The oxidative stress response in Bacillus subtilis

Abstract Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide. Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein. Induced protection against higher concentrations (10–30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant. Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide. A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide. This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type. The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.     

Ultrastructural localization of alkaline phosphatase in the cyanobacteriaCoccochloris peniocytis andAnabaena cylindrica

Summary Histochemical techniques applied at the ultrastructural level have established the periplasmic space as the site of cell bound alkaline phosphatase activity inAnabaena cylindrica andCoccochloris peniocytis. For localization of activity unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron dense lead phosphate. The majority of cell bound activity appeared to be associated with layer 3 of the cell wall. InA. cylindrica a secondary site of cell bound activity appeared to be in the sheath. Placement in a phosphate free medium caused a substantial increase in the enzyme activity ofA. cylindrica while the activity present in log phase cells ofC. peniocytis was similar to that found in phosphate starved cells.C. peniocytis also secretes the enzyme into the surrounding medium.     

No Evidence for Adaptive Micro-Evolution to a Decrease in Phosphorus-Loading of a Daphnia Population Inhabiting A Pre-Alpine Lake

Many polluted lakes in Europe are being restored and phosphorus concentrations have dropped dramatically in these lakes. We studied the genetic structure of Daphnia galeata over the past 30 years in Lake Greifensee, Switzerland, a period during which the phosphorus concentration in the lake reduced dramatically. Distinct genotypes of D. galeata were hatched from diapausing eggs extracted from six different time horizons in dated sediments. We compared juvenile growth, size and egg production of D. galeata reared on high-P and low-P algae to investigate whether Daphnia have evolved to grow better on phosphorus-limited algae. Our results indicate that life histories of D. galeata differed significantly between both food types. We also found significant clone effects for size and egg number. But we found no significant interaction between the depth from which the clones were selected and food quality. This means that we found no evidence for adaptive micro-evolution in response to P reduction in the lake. We discuss our results in relation to other studies that found evidence for adaptive micro-evolution in comparable time frames.     

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60 c-src activity

pp60 ^c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60 ^c-src but failed to increase hormone independent (basal) pp60 ^c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60 ^c-src was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60 ^c-src in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60 ^c-src autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60 ^c-src was reduced 54±16% compared to controls. Hormone-independent pp60 ^c-src kinase activity was unchanged by expression of the PTPase. pp60 ^c-src was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr⁵²⁷) and kinase domain (Tyr⁴¹⁶) residues. In addition,in vitro dephosphorylation by CD45 increased pp60 ^c-src activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60 ^c-src was not. The lack of activation of pp60 ^c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase phosphotyrosine phosphatase - PDGF platelet-derived growth factor - PMSF phenylmethylsulfonyl fluoride - LCA, CD45 leukocyte common antigen - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - DTT dithiothreitol - Na₃VO₄ sodium orthovanadate - PV pervanadate - -ME -mercaptoethanol     

Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii

Nonreducing O-linked oligosaccharides were obtained from the peptidorhamnomannan of mycelia of Pseudallescheria boydii by alkaline beta-elimination under reducing conditions. They were separated by gel filtration chromatography to give three oligosaccharide fractions. The major oligosaccharide from fraction 1 was characterized by a combination of techniques including electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), nuclear magnetic resonance (NMR), and methylation gas-liquid chromatography-mass spectrometry (GC-MS) analysis. It was branched, with a principal chain of alpha-Rhap-(1 --> 3)-alpha-Rhap-(1 --> 3)-alpha-Manp-(1 --> 2)-Man-ol substituted at O-6 of mannitol with an alpha-Glcp-(1 --> 4)-beta-Galp group. Species containing one and two additional alpha-Glcp-(1 --> 4) substituents in the rhamnose branch were also present. The major component of fraction 2 was a substructure of oligosaccharide-1, lacking a hexose from the Glc-Gal branch. Fraction 3 contained a mixture of smaller, unbranched, oligosaccharides. In hapten inhibition tests, fractions 1 and 2 blocked the reaction between peptidorhamnomannan (PRM) and rabbit anti-P. boydii mycelium hyperimmune serum by approximately 75%, whereas fraction 3 inhibited by approximately 55%.     

Short-term variation of nutritive and metabolic parameters in <Emphasis Type="Italic">Temora longicornis</Emphasis> females (Crustacea,Copepoda) as a response to diet shift and starvation

Changes in fatty acid patterns, digestive and metabolic enzyme activities and egg production rates (EPR) were studied in the small calanoid copepod Temora longicornis. Female copepods were collected in spring 2005 off Helgoland (North Sea). In the laboratory one group of copepods was fed with the cryptophycean Rhodomonas baltica for a period of 3 days. Another group of copepods was maintained without food. According to the fatty acid patterns, animals from the field were feeding on a more detrital, animal-based and to a minor extent to a diatom-based diet. Under laboratory conditions, females rapidly accumulated fatty acids such as 18:4 (n-3), 18:3 (n-3) and 18:2 (n-6) which are specific of R. baltica. Diatom-specific fatty acids such as 16:1 (n-7) were strongly reduced. In fed animals the activities of digestive and metabolic enzymes remained constant and egg production rates were highest on day 2. Starving animals, in contrast, showed significantly reduced faecal pellet production and EPR. Proteolytic enzyme activity decreased rapidly within 24 h and remained at a low level until the end of the experiment. Citrate synthase decreased continuously as well. T. longicornis rapidly reacts to dietary changes and food depletion. It has limited energy stores and, thus, strongly depends on continuous food supply.     

