The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication

BACKGROUND: The centrosome is composed of a centriole pair and pericentriolar material (PCM). By marking the site of PCM assembly, the centrioles define the number of centrosomes present in the cell. The PCM, in turn, is responsible for the microtubule (MT) nucleation activity of centrosomes. Therefore, in order to assemble a functional bipolar mitotic spindle, a cell needs to control both centriole duplication and PCM recruitment. To date, however, the molecular mechanisms that govern these two processes still remain poorly understood. RESULTS: Here we show that SPD-2 is a novel component of the C. elegans centrosome. SPD-2 localizes to the centriole throughout the cell cycle and accumulates on the PCM during mitosis. We show that SPD-2 requires SPD-5 for its accumulation on the PCM and that in the absence of SPD-2, centrosome assembly fails. We further show that centriole duplication is also defective in spd-2(RNAi) embryos, but not in spd-5(RNAi) embryos, where PCM recruitment is efficiently blocked. CONCLUSIONS: Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.     

Molecular diagnosis of neonatal diabetes mellitus using next-generation sequencing of the whole exome

Background

Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis.

Methodology/Principal Findings

We carried out WES for a patient presenting with permanent NDM, for whom mutations in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patient''s mother, father and young brother, all of whom are normoglycemic.

Conclusions/Significance

WES identified a novel de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.     

Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant.

Summary Preincubation at 42^o, before infection at permissive temperature by phage , of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage. Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair. This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating DNA, probably due to induction of diffusible products.     

In vitro stem elongation of sweet pepper in media containing 24-epi-brassinolide

In vitro regeneration of sweet pepper (Capsicum annuum L. cvs Jupiter and Pimiento Perfection) has been performed via direct organogenesis. The resulting shoot-buds were placed on media containing 24-epi-brassinolide (EBR) 0.1 μM, a plant steroid lactone, in the presence or absence of zeatin 9.1 μM plus GA3 5.2 μM for further stem elongation. Different responses to these treatments were recorded depending upon the protocols used and the genotypes tested. It appears that EBR does not always act directly on stem elongation but may be an elicitor and/or an enhancer of elongation in concert with endogenous and other exogenously added growth regulators. Elongated shoots were easily rooted with alpha-naphtalenacetic acid 0.5 μM (0.1 mgl-1) and transfered to soil, and following acclimation were taken to maturity in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

An AP site can protect against the mutagenic potential of 8-oxoG when present within a tandem clustered site in E. coli

Ionizing radiation induces clustered DNA damaged sites, defined as two or more lesions formed within one or two helical turns of the DNA through passage of a single radiation track. It is now established that clustered DNA damage sites are found in cells and present a challenge to the repair machinery of the cell but to date, most studies have investigated the effects of bi-stranded lesions. A subset of clustered DNA damaged sites exist in which two or more lesions are present in tandem on the same DNA strand. In this study synthetic oligonucleotides containing an AP site 1, 3 or 5 bases 5' or 3' to 8-oxo-7,8-dihydroguanine (8-oxoG) on the same DNA strand were synthesized as a model of a tandem clustered damaged sites. It was found that 8-oxoG retards the incision of the AP site by exonuclease III (Xth) and formamidopyrimidine DNA glycosylase (Fpg). In addition the rejoining of the AP site by xrs5 nuclear extracts is impaired by the presence of 8-oxoG. The mutation frequency arising from 8-oxoG within a tandem clustered site was determined in both wild type and mutant E. coli backgrounds. In wild-type, nth, fpg and mutY null E. coli, the mutation frequency is slightly elevated when an AP site is in tandem to 8-oxoG, compared with when 8-oxoG is present as a single lesion. Interestingly, in the double mutant mutY/fpg null E. coli, the mutation frequency of 8-oxoG is reduced when an AP site is present in tandem compared with when 8-oxoG is present as a single lesion. This study demonstrates that tandem lesions can present a challenge to the repair machinery of the cell.     

The palisade endings of cat extraocular muscles: a light and electron microscope study.

The palisade endings (PEs), a particular type of nerve ending found only in extraocular muscles of mammals, have been studied using both silver-stained teased preparations and electron microscope techniques. They have been found, in act, in both the proximal and distal muscle insertions of the four recti and the two oblique mucles. PEs are exclusively associated with some of the mitochondria-poor, multiply-innervated muscle fibres present in the globar layer os these muscles, and consist of a multitude of terminal branches embracing the extremity of the muscle fibre and penetrating the infoldings formed by the muscle fibre at its tendinous attachment. The whole formation is surrounded by a thin capsule. These nerve endings present striking similarities to the developing Golgi tendon organ; the terminal branches lying among the collagen fibrils and occasionally making 'sensory-like' close contacts with the muscle fibre are disposed in such a way that they could easily have a sensory role. It was concluded that PEs present sufficient morphological evidence to be considered as sensory, encapsulated, myotendinous receptors, each related to a single multiply-innervated muscle fibre.     

