Biomedical subjects
Publications and source records attributed to M Gross.
An unusual case of lipoma arising in the floor of the mouth of a 70-year-old man is presented. The unusual appearance suggests that the tumour be included as a rare possibility in the differential diagnosis of swellings in the floor of the mouth that can also adversely affect lower denture stability.
Levansucrase (EC 2.4.1.10), an exoenzyme of Pseudomonas syringae pv. phaseolicola, was purified to homogeneity from the cell supernatant by chromatography on TMAE-Fraktogel and butyl-Fraktogel. The enzyme has molecular masses of 45 kDa under denaturing conditions and 68 kDa during gel filtration of the native form. In isoelectric focusing, active bands appeared at pH 3.55 and 3.6. Maximum sucrose cleaving activities were measured at pH 5.8 to 6.6 and 60 degrees C. The enzyme was highly tolerant to denaturing agents, proteases, and repeated freezing and thawing. The molecular weight of the produced levan depended on temperature, salinity, and sucrose concentration. The enzyme had levan-degrading activity and did not accept raffinose as a substrate. Comparison of the N-terminal amino acid sequence with the predicted amino acid sequence of levansucrases from Erwinia amylovora and Zymomonas mobilis showed 88 and 69% similarity, respectively, in amino acids 5 to 20. No similarity could be detected to levansucrases of gram-positive bacteria in the first 20 amino acids. By comparison of all levansucrases which have been sequenced to date, the enzyme seems to be conserved in the gram-negative bacteria. The rheological behavior of the product levan prompted a new assessment of the enzyme's role in pathogenesis. Depending on formation conditions, levan solutions exclude other polymer solutions. This behavior supports the presumption that the levansucrase is important in the early phase of infection by creating a separating layer between bacteria and plant cell wall to prevent the pathogen from recognition.
Reshaped human MAb RSHZ19, which is specific for the surface fusion protein of respiratory syncytial virus (RSV) is in clinical development for the prevention and treatment of RSV-induced disease in human infants. The current studies profile lung virus clearance and evaluate lung histopathology in MAb-treated, RSV-infected cotton rats, a well characterized model of RSV infection. The highest dose of this MAb (10 mg/kg) administered parenterally 24 h before infection decreased subgroup A or B RSV lung titers to below detectable levels (> or = 2.3 log10 reduction), and significantly reduced lung virus titers (> or = 2.0 log10 reduction) when administered 96 h postinfection. Prophylactic administration of 10 mg/kg RSHZ19 was significantly more protective than 1000 mg/kg conventional human immune serum globulin (HSIg), and protective serum-neutralizing titers in MAb-treated animals (1:32, which correlated with approximately 40 micrograms/ml determined by anti-idiotype ELISA) were significantly lower than those reported previously for HSIg or for convalescent human serum (1:200-1:400). MAb concentration in lung lavages was determined by ELISA to be approximately 1% of the serum MAb concentration, but was not detectable by neutralization assay. The degree of lung histopathology in MAb-treated cotton rats was proportional to lung virus titer, and inversely proportional to the RSHZ19 dose administered. There was no evidence of exacerbated disease in the lungs of MAb-treated animals. These studies thus support the potential clinical utility of RSHZ19 MAb in the prevention and treatment of RSV-induced disease in humans.
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Voice quality was assessed in 44 laryngectomized patients using 13 subjective and 8 objective criteria. All patients were using either esophageal voice. Servox vibrator or a voice prosthesis. Five of eight patients with voice prostheses had secondary tracheo-esophageal punctures. Subjective assessment showed better results for most of the criteria in favor of the voice prosthesis. With objective measurements, maximum phonation times and maximum numbers of syllables per intake of air were significantly higher in patients with voice prostheses compared with other techniques for voices restoration. Overall intelligibility in the postlaryngectomy "telephone test" was obviously better in the patients using the prostheses than in those with esophageal voice or Servox vibrator voice, while no significant differences were found among prostheses and esophageal voices in the medium speed of speaking, various sound pressure levels and medium habitual pitch range. In patients with primary voice prostheses maximum phonation time was longer (average, 2.5 s) than in patients with secondary voice prostheses. Overall intelligibility of the different groups was not significantly influenced by age. The need for postoperative voice rehabilitation was significantly higher in patients with esophageal voices compared with those using Servox vibrator voice or a voice prostheses. Our results support in principle primary tracheo-esophageal puncture for a voice prostheses as best treatment of the laryngectomized patient.
