Efficient cleavage of pre-tRNAs by E. coli RNAse P RNA requires the 2'-hydroxyl of the ribose at the cleavage site.

RNAse P cleaves pre-tRNAs to liberate 5'-flanks and 5'-matured, 5'-phosphorylated tRNAs. It is not evident if the 2'-hydroxyls of the ribose moieties in the substrate are involved in the reaction. To study their influence in two different pre-tRNAs, we have modified specifically the 2'-hydroxyl groups at the cleavage site and in neighbouring positions. We have shown that these hydroxyls are important but not essential for the processing of these substrates by E. coli RNase P RNA (M1 RNA). The reduction in the catalytic efficiency was moderate for 2'-deoxy and severe for 2'-methoxy substitutions at the cleavage site. Additional effects of modifications in neighbouring positions were smaller. Based on our data we suggest that the modifications do not interfere with binding of the substrate, whereas they prevent an optimal steric arrangement for the hydrolysis reaction.     

Taxonomic revision of the genus Carasobarbus Karaman, 1971 (Actinopterygii,Cyprinidae)

Representatives of the fish genus Carasobarbus Karaman, 1971 (Actinopterygii: Cyprinidae) from the Middle East and North Africa were previously placed in 14 different genus-group taxa (Barbellion, Barbus, Barynotus, Capoeta, Carasobarbus, Cyclocheilichthys, Kosswigobarbus, Labeobarbus, Luciobarbus, Pseudotor, Puntius, Systomus, Tor and Varicorhinus). The generic assignment of several species changed frequently, necessitating a re-evaluation of their taxonomic status. In this study, the genus Carasobarbus is revised based on comparative morphological examinations of about 1300 preserved specimens from collections of several museums and freshly collected material. The species Carasobarbus apoensis, Carasobarbus canis, Carasobarbus chantrei, Carasobarbus exulatus, Carasobarbus fritschii, Carasobarbus harterti, Carasobarbus kosswigi, Carasobarbus luteus and Carasobarbus sublimus form a monophyletic group that shares the following combination of characters: medium-sized barbels with a smooth last unbranched dorsal-fin ray, nine or 10 branched dorsal-fin rays and six branched anal fin-rays; scales large, shield-shaped, with many parallel radii; the lateral line containing 25 to 39 scales; the pharyngeal teeth hooked, 2.3.5-5.3.2 or 2.3.4-4.3.2; one or two pairs of barbels. The species are described in detail, their taxonomic status is re-evaluated and an identification key is provided. A lectotype of Systomus luteus Heckel, 1843 is designated. Carasobarbus Karaman, 1971, Kosswigobarbus Karaman, 1971, and Pseudotor Karaman, 1971 are subjective synonyms, and acting as First Reviser we gave precedence to the name Carasobarbus.     

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Reactivity pattern of 15 HLA-Dw1 homozygous typing cells in primary mixed lymphocyte culture

Summary The Dwl specificity was highly correlated with the serologically determined HLA-DR1 antigen in the Eighth International Histocompatibility Workshop 1980. By testing a large number of HLA-Dwl, DR1 defined homozygous typing cells (HTC) in a checkerboard primary mixed lymphocyte reaction, on a panel of about 30 HLA-DR1 heterozygous individuals, and in family segregation, three Dwl subtypes could be defined in association with certain HLA-A, -B, and -C-antigens. HTC TA, FRI, and FRA carrying the HLA haplotypes A11, B35, Cw4 or A3, B35, Cw4 in the homozygous state gave positive typing results with most HLA-DR1 positive panel members and stimulated only four other Dw1 HTCs (SRR35%). In contrast to this operationally broad specificity, Dw1-HTC-HEN (HLA-A2, B44, C-, homozygous) was non-stimulatory to all HTCs except one, but gave high responses against these, leading to the definition of a narrow specificity included in the broad one. Another such narrow specificity was represented by HTC FEE (HLA-A2, B27, Cw2 homozygous). Typing patterns with FEE were mostly different from those defined with other HTC. In family studies a specific typing pattern for this HTC could be shown to segregate with HLA. However, within some of these responses a contribution of the HLA haplotype in the trans position must be assumed.Supported in part by DFG grant Ri 164/14     

