Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism

The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion. The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99). Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group. The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.     

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility

The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.     

High-throughput expression,purification, and characterization of recombinant Caenorhabditis elegans proteins

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.     

Influence of flowering cover crops on Anagrus parasitoids (Hymenoptera: Mymaridae) and Erythroneura leafhoppers (Homoptera: Cicadellidae) in New York vineyards

Abstract 1 We tested the hypothesis that providing nectar‐producing cover crops will enhance the biological control of grape leafhoppers (Erythroneura spp.) by Anagrus wasps in commercial vineyards in New York, U.S.A. 2 We established three cover crops between vine rows in a commercial vineyard: buckwheat (Fagopyrum esculentum (Moench)), clover (Trifolium repens L.) and mowed sod (Dactylis glomerata L.). 3 There was no effect of cover crop on adult Anagrus in 1996, whereas in 1997 adults were more abundant within edge vines with buckwheat compared to vines with clover or sod; adults were more abundant at the vineyard edge, especially early in the season. 4 Parasitism of ‘sentinel’ leafhopper eggs was higher on vines with buckwheat compared to parasitism on vines with clover or sod in 1996; a similar, non‐significant trend, was observed in 1997. 5 Neither the abundance nor the distribution of leafhoppers was influenced by cover crops, although in 1997 there was a trend toward greater numbers of nymphs on edge vines with buckwheat. 6 In a cage experiment, parasitism by Anagrus of leafhopper eggs on grapes was greater when adults had access to flowering buckwheat rather than buckwheat without flowers. 7 In a laboratory study, longevity of female Anagrus was increased when provided with honey or sugar water compared to water only or nothing. 8 Our results suggest that parasitism of grape leafhoppers by Anagrus may be enhanced by providing floral resources within vineyards in New York, although it is unclear whether this will produce meaningful reductions in pest abundance.     

Treatment of chronic endometritis in dairy cows with an intrauterine application of enzymes. A field trial

The use of proteolytic enzymes has been established in the non-antibiotic treatment of mastitis in dairy cattle. The objective of this study was to evaluate, if enzymes are efficacious in the treatment of chronic endometritis. In a controlled field trial, cows with vaginal discharge 21-27 days in milk (DIM) were randomly assigned to two treatment groups. Endometritis was classified into three categories, depending on the type of vaginal discharge: clear mucus with flakes of pus (E1), mucopurulent discharge or fluctuating contents in the uterus (E2), and purulent discharge (E3). In group ENZYMES (n=191), cows received an intrauterine treatment with a salve containing the enzymes trypsin (16 mg), chymotrypsin (16 mg), and papain (8 mg). Cows in group PGF (n=225) were treated with 0.5mg of cloprostenol. Cows that did not show any clinical signs of chronic endometritis were regarded as healthy control group (HC, n=699). In groups ENZYMES and PGF, all cows were re-examined 35-41 DIM. In group ENZYMES, cows were re-treated with enzymes if signs of endometritis were found, while in group PGF all cows received a second dose of cloprostenol, regardless of their clinical findings. Cure rate after the first treatment, defined as the absence of vaginal discharge at the re-examinations, was 59.7 and 68.0% in groups ENZYMES and PGF, respectively (P>0.05). Reproductive performance measures showed no significant differences between the two treatment groups. Service rate was significantly lower for ENZYMES and PGF, respectively, compared to HC. Conception rates to all services and percentages of cows pregnant by 250 DIM were significantly lower in group ENZYMES compared to HC, while no further differences were found between PGF and HC. In both treatment groups, cure rate and reproductive performance measures were better for cows categorized E1 or E2, than for cows categorized E3, respectively. Conception rate to all services for cows with endometritis category E1 was higher in group PGF than in group ENZYMES (P<0.05). The results of this field trial suggest that prostaglandin F(2alpha) is still the treatment of choice for chronic endometritis in dairy cattle.     

Pigment interactions in chlorosomes of various green bacteria

Resonance Raman experiments were performed on different green bacteria. With blue excitation, i.e. under Soret resonance or preresonance conditions, resonance Raman contributions were essentially arising from the chlorosome pigments. By comparing these spectra and those of isolated chlorosomes, it is possible to evaluate how the latter retain their native structure during the isolation procedures. The structure of bacteriochlorophyll oligomers in chlorosomes was interspecifically compared, in bacteriochlorophyllc- and bacteriochlorophylle- synthesising bacteria. It appears that interactions assumed by the 9-keto carbonyl group are identical inChlorobium limicola, Chlorobium tepidum, andChlorobium phaeobacteroides. In the latter strain, the 3-formyl carbonyl group of bacteriochlorophylle is kept free from intermolecular interactions. By contrast, resonance Raman spectra unambiguously indicate that the structure of bacteriochlorophyll oligomers is slightly different in chlorosomes fromChloroflexus auranticus, either isolated or in the whole bacteria.     