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AMP deaminase (AMPD) is a complex allosteric enzyme encoded by a multigene family in higher eukaryotes. The amino terminus of each AMPD gene is unique, while the mild and carboxy termini have been highly conserved among all the AMPD genes. Mutational analyses of the AMPD1 gene demonstrate that the catalytic site and a regulatory site, likely an ATP binding site, are located in the highly conserved carboxy terminus. Deletion mutants and a normal splice variant of AMPD1 demonstrate that the amino terminus has a profound influence on catalytic activity of AMPD and by inference from prior studies this region also influences binding of AMPD1 to myosin. Results of these studies suggest a regulatory model in which alternative splicing in the amino terminal region of AMPD1 generates isoforms of AMPD that exhibit differential sensitivity to effector molecules such as ATP.
The conformation of a three-disulphide derivative of bovine alpha-lactalbumin bound to the molecular chaperone GroEL has been investigated by monitoring directly its hydrogen exchange kinetics using electrospray ionization mass spectrometry. The bound protein is weakly protected from exchange to an extent closely similar to that of an uncomplexed molten globule state of the three-disulphide protein. Binding to GroEL in this system appears to involve relatively disordered partly folded states resembling intermediates formed in the very early stages of kinetic folding of many proteins in vitro.
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We have purified a soluble rabbit reticulocyte protein, previously termed the supernatant factor, that reverses the inhibition of protein synthesis in hemin-deficient lysate by promoting the inactivation of the hemin-controlled eIF-2 alpha kinase (HCR) mediating the effect of hemin deficiency. We have identified the supernatant factor as a member of the heat shock protein 70 family, denoted hsp 70(R), based upon its size (72 kDa), specific reaction to a monoclonal antibody against eukaryotic hsp 70, strong binding affinity for ATP, and endogenous ATPase activity. We have investigated the role of hsp 70(R) and hemin in the regulation of the activation of HCR from its latent precursor (ProHCR) and the translational control of protein synthesis in rabbit reticulocyte lysate. We find that autophosphorylation of Pro-HCR is reduced by about 75% by adding saturating hsp 70(R) and almost completely reduced by adding either saturating hemin or limiting hemin plus limiting hsp 70(R). In contrast, autophosphorylation of HCR, which is similar in magnitude to that of ProHCR, is unaffected by adding either saturating hsp 70(R), saturating hemin, or limiting amounts of both. The activation of HCR (measured by inhibition of protein synthesis) from isolated ProHCR is completely prevented by hsp 70(R) in the presence, but not absence, of dithiothreitol. This suppression appears to be due to the association of hsp 70(R) with ProHCR, since hsp 70(R) action is prevented by ATP/Mg2+ and because activation of HCR from less purified ProHCR, that has associated hsp 70(R), is suppressed by dithiothreitol alone. This association is confirmed by sucrose gradient centrifugation, which shows co-sedimentation of some hsp 70(R) with ProHCR following preincubation together that is prevented by ATP/Mg2+ and does not occur after conversion of ProHCR to HCR. Limiting hsp 70(R) reduces the concentration of hemin required to prevent activation of HCR from isolated ProHCR from 0.75 to 0.15 microM and the optimal hemin concentration needed to maintain protein synthesis in reticulocyte lysate from 25 to 10 microM. Limiting hsp 70(R) also allows the delayed addition of hemin to suppress activation of HCR from ProHCR and to reverse inhibition of protein synthesis in hemin deficient lysate. The association of hsp 70(R) with ProHCR also underlies the observation that much more protein is synthesized in reticulocyte lysate in the absence of hemin at 25 degrees C than at temperatures of 30 degrees C or greater. These observed effects may be specific to hsp 70(R), since they are not observed with rabbit reticulocyte eIF-2 or eIF-2B, and since the comparable hsp 70 from bovine brain is incapable of maintaining or restoring protein synthesis in hemin-deficient lysate.
