Leu-Enkephalin Generally Labeled with Tritium in Studying the Selank Inhibiting Effect on the Enkephalin-Degrading Enzymes of Human Blood Plasma

A method of analysis of enkephalinase activity in blood plasma based on the application of Leu-enkephalin generally labeled with tritium at all its amino acid residues was developed. The method allows the simultaneous estimation of activity of several peptidases in microquantities of tissues. [G-³H]Leu-enkephalin was prepared by the method of solid phase catalytic isotope exchange (120 Ci/mmol) and subjected to proteolysis by the treatment with blood plasma. The resulting radioactive metabolites were separated by HPLC in the presence of the mixture of unlabeled fragments of Leu-enkephalin as internal standards. It was shown that aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases respond for approximately 80%, 2%, and 10% of the total enzymatic activity, respectively. The new pathway of degradation of Leu-enkephalin by carboxypeptidase that provides for 6% of the total enkephalin-degrading activity was discovered. Bestatin was shown to predominantly inhibit aminopeptidases and carboxypeptidases, whereas selank is more specific for carboxypeptidases and dicarboxypeptidases.     

Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes

Environmental stress deleteriously affects every aspect of an ectotherm's biological function. Frequent exposure of terrestrial insects to temperature variation has thus led to the evolution of protective biochemical and physiological mechanisms. However, the physiological mechanisms underlying the positive impact of fluctuating thermal regimes (FTRs) on the fitness and survival of cold-exposed insects have not been studied. We have thus investigated the metabolic changes in adults of the beetle Alphitobius diaperinus in order to determine whether FTRs trigger the initiation of a metabolic response involving synthesis of protective compounds, such as free amino acids (FAAs) and polyols. The metabolic profile was analyzed during constant fluctuating thermal regimes (the beetles had daily pulses at higher temperatures that enabled them to recover) and compared with constant cold exposure and untreated controls. The increase of several essential amino acids (Lys, Iso, Leu, Phe and Trp) in cold-exposed beetles supports the conclusion that it results from the breakdown of proteins. Some FAAs have been shown to have cryoprotective properties in insects, but the relationship between FAAs, cold tolerance and survival has not yet been well defined. Instead of considering FAAs only as a part of the osmo- and cryoprotective arsenal, they should also be regarded as main factors involved in the multiple regulatory pathways activated during cold acclimation. Under FTRs, polyol accumulation probably contributes to the increased duration of survival in A. diaperinus.     

The evolutionary emergence of stochastic phenotype switching in bacteria

Stochastic phenotype switching - or bet hedging - is a pervasive feature of living systems and common in bacteria that experience fluctuating (unpredictable) environmental conditions. Under such conditions, the capacity to generate variable offspring spreads the risk of being maladapted in the present environment, against offspring likely to have some chance of survival in the future. While a rich subject for theoretical studies, little is known about the selective causes responsible for the evolutionary emergence of stochastic phenotype switching. Here we review recent work - both theoretical and experimental - that sheds light on ecological factors that favour switching types over non-switching types. Of particular relevance is an experiment that provided evidence for an adaptive origin of stochastic phenotype switching by subjecting bacterial populations to a selective regime that mimicked essential features of the host immune response. Central to the emergence of switching types was frequent imposition of 'exclusion rules' and 'population bottlenecks' - two complementary faces of frequency dependent selection. While features of the immune response, exclusion rules and bottlenecks are likely to operate in many natural environments. Together these factors define a set of selective conditions relevant to the evolution of stochastic switching, including antigenic variation and bacterial persistence.     

Infectious Epstein-Barr virus lacking major glycoprotein BLLF1 (gp350/220) demonstrates the existence of additional viral ligands

The binding of the viral major glycoprotein BLLF1 (gp350/220) to the CD21 cellular receptor is thought to play an essential role during infection of B lymphocytes by the Epstein-Barr virus (EBV). However, since CD21-negative cells have been reported to be infectible with EBV, additional interactions between viral and cellular molecules seem to be probable. Based on a recombinant genomic EBV plasmid, we deleted the gene that encodes the viral glycoprotein BLLF1. We tested the ability of the viral mutant to infect different lymphoid and epithelial cell lines. Primary human B cells, lymphoid cell lines, and nearly all of the epithelial cell lines that are susceptible to wild-type EBV infection could also be successfully infected with the viral mutant in vitro, although the efficiency of infection with BLLF1-negative virus was clearly lower than the one observed with wild-type EBV. Our studies show that the interaction between BLLF1 and CD21 is not absolutely required for the infection of lymphocytes and epithelial cells, indicating that viral molecules other than BLLF1 can mediate the binding of EBV to its target cells. In this context, our results further suggest the hypothesis that additional cellular molecules, apart from CD21, allow virus entry into these cells.     

