Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor

A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.     

Pleiotropic regulatory genes bldA,adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA^Leu_UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.     

The role of posttranslational modification in moonlighting glyceraldehyde-3-phosphate dehydrogenase structure and function

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting protein exhibiting distinct activities apart from its classical role in glycolysis. Regulation of its moonlighting functions and its subcellular localization may be dependent on its posttranslational modification (PTM). The latter include its phosphorylation, which is required for its role in intermembrane trafficking, synaptic transmission and cancer survival; nitrosylation, which is required for its function in apoptosis, heme metabolism and the immune response; acetylation which is necessary for its modulation of apoptotic gene regulation; and N-acetylglucosamine modification which may induce changes in GAPDH oligomeric structure. These findings suggest a structure function relationship between GAPDH posttranslational modification and its diverse moonlighting activities.

     

Structures and functional roles of the sugar chains of human erythropoietins.

Erythropoietin (EPO) is a haemopoietic hormone specific to cells of erythroid lineage. EPO has recently become available for the treatment of anaemia as the first human recombinant biomedicine produced in heterologous mammalian cells. Human EPO is characterized by its large carbohydrate chains, which occupy close to 40% of its total mass. These sugar moieties were thought to be important for the biological activity of EPO, but detailed studies were not performed until the structures were elucidated. The variety of roles for the sugar chains were then immediately found once the structures were known. EPO is an excellent model for investigating the roles of sugar chains on glycoproteins, since its gene and its multiple glycoforms are available, as well as sensitive bioassays for testing. In this review, we will first summarize the known sugar chain structures of EPO from different host cells, and then discuss the host-cell dependent and peptide structure-dependent glycosylation of glycoproteins. We will then address how one investigates the roles of sugar chains of glycoproteins, show several examples of such investigations, and discuss the functional roles of HuEPO's sugar chains in its biosynthesis and secretion, its in vitro and in vivo biological activities, and its half-life in blood circulation.     

Scope of atomic force microscopy in the advancement of nanomedicine

One of the most exciting fields of current research is nanomedicine, but its definition and landscape remains elusive due to its continuous expansion in all directions and thus constantly eroding its boundaries and defying definitions. This lack of conceptual framework and confusing definitions was a hurdle for policy makers to enunciate credible goals and allocate resources for the advancement of the field. In this mini review, we have provided a broad framework of nanomedicine which defines its elusive landscape, and we hope this framework will accommodate its explosive growth in the future. Also, we have highlighted the role and scope of atomic force microscopy techniques in the advancement of nanomedicine. For improving health care of all that eventually would require successful intervention at fundamental biological processes, the importance of understanding the structure-function relationship of biomolecules cannot be over emphasized. In this context, AFM and its variants play a pivotal role in contributing towards the nanomedicine knowledge-base that is required for fruitful developments in nano-diagnostics and nano-therapeutics.     

Investigations of Photosensitive Proteins by Serial Crystallography

This review contains recent data on serial femtosecond X-ray crystallography (SFX), based on a femtosecond X-ray free electron laser, as well as on the possibilities of its application for studying photosensitive proteins. Development of this method began rather recently, and it has already shown its effectiveness and some unique advantages over conventional X-ray structural analysis. This technology is especially promising for structural studies of membrane proteins and for kinetic studies. The main principle of the method, the possibility of its application in structural biology, its advantages and disadvantages, as well as its prospects for further development are analyzed in this review. Special attention is given to publications in which the SFX method has been used to study photosensitive proteins.     

Evaluation of the cytogenetic effect of the herbicide treflan and of a number of its metabolites on mammalian somatic cells

Investigation of the cytogenetic effect of two samples of trifluralin, a herbicide, (chemically pure active ingredient and 25% emulsion concentrate) and its eight metabolites in the culture of human peripheral lymphocytes in vitro and in the bone marrow cells of mice in vivo has revealed that trifluralin itself does not induce chromosome aberrations both in vitro and in vivo, but some of its derivatives possess moderate (metabolites III and VII) or weak (metabolites II and XI) clastogenic activity. The results obtained should be taken into account for the hygienic regulation of trifluralin use allowing for the reality of entrance of its mutagenic derivatives into the human organism and for the degree of its genetic danger.     

