A rapid assay for ribokinase.

A rapid method capable of detecting low levels of ribokinase is given. [γ-³²P]ATP is converted to ribose 5-[³²P]phosphate which is not absorbable onto charcoal. The assay is linear in enzyme concentration to a lower limit of at least 4 × 10^?2 mg of enzyme/ml.     

Overexpression of a diacylglycerol acyltransferase gene in Phaeodactylum tricornutum directs carbon towards lipid biosynthesis

Under nutrient deplete conditions, diatoms accumulate between 15% to 25% of their dry weight as lipids, primarily as triacylglycerols (TAGs). As in most eukaryotes, these organisms produce TAGs via the acyl‐CoA dependent Kennedy pathway. The last step in this pathway is catalyzed by diacylglycerol acyltransferase (DGAT) that acylates diacylglycerol (DAG) to produce TAG. To test our hypothesis that DGAT plays a major role in controlling the flux of carbon towards lipids, we overexpressed a specific type II DGAT gene, DGAT2D, in the model diatom Phaeodactylum tricornutum. The transformants had 50‐ to 100‐fold higher DGAT2D mRNA levels and the abundance of the enzyme increased 30‐ to 50‐fold. More important, these cells had a 2‐fold higher total lipid content and incorporated carbon into lipids more efficiently than the wild type (WT) while growing only 15% slower at light saturation. Based on a flux analysis using ¹³C as a tracer, we found that the increase in lipids was achieved via increased fluxes through pyruvate and acetyl‐CoA. Our results reveal overexpression of DAGT2D increases the flux of photosynthetically fixed carbon towards lipids, and leads to a higher lipid content than exponentially grown WT cells.     

Cytological evidence of defective template in the fragile X chromosome

In cells of fragile X patients, the changed X segment may appear as a poorly staining region or a gap, or as a deletion, involving one or both chromatids. To find out whether the fragile site represents ah incompletely replicated DNA sequence, as has been suggested recently, we analyzed the four chromatids of methotrexate-induced endoreduplicated fragile X chromosomes. Our main observations were: (1) a deleted chromatid was never internal to a poorly staining one; (2) an endoreduplicated X chromosome with a fragile site never included a normal chromatid. These results can be explained by assuming that DNA at the fragile site, when replicated in the presence of methotrexate, may undergo defective replication and give rise to improperly packaged chromatin, appearing as a chromatid with a poorly staining region or a gap in the following metaphase. The same DNA may fail to function as a template in the following S-phase and give rise to a chromatid with a single-stranded segment, appearing as a deleted chromatid in the following metaphase.Dedicated to the memory of Professor Menashe Marcus, teacher, colleague, arid friend     

Diet and feeding strategy of smooth butterfly ray Gymnura micrura in northeastern Brazil

The purpose of the present study was to analyze the diet of the smooth butterfly ray Gymnura micrura using qualitative and quantitative approaches to describe the feeding spectrum as well as ecological aspects related to feeding and niche width. The rays were obtained through monthly sampling as by‐catch from bottom trawl fisheries in northeastern Brazil from August 2007 to July 2008. A total of 286 stomachs were sampled, of which 176 contained food items. G. micrura is a predator in the region that feeds intermittently on large whole prey items, followed by a long period of digestion with simply little or no feeding activity. A low diversity of food items was found in the G. micrura diet, with an absolute predominance of teleosts, especially the roughneck grunt Pomadasys corvinaeformis. It is suggested that the species has a narrow feeding niche and a high degree of specialization. This presumed high degree of feeding specialization is discussed and the findings are compared in light of similar studies for closely related species, allowing to propose the family as primarily constituting piscivorous rays.     

Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.     

An innovative appendage invagination procedure to reduce thrombus formation – a numerical model

Invagination is an innovative technique for closing the left atrial appendage (LAA) to reduce the risk of thrombi formation. The influence of LAA invagination on the flow fields in the atria was investigated based on a computational fluid dynamics. The simulation results demonstrated that the novel invagination process can eliminate low velocities (blood stasis) and low shear rate and thus decrease the risk of thrombus formation during atrial fibrillation. This innovative technique may enhance the clinical treatment of patients with atrial fibrillation by improving the atrial flow field while lowering the risk of creating emboli.     

Rapid fluorescent assay for screening drugs on Leishmania amastigotes

A rapid fluorescent viability assay employing alamarBlue was optimized for use with Leishmania axenic amastigotes, the stage of the parasite responsible for disease pathology. The activity of two protein kinase inhibitors, Staurosporine and H-89, as well as Amphotericin B, on promastigotes and amastigotes of Leishmania donovani and Leishmania tropica was compared. Both protein kinase inhibitors inhibited promastigote growth at lower concentrations than amastigotes, while the GI(50) for Amphotericin B on both stages was similar. This assay only requires a limited number of axenic amastigotes (50,000 cells/well) and can be used to rapidly screen large chemical or natural product libraries for activity against amastigotes.     

Genes coding for integration host factor are conserved in gram-negative bacteria.

A genetic system for the selection of clones coding for integration host factor and HU homologs is described. We demonstrate that the himA and hip genes of Serratia marcescens and Aeromonas proteolytica can substitute for the Escherichia coli genes in a variety of biological assays. We find that the sequence and genetic organization of the himA and hip genes of S. marcescens are highly conserved.     

DNA hypomethylation causes an increase in DNase-I sensitivity and an advance in the time of replication of the entire inactive X chromosome

Summary We have examined the effect of 5-azacytidine (5-aza-C) induced hypomethylation of DNA on the time of replication and DNase I sensitivity of the X chromosomes of female Gerbillus gerbillus (rodent) lung fibroblast cells. Using in situ nick translation to visualise the potential state of activity of large regions of metaphase chromosomes we show that 5-aza-C causes a dramatic increase in the DNase-I sensitivity of the entire inactive X chromosome of female G. gerbillus cells and this increase in nuclease sensitivity correlates with a large shift in the time of replication of the inactive X chromosome from late S phase to early S phase. These effects of 5-aza-C on the inactive X chromosome are associated with a 15% decrease in DNA methylation. Our results indicate that DNA methylation concomitantly affects both the time of replication and the chromatin conformation of the inactive X chromosome.