Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.     

Dephosphorylation of tau protein by calcineurin triturated into neural living cells

Alzheimer disease and related dementia are characterized by the presence of hyperphosphorylated tau aggregated into filaments. The role of tau phosphorylation in the fibrillogenesis has not yet been unraveled. Therefore, it is important to know which phosphatases can dephosphorylate tau protein in vivo. The effect of recombinant purified calcineurin (CN(PP2B)) and several calcineurin mutants on tau phosphorylation was studied in two neuronal like cell lines PC12 and SH-SY5Y. The modulation of tau phosphorylation at Ser199/Ser202, Ser396/Ser404, Ser262/Ser356, and Thr181 sites was examined in these cell lines using the phosphorylation state-dependent antitau antibodies Tau 1, PHF1, 12E8, and AT270. The results have shown that CN directly dephosphorylates all of those sites of tau protein. Recombinant calcineurin introduced into cells that have previously been treated with okadaic acid and cyclosporin A, which are inhibitors of phosphatases (PP1/PP2A and PP2B), has a direct effect on the phosphorylation status on all phosphorylation sites studied. We conclude that calcineurin is (besides PP2A) a important modulator of tau phosphorylation in vivo.     

Two populations of node monocilia initiate left-right asymmetry in the mouse

The vertebrate body plan has conserved handed left-right (LR) asymmetry that is manifested in the heart, lungs, and gut. Leftward flow of extracellular fluid at the node (nodal flow) is critical for normal LR axis determination in the mouse. Nodal flow is generated by motile node cell monocilia and requires the axonemal dynein, left-right dynein (lrd). In the absence of lrd, LR determination becomes random. The cation channel polycystin-2 is also required to establish LR asymmetry. We show that lrd localizes to a centrally located subset of node monocilia, while polycystin-2 is found in all node monocilia. Asymmetric calcium signaling appears at the left margin of the node coincident with nodal flow. These observations suggest that LR asymmetry is established by an entirely ciliary mechanism: motile, lrd-containing monocilia generate nodal flow, and nonmotile polycystin-2 containing cilia sense nodal flow initiating an asymmetric calcium signal at the left border of the node.     

Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs.     

Structural flexibility of Aib-containing peptides: the N-terminal tripeptide of trichotoxin

The sequence Aib-Gly-Aib which corresponds to the N-terminus of the microheterogeneous peptide antibiotic trichotoxin has been studied crystallographically in the context of different protecting groups. Peptides Ac-Aib-Gly-Aib-OH (A) and Z-Aib-Gly-Aib-OH (B) form beta-turns. Both peptides show a remarkable conformational flexibility forming a large variety of beta-turns of different types.     

<Emphasis Type="Italic">Wolbachia</Emphasis> endosymbiont infection in two Indian butterflies and female-biased sex ratio in the Red Pierrot, <Emphasis Type="Italic">Talicada nyseus</Emphasis>

The maternally inherited obligate bacteria Wolbachia is known to infect various lepidopteran insects. However, so far only a few butterfly species harbouring this bacterium have been thoroughly studied. The current study aims to identify the infection status of these bacteria in some of the commonly found butterfly species in India. A total of nine butterfly species belonging to four different families were screened using PCR with Wolbachia-specific wsp and ftsZ primers. The presence of the Wolbachia super group ‘B’ in the butterflies Red Pierrot, Talicada nyseus (Guerin) (Lepidoptera: Lycaenidae) and Blue Mormon, Papilio polymnestor Cramer (Papilionidae), is documented for the first time in India. The study also gives an account on the lifetime fecundity and female-biased sex ratio in T. nyseus, suggesting a putative role for Wolbachia in the observed female-biased sex ratio distortion.