Comparison of nutritional status of field and laboratory reared Balanus amphitrite Darwin (Cirripedia: Thoracica) larvae and implication of starvation

Experiments were carried out to evaluate the influence of rearing temperature and food concentration (20 and 30 °C, 1×10⁵ and 2×10⁵ cells ml⁻¹) on the starvation threshold and nucleic acid content of the larvae of Balanus amphitrite. The larvae were also field-reared using micro-enclosures. Laboratory-reared larvae were larger in size than the field-reared larvae. An increase in size, DNA content and instar index of the starved II instar larvae was observed indicating that the absence of food may not be fatal to this early instar. The temperature at which larvae were raised and the food concentration had variable influence on the capacity to withstand starvation. Exposure to increased temperatures during starvation eliminated the effect of doubling food concentration during their feeding period prior to starvation. The larvae reared at 20 °C had comparatively lower nucleic acid content. The laboratory-reared larvae had ca. 1.7 times greater RNA:DNA ratio than larvae raised at comparable temperature in the field.     

IL-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts.

IL-12 is a 75-kDa heterodimeric cytokine composed of disulfide-bonded 35-kDa and 40-kDa subunits. Included among the biologic activities mediated by IL-12 is induction of proliferation of PHA-activated human PBL. The concentration of IL-12 required to stimulate maximum proliferation of PHA-activated lymphoblasts is 50 to 100 pM. In this study, highly purified 125I-labeled IL-12 (7 to 15 microCi/microgram; 50 to 100% bioactive) was used to characterize the receptor for IL-12 on 4-day PHA-activated lymphoblasts. The binding of 125I-labeled IL-12 to PHA-activated lymphoblasts was saturable and specific because the binding of radiolabeled ligand was only inhibited by IL-12 and not by other cytokines. The kinetics of [125I]IL-12 binding to PHA-activated lymphoblasts was rapid at both 4 degrees C and 22 degrees C; reaching equilibrium within 60 min. At 22 degrees C, the rate of dissociation of [125I]IL-12 was slow in the absence of competing IL-12 (t1/2 = 5.9 h) and more rapid in the presence of 25 nM competing IL-12 (t1/2 = 2.5 h). The kinetically derived equilibrium dissociation constant ranged from 10 to 83 pM. Analysis of steady state binding data by the method of Scatchard identified a single binding site with an apparent equilibrium dissociation constant of 100 to 600 pM and 1000 to 9000 sites/lymphoblast. The equilibrium dissociation constant for competing ligands and sites per cell calculated from unlabeled IL-12 competition experiments ranged from 164 to 315 pM and 1067 to 3336, respectively, which is in good agreement with the values determined from steady state binding. The variations in KD and sites per cell were dependent on the individual preparations of lymphoblasts. Although the steady state binding data were consistent with a single class of high affinity binding sites, the kinetic dissociation data indicates a cooperative interaction between receptors on PHA-activated lymphoblasts. Affinity cross-linking of surface bound [125I]IL-12 to PHA-activated lymphoblasts at 4 degrees C identified a major complex of approximately 210 to 280 kDa. Anti-IL-12 antibodies also immunoprecipitated a complex of approximately 210 to 280 kDa that was produced by cross-linking unlabeled IL-12 to 125I-labeled lymphoblast cell-surface proteins. Cleavage of this complex with reducing agent identified one radiolabeled protein of approximately 110 kDa. These data suggest that the IL-12 binding site on PHA-activated lymphoblasts may be composed of a single protein of approximately 110 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)     

To ∼P or Not to ∼P? Non‐canonical activation by two‐component response regulators

Bacteria sense and respond to their environment through the use of two‐component regulatory systems. The ability to adapt to a wide range of environmental stresses is directly related to the number of two‐component systems an organism possesses. Recent advances in this area have identified numerous variations on the archetype systems that employ a sensor kinase and a response regulator. It is now evident that many orphan regulators that lack cognate kinases do not rely on phosphorylation for activation and new roles for unphosphorylated response regulators have been identified. The significance of recent findings and suggestions for further research are discussed.     

Central insulin sensitivity in male and female juvenile rats

The incidence of juvenile obesity is increasing at an alarming rate. In adults, central insulin administration decreases hypothalamic orexigenic neuropeptides, food intake and body weight more effectively in males than females. Mechanisms regulating energy balance in juvenile animals are inherently different from those in adults due to differences in growth rates and hormonal milieu. Therefore, we sought to determine if central insulin treatment in juvenile rats (4 wk) would have similar sex-dependent effects on food intake as those reported in adult rats. Twenty-four hour food intake was measured following icv saline or insulin (0.01 or 0.1 U) prior to the onset of dark phase of the light cycle. An additional set of animals was used to assess the effects of central insulin on hypothalamic orexigenic (NPY, AgRP) and anorexigenic (POMC) neuropeptide mRNA expression. In both males and females, insulin reduced meal size initially (first 4 h) and later decreased meal frequency (4-24 h) to reduce cumulative food intake. Consistent with this, central insulin decreased hypothalamic NPY and AgRP and increased POMC mRNA expression. In contrast to adult studies, there were no demonstrated sex differences. These studies indicate that juvenile females and males are equally sensitive to central insulin anorexigenic effects, perhaps due to a lack of circulating gonadal hormones. The anorexigenic responsiveness of both genders suggests a potential pharmacologic approach to childhood obesity.     

