Identification of elements in human long 3′ UTRs that inhibit nonsense-mediated decay

The nonsense-mediated mRNA decay (NMD) pathway serves an important role in gene expression by targeting aberrant mRNAs that have acquired premature termination codons (PTCs) as well as a subset of normally processed endogenous mRNAs. One determinant for the targeting of mRNAs by NMD is the occurrence of translation termination distal to the poly(A) tail. Yet, a large subset of naturally occurring mRNAs contain long 3′ UTRs, many of which, according to global studies, are insensitive to NMD. This raises the possibility that such mRNAs have evolved mechanisms for NMD evasion. Here, we analyzed a set of human long 3′ UTR mRNAs and found that many are indeed resistant to NMD. By dissecting the 3′ UTR of one such mRNA, TRAM1 mRNA, we identified a cis element located within the first 200 nt that inhibits NMD when positioned in downstream proximity of the translation termination codon and is sufficient for repressing NMD of a heterologous reporter mRNA. Investigation of other NMD-evading long 3′ UTR mRNAs revealed a subset that, similar to TRAM1 mRNA, contains NMD-inhibiting cis elements in the first 200 nt. A smaller subset of long 3′ UTR mRNAs evades NMD by a different mechanism that appears to be independent of a termination-proximal cis element. Our study suggests that different mechanisms have evolved to ensure NMD evasion of human mRNAs with long 3′ UTRs.     

The G protein-coupled 5-HT1A receptor causes suppression of caspase-3 through MAPK and protein kinase Calpha

Several studies have analysed aromatic interactions, involving mostly phenylalanine, tyrosine and tryptophan. Only a few studies have considered histidine as an interacting aromatic residue. An extensive analysis of aromatic His-X interactions is performed here on a data set of 593 PDB structures: 68% of the histidine are involved in aromatic pairs and 1271 non-redundant His-X pairs were analysed. Thirty percent of these pairs involve an aromatic partner less than 6 apart in the sequence. These near-sequence pairs correspond to conformations which stabilise secondary structures, mainly alpha-helices when the residues are 4 apart and beta-strands when they are 2 apart in the sequence. The partners of the other His-X pairs (887, 70%) are more than 5 apart in the sequence. Of these far-sequence pairs, 35% bridge beta strands and only 9% helices. The near-sequence pairs are sterically constrained as supported by conformer distribution. The X partners of far-sequence His-X pairs are mainly "above" the histidine ring with tilted and normal rings, corresponding to a "T shape; face to edge" orientation. Phenylalanine, the only aromatic residue with no heteroatom, is a disfavoured partner, whereas histidine is the preferred one. Heteroatom-heteroatom interactions are favoured in near-sequence as well as in far-sequence His-His, His-Trp and His-Tyr pairs.     

Molecular characterization of Vibrio harveyi bacteriophages isolated from aquaculture environments along the coast of India

Seven bacteriophages specific to Vibrio harveyi, the causative agent of luminous vibriosis in shrimp, were isolated from coastal aquaculture systems like shrimp farms, hatcheries and tidal creeks along the east and west coast of India. All the seven phages were found to have the typical head and tail morphology with double-stranded DNA as genetic material. Morphologically, six phages were grouped under family Siphoviridae and one under Myoviridae. These phages were further characterized with respect to host range, morphology and structural proteins. Genomic fingerprinting was carried out using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD). Major capsid proteins of all the phages detected by SDS-PAGE were distinct from one another. All the phages were found to be highly lytic for V. harveyi and had different lytic spectrum for the large number of isolates tested. Six of the seven phages isolated had a broad lytic spectrum and could be potential candidates for biocontrol of V. harveyi in aquaculture systems.     

Hormonal regulation of sialic acid-binding (SAS) protein synthesis of rat uterus

Sialic acid binding proteins (SAS) of rat uteri have been found in all three stages of the estrous cycle. To study the control of synthesis of these proteins two different animal models were used I-immature female rats (25 d) where the hormones estradiol (E₂) and progesterone (P₄) were given separately and together, and II-adult female rats where hormone treatment commenced 14 days after ovariectomy. The data indicated that E₂ initiated the synthesis of SAS proteins in the immature animals, while P₄ could inhibit SAS synthesis, either given alone or together with E₂. However, prior priming of the rat with E₂ and subsequent administration of P₄ stimulated SAS protein synthesis.     

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small ''bag'' called the spermatheca. Later on, hermaphrodites continually produce oocytes¹. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for >50% of the total cells in a typical adult worm². Therefore, isolating sperm from males is easier than from that of hermaphrodites.Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome³. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population³.Males can be easily distinguished from hermaphrodites by observing the tail morphology⁴. Hermaphrodite''s tail is pointed, whereas male tail is rounded with mating structures.Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids².Males are directly dissected on a small drop of ''Sperm Medium''. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes⁵, and Nelson⁶ previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process⁷. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.Download video file.^{(33M, flv)}     

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Aims:  The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp.
Methods and Results:  The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli . Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila . Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations.
Conclusions:  The ompW -based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish.
Significance and Impact of the Study:  To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila . Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas . Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.     

Synthesis of anticancer beta-lactams: mechanism of action

Synthesis of the trans 1-N-chrysenyl and 1-N-phenanthrenyl 3-acetoxy-4-phenyl-2-azetidinones has been achieved. Microwave-assisted reaction has proved useful in the synthesis of these compounds. Cell growth inhibition study has indicated selective anticancer activity against two leukemia and colon carcinoma cell lines. A mechanistic correlation of their anticancer activity has been described. Striking G2 blockade that is clearly distinct in cell cycle analysis and demonstrated only in sensitive cell lines has been observed. They do not induce apoptosis in sensitive or resistant lines. They also do not inhibit topoisomerases. Ames test has shown they are nonmutagenic.