Extra terrestrial abiogenic organization of organic matter: The hollow spheres of the Orgueil meteorite

Fragments of the Orgueil meteorite were macerated in mineral acids (HNO₃-HF-HNO₃) to dissolve the mineral matrix and separate the acid-resistant organic residues; a routine procedure in the extraction of pollen and spores from terrestrial sediments. Numerous spherical hollow objects were found, optically resembling the brown amorphous residual organic matrix of the meteorite. Their morphology, size-distribution, and chemical composition, revealed by electron microprobe with reference to carbon and phosphorus, are described, and evaluated in connection with criteria of biogenicity. The intrinsic criteria are satisfactorily met, but the extrinsic requirement of a sedimentary environment is not met. A review of the literature concerning the meteoritic environment suggests an explanation of these spheres based on the environment of their formation. It is proposed that they are organic coatings on olivine microchondrules, magnetite and glass globules, the mineral component of which has been dissolved by the acid maceration. They could have initially resulted from the polymerization of dispersed small organic molecules condensing on the surface of the microchrondrules. The latter were injected from a volcanic nuée ardente into the dispersed cold primordial cosmic dust of hydrated silicates and organic molecules, around the meteorite parent-body. This presumably occurred before the cosmic dust accreted as the carbonaceous chondritic outer layer of the parent-body. Upsurging reducing hot gases from the nuée ardente would polymerize part of the dispersed organic matter as the insoluble brown amorphous matrix, possibly the sticking agent when the cosmic dust accreted. The spiraled form of several of the organic structures described here are suggestive of atmospheric heat microturbulences. Organic membranes and comet-form tails of spherical coatings suggest polymerization in the wake of injected microchondrules. These diverse organic structures would result in our view from the abiogenic thermal organization of organic matter in an extraterrestrial gas-solid system.

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Résumé Plusieurs fragments de la météorite d'Orgueil ont été macérés dans des acides minéraux (HNO₃, HF, HNO₃), afin de dissoudre la fraction minérale et isoler la fraction résiduelle résistant aux acides. C'est là un procédé utilisé couramment en palynologie pour extraire les grains de pollen et les spores des sédiments terrestres.De nombreux objets microscopiques, sphériques et creux, ont été mis en évidence. Ils sont optiquement similaires au résidu organique brun, amorphe, dans lequel ils sont enrobés. Leur morphologie, leur répartition en fonction de leur taille, et leur composition chimique élémentaire, analysée par la microsonde électronique, qui révèle la présence de carbone et de phosphore, sont décrites, puis évaluées en fonction des critères disponibles d'une éventuelle origine biologique. Les critères intrinsèques aux objets sont bien satisfaits, mais non le critère extrinsèque d'un environnement sédimentaire convenable.L'analyse des hypothèses qui ont été avancées pour décrire l'environnement originel de la météorite, permet de suggérer une explication de ces sphères creuses organiques, qui repose entièrement sur cet environnement à l'époque de leur formation. Ce sont des revêtements organiques à la surface de microchondrules d'olivine, de globules de verre et de magnétite, minéraux de haute température appartenant à la fraction minérale de la météorite qui a été dissoute par la macération acide.Ces coques organiques résulteraient de la polymérisation de petites molécules organiques dispersées, qui se seraient condensées à la surface de gouttelettes minérales en fusion. Ces dernières ont pu être éjectées par une nuée ardente volcanique issue du corps parent de la météorite, et projetées dans la poussière cosmique primitive froide en suspension autour de ce corps parent, composée de silicates hydratés et de petites molécules organiques.C'est ensuite seulement que cette suspension de poussière primitive aurait subi l'accrétion pour former finalement la couche extérieure froide de matière étéoritique carbonée du corps parent. En outre, des gaz réducteurs à haute température, s'élevant de la nuée ardente, ont pu polymériser en partie la matière organique en suspension, pour former la matière météoritique organique amorphe, résistant aux acides, qui a peut-être été l'agent agglomérant lors de l'accrétion.Les formes spiralées de plusieurs des structures organiques décrites ici suggèrent des microturbulences atmosphériques dûes à la chaleur. Des membranes organiques, et l'appendice en forme de queue de comète d'une sphérule, suggèrent une polymérisation organique dans le sillage de la trajectoire de microchondrules. Selon notre opinion, ces diverses structures organiques résultent donc de l'organisation abiogénique sous l'effet de la température, de matière organique préexistante, plus simple, dans un système solide-gaz extraterrestre.