Activation of human cyclin-dependent kinases in vitro.

We have analyzed the activation of human cyclin-dependent kinases in a cell-free system. Human CDC2, cyclin-dependent kinase 2 (CDK2), cyclin A, and cyclin B1 were produced in insect cells by infection with recombinant baculoviruses. CDC2 or CDK2 monomers in lysates of infected cells could be activated by the addition of lysates containing cyclin A or B1. CDC2 activation by cyclin B1, as well as CDK2 activation by cyclins A and B1, was accompanied by the formation of high molecular weight complexes. In contrast, CDC2 did not bind effectively to cyclin A. CDC2 activation by cyclin B1 was studied in detail and was found to be accompanied by phosphorylation of CDC2 on Threonine 161. The binding of CDC2 to cyclin B1 also occurred under conditions where CDC2 phosphorylation was prevented, resulting in an inactive complex that could then be phosphorylated and activated on addition of cell extract. Highly purified CDC2 and cyclin B1 also formed inactive complexes that could be activated in an ATP-dependent fashion by unidentified components in crude cell extracts. These data suggest that the CDC2 activation process begins with cyclin binding, after which CDC2 phosphorylation, catalyzed by a separate enzyme, leads to activation.     

Distinct sub-populations of the retinoblastoma protein show a distinct pattern of phosphorylation.

Phosphorylation of the retinoblastoma protein (pRB) is assumed to regulate its growth-controlling function. Moreover, hypophosphorylated and hyperphosphorylated forms of pRB can be distinguished by virtue of the distinct affinities with which they bind to the cell nucleus. This property allows the identification of individual cell nuclei that contain pRB in one or the other form. We show here that after cells emerge from a quiescent (G0) state, conversion of their complement of pRB into a hyperphosphorylated form occurs in late G1, preceding entry into S phase by several hours. Thus, contrary to earlier reports, pRB phosphorylation is not co-ordinated with the G1-S transition and may not directly regulate it. A distinct set of phosphopeptides is found exclusively in those forms of pRB that show the loose nuclear association characteristic of the hyperphosphorylated form of pRB. Another set of phosphopeptides is found with both hypophosphorylated and hyperphosphorylated forms. This suggests the existence of distinct patterns of phosphorylation that are associated with different subsets of pRB molecules. We conclude that substantial phosphorylation of pRB exists in G1 even prior to the hyperphosphorylation point. Cyclin-dependent kinases can cause a liberation of pRB from cell nuclei in vitro. Phosphorylation by members of this kinase family is therefore likely to be directly involved in the change in nuclear affinity in vivo and the associated changes in pRB functioning.     

DNA Binding and Condensation Properties of the Herpes Simplex Virus Type 1 Triplex Protein VP19C

Herpesvirus capsids are regular icosahedrons with a diameter of a 125 nm and are made up of 162 capsomeres arranged on a T = 16 lattice. The capsomeres (VP5) interact with the triplex structure, which is a unique structural feature of herpesvirus capsid shells. The triplex is a heterotrimeric complex; one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent capsomeres. VP19C interacts with VP23 and with the major capsid protein VP5 and is required for the nuclear localization of VP23. Mutation of VP19C results in the abrogation of capsid shell synthesis. Analysis of the sequence of VP19C showed the N-terminus of VP19C is very basic and glycine rich. It was hypothesized that this domain could potentially bind to DNA. In this study an electrophoretic mobility shift assay (EMSA) and a DNA condensation assay were performed to demonstrate that VP19C can bind DNA. Purified VP19C was able to bind to both a DNA fragment of HSV-1 origin as well as a bacterial plasmid sequence indicating that this activity is non-specific. Ultra-structural imaging of the nucleo-protein complexes revealed that VP19C condensed the DNA and forms toroidal DNA structures. Both the DNA binding and condensing properties of VP19C were mapped to the N-terminal 72 amino acids of the protein. Mutational studies revealed that the positively charged arginine residues in this N-terminal domain are required for this binding. This DNA binding activity, which resides in a non-conserved region of the protein could be required for stabilization of HSV-1 DNA association in the capsid shell.     

From genomics to metagenomics

Next-generation sequencing has changed metagenomics. However, sequencing DNA is no longer the bottleneck, rather, the bottleneck is computational analysis and also interpretation. Computational cost is the obvious issue, as is tool limitations, considering most of the tools we routinely use have been built for clonal genomics or are being adapted to microbial communities. The current trend in metagenomics analysis is toward reducing computational costs through improved algorithms and through analysis strategies. Data sharing and interoperability between tools are critical, since computation for metagenomic datasets is very high.     

Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY

Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non‐invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non‐invasive r‐ L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein‐expressing Caco‐2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens.