Genetics of seed-coat colour in Citrullus lanatus (Thunb.) Mansf.

Summary Intervarietal crosses in watermelon, Citrullus lanatus (Thunb.) Mansf., involving six parents with black (J18-1 and J 75), brown (J56-1 and N.H. Midget), red (Bykovski-199) or light cream (Red Nectar) seed-coat colour were made. Parents, F₁, F₂ and backcross populations were evaluated for their phenotypic expressions with regard to the seed-coat colours involved. Black colour was monogenically dominant over brown light cream and red colour of seed-coat separately or independently. Red colour was dominant over light cream colour of seed-coat by a single pair of genes. The light cream colour was recessive to the brown seed-coat colour of watermelon where a single pair of genes was involved.     

Solution structure and phosphopeptide binding to the N-terminal domain of Yersinia YopH: comparison with a crystal structure

Virulence of pathogenic bacteria of the genus Yersinia requires the injection of six effector proteins into the cytoplasm of host cells. The amino-terminal domain of one of these effectors, the tyrosine phosphatase YopH, is essential for translocation of YopH, as well as for targeting it to phosphotyrosine-containing substrates of the type pYxxP. We report the high-resolution solution structure of the N-terminal domain (residues 1-129) from the Yersinia pseudotuberculosis YopH (YopH-NT) in complex with N-acetyl-DEpYDDPF-NH(2), a peptide derived from an in vivo protein substrate. In contrast to the domain-swapped dimer observed in a crystal structure of the same protein (Smith, C. L., Khandelwal, P., Keliikuli, K., Zuiderweg, E. R. P., and Saper, M. A. (2001) Mol. Microbiol. 42, 967-979), YopH-NT is monomeric in solution. The peptide binding site is located on a beta-hairpin that becomes the crossover point in the dimer structure. The binding site has several characteristics that are reminiscent of SH2 domains, which also bind to pYxxP sequences.     

Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.     

Effect of overexpression and nuclear translocation of constitutively active PKB-alpha on cellular survival and proliferation in HepG2 cells

Protein kinase B (Akt/PKB) is a key component in the PI 3-kinase mediated cell survival pathway and has oncogenic transformation potential. Although the over-expression of PKB-alpha can prevent cell death following growth factor withdrawal, the long-term effects of stable over-expression of PKB-alpha on cell survival in the absence of growth factors remain to be resolved. In the present study, we generated HepG2 cells with stable expression of active PKB-alpha and compared its characteristics with HepG2 cells. Basal as well as insulin-stimulated levels of Ser(473) and Thr(308) phosphorylation in PKB-alpha transfected HepG2 cells were much higher than HepG2 cells. Constitutive expression of active PKB-alpha enabled HepG2 cells to survive up to 96 h without serum in growth media while HepG2 cells fail to survive after 48 h of serum withdrawal. A strong positive correlation (R(2) = 0.71) between cell proliferation and phosphorylated form of PKB-alpha at Thr(308) was observed along with higher levels of phosphorylated 3'-phosphoinositide-dependent kinase-1 (PDK-1). HepG2 cells with constitutive expression of active PKB-alpha also showed higher levels of phosphorylated p65 subunit of nuclear factor-kappaB (NFkappaB) in comparison with HepG2 cells. Predominant nuclear localization of phosphorylated PKB-alpha was observed in stably transfected HepG2 cells. These results indicate that constitutive expression of active PKB-alpha renders HepG2 cells independent of serum based growth factors for survival and proliferation.     

Hepatic glycogen metabolism in the db/db mouse

Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in V_max, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Improved synthesis of histone deacetylase inhibitors (HDIs) (MS-275 and CI-994) and inhibitory effects of HDIs alone or in combination with RAMBAs or retinoids on growth of human LNCaP prostate cancer cells and tumor xenografts

We have developed new, simple, and efficient procedures for the synthesis of two promising histone deacetylase inhibitors (HDIs), CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide), and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide) from commercially available acetamidobenzoic acid and 3-(hydroxymethyl)pyridine, respectively. The procedures provide CI-994 and MS-275 in 80% and 72% overall yields, respectively. We found that the combination of four HDIs (CI-994, MS-275, SAHA, and TSA) with retinoids all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (13-CRA) or our atypical retinoic acid metabolism blocking agents (RAMBAs) 1 (VN/14-1) or 2 (VN/66-1) produced synergistic anti-neoplastic activity on human LNCaP prostate cancer cells. The combination of 2 and SAHA induced G1 and G2/M cell cycle arrest and a decrease in the S phase in LNCaP cells. 2+SAHA treatment effectively down-regulated cyclin D1 and cdk4, and up-regulated pro-differentiation markers cytokeratins 8/18 and pro-apoptotic Bad and Bax. Following subcutaneous administration, 2, SAHA or 2+SAHA were well tolerated and caused significant suppression/regression of tumor growth compared with control. These results demonstrate that compound 2 and its combination with SAHA are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.     

Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K⁺ channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.     

Genetic incorporation of human metallothionein into the adenovirus protein IX for non-invasive SPECT imaging

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of (99m)Tc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a (99m)Tc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo.