Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.     

Delineating the conformational dynamics of intermediate structures on the unfolding pathway of β-lactoglobulin in aqueous urea and dimethyl sulfoxide

Abstract

The funnel shaped energy landscape model of the protein folding suggests that progression of folding proceeds through multiple pathways, having the multiple intermediates which leads to multidimensional free-energy surface. Herein, we applied all-atom MD simulation to conduct a comparative study on the structure of β-lactoglobulin (β-LgA) in aqueous mixture of 8?M urea and 8?M dimethyl sulfoxide (DMSO), at different temperatures. The cumulative results of multiple simulations suggest a common unfolding pathway of β-LgA, occurred through the stable and meta-stable intermediates (I), in both urea and DMSO. However, the free-energy landscape (FEL) analyses show that the structural transitions of I-states are energetically different. In urea, FEL shows distinct ensemble of intermediates, I1 and I2, separated by the energy barrier of ～3.0?kcal mol^?1. Similarly, we find the population of two distinct I1 and I2 states in DMSO, however, the I1 appeared transiently around ～30–35?ns and is short-lived. But, the I2 ensemble is observed structurally compact and long-lived (～50–150?ns) as compared to unfolding in urea. Furthermore, the I1 and I2 are separated through a high energy barrier of ～6.0?kcal mol^?1. Thus, our results provide the structural insights of intermediates which essentially bear the signature of a different unfolding pathway of β-LgA in urea and DMSO.

Abbreviations β-LgA β-lactoglobulin

DMSO dimethyl sulfoxide

FEL free-energy landscape

GdmCl guanidinium chloride

I intermediate state

MG molten globule state

PME particle mesh Ewald

Q fraction of native contacts

RMSD root mean square deviation

RMSF root mean square fluctuation

R_g radius of gyration

SASA solvent Accessible Surface Area

scSASA the side chain SASA

Trp tryptophan

Communicated by Ramaswamy H. Sarma     

Nitrogen fertiliser and establishment method affect growth,yield and nitrogen use efficiency of rice under alternate wetting and drying irrigation

Sustainable rice production through selection of best suitable cultivar, water-efficient crop establishment method and optimum nitrogen (N) management practice is needed to feed the growing world population. Two polyhouse experiments were conducted to evaluate the response of rice to different rates and schedules of N application under different establishment methods subjected to alternate wetting and drying (AWD) irrigation. In the first experiment, the response of two rice cultivars (Pathumthani 1, RD57) under three establishment methods (transplanting [TP], wet direct seeding [WDS], dry direct seeding [DDS]) and five N rates (0 [N₀], 30 [N₃₀], 60 [N₆₀], 90 [N₉₀], 120 [N₁₂₀] kg ha⁻¹) was evaluated. The second experiment consisted of the same cultivars and establishment methods, but with four N application schedules (T₁: 100% at basal; T₂: 75% + 25% at basal and at active tillering, respectively; T₃: 50% + 25% + 25% at basal, at active tillering and at panicle initiation, respectively; and T₄: 25% + 25% + 25% + 25% each at basal, at active tillering, at panicle initiation and at early flowering or just before heading starts) applied at the rate of N₆₀ kg ha⁻¹. Plants were maintained under AWD irrigation (soil was saturated by applying water whenever soil water potential drops to −5 kPa during the implementation period) in both experiments. RD57 performed better than Pathumthani 1 having higher shoot dry matter, panicle number, grain yield, total N uptake and apparent N recovery efficiency. TP gave better response than WDS and DDS regardless of cultivars. Application of N₁₂₀ resulted in better growth, yield and its components and total N uptake regardless of establishment methods and cultivars. Increasing N rate decreased N use efficiency (NUE). Scheduling the interval of N application to two or three times (T₂ or T₃) for RD57 and Pathumthani 1, respectively, provided overall better results, and could be recommended for the tested rice cultivars. An optimal N rate and selection of critical growth stages for N application would be very effective for maximising yield and NUE under the water-saving cultivation technique of AWD irrigation.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Effects of X-rays and Colchicine on Pollen of Trichosanthes anguina L. (Cucurbitaceae)

Pollen grains of M, and C, generation plants of Trichosanthes anguina L. were studied after treatment of seeds with X-rays and colchicine respectively. Pollen sterility increased with increase in X-ray dose and colchicine concentration. The polar axis of X-irradiated populations was shorter than that of controls. But colchicine treatment resulted in larger pollen grains than the controls, although the exine did not increase as much. Ex-perimentally-produced tetraploids had larger pollen grains than diploids and triploids. The triploids were characterised by changes in pore morphology. Of 293 treated plants examined, 11 aberrant plants were isolated in the M, and C, generations. These plants had large and small fertile pollen grains, and also showed treatment effects in external morphology and chromosomal aberration during meiosis. The changes in polar diameter were not associated with variation in chromosomal material.     

Non-monotonic Response to Monotonic Stimulus: Regulation of Glyoxylate Shunt Gene-Expression Dynamics in Mycobacterium tuberculosis

Understanding how dynamical responses of biological networks are constrained by underlying network topology is one of the fundamental goals of systems biology. Here we employ monotone systems theory to formulate a theorem stating necessary conditions for non-monotonic time-response of a biochemical network to a monotonic stimulus. We apply this theorem to analyze the non-monotonic dynamics of the σ^B-regulated glyoxylate shunt gene expression in Mycobacterium tuberculosis cells exposed to hypoxia. We first demonstrate that the known network structure is inconsistent with observed dynamics. To resolve this inconsistency we employ the formulated theorem, modeling simulations and optimization along with follow-up dynamic experimental measurements. We show a requirement for post-translational modulation of σ^B activity in order to reconcile the network dynamics with its topology. The results of this analysis make testable experimental predictions and demonstrate wider applicability of the developed methodology to a wide class of biological systems.     

Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks

In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm) isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra) analysis plots standardized (z-score) tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r²). This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV) serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993–1994 and a single case from 2011 as well as links between two isolates from over 30 years ago. The identification of these potential links shows that JSpecies Tetra analysis can be a useful tool in rapidly assessing genetic relatedness of Lm isolates during outbreak investigations and for comparing historical isolates. Our analyses led to the identification of a highly related clonal group involved in two separate outbreaks, stone fruit and caramel apple, and suggests the possibility of a new genotype that may be better adapted for certain foods and/or environment.