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Biomedical subjects

A Mayer

Publications and source records attributed to A Mayer.

At least 91 records · Page 5Linked to original sources
PubMed1998

cis and trans sites of the TOM complex of mitochondria in unfolding and initial translocation of preproteins.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. Our previous studies led to the concept of two preprotein binding sites acting in series, the surface-exposed cis site and the trans site exposed to the intermembrane space. We report here that preproteins are bound to the cis site in a labile fashion even at low ionic strength, whereas intermediates arrested at the trans site remained firmly bound at higher salt concentration. The stability of the trans site intermediate results from interactions of both the presequence and unfolded parts of the mature part of the preprotein with the TOM complex. Binding to the trans site proceeded at rates comparable with those of unfolding of the mature domain and appeared to be kinetically limited by the unfolding reaction. Efficient binding to the trans site and unfolding were observed with both outer membrane vesicles and intact mitochondria whose membrane potential, DeltaPsi, was dissipated. Upon re-establishing DeltaPsi, trans site-bound preprotein resumed translocation into the matrix. The rates of unfolding and binding to the trans site were the same as those for translocation into intact energized mitochondria. We conclude that preprotein unfolding in intact mitochondria can take place without the involvement of the translocation machinery of the inner membrane and, in particular, the matrix Hsp70 chaperone. Further, preprotein unfolding at the outer membrane can be a rate-limiting step for formation of the trans site intermediate and for the entire translocation reaction.

Animals↗
PubMed1998

The Y-chromosomal genes SRY and ZFY are transcribed in adult human brain.

Sexual differentiation of the brain is thought to be regulated by hormonal signals from the developing male gonad. However, more-recent experimental and clinical data throw some doubt on the general validity of the "classical" steroid hypothesis and suggest that additional intervening factors or mechanisms need to be considered. In particular, it is now envisaged that neurons are capable of acquiring sex-specific properties independently of their hormonal environment. Here we show that two Y-chromosomal genes involved in sex determination of the gonad, SRY and ZFY, are transcribed in hypothalamus, and frontal and temporal cortex of the adult male human brain. These genes are candidates for male-specific transcriptional regulators that could confer upon human brain cells the potential for hormone-independent realization and maintenance of genetic sex.

Aged↗
PubMed1998

Exemplifying guidelines for preparation of recombinant DNA products in phase I trials in cancer: preparation of a genetically engineered anti-CEA single chain Fv antibody.

Products of recombinant DNA technology have potential for the diagnosis or treatment of cancer. There is a need to investigate whether they function by the intended mechanism in small phase I clinical trials before their suitability for more extensive studies can be assessed. Quality and safety of these products should be assured prior to their use in humans in a way which is appropriate to the preliminary nature of the trials but not inhibitory to progress. The Cancer Research Campaign control recommendations for products derived from recombinant DNA technology (Begent RHJ and associates. Eur J Cancer 1993, 29A, 13, 1907-1910) provide guidelines for the production of new biotechnology products in academic research units within a relatively short time, while ensuring appropriate quality and safety. The practical application of the guidelines requires that solutions are found for the quality and safety issues during the production of recombinant products. We describe an approach to the relevant quality and safety issues during and after the production and purification of a genetically engineered anti-carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody for a phase I trial of radioimmunoguided surgery with the intention of providing a model for other products.

Biotechnology↗
PubMed1998

S-layered Aneurinibacillus and Bacillus spp. are susceptible to the lytic action of Pseudomonas aeruginosa membrane vesicles.

When S-layered strains of Bacillus stearothermophilus and Aneurinibacillus thermoaerophilus, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from P. aeruginosa during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact. meso-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease.

Bacillus↗
PubMed1998

Extending a teleradiology system by tools for 3D-visualization and volumetric analysis through a plug-in mechanism.

