Development of a radiometric metabolic viability testing method for human and porcine skin

A radiometric viability assay based upon the conversion of [¹⁴C]glucose into ¹⁴CO₂ by the viable cells on the dermal side of whole skin has been developed. The assay proved to be sensitive, reproducible, and practical, and was based upon the use of a microbiological growth detection system commonly used in many hospitals and laboratories. Relatively small samples of skin (0.25–1.00 g) were used in the test, and it was found that microbiological contamination did not interfere with the assay under normal conditions. The linear proportionality of the assay with both time and amount of skin assayed precluded the difficulties of nonlinear proportionality in other systems, allowing direct comparisons to be made between skin samples of different sizes and different incubation times. The assay could also detect ¹⁴CO₂ released from many radiolabeled substrates, including glucose, aspartate, glutamate, ornithine, orotic acid, and glycerol. Thus, the method could be used to test a number of cellular functions necessary for viability, including glycolysis, the functioning of the citric acid cycle and the pentose phosphate pathway, sugar and amino acid metabolism, pyrimidine biosynthesis, and cryopreservative agent metabolism. Since any of these tests could be performed in 4 hr, a viability assay based upon glycolysis alone, or in combination with any of the other tested substrates, could be carried out after allograft skin procurement before a decision needed to be made on skin cryopreservation.     

Simian virus 40 T antigen activates the late promoter by modulating the activity of negative regulatory elements.

Late promoter activity measured before viral DNA replication results from a complex involvement of negative and positive cis-acting elements located both in the enhancer and in the 21-bp repeats. GC motifs located within the 21-bp repeats act in cooperation with sequences overlapping the early TATA box to down-regulate the late promoter activity. Analysis of insertion mutants indicates that the late promoter might be negatively regulated at least partially by the early promoter machinery. The GTI motif located within the enhancer as well as the GC motifs lose the ability to down-regulate the late promoter in the presence of T antigen. Results obtained with tsA58 protein indicate that two different domains of T antigen are involved in the negative autoregulation of the early promoter activity and in the release of the down-regulation of the late promoter by the GC motifs.     

Galanin attenuates retention of one-trial reward learning.

Galanin is a neuropeptide that coexists with acetylcholine in the septohippocampal pathway. Galanin appears to have a negative modulating influence on cholinergic transmission, suggesting that it might interfere with memory formation on a one-trial discriminative reward learning task. The apparatus was a starburst maze with five radiating alleys, one an ascending baited alley. The subjects were 38 two to three month old Sprague-Dawley rats cannulated in the body of the lateral ventricles and deprived to 80% of initial weight. Ten rats were infused i.c.v. over six mins. with 8 micrograms galanin in 24 microliters saline and 10 with saline alone. Twenty mins. after completion of infusion, each rat was placed in the maze and observed under "blind" conditions for number of errors (blind alleys entered) and latency to reach reward. Each rat's speed score was 100 sec./latency. One day later, each rat was retested in the maze. Each rat's retention scores were its decrease in errors and increase in speed between the single training trial and the retention trial. Galanin infused rats showed significantly less retention by both measures. In a second experiment, either the same dose of galanin or saline alone was infused 20 mins. before the retention trial. There was no significant effect, suggesting that galanin may interfere with memory formation rather than memory retrieval or task performance.     

Paleoecology of Upper Eifelian and Lower Givetian coral limestones in the northwestern Sauerland (Devonian; Rhenish Massif)

Summary The prevailing sandy/silty lower part of the Middle Devonian in the northwestern Sauerland includes two coral limestone horizons, which contain a rich fauna of corals, stromatoporoids, and calcareous algae. The Ihmert-Formation is subdvided into three parts. The older coral limestone horizon is the Grünewiese-Member of the Ihmert-Formation (uppermost Eifelian), the younger is in the Bredenbruch-Member of the Unterhonsel-Formation (lower Lower Givetian). Conclusions about the environmental constraints are drawn from the sedimentology and the fossil content of the coral limestones. Predominant biostromes are built between storm wave base and normal wave base. Only the few bioherms grew above the normal wave base. These coral limestones were deposited in a tropical or subtropical normal marine environment in the shallow euphotic zone. Among the reef-builders epoecism is very frequent, and until now this phenomenon has not been investigated in detail. Fragile rugose and tabulate corals lived as commensals with stromatoporoids. Some other aspects of paleoecology are concisely presented.     

