Two-dimensional protein electrophoretic analysis of postponed aging inDrosophila

Five populations ofDrosophila melanogaster that had been selected for postponed aging were compared with five control populations using two-dimensional protein gel electrophoresis. The goals of the study were to identify specific proteins associated with postponed aging and to survey the population genetics of the response to selection. A total of 321 proteins were resolvable per population; these proteins were scored according to their intensity. The resulting data were analyzed using resampling, combinatoric, and maximum parsimony methods. The analysis indicated that the populations with postponed aging were different from their controls with respect to specific proteins and with respect to the variation between populations. The populations selected for postponed aging were more heterogeneous between populations than were the control populations. Maximum parsimony trees separate the selected populations, as a group, from their controls, thereby exhibiting a homoplastic pattern.     

Understorey vegetation moderates climate in open forests: The role of the skirt-forming grass tree Xanthorrhoea semiplana F.Muell

Microsites are created by abiotic and biotic features of the landscape and may provide essential habitats for the persistence of biota. Forest canopies and understorey plants may moderate wind and solar radiation to create microclimatic conditions that differ considerably from regional climates. Skirt-forming plants, where senescent leaves create hut-like cavities around the stem, create microsites that are sheltered from ambient conditions and extreme weather events, constituting potential refuges for wildlife. We investigate day and night temperatures and humidity for four locations (grass tree cavities, soil, 20 cm above-ground, 1 m above-ground) in a South Australian forest with relatively open canopy of stringybark eucalypts (Eucalyptus baxteri, E. obliqua) and an understorey of skirt-forming grass trees (Xanthorrhoea semiplana) at 5, 10, 20, and 40 m from the forest edge. We also measured the percentage of canopy and understorey covers. Generally, temperature and humidity differed significantly between more sheltered (grass tree cavities, soil) and open-air microsites, with the former being cooler during the day and warmer and more humid during the night. Furthermore, our results suggest that canopy cover tends to decrease, and understorey cover tends to increase, the temperature of microsites. Distance to the edge was not significantly related to temperature for any microsite, suggesting that the edge effect did not extend beyond 10 m from the edge. Overall, grass trees influenced microclimatic conditions by forming a dense understorey and providing cavities that are relatively insulated. The capacity of grass tree cavities to buffer external conditions increased linearly with ambient temperatures, by 0.46°C per degree increase in maximum and 0.25°C per degree decrease in minimum temperatures, potentially offsetting climate warming and enabling persistence of fauna within their thermal limits. These climate moderation properties will make grass trees increasingly important refuges as extreme weather events become more common under anthropogenic climate change.     

Isolation and properties of a soluble sialidase from the culture fluid of Chinese hamster ovary cells

A Soluble sialidase that can degrade recombinant glycoproteinsexpressed in Chinese hamster ovary (CHO) cells has been isolatedand purified to near homogeneity from the cell culture fluidof this host. Purification of     

Am improved procedure for the isolation of chloroplast DNA fromChlamydomonas reinhardtii

The isolation of chloroplast DNA fromChlamydomonas reinhardtii requires the efficient separation of this AT-rich genome from the GC-rich nuclear genome by density-gradient centrifugation. We describe a simple and efficient method for separating these DNA fractions by using a sodium iodide gradient in combination with the DNA-binding dye, bisbenzimide. The yield of chloroplast DNA is close to the theoretical maximum and the DNA is suitable for restriction enzyme analysis and cloning. This method is applicable to the isolation of AT-rich plastid genomes from other organisms and may be appropriate as a general method for separating species of DNA that differ in their AT/GC ratios. An erratum to this article is available at .     

Selected contribution: Effects of aging on cerebrovascular tone and [Ca2+]i.