This paper describes ongoing research concerning interactive volume visualization coupled with tools for volumetric analysis. To establish an easy to use application, the 3D-visualization has been embedded in a state of the art teleradiology system, where additional functionality is often desired beyond basic image transfer and management. Major clinical requirements for deriving spatial measures are covered by the tools, in order to realize extended diagnosis support and therapy planning. Introducing the general plug-in mechanism this work exemplarily describes the useful extension of an approved application. Interactive visualization was achieved by a hybrid approach taking advantage of both the precise volume visualization based on the Heidelberg Raytracing Model and the graphics acceleration of modern workstations. Several tools for volumetric analysis extend the 3D-viewing. They offer 3D-pointing devices to select locations in the data volume, measure anatomical structures or control segmentation processes. A haptic interface provides a realistic perception while navigating within the 3D-reconstruction. The work is closely related to research work in the field of heart, liver and head surgery. In cooperation with our medical partners the development of tools as presented proceed the integration of image analysis into clinical routine.

Humans↗
PubMed1998

Technical aspects of virtual liver resection planning.

Operability of a liver tumor is depending on its three dimensional relation to the intrahepatic vascular trees which define autonomously functioning liver (sub-)segments. Precise operation planning is complicated by anatomic variability, distortion of the vascular trees by the tumor or preceding liver resections. Because of the missing possibility to track the deformation of the liver during the operation an integration of the resection planning system into an intra-operative navigation system is not feasible. So the main task of an operation planning system in this domain is a quantifiable patient selection by exact prediction of post-operative liver function and a quantifiable resection proposal. The system quantifies the organ structures and resection volumes by means of absolute and relative values. It defines resection planes depending on security margins and the vascular trees and presents the data in visualized form as a 3D movie. The new 3D operation planning system offers quantifiable liver resection proposals based on individualized liver anatomy. The results are visualized in digital movies as well as in quantitative reports.

Humans↗
PubMed1997

A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes.

Mucosally induced immunological tolerance is an attractive strategy for preventing or treating illnesses resulting from untoward inflammatory immune reactions against self- or non-self-antigens. Oral administration of relevant autoantigens and allergens has been reported to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders. However, the approach often requires repeated feeding of large amounts of tolerogens over long periods and is only partly effective in animals already systemically sensitized to the ingested antigen such as in animals already harboring autoreactive T cells, and thus presumably also in humans with an autoimmune disease. We have recently shown that oral administration of microgram amounts of antigen coupled to cholera toxin B subunit (CTB), can effectively suppress systemic T cell reactivity in naive as well as in immune animals. We now report that feeding small amounts (2-20 microg) of human insulin conjugated to CTB can effectively suppress beta cell destruction and clinical diabetes in adult nonobese diabetic (NOD) mice. The protective effect could be transferred by T cells from CTB-insulin-treated animals and was associated with reduced lesions of insulitis. Furthermore, adoptive co-transfer experiments involving injection of Thy-1,2 recipients with diabetogenic T cells from syngeneic mice and T cells from congenic Thy-1,1 mice fed with CTB-insulin demonstrated a selective recruitment of Thy-1,1 donor cells in the peripancreatic lymph nodes concomitant with reduced islet cell infiltration. These results suggest that protection against autoimmune diabetes can be achieved by feeding minute amounts of a pancreas islet cell autoantigen linked to CTB and appears to involve the selective migration and retention of protective T cells into lymphoid tissues draining the site of organ injury.

Administration, Oral↗
PubMed1997

Ubiquitin-dependent degradation of certain protein substrates in vitro requires the molecular chaperone Hsc70.

Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin and degradation of the tagged substrate. Conjugation is catalyzed via a three-step mechanism that involves three distinct enzymes that act successively: E1, E2, and E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate is specifically bound, catalyzes formation of a polyubiquitin chain that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we demonstrate that this protein is required for the degradation of actin, alpha-crystallin, glyceraldehyde-3-phosphate dehydrogenase, alpha-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is Hsc70-independent. Mechanistic analysis revealed that the chaperone is required for the conjugation reaction; however, it does not substitute for E3. Involvement of the chaperone in the proteolytic process requires complex formation with the substrate. Formation of this complex appears to be essential in the proteolytic process. In addition, the proper function of the chaperone in the proteolytic process requires the presence of K+, which allows rapid cycles of dissociation and association of the complex. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site. In addition, it can also act directly on the ubiquitination machinery.

