Isolation, structure, and antibacterial activity of thiazomycin A, a potent thiazolyl peptide antibiotic from Amycolatopsis fastidiosa

Thiazolyl peptides are a class of thiazole-rich macrocyclic potent antibacterial agents. Recently, we described thiazomycin, a new member of thiazolyl peptides, discovered by a thiazolyl peptide specific chemical screening. This method also allowed for the discovery of a new thiazolyl peptide, thiazomycin A, which carries modification in the oxazolidine ring of the amino sugar moiety. Thiazomycin A is a specific inhibitor of protein synthesis (IC(50) 0.7 microg/mL) and a potent Gram-positive antibacterial agent with minimum inhibitory concentration (MIC) ranging 0.002-0.25 microg/mL. The isolation and structure elucidation and biological activities of thiazomycin A are described.     

Protein-template-directed synthesis across an acrolein-derived DNA adduct by yeast Rev1 DNA polymerase

Acrolein is generated as the end product of lipid peroxidation and is also a ubiquitous environmental pollutant. Its reaction with the N2 of guanine leads to a cyclic gamma-HOPdG adduct that presents a block to normal replication. We show here that yeast Rev1 incorporates the correct nucleotide C opposite a permanently ring-closed form of gamma-HOPdG (PdG) with nearly the same efficiency as opposite an undamaged G. The structural basis of this action lies in the eviction of the PdG adduct from the Rev1 active site, and the pairing of incoming dCTP with a "surrogate" arginine residue. We also show that yeast Polzeta can carry out the subsequent extension reaction. Together, our studies reveal how the exocyclic PdG adduct is accommodated in a DNA polymerase active site, and they show that the combined action of Rev1 and Polzeta provides for accurate and efficient synthesis through this potentially carcinogenic DNA lesion.     

Ranking of binding and nonbinding peptides to MHC class I molecules using inverse folding approach: Implications for vaccine design

T cell recognition of the peptide–MHC complex initiates a cascade of immunological events necessary for immune responses. Accurate T-cell epitope prediction is an important part of the vaccine designing. Development of predictive algorithms based on sequence profile requires a very large number of experimental binding peptide data to major histocompatibility complex (MHC) molecules. Here we used inverse folding approach to study the peptide specificity of MHC Class-I molecule with the aim of obtaining a better differentiation between binding and nonbinding sequence. Overlapping peptides, spanning the entire protein sequence, are threaded through the backbone coordinates of a known peptide fold in the MHC groove, and their interaction energies are evaluated using statistical pairwise contact potentials. We used the Miyazawa & Jernigan and Betancourt & Thirumalai tables for pairwise contact potentials, and two distance criteria (Nearest atom ≫ 4.0 Å & C-beta ≫ 7.0 Å) for ranking the peptides in an ascending order according to their energy values, and in most cases, known antigenic peptides are highly ranked. The predictions from threading improved when used multiple templates and average scoring scheme. In general, when structural information about a protein-peptide complex is available, the current application of the threading approach can be used to screen a large library of peptides for selection of the best binders to the target protein. The proposed scheme may significantly reduce the number of peptides to be tested in wet laboratory for epitope based vaccine design.     

Microbial Diversity in Ngari,Hentak and Tungtap,Fermented Fish Products of North-East India

Ngari, hentak and tungtap are traditional fermented fish products of North-East India. Eighteen samples of ngari, hentak and tungtap were collected and were analysed for microbial load. Lactic acid bacteria, endospore-forming rods, yeasts and aerobic mesophilic counts ranged from 4.0 to 7.2, 3.3–4.6, <1–3.5 and 4.3–7.3 log c.f.u./g, respectively. Lactic acid bacteria were identified as Lactococcus lactis subsp. cremoris, Lactococcus plantarum, Enterococcus faecium, Lactobacillus fructosus, Lactobacillus amylophilus, Lactobacillus coryniformis subsp. torquens and Lactobacillus plantarum. Endospore-forming rods were identified as Bacillus subtilis and Bacillus pumilus, aerobic coccal strains were identified as Micrococcus. Yeasts were identified as species of Candida and Saccharomycopsis. Pathogenic contaminants were detected in all samples, however, none of the sample contained more than 10² c.f.u./g of Bacillus cereus, 10³ c.f.u./g of Staphylococcus aureus and enterobacteriaceae population, respectively. Enzymatic and antimicrobial activities of the isolates were tested. None of the strains produced biogenic amines in the method applied. Most strains of LAB had a high degree of hydrophobicity, indicating their ‘probiotic’ characters. This study has demonstrated the microbial diversity within the species of lactic acid bacteria, Bacillus and yeasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Study of NADP+- linked Isocitrate Dehydrogenase from Germinating Mung Bean (Vigna radiata)

