The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli

The small subunit (SSU) of the ribosome of E. coli consists of a core of ribosomal RNA (rRNA) surrounded peripherally by ribosomal proteins (r-proteins). Ten of the 15 universally conserved SSU r-proteins possess nonglobular regions called extensions. The N-terminal noncanonically structured extension of S12 traverses from the solvent to intersubunit surface of the SSU and is followed by a more C-terminal globular region that is adjacent to the decoding center of the SSU. The role of the globular region in maintaining translational fidelity is well characterized, but a role for the S12 extension in SSU structure and function is unknown. We examined the effect of stepwise truncation of the extension of S12 in SSU assembly and function in vitro and in vivo. Examination of in vitro assembly in the presence of sequential N-terminal truncated variants of S12 reveals that N-terminal deletions of greater than nine amino acids exhibit decreased tRNA-binding activity and altered 16S rRNA architecture particularly in the platform of the SSU. While wild-type S12 expressed from a plasmid can rescue a genomic deletion of the essential gene for S12, rpsl; N-terminal deletions of S12 exhibit deleterious phenotypic consequences. Partial N-terminal deletions of S12 are slow growing and cold sensitive. Strains bearing these truncations as the sole copy of S12 have increased levels of free SSUs and immature 16S rRNA as compared with the wild-type S12. These differences are hallmarks of SSU biogenesis defects, indicating that the extension of S12 plays an important role in SSU assembly.     

Aerobic Degradation of N-Methyl-4-Nitroaniline (MNA) by Pseudomonas sp. Strain FK357 Isolated from Soil

N-Methyl-4-nitroaniline (MNA) is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and 1, 2, 4-benzenetriol (BT) as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH₂ substituent) reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway.     

Antagonistic and plant growth promoting activities of endophytic and soil actinomycetes

The objective of this study was the isolation and screening of actinomycete isolates for antagonistic potential and plant growth promoting activities. A total of 321 isolates were recovered from different plants, their rhizospheric soils and non-rhizospheric soils of Punjab and Himachal Pradesh regions. Out of these, 62 were endophytic, 156 were rhizospheric and 103 were non-rhizospheric isolates. In primary screening (dual culture assay), 83 isolates antagonised one or more test phytopathogenic fungi. From these active isolates, 20 were found to be antagonistic in well diffusion assay (secondary screening) and most of them demonstrated broad spectrum inhibitory activity against five to six test fungi. Studies on plant growth promoting activities revealed that 12 showed abilities to produce indole acetic acid, 10 produced siderophores and 12 showed ammonia production. Phosphate solubilisation was observed in five isolates and four fixed atmospheric N₂. In addition, production of hydrolytic enzymes such as chitinase, amylase, cellulase and protease was demonstrated by five, twenty, eleven and eleven isolates, respectively. The results of this study indicate that these isolates may be used as biocontrol and plant growth promoting agents. Morphological and chemotaxonomic studies revealed that all the active isolates belonged to the genus Streptomyces     

Metabolism of 2-Chloro-4-Nitroaniline via Novel Aerobic Degradation Pathway by Rhodococcus sp. Strain MB-P1

2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium.     

Severe Painful Vaso-Occlusive Crises and Mortality in a Contemporary Adult Sickle Cell Anemia Cohort Study

Background

Frequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort.

Methods

Two hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation.

Results

Higher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p < 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p < 0.0001) were also independent risk factors for mortality.

Conclusions

Severe painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies.

Trial Registration

ClinicalTrials.gov NCT00011648 http://clinicaltrials.gov/     

Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 µM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.     

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH₄), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH₄ were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH₄ to kill E. coli, by using PMNs from NOS2^?/? mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH₄ and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.     

Glutamine Metabolism Regulates Proliferation and Lineage Allocation in Skeletal Stem Cells

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Effect of Vanadium Supplementation on Production Performance,Nutrient Utilization,Plasma Mineral Concentration,and Mineral Balance in Lactating Goats

Biological Trace Element Research - Vanadium (V) has not been elucidated as an essential mineral in ruminants, though in lower organisms and rat model, its role is well known as insulin—a...