Actins↗
PubMed1997

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233-1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.

Animals↗
PubMed1997

Divergence of HIV-1 quasispecies in an epidemiologic cluster.

BACKGROUND: During treatment with blood components prepared from an HIV-infected donation, two recipients became infected in 1985. One recipient infected her sexual partner. OBJECTIVE: To evaluate the evolution of the originally-shared HIV-1 quasispecies in different human hosts over time, sequence data were obtained from serum from the actual donation sample of blood, and from plasma samples collected from the four members of the epidemiologic cluster over a period extending from 1986 to 1993. METHODS: The V3 hypervariable region of env and the gag p17 gene were analysed. CD4 and CD8 counts, as well as HIV RNA burden data, were collected. RESULTS: One patient died from AIDS during the study. This patient showed a greater degree of diversity in the V3 region, with a higher positive charge over time, than the other individuals. Phylogenetic analysis revealed that the V3 sequences from each of the four individuals occupied separate branches of a phylogenetic reconstruction (tree). Two distinct subgroups evolved in the donor, one with GPGR and the other with GSGR/GSGK at the tip of the V3 loop. This latter group was not detected in the other individuals. The sequences in the sexual partner were no more related to those in the infecting transfusion recipient than to sequences from the other members of the cluster, consistent with sexual transmission having occurred at a time shortly after the recipient was infected. CONCLUSION: The shared HIV-1 quasispecies in this epidemiologic cluster diverged in an individual-specific manner.

Amino Acid Sequence↗
PubMed1997

A heterodimer of thioredoxin and I(B)2 cooperates with Sec18p (NSF) to promote yeast vacuole inheritance.

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between salt-washed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as I(B)2, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo and for the LMA1 activity in cell extracts. Each subunit acts via a novel mechanism, as the thioredoxin subunit is not acting through redox chemistry and LMA1 is still needed for the fusion of vacuoles which do not contain proteinase B. Both Sec18p and LMA1 act at an early stage of the in vitro reaction. Though LMA1 does not stimulate Sec18p-mediated Sec17p release, LMA1 cannot fulfill its function before Sec18p. Upon Sec17p/Sec18p action, vacuoles become labile but are rapidly stabilized by LMA1. The action of LMA1 and Sec18p is thus coupled and ordered. These data establish LMA1 as a novel factor in trafficking of yeast vacuoles.

Adenosine Triphosphatases↗
PubMed1997

Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF).

Vacuole inheritance in yeast involves the formation of tubular and vesicular "segregation structures" which migrate into the bud and fuse there to establish the daughter cell vacuole. Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion. We have developed biochemical and microscopic assays for the docking step of in vitro vacuole fusion and characterized its requirements. The vacuoles must be primed for docking by the action of Sec17p (alpha-SNAP) and Sec18p (NSF). Priming is necessary for both fusion partners. It produces a labile state which requires rapid docking in order to lead productively to fusion. In addition to Sec17p/Sec18p, docking requires the activity of the Ras-like GTPase Ypt7p. Unlike Sec17p/Sec18p, which must act before docking, Ypt7p is directly involved in the docking process itself.

Adenosine Triphosphatases↗
PubMed1997

Heat shock- and ethanol-induced ionic changes in C6 rat glioma cells determined by NMR and fluorescence spectroscopy.

