Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.     

Darwinian gradualism and its limits: The development of Darwin's views on the rate and pattern of evolutionary change

Conclusion The major tenets of the recent hypothesis of punctuated equilibrium are explicit in Darwin's writing. His notes from 1837–1838 contain references to stasis and rapid change. In the first edition of the Origin (1859), Darwin described the importance of isolation of local varieties in the process of speciation. His views on the tempo of speciation were influenced by Hugh Falconer and also, perhaps, by Edward Suess (1831–1914). It is paradoxical that, although both topics were recorded in his unpublished notes of 1837–1838, the second was not explicitly and fully discussed until the fourth edition of the Origin (1866). While no wholly satisfactory explanation of this paradox suggests itself, it seems probable that Falconer's work on the persistence of fossil species of elephant helped Darwin to see the wider significance of the tempo of evolution for his general theory.     

Energetics and intermediates of the assembly of Protein OmpA into the outer membrane of Escherichia coli

OmpA is a major protein of the outer membrane of Escherichia coli. It is made as a larger precursor, pro-OmpA, which requires a membrane potential for processing. We now show that pro-OmpA accumulates in the cytoplasm of cells treated with carbonyl cyanide m-chlorophenylhydrazone, an uncouple which lowers the membrane potential. Upon restoration of the potential, this pro-OmpA is secreted, processed, and assembled into the outer membrane. Pro-OmpA made in vitro is also recovered with the postribosomal supernatant. It is efficiently processed to OmpA by liposomes which have bacterial leader peptidase that is exclusively internally oriented. These experiments show that: (i) the insertion of pro-OmpA into the plasma membrane is not coupled to its synthesis; (ii) insertion is promoted by the transmembrane electrochemical potential; (iii) pro-OmpA can cross a bilayer spontaneously; and (iv) pro-OmpA is processed by the same leader peptidase which converts M13 procoat to coat.     

Prolonged delirium without anticholinergic signs following amitriptyline overdose.

In a case of acute intoxication with a tricyclic antidepressant (amitriptyline) delirium was prolonged without there being prominent peripheral anticholinergic or electrocardiographic signs. Administration of physostigmine, repeated when necessary, reversed the delirium.     

Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.

We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

Effects of dexamethasone on carnitine metabolism in liver and extrahepatic tissues

The in vivo effects of dexamethasone administration on liver and extrahepatic tissue carnitine concentrations were assessed in 48-h-starved rats. In heart and kidney, but not in liver, dexamethasone significantly increased total carnitine concentration. Acute (2.5 h) treatment with 2-tetradecylglycidate (TDG), a specific inhibitor of carnitine palmitoyl transferase 1, not only increased total hepatic carnitine concentrations, but also permitted an effect of dexamethasone (a further increase in hepatic carnitine concentration). The results are discussed in terms of acute (substrate-mediated) and chronic (hormonal) control of carnitine turnover.     

Evidence for the presence of lipopolysaccharide in Treponema phagedenis (biotype Reiterii) but not in Treponema pallidum (Nichols)

Abstract Immunoblotting profiles of whole or protease-K-digested organisms with homologous antisera demonstrated the presence of a characteristic ladder pattern of smooth LPS in Treponema phagedenis . Periodic acid silver staining of SDS-PAGE gels confirmed these findings. However, when heterologous or homologous serum was reacted with Treponema pallidum , no such pattern or cross-reactions were observed. The significance of apparent absence of LPS in T. pallidum is discussed.     

Within-tree spatial variation in the leaf monoterpenes of Sequoia sempervirens

Location within a tree was analyzed as a source of variation in Sequoia sempervirens leaf monoterpenes. No differences were found for quantitative composition or total yield/dry wt among lower, middle and upper canopy positions. The awlshaped, spirally arranged leaves of vigorous upper shoots showed small quantitative compositional differences, but not differences in total yield. The intermediate leaf form of young sprouts had the most different monoterpene quantitative composition and about three times the total yield of the above two leaf forms. Analysis of a clonal ring of 17 adult trees resulted in coefficients of variation similar to those for samples collected from different canopy levels of the same shoot. Results revealed the sources and magnitudes of experimental error in comparative studies of this species' leaf monoterpenes, and did not support the concept that somatic mutation provides an important source of variation in a large, long-lived organism such as coast redwood.     

Partial-digest DNA sequencing.

A technique called partial-digest sequencing that permits DNA of 4-6 kb in length to be sequenced without subcloning is described. The method exploits the specific cuts introduced by partial digestion with restriction endonucleases that have 4-base recognition sites to produce ordered ladders of PCR-amplified fragments. The staggered ends contain PCR primers and can thus be individually sequenced using conventional methods to yield overlapping sequences covering the entire region. This method should have significant impact on both large and small DNA sequencing projects and find many applications in general manipulations in which ordered sets of deletions need to be produced.