蔓越橘的营养成分及其药理作用研究综述

蔓越橘中含有多种酚类物质,具有预防尿路感染复发、清除自由基、促进心血管健康等独特的保健功效,本文就其在抑制细菌黏附、抗氧化、抗肿瘤等方面的药理作用及部分作用机理进行综述。     

Structural characteristics of the patagium of Ptychozoon kuhli (Reptilia: Gekkonidae) in relation to parachuting locomotion

Ptychozoon kuhli is known for its parachuting/gliding capabilities. In this contribution, we document the allometric scaling properties of its patagium, accessory flaps and folds, and its total body surface area and compare them to similar attributes of the agamine lizard Draco volans. Ptychozoon kuhli has passive patagia that lack skeletal support and muscular control. Patagial area in P. kuhli is smaller than that in D. volans for individuals of identical snout-vent length, but the accessory folds and flaps compensate for this shortfall and overall P. kuhli has equivalent total body surface area to D. volans for equally sized individuals. The structure of the patagium of Ptychozoon kuhli was investigated in terms of its scalation patterns and structural integrity, its relationship to the body wall and its mechanism of erection, and the distribution of mechanoreceptive sensilla across its surface. Scalation patterns and the internal architecture of the patagium indicate how its shape and form are maintained as it erects and becomes exposed to air flow. Its cross-sectional shape, together with that of the entire body indicates how it is able to behave as an airfoil. The distribution of sensilla across the patagial surface reflect positioning indicative of the monitoring of scale-to-scale contact on the dorsal surface, and possibly air pressure and flow on the ventral surface.     

Properties and specificity of the major anionic trypsin-like enzyme in the keratinolytic larvae of the webbing clothes moth.

The major form of the trypsin-like proteinases from the larvae of the webbing clothes moth Tineola bisselliella has been further purified and some of its properties investigated. It differs from bovine trypsin in several respects. It is anionic at neutral pH, is very stable at alkaline pH, has no requirement for calcium ions for this stability and is very sensitive to urea. It resembles vertebrate trypsins in its complete inhibition by diisopropylfluorophosphate, its pH optimum of 8.5 for hydrolysis of benzoyl-arginine p-nitroanilide and its cleavage specificity against glucagon and the beta-chain of S-carboxymethyl insulin.     

入侵害虫螺旋粉虱及其在我国的适生区预测

主要介绍热带亚热带入侵害虫螺旋粉虱Aleurodicus dispersus Russell的形态特征、分布、寄主与生物学特性;利用DYMEX软件分析螺旋粉虱在我国潜在的分布区。模型分析结果表明,最适宜的潜在的适生区主要集中在中国的南部,包括海南、广东、广西以及台湾等的大部分区域;在云南只有零散的潜在适生区;而广大的西北和东北不利于该虫的生存;依据适生性风险预测结果,提出了应急处理措施。     

Regulation of ubiquinone metabolism

Interest in ubiquinone (UQ) has increased during recent years, mainly because of its antioxidant function and its use as a dietary supplement. However, our knowledge of the biosynthesis, catabolism, and regulation of this lipid in mammalian tissues is quite limited. UQ exhibits a high rate of turnover in all tissues indicating that cells possess efficient metabolic pathways for handling this compound and controlling its tissue levels. Besides reviewing the generally accepted metabolic pathway, alternative synthetic mechanisms are described. The lack of data concerning catabolism and regulation of this compound is emphasized. Reasons for the rather limited uptake of dietary UQ are discussed and alternative mechanisms for its beneficial effects on organ function are suggested. Since appropriate tissue uptake of dietary UQ probably only occurs in deficient states, the definition of partial UQ deficiency and its consequences is urgently needed. The possibility of raising tissue UQ levels by drug treatment or natural metabolites is raised as a choice of preference for the future.     

Development and evaluation of a thermoreversible ovule formulation of stampidine,a novel nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide

Stampidine [2',3'-didehydro-2',3'-dideoxythymidine 5'-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions. Stampidine was solubilized in a thermoreversible ovule formulation composed of polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80. Does were exposed intravaginally for 14 days to an ovule formulation with and without 0.5%, 1%, or 2% stampidine corresponding to 1 x 107- to 4 x 107-fold higher than its in vitro anti-HIV IC50 value. Vaginal tissues harvested on Day 15 were evaluated for mucosal toxicity and cellular inflammation. Additionally, does were exposed intravaginally to stampidine, and plasma collected at various time points was assayed by analytical HPLC for the prodrug and its bioactive metabolites. Stampidine did not cause mucosal inflammation. The vaginal irritation scores for 0.5-2% stampidine were within the acceptable range for clinical trials. The prodrug and its major metabolites were undetectable in the blood plasma. The marked stability of stampidine at acidic pH, its rapid spreadability, together with its lack of mucosal toxicity or systemic absorption of stampidine via a thermoreversible ovule may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.     