The effects of two different stressors, heat shock (HS; 44 degrees C, 20 min) and ethanol (1.2 M, 60 min), on ion content and membrane potential were investigated in C6 rat glioma cells. Both treatments were previously shown to induce the HS response [26]. Intracellular pH (pH(i)), sodium ion concentration ([NA+]i), potassium ion concentration ([K+]i) and membrane potential were determined by means of continuous 31P and 23Na nuclear magnetic resonance (NMR), continuous fluorescence spectroscopy and 86Rb uptake. Lactate extrusion was determined in addition with respect to pH(i) regulation. The aim of this study was a detailed picture of HS and ethanol-induced ion changes in a single cell type, because stress-induced changes in the intracellular ionic balance may be important factors for determining proliferation, stress response and apoptosis. HS lowered the pH(i) from 7.38 +/- 0.04 to about 7.05 +/- 0.04. [Na+]i decreased during HS to 50% of the control and recovered to normal level 95 min after HS treatment. During HS, [K+]i remained constant but increased after HS. The membrane potential hyperpolarized from -83 mV to -125 mV and returned to initial values during HS treatment. Lactate extrusion increased 3-fold after HS. Ethanol (1.2 M) lowered the pH(i) from pH 7.38 +/- 0.04 to pH 7.0 +/- 0.04, but in contrast to heat strongly increased [Na]i. It hyperpolarized the membrane potential from -83 to -125 mV. Ethanol also increased lactate extrusion similar to HS. Also in contrast to the effect of HS, the potassium concentration decreased during ethanol treatment. The Na(+)-H+ exchanger monensin was used to overcome the apparent inhibition of the cellular Na(+)-H+ exchanger by HS. At normal pH(e) (7.4) monensin increased [Na+]i and pH(i) considerably. A subsequent HS reduced [Na+]i only minimally. Acidification of the cells by low pH(e) (6.2) prior to HS did not abolish the HS-induced drop of pH(i), indicating that the Na(+)-H+ exchanger was also inhibited at low pH(i). At low pH(e), monensin transports H+ into the cell. A subsequent HS decreased pH(i) only little, showing the importance of inhibition of the Na(+)-H+ exchanger for the HS-induced pH(i) decrease. 100 microM amiloride reduced pH(i) and [Na+]i in a similar way as HS, but did not change pH(i) and [Na+]i much during a HS. These results indicate that some of the HS-induced ionic changes are mediated by inhibition of the Na(+)-H+ exchanger, activation of Na(+)-K(+)-ATPase and changes of membrane conductance for ions.

Animals↗
PubMed1997

The effect of nigral implantation on sensitization to dopamine agonists in 6-hydroxydopamine-lesioned rats.

The implantation of fetal nigral tissue into the striatum of patients with Parkinson's disease is a promising approach to treatment which may produce clinical benefit partly by influencing drug responsiveness. The purpose of the present study was to determine the pharmacological mechanisms which drug response changes by measuring to what extent sensitization produced by repeated apomorphine treatment was attenuated by tissue implantation in rats with nigrostriatal lesions. Prior to implantation of nigral cell suspensions, the daily administration of apomorphine to rats with unilateral 6-hydroxydopamine lesions produced a progressive increase in the magnitude and duration of rotational behaviour. After implantation, apomorphine-induced rotational effects were reduced to levels observed upon the initial exposure to drug and did not increase following repeated treatment. Attenuated responses to selective D1 and D2 agonists were also observed after implantation. In vehicle-implanted rats, the initial response to apomorphine was attenuated but then increased following repeated apomorphine administration. No attenuation in responses to selective D1 and D2 agonists was observed in this group. Cell suspensions prepared from fresh and cyropreserved tissue produced similar behavioural effects, even though the volume of transplanted striatum exhibiting tyrosine hydroxylase activity was greater with fresh tissue. The duration of rotational behaviour induced by apomorphine was not affected by cell implantation. These findings suggest that the expression of sensitization in an animal model of parkinsonism may disappear after a period without drug treatment. Implantation of nigral tissue may produce beneficial results in parkinsonism by limiting the development of dopamine agonist-induced sensitization.

Animals↗
PubMed1997

Host-specific driving force in human immunodeficiency virus type 1 evolution in vivo.