Prey-capture in the African clawed toad (Xenopus laevis): comparison of turning to visual and lateral line stimuli

Separately delivered visual and lateral line stimuli elicit similar but not identical orientation and approach by intact, sighted Xenopus. Response frequencies for visual stimuli declined sharply for distant or caudal stimuli while those for lateral line stimuli changed little. Turn angles correlated highly with stimulus angles but were smaller on average, so regression slopes were less than one. Regression slopes were smaller for visual than for lateral line stimuli, but this apparent difference was due to different distributions of stimulus distance interacting with the toad’s rotation center. Errors in final headings, most often under-rotations, did not differ by modality. Frequencies of lunges and arm capture movements were higher for visual stimuli both overall and especially for rostral proximal stimuli. The results demonstrate accurate orientation by sighted Xenopus to visual and lateral line stimuli; they are consistent with expectations based on in-register tectal maps. Orientation to lateral line stimuli is similar to previous results with blinded animals, revealing no heightened acuity in the latter. Modality differences indicate that the lateral line system is better for omnidirectional orientation and approach to distant stimuli whereas the visual system is more attuned to nearby rostral stimuli and more apt to mediate strikes.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

Protection against glucose-induced neuronal death by NAAG and GCP II inhibition is regulated by mGluR3

Glutamate carboxypeptidase II (GCP II) inhibition has previously been shown to be protective against long-term neuropathy in diabetic animals. In the current study, we have determined that the GCP II inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA) is protective against glucose-induced programmed cell death (PCD) and neurite degeneration in dorsal root ganglion (DRG) neurons in a cell culture model of diabetic neuropathy. In this model, inhibition of caspase activation is mediated through the group II metabotropic glutamate receptor, mGluR3. 2-PMPA neuroprotection is completely reversed by the mGluR3 antagonist (S)-alpha-ethylglutamic acid (EGLU). In contrast, group I and III mGluR inhibitors have no effect on 2-PMPA neuroprotection. Furthermore, we show that two mGluR3 agonists, the direct agonist (2R,4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) and N-acetyl-aspartyl-glutamate (NAAG) provide protection to neurons exposed to high glucose conditions, consistent with the concept that 2-PMPA neuroprotection is mediated by increased NAAG activity. Inhibition of GCP II or mGluR3 may represent a novel mechanism to treat neuronal degeneration under high-glucose conditions.     

Evaluation of portable point-of-care CD4 counter with high sensitivity for detecting patients eligible for antiretroviral therapy

Background

Accurate, inexpensive point-of-care CD4+ T cell testing technologies are needed that can deliver CD4+ T cell results at lower level health centers or community outreach voluntary counseling and testing. We sought to evaluate a point-of-care CD4+ T cell counter, the Pima CD4 Test System, a portable, battery-operated bench-top instrument that is designed to use finger stick blood samples suitable for field use in conjunction with rapid HIV testing.

Methods

Duplicate measurements were performed on both capillary and venous samples using Pima CD4 analyzers, compared to the BD FACSCalibur (reference method). The mean bias was estimated by paired Student''s t-test. Bland Altman plots were used to assess agreement.

Results

206 participants were enrolled with a median CD4 count of 396 (range; 18–1500). The finger stick PIMA had a mean bias of −66.3 cells/µL (95%CI −83.4−49.2, P<0.001) compared to the FACSCalibur; the bias was smaller at lower CD4 counts (0–250 cells/µL) with a mean bias of −10.8 (95%CI −27.3−+5.6, P = 0.198), and much greater at higher CD4 cell counts (>500 cells/µL) with a mean bias of −120.6 (95%CI −162.8, −78.4, P<0.001). The sensitivity (95%CI) of the Pima CD4 analyzer was 96.3% (79.1–99.8%) for a <250 cells/ul cut-off with a negative predictive value of 99.2% (95.1–99.9%).

Conclusions

The Pima CD4 finger stick test is an easy-to-use, portable, relatively fast device to test CD4+ T cell counts in the field. Issues of negatively-biased CD4 cell counts especially at higher absolute numbers will limit its utility for longitudinal immunologic response to ART. The high sensitivity and negative predictive value of the test makes it an attractive option for field use to identify patients eligible for ART, thus potentially reducing delays in linkage to care and ART initiation.