Enantiospecificity of cholesterol function in vivo

The importance of the absolute configuration of cholesterol for its function in vivo is unknown. To directly test this question in vivo, we synthesized the enantiomer of cholesterol (ent-cholesterol) and tested its ability to substitute for natural cholesterol (nat-cholesterol) in the growth, viability, and behavior of Caenorhabditis elegans, a cholesterol auxotroph. First-generation animals grown on ent-cholesterol were viable with only mild behavioral defects. However, ent-cholesterol produced 100% lethality/arrest of their second generation progeny. Isotopically labeled ent-cholesterol incorporated into animals, indicating that its lethality was not secondary to cholesterol starvation. When mixed with nat-cholesterol, ent-cholesterol was not inert; rather, it antagonized the activity of nat-cholesterol. These results demonstrate for the first time that the absolute configuration of cholesterol, not just its physical properties, is essential for its functions in vivo.     

Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.     

Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440

A major challenge in microbiology is the elucidation of the genetic and ecophysiological basis of habitat specificity of microbes. Pseudomonas putida is a paradigm of a ubiquitous metabolically versatile soil bacterium. Strain KT2440, a safety strain that has become a laboratory workhorse worldwide, has been recently sequenced and its genome annotated. By drawing on both published information and on original in silico analysis of its genome, we address here the question of what genomic features of KT2440 could explain or are consistent with its ubiquity, metabolic versatility and adaptability. The genome of KT2440 exhibits combinations of features characteristic of terrestrial, rhizosphere and aquatic bacteria, which thrive in either copiotrophic or oligotrophic habitats, and suggests that P. putida has evolved and acquired functions that equip it to thrive in diverse, often inhospitable environments, either free-living, or in close association with plants. The high diversity of protein families encoded by its genome, the large number and variety of small aralogous families, insertion elements, repetitive extragenic palindromic sequences, as well as the mosaic structure of the genome (with many regions of 'atypical' composition) and the multiplicity of mobile elements, reflect a high functional diversity in P. putida and are indicative of its evolutionary trajectory and adaptation to the diverse habitats in which it thrives. The unusual wealth of determinants for high affinity nutrient acquisition systems, mono- and di-oxygenases, oxido-reductases, ferredoxins and cytochromes, dehydrogenases, sulfur metabolism proteins, for efflux pumps and glutathione-S-transfereases, and for the extensive array of extracytoplasmatic function sigma factors, regulators, and stress response systems, constitute the genomic basis for the exceptional nutritional versatility and opportunism of P. putida , its ubiquity in diverse soil, rhizosphere and aquatic systems, and its renowned tolerance of natural and anthropogenic stresses. This metabolic diversity is also the basis of the impressive evolutionary potential of KT2440, and its utility for the experimental design of novel pathways for the catabolism of organic, particularly aromatic, pollutants, and its potential for bioremediation of soils contaminated with such compounds as well as for its application in the production of high-added value compounds.     

Dynamics of albumin transport across the venule walls of the rat mesentery

FITC-Albumin was injected into the circulation for estimating transport possibilities of blood microvessels and then investigating its maintenance near vessel wall with the help of the photometry. Velocity of albumin entrance into the interstitium and its top concentration near the wall of consecutive links of venules (20-60 mm) raised. Our data show that it may be caused by enhancing vessel surface and increasing its permeability for this protein.     

The postcranial dimensions of the La Chapelle-aux-saints 1 Neandertal

The La Chapelle-aux-Saints 1 Neandertal has figured prominently in considerations of Neandertal body size and proportions. In this context, a reassessment of its major long bones and a reassembly of its principal pelvic elements (sacrum and right ilium) was undertaken. There are secure measurements for its humeral and radial lengths and its femoral head diameter, but the femoral and tibial lengths were almost certainly greater than previous values. The resultant humeral, femoral and tibial lengths are similar to those of other male Neandertals, its femoral head diameter is among the largest known for Middle and Late Pleistocene humans, but its radial length is relatively short. The pelvic assembly provides modest bi-iliac and inlet transverse diameters compared with the few sufficiently complete and undistorted Middle and Late Pleistocene archaic human pelves, but its dimensions are similar to those of large male early modern humans.