Cyclosporin A does not inhibit the PHA-stimulated increase in intracellular Ca2+ concentration but inhibits the increase in E-rosette receptor (CD2) expression and appearance of interleukin-2 receptors (CD25)

The immunosuppressive drug cyclosporin A (CsA) inhibits mixed lymphocyte responses, blocks the generation of cytotoxic T lymphocytes, and inhibits the T lymphocyte proliferative response stimulated by polyclonal activators such as phytohemagglutinin (PHA). Nevertheless, there have been contradictory reports attempting to explain the mechanism(s) for this immunosuppressive activity. In the current studies, human peripheral blood mononuclear cells (PBM) were stimulated with PHA in the presence or absence of CsA. Flow cytometric examination of PBM loaded with the Ca2+-sensitive dye Indo-1 showed that concentrations of CsA sufficient to inhibit 90-100% of tritiated thymidine incorporation had no effect on the PHA-stimulated increase in the intracellular Ca2+ concentration ([Ca2+]i). Likewise, inhibitory amounts of CsA had virtually no effect on the increase in cell volume that occurs during T lymphocyte activation. These results were not altered by pretreating the PBM with CsA for 30 min at 37 degrees C prior to adding the PHA. On the other hand, inhibitory concentrations of CsA prevented the expression of receptors for T cell growth factor (interleukin-2, IL-2), as measured by monoclonal antibodies to CD25 after 16-24-hr incubation. In like manner, CsA also prevented the increase in the expression of the E-rosette receptor (CD2) on these same cells. If cultures containing PHA and inhibitory amounts of CsA were incubated for 40-72 h, there was partial recovery both of proliferative activity and of the expression of CD25 and CD2. Thus, CsA does not appear to affect the initial activation signal(s), but does interfere with one or more subsequent events necessary to initiate the appearance of "activation antigens."     

The requirement for proteinase activity for human lymphocyte-mediated natural cytotoxicity (NK): evidence that the proteinase is serine dependent and has aromatic amino acid specificity of cleavage

We used reagents specific for serine-dependent proteinases to verify that a proteinase of this class is necessary for natural cytotoxicity (NK). NK was inhibited by phenylmethylsulfonylfluoride (PMSF), by diisopropylfluorophosphate (DFP), and by the plasma antiproteinase alpha-1-antichymotrypsin (alpha-1-X), all of which are specific for serine-dependent proteinases. Substrate specificity was then determined on the basis of the specificity of the plasma and fungal anti-proteinases and synthetic alternate substrates that affected NK. alpha-1-X, which inhibits only serine proteinases with aromatic amino acid specificity, blocked NK. Chymostatin, but not other fungal inhibitors, also blocked NK activity. Furthermore, the only synthetic substrates that effectively reduced NK were those derived from aromatic amino acids. The ester derivatives of these substrates inhibited NK better than the amides. NK inhibition with these alternate substrates was also stereospecific, with the L forms twofold more active than the D forms. These reagents did not block initial lymphocyte-target cell binding. Therefore we propose that the "NK-proteinase" is involved in either the initiation of cytolysis, perhaps as part of stimulus and secretion of cytolytic molecules, or in the cascade of events that may lead to the formation of final lytic substance.     

Seasonal Variation of Western White Pine (Pinus monticola D. Don) Foliage Proteins