To investigate the process of human immunodeficiency virus type 1 (HIV-1) evolution in vivo, a total of 179 HIV-1 V3 sequences derived from cell-free plasma were determined from serial samples in three epidemiologically linked individuals (one infected blood donor and two transfusion recipients) over a maximum period of 8 years. A systematic analysis of pairwise comparisons of intrapatient sequences, both within and between each sample time point, revealed a preponderance and accumulation of nonsynonymous rather than synonymous substitutions in the V3 loop and flanking regions as they diverged over time. This strongly argues for the dominant role that positive selection for amino acid change plays in governing the pattern and process of HIV-1 env V3 evolution in vivo and nullifies hypotheses of purely neutral or mutation-driven evolution or completely chance events. In addition, different rates of evolution of HIV-1 were observed in these three different individuals infected with the same viral strain, suggesting that the degree of positive pressure for HIV-1 amino acid change is host dependent. Finally, the observed similar rate of accumulation in divergence within and between infected individuals suggests that the process of genetic divergence in the HIV epidemic proceeds regardless of host-to-host transmission events, i.e., that transmission does not reset the evolutionary clock.

Amino Acid Sequence↗
PubMed1997

Latency versus persistence or intermittent recurrences: evidence for a latent state of murine cytomegalovirus in the lungs.

The state of cytomegalovirus (CMV) after the resolution of acute infection is an unsolved problem in CMV research. While the term "latency" is in general use to indicate the maintenance of the viral genome, a formal exclusion of low-level persistent productive infection depends on the sensitivity of the assay for detecting infectious virus. We have improved the method for detecting infectivity by combining centrifugal infection of permissive indicator cells in culture, expansion to an infectious focus, and sensitive detection of immediate-early RNA in the infected cells by reverse transcriptase PCR. A limiting-dilution approach defined the sensitivity of this assay. Infectivity was thereby found to require as few as 2 to 9 virion DNA molecules of murine CMV, whereas the standard measure of infectivity, the PFU, is the equivalent of ca. 500 viral genomes. Since murine CMV forms multicapsid virions in most infected tissues, the genome-to-infectivity ratio is necessarily >1. This assay thus sets a new standard for investigating CMV latency. In mice in which acute infection was resolved, the viral DNA load in the lungs, a known organ site of CMV latency and recurrence, was found to be 1 genome per 40 lung cells, or a total of ca. 1 million genomes. Despite this high load of CMV DNA, infectious virus was not detected with the improved assay, but recurrence was inducible. These data provide evidence against a low-level persistent productive infection and also imply that intermittent spontaneous recurrence is not a frequent event in latently infected lungs.

Animals↗
PubMed1997

Bone marrow failure by cytomegalovirus is associated with an in vivo deficiency in the expression of essential stromal hemopoietin genes.

Bone marrow (BM) failure associated with cytomegalovirus (CMV) infection is a feared complication after clinical BM transplantation. Experiments in long-term BM cultures have indicated that BM stromal cells (BMSC) are targets of productive CMV infection, but an in situ infection of BM stroma remained to be documented, and the pathomechanism is open to question. Here we describe a murine in vivo model of lethal CMV aplastic anemia (CMV-AA). The reconstitution of hematopoietic progenitor cells expressing stem cell factor (SCF) receptor was found to be defective in CMV-AA. While murine CMV replication in permissive parenchymal tissues is cytolytic, the hematopoietic cord was found to be a site of very limited virus production with foci of reticular BMSC expressing the intranuclear viral IE1 protein, but with only a few BMSC positive for viral genome in the in situ hybridization. XX-XY BM chimeras were established in order to quantitate Y-chromosome-tagged BMSC by a PCR specific for the male-sex-determining gene Tdy. This approach revealed that murine CMV infection is not associated with a significant loss of BMSC. Despite the physical integrity of the stromal network, the functional integrity of the stroma was impaired. While housekeeping genes were expressed normally in BMSC of infected mice, the expression of genes encoding the essential hemopoietins SCF, granulocyte colony-stimulating factor, and interleukin-6 was markedly reduced. In conclusion, the mechanism of BM failure is not a stromal lesion but an insufficient stromal function. These findings explain CMV-AA as a manifestation of multiple hemopoietin deficiency.

Anemia, Aplastic↗