NADP⁺- linked isocitrate dehydrogenase has been purified to apparent homogeneity from 36 h germinated mung beans by ammonium sulphate fractionation, heat treatment, acid treatment, and DEAE — Cellulose column chromatography. The enzyme was purified to 150 fold with 15% recovery. The preparation showed single protein band on native PAGE and was free from bound nucleotides and coloured pigments (A₂₈₀/A₂₆₀ = 1.4). The molecular weight was found to be 141,000 and was made of four identical subunits (mol wt 36,000). Thermal inactivation at 50, 53, and 55 °C revealed simple first order kinetics and t_1/2 was found to be 38, 10, and 3 min, respectively. The enzyme exhibited absolute specificity for NADP⁺ and substrate. The K_m for isocitrate and NADP⁺ was 28.57 µM and 70 µM, respectively. The enzyme appeared to be regulated by various metabolites of Krebs’ cycle intermediates.     

Chromium accumulation and toxicity in aquatic vascular plants

Chromium poisoning among leather tanners has long been known. The workers have been found to suffer from ulcers, allergic dermatitis, lung cancer, and liver necrosis due to prolonged contact with chromium salts. One of the highly catastrophic incidences of lung cancer as a result of inhaling dust containing Cr (VI) was reported in 1960 from the Kiryama factory of the Nippon-Denko concern on the island of Hokkaido, Japan. Pollution of water resources, both surface and underground, by indiscriminate discharge of spent wastes of chromium-based industries has become a serious global concern, for it has created an acute scarcity of safe drinking water in many countries. In August 1975 it was observed that underground drinking water in Tokyo near the chromium (VI))-containing spoil heaps contained more than 2000 times the permissible limit of chromium. In Ludhiana and Chennai, India, chromium levels in underground water have been recorded at more than 12 mg/L and 550–1500 ppm/L, respectively. Chromium is widely distributed in nature, occupying 21st position in the index of most commonly occurring elements in the earth’s crust. Chromium occurs in nature in the form of a compound (chromium + oxygen + iron) known as “chromite.” The geographical distribution of chromite mines is uneven. Over 95% of economically viable chromite ores are situated in the southern part of Africa. Its annual global production is ca. 9 million tons, mainly mined in the former Soviet Union, Albania, and Africa. In India, over 90% of chromite deposits are located in Sukinda Valley of Orissa. Chromium occurs in several oxidation states, ranging from Cr²⁺ to Cr⁶⁺, with trivalent and hexavalent states being the most stable and common in the terrestrial environment. Chromium (III) is used for leather tanning because it forms stable complexes with amino groups in organic material. In the presence of excessive oxygen, chromium (III) oxidizes into Cr (VI), which is highly toxic and more soluble in water than are other forms. Chromium (VI) can easily cross the cell membrane, whereas the phosphate-sulphate carrier also transports the chromite anions. On the other hand, Cr (III) does not utilize any specific membrane carrier and hence enters into the cell through simple diffusion. The diffusion is possible only after the formation of appropriate lipophilic ligands. Use of chromium as industrial material was discovered only 100 years ago. It was used for the first time in the production of corrosion-resistant steel (stainless steel) and coatings. Subsequently, chromium was widely deployed in various industries; namely, electroplating, dyes and pigments, textiles, photography, and wood processing. The tanning industry is one of the major users of chromium (III) salts. During leather processing the conversion of putrefactive proteinaceous matter, skin, into non-putricible is carried out by the treatment of chromium sulphate solution. According to an estimate, ca. 32 tons of chromium sulphate salts are used annually in Indian tanneries. As a result of unplanned disposal of spent tannery wastes, ca. 2000–3200 tons of chromium as element escapes into the environment. This has raised severe ecological concern and reduced the forest cover considerably. Aquatic vascular plants play an important role in the uptake, storage, and recycling of metals. The uptake of metals depends on the chemical form present in the system and on the life form of the macrophytes (floating, free floating, well rooted, or rootless). The free-floating species (Eichhornia, Lemna, Pistia) absorb elements through the roots/leaves, whereas the rootless speciesCeratophyllum demersum absorbs mainly through the finally divided leaves. Submerged species showed higher chromium