Enhancement of gentamicin-induced nephrotoxicity by Mg deficiency in non-pregnant rats. Morphological, enzyme histochemical and immunohistochemical studies

Non-pregnant rats fed an Mg-deficient diet showed some degenerated and calcified proximal tubules in the inner region of the medullary rays accompanied by reduced or absent enzyme activities. After gentamicin treatment some damaged convoluted proximal tubules occurred. Enzyme histochemistry revealed decreased activities for brush border and lysosomal hydrolases; using immunohistochemistry lesions were detectable for the cytoskeletal proteins keratin and vimentin. Administration of gentamicin to Mg-deficient rats led to a further decrease of hydrolase activities in obviously intact proximal tubules and drastic structural and enzymatic defects as well as alterations of the cytoskeletal proteins in the convoluted and straight segments of other proximal tubules and to a lesser degree also in glomeruli and further portions of the tubular apparatus including the collecting ducts.     

Fast optical monitoring of microscopic excitation patterns in cardiac muscle.

Many vital processes depend on the generation, changes, and conduction of cellular transmembrane potentials. Optical monitoring systems are well suited to detect such cellular electrical activities in networks of excitable cells and also tissues simultaneously at multiple sites. Here, an exceptionally fast array system (16 x 16 photodiodes, up to 4,000,000 samples per second, 12-bit resolution) for imaging voltage-sensitive dye fluorescence, permitted real time measurements of excitation patterns at a microscopic size scale (256 pixels within an area of 1.8-8 mm2), in rat cardiac muscle in vitro. Results emphasize a recent hypothesis for cardiac impulse conduction, based on cardiac structural complexities, that is contradictory to all continuous cable theory models.     

Molecular dynamics of the alpha-helical epitope of a novel synthetic lipopeptide foot-and-mouth disease virus vaccine

A novel synthetic foot-and-mouth disease virus (FMDV) peptide vaccine consisting of a synthetic B-cell and macrophage activator covalently linked to an amphiphilic alpha-helical T-cell epitope was developed. The low molecular weight vaccine of 3400 daltons is composed of virus VP1 antigenic determinant and the immunologically active lipotripeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P3CSS) as built-in adjuvant. The vaccine, tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl-FMDV-VP1 (VP1 = serotype O1K 135-154) induces protection against homologous challenge and serotype-specific virus neutralizing antibodies in guinea pigs after single administration without further adjuvants or carriers. A P3CSS conjugate with the FMDV-VP1 segment 135-154 of strain O Wuppertal produced only poor cross-protection against challenge with O1K virus. The antigenic determinant VP1(135-154) is an amphiphilic alpha-helix, as shown by CD. Molecular dynamics simulations (MDS) carried out using the highly homologous alpha-helical alcohol dehydrogenase (ADH) segment H3 as starting conformation for VP1(138-149) suggest that the FMDV segment 138-149 may adopt alpha-helical conformation during binding to its T-cell receptor, and that the development of the system during MDS may be considered as the dissociation step of the complex.     

Reactive extraction of penicillin G from mycel-containing broth in a countercurrent extraction decanter

Penicillin was recovered from mycel-containing fermentation broth by direct reactive extraction into a counter-current extraction decanter, Type CA 226-290 of the Westfalia Separator Co., at room temperature via steady state operation. Penicillin concentrations in the feed varied from 3 to 41 g L(-1), Amberlite LA-2 carrier concentrations from 7 to 20 g L(-1)and/or DITDA carrier concentrations from 7.2 to 84 g L(-1), the LA-2-to-penicillin mole concentration ratio from 4 to 6.4, and/or the DITDA-to-penicillin mole concentration ratio was maintained at 2. The throughputs of the fermentation broth (520 to 880 L h(-1)) of the solvent phase (200 to 860 L h(-1)) and the over all throughput (800 to 1750 L h(-1)) were high. Extraction degrees of 72 to 96% were achieved between pH 4.6 and 5.1. Without carriers in the same pH range, extraction degrees of only 17 to 19% were attained. By reducing the pH to 2.3 and in the absence of carriers, the degree of extraction was increased to 61%. However, during the extraction, 6.5% of the penicillin decomposed. At these high throughputs, the steady state was attained within 1 to 4 min. Through the mechanical stress, the length of the hyphae was reduced and the protein content of the broth was increased by 50 to 100%. However, this protein content had no appreciable influence on the phase separation.     

Chronic immune thrombocytopenic purpura--immunological analyses of a patient pre- and post-HIV seroconversion

A seven year follow-up of immune parameters is reported for a patient with chronic immune thrombocytopenic purpura (ITP) pre and post human immunodeficiency virus (HIV) seroconversion. Therapies such as intravenous IgG, prednisone, vincristine, or Ciclosporin A had no clear-cut beneficial effect on platelet counts. A long-term normalization of platelet counts was achieved by splenectomy. At splenectomy the patient was seropositive for HIV, most likely transmitted by blood products received half a year prior to laparatomy. Mean plasma levels of the second component of complement, C2, were half of the normal values prior to and within the lowest normal range post HIV seroconversion. Nevertheless, the T cell-dependent B cell response to HIV, which is dependent on the activation of C3 via the classical pathway of complement, was normal: Western blot analysis of total IgG and of IgG subclass responses to individual HIV antigens proved to be unimpaired.     

Antiviral T cell competence and restriction specificity of mixed allogeneic (P1 + P2----P1) irradiation chimeras

Mixed irradiation bone marrow chimeras were prepared by reconstituting lethally irradiated C57BL/10 (B10) or B10.D2 mice with T cell-depleted bone marrow cells of B10 plus B10.D2 origin. These chimeras were healthy and survived well under conventional housing conditions and after experimental laboratory infections. Of a total of 17 chimeras tested, 2 died spontaneously or from the injected virus. Twelve of fifteen chimeras mounted a measurable cytotoxic T cell response to virus. Despite approximately equal percentages of B10 and B10.D2 lymphocytes in chimeras, cytotoxic T cell responses to vaccinia virus and lymphocytic choriomeningitis virus were mediated variably by either syngeneic or allogeneic donor lymphocytes; thus the H-2 type of effector T cells frequently did not correspond to the 50:50 distribution of spleen or peripheral blood lymphocytes. Cytotoxic responses were restricted exclusively to recipient H-2 type. All mixed chimeras examined were able to mount a good IgG response to vesicular stomatitis virus. These results confirm previous data suggesting that such mixed chimeras are healthy and immunocompetent and demonstrate strict recipient-determined restriction specificity of effector T cells; they also suggest that if T help is necessary for induction of virus-specific cytotoxic T cells, it does not require host-restricted interactions between helper T cells and precursor cytotoxic T cells.     

Focusing water shock waves for lithotripsy by various ellipsoid reflectors

A spherical shock wave generated in the first focus of an ellipsoid is focused in the second focus after reflection. Shadowgraphs provide an impression of the converging process, while the pressure field ist measured with speciality designed pressure probes having a high temporal and spatial resolution. Two different types of reflectors have been investigated. The first is like a spherical mirror with a short focal length, the second is a deep reflector "surrounding" the generation point. Variation of the parameters, size of the reflector, and incident shock energy reveal their influence on the focusing process and the point of maximal pressure, which in the case of deep ellipsoids can deviate from the geometrical focus. Here, non-linear propagation effects on the long distances to the focus lead to an increase in the size of the focal region. Although these reflectors utilize much more of the primary shock front, their peak pressure of 400 bar is low compared with the "shallow" reflectors with 1200 bar and their small focus. The effect of the deep type on kidney stones is however greater. The desintegration process and the reaction of the generated cavitation bubbles have been studied with a high speed camera. It can be shown that stone splinters do not injure the kidney tissue, but liquid jets generated by oscillating cavitation bubbles lead to tissue damage.     

Isolation of heparin-binding growth factors from bovine, porcine and canine hearts

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.     

A novel L-glutamate oxidase from Streptomyces endus. Purification and properties

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2. The Km value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.