Identification of Paenibacillus larvae to the subspecies level: an obstacle for AFB diagnosis

This study was initially aimed at developing a PCR-test to differentiate between the pathogenic agent of American foulbrood (Paenibacillus larvae subsp. larvae) and powdery-scale disease (P. larvae subsp. pulvifaciens) of the honeybee. The test was based on the "insert of clone 9" (iC9), referring to a cloned 1.9 kB HaeIII fragment that occurs only in the P. larvae subsp. larvae reference strains and possibly correlates with American foulbrood virulence. It was shown that an iC9-based PCR-test discriminates between the BCCM/LMG reference strains of both subspecies. However, the screening of 179 Belgian field strains revealed five isolates that gave no iC9-based amplicon, thus rather resembling to P. larvae subsp. pulvifaciens. In addition, they all produced acid from mannitol, a characteristic previously assigned to the pulvifaciens subspecies. Because the reference strains gave conflicting data, this carbohydrate acidification was not conclusive. Therefore, the exact taxonomic position of the five retained strains was determined by a polyphasic approach using SDS-PAGE, AFLP, and ERIC-based PCR. Four iC9-negative field strains could be identified as P. larvae subsp. larvae; the taxonomic position of the fifth field strain remained ambiguous. The latter was provisionally classified as a subspecies pulvifaciens strain on the basis of SDS-PAGE. The present paper demonstrates the existence of field strains that do not fit well in the subdivision of the species P. larvae into two subspecies. Knowing that only one of both subspecies represents the pathogenic agent of AFB, this is a serious obstacle for the diagnosis of this honeybee disease.     

Sustaining Plants and People: Traditional Q’eqchi’ Maya Botanical Knowledge and Interactive Spatial Modeling in Prioritizing Conservation of Medicinal Plants for Culturally Relative Holistic Health Promotion

Ethnobotanical surveys were conducted to locate culturally important, regionally scarce, and disappearing medicinal plants via a novel participatory methodology which involves healer-expert knowledge in interactive spatial modeling to prioritize conservation efforts and thus facilitate health promotion via medicinal plant resource sustained availability. These surveys, conducted in the Maya Mountains, Belize, generate ethnobotanical, ecological, and geospatial data on species which are used by Q’eqchi’ Maya healers in practice. Several of these mountainous species are regionally scarce and the healers are expressing difficulties in finding them for use in promotion of community health and wellness. Based on healers’ input, zones of highest probability for locating regionally scarce, disappearing, and culturally important plants in their ecosystem niches can be facilitated by interactive modeling. In the present study, this is begun by choosing three representative species to train an interactive predictive model. Model accuracy was then assessed statistically by testing for independence between predicted occurrence and actual occurrence of medicinal plants. A high level of accuracy was achieved using a small set of exemplar data. This work demonstrates the potential of combining ethnobotany and botanical spatial information with indigenous ecosystems concepts and Q’eqchi’ Maya healing knowledge via predictive modeling. Through this approach, we may identify regions where species are located and accordingly promote for prioritization and application of in situ and ex situ conservation strategies to protect them. This represents a significant step toward facilitating sustained culturally relative health promotion as well as overall enhanced ecological integrity to the region and the earth.     

The role of nocturnal vision in mate choice: females prefer conspicuous males in the European tree frog (Hyla arborea)

Nocturnal frog species rely extensively on vocalization for reproduction. But recent studies provide evidence for an important, though long overlooked, role of visual communication. In many species, calling males exhibit a conspicuous pulsing vocal sac, a signal bearing visually important dynamic components. Here, we investigate female preference for male vocal sac coloration—a question hitherto unexplored—and male colour pattern in the European tree frog (Hyla arborea). Under nocturnal conditions, we conducted two-choice experiments involving video playbacks of calling males with identical calls and showing various naturally encountered colour signals, differing in their chromatic and brightness components. We adjusted video colours to match the frogs'' visual perception, a crucial aspect not considered in previous experiments. Females prefer males with a colourful sac and a pronounced flank stripe. Both signals probably enhance male conspicuousness and facilitate detection and localization by females. This study provides the first experimental evidence of a preference for specific vocal sac spectral properties in a nocturnal anuran species. Vocal sac coloration is based on carotenoids and may convey information about male quality worthwhile for females to assess. The informative content of the flank stripe remains to be demonstrated.