Antigenic determinants of T lymphocyte alpha beta receptor and other leukocyte surface proteins as differential markers of skeletal muscle regeneration: detection of spatially and timely restricted patterns by MAM microscopy.

Different stages of human muscle regeneration have been identified by multiple antigen-mapping (MAM) microscopy at the level of a novel marker system. This immunofluorescence method allows the selective imaging of numerous antigen signals from cells or tissue sections. It is shown that 2 monoclonal antibodies (mAbs) against the common region of the alpha beta T lymphocyte antigen receptor (alpha beta and alpha TCR chains) and 3 mAbs against the leukocyte surface antigens Leu8, OKM5 and Leu19 recognize regenerating muscle cells at different time points of regeneration in human muscle sections. Paradoxically, these epitopes are not expressed by T lymphocytes or other mononuclear leukocytes invading regenerating muscle. Hence, presence of the corresponding antigens in muscle fibers may exclude their expression by muscle-invasive immune cells suggesting a function in muscle-specific cell-to-cell recognition. Simultaneous localization of these epitopes in Duchenne dystrophy reveals 10 different phenotypes of regenerating and normal infantile muscle cells due to different developmental stages during the myocyte differentiation. In adult muscle (mitochondrial myopathy) segmental muscle fiber necrosis is accompanied by high concentration of alpha beta/alpha TCR epitopes in the intact fiber ends, which are the target sites of myogenesis. The same sites are invaded by OKM5+ endomysial capillary sprouts that terminate at the tip of the alpha beta/alpha TCR reactive fiber ends. These hitherto unrecognized initial events of segmental muscle regeneration seem to be followed by fragmentation of the invasive capillaries into single endothelial cells, which then switch from the OKM5+ Leu19- through the OKM5+ Leu19+ to the OKM5- Leu19+ phenotype. This cell type exhibits the typical features of Leu19+ myogenic stem cells described earlier. The findings may give rise to a concept of muscle repair, in which the alpha beta/alpha TCR-related antigen, concentrated in fiber stumps, might provide positional information for invading endothelial cells. These cells appear to be a source of myogenic stem cells for regeneration.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGFβ), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGFβ and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGFβ to bFGF determines the magnitude of the Schwann cell response.     

Alteration by centric fission of the diploid chromosome number in Vicia faba L.

An individual with a diploid chromosome complement of 2n=14 instead of 2n=12 is deseribed for the broad bean Vicia faba L. This karyotypic deviation resulted from centromere splitting of the wildtype metacentric satellite chromosome pair as demonstrated by Giemsa banding pattern. It is the first case of centric fission observed in this species. Our data support the hypothetic mechanism of Robertsonian rearrangements suggested by Holmquist and Dancis (Genetica 52–53: 151–163, 1980).     

Diffusional limitations of immobilized Escherichia coli in hollow-fiber reactors: influence on 31P NMR spectroscopy

Escherichia coli cells were immobilized and grown in hollow-fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using (35)S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 degrees C were shown to be severe, with regions of starved cells occupying up to 80% of the cell-containing region. Phosphorus-31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume-averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur-35 autoradiographs of a reactor operated at 16 degrees C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near-normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopic experiments.     