Nanotube-mediated cross-feeding couples the metabolism of interacting bacterial cells

Bacteria frequently engage in cross-feeding interactions that involve an exchange of metabolites with other micro- or macroorganisms. The often obligate nature of these associations, however, hampers manipulative experiments, thus limiting our mechanistic understanding of the ecophysiological consequences that result for the organisms involved. Here we address this issue by taking advantage of a well-characterized experimental model system, in which auxotrophic genotypes of E. coli derive essential amino acids from prototrophic donor cells using intercellular nanotubes. Surprisingly, donor–recipient cocultures revealed that the mere presence of auxotrophic genotypes was sufficient to increase amino acid production levels of several prototrophic donor genotypes. Our work is consistent with a scenario, in which interconnected auxotrophs withdraw amino acids from the cytoplasm of donor cells, which delays feedback inhibition of the corresponding amino acid biosynthetic pathway and, in this way, increases amino acid production levels. Our findings indicate that in newly established mutualistic associations, an intercellular regulation of exchanged metabolites can simply emerge from the architecture of the underlying biosynthetic pathways, rather than requiring the evolution of new regulatory mechanisms.     

Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase.

To test the hypothesis that Na+/K+-ATPase works as an (alpha beta)2-diprotomer with interacting catalytic alpha-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na+-like 80-kDa as well as K+-like 60-kDa peptides and N-terminal 40-kDa peptides of the alpha-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4-8-N3-ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+-ATPase with an inactivation rate constant (k2) of 12 x 10-3.min-1 at 37 degrees C and a dissociation constant (Kd) of 207 +/- 28 microm. Tryptic digestion of the [gamma32P]Co(NH3)4-8-N3-ATP-inactivated and photolabelled alpha-subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+-like C-terminal 60-kDa fragment which was labelled in addition to an unlabelled Na+-like C-terminal 80-kDa fragment. Tryptic digestion of [alpha32P]-or [gamma32P]Cr(H2O)4ATP - bound to the E1ATP (high affinity) site - led to the labelling of a Na+-like 80-kDa fragment besides the immediate formation of an unlabelled K+-like N-terminal 40-kDa fragment and a C-terminal 60-kDa fragment. Because a labelled Na+-like 80-kDa fragment cannot result from an unlabelled K+-like 60-kDa fragment, and because unlabelled alpha-subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+- and K+-like conformations are stabilized by tight binding of substitution-inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (alphabeta)2-diprotomeric Na+/K+-ATPase.     

Regulatory T cells and control of the germinal centre response

Germinal centres (GCs) are specialised lymphoid microenvironments that form in secondary B-cell follicles upon exposure to T-dependent antigens. In the GC, clonal expansion, selection and differentiation of GC B cells result in the production of high-affinity plasma cells and memory B cells that provide protection against subsequent infection. The GC is carefully regulated to fulfil its critical role in defence against infection and to ensure that immunological tolerance is not broken in the process. The GC response can be controlled by a number of mechanisms, one of which is by forkhead box p3 expressing regulatory T (Treg) cells, a suppressive population of CD4⁺ T cells. A specialised subset of Treg cells – follicular regulatory T (Tfr) cells – form after immunisation and are able to access the GC, where they control the size and output of the response. Our knowledge of Treg cell control of the GC is expanding. In this review we will discuss recent advances in the field, with a particular emphasis on the differentiation and function of Tfr cells in the GC.     

Cytoskeleton in plant development

The plant cytoskeleton has crucial functions in a number of cellular processes that are essential for cell morphogenesis, organogenesis and development. These functions have been intensively investigated using single cell model systems. With the recent characterization of plant mutants that show aberrant organogenesis resulting from primary defects in cytoskeletal organization, an integrated understanding of the importance of the cytoskeleton for plant development has begun to emerge. Newly established techniques that allow the non-destructive visualization of microtubules or actin filaments in living plant cells and organs will further advance this understanding.     

Remodelling of membrane phospholipids during transition to diapause and cold-acclimation in the larvae of Chymomyza costata (Drosophilidae)

The composition of molecular species of phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) was analysed in fat body and muscle tissues of Chymomyza costata larvae of different physiological states that markedly differed in their level of freeze-tolerance. Actively moving and feeding 3rd instar larvae had low (zero) capacity of freeze-tolerance and similar phospholipid (PL) compositions irrespective of their developmental destiny (non-diapause vs. diapause). Extensive remodelling of PL composition was found in these larvae in response to: (a) chilling of non-diapause larvae at 5 degrees C for 1 month; (b) developmental transition to diapause; and (c) chilling of diapause larvae. Transition to diapause and chilling led to an increase in freeze-tolerance. The increase in molar proportion of molecular species containing palmitic/linoleic (16:0/18:2) fatty acyls (FAs) esterified to sn-1/sn-2 positions of glycerol was the most prominent change, which was tightly statistically correlated with increasing freeze-tolerance. The increase of PLs with combination of 16:0/18:2 FAs was registered consistently in PEs and PCs in fat body and muscle tissues in response to chilling and to diapause onset. This increase was countered by a decreases of various molecular species, depending on tissue and lipid class. Most decreasing species shared one common theme: they had a saturated FA (palmityl, margaryl, stearyl) esterified at sn-1 position and a monounsaturate (palmitoleyl, oleyl) esterified at sn-2 position of glycerol. Possible adaptive meaning of PL molecular species remodelling is discussed.