The human pyridoxal kinase, a plausible target for ginkgotoxin from Ginkgo biloba

Ginkgotoxin (4'-O-methylpyridoxine) occurring in the seeds and leaves of Ginkgo biloba, is an antivitamin structurally related to vitamin B(6). Ingestion of ginkgotoxin triggers epileptic convulsions and other neuronal symptoms. Here we report on studies on the impact of B(6) antivitamins including ginkgotoxin on recombinant homogeneous human pyridoxal kinase (EC 2.7.1.35). It is shown that ginkgotoxin serves as an alternate substrate for this enzyme with a lower K(m) value than pyridoxal, pyridoxamine or pyridoxine. Thus, the presence of ginkgotoxin leads to temporarily reduced pyridoxal phosphate formation in vitro and possibly also in vivo. Our observations are discussed in light of Ginkgo medications used as nootropics.     

PhyloGena--a user-friendly system for automated phylogenetic annotation of unknown sequences

MOTIVATION: Phylogenomic approaches towards functional and evolutionary annotation of unknown sequences have been suggested to be superior to those based only on pairwise local alignments. User-friendly software tools making the advantages of phylogenetic annotation available for the ever widening range of bioinformatically uninitiated biologists involved in genome/EST annotation projects are, however, not available. We were particularly confronted with this issue in the annotation of sequences from different groups of complex algae originating from secondary endosymbioses, where the identification of the phylogenetic origin of genes is often more problematic than in taxa well represented in the databases (e.g. animals, plants or fungi). RESULTS: We present a flexible pipeline with a user-friendly, interactive graphical user interface running on desktop computers that automatically performs a basic local alignment search tool (BLAST) search of query sequences, selects a representative subset of them, then creates a multiple alignment from the selected sequences, and finally computes a phylogenetic tree. The pipeline, named PhyloGena, uses public domain software for all standard bioinformatics tasks (similarity search, multiple alignment, and phylogenetic reconstruction). As the major technological innovation, selection of a meaningful subset of BLAST hits was implemented using logic programming, mimicing the selection procedure (BLAST tables, multiple alignments and phylogenetic trees) are displayed graphically, allowing the user to interact with the pipeline and deduce the function and phylogenetic origin of the query. PhyloGena thus makes phylogenomic annotation available also for those biologists without access to large computing facilities and with little informatics background. Although phylogenetic annotation is particularly useful when working with composite genomes (e.g. from complex algae), PhyloGena can be helpful in expressed sequence tag and genome annotation also in other organisms. AVAILABILITY: PhyloGena (executables for LINUX and Windows 2000/XP as well as source code) is available by anonymous ftp from http://www.awi.de/en/phylogena.     

In vivo labeling with stable isotopes as a tool for the identification of unidentified peaks in the metabolome analysis of Corynebacterium glutamicum by GC/MS

Of the hyphenated techniques used for metabolic profiling of cell and tissue extracts, GC/MS is in some ways advantageous as it allows the simultaneous fingerprinting of chemically very different metabolites, and the electron impact mass spectra recorded in many cases lead to unambiguous identification of the compounds. However, prior to chromatography, the hydrophilic substances of the cell extracts have to be converted to vaporizable derivatives, the mass spectra of which often are not known or not listed in the available spectral libraries, even if they are derived from simple biochemicals. Thus, numerous chromatographic peaks remain as yet unidentified. Attempts to identify these peaks afford the acquisition of more data on these compounds. The value of in vivo labeling of metabolites with (13)C and (15)N for this purpose is described.     

Response of urinary purine derivatives excretion to different levels of ruminal glucose infusion in heifers

This study investigated the response of urinary purine derivatives (PD) excretion to increasing levels of intraruminal glucose infusion to evaluate how well this indicator reflects induced changes in microbial crude protein flow. Four rumen-cannulated heifers (482 ± 25 kg body weight) were fed at maintenance energy level with a basal diet (on fresh matter basis) of 4 kg/d hay, 1.5 kg/d concentrate and 60 g/d minerals in two equal meals. The trial comprised a control period (Control I) without glucose infusion followed by four consecutive periods in which all animals received 125 g, 250 g, 500 g or 1000 g/d of glucose, respectively. For this, daily dosages of glucose and urea (90 g/d during all periods) were divided into three portions that were dissolved in water and directly administered into the rumen during morning and afternoon feedings and once during noon. After the highest glucose dosage, a second control period was carried out (Control II). Urinary PD excretion increased with glucose infusion of 125 g/d (71.4 mmol/d) and 1000 g/d (74.2 mmol/d) over the level at Control I (53.9 mmol/d (standard error of the mean (SEM) 3.4; p = 0.012). After withdrawing glucose infusion, PD excretion (79.0 mmol/d) did not return to Control I level (p = 0.001). In contrast, faecal nitrogen (N) excretions linearly increased with incremental glucose infusion (p < 0.001) from 33.9 g/d at Control I to 39.7 g/d (SEM 0.5) at 1000 g/d of glucose and were similar in Control I and II (p = 0.086). The contradicting responses in the excretions of faecal N and urinary PD to increasing glucose infusions highlight the limited accuracy of the PD excretion as a non-invasive indicator when incremental dosages of rapidly fermentable carbohydrates are supplied.     

Distinct effects of two HIV-1 capsid assembly inhibitor families that bind the same site within the N-terminal domain of the viral CA protein

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.     

Intelectin: a novel lipid raft-associated protein in the enterocyte brush border

Intelectin is a mammalian Ca2+-dependent, D-galactosyl-specific lectin expressed in Paneth and goblet cells of the small intestine and proposed to serve a protective role in the innate immune response to parasite infection. In addition, it is structurally identical to the intestinal lactoferrin receptor known to reside in the enterocyte brush border. To clarify this apparent discrepancy with regard to localization, the aim of this work was to study the cellular and subcellular distribution of small intestinal intelectin by immunofluorescence and immunogold electron microscopy. Secretory granules of lysozyme-positive Paneth cells in the bottom of the crypts as well as goblet cells along the crypt-villus axis were intensively labeled with intelectin antibodies, but quantitatively, the major site of intelectin deposition was the enterocyte brush border. This membrane is organized in stable glycolipid-based lipid raft microdomains, and like the divalent lectin galectin-4, intelectin was enriched in microvillar "superrafts", i.e., membranes that resist solubilization with Triton X-100 at 37 degrees C. This strategic localization suggests that the trimeric intelectin, like galectin-4, serves as an organizer and stabilizer of the brush border membrane, preventing loss of digestive enzymes to the gut lumen and protecting the glycolipid microdomains from pathogens.     

Song differences between populations of seven subspecies of the African forest weaver Ploceus bicolor Vieillot (Aves: Passeriformes)

We present an analysis of song type differences between populations of seven forest weaver (Ploceus bicolor) subspecies. Phonology and syntax of the melodious parts of song types differ between these populations to the same extent as they differ between population-specific dialects within a subspecies. Phonological and syntactical song features are socially transmitted; they can be transmitted even between representatives of taxonomically different subspecies. The Harsh Call, a complex element embedded in the melody, is of similar structure in populations of four South African subspecies, but shows markedly different characteristics in populations of the subspecies from Kenya and Cameroon. Song differences between populations are suggested to result from cultural drift and geographic isolation. A correspondence between genetically determined morphological structures on the one hand and socially transmitted song structure on the other seems to be a coincidence due to geographic separation.