The Yeast Rad50 Gene Encodes a Predicted 153-Kd Protein Containing a Purine Nucleotide-Binding Domain and Two Large Heptad-Repeat Regions

The RAD50 gene of Saccharomyces cerevisiae is required for chromosome synapsis and recombination during meiosis and for repair of DNA damage during vegetative growth. The precise role of the RAD50 gene product in these processes is not known. Most rad50 mutant phenotypes can be explained by the proposal that the RAD50 gene product is involved in the search for homology between interacting DNA molecules or chromosomes, but there is no direct evidence for this model. We present here the nucleotide sequence of the RAD50 locus and an analysis of the predicted 153-kD RAD50 protein. The amino terminal region of the predicted protein contains residues suggestive of a purine nucleotide binding domain, most likely for adenine. The remaining 1170 amino acids consist of two 250 amino acid segments of heptad repeat sequence separated by 320 amino acids, plus a short hydrophobic carboxy-terminal tail. Heptad repeats occur in proteins such as myosin and intermediate filaments that form alpha-helical coiled coils. One of the two heptad regions in RAD50 shows similarity to the S-2 domain of rabbit myosin beyond that expected for two random coiled coil proteins.     

Human breast milk stimulates bile acid secretion by fetal rabbit liver in organ culture

Fetal livers from rabbits at 30 days of gestation were grown in organ culture and the effect of human milk added to the culture medium on the ability of liver to excrete bile acids (cholylglycine) was examined. Human breast milk promoted a concentration related increase in cholylglycine accumulation in the medium. The factor(s) present in milk responsible for this effect appear to be non-protein in nature and is associated with the floating lipid fraction. Furthermore, milk enhances the integrity of liver explants, as established by light microscopy.     

Abnormal bile acid pool and composition in neonates of spontaneously diabetic Wistar BB rats and its change during development

Streptozotocin-induced diabetes during pregnancy in rats causes a decrease in primary bile acid pool in neonates. To rule out direct drug effect on the fetus as the basis for this change, studies of bile acid pool and composition at birth and during subsequent development was carried out in neonates of spontaneously diabetic Wistar BB rats and compared to control neonates. The cholic acid pool in neonates of diabetic rats was lower when compared to control neonates at birth. The pool of secondary bile acids was markedly increased in neonates of diabetic rats, with increases in lithocholic and 3 beta,12 alpha-dihydroxycholanoic acid. With age, the cholic acid pool of neonates from diabetic rats was increased and at 3 months of age it was actually higher than in control neonates. The pool of chenodeoxycholic at diabetes onset age was lower in neonates of diabetic rats. HDL-cholesterol was lower in neonates of diabetic rats at 1 week, but this reversed at 3 months of age. These studies firmly establish that neonates of diabetic rats have abnormal bile acid pool and composition at birth which changes to adult diabetic pattern with age.     

Isolation and characterisation of a new mosquitocidal bacterium strain of Enterobacter cloacae VCRC-B519 from marine soil

A novel mosquitocidal bacterium was isolated from marine soil. 16S rRNA gene sequence alignment depicted that this isolate belonged to the strain, Enterobacter cloacae VCRC-B519 (NCBI: KC119193). Biochemical studies such as bacterial growth, biomass production and protein (toxin) synthesis showed that the strain is plausibly useful for mosquito control. It showed an increasing pattern of toxicity for Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti, without negative effects for non-targeted organisms Chironomus riparius, Daphnia cephalata and Notonecta glauca. The qualitative analysis of the E. cloacae showed that three polypeptides (M.wt: 25, 30 and 50 kDa) were associated to the toxicity observed. The characterisation of these polypeptides (M/S MALDI-TOF)showed that they are enzymatic in nature. Consequently, the peptide sequences are identified to be polysugar degrading enzymes (25 kDa), cell wall associated hydrolases (30 kDa) and amino peptidase (50 kDa). Phylogenetic analyses of 16S rDNA gene sequence of E. cloacae revealed the occurrence of homology with closely related Enterobacter species. Therefore, it is concluded that the marine bacterium (Enterobacter cloacae)is possibly of use for the biological control of mosquito immatures.     

Complete Genome Sequence and Pathogenicity of Two Swine Parainfluenzavirus 3 Isolates from Pigs in the United States

