Two major classes of mitogen-reactive B lymphocytes defined by life span

The relative numbers of short- and long-lived mitogen-reactive B cells in the peripheral pool were evaluated by studying the decay of lipopolysaccharide (LPS)-reactive B lymphocytes in LPS nonresponder, histocompatible hosts for periods of up to 3 wk after cell transfer. The results obtained demonstrate the existence of two major classes of mitogen-reactive B cells defined by life span. The "short-lived" cell component comprises about 80 to 90% of the reactive cells and decays with a life expectancy of 18 to 24 hr. The long-lived cell component, with life expectancies of 10 to 20 days, comprises about 10 to 20% of the reactive cells and is preferentially enriched in circulating B cells. The present ratio for short- and long-lived B cells implies a highly dynamic state for the immune system which must be advantageous in the selection of available repertoires.     

Coated-vesicle formation in vitro: conflicting results using different assays

Cell-free systems provide essential tools for elucidating the molecular mechanisms underlying complex cellular processes such as vesicular transport. The biochemical utility of these model systems is strengthened by assays that allow rapid, quantitative detection of the events being studied. Two model systems have recently been developed to reconstitute coated-vesicle budding, and two different biochemical assays are used to detect this event. Striking differences in the biochemical requirements for 'coated-vesicle budding' are detected by these two assays, suggesting that two distinct events are being measured. These findings have wide implications for the use of cell-free assay systems in cell biology.     

Life span of queens in the antFormica exsecta

Summary The antFormica exsecta commonly has two types of colonies: either polygynous and polydomous or monogynous and monodomous. The longevity of queens was studied in monogynous colonies in southern Finland by indirect methods using genetic markers; these data were also used to estimate the number of matings and queen replacement. The average genetic relatedness among worker nest mates was 0.72. Taking inbreeding into account (the inbreeding coefficient wasF=0.16), this value agrees with the assumption that 40% of the queens mated with one male and 60% with two males. The distribution of genotypes within colonies remained stable in successive years, indicating that queen replacement did not occur or was extremely rare. This means that the life span of nests reflects directly the life span of the queens. Eleven of the 16 nests found in 1979 were still alive ten years later. This corresponds to an annual mortality of 3.7% and a mean life span of 27 years. A total of 57 colonies were mapped in the population over a period of ten years. Averaging over the years, the annual mortality was estimated to be 4.9%. This represents a mean life span of 20 years if mortality was independent of age.     

The in vitro life span and fate of murine B lymphocytes stimulated by endotoxin.

The in vitro life span of murine spleen lymphocytes stimulated by endotoxin (LPS) was determined. Lymphocytes synthesizing DNA spontaneously in culture and those stimulated to DNA synthesis early (24 hr) and later (48 hr) in culture by LPS had half-lives of approximately 24 hr. The continuing presence of LPS in culture did not prolong cell longevity nor did free LPS have to be present to allow successive rounds of DNA synthesis in committed cells. Once activated to DNA synthesis, blast cells and lymphoblast-like cells did not revert to small lymphocytes.     

Proteomic basis for the possible use of lymphocytes for metabolic screenings

Summary. The advent of proteomics has provided a tool for the concomitant identification and determination of a large series of proteins using two-dimensional gel electrophoresis with subsequent mass spectrometrical analysis. We tried an approach to analyse the high abundance enzyme proteome of a lymphocytic cell line.Immortalised lymphocytes were grown in RPMI 1640 in the presence of glutamine, harvested and the 100,000×g supernatant of the homogenate was applied on two-dimensional gel electrophoresis with subsequent in-gel digestion of protein spots and MALDI-TOF (Matrix-associated laser desorption/ionization mass spectroscopy) analysis of resulting peptides using specific software.A series of 57 metabolic enzymes were identified including enzymes of carbohydrate, amino acid, purine and intermediary metabolism.We are presenting a tool for the analysis of metabolic systems including enzyme deficiencies at the protein level with the advantage of unambiguous identification of proteins and thus complementing enzyme activity determinations.J. E. Oh and K. Krapfenbauer have been equally contributing to the work.     

Optimal codon identities in bacteria: implications from the conflicting results of two different methods

A correlation method was recently adopted to identify selection-favored 'optimal' codons from 675 bacterial genomes. Surprisingly, the identities of these optimal codons were found to track the bacterial GC content, leading to a conclusion that selection would generally shape the codon usages to the same direction as the overall mutation does. Raising several concerns, here we report a thorough comparative study on 203 well-selected bacterial species, which strongly suggest that the previous conclusion is likely an illusion. Firstly, the previous study did not preclude species that are suffering weak or no selection pressures on their codon usages. For these species, as showed in this study, the optimal codon identities are prone to be incorrect and follow GC content. Secondly, the previous study only adopted the correlation method, without considering another method to test the reliability of inferred optimal codons. Actually by definition, optimal codons can also be identified by simply comparing codon usages between high- and low-expression genes. After using both methods to identify optimal codons for the selected species, we obtained highly conflicting results, suggesting at least one method is misleading. Further we found a critical problem of correlation method at the step of calculating gene bias level. Due to a failure of accurately defining the background mutation, the problem would result in wrong optimal codon identities. In other words, partial mutational effects on codon choices were mistakenly regarded as selective influences, leading to incorrect and biased optimal codon identities. Finally, considering the translational dynamics, optimal codons identified by comparison method can be well-explained by tRNA compositions, whereas optimal codons identified by correlation method can not be. For all above reasons, we conclude that real optimal codons actually do not track the genomic GC content, and correlation method is misleading in identifying optimal codons and better be avoided.     

