Development of a CrN/Cu nanocomposite coating on titanium-modified stainless steel for antibacterial activity against Pseudomonas aeruginosa

A relatively simple method was developed to fabricate CrN/Cu nanocomposite coatings using pulsed DC magnetron sputtering for application in antibacterial activity. These nanocomposite coatings were applied on titanium (Ti)-modified stainless steel substrata (D-9 alloy) and the antibacterial activity of these coating with respect to the Gram-negative bacterium Pseudomonas aeruginosa was investigated qualitatively and quantitatively. Scanning electron microscopy, epifluorescence microscope analyses, and total viable counts confirmed that inclusion of copper in the CrN/Cu nanocomposite coatings provided antibacterial activity against P. aeruginosa. The quantitative examination of the bacterial activity of P. aeruginosa was estimated by the survival ratio as calculated from the number of viable cells which formed colonies on nutrient agar plates.     

Isolation of proline-based cyclic dipeptides from Bacillus sp. N strain associated with rhabitid entomopathogenic nematode and its antimicrobial properties

Entomopathogenic nematodes (EPN) are well-known as biological control agents and are found to have associated bacteria which can produce a wide range of bioactive secondary metabolites. We report herewith isolation of six proline containing cyclic dipeptides cyclo(d-Pro-l-Leu), cyclo(l-Pro-l-Met), cyclo(d-Pro-l-Phe), cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Tyr) and cyclo(l-Pro-d-Tyr) from ethyl acetate extract of the Luria Broth (LB) cell free culture filtrate of Bacillus sp. strain N associated with a new EPN Rhabditis sp. from sweet potato weevil grubs collected from Central Tuber Crops Research Institute farm. Antimicrobial studies of these 2,5-diketopiperazines (DKPs) against both medicinally and agriculturally important bacterium and fungi showed potent inhibitory values in the range of μg/mL. Cyclic dipeptides showed significantly higher activity than the commercial fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani, and Pencillium expansum. The highest activity of 2 μg/mL by cyclo(l-Pro-l-Phe) was recorded against P. expansum, a plant pathogen responsible for causing post harvest decay of stored apples and oranges. To our knowledge, this is the first report on the isolation of these DKPs from Rhabditis EPN bacterial strain Bacillus sp.     

Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G₀/G₁ cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G₀/G₁ cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.     

Endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein.

On the assumption that dephosphorylation of the neurotropic coronavirus JHM (JHMV) nucleocapsid protein (N) may be connected with initiation of the infectious cycle we searched for a relevant host enzyme activity. Analysis of subcellular fractions from L-2 murine fibroblasts, separated by dual Percoll density gradients, revealed the presence of a phosphoprotein phosphatase (PPPase), co-sedimenting with the endososomal/prelysosomal material, which possesses high activity against N. With purified [32P]N as substrate it was demonstrated that this PPPase, distinguishable from acid and alkaline phosphatases, acts optimally at neutral pH in the presence of Mn2+ following treatment with a detergent. Complete inhibition with okadaic acid at 0.9-4.5 microM but not at 1-10 nM relegates this PPPase to a type 1 protein phosphatase. Similar PPPase activity for N was present in the endosome fraction of a rat Roc-1 astrocytoma-oligodendrocyte cell line and in homogenates of brain and cultured oligodendrocytes. Our data suggest that the phosphorylated N of the inoculum may be modified by the endosomal PPPase in host cells, including those from the CNS so as to facilitate the JHMV infectious process.     

Nitrogen fixation in tomato (Lycopersicon esculentum Mill ‘Pusa Ruby’)

Nitrogen fixation (acetylene reduction) was found in intact tomato (Lycopersicon esculentum Mill ‘Pusa Ruby’) plants in the field, in pots and also in aseptic cultures. The unsterilized as well as sterilized rhizoplane and phylloplane of the plant when assayed separately also responded to the test. From root bits of tomato sterilized upto 20 minutes with 0.1% mercuric chloride, growth of the bacterium from the interior of the root into the medium was observed thereby indicating their presence within the endorhizosphere. Phase contrast and electron microscopic studies of the root system of tomato revealed the presence of bacterial colonies in the epidermis, cortex and vascular bundles. Bacterial numbers in the endosphere, of root and leaf were 30×10⁴ and 12×10⁴, respectively, per gram fresh weight of tissue. The bacteria were predominantly rod-shaped 1.4–4.8×0.9–1.95 μm in 24-h-old cultures, pleomorphic, polar or bipolary flagellated having β-hydroxy butyrate granules. The bacterium has been identified as a new species of Azospirillum.     

Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.     

Chromosome mapping of the murine syndecan gene.

The chromosomal localization of the murine syndecan gene was determined by analysis of DNA from a panel of mouse-hamster cell hybrids containing various mouse chromosomes, detection of immunoreactive syndecan in culture medium of these cells, and linkage analysis of a mouse interspecific backcross. Southern analysis of the mouse-hamster cell hybrid DNA shows two distinct hybridizing sequences, one on mouse Chromosome 12 and the other on the X chromosome. Localization of the syndecan gene to mouse Chromosome 12 was determined by detection of immunoreactive syndecan in the culture medium of cell hybrids containing mouse Chromosome 12. Hybrids containing other mouse chromosomes were negative. Linkage analysis by Southern hybridization of DNA from a mouse interspecific backcross using a syndecan-specific probe localized the syndecan gene locus, Synd, to the proximal end of Chromosome 12, tightly linked to the Pomc-1 and Nmyc loci. The syndecan gene is likely on human Chromosome 2 because this region shows conservation of synteny between mouse and human chromosomes.     

Assignment of a serotonin 5HT-2 receptor gene (HTR2) to human chromosome 13q14-q21 and mouse chromosome 14

A gene for serotonin 5HT-2 receptor (HTR2) is assigned to human chromosome 13 by somatic cell hybrids and to region 13q14-q21 by in situ hybridization. It is assigned to mouse chromosome 14 by somatic cell hybrid analysis.     

Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branchpoints for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism by which alternative promoters can be coordinated with downstream alternative splicing.