Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice

Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like alpha-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves the alpha-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production of capsular glucan and glycogen, respectively. Attempts to disrupt Rv3032 in the glgA mutant were unsuccessful, suggesting that a functional copy of at least one of the two alpha-1,4-glucosyltransferases is required for growth. Importantly, the glgA mutant was impaired in its ability to persist in mice, suggesting a role for the capsular glucan in the persistence phase of infection. Unexpectedly, GlgB was found to be an essential enzyme.     

Action of multiple base excision repair enzymes on the 2'-deoxyribonolactone

Free radical attack on the sugar-phosphate backbone generates oxidized apurinic/apyrimidinic (AP) residues in DNA. 2'-deoxyribonolactone (dL) is a C1'-oxidized AP site damage generated by UV and gamma-irradiation, and certain anticancer drugs. If not repaired dL produces G-->A transitions in Escherichia coli. In the base excision repair (BER) pathway, AP endonucleases are the major enzymes responsible for 5'-incision of the regular AP site (dR) and dL. DNA glycosylases with associated AP lyase activity can also efficiently cleave regular AP sites. Here, we report that dL is a substrate for AP endonucleases but not for DNA glycosylases/AP lyases. The kinetic parameters of the dL-incision were similar to those of the dR. DNA glycosylases such as E. coli formamidopyrimidine-DNA glycosylase, mismatch-specific uracil-DNA glycosylase, and human alkylpurine-DNA N-glycosylase bind strongly to dL without cleaving it. We show that dL cross-links with the human proteins 8-oxoguanine-DNA (hOGG1) and thymine glycol-DNA glycosylases (hNth1), and dR cross-links with Nth and hNth1. These results suggest that dL and dR induced genotoxicity might be strengthened by BER pathway in vivo.     

Imaging cell interactions with native and crosslinked polyelectrolyte multilayers

The adhesion of primary chondrocytes to polyelectrolyte multilayer films, made of poly(l-lysine) (PLL) and hyaluronan (HA), was investigated for native and crosslinked films, either ending by PLL or HA. Crosslinking the film was achieved by means of a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide. The adhesion of macrophages and primary chondrocytes was investigated by microscopical techniques (optical, confocal, and atomic), providing useful information on the cell/film interface. Native films were found to be nonadhesive for the, primary chondrocytes, but could be degraded by macrophages, as could be visualized by confocal laser scanning microscopy after film labeling. Confocal microscopy images show that these films can be deformed by the condrocytes and that PLL diffuses at the chondrocyte membrane. In contrast, the cells adhered and proliferated well on the crosslinked films, which were not degraded by the macrophages. These results were confirmed by a MTT test over a 6-d period and by atomic force microscopy observations. We thus prove that chemical crosslinking can dramatically change cell adhesion properties, the cells being more stably anchored on the crosslinked films. Both authors kcontributed equally.     

Immunobiology of hookworm infection

Hookworms infect almost one billion people and are a major cause of iron-deficiency anaemia in developing countries of the tropics. Despite their prevalence and the morbidity they cause, little is known about the immune response to this complex eukaryotic parasite. Recent publications have shed light on the human cellular immune responses to hookworms, as well as mechanisms that hookworms utilize to skew the immune response in its favour. Unlike most other human helminth infections, neither age- nor exposure-related immunity develops in the majority of infected people. A vaccine is therefore a highly desirable goal. To this end, gene sequencing efforts have resulted in the deposition of more than 10,000 hookworm cDNA sequences in the public domain, providing a molecular snapshot of this intriguing parasite and providing novel tools for the development of new control strategies. Significant progress has been made in the development of anti-hookworm recombinant vaccines, and clinical trials are expected to begin in the near future.     

Photoreactive threading agent that specifically binds to abasic sites in DNA

We report the synthesis and study of a photoreactive nitrobenzamide containing acridine that specifically interacts at abasic site in DNA by threading intercalation and introduces under irradiation a lesion on the opposite strand at the unpaired pyrimidine.     

