Intragenomic heterogeneity and intergenomic recombination among haloarchaeal rRNA genes

More than one copy of rRNA operons, which code for both the small-subunit (SSU) and large-subunit (LSU) rRNA, are often found in prokaryotes. It is generally assumed that all rRNA operons within a single cell are almost identical. A notable exception is the extremely halophilic archaeal genus Haloarcula, most species of which are known to harbor highly divergent rRNA operons that differ at approximately 5% of the nucleotide positions in the SSU gene and at 1 to 2% of the nucleotide positions in the LSU gene. We report that such intragenomic heterogeneity is not unique to Haloarcula, as high levels of intragenomic sequence variation have been observed for the SSU genes of two other genera of extreme halophiles, Halosimplex and Natrinema. To investigate this in detail, the two rRNA operons of Halosimplex carlsbadense and the four operons of Natrinema sp. strain XA3-1 were cloned and completely sequenced. The SSU and LSU genes of H. carlsbadense show the highest levels of intragenomic heterogeneity observed so far in archaea (6.7 and 2.6%). The operons of Natrinema sp. strain XA3-1 have additional unusual characteristics, such as identical internal transcribed spacers, while one of four SSU genes is 5% divergent and all LSU genes differ from each other by 0.9 to 1.9%. The heterogeneity among the Natrinema sp. strain XA3-1 LSU genes is localized in hot spots, and one of these regions is shown to be the result of a recombination event with a distantly related halophile. This is the first example of interspecies recombination between rRNA genes in archaea, and the recombination occurred over one of the largest phylogenetic distances ever reported for such an event. We suggest that intragenomic heterogeneity of rRNA operons is an ancient and stable trait in several lineages of the Halobacteriales. The impact of this phenomenon on the taxonomy of extremely halophilic archaea is discussed.     

Autocrine transforming growth factor alpha regulates cell adhesion by multiple signaling via specific phosphorylation sites of p70S6 kinase in colon cancer cells

Recently, we showed that autocrine transforming growth factor alpha (TGFalpha) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin alpha2, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin alpha2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861-19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin alpha2 function in HCT116 cells. Selective MEK inhibitors attenuated TGFalpha-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin alpha2 expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGFalpha regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells.     

Papillary thyroid carcinoma and its variants in fine needle aspiration smears. A cytomorphologic study with special reference to tall cell variant

OBJECTIVE: To study the fine needle aspiration (FNA) cytologic features of papillary thyroid carcinoma (PTC) with special reference to its tall cell variant (TCV), which is the most aggressive of the variants. STUDY DESIGN: Fifty-four PTC cases were classified into variants, and the frequency of well-known morphologic criteria was determined. Four parameters were quantitatively analyzed based on a study of 200 consecutive neoplastic follicular cells: shape of cells, color of cytoplasm, intranuclear cytoplasmic inclusion (INCI) and nuclear grooves. RESULTS: The PTC cases included 6 TCV (> or = 30% tall cells), 8 cases with a significant tall cell component (sig. TCC) having 10-29% tall cells, 17 usual variant (UV), 17 follicular variant (FV) and 6 miscellaneous variants. TCV differed significantly from UV and FV in having a higher tall cell count, higher count of cells with reddish cytoplasm and INCI, and higher frequency of cases with lymphocytic infiltration. PTC (with significant tall cell component [TCC]) differed significantly from TCV with regard to tall cell count and lymphocytic infiltration, from UV with respect to tall cell count and monolayered sheets, and from FV with respect to tall cells, INCI, grooved nuclei, acinar formation, fire-flare appearance and giant cells. CONCLUSION: TCV was cytologically distinct from other variants. The biologic behavior of PTC cases with significant TCC, which morphologically seem to be a group intermediate between TCV on the one hand and UV and FV on the other, however, needs to be carefully monitored.     

Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana

Anthocyanidin reductase (ANR), encoded by the BANYULS gene, is a newly discovered enzyme of the flavonoid pathway involved in the biosynthesis of condensed tannins. ANR functions immediately downstream of anthocyanidin synthase to convert anthocyanidins into the corresponding 2,3-cis-flavan-3-ols. We report the biochemical properties of ANRs from the model legume Medicago truncatula (MtANR) and the model crucifer Arabidopsis thaliana (AtANR). Both enzymes have high temperature optima. MtANR uses both NADPH and NADH as reductant with slight preference for NADPH over NADH. In contrast, AtANR only uses NADPH and exhibits positive cooperativity for the co-substrate. MtANR shows preference for potential anthocyanidin substrates in the order cyanidin>pelargonidin>delphinidin, with typical Michaelis-Menten kinetics for each substrate. In contrast, AtANR exhibits the reverse preference, with substrate inhibition at high concentrations of cyanidin and pelargonidin. (+)-Catechin and (+/-)-dihydroquercetin inhibit AtANR but not MtANR, whereas quercetin inhibits both enzymes. Possible catalytic reaction sequences for ANRs are discussed.     

The formation of straight and twisted filaments from short tau peptides

We studied fibril formation in a family of peptides based on PHF6 (VQIVYK), a short peptide segment found in the microtubule binding region of tau protein. N-Acetylated peptides AcVYK-amide (AcVYK), AcIVYK-amide (AcPHF4), AcQIVYK-amide (AcPHF5), and AcV-QIVYK-amide (AcPHF6) rapidly formed straight filaments in the presence of 0.15 m NaCl, each composed of two laterally aligned protofilaments approximately 5 nm in width. X-ray fiber diffraction showed the omnipresent sharp 4.7-A reflection indicating that the scattering objects are likely elongated along the hydrogen-bonding direction in a cross-beta conformation, and Fourier transform IR suggested the peptide chains were in a parallel (AcVYK, AcPHF6) or antiparallel (AcPHF4, AcPHF5) beta-sheet configuration. The dipeptide N-acetyl-YK-amide (AcYK) formed globular structures approximately 200 nm to 1 microm in diameter. The polymerization rate, as measured by thioflavin S binding, increased with the length of the peptide going from AcYK --> AcPHF6, and peptides that aggregated most rapidly displayed CD spectra consistent with beta-sheet structure. There was a 3-fold decrease in rate when Val was substituted for Ile or Gln, nearly a 10-fold decrease when Ala was substituted for Tyr, and an increase in polymerization rate when Glu was substituted for Lys. Twisted filaments, composed of four laterally aligned protofilaments (9-19 nm width, approximately 90 nm half-periodicity), were formed by mixing AcPHF6 with AcVYK. Taken together these results suggest that the core of PHF6 is localized at VYK, and the interaction between small amphiphilic segments of tau may initiate nucleation and lead to filaments displaying paired helical filament morphology.     

Abo blood groups and completed reproductive performance of rural Haryanavi couples: analysing measures of selection intensities

The possible differential effects of ABO blood group materno-paternal (fetal) incompatibility on completed reproductive performance were investigated on a sample of 100 couples (100 fathers and 100 mothers) from three villages in the Jind district of Haryana state, India. The average number of live births per mating couple was slightly higher for the incompatible matings (5.32) than the compatible ones (5.05). This advantage was offset by higher postnatal mortality in the former. Consequently, the average number of living children in the compatible matings (4.64) was higher than in the incompatible ones (4.18). With reference to individual ABO matings, the index of relative fertility (Irf) was the highest in A x AB followed by B x A type of incompatible matings. No decrease in live births in O x A and O x B incompatible matings was observed compared with their reciprocal compatible ones, i.e. A x O and B x O matings, as has been hypothesized in previous studies. The total pregnancy wastage was substantially higher in ABO-incompatible matings (24.59%) than compatible matings (8.45%). About 71% of the postnatal deaths took place within one year of the birth in the case of incompatible matings compared with 50% in the case of compatible matings. The study supports the hypothesis that selection is operative at the ABO locus as revealed by the measures of selection intensity. The loss of fitness in the present sample was associated with differential mortality. There were no differences in the proportions of average number of male live births in the compatible (0.55) and incompatible matings (0.58). However, in the individual mating types, there was some evidence of higher or lower proportions of male live births.     