Low-resolution structure of the tetrameric phenylalanyl-tRNA synthetase from Escherichia coli. A neutron small-angle scattering study of hybrids composed of protonated and deuterated protomers

Escherichia coli phenylalanyl-tRNA synthetase is a tetrameric protein composed of two types of protomers. In order to resolve the subunit organization, neutron small-angle scattering experiments have been performed in different contrasts with all types of isotope hybrids that could be obtained by reconstituting the alpha 2 beta 2 enzyme from the protonated and deuterated forms of the alpha and beta subunits. Experiments have been also made with the isolated alpha promoter. A model for the alpha 2 beta 2 tetramer is deduced where the two alpha promoters are elongated ellipsoids (45 x 45 x 160 A3) lying side by side with an angle of about 40 degrees between their long axes and where the two beta subunits are also elongated ellipsoids (31 x 31 x 130 A3) with an angle of 30 degrees between their axes. This model was obtained by assuming that the two pairs of subunits are in contact in an orthogonal manner and by taking advantage of the measured distance between the centers of mass of the alpha 2 and beta 2 pairs (d = 23 +/- 2 A).     

Localization of a reactive exofacial sulfhydryl on the glucose carrier of human erythrocytes

Tryptic digestion studies of the human erythrocyte glucose carrier have shown that a reactive and transport-sensitive exofacial sulfhydryl is located in the carboxy-terminal half of the molecule, corresponding to Cys347, Cys421, or Cys429. In the present studies, the erythrocyte glucose carrier labeled on the exofacial sulfhydryl with bis(maleimidomethyl) ether-L-[35S]cysteine was chemically cleaved, either at tryptophans by N-bromosuccinimide or at nonalkylated cysteines by 2-nitro-5-thiocyanobenzoic acid. The resulting fragments were separated by linear gradient polyacrylamide gel electrophoresis, and the labeled fragments were identified by their apparent molecular weight, and by immunoblotting with antibodies to specific regions of the carrier protein. All of the labeled fragments were recognized by an antibody to the carboxy terminus of the carrier, but not by an antibody to a cytoplasmic loop on the C-terminal half of the carrier. The labeled exofacial sulfhydryl was assigned to Cys429, since this is the only residue of the three possibilities which is beyond the expected cleavage sites of the two reagents in the carrier sequence. These results concur with the predictions of hydropathy analysis and will be relevant for studies of how modification of this sulfhydryl affects carrier function, particularly since several other known carrier isoforms lack a corresponding cysteine.     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

Isolation and characterisation of the Xenopus laevis albumin genes: loss of 74K albumin gene sequences by library amplification.

The blood of the frog X.laevis contains 2 albumins of 68,000 and 74,000 daltons which are encoded in the liver by two related mRNAs. When an amplified X.laevis DNA library was screened with cloned albumin cDNA only 68,000 dalton albumin gene sequences were isolated. Hybridisation of the albumin cDNA to Southern-blots of Eco R1 digested X.laevis DNA showed that the sequences present in the recombinants did not account for all the fragments which hybridised on the Southern-blots. This indicated that 74K albumin gene sequences exist but that they are not present in the amplified DNA library. A X.laevis genomic library was therefore constructed and screened for albumin genes without amplification. Both 68K and 74K albumin gene sequences were isolated. Recombinants containing 74K albumin gene sequences grew extremely poorly and this probably explains why the 74K albumin sequences were ot isolated from the amplified library. Characterisation of the cloned DNA indicates that there is one sequence coding for the 68K albumin but two different sequences coding for the 75K albumin.     

Biofeedback treatment of dysmenorrhea

Nine dysmenorrheic women were run in EMG and thermal biofeedback procedures with concurrent autogenic relaxation practice. Significant reductions in subjective estimates of symptomology associated with dysmenorrhea were noted in all subjects. EMG levels correlated positively with the reductions in symptoms. Thermal levels did not correlate with EMG. In fact no consistent patterns in thermal measures were noted. However, thermal biofeedback cannot be ruled out as an effective treatment for dysmenorrhea since reductions in symptoms occurred during thermal biofeedback training. Another significant aspect of the present study is the effectiveness of long treatment procedures. A six month period was employed and significant reductions in symptoms were noted following two months of biofeedback treatment. Finally, the importance of beginning biofeedback treatment prior to onset of menstrual symptoms is indicated.