Differential Gene Expression and Characterization of Tissue-specific cDNA Clones in Oil Palm using mRNA Differential Display

The mRNA differential display method was utilized to study the differential expression and regulation of genes in two species of oil palm, the commercially grown variety Elaeis guineensis, var. tenera and the South American species, Elaeis oleifera. We demonstrated the differential expression of genes in the mesocarp and kernel at the week of active oil synthesis (15 week after anthesis) during fruit development as compare to the roots and leaves and the isolation of tissue-specific and species-specific cDNA clones. A total of eight specific cDNA clones were isolated and their specificities were confirmed by Northern hybridization and classified into three groups. Group one contains four clones (KT3, KT4, KT5 and KT6) that are kernel-specific for E. guineensis, tenera and E. oleifera. The second group represents clone FST1, which is mesocarp and kernel-specific for E. guineensis, tenera and E. oleifera. The third group represents clones MLT1, MLT2 and MLO1 that are mesocarp and leaf-specific. Northern analysis showed that their expressions were developmentally regulated. Nucleotide sequencing and homology search in GenBank data revealed that clones KT3 and KT4 encode for the same maturation protein PM3. While clones MLT1 and MLT2 encode for S-ribonuclease binding protein and fibrillin, respectively. The other clones (KT5, KT6, FST1 and MLO1) did not display any significant homology to any known protein.     

T-cadherin GPI-anchor is insufficient for apical targeting in MDCK cells

T-cadherin is a 95kDa glycoprotein member of the cadherin family of adhesion molecules attached to the extracellular surface of the cell membrane through a glycosyl-phosphatidylinositol (GPI)-anchor. Whether a T-cadherin ectodomain apical targeting signal or the GPI-anchor itself targets this protein to the apical membrane is not known. Chimeras of the reporter EGFP and T-cadherin have demonstrated that a minimal construct consisting of the C-terminal 25 amino acids including the N690 (omega-site) of T-cadherin was sufficient to GPI-anchor the EGFP protein. However, efficient GPI-anchor with minimal secretion of the protein required an additional 5 residues (omega-1 to omega-5). The GPI-anchored chimeras fractionated to the Triton X-100 detergent insoluble fraction and were released to the cell culture supernatant by phosphoinositide-specific phospho-lipase C digestion. When expressed in MDCK cells, all GPI-anchored chimeras targeted to the basolateral membrane, while the T/N-chimera and the wild-type T-cadherin targeted to the apical membrane. Therefore, T-cadherin is an example of another rare GPI-anchored protein where the anchor itself is not sufficient for apical targeting in MDCK cells.     

Naive CD4+ T cells from lupus-prone Fas-intact MRL mice display TCR-mediated hyperproliferation due to intrinsic threshold defects in activation

Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.     

Growth and regenerability of long-term suspension cultures of the U.S. rice cultivar Mercury

Suspension cultures of the U.S. rice cultivar Mercury have been maintained in modified General Medium for more than 3 years. These suspensions have continued to have high and relatively stable regeneration rates. Two different explants, immature panicles and seeds, were compared during the development of these embryogenic suspensions. Initial formation of secondary embryogenic callus from immature panicles on induction medium was greater than that from seeds. Suspensions of these two cell lines, however, did not differ morphologically and maintained similar regeneration rates. After 5 months in culture the rates of regeneration began to decline. The suspensions were plated onto regeneration medium without growth regulators for 2 weeks and then embryogenic cells were manually selected and used to develop secondary suspensions. Through this simple rejuvenation procedure, the suspensions retained high and stable regeneration rates. Variability in suspension growth, however, was observed during the culture period. Slower growth occurred at weeks 13, 15, 27, and 29 and was associated with a decrease in regeneration rates. Reproductive fertility of regenerated plants remained high for 3.5 years but then declined.Abbreviations CH casein (acid hydrolysate) - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige & Skoog basal medium - SE standard error     

LONG-TERM EXPERIMENTAL EVOLUTION IN ESCHERICHIA COLI. III. VARIATION AMONG REPLICATE POPULATIONS IN CORRELATED RESPONSES TO NOVEL ENVIRONMENTS

Twelve populations of Escherichia coli were founded from a single clone and propagated for 2000 generations in identical glucose-limited environments. During this time, the mean fitnesses of the evolving populations relative to their common ancestor improved greatly, but their fitnesses relative to one another diverged only slightly. Although the populations showed similar fitness increases, they may have done so by different underlying adaptations, or they may have diverged in other respects by random genetic drift. Therefore, we examined the relative fitnesses of independently derived genotypes in two other sugars, maltose and lactose, to determine whether they were homogeneous or heterogeneous in these environments. The genetic variation among the derived lines in fitness on maltose and lactose was more than 100-times greater than their variation in fitness on glucose. Moreover, the glucose-adapted genotypes, on average, showed significant adaptation to lactose, but not to maltose. That pathways for use of maltose and glucose are virtually identical in E. coli, except for their distinct mechanisms of uptake, suggests that the derived genotypes have adapted primarily by improved glucose transport. From consideration of the number of generations of divergence, the mutation rate in E. coli, and the proportion of its genome required for growth on maltose (but not glucose), we hypothesize that pleiotropy involving the selected alleles, rather than random genetic drift of alleles at other loci, was the major cause of the variation among the derived genotypes in fitness on these other sugars.     

Immunological characterization of diphtheria toxin recovered from Corynebacterium pseudotuberculosis

Diphtheria toxin (DT) is a potent toxin produced by the so-called diphtheria group which includes Corynebacterium diphtheriae (C. diphtheriae), Corynebacterium ulcerans (C. ulcerans), and Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The present investigation is aimed to study in detail the production of DT by C. pseudotuberculosis. Twenty isolates were obtained from sheep diseased with caseous lymphadenitis (CLA) and twenty-six isolates were obtained from 26 buffaloes diseased with oedematous skin disease (OSD). All isolates were identified by standard microbiological and DT production was assayed serologically by modified Elek test and immunoblotting. All sheep isolates were nitrate negative, failed to hydrolyze starch and could not produce DT, while all buffalo isolates (biotype II) revealed positive results and a specific band of 62 kDa, specific to DT, was resulted in all concentrated cell fractions (CF), but was absent from non-toxigenic biotype I isolates. At the same time, another band of 31 kDa specific to the PLD gene was obtained with all isolates of biotype I and II. Moreover, all isolates showed positive synergistic hemolytic activity and antagonistic hemolysis with β-hemolytic Staphylococci. The obtained results also indicated that C. pseudotuberculosis could be classified into two strains; non-toxigenic biotype I strain, which failed to produce DT as well as being negative to nitrate and starch hydrolysis, and toxigenic biotype II strain, which can reduce nitrate, hydrolyze starch as well as produce DT.