Recently, a western white pine protein, Pin m III, was shownto be associated with overwintering and frost hardiness of westernwhite pine foliage. To examine whether Pin m III is directlyinvolved in frost hardiness by functioning as an antifreezeprotein, work is underway to clone the gene encoding this proteinand to assess the function of this gene in freezing toleranceby incorporating the gene in a test plant, such as tobacco.Here, we examined in more detail, by SDS-PAGE and also by twodimensional gel electrophoresis, the seasonal variation of additionalproteins in western pine foliage. SDS-PAGE analysis of threeseedlots showed that different proteins reached a maximum levelin different months, although most proteins (5 to 11) reacheda maximum level in winter months (December, January and February).The 2-D gel analysis of foliage sampled on three harvest dates(October, January and April) of one seedlot revealed a seasonalvariation of a large number proteins (76 to 184). Of the seasonallyvaried proteins, the amino terminal sequence of several proteinsincluding Pin m III was determined. One of the sequences wasidentified by homology to that of the small subunit of ribulosebiphosphate carboxylase, whose level increased substantiallyfrom fall to spring. The amino terminal sequence of Pin m IIIhad 89% homology to a sugar pine protein, Pin 11. The anti-photosystemII antibody was used to monitor the annual variation of theextrinsic 23-kDa photosystem II protein. The level of the extrinsic23-kDa photosystem II protein decreased slowly as fall progressedand reached its lowest level in December and then increasedin early spring indicating that this variation is due to photosyntheticactivity of the foliage during the season. (Received July 29, 1995; Accepted March 5, 1996)     

10CM短柱快速分离3-MH的实验研究

本研究在改进后短程序基础上,对氨基酸分离柱进行了改进。改进后的分离柱长为10cm。比原来20cm长柱分离3—MH的时间缩短了近1/2。实验所得的(回收率为97.59%,分离度0.89±0.02。变异系数1.17)这些指标较国外用其它方法所得的结果有良好的相关性。多次测定结果说明长柱与短柱比较无明显差异。证明了短柱对3—MH含量无影响。这一改进所建立的方法大大地缩短了样品的分析时间,节约了大量进口试剂,开展这方面的工作将有利益提高严重烧伤、创伤后蛋白质代谢和营养学等方面的研究水平。     

A surplus no more? Variation in krill availability impacts reproductive rates of Antarctic baleen whales

The krill surplus hypothesis of unlimited prey resources available for Antarctic predators due to commercial whaling in the 20th century has remained largely untested since the 1970s. Rapid warming of the Western Antarctic Peninsula (WAP) over the past 50 years has resulted in decreased seasonal ice cover and a reduction of krill. The latter is being exacerbated by a commercial krill fishery in the region. Despite this, humpback whale populations have increased but may be at a threshold for growth based on these human-induced changes. Understanding how climate-mediated variation in prey availability influences humpback whale population dynamics is critical for focused management and conservation actions. Using an 8-year dataset (2013–2020), we show that inter-annual humpback whale pregnancy rates, as determined from skin-blubber biopsy samples (n = 616), are positively correlated with krill availability and fluctuations in ice cover in the previous year. Pregnancy rates showed significant inter-annual variability, between 29% and 86%. Our results indicate that krill availability is in fact limiting and affecting reproductive rates, in contrast to the krill surplus hypothesis. This suggests that this population of humpback whales may be at a threshold for population growth due to prey limitations. As a result, continued warming and increased fishing along the WAP, which continue to reduce krill stocks, will likely impact this humpback whale population and other krill predators in the region. Humpback whales are sentinel species of ecosystem health, and changes in pregnancy rates can provide quantifiable signals of the impact of environmental change at the population level. Our findings must be considered paramount in developing new and more restrictive conservation and management plans for the Antarctic marine ecosystem and minimizing the negative impacts of human activities in the region.     

Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda)

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.     

Functional analysis of the iron-stress induced CP 43′ polypeptide of PS II in the cyanobacterium Synechococcus sp. PCC 7942