Leishmania trypanothione synthetase-amidase structure reveals a basis for regulation of conflicting synthetic and hydrolytic activities

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.     

Volume regulation by human lymphocytes: Characterization of the ionic basis for regulatory volume decrease

The mechanism of volume regulation in hypotonic media was analysed in human peripheral blood mononuclear (PBM) cells. Electronic cell sizing showed that hypotonic swelling is followed by a regulatory volume decrease (RVD) phase. This was confirmed by both electron microscopy and by cellular water determinations. The rate of regulatory shrinking was proportional to the degree of hypotonicity in the 0.5–0.9 X isotonic range. Cell viability was only marginally affected in this range. The content of cellular K⁺ decreased during RVD, while Na⁺ content remained unchanged. Similarly, the efflux of ⁸⁶Rb (used as a K⁺ analog) increased upon dilution, whereas ²²Na efflux was not altered. ⁸⁶Rb uptake was enhanced by hypotonic stress and both ouabain-sensitive and -insensitive components were affected. A ouabain-sensitive stimulation was also seen in Na⁺- free media. Ouabain partially inhibited RVD only if added to the cells hours before hypotonic challenge. A normal shrinking response was observed in K⁺-free media, and also in Na⁺-free media when Li⁺, choline⁺, or Tris⁺ were the substitutes. In high K⁺ or Rb⁺ hypotonic media shrinking was absent and a second swelling phase was observed. Cs⁺ displayed an intermediate behavior, with shrinking observed at lower dilutions and secondary swelling at higher ones. The direction and magnitude of the response also changed when the external K⁺ concentration was varied and, with 50 mM K⁺, no regulatory volume change occurred following hypotonic stress. These findings suggest that RVD occurs largely by a passive loss of cellular K⁺, resulting from a selective increase in permeability to this ion. In addition, the (Na-K) pump appears to be activated upon cell swelling by a mechanism other than Na⁺ entry into the cell, but this activation is not essential for RVD.     

Gluconeogenesis in the ruminant fetus: evaluation of conflicting evidence from radiotracer and other experimental techniques

Conflicting evidence exists as to whether the gluconeogenetic process is active in the late gestation fetal lamb. In vitro evidence based on measurements of enzyme activity and substrate flux into glucose indicates that the capacity for gluconeogenesis exists in fetal liver. The in vivo conversion of [14C]lactate and [14C]alanine into glucose in the lamb fetus has been demonstrated. Lactate and alanine account for 49 and 2.3% of the fetal glucose pool, respectively. Although gluconeogenesis can occur in the fetal lamb, alterations in net rates of umbilical uptake of glucose or lactate, fetal blood glucose concentrations, fetal or maternal glucose replacement rates, or maternal nutrition may alter the observed rates of fetal gluconeogenesis.     

Functional transcriptomics: An experimental basis for understanding the systems biology for cancer cells

We review gene signatures associated with multi-drug resistance and RAS oncogene-mediated transformation as established by microarray analysis and cDNA subtraction. In view of the complexity and diversity of gene expression patterns, we discuss experimental strategies based on signaling pertubation and target gene silencing by RNA interference to narrow down the number of critical genes and to assess their function individually. In addition, we address the question of how oncogenic signaling pathways and the genetic program are wired. DNA methylation of target gene promoters was recognized as one of the critical mechanisms for gene repression, particularly for target genes capable of constraining malignant growth.     

Interactions between B lymphocyte subpopulations. Augmentation of the responses of resting B lymphocytes by activated B lymphocytes

Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.     

Parasite-host specificity: experimental studies on the basis of parasite adaptation

Specificity in parasitic interactions can be defined by host genotypes that are resistant to only a subset of parasite genotypes and parasite genotypes that are infective on a subset of host genotypes. It is not always clear if specificity is determined by the genotypes of the interactors, or if phenotypic plasticity (sometimes called acclimation) plays a larger role. Coevolutionary outcomes critically depend on the pervasiveness of genetic interactions. We studied specificity using the bacterial parasite Pasteuria ramosa and its crustacean host Daphnia magna. First, we tested for short-term adaptation of P. ramosa lines that had been rapidly shifted among different host genotypes. Adaptation at this time-scale would demonstrate the contribution of phenotypic plasticity to specificity. We found that infectivity was stable across lines irrespective of recent passage history, indicating that in the short term infection outcomes are fixed by genetic backgrounds. Second, we studied longer-term evolution with two host clones and two parasite lines. In this experiment, P. ramosa lines had the possibility to evolve adaptations to the host genotype (clone) in which they were serially passaged, which allowed us to test for a genetic component to specificity. Substantial differences arose in the two passaged lines: one parasite line gained infectivity on the host clone it was grown on, but it lost infectivity on the other host genotype (this line evolved specificity), while the other parasite line evolved higher infectivity on both host clones. We crossed the two host genotypes used in the serial passage experiment and found evidence that the number of host genes that underlies resistance variation is small. In sum, our results show that P. ramosa specificity is a stably inherited trait, it can evolve rapidly, and it is controlled by few genes in the host. These findings are consistent with the idea of a rapid, ongoing arms race between the bacterium and its host.