Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the -isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC- is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC- (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC- showed monolayer injury as indicated by decreased native PKC- activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC- was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC- was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-/tubulin complex. Second, DN transfection to inhibit the endogenous PKC- led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC- led to a mostly cytosolic distribution of -isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC- isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC- involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-. microtubules; tubulin; occludin; epithelial barrier permeability; protein kinase C isoform     

Transition metal acetylsalicylates and their anti-inflammatory activity

Mononuclear and binuclear transition metal [Co(II), Cu(II), Ni(II) and Zn(II)] acetylsalicylates of the type [M(L)2], [M(L)2Cl2] and [(M)2(L)4] have been prepared and characterized on the basis of their physical, spectral and analytical data. The complexes have been investigated in an in vivo animal model for anti-inflammatory activity and show a better effect and a more potent action than acetylsalicylic acid.     

The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data

A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.     

An evaluation of the assembly of an approximately 15-Mb region on human chromosome 13q32-q33 linked to bipolar disorder and schizophrenia

The human 13q32-q33 region has been linked to both bipolar disorder and schizophrenia. Before completion of the draft sequences, we developed an approximately 15-Mb comprehensive map for the region extending from D13S1300 to ATA35H12. This map was assembled using publicly available mapping data and sequence-tagged site (STS)-based PCR confirmation. We then compared this map with the NCBI, Celera Genomics, and UCSC Golden Path data in February, June, and September 2001. All data sets showed gaps, misassignment of STSs, and errors in orientation and marker order. Surprisingly, the completed sequences of many bacterial artificial chromosomes (BACs) had been truncated. Of 21 gaps that were detected, 4 were present in both the NCBI and Celera databases. All gaps could be filled using 1-2 BAC clones. A total of 39 loci mapped to additional sites within the human genome, providing evidence of segmental duplications. Additionally, 61 unique cDNA clones were sequenced to increase available transcribed sequence, and 11,353 reference single-nucleotide polymorphisms (SNPs) with an average density of 1 SNP/3720 bases were identified. Overall, integration of the data from multiple sources is still needed for complete assembly of the 13q32-q33 region. (c)     

Trans complementation of variant Cre proteins for defects in cleavage and synapsis

The Cre recombinase is a member of the integrase family of conservative site-specific recombinases. These proteins share five conserved catalytic residues, one of which is a tyrosine that acts as the nucleophile to attack the scissile phosphodiester bond in the DNA target. Recombination by the Cre recombinase takes place in a supramolecular structure called a synapse that consists of four molecules of Cre bound to two DNA target sequences called lox sites. The synapse is held together by an intricate network of protein-protein interactions. They bend the two sites into square planar structure that resembles a Holliday intermediate. We have studied three mutant Cre proteins that appear to have defects in synapsis (Cre A36V, Cre T41F, and Cre G314R). We found that they were unable to carry out strand cleavage but that cleavage occurred if they were mixed with a cleavage-defective Cre protein that lacks the catalytic nucleophilic tyrosine residue. The three variant proteins could also be complemented for the formation of a novel structure ("complexV"), which may be a cleaved synaptic intermediate. We suggest that these three mutant proteins have a defect in DNA bending and discuss the relationship between bending, synapsis, and cleavage.     

Tightly clustered 11q23 and 22q11 breakpoints permit PCR-based detection of the recurrent constitutional t(11;22)

Palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11 at the constitutional t(11;22) breakpoint are predicted to induce genomic instability, which mediates the translocation. A PCR-based translocation-detection system for the t(11;22) has been developed with PCR primers flanking the PATRRs of both chromosomes, to examine the involvement of the PATRRs in the recurrent rearrangement. Forty unrelated carriers of the t(11;22) balanced translocation, plus two additional, independent cases with the supernumerary-der(22) syndrome, were analyzed to compare their translocation breakpoints. Similar translocation-specific junction fragments were obtained from both derivative chromosomes in all 40 carriers of the t(11;22) balanced translocation and from the der(22) in both of the offspring with unbalanced supernumerary-der(22) syndrome, suggesting that the breakpoints in all cases localize within these PATRRs and that the translocation is generated by a similar mechanism. This PCR strategy provides a convenient technique for rapid diagnosis of the translocation, indicating its utility for prenatal and preimplantation diagnosis in families including carriers of the balanced translocation.     

Are developing countries prepared to face Ebola-like outbreaks?

<正>Ebola virus disease has caused havoc in West Africa,with 11,162deaths and more than 27,181 cases(as of May 31,2015)being reported since the virus emerged in early2014 in Guinea.The maximum number of cases has been reported in Sierra Leone(12,827),while most of the reported deaths have occurred in Liberia(4,806),according to the Center for Disease Control and     

Initial Data on the Molecular Epidemiology of Cryptosporidiosis in Lebanon

Cryptosporidium spp. represent a major public health problem worldwide and infect the gastrointestinal tract of both immunocompetent and immunocompromised persons. The prevalence of these parasites varies by geographic region, and no data are currently available in Lebanon. To promote an understanding of the epidemiology of cryptosporidiosisin this country, the main aim of this study was to determine the prevalence Cryptosporidium in symptomatic hospitalized patients, and to analyze the genetic diversity of the corresponding isolates. Fecal specimens were collected in four hospitals in North Lebanon from 163 patients (77 males and 86 females, ranging in age from 1 to 88 years, with a mean age of 22 years) presenting gastrointestinal disorders during the period July to December 2013. The overall prevalence of Cryptosporidium spp. infection obtained by modified Ziehl-Neelsen staining and/or nested PCR was 11%, and children <5 years old showed a higher rate of Cryptosporidium spp. The PCR products of the 15 positive samples were successfully sequenced. Among them, 10 isolates (66.7%) were identified as C. hominis, while the remaining 5 (33.3%) were identified as C. parvum. After analysis of the gp60 locus, C. hominis IdA19, a rare subtype, was found to be predominant. Two C. parvum subtypes were found: IIaA15G1R1 and IIaA15G2R1. The molecular characterization of Cryptosporidium isolates is an important step in improving our understanding of the epidemiology and transmission of the infection.