Under conditions of iron-stress, the Photosystem II associated chlorophyll a protein complex designated CP 43, which is encoded by the isiA gene, becomes the major pigment-protein complex in Synechococcus sp. PCC 7942. The isiB gene, which is located immediately downstream of isiA, encodes the protein flavodoxin, which can functionally replace ferredoxin under conditions of iron stress. We have constructed two cyanobacterial insertion mutants which are lacking (i) the CP 43 apoprotein (designated isiA ^–) and (ii) flavodoxin (designated isiB ^–). The function of CP 43 was studied by comparing the cell characteristics, PS II functional absorption cross-sections and Chl a fluorescence parameters from the wild-type, isiA ^– and isiB ^– strains grown under iron-stressed conditions. In all strains grown under iron deprivation, the cell number doubling time was maintained despite marked changes in pigment composition and other cell characteristics. This indicates that iron-starved cells remained viable and that their altered phenotype suggests an adequate acclimation to low iron even in absence of CP 43 and/or flavodoxin. Under both iron conditions, no differences were detected between the three strains in the functional absorption crossection of PS II determined from single turnover flash saturation curves of Chl a fluorescence. This demonstrates that CP 43 is not part of the functional light-harvesting antenna for PS II. In the wild-type and the isiB ^– strain grown under iron-deficient conditions, CP 43 was present in the thylakoid membrane as an uncoupled Chl-protein complex. This was indicated by (1) an increase of the yield of prompt Chl a fluorescence (F_o) and (2) the persistence after PS II trap closure of a fast fluorescence decay component showing a maximum at 685 nm.Abbreviations Chl chlorophyll - CP 43, CP 47 and CP 43 Chl a binding protein complexes of indicated molecular mass - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_m and F_m fluorescence when all PS II reaction centers are dosed in dark- and light-acclimated cells, respectively - F_o fluorescence when all PS II reaction centers are open in dark acclimated cells - F_v variable fluorescence after dark acclimation (F_m–F_o)     

In vitro studies of the rabbit immune system. VI. Rabbit anti-mouse cytotoxic T effector cells are inhibited by anti-rabbit T cell serum in the absence of complement.

Xenogeneic rabbit anti-mouse cell-mediated cytotoxic activity could be generated by culturing lymphoid cells from mesenteric lymph nodes (MLN), spleen, or peripheral blood of rabbits primed 2 to 8 weeks earlier with mouse tumor or spleen cells. MLN cells, which provided the best source of activity after being cultured with 5 to 10 X 10(6) mitomycin C-treated mouse spleen cells for 4 to 6 days, produced 30 to 90% specific isotope release after 4 to 7 hr incubation with 15Cr-labeled tumor target cells. Xenogeneic cytotoxic activity was primarily H-2 specific and could not be blocked by immune complexes but was abrogated by treatment with goat anti-rabbit thymocyte serum plus complement (ATS + C) before or after culture. Therefore, the activity appeared to be mediated by cytotoxic T lymphocytes (CTL). Furthermore, ATS without C abrogated cytotoxic activity when included in the CTL assay at concentrations of 5 to 15 microliter/10(7) effector cells. The inhibitory activity of ATS was directed to the rabbit effector population and could be absorbed completely by rabbit thymocytes. Antisera to mouse T cells with comparable cytolytic activity in the presence of C did not inhibit murine allogeneic CTL.     

Influence of single and concurrent clofibrate and phenobarbital administration on cytochrome P450-dependent mixed function oxidase activities and peroxisome proliferation in male rat liver

The influence of both single and concurrent administration of phenobarbital and clofibrate on hepatomegaly, cytochrome P450-depen-dent mixed function oxidase activities, and peroxisome proliferation in male rat liver have been studied. Both xenobiotics separately increase the liver :body weight ratio and their combined administration results in greater hepatomegaly than either compound alone. Both compounds induce NADPH-cytochrome c(P450) reductase activity and laurate ω- and ω-1-hydroxylase activities, but only phenobarbital induces pentoxyresorufin-O-de-alkylase. None of the drug treatments induced microsomal cytochrome b5. Phenobarbital did not cause peroxisome proliferation and inhibited the corresponding clofibrate-dependent proliferation. Taken collectively, our studies have demonstrated that concomitant treatment with phenobarbital and clofibrate are largely permissive with respect to the hepatic mixed function oxidase system but have opposing effects on the phenomenon of peroxisome proliferation in the same tissue.