Dexamethasone stimulates insulin receptor synthesis in cultured rat hepatocytes

The ability of the glucocorticoid dexamethasone to modulate the insulin receptor was examined directly in primary cultures of hepatocytes prepared from adult male rats. Hepatocytes were cultured in a defined medium in the presence and absence of dexamethasone, 0.1 microM. The exposure of hepatocytes to dexamethasone resulted in a time-dependent (steady state by 32 h) increase in insulin binding in both intact hepatocytes and Triton X-100-soluble extracts (total insulin receptor content). The enhanced insulin binding found in soluble extracts of dexamethasone-treated hepatocytes was the result of an increase in insulin receptor number without a change in receptor affinity. In order to assess the mechanism by which dexamethasone "up-regulates" the insulin receptor, the heavy isotope density-shift technique was used to analyze insulin receptor turnover in control and dexamethasone-treated hepatocytes. Hepatocytes were initially cultured for 32 h in standard culture media containing only "light" (14C, 12C, 1H) amino acids. In hepatocytes exposed to dexamethasone, a 417% increase in insulin binding in Triton X-100-soluble extracts was observed. After 32 h, when steady state binding is achieved in dexamethasone-treated cultures, parallel cultures of hepatocytes incubated in the absence and presence of dexamethasone were washed and subsequently cultured in media containing "heavy" amino acids (15N, 13C, 2H). The time-dependent disappearance of light insulin receptor (receptor degradation) and appearance of heavy insulin receptor (receptor synthesis) were monitored using CsCl gradients to resolve the two density species of receptor. At steady state, the rate of receptor synthesis (k8) was 2.94 and 0.62 fmol of insulin bound h-1 in dexamethasone-treated and control hepatocytes, respectively. In contrast to this large increase in the rate of receptor synthesis observed in dexamethasone-treated cells, the first order rate constant for decay (k d) was the same in dexamethasone-treated (0.074 h-1) and in control (0.077 h-1) hepatocytes. We therefore conclude that glucocorticoid-induced up-regulation of the insulin receptor in the liver is due to stimulation of insulin receptor synthesis.     

Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.     

Long-term, low-level microwave irradiation of rats.

Our goal was to investigate effects of long-term exposure to pulsed microwave radiation. The major emphasis was to expose a large sample of experimental animals throughout their lifetimes and to monitor them for effects on general health and longevity. An exposure facility was developed that enabled 200 rats to be maintained under specific-pathogen-free (SPF) conditions while housed individually in circularly-polarized waveguides. The exposure facility consisted of two rooms, each containing 50 active waveguides and 50 waveguides for sham (control) exposures. The experimental rats were exposed to 2,450-MHz pulsed microwaves at 800 pps with a 10-microseconds pulse width. The pulsed microwaves were square-wave modulated at 8-Hz. Whole body calorimetry, thermographic analysis, and power-meter analysis indicated that microwaves delivered at 0.144 W to each exposure waveguide resulted in an average specific absorption rate (SAR) that ranged from 0.4 W/kg for a 200-g rat to 0.15 W/kg for an 800-g rat. Two hundred male, Sprague-Dawley rats were assigned in equal numbers to radiation-exposure and sham-exposure conditions. Exposure began at 8 weeks of age and continued daily, 21.5 h/day, for 25 months. Animals were bled at regular intervals and blood samples were analyzed for serum chemistries, hematological values, protein electrophoretic patterns, thyroxine, and plasma corticosterone levels. In addition to daily measures of body mass, food and water consumption by all animals, O2 consumption and CO2 production were periodically measured in a sub-sample (N = 18) of each group. Activity was assessed in an open-field apparatus at regular intervals throughout the study. After 13 months, 10 rats from each group were euthanatized to test for immunological competence and to permit whole-body analysis, as well as gross and histopathological examinations. At the end of 25 months, the survivors (11 sham-exposed and 12 radiation-exposed rats) were euthanatized for similar analyses. The other 157 animals were examined histopathologically when they died spontaneously or were terminated in extremis.