Enterotoxigenic Bacteria in Food and Water from an Ethiopian Community

Food and water samples from an Ethiopian community were screened for the presence of enterotoxin-producing bacteria. Using the Chinese hamster ovary cell assay, 40 of 213 isolates (18.8%) produced heat-labile (LT) enterotoxin. These LT-producing isolates comprised 33 of 177 (18.6%) strains from 24 of 68 food samples (35.3%) and 7 of 36 (19.4%) isolates of 4 of 17 water samples (23.5%). One LT-producing strain each of Salmonella emek and of Shigella dysenteriae was found. Three pseudomonads, all LT producers, produced heat-stable enterotoxin as gauged by the suckling mouse test. Two strains of LT-enterotoxigenic Escherichia coli O68 were found in water samples. No enterotoxigenic E. coli were isolated from food samples, but 13 of the LT-producing strains were Enterobacter, Klebsiella, Serratia, and Proteus species, and 7 food samples yielded more than one species of enterotoxigenic bacterium. Of the enterotoxigenic isolates from food, 15 were oxidase-positive strains of the genera Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Vibrio. LT-enterotoxigenic Enterobacter, Acinetobacter, Klebsiella, Proteus, Providencia, and Serratia species represented 20 of the food and water isolates. Culture supernatant fluids of representative strains of oxidase-positive and oxidase-negative species giving positive reactions in Chinese hamster ovary cell tests induced fluid accumulation in rabbit ileal loops. Eight of the food samples and two of the water samples contained more than one isolate or species of enterotoxigenic bacterium. The stability of the LT production by oxidase-positive bacteria and non-E. coli strains was assessed by the rabbit skin and adrenal cell tests after 9 months and 1 year of storage, respectively, in Trypticase soy broth with glycerol at −70°C. Only 33% of the oxidase-positive strains were still LT enterotoxigenic. Of the oxidase-negative strains, 50 and 33% were LT producing at 9 months and 1 year, respectively. None of the E. coli isolates, both enterotoxigenic and nonenterotoxigenic, possessed K88, K99, or colonization factor antigen. The survey demonstrates the presence in food and water of enterotoxigenic bacteria of the same species as those isolated from cases of infantile diarrhea in the same community, although a correlation between these sources and infantile diarrhea remains to be established.     

Enzymes of amide and ureide biogenesis in developing soybean nodules

Amide and ureide biogenic enzymes were measured in the plant fraction of soybean (Glycine max) nodules during the period 11 to 23 days after inoculation with Rhizobium japonicum (USDA 3I1b142). Enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, and aspartate aminotransferase, showed substantial increases in their specific activities over the time course. These increases paralleled the induction of nitrogenase activity in the bacteroid and leghemoglobin synthesis in the plant fraction. The specific activity of asparagine synthetase, however, showed a rapid decline after an initial increase in specific activity. Following the initial increases in the ammonia assimilatory enzymes, there was an increase in the activity of 5-phosphoribosylpyrophosphate amidotransferase, the enzyme which catalyzes the first committed step of de novo purine biosynthesis. This was followed by a dramatic increase in the purine oxidative enzymes, xanthine dehydrogenase and uricase. Smaller increases were observed in the activities of enzymes associated with the supply of metabolites to the purine biosynthetic pathway: phosphoglycerate dehydrogenase, serine hydroxymethylase, and methylene tetrahydrofolate dehydrogenase.     

Structure and origin of a snapback defective interfering particle RNA of vesicular stomatitis virus

The nucleotide sequence of the region which covalently links the complementary strands of the "snapback" RNA of vesicular stomatitis virus, DI011, is (Formula: see text). Both strands of the defective interfering (DI) particle RNA were complementary for their full length and were covalently linked by a single phosphate group. Because the strands were exactly the same length and complementary, template strand and daughter strand nucleocapsids generated during replication of DI 011 were undistinguishable on the basis of sequence, a property not shared by other types of DI particle RNAs. Treatment of the RNA with RNase T1 in high-ionic-strength solutions cleaved the RNA only between positions 1 and 1'. These results and the availability of the guanosine residue in position 1' to kethoxal, a reagent that specifically derivatizes guanosines of single-stranded RNA, suggest that steric constraints keep a small portion of the "turnaround" region in an open configuration. The sequence of the turnaround region was not related in any obvious way to the sequences at the 3' and 5' termini and limited the number of possible models for the origin of this type of DI particle RNA. Two models for the genesis of DI 011 RNA are discussed. We favor one in which the progenitor DI 011 RNA was generated by replication across a nascent replication fork.     

Ammonia Assimilation in Alnus glutinosa and Glycine max: SHORT-TERM STUDIES USING [N]AMMONIUM

The pattern of assimilation of NH₄⁺ by Alnus glutinosa, a N₂-fixing, nonleguminous angiosperm, was examined. Detached nodules, roots, and nodulated roots of intact plants were exposed to ¹³NH₄⁺ for up to 15 minutes. Glutamine was the most highly labeled compound at all times; the only other compound labeled significantly was glutamate. Similar results were obtained after incubating soybean (L. merr) nodules and roots with ¹³NH₄⁺. These observations and the results of pulse-labeling and inhibitor studies with nodules of Alnus were distinctly different from those predicted for the assimilation of NH₄⁺ via glutamine synthetase and glutamate synthase and suggest that glutamate dehydrogenase may play a major role in the assimilation of exogenously supplied NH₄⁺.