RPA alleviates the inhibitory effect of vinylphosphonate internucleotide linkages on DNA unwinding by BLM and WRN helicases

Bloom (BLM) and Werner (WRN) syndrome proteins are members of the RecQ family of SF2 DNA helicases. In this paper, we show that restricting the rotational DNA backbone flexibility, by introducing vinylphosphonate internucleotide linkages in the translocating DNA strand, inhibits efficient duplex unwinding by these enzymes. The human single-stranded DNA binding protein replication protein A (RPA) fully restores the unwinding activity of BLM and WRN on vinylphosphonate-containing substrates while the heterologous single-stranded DNA binding protein from Escherichia coli (SSB) restores the activity only partially. Both RPA and SSB fail to restore the unwinding activity of the SF1 PcrA helicase on modified substrates, implying specific interactions of RPA with the BLM and WRN helicases. Our data highlight subtle differences between SF1 and SF2 helicases and suggest that although RecQ helicases belong to the SF2 family, they are mechanistically more similar to the SF1 PcrA helicase than to other SF2 helicases that are not affected by vinylphosphonate modifications.     

Comparison of PSA-specific CD8<Superscript>+</Superscript> CTL responses and antitumor immunity generated by plasmid DNA vaccines encoding PSA-HSP chimeric proteins

The ability of heat shock proteins (HSPs) to increase the potency of protein- and DNA-based vaccines has been previously reported. We have constructed several plasmid-based vectors encoding chimeric proteins containing prostate-specific antigen (PSA) fused to Mycobacterium tuberculosis hsp70, M. bovis hsp65, Escherichia coli DnaK (hsp70), or human hsp70. Immunizing mice with these plasmids induced CD8⁺ cytotoxic T lymphocytes (CTLs) specific to human PSA and protected mice from a subsequent subcutaneous challenge with PSA-expressing tumors. We did not observe a significant difference either in the levels of PSA-specific CTLs or in protection against tumor challenge in mice immunized with plasmids expressing PSA-HSP chimeric proteins, as compared to mice receiving a conventional PSA-expressing DNA plasmid. Our data indicate that using HSPs as fusion partners for tumor-specific antigens does not always result in the enhancement of antigen-specific CTL responses when applied in the form of DNA vaccines.     

Phylogenetic distribution of TTAGG telomeric repeats in insects.

We examined the presence of TTAGG telomeric repeats in 22 species from 20 insect orders with no or inconclusive information on the telomere composition by single-primer polymerase chain reaction with (TTAGG)6 primers, Southern hybridization of genomic DNAs, and fluorescence in situ hybridization of chromosomes with (TTAGG)n probes. The (TTAGG)n sequence was present in 15 species and absent in 7 species. In a compilation of new and published data, we combined the distribution of (TTAGG)n telomere motif with the insect phylogenetic tree. The pattern of phylogenetic distribution of the TTAGG repeats clearly supported a hypothesis that the sequence was an ancestral motif of insect telomeres but was lost repeatedly during insect evolution. The motif was conserved in the "primitive" apterous insect orders, the Archaeognatha and Zygentoma, in the "lower" Neoptera (Plecoptera, Phasmida, Orthoptera, Blattaria, Mantodea, and Isoptera) with the exception of Dermaptera, and in Paraneoptera (Psocoptera, Thysanoptera, Auchenorrhyncha, and Sternorrhyncha) with the exception of Heteroptera. Surprisingly, the (TTAGG)n motif was not found in the "primitive" pterygotes, the Palaeoptera (Ephemeroptera and Odonata). The Endopterygota were heterogeneous for the occurrence of TTAGG repeats. The motif was conserved in Hymenoptera, Lepidoptera, and Trichoptera but was lost in one clade formed by Diptera, Siphonaptera, and Mecoptera. It was also lost in Raphidioptera, whereas it was present in Megaloptera. In contrast with previous authors, we did not find the motif in Neuroptera. Finally, both TTAGG-positive and TTAGG-negative species were reported in Coleoptera. The repeated losses of TTAGG in different branches of the insect phylogenetic tree and, in particular, in the most successful lineage of insect evolution, the Endopterygota, suggest a backup mechanism in the genome of insects that enabled them frequent evolutionary changes in telomere composition.     

Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution

There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+ current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+ current (I(Ca,L)) in myocytes dialyzed with 10 mM K+ solution and superfused with Tyrode's solution. Although depolarizations from holding potential (Vhp) -40 to 0 mV elicited relatively small inward I(Ca,L) in these myocytes, removal of external K+ or addition of 0.2 mM Ba2+ more than doubled the amplitude of the current. The basis of the enhancement of I(Ca,L) was the suppression of a large transient outward K+ current. Similar enhancement was observed when Vhp was moved to -80 mV and test depolarizations were preceded by short prepulses to -40 mV. Investigation of the time and voltage properties of the outward K+ transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+ solution, accelerated in myocytes dialyzed with 100 microM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic "time-independent" inwardly rectifying K+ current.     

Multivariate discrimination among cryptic species of the mite genus Chaetodactylus (Acari: Chaetodactylidae) associated with bees of the genus Lithurgus (Hymenoptera: Megachilidae) in North America

Twenty-seven morphological characters from 111 heteromorphic deutonymphs of the mite genus Chaetodactylus Rondani (Acari: Chaetodactylidae) were analyzed. The mites were collected from four species of bees of the genus Lithurgus Berthold (Hymenoptera: Megachilidae) in continental North America. Principal component and canonical variates analyses on Darroch and Mosimann shape and size-and-shape variables revealed the presence of three cryptic species. Chaetodactylus gibbosi sp. n. (Florida) is geographically isolated from C. lithurgi sp. n. distributed in Texas, New Mexico, Arizona, Colorado, and Idaho. Sympatric C. lithurgi and C. abditus sp. n. (USA: Arizona, Mexico: Socorro Is.) are seasonally isolated in Arizona. Chaetodactylus gibbosi is associated with a single bee species, Lithurgus gibbosus Smith in Florida. The host range of C. lithurgi includes bees flying predominantly in the spring: L. apicalis Cresson, L. littoralis Cockerell, and western L. gibbosus. Chaetodactylus abditus sp. n. is associated with L. planifrons Friese and L. echinocacti Cockerell, flying predominantly in the fall in Arizona. No distinct groups separated by geographic locality or size were detected in any species. A six-variable model developed by the canonical variates analysis and estimated using jackknife resampling and external validation (n=100) is capable of classifying the three species with 100% accuracy. Factors that influenced speciation of cryptic species of Chaetodactylus associated with Lithurgus are discussed. Based on morphological and geographical data and data on mite associates, the western and eastern populations of the bee L. gibbosus are distinct. Therefore, the taxonomic status of L. gibbosus s. lat. should be reevaluated.     

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.     

Costal cartilages--a clue for determination of sex

Mineralization and ossification in the human costal cartilages were studied radiologically. The aim of our study was to evaluate differences between males and females with respect to patterns of costal cartilage calcification and also with respect to ageing. Material for this study consists of 1044 chest and abdominal radiograms of the Czech population from the Department of Radiology (537 males and 507 females). Further radiograms of 18 chest plates were obtained at routine necropsy of cadavers. The radiograms were examined for pattern of ossification of the costal cartilage. The first rib cartilages were not considered because there are no sex differences. The lower ribs exhibit sexual dimorphism. Mineralization and ossification changes appear at the end of puberty and their occurrence increases with age. The sexual difference in pattern of human costal cartilages is statistically significant and thus highly predictive of sex determination.