The lower limits of cerebral blood flow autoregulation shift toward high pressures in aged compared with young rats. Intraluminal pressure stimulates contractile mechanisms in cerebral arteries that might, in part, cause an age-dependent shift in autoregulation. The present project tested two hypotheses. First, cerebral artery tone is greater in isolated arteries from aged compared with mature adult rats. Second, aging decreases the modulatory effect of endothelium-derived nitric oxide (NO) and increases vascular smooth muscle Ca2+ sensitivity. Isolated segments of middle cerebral arteries from male 6-, 12-, 20-, and 24-mo-old Fischer 344 rats were cannulated and loaded with fura-2. Diameter and Ca2+ responses to increasing pressure were measured in HEPES, during NO synthase inhibition [NG-nitro-l-arginine methyl ester (l-NAME)], and after removal of the endothelium. Cerebral artery tone (with endothelium) increased with age. Only at the lowest pressure (20 and 40 mmHg) was intracellular Ca2+ concentration ([Ca2+]i) greater in arteries from 24-mo-old rats compared with the other age groups. l-NAME-sensitive constriction increased significantly in arteries from 6- to 20-mo-old rats but declined significantly thereafter in arteries from 24-mo-old rats. [Ca2+]i was less in arteries from 24-mo-old rats compared with the other groups after treatment with l-NAME. Another endothelial-derived factor, insensitive to l-NAME, also decreased significantly with age. For example, at 60 mmHg, the l-NAME-insensitive constriction decreased from 47 +/- 10, 42 +/- 5, 21 +/- 2, and 3 +/- 1 microm in 6-, 12-, 20-, and 24-mo-old rats, respectively. Our data suggest that aging alters cerebral artery tone and [Ca2+]i responses through endothelial-derived NO synthase-sensitive and -insensitive mechanisms. The combined effect of greater cerebral artery tone with less endothelium-dependent modulation may in part contribute to the age-dependent shift in cerebral blood flow autoregulation.     

Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells

In normal rats treated with 1,25(OH)₂D₃ or 24,25(OH)₂D₃, serum Ca²⁺, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D₃ analogues on target organ function, rat UMR 106 osteosarcoma and GH₃ pituitary adenoma cells in monolayer culture were exposed accordingly.Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)₂D₃ or 24,25(OH)₂D₃ did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)₂D₃ exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G₃₆ and G_q/11, respectively. In contrast, 24,25(OH)₂D₃ remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G₃₆. TRH- and Gpp(NH)p-elicited PLC activities in the GH₃ cells were significantly reduced by 1,25(OH)₂D₃ with a concurrent reduction in cellular amounts of G_q/11, however, 24,25(OH)₂D₃ did not significantly alter any signalling systems nor G-proteins analyzed.It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)₂D₃ and 24,25(OH)₂D₃, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca²⁺ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D₃ treated rats.Abbreviations AC adenylate cyclase - ALP alkaline phosphatase - BGP osteocalcin - BSA bovine serum albumin - DA dopamine - DAG diacylglycerol - GH growth hormone - GHRH growth hormone releasing hormone - Gpp(NH)p guanosine 5-[-imido]triphosphate - G-protein guanine nucleotide-binding regulatory protein - G_s etc. G_s protein -subunit - IP₃ inositol 1,4,5 trisphosphate - OAF osteoclast activating factor - PGE₂ prostaglandin E₂ - PKA & PKC protein kinase A & C - PLC phospholipase C - PRL prolactin - PTH parathyroid hormone - SRIF somatostatin - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide - 25(OH)D₃ 25 hydroxy vitamin D₃ - 1,25(OH)₂D₃ 1·25 dihydroxy vitamin D₃ - 24,25(OH)₂D₃ 24,25 dihydroxy vitamin D₃     

Detection of a 65 kDaras binding protein in rat and sheep brain cytosol using a chemical cross linking agent

The ability of aras protein to associate with proteins present in rat brain cytosolin vitro was investigated using chemical cross-linking agents and the¹²⁵I-labelled v-H-ras protein. Two iodinated protein complexes with apparent molecular weights of 40 and 85 kDa were observed when a mixture of rat brain cytosol and [¹²⁵I]ras was treated with the cross-linking agent disuccinimidyl suberate and subjected to SDS-PAGE. Formation of the [¹²⁵I] 85 kDa complex was enhanced by a high concentration of EDTA while generation of the 40 kDa species was abolished by this treatment. Formation of the [¹²⁵I] 85 kDa complex was inhibited by unlabelledras protein, GTP, GTPS, and GDP but not by ATPS and GMP.Chromatography of the cross-linked brain cytosol-[¹²⁵I]ras mixture on DEAE cellulose partially resolved the [¹²⁵I] 85 kDa complex from the [¹²⁵I]ras protein. The [¹²⁵I] 85 kDa complex (formed using ethyleneglycolbis (succinimidylsuccinate) as the cross-linking agent) could be immunoprecipitated using a rabbit anti-ras polyclonal antibody. Treatment of the immunoprecipitate with hydroxylamine to cleave the cross-link yielded [¹²⁵I]-labelledras. A substantial enrichment of the proportion of the [¹²⁵I] 85 kDa complex in the cross-linked extract was achieved by preparative SDS-PAGE. It is concluded that thein vitro chemical cross-linking approach employed here has detected tworas binding proteins in rat brain cytosol: a 65 kDa heat-sensitive and a 20 kDa heat-stable protein. The possibility that the 65 kDaras binding protein is aras regulatory orras effector protein which has not so far been characterised is briefly discussed.Abbreviations DSS disuccinimidyl suberate - EGS ethyleneglycolbis (succinimidylsuccinate) - GTPS guanosine 5-[-thio] triphosphate - ATPS adenosine 5-[-thio] triphosphate     

