlea of the mole rat Cryptomys hottentotus was investigated with physiological and anatomical methods. In order to reveal the place-frequency map of the cochlea, iontophoretic HRP-applications were made in the cochlear nucleus at physiologically characterized locations. Subsequent HRP-transport in auditory nerve fibres and labeling patterns of spiral ganglion cells within the cochlea were evaluated. A cochlear place-frequency map was constructed from 17 HRP-applications in the cochlear nucleus at positions where neurons had characteristic frequencies between 0.1 and 12.6 kHz. As in other mammals, high frequencies were found to be represented at the cochlear base, low frequencies at the cochlear apex. The place-frequency map had three distinct parts which were characterized by their different slopes. A clear over-representation of the frequencies between 0.6 and 1 kHz was revealed, in this frequency range the slope of the place-frequency map amounted to 5.3 mm/octave. As calculated from the regression analysis, below 0.6 kHz the slope of the cochlear place-frequency map amounted to 0.24 mm/octave, above 1 kHz to 0.9 mm/octave. As in other mammals width of the basilar membrane (BM) increased from the cochlear base towards the cochlear apex. Also in concordance with the findings in other mammals, BM-thickness decreased from the cochlear base to the apex. However, it was remarkable to find that there was no or little change in BM-width and thickness between 40 and 85% BM-length. It was also revealed that scala tympani was only 1/10th the size found in the rat or other mammals of similar body size.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

ConA induced changes in energy metabolism of rat thymocytes.

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na+K(+)-ATPase, Ca(2+)-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na+K(+)-ATPase with about 20% each of total oxygen consumption, while Ca(2+)-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.

Animals↗

PubMed1992

Immunohistological detection of tissue factor in normal and abnormal human mammary glands using monoclonal antibodies.

Tissue factor (TF) is the primary cell-bound initiator of the coagulation protease cascade. The cytological distribution of TF in various tissues may be described on the basis of immunohistochemistry with epitope-defined monoclonal antibodies and the extravascular distribution of TF apparently represents a haemostatic envelope ready to activate coagulation when vascular integrity is disrupted. The present study localized TF in human breast cancer tissues when compared with normal breast gland tissues and benign disorders of the mammary gland. By use of a cocktail of three epitope-defined monoclonal antibodies, TF was detected only in the myoepithelia of the resting breast gland. In proliferating disorders like fibrocystic disease or in fibroadenomas, both myoepithelia and luminal epithelia showed TF expression. Of 115 breast cancers 93 reacted with anti-TF, in an inhomogeneous manner in terms of intensity and number of positive cells. There was a tendency for more positive and intensely stained cells to be found in well-differentiated structures such as tubules. Invasive ductal carcinomas exhibiting more positive and more strongly stained cells were less commonly metastatic to lymph nodes when compared with the tumours with no detectable or very low TF immunostaining. A semi-quantitatively recorded score of TF immunostaining correlated with the procoagulatory activity measured (7 fibroadenomas and 24 carcinomas). The results of this study suggest that proliferation and differentiation of the mammary gland is associated with enhanced TF expression in the epithelia which are negative for TF staining in the resting gland. Malignant growth is characterized by randomly expressed epithelial TF, which expression is enhanced and more frequent in well-differentiated tumour cells.

Antibodies, Monoclonal↗

PubMed1992

ConA induced changes in energy metabolism of rat thymocytes.

Adenosine Triphosphate↗

PubMed1992

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.

Animals↗