Vaccination with SIVmac239Δnef activates CD4+ T cells in the absence of CD4+ T-cell loss

Background  Pathogenic HIV and SIV infections characteristically deplete central memory CD4⁺ T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4⁺ T-cell play roles in protection against wild-type virus challenge.
Methods  Rhesus macaques were vaccinated with SIVmac239Δnef and quantitative and phenotypic polychromatic flow cytometry analyses were performed on mononuclear cells from blood, lymph nodes and rectal biopsies.
Results  Animals vaccinated with SIVmac239Δnef demonstrated no loss of CD4⁺ T cells in any tissue, and in fact CCR5⁺ and CD28⁺CD95⁺ central memory CD4⁺ T cells were significantly increased. In contrast, CD4⁺ T-cell numbers and CCR5 expression significantly declined in unvaccinated controls challenged with SIVmac239. Also, intracellular Ki67 increased acutely as much as 3-fold over baseline in all tissues after SIVmac239Δnef vaccination then declined following primary infection.
Conclusion  We demonstrated in this study that SIVmac239Δnef vaccination did not deplete CD4⁺ T cells but transiently activated and expanded the memory cell population. However, increases in numbers and activation of memory CD4⁺ T cells did not appear to influence protective immunity.     

Half-life of tRNA Containing N6(Δ2-isopentenyl)adenosine in Lactobacillus acidophilus ATCC 4963

tRNA containing N⁶-(Δ²-isopentenyl)adenosine may be precursors for the plant hormone cytokinin. To discriminate between tRNA containing and not containing cytokinin nucleotides, double labelling experiments were made by the use of [2¹⁴C]-mevalonic acid and [³H-methyl]-methionine. At a generation cycle of 2 h for Lactobacillus acidophilus ATCC 4963, the half-lives of tRNA labelled with [³H-methyl]-methionine and [2-¹⁴C]-mevalonic acid are similar, namely 3 h. Isopentenylation of tRNA could be measured to be maximally 1:10.     

Expression of Δ1-pyrroline-5-carboxylate dehydrogenase and proline/arginine homeostasis in Solanum tuberosum

Polyclonal antibodies raised in mouse against purified potato Δ ¹-pyrroline-5-carboxylate dehydrogenase (P5C-DH, EC 1.5.1.12), which catalyses the last step in the catabolism of both proline and arginine, were used to investigate the expression of this enzyme. Distribution of P5C-DH in potato ( Solanum tuberosum L. cv. Desiree) organs was studied at different stages during plant development. Variations in enzyme level were determined in axenically grown plantlets following the addition of exogenous proline, and in cell suspension cultures under hyperosmotic stress and after its relief. Free proline and arginine levels were also quantified, and compared to those of the enzyme. Results were consistent with a developmental, but not with an environmental, control of P5C-DH expression. The possible involvement of specific isozymes in proline and arginine oxidation is discussed.     

Diurnal variation of Δ13CO2, ΔC18O16O and evaporative site enrichment of δH218O in Piper aduncum under field conditions in Trinidad

Concurrent measurements of gas exchange, instantaneous isotope discrimination (Δ) against ¹³CO₂ and C¹⁸O¹⁶O, and extent of ¹⁸O enrichment in H₂O at the evaporative sites, were followed in a tropical forest pioneer, Piper aduncum, on two different days in Trinidad during February 1995. Δ¹³CO₂ differed from that predicted from measurements of internal:external CO₂ concentration (C_i/C_a) and showed a wide range of values which decreased throughout the course of the day. Derivation of C_c (the CO₂ concentration at the carboxylation site) was not possible using carbon isotope discrimination under field conditions in situ and was derived assuming a constant value of internal transfer conductance (g_w). Under low rates of assimilation the derived C_c/C_a, like C_i/C_a, remained relatively stable over the course of both days and ΔC¹⁸O¹⁶O followed evaporative demand. Lower values of ΔC¹⁸O¹⁶O on day 2 occurred in response to the indirect effect of increased leaf-to-air vapour pressure deficits (VPD) and reduced stomatal conductance. For the first time, direct determination of the δH₂¹⁸O of transpired water vapour (δ_t) allowed derivation of evaporative site enrichment without the prerequisite of isotopic steady state (ISS) defined in the Craig and Gordon model. Generally, δ_t was less enriched than the source water (δ_s) in the morning and more enriched in the afternoon, which would be predicted from an increase and decrease in ambient VPD, respectively. On both days, leaves of P. aduncum approached ISS (indicated where δ_t≈δ_s) between 1300 and 1500 h. Evaporative site enrichment was maintained into the late afternoon, despite a decrease in ambient VPD. The data presented provide a greater insight into the natural variation in isotopic discrimination under field conditions, which may help to refine models of terrestrial biome discrimination.     