Crystal structure of schistatin, a disintegrin homodimer from saw-scaled viper (Echis carinatus) at 2.5 A resolution

This is the first structure of a biological homodimer of disintegrin. Disintegrins are a class of small (4-14 kDa) proteins that bind to transmembrane integrins selectively. The present molecule is the first homodimer that has been isolated from the venom of Echis carinatus. The monomeric chain contains 64 amino acid residues. The three-dimensional structure of schistatin has been determined by the multiple isomorphous replacement method. It has been refined to an R-factor of 0.190 using all the data to 2.5 A resolution. The two subunits of the disintegrin homodimer are related by a 2-fold crystallographic symmetry. Thus, the crystallographic asymmetric unit contains a monomer of disintegrin. The monomer folds into an up-down topology with three sets of antiparallel beta-strands. The structure is well ordered with four intramolecular disulfide bonds. the two monomers are firmly linked to each other through two intermolecular disulfide bridges at their N termini together with several other interactions. This structure has corrected the error in the disulfide bond pattern of the two intermolecular disulfide bridges that was reported earlier using chemical methods. Unique sequence and structural features of the schistatin monomers suggest that they have the ability to bind well with both alphaIIb beta3 and alphav beta3 integrins. The N termini anchored two chains of the dimer diverge away at their C termini exposing the Arg-Gly-Asp motif into opposite directions thus enhancing their binding efficiency to integrins. This is one of the unique features of the present disintegrin homodimer and seems to be responsible for the clustering of integrin molecules. The homodimer binds to integrins apparently with a higher affinity than the monomers and also plays a role in the signaling pathway.     

Long-lived poxvirus immunity, robust CD4 help, and better persistence of CD4 than CD8 T cells

The currently used smallpox vaccine is associated with a high incidence of adverse events, and there is a serious need for a safe and effective alternative vaccine. Here, we carried out a longitudinal evaluation of vaccinia virus-specific CD4 and CD8 T cells in smallpox-vaccinated individuals by using a highly sensitive intracellular cytokine staining assay. Our results demonstrate that, in addition to the CD8 response, the smallpox vaccinations raised a robust CD4 response with a Th1-dominant cytokine profile. These CD4 T cells were stable and exhibited only a twofold contraction between peak effector and memory phases compared with an approximate sevenfold contraction for CD8 cells. A significant proportion of vaccinated individuals lost detectable CD8 memory while maintaining CD4 memory. After a booster immunization, these individuals generated a robust CD8 response, which some of them rapidly lost. Thus, the current smallpox vaccine provides long-lasting CD4 help that may be critical for long-lived B-cell memory. We suggest that the provision of adequate CD4 help for CD8 and humoral effector functions will be critical to the success of the next generation of smallpox vaccines.     

Coordinated expression and immunogenicity of an outer membrane protein from Salmonella enterica serovar Typhi under iron limitation, oxidative stress and anaerobic conditions

Summary Successful pathogens overcome the environmental stresses by the coordinated expression of various genes and eventually proteins. Since, the surface of the microbe is likely to come in contact with the host initially, an attempt was made to identify the outer membrane proteins (OMPs), if any, which may get expressed under more than one environmental conditions simulating the in vivo ones. In the present study, Salmonella enterica serovar Typhi was grown under iron-limited, oxidative stress as well as anaerobic conditions and the OMP profiles were compared. A 69 kDa OMP was found to express with enhanced intensity under the selected stress conditions in comparison to normal conditions. The phenotypic similarity among the proteins was assessed on the basis of their molecular weight, cross reactivity and HPLC. The protein expressed under oxidative stress and anaerobic conditions reacted with the antibodies raised against iron-regulated outer membrane protein (IROMP), indicating the sharing of at least some of the epitopes. A single peak observed after subjecting the pooled 69 kDa protein sample and appearance of a single band on SDS-PAGE thereafter, confirmed the purity and phenotypic similarity of the 69 kDa OMP. Reactivity of pooled 69 kDa protein with 85% of sera from typhoid patients revealed the in vivo expression of this protein. The results of this study indicate the coordination of this phenotype under iron stress, oxidative stress and anaerobic conditions. In view of the expression of the 69 kDa protein under the selected stress conditions and their in vivo immunogenicity, these findings may be relevant for the better understanding of the host–microbe interactions and for the further development of diagnostic and preventive strategies.