The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat.

In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase (hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.     

Effect of Hydrogenase and Mixed Sulfate-Reducing Bacterial Populations on the Corrosion of Steel

The importance of hydrogenase activity to corrosion of steel was assessed by using mixed populations of sulfate-reducing bacteria isolated from corroded and noncorroded oil pipelines. Biofilms which developed on the steel studs contained detectable numbers of sulfate-reducing bacteria (10⁴ increasing to 10⁷/0.5 cm²). However, the biofilm with active hydrogenase activity (i.e., corrosion pipeline organisms), as measured by a semiquantitative commercial kit, was associated with a significantly higher corrosion rate (7.79 mm/year) relative to noncorrosive biofilm (0.48 mm/year) with 10⁵ sulfate-reducing bacteria per 0.5 cm² but no measurable hydrogenase activity. The importance of hydrogenase and the microbial sulfate-reducing bacterial population making up the biofilm are discussed relative to biocorrosion.     

Enzymological characterization of a putative canine analogue of primary hyperoxaluria type 1.

This paper concerns an enzymological investigation into a putative canine analogue of the human autosomal recessive disease primary hyperoxaluria type 1 (alanine:glyoxylate/serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and serine:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephropathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including D-glycerate dehydrogenase/glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate/serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal human liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is analogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

Improvement of regeneration of Lycopersicon pennellii protoplasts by decreasing ethylene production

Summary Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplasts could be correlated with high ethylene production and an increased cell sap osmolality. The ethylene action inhibitor silver thiosulphate improved protoplast yield and viability, especially when using donor tissue, germinated and cultured on medium containing silver thiosulphate (type III plants). Moreover, the choice of cell wall degrading enzymes influenced protoplast viability, since ethylene release was significantly lower using Cellulase R 10 than Cellulysin. All improvements together resulted in an efficient protocol for the isolation and regeneration of Lycopersicon pennellii protoplasts.Abbrevations ACC 1-Aminocyclopropane-1-carboxylic acid - FW Fresh Weight - Mes -Morpholino ethane sulphonic acid - NMU N-Nitroso-N-Methyl-Urea - PE Plating Efficiency = Number of calli / number of protoplasts x 100% - Pps protoplasts - STS Silver thiosulfate     

Analysis of respiratory neuronal activity in fetal sheep.

We developed a new method to monitor fetal medullary respiratory neurons utilizing a two-stage approach. At 129-133 days of gestation, sheep were anesthetized, and a window was placed over the area of the fourth ventricle. After a recovery period of 3-5 days, the fetus was exteriorized into a saline bath under maternal spinal anesthesia, and the head was connected rigidly to a stereotaxic frame. Microelectrodes were inserted into the area of the nucleus tractus solitarius during rapid-eye-movement sleep, and extracellular recordings of 223 respiratory neurons were analyzed: 76% were inspiratory, 9% expiratory, and 15% phase spanning, as classified by visual and computer correlation to diaphragmatic activity. More detailed analysis of 100 neurons was done to assess the respiratory component (eta 2) by use of a modification of the method developed by Orem and Dick (J. Neurophysiol, 50: 1098-1107, 1983). With use of cohorts of 25 breaths, fetal respiratory neurons were found to frequently change their phase relationship to diaphragmatic activity. The eta 2 statistic of fetal respiratory neurons was not a stable characteristic but changed over time. This could be a reflection of an immature central respiratory system before birth or the lack of major sensory inputs.     

Interleukin-4. A regulatory protein

Since its discovery in 1982, numerous biological activities of interleukin-4 (IL-4) have been described. Like other cytokines, IL-4 is highly pleiotropic, both with respect to the number of different target cells that are responsive to it and with respect to the number of different biological responses it elicits. Interleukin-4 was initially described as a costimulant for the proliferation of B lymphocytes stimulated with anti-IgM antibody. Synonyms for this cytokine are B cell growth factor-1 (BCGF-1) and B cell stimulatory factor-1 (BSF-1). After cloning of both the murine and human IL-4, the use of recombinant IL-4 enabled detailed studies of its biological functions. Many cell types, mainly of hematological origin, express receptors for IL-4. Accordingly, effects of IL-4 have been described on B lymphocytes, T lymphocytes, NK cells, mononuclear phagocytes, mast cells, fibroblasts and hematopoietic progenitor cells. Currently, there are three major areas in which IL-4 appears to play an important role: 1) regulation of B cell growth and of antibody isotype expression. In this context, a possible role for IL-4 in allergic reactions is of special interest. 2) Stimulation of T cell growth and the generation of cytotoxic T lymphocytes. In addition to the suppressive effects on the induction of non HLA-restricted cellular cytotoxicity by natural killer- (NK) and lymphokine-activated killer (LAK) cells, this suggests a role for IL-4 in the regulation of cellular immune responses. 3) Regulation of the growth and differentiation of hematopoietic bone marrow stem cells. IL-4 itself does not induce proliferation of hematological progenitor cells but it can modulate the growth-factor dependent proliferation of these cells. In this review the biological functions of IL-4, reported until present, are discussed.     

Distribution of chylomicron degradation products in the isolated,perfused rat heart

Chylomicron degradation by hearts from fed and fasted rats was studied using a perfusion technique, which allows the separate collection of coronary (Q_rv) and interstitial effluent (Q_i). Upon perfusion with [³H]-cholesterol-containing chylomicrons the tissue recovery of label was highest in the fasted state, while label recovered in Q_i was highest in the fed state. Density gradient centrifugation of Q_i indicated that the label was recovered in lipoproteins with higher densities: low density lipoproteins (1.019<d<1.050), high density lipoproteins (1.050<d<1.21) and a fraction of d>1.21. These particles probably represent chylomicron degradation products (remnants and “surface fragments”). Our results indicate that tissue cholesterol uptake during chylomicron degradation may be inhibited in the fed state. Furthermore, the role of the myocyte (or interstitial) lipoprotein lipase in chylomicron degradation is discussed.     

Mutually synchronized relaxation oscillators as prototypes of oscillating systems in biology

Summary This paper discusses the analogy between phenomena in populations of coupled biological oscillators and the behaviour of systems of synchronized mathematical oscillators. Frequency entrainment in a set of coupled relaxation oscillators is investigated with perturbation methods. This analysis leads to quantitative results for entrainment and explains phenomena such as travelling waves in systems of spatially distributed oscillators.     

The role of auditory stimuli in the courtship of Drosophila melanogaster

Simulated courtship song of male Drosophila melanogaster was played to males or females of this species. Upon receiving the song males increase their locomotor activity and start courting each other, whereas females reduce their locomotor activity. In wingless males the locomotor activity difference between the silent control and the experimental sound situation is much larger than in winged males, due to the inactivity of wingless males in the control situation. Males which had been kept singly up to the time of the experiment exhibit higher locomotor and sexual activity than group housed males. A second component of the male courtship song ‘sine song’ is described, together with experiments which investigate the sensory basis of the effect male courtship song has on males.