Two novel paramyxoviruses, 81-19252 (Texas81) and 92-7783 (ISU92), isolated from the brains of pigs in the United States in the 1980s and 1990s, were characterized. The complete genome of Texas81 virus was 15,456 nucleotides (nt) in length, that of ISU92 was 15,480 nt, and both genomes consisted of six nonoverlapping genes, predicted to encode nine proteins, with conserved and complementary 3′ leader and 5′ trailer regions and conserved gene starts, gene stops, and trinucleotide intergenic sequences similar to those in paramyxoviruses. The corresponding genes from these two viruses were similar in length, except for the F genes, of which the ISU92 form had an additional 24-nt U-rich 3′ untranslated region. The P genes of swine viruses were predicted to produce V and D mRNAs by RNA editing (one to four G insertions in Texas81 and one to nine G insertions in ISU92) or C mRNA by alternative translation initiation. Sequence-specific features related to virus replication and host-specific amino acid signatures indicated that these viruses originated from bovine parainfluenzavirus 3 (bPIV3). Phylogenetic analysis of individual genes suggested that these viruses are novel members of the genus Respirovirus of the Paramyxovirinae subfamily and may be grouped into two subgenotypes of genotype A of bPIV3. Our comprehensive studies revealed that these swine PIV3 are variants of bPIV3 and were possibly transferred from cattle to pigs but failed to establish an active enzootic state. These two viruses were mildly pathogenic to conventionally reared pigs, and results from a limited enzyme-linked immunosorbent assay-based serosurvey of swine farms in Minnesota and Iowa in 2007 and 2008 were negative.Outbreaks of infections with many novel paramyxoviruses causing catastrophic illnesses have been reported all over the world in the last few decades. A large number of diverse host species have been involved, including avian, porcine, canine, bovine, equine, ovine, human, reptilian, and aquatic species (22, 29, 40, 50, 51). Cases of cross-species transmission and pathogen jumping to humans were also reported (10, 20), demonstrating the value of characterizing new animal pathogens, even if their pathogenic potential is currently unknown. Prior to the 1990s, only La Piedad Michoacán paramyxovirus had been well studied as a neurotropic paramyxovirus isolated from pigs. Many paramyxovirus porcine pathogens have been reported since the 1950s in numerous countries, including Japan (55), Canada (18), and Israel (32), as well as the United States (25, 32). There was also a case of concurrent infection with a porcine reproductive and respiratory syndrome virus and a paramyxovirus which was subsequently named SER virus (70) in Germany in the 1990s (28). Four bat-associated paramyxoviruses were reported to cause disease in animals and humans in 1994 (72). Hendra virus and Nipah virus, which caused severe respiratory disease and death in horses and their trainer and severe febrile encephalitis and death in pigs and farmers, respectively, have been classified as members of the genus Henipavirus in the subfamily Paramyxovirinae (7, 9, 20, 30, 48). Some recently isolated viruses, such as Menangle virus (55), Tupaia paramyxovirus (69), Tioman virus (11), Mossman virus (47), J-virus (31, 33), Beilong virus (42), Mapuera virus (34), Tursiops truncatus parainfluenzavirus 1 (PIV1), isolated from bottlenose dolphins (50), and Atlantic salmon paramyxovirus (51), remain unclassified below the subfamily level. All members of the subfamily Paramyxovirinae have six genes in the following order: 3′-N-P-M-F-A-L-5′, where N, P, M, F, A, and L indicate the genes for the nucleocapsid protein, the phosphoprotein, and the matrix, fusion, attachment, and large polymerase proteins, respectively (40).Recently, we reported the antigenic and molecular characterization of glycoprotein genes from two novel swine PIV3 (sPIV3) isolates from the brains of pigs that experienced respiratory and central nervous system disease (57). These two sPIV3 strains were antigenically and genetically very closely related to bovine PIV3 (bPIV3) in the genus Respirovirus (57). However, the pathogenicity of these sPIV3 strains in conventionally reared pigs and the complete genome sequences of these isolates are presently unknown.In bovines, bPIV3 infection results in asymptomatic to severe respiratory disease, but no neurological disease has been reported (16). Limited sequence polymorphism among the bPIV3 strains was detected previously (12, 66). Recently, after an analysis of Australian isolates of bPIV3, two distinct genotypes of bPIV3, A and B, were proposed (29). In this study, we have performed a complete genome sequence analysis of these swine isolates and determined their pathogenicity in conventionally reared pigs. Our analysis indicated that there are two distinct genetic groupings discernible within genotype A, represented by bPIV3 shipping fever strain (bPIV3-SF)-like and bPIV3 strain 910N (bPIV3-910N)-like viruses, with one swine isolate in each of these groups. Several amino acid residues that may reflect the minor population variations in the new host due to cross-species infection were identified. But both swine viruses induced a very mild respiratory illness without any neurological signs in young piglets, suggesting that coinfection with other infectious agents or the presence of other environmental factors may be required to precipitate clinical disease.     

Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic. Here, we present evidence that genetically modified, recombinant NDV strains are cytotoxic to human tumor cell lines of ecto-, endo-, and mesodermal origin. We show that cytotoxicity against tumor cells is due to multiple caspase-dependent pathways of apoptosis independent of interferon signaling competence. The signaling pathways of NDV-induced, cancer cell-selective apoptosis are not well understood. We demonstrate that NDV triggers apoptosis by activating the mitochondrial/intrinsic pathway and that it acts independently of the death receptor/extrinsic pathway. Caspase-8-methylated SH-SY5Y neuroblastoma cells are as sensitive to NDV as other caspase-8-competent cells. This demonstrates that NDV is likely to act primarily through the mitochondrial death pathway. NDV infection results in the loss of mitochondrial membrane potential and the subsequent release of the mitochondrial protein cytochrome c, but the second mitochondrion-derived activator of caspase (Smac/DIABLO) is not released. In addition, we describe early activation of caspase-9 and caspase-3. In contrast, cleavage of caspase-8, which is predominantly activated by the death receptor pathway, is a TNF-related, apoptosis-inducing ligand (TRAIL)-induced late event in NDV-mediated apoptosis of tumor cells. Our data, therefore, indicate that the death signal(s) generated by NDV in tumor cells ultimately converges at the mitochondria and that it acts independently of the death receptor pathway. Our cytotoxicity studies demonstrate that recombinant NDV could be developed as a cancer virotherapy agent, either alone or in combination with therapeutic transgenes. We have also shown that trackable oncolytic NDV could be developed without any reduction in oncolytic efficacy.