An aminoquinazoline inhibitor of the essential bacterial cell wall synthetic enzyme GlmU has a unique non-protein-kinase-like binding mode

GlmU is a bifunctional enzyme with acetyltransferase and uridyltransferase activities, and is essential for the biosynthesis of the bacterial cell wall. Inhibition results in a loss of cell viability. GlmU is therefore considered a potential target for novel antibacterial agents. A HTS (high-throughput screen) identified a series of aminoquinazolines with submicromolar potency against the uridyltransferase reaction. Biochemical and biophysical characterization showed competition with UTP binding. We determined the crystal structure of a representative aminoquinazoline bound to the Haemophilus influenzae isoenzyme at a resolution of 2.0 ?. The inhibitor occupies part of the UTP site, skirts the outer perimeter of the GlcNAc1-P (N-acetylglucosamine-1-phosphate) pocket and anchors a hydrophobic moiety into a lipophilic pocket. Our SAR (structure-activity relationship) analysis shows that all of these interactions are essential for inhibitory activity in this series. The crystal structure suggests that the compound would block binding of UTP and lock GlmU in an apo-enzyme-like conformation, thus interfering with its enzymatic activity. Our lead generation effort provides ample scope for further optimization of these compounds for antibacterial drug discovery.     

Zooplankton (Cladocera, Ostracoda), Chironomidae and other benthic faunal remains in sediment cores from nine North African wetland lakes: the CASSARINA Project

Palaeolimnological studies were carried out on sediment cores from nine North African wetland lakes. The lakes represented a variety of habitats ranging from freshwater to mixohaline conditions and with and without direct connections to the sea. Sediment cores were examined for records of recent environmental change during the 20th century period. Faunal remains analysed in the sediments included those of Cladocera, Ostracoda, Chironomidae, Mollusca and, at the sites with marine connections, Foraminifera. Cyprideis torosa littoralis was the most common ostracod, occurring in sediments from the seven sites linked with the sea and also in brackish water Sidi Bou Rhaba. In acid Megene Chitane ostracods were scarce, being represented by a single species (Cypria ophtalmica). Candona neglecta completely disappeared at two sites (Sidi Bou Rhaba and Bokka) during the 20th century. Of the Cladocera, Chydorus sphaericus was common in the moderately mixohaline sites and but Heterocypris salina occurred only where marine salinities were occasionally achieved (in Zerga, Ichkeul and Korba). Microinvertebrate assemblages in the Nile Delta lake cores and to a less extent in Zerga showed a clear response indicative of more fresher conditions occurring during the latter part of the 20th century. However the freshening trend began prior to construction of the Aswan High Dam (mid 1960s).With the exception of the most saline site (Korba), microinvertebrate communiuties at all the CASSARINA sites have experienced major disturbances during the 20th century. Changes in freshwater availability associated with increased human usage of water resources is perceived as a major factor regulating the abundance and occurrence of aquatic microinvertebrate species at these sites     

Sexual Orientation Disparities in Weight Status in Adolescence: Findings From a Prospective Study

A growing number of studies among adult women have documented disparities in overweight adversely affecting lesbian and bisexual women, but few studies have examined sexual orientation–related patterns in weight status among men or adolescents. We examined sexual orientation group trends in BMI (kg/m²), BMI Z‐scores, and overweight using 56,990 observations from 13,785 adolescent females and males in the Growing Up Today Study (GUTS), a large prospective cohort of US youth. Participants provided self‐reported information from six waves of questionnaire data collection from 1998 to 2005. Gender‐stratified linear regression models were used to estimate BMI and BMI Z‐scores and modified Poisson regression models to estimate risk ratios for overweight, controlling for age and race/ethnicity, with heterosexuals as the referent group. Among females, we observed fairly consistently elevated BMI in all sexual orientation minority groups relative to heterosexual peers. In contrast, among males we documented a sexual‐orientation‐by‐age interaction indicating steeper increases in BMI with age from early‐to‐late adolescence in heterosexuals relative to sexual orientation minorities. Additional prospective research is needed to understand the determinants of observed sexual orientation disparities and to inform appropriate preventive and treatment interventions. The long‐term health consequences of overweight are well‐documented and over time are likely to exact a high toll on populations with elevated rates.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

Summary The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10^–7 to 10^–8 after 12 h of fermentation. These frequencies were 60–400 times lower than in unstirred M17 broth and 100 000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10^–5–10^–6. Correspondence to: C.-Y. Boquien     

Myoblast Fusion Requires Fibronectin Degradation by Exteriorized m-Calpain

We recently reported that when myoblasts fuse, m-calpain could be exteriorized. Indeed, at present a number of works support this hypothesis because this enzyme was localized intercellularly and more particularly associated to extracellular matrix components. Knowing that the cell surface of the fusing myoblast is supposed to undergo many changes, we addressed the question whether m-calpain could be involved in the phenomenon of fusion via fibronectin cleavage or degradation. Using different digestion experiments, we demonstrated that soluble purified fibronectin and highly insoluble fibronectin fibrils represent very good substrates for this proteinase; moreover, at the burst of fusion, fibronectin proteolytic fragments could be identified. On the other hand, we have conducted biological assays on cultured myoblasts using a defined medium supplemented by exogenous factors capable of stimulating or inhibiting m-calpain activity. The effects of such factors on rat myoblast fusion and concomitantly on the targeted glycoprotein were analyzed and quantified. When m-calpain activity and the phenomenon of fusion were reduced (defined medium without insulin), the amount of the 220-kDa fibronectin band was increased by 43%. When m-calpain activity and myoblast fusion were prevented by addition of antibodies to m-calpain or calpain inhibitor II, the fibronectin concentration was higher since it was increased by 67 and 71%, respectively. In addition, when observed at the ultrastructural level, m-calpain seems to be localized at the potential fusion site of myoblasts and more particularly associated to the extracellular matrix when muscle cells were initially treated by anti-m-calpain IgG. Taken together, these results support the hypothesis that exteriorized m-calpain could be, in part, involved in myoblast fusion via fibronectin alteration or degradation.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

The effect of plasmid introduction into Lactococcus lactis subsp. lactis IL2661 on the growth of this strain and on plasmid stability was studied in pure batch cultures. The plasmids used (coding for erythromycin or chloramphenicol resistance) were the following: pIL205 (42 kb), pIL252 (4.6 kb, 6-9 copies), pIL253 (4.8 kb, 45-85 copies) and pE194 (inserted in the chromosome). Growth and acidification of L. lactis subsp. lactis IL2661 were similar to those of the derived recombinant lactococci. The maximal population at the end of the fermentation (9 h) was about 1.1 +/- 0.3 x 10(10) cfu/ml, and maximal growth rate 0.92 +/- 0.07 h-1. Growth yield and lactic acid concentrations were 3.9 +/- 0.8 x 10(11) cfu/g lactose consumed and 25.6 +/- 2.3 g/l, respectively. Different levels of plasmid stability were detected. Plasmid pE194, and plasmids pIL252 and pIL253 in the absence of pIL205, were stable after 10 h of culture. A slight loss (1-2%) of pIL205 was observed in all strains. In the presence of pIL205, plasmids pIL252 and pIL253 were maintained in only 56-95% of the cells. This result suggested an incompatibility between pIL205 and pIL252 or pIL253.