accumulation than do floating and emergent ones. The order is:Elodea canadensis > Lagarosiphon major > Potamogeton crispes > Trapa natans > Phragmitis communis. Roots of water hyacinth (Eichhornia crassipes) showed an accumulation of 18.92 μmol (g dry tissue wt^-1) Cr. AlthoughCeratophyllum demersum andHydrodictyon reticulatum showed lower levels of chromium accumulation, their bioconcentration factor values were very high. Floating-species duckweeds (Lemna, Spirodela) are potential accumulators of heavy metals. They have bioconcentrated Fe and Cu, as high as 78 times their concentration in wastewater. Duckweeds have also shown the ability to accumulate chromium substantially. Although duckweeds attain higher concentrations of chromium in their tissues than do other macrophytes, their bioconcentration factor (BCF) values were much lower than those reported in other aquatic species. A moderate accumulation of chromium has been found in emergent species. Plants ofScirpus validatus andCyperus esculentus accumulated 0.55 kg and 0.73 kg^-1 Cr, respectively. InBacopa monnieri andScirpus lacustris accumulations of 1600 and 739 μg g^-1 dw Cr, respectively, have been reported when exposed to 5 mg L^-1 Cr for 168 hours in solution culture. The accumulation of Cr was greater in the root than the shoot. Higher accumulations of chromium in roots and least in shoots of emergent species have also been recorded. Phytotoxicity of chromium in aquatic environment has not been studied in detail. The mechanism of injury in terms of ultrastructural organization, biochemical changes, and metabolic regulations has not been elucidated. It has been pointed out that while considering the toxicity of heavy metals, a distinction should be made between elements essential to plants and metals that have no proven beneficial biochemical effects. For example, an increased level of chromium may actually stimulate growth without being essential for any metabolic process. In aquatic species—namely,Myriophyllum spicatum— the maximum increase in shoot length was found at 50 μgl^-1 Cr. Higher concentrations up to 1000 μ gl^-1 caused an almost linear reduction both in shoot weight and length. Duckweeds showed relatively greater tolerance to chromium. However, an inhibition of growth inSpirodela andLemna was found at 0.02 mM and 0.00002 mM Cr concentrations, respectively. Mortality ofL. aequinoctialis was found at 0.005 mM Cr and higher concentrations. The effective chromium concentrations (EC-50) for some aquatic species have been reported as follows:Lemna minor, 5.0 mg L^-1, 14 days EC;L. Paucicostata, 1.0 mg L-¹, 20 days EC;Myriophyllum spicatum, 1.9 mg L^-1, 32 days EC; andSpirodela polyrrhiza, 50 mg L^-1, 14 days EC. Chromium toxicity on biochemical parameters showed a reduction in photosynthetic rate at 50 μgl^-1 Cr inMyriophyllum spicatum. Decrease in chlorophyll and protein contents were also recorded inNajas indica, Vallisneria spiralis, andAlternanthera sessilis with an increase in chromium concentration. InLimnanthemum cristatum, a slight reduction in chlorophyll and almost no change in control were found due to chromium toxicity. Submerged species (Ceratophyllum demersum, Vallisneria spiralis) and an emergent one (Alternanthera sessilis) showed decreases in chlorophyll as well as in protein contents when treated with chromium. Chromium-induced morphological and ultrastructural changes have been reported in several aquatic vascular plants: InLemna minor andCeratophyllum demersum, chromium-induced changes in chloroplast fine structure disorganized thylakoids with loss of grain and caused formation of many vesicles in the chloroplast. Chromium (VI) has caused stunting and browning of roots produced from the chromium-treated excised leaves ofLimnanthemum cristatum. At 226 μg/g Cr dry wt leaf tissue concentration, development of brown coloration in the hydathodes of juvenile leaves ofLimnanthemum cristatum is a characteristic chromiuminduced alteration. Aquatic vascular plants and algae may serve as effective bioindicators in respect to metals in aquatic environments. Chromium-induced morphological and ultrastructural changes inLimnanthemum cristatum have significant indicator values and could be used for assessing the level of chromium in ambient water.Wolffia globosa, a rootless duckweed, showed substantial chromium accumulation and high concentration factor (BCF) value at very low ambient chromium concentrations, suggesting its feasibility in detecting chromium pollution in water resources. Methylene blue-stained cells ofScenedesmus acutus become uniformly dark blue during chromium (VI) treatment. This may serve as an indicator of chromium pollution.     