Morphology of the terminalia of the stick insect Dajaca napolovi from Vietnam (Insecta: Phasmatodea)

Aschiphasmatinae is a small group of stick insects from the Oriental region whose genital morphology has been rarely described in detail. The subfamily is of particular interest, as phylogenetic studies have shown Aschiphasmatinae to be the sister group to the remaining Euphasmatodea. In this paper, the male and female terminalia are described for the first time in Dajaca napolovi Brock, a little known aschiphasmatine species from Vietnam. In the male, the transversally undivided abdominal sternum IX and gently incurved cerci with a conspicuous apical tooth represent apomorphies of Aschiphasmatinae. Thorn pads on the hind margin of abdominal tergum X consist of only a single row of 6–7 ventrally oriented teeth. The simple thorn pad structure of Aschiphasmatinae can represent an ancestral condition for Euphasmatodea. The vomer on venter X is well‐developed and features two unusually large basal apodemes and a strongly developed apical spine showing a specialized streaked surface micropattern. Female terminalia are characterized by an unkeeled abdominal sternum VIII covering the reduced primary ovipositor. Gonapophysis VIII does not extend beyond the tip of gonapophysis IX. An asymmetry concerning the size of the paired gonapophyses is reported for the first time in Phasmatodea.     

Conformational stabilization at the active site of molluskan (Rapana thomasiana) hemocyanin by a cysteine-histidine thioether bridge A study by mass spectrometry and molecular modeling

In some type-3 copper proteins (molluskan hemocyanin, catechol oxidase and fungal tyrosinase) one of the histidine residues, liganding the Cu(A) atom of the dinuclear copper active site, is covalently linked to a cysteine residue by a thioether bridge. The purpose of this study was to disclose the function of this bridge. Mass spectral analysis of a peptide, isolated from Rapana thomasiana (gastropodan mollusk) hemocyanin, indicated a stabilization of the peptide structure in the region of the bridge. Molecular modeling of three thioether containing type-3 copper proteins using the dead-end elimination method showed that the concerned histidine would be very flexible if not linked to the cysteine. Also, the side chain orientation of the histidine is rather exceptional, as evidenced by statistical data from the protein databank. It is suggested that the role of the bridge is to fix the histidine in an orientation that is optimal for coordination of the Cu(A) atom.     

Abasic DNA structure, reactivity, and recognition

Loss of a base in DNA, i.e., creation of an abasic site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously, or under the action of radiations and alkylating agents, or enzymatically as an intermediate in the repair of modified or abnormal bases. The abasic site lesion is mutagenic or lethal if not repaired. From a chemical point of view,the abasic site is an alkali-labile residue that leads to strand breakage through beta- and delta- elimination. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of synthetic abasic duplexes. Several efficient synthetic methods have thus been developed to introduce the lesion (or a stable analogue) at defined position in the sequence. Physicochemical and spectroscopic examination of such duplexes, including calorimetry, melting temperature, high-field nmr and molecular modeling indicate that the lesion strongly destabilizes the duplex, although remaining in the canonical B-form with structural modifications strictly located at the site of the lesion. Probes have been developed to titrate the damage in DNA in vitro. Series of molecules have been devised to recognize specifically the abasic site, exhibiting a cleavage activity and mimicking the AP nucleases. Others have been prepared that bind strongly to the abasic site and show promise in potentiating the cytotoxic and antitumor activity of the clinically used nitrosourea (bis-chloroethylnitrosurea).     

Metabolic compensation constrains the temperature dependence of gross primary production

Gross primary production (GPP) is the largest flux in the carbon cycle, yet its response to global warming is highly uncertain. The temperature dependence of GPP is directly linked to photosynthetic physiology, but the response of GPP to warming over longer timescales could also be shaped by ecological and evolutionary processes that drive variation in community structure and functional trait distributions. Here, we show that selection on photosynthetic traits within and across taxa dampens the effects of temperature on GPP across a catchment of geothermally heated streams. Autotrophs from cold streams had higher photosynthetic rates and after accounting for differences in biomass among sites, biomass‐specific GPP was independent of temperature in spite of a 20 °C thermal gradient. Our results suggest that temperature compensation of photosynthetic rates constrains the long‐term temperature dependence of GPP, and highlights the importance of considering physiological, ecological and evolutionary mechanisms when predicting how ecosystem‐level processes respond to warming.