Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array

Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H⁺)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.     

Toxicity of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> LP1-G Against Susceptible and Resistant <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> and the Cloning of the Mosquitocidal Toxin Gene

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.Received: 7 October 2002 / Accepted: 4 November 2002     

New minor taxanes analogues from the needles of Taxus canadenis

Seven new taxanes were isolated from the needles of the Canadian yew: unusual functional groups, positions and/or stereochemical features are described. Their chemical structures were rigorously characterized by detailed high resolution NMR analyses and confirmed by high resolution Fast Atom Bombardment Mass Spectrometry. Unlike paclitaxel and taxuspine D, these taxanes had no effect on tubulin assembly.     

Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.     

Lymphocyte reduction induced by hindlimb unloading： distinct mechanisms in the spleen and thymus

Hindlimb unloading (HU) in rodent is a well-accepted ground-based model used to simulate some of the condi-tions of space flight and reproduce its deleterious effects on the musculoskeletal, cardiovascular and immune systems. In this study, the effects of HU on lymphocyte homeostasis in the spleen and thymus of mice were examined. HU was found to drastically deplete various cell populations in the spleen and thymus. These changes are likely to be mediated by apoptosis, since DNA strand breaks indicative of apoptosis were detected by terminal deoxynucleotidyl transferase-mediated nick end-labeling in both splenocytes and thymocytes. Surprisingly, administration of opioid antagonists or interference with the Fas-FasL interaction was able to block HU-induced reductions of splenocytes, but not thymocytes. On the other hand, steroid receptor antagonists blocked the reduction of lymphocyte numbers in both spleen and thymus. Therefore, the effects of HU on the homeostasis of splenocytes and thymocytes must be exerted through distinct mechanisms.     

Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are necessary for lipoprotein assembly. ApoB consists of five structural domains, betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3). We propose that MTP contains three structural motifs (N-terminal beta-barrel, central alpha-helix, and C-terminal lipid cavity) and three functional domains (lipid transfer, membrane associating, and apoB binding). MTP's lipid transfer activity is required for the assembly of lipoproteins. This activity renders nascent apoB secretion-competent and may be involved in the import of triglycerides into the lumen of endoplasmic reticulum. In addition, MTP binds to apoB with high affinity involving ionic interactions. MTP interacts at multiple sites in the N-terminal betaalpha(1) structural domain of apoB. A novel antagonist that inhibits apoB-MTP binding decreases apoB secretion. Furthermore, site-directed mutagenesis and deletion analyses that inhibit apoB-MTP binding decrease apoB secretion. Lipids modulate protein-protein interactions between apoB and MTP. Lipids associated with MTP increase apoB-MTP binding whereas lipids associated with apoB decrease this binding. Thus, specific antagonist, site-directed mutagenesis, deletion analyses, and modulation studies support the notion that apoB-MTP binding plays a role in lipoprotein biogenesis. However, specific steps in lipoprotein assembly that require apoB-MTP binding have not been identified. ApoB-MTP binding may be important for the prevention of degradation and lipidation of nascent apoB.     

Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite

AMP-activated kinase (AMPK) is a fuel-sensing enzyme present in most mammalian tissue. In response to a decrease in the energy state of a cell AMPK is phosphorylated and activated by still poorly characterized upstream events. Exposure of bovine aortic endothelial cells (BAEC) to chemically synthesized ONOO- acutely and significantly increased phosphorylation of c-Src, PDK1, AMPK, and its downstream target, acetyl-CoA carboxylase (ACC), without affecting cellular AMP. This novel pathway for AMPK activation was confirmed by the use of pharmacological inhibitors and dominant-negative mutants. Exposure of BAEC to hypoxia-reoxygenation (H/R) caused a biphasic increase in AMPK and ACC phosphorylation, which was prevented by adenoviral overexpression of superoxide dismutase (SOD) or inhibition of nitric-oxide synthase (NOS) implicating a role of ONOO- formed during H/R. Furthermore, dominant-negative mutants of c-Src or kinase-defective PDK1 also blocked H/R-induced AMPK activation indicating that, as with addition of exogenous ONOO-, both c-Src and PI 3-kinase are upstream of AMPK. Moreover, H/R, like ONOO-, significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO- favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO- activates AMPK in a c-Src-mediated, PI 3-kinase-dependent manner, and suggest that ONOO--induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC.     

Nanosecond dynamics of the mouse acetylcholinesterase cys69-cys96 omega loop

The paradox of high substrate turnover occurring within the confines of a deep, narrow gorge through which acetylcholine must traverse to reach the catalytic site of acetylcholinesterase has suggested the existence of transient gorge enlargements that would enhance substrate accessibility. To establish a foundation for the experimental study of transient fluctuations in structure, site-directed labeling in conjunction with time-resolved fluorescence anisotropy were utilized to assess the possible involvement of the omega loop (Omega loop), a segment that forms the outer wall of the gorge. Specifically, the flexibility of three residues (L76C, E81C, and E84C) in the Cys69-Cys96 Omega loop and one residue (Y124C) across the gorge from the Omega loop were studied in the absence and presence of two inhibitors of different size, fasciculin and huperzine. Additionally, to validate the approach molecular dynamics was employed to simulate anisotropy decay of the side chains. The results show that the Omega loop residues are significantly more mobile than the non-loop residue facing the interior of the gorge. Moreover, fasciculin, which binds at the mouth of the gorge, well removed from the active site, decreases the mobility of 5-((((2-acetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid reporter groups attached to L76C and Y124C but increases the mobility of the reporter groups attached to E81C and E84C. Huperzine, which binds at the base of active-site gorge, has no effect on the mobility of reporter groups attached to L76C and Y124C but increases the mobility of the reporter groups attached to E81C and E84C. Besides showing that fluctuations of the Omega loop residues are not tightly coupled, the results indicate that residues in the Omega loop exhibit distinctive conformational fluctuations and therefore are likely to contribute to transient gorge enlargements in the non-liganded enzyme.