In man, there are at least four isoforms of adenosine monophosphate deaminase (AMPD): myoadenylate deaminase in skeletal muscle, the L isoform in liver, and the E1 and E2 isoforms in erythrocytes. Myoadenylate deaminase is encoded by the AMPD1 gene located on chromosome 1 p13-p21, the L isoform by the AMPD2 gene, and both isoforms in erythrocytes by the AMPD3 gene. Myoadenylate deaminase deficiency is found in 2-3% of all muscle biopsies. The inborn type of myoadenylate deaminase deficiency is caused by a single mutant allele harbouring two mutations: C34-->T (Gln-->Stop) and C143-->T (Pro-48-->Leu). Population studies revealed a frequency of the mutant allele of 0.12 in Caucasian Americans and Germans. The C34-->T mutation is located in exon 2, which is alternatively spliced in part of the AMPD1 transcript in human muscle. Since the second mutation does not affect enzyme function, alternatively spliced mRNA encodes a catalytically active enzyme. Only one patient with a disorder linked to liver AMPD has been described so far. In this patient the decreased inhibition of this enzyme by GTP resulted in uric acid overproduction and gout. A complete lack of erythroyte AMPD activity is found in asymptomatic subjects. The molecular basis of both disorders is not yet known.
Oceans not only cover the major part of the earth's surface but also reach into depths exceeding the height of the Mt Everest. They are populated down to the deepest levels (approximately 11,800 m), which means that a significant proportion of the global biosphere is exposed to pressures of up to 120 MPa. Although this fact has been known for more than a century, the ecology of the 'abyss' is still in its infancy. Only recently, barophilic adaptation, i.e. the requirement of elevated pressure for viability, has been firmly established. In non-adapted organisms, increased pressure leads to morphological anomalies or growth inhibition, and ultimately to cell death. The detailed molecular mechanism of the underlying 'metabolic dislocation' is unresolved. Effects of pressure as a variable in microbiology, biochemistry and biotechnology allow the structure/function relationship of proteins conjugates to be analyzed. In this context, stabilization by cofactors or accessory proteins has been observed. High-pressure equipment available today allows the comprehensive characterization of the behaviour of proteins under pressure. Single-chain proteins undergo pressure-induced denaturation in the 100-MPa range, which, in the case of oligomeric proteins or protein assemblies, is preceded by dissociation at lower pressure. The effects may be ascribed to the positive reaction volumes connected with the formation of hydrophobic and ionic interactions. In addition, the possibility of conformational effects exerted by moderate, non-denaturing pressures, and related to the intrinsic compressibility of proteins, is discussed. Crystallization may serve as a model reaction of protein self-organization. Kinetic aspects of its pressure-induced inhibition can be described by a model based on the Oosawa theory of molecular association. Barosensitivity is known to be correlated with the pressure-induced inhibition of protein biosynthesis. Attempts to track down the ultimate cause in the dissociation of ribosomes have revealed remarkable stabilization of functional complexes under pseudo-physiological conditions, with the post-translational complex as the most pressure-sensitive species. Apart from the key issue of barosensitivity and barophilic adaptation, high-pressure biochemistry may provide means to develop new approaches to nonthermic industrial processes, especially in the field of food technology.
In an attempt to generalize previous observations (Jaenicke et al., Appl. Environ. Microbiol. 1988, 54, 2375-2380) and to find a convenient model system for studies of the pressure response, we tested the suitability of Escherichia coli and Thermotoga maritima (bacteria), and of five different eukaryotic species including the filamentous fungi Asteromyces cruciatus and Dendryphiella salina, and the marine yeasts Debaryomyces hansenii, Rhodosporidium sphaerocarpum, and Rhodotorula rubra. Using two-dimensional polyacrylamide gel electrophoresis, detailed investigations on the pressure response were carried out with E. coli and Rhodosporidium sphaerocarpum. In the former organism, major pressure response proteins could not be detected, although there are significant differences in expression of some proteins as well as some minor components that are found in all of the high pressure cell extracts but not in extracts from cultures grown at atmospheric pressure. In Rhodosporidium sphaerocarpum, no change in protein expression patterns was observed between 0.1 and 20 MPa. However, approaching the limit of viability of 50 MPa, additional protein spots became detectable at 45 MPa. This finding correlates with the observation of abnormal growth forms of the organism at this pressure (Lorenz, R. et al. manuscript in preparation).
The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK. Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.