Changes in leaf Δ13C of herbarium plant species during the last 3 centuries of CO2 increase

Δ¹³C were determined for herbarium specimens of 12 C₃ plants (trees, shrubs and herbs) collected during the last 240 years in Catalonia, an area with a Mediterranean climate. Values were 19.91 (S.E. = 0.32, n= 21) for 1750–1760, 19–86 (S.E. = 0.21, n= 49) for 1850–1890 and 19.95 (S.E. = 0.29, n= 25) for 1925–1950, and decreased significantly to 18.87 (S.E. = 0.31, n= 29) for 1982–1988. More irregular temporal changes were found in Δ¹³C of two C₄ species, but they also suggest a decrease in discrimination in recent decades. These results suggest that either carbon assimilation rates have increased or stomatal conductance has decreased, and therefore, that there has been an increase in water use efficiency over the last few decades.     

Correlating δ13C and δ18O in cellulose of trees

We measured the carbon and oxygen isotopic composition of stem cellulose of Pinus sylvestris, Picea abies, Fagus sylvatica and Fraxinus excelsior. Several sites along a transect of a small valley in Switzerland were selected which differ in soil moisture conditions. At every site, six trees per species were sampled, and a sample representing a mean value for the period from 1940 to 1990 was analysed. For all species, the mean site δ¹³C and δ¹⁸O of stem cellulose are related to the soil moisture availability, whereby higher isotope ratios are found at drier sites. This result is consistent with isotope fractionation models when assuming enhanced stomatal resistance (thus higher δ¹³C of incorporated carbon) and increased oxygen isotope enrichment in the leaf water (thus higher δ¹⁸O) at the dry sites. δ¹⁸ O-δ¹³C plots reveal a linear relationship between the carbon and oxygen isotopes in cellulose. To interpret this relationship we developed an equation which combines the above-mentioned fractionation models. An important new parameter is the degree to which the leaf water enrichment is reflected in the stem cellulose. In the combined model the slope of the δ¹⁸O-δ¹³C plot is related to the sensitivity of the p_i/p_a of a plant to changing relative humidity.     

纯化标签的不同位置对Δ6脂肪酸脱饱和酶异源表达的影响

【背景】目前利用酵母表达系统已鉴定了多种物种中的Δ6脂肪酸脱饱和酶(FADS6)。由于FADS6是一种具有多个跨膜螺旋的膜蛋白,使得其大量表达和纯化具有挑战性。【目的】探索FADS6的高效表达策略,研究纯化标签添加的位置对高山被孢霉FADS6I (Ma FADS6I)重组表达效率的影响。【方法】在毕赤酵母表达载体中插入串联亲和标签HRV 3C-Protein A-His,利用改造后的载体构建带有N端或C端标签的Ma FADS6I表达载体;通过电转化获得毕赤酵母重组表达菌株;利用斑点印迹杂交(DotBlot)、聚丙烯酰胺凝胶电泳(SDS-PolyacrylamideGelElectrophoresis,SDS-PAGE)和免疫印迹(Western Blot)分析重组蛋白的表达水平,并利用气相色谱-质谱(Gas Chromatography-Mass Spectrometry,GC-MS)分析检测Ma FADS6I催化生成的脂肪酸。【结果】通过大量的毕赤酵母转化子筛选,最终获得高效表达Ma FADS6I的毕赤酵母重组菌,证实各转化子的表达具有差异性,Ma FADS6I的C端带有纯化标签较N端更有利于表达。【结论】在Ma FADS6I的C端添加纯化标签比在N端添加更有利于该蛋白在酵母系统中的表达以及底物的转化,为进一步探究FADS6高效表达和结构功能奠定了基础。     

Transmembrane 36CI− Flux Measurements and Desensitization of the γ-Aminobutyric AcidA Receptor

Abstract: Some data on the concentration range of response and the concentration for half-response (EC₅₀) of γ-aminobutyric acid (GABA) for the GABA_A receptor are reviewed and compared. An analysis of the ³⁶CI⁻ flux assay demonstrates that both the EC₅₀ and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the ³⁶CI⁻ concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC₅₀ can be decreased by orders of magnitude by increasing the assay time. The EC₅₀ measured in a finite time is less than the half-response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC₅₀ depends on the rate of receptor desensitization. The computer simulations showed that a GABA_A receptor with a half-response concentration of 100 μ M GABA can give ³⁶CI⁻ flux measurements with an EC₅₀ value 100-fold lower.     

Effect of digestion on the δ13C and δ15N of fish-gut contents

Gut contents of sand goby Pomatoschistus minutus showed higher C and N isotope values than the food before consumption. This enrichment was more pronounced in the hindgut than in the foregut, probably because of preferential assimilation of ¹²C and ¹⁴N along the gastro-intestinal tract. The results indicated that the shift towards higher values in the alimentary canal occurs in the first 2 h after feeding.     

Mutations in genes downstream of the rpoN gene (encoding σ54) of Klebsiella pneumoniae affect expression from σ54-dependent promoters

Two open reading frames (ORFs), designated ORF95 and ORF162, downstream of the Klebsiella pneumoniae sigma 54 structural gene (rpoN) have been sequenced and shown to encode polypeptides of 12 kD and 16 kD, respectively. ORFs homologous to ORF95 are present downstream of four out of five rpoN genes sequenced to date from a range of Gram-negative bacteria, and ORF162 is also conserved, at least in Pseudomonas putida. Chromosomal mutations have been created in each gene using a kan cassette and both have the same phenotype, i.e. they cause an increase in the level of expression from sigma 54-dependent promoters. We propose that the products of both genes function to modulate the activity of E sigma 54, although a physiological role for these proteins has not yet been identified.     

INTRACRANIAL CONVERSION OF LINOLEIC ACID TO ARACHIDONIC ACID: EVIDENCE FOR LACK OF Δ8 DESATURASE IN THE BRAIN

Eleven-day old rats were given intracranial injection of [1-¹⁴C]linoleic acid (all cis 9,12 octadecadienoic acid) and sacrificed after 8 h. Analysis of brain fatty acids showed that 16:0, 18:2, 20:2,20:3 and 20:4 were labeled. Separation by AgN03:Si02 TLC plates followed by reductive ozonolysis characterized thc polyunsaturated fatty acids as 18:2 (Δ9,12), 20:2 (Δ11,14), 20:3 (Δ8,11,14) and 20:4 (Δ5,8,11,14). A smaller amount of 18:3 (Δ6,9,12) was also identified. This initially suggested 20:2 (A1 1,14) as an intermediate in the optional pathway of biosynthesis of arachidonate. However, when [l-14C]eicosadienoic acid (Δ1 1,141 itself was injected in the brain it was converted to 20:3 (Δ5,11,14) (a non-methylene interrupted double bond system) rather than the expected 20:3 (Δ8,11,14). Only a small amount of arachidonate was formed from 20:2 (Δ11,14). Thus it was concluded that 20:2 (Δ11,14) was not an intermediate in the pathways of arachidonate biosynthesis due to lack of Δ⁵ desaturase in thc brain which agrees with the findings of SPKECRER & LEE (1975) in rat liver.