Identification, physical map location and sequence of the denV gene from bacteriophage T4

The denV gene from bacteriophage T4, which codes for endonuclease V, a small DNA repair enzyme, has been cloned and identified by an approach combining DNA sequencing and genetics, independent of the phenotypic effect of the cloned gene. Appropriate DenV+ and DenV- deletion mutants were mapped physically to define precisely a region encompassing the denV gene. This region was sequenced in order to identify a protein-coding sequence of the correct size for the denV gene (400-500 bp). Finally, identification was confirmed by sequencing the corresponding fragments cloned from four genetically and phenotypically well-characterized denV mutants. The denV gene is located at 64 kb on the T4 genome, adjacent to the ipII gene, and codes for a basic protein of 138 amino acids with a deduced molecular weight of 16,078.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

Influence of sialic acid on cell surface properties in I-cell disease fibroblasts

Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing. This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1, National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health.     

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman     

Chromosome replication in aging human diploid fibroblasts

The patterns of termination of DNA replication in human embryonic MRC-5 fibroblasts at four passage levels have been examined by autoradiography. Only chromosome 9 showed statistically significant differences in the time of replication among cultures of different ages. This chromosome terminated replication earlier at later passages than at earlier passages, primarily because of differences in the time of replication of the centromere region. Because very few differences were observed at different passage levels, we conclude that changes in the order of chromosome replication are unlikely to contribute to the phenomenon of in vitro senescence.     

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue,cell, and model membrane preparations

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.     

Oxidation of methane in boreal forest soils: a comparison of seven measures

Methane oxidation rates were measured in boreal forest soils using seven techniques that provide a range of information on soil CH₄ oxidation. These include: (a) short-term static chamber experiments with a free-air (1.7 ppm CH₄) headspace, (b) estimating CH₄ oxidation rates from soil CH₄ distributions and (c)²²²Rn-calibrated flux measurements, (d) day-long static chamber experiments with free-air and amended (+20 to 2000 PPM CH₄) headspaces, (e) jar experiments on soil core sections using free-air and (f) amended (+500 ppm CH₄) headspaces, and (g) jar experiments on core sections involving tracer additions of¹⁴CH₄. Short-term unamended chamber measurements,²²²Rn-calibrated flux measurements, and soil CH₄ distributions show independently that the soils are capable of oxidizing atmospheric CH₄ at rates ranging to < 2 mg m^–2 d^–1. Jar experiments with free-air headspaces and soil CH₄ profiles show that CH₄ oxidation occurs to a soil depth of 60 cm and is maximum in the 10 to 20 cm zone. Jar experiments and chamber measurements with free-air headspaces show that CH₄ oxidation occurs at low (< 0.9 ppm) thresholds. The¹⁴CH₄-amended jar experiments show the distribution of end products of CH₄ oxidation; 60% is transformed to CO₂ and the remainder is incorporated in biomass. Chamber and jar experiments under amended atmospheres show that these soils have a high capacity for CH₄ oxidation and indicate potential CH₄ oxidation rates as high as 867 mg m^–2 d^–1. Methane oxidation in moist soils modulates CH₄ emission and can serve as a negative feedback on atmospheric CH₄ increases.     

Competition by Estrogens for Catecholamine Receptor Binding In Vitro

Abstract: We have examined the ability of various steroids to compete for high-affinity binding of ³H-labeled ligands to catecholamine receptors in membranes prepared from rat cerebral cortex, striatum, and anterior pituitary. Ligands employed were: [³H]WB4101, [³H]prazosin, [³H]yohimbine, and [³H]clonidine (alpha-noradrenergic); [³H]dihydroalprenolol (beta-noradrenergic); [³H]spiperone and [³H]ADTN (dopaminergic). Only the 17β estrogens were effective and only binding of [³H]spiperone and [³H]ADTN in striatum and [³H]WB4101 and [³H]prazosin in cerebral cortex was reduced. Thus putative dopaminergic and alpha₁-noradrenergic sites alone appear to recognize estrogens. A slight competitive effect on [³H]spiperone binding to anterior pituitary membranes was also observed. Among the 17β estrogens tested, the most effective in all cases was the catechol estrogen 2-hydroxyestradiol (2-OHE₂). The ability of 2-OHE₂ (IC₅₀= 20–30 μM) to inhibit ligand binding to alpha₁ receptors was comparable to that of norepinephrine (IC₅₀= 10–20 μM), whereas for dopamine receptors in striatum and pituitary 2-OHE₂ was an order of magnitude less effective than dopamine (IC₃₀= 12 μM) in reducing binding of ³H ligands. Estradiol-17β and 2-hydroxyestrone were also able to inhibit binding, but the order of steroid potency was different for alpha₁ and dopaminergic receptors. Progesterone, testosterone, and corticosterone were without effect in all cases. These results show that there is specificity of steroid interactions with catecholamine receptors in the brain, both in terms of steroid structure and receptor type. The possible relevance of these interactions to neuroendocrine function is discussed.     

Demonstration of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Cultured Human Schwann Cells

Abstract: Schwann cell cultures were established from adult human sural nerve biopsies. 2'3'-Cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was estimated in the homogenates of those cells by a sensitive isotope assay using [³H]2',3'-cyclic AMP as substrate. A high level of CNPase activity was observed in cultured Schwann cells, whereas cultured human muscle and skin fibroblasts contained negligible levels of CNPase activity. CNPase of human Schwann cells followed typical enzyme-substrate kinetics, with an apparent K _m of 1.6 m M for 2',3'-cyclic AMP, and the enzyme was stimulated by detergents such as Triton X-100 and deoxycholate. It was inhibited by p -chloromercuricbenzoate and 2'-AMP. These properties are typical of CNPase isolated from adult brain and spinal cord. CNPase can serve as a new biochemical marker of normal cultured human Schwann cells and can be useful in analyzing the properties of cultured Schwann cells from patients with dysschwannian neuropathies.     

Pathological changes in the nephrocytes of the shore crab, Carcinus maenas, following injection of bacteria

The gills of Carcinus maenas were examined by light and electron microscopy following injection of either sterile saline or the bacteria Bacillus cereus and Moraxella sp., to determine any role(s) for the nephrocytes in the host defense reactions. The results showed that although intact bacteria were not sequestered to the nephrocytes, these cells were active in the removal of large quantities of cell debris from the hemolymph. Much of this material was derived from the breakdown of the hemocytes in response to the presence of bacteria and it's accumulation in the central vacuoles of the nephrocytes resulted in the degradation of these cells. It is proposed that while nephrocytes do not phagocytose intact bacteria, they augment the host defenses by clearing much of the hemocyte and associated bacterial debris from the gills, thus preventing blockage of the lamellar sinuses and subsequent impairment of respiration.