We expressed gram amounts of full-length and mutant rat brain calcium-binding proteins (calbindins-D28K) lacking one or two "EF-hand" motifs in a bacterial expression system. The cDNA for the full-length rat calcium-binding protein was cloned into the NdeI and BamHI sites of the pET3a vector. Additionally, constructs of the rat brain calcium-binding protein lacking EF-hand 2 (delta 2 mutant), EF-hand 6 (delta 6 mutant), and EF-hands 2 and 6 (delta 2, 6 mutant) were constructed using the same vector. These chimeric plasmids were used to transfect BL21 (DE3) pLysS Escherichia coli cells. Following transformation, the cells were grown in the presence of isopropylthiogalactoside in order to induce bacterial T7 polymerase, which resulted in the production of large amounts of the proteins of interest in the bacterial cytosol. Expressed full-length and delta 2 and delta 2,6 mutant proteins represented 50% or more of total bacterial protein. The delta 6 protein was not expressed. Cell lysis followed by purification of the proteins on DEAE-cellulose routinely resulted in gram yields of the proteins. The purified proteins displayed the appropriate amino acid composition and amino-terminal amino acid sequence. When analyzed by matrix-assisted laser desorption mass spectrometry the proteins were found to have the appropriate molecular weights (within the accuracy limits of the instrument). The expressed proteins bound to a polyclonal antiserum raised against chick intestinal calcium-binding protein. In addition, the full-length, delta 2, and delta 2,6 mutants bound calcium as assessed by a 45Ca blotting procedure. The production of large amounts of readily purified vitamin D-dependent calcium-binding proteins should be useful in biophysical studies of the proteins.
A 42 amino acid peptide, Pf EBA-175 (1062-1103), also called EBA-peptide 4 of the 175-kDa Plasmodium falciparum sialic acid binding protein, a putative merozoite invasion ligand, has been shown to be a target of parasite growth inhibitory antibodies. We expressed and purified a recombinant protein, NS1-Pf EBA-175 (946-1133) which included the 42 amino acid peptide, and compared antibodies induced by immunization with the protein to antibodies raised against the 42 amino acid peptide. Sera from rabbits immunized with the recombinant protein and the synthetic peptide immunoprecipitated authentic EBA-175, and had comparable ELISA titers against peptide Pf EBA-175 (1062-1103). However, IFAT titers against infected erythrocytes and growth inhibitory activity were substantially higher in sera from animals immunized with the 42 amino acid synthetic peptide. Epitope mapping of the 42 amino acid peptide identified a 19 amino acid peptide, Pf EBA-175 (1069-1087), which blocked the ability of antibodies against the 42 amino acid peptide to (1) immunoprecipitate EBA-175, (2) bind to the 42 amino acid peptide in an ELISA, and (3) recognize infected parasites in an IFAT. Sera from rabbits immunized with the 19 amino acid peptide conjugated to KLH had excellent parasite growth inhibitory activity (at 1:5 serum dilution, 49.9 +/- 7.4%, mean +/- SD of three separate assays), but the activity was lower in each of the three assays than that of sera from rabbits immunized with the 42 amino acid peptide (67.8 +/- 24.8%). These data indicate that the activity of antibodies raised against the linear 42 amino acid peptide, Pf EBA-175 (1062-1103) are primarily, if not exclusively, directed against 19 of the 42 amino acids, and identify this region of Pf EBA 175 as a target for vaccine development.
This study examines the hypothesis that whole gut irrigation with polyethylene glycol-electrolyte gavage solution (PEG-ELS) increases intravascular volume. Seventeen patients drank 6 to 8 liters of PEG-ELS in preparation for elective colonoscopy. The patients were weighed and serum electrolytes, albumin, hematocrit, blood urea nitrogen, and creatinine were obtained prior to the gavage and 8 hr following gavage. Plasma volume was measured before and after gavage using an isotope dilution technique involving 125I-human serum albumin. No patients developed symptoms of intravascular volume excess or depletion following gavage. There was no significant change in body weight, serum sodium, chloride, potassium, bicarbonate, blood urea nitrogen, or creatinine following gavage with PEG-ELS. When measured by the isotope dilution technique, the mean plasma volume increased from 3174 +/- 117 ml before gavage to 3365 +/- 160 ml following gavage (P = 0.03). This represented a mean percentage change in plasma volume of 5.88 +/- 2.4%. The percentage change in plasma volume associated with gavage ranged from -9.8 to +29.8%. This data supports the hypothesis that gavage with polyethylene glycol-electrolyte solution is associated with an increase in plasma volume. Although in most patients the increase in plasma volume is minimal, there is significant variability in this response, with some patients experiencing substantial increases in plasma volume.
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