Anterior-posterior graded response to Otx2 controls proliferation and differentiation of dopaminergic progenitors in the ventral mesencephalon

Meso-diencephalic dopaminergic (mdDA) neurons control voluntary movement, cognition and the reward response, and their degeneration is associated with Parkinson's disease (PD). Prospective cell transplantation therapies for PD require full knowledge of the developmental pathways that control mdDA neurogenesis. We have previously shown that Otx2 is required for the establishment of the mesencephalic field and molecular code of the entire ventral mesencephalon (VM). Here, we investigate whether Otx2 is a specific determinant of mesencephalic dopaminergic (mesDA) neurogenesis by studying mouse mutants that conditionally overexpress or lack Otx2. Our data show that Otx2 overexpression in the VM causes a dose-dependent and selective increase in both mesDA progenitors and neurons, which correlates with a remarkable and specific enhancement in the proliferating activity of mesDA progenitors. Consistently, lack of Otx2 in the VM specifically affects the proliferation of Sox2+ mesDA progenitors and causes their premature post-mitotic transition. Analysis of the developmental pathway that controls the differentiation of mesDA neurons shows that, in the absence of Otx2, the expression of Lmx1a and Msx1, and the proneural genes Ngn2 and Mash1 is not activated in Sox2+ mesDA progenitors, which largely fail to differentiate into Nurr1+ mesDA precursors. Furthermore, proliferation and differentiation abnormalities exhibit increasing severity along the anterior-posterior (AP) axis of the VM. These findings demonstrate that Otx2, through an AP graded effect, is intrinsically required to control proliferation and differentiation of mesDA progenitors. Thus, our data provide new insights into the mechanism of mesDA neuron specification and suggest Otx2 as a potential target for cell replacement-based therapeutic approaches in PD.     

<Emphasis Type="Italic">Arabidopsis HOG1</Emphasis> gene and its petunia homolog <Emphasis Type="Italic">PETCBP</Emphasis> act as key regulators of yield parameters

Plant hormones influence the key parameters that contribute to crop yield, including biomass, branching and seed number. We tested manipulation of cytokinin signaling as an avenue for influencing these growth parameters. Here we report a full-length cDNA coding for a cytokinin binding protein, Petunia cytokinin binding protein (PETCBP) from Petunia hybrida cv. Mitchell. PETCBP encodes for a protein that exhibits high sequence similarity to S-adenosyl-L: -homocysteine hydrolase (SAHH). Transgenic petunia plants expressing this gene in antisense orientation displayed profuse branching, delayed flowering and delayed shoot bud induction from leaf explants in vitro. Homologs were also isolated from Arabidopsis thaliana homology-dependent gene silencing 1 (HOG1) and Orzya sativa (OsCBP). Arabidopsis HOG1 showed high affinity cytokinin binding activity and modified plant architecture similar to PETCBP. Transgenic Arabidopsis plants overexpressing HOG1 showed early flowering with a significantly reduced plant biomass and number of leaves. In contrast, profuse branching, delayed flowering, increased leaf size and higher seed yield were the major phenotypes observed in the antisense suppression lines. These results suggest that genetic manipulation of this cytokinin binding protein or its orthologs could be used for improving crop biomass and seed yield.