Autoradiographic localization of ornithine decarboxylase in mouse kidney by use of radiolabeled α-difluoromethylornithine

Summary Ornithine decarboxylase, a key enzyme in polyamine biosynthesis and cell growth, has been localized in mouse kidney by autoradiography after administration of radiolabeled -difluoromethylornithine. This drug is an enzyme-activated irreversible inhibitor of ornithine decarboxylase and forms a covalent bond with the enzyme. It was found that ornithine decarboxylase is present in all cell types studied but that the highest content occurs in the proximal convoluted tubules followed by the distal convoluted tubules and the collecting tubules. The majority of the enzyme is located in the cytoplasm but about 10–15% is present in the nuclei (often associated with nucleolus-like components) of the cells of the proximal and distal convoluted tubules. The labeled ornithine decarboxylase was lost rapidly from both nucleus and cytoplasm of all the cell types examined, and labeling by radioactive -difluoromethylornithine was greatly reduced if the mice were pretreated for 5 h with cycloheximide to block protein synthesis. These results indicate that ornithine decarboxylase turns over rapidly in all of the cells.     

Characterization of group H streptococcal temperate bacteriophage phi 227.

phi 227, a temperate phage from a group H streptococcus (Streptococcus sanguis), was propagated vegetatively in group H strain Wicky 4-EryR, and its characteristics were determined. A procedure dependent on multiplicity of infection, incubation time, and treatment of crude lysates with diatomaceous earth was found to optimize phage yield, resulting in titers of 1 X 10(10) to 2 X 10(10) PFU/ml. Without prior treatment with diatomaceous earth, subsequent purification procedures (methanol, ammonium sulfate, polyethylene glycol) gave recoveries of less than 1% of crude lysate titers. Adsorption of phi227 to host cells was relatively unaffected by the medium, but calcium (not substituted by magnesium) was required for formation of infectious centers. The phage receptor was present on purified cell walls, resisted trypsin and heat, and was removed ty hydrochloric acid, trichloracetic acid, and hot formamide: however, formamide-extracted material failed to inactivate phage, and the nature of the receptor is unknown. Single-step growth experiments showed a latent period of 39 min and a burst size of 100 PFU/infectious center; results were unaffected by omission of supplemental Ca2+, by supplementation with Mg2, addition of glucose, or changes of pH between 6.35 and 8.0; but increased temperature (40 to 43 degrees C) shortened the latent period and decreased the burst size. The latent period was prolonged in genetically competent host cells and in chemically defined medium; and in the latter, the burst size was smaller. Phage replication was sensitive to those metabolic inhibitors which inhibited the host streptococcus: these included rifampin, fluorodeoxyuridine, hydroxyurea, dihydrostreptomycin, and 6-P-hydroxyphenylazouracil. The data suggest that phi227 does not code for a rifampin-resistant RNA polymerase. However, in a rifampin-resistant host strain, phage replication and lysogen formation were both decreased suggesting that altered host core polymerase had less affinity for (some) promotors on the phi227 template. In transfection, a Ca2+-dependent stabilization step that was inhibited by Mg2+ was demonstrated; transformation was not affected by either Ca2+ or Mg2+, and the site and nature of the stabilization are unknown. More than one molecule of DNA was required for plaque formation. Biophysical characterization showed a type B phage of buoyant density (CsCl) 1.50, containing five proteins and 54.8% DNA. The duplex linear DNA had a molecular weight (calculated from contour length) of 23.2 X 10(6) and a guanine plus cytosine content (calculated from melting point) of 42.3 mol%. Similar characterizations of streptococcal phages, including biophysical data, have not been previously available.     

The nature of the genetic determinant for the SHV-1 beta-lactamase.

Summary In two unrelated plasmids of incompatibility groups FII and N the gene for the SHV-1 -lactamase exists as part of a transposable element of molecular weight 9.5 megadaltons. This transposon has moved onto plasmids of at least three incompatibility groups; PI, I and J. This confirms the suggestion that the recent spread of the SHV-1 -lactamase has been associated with the transposition of the genetic determinant of this enzyme between unrelated plasmids.