Drug-efficacy depends on the inhibitor type and the target position in a metabolic network--a systematic study

Drug discovery usually focuses on candidate molecules that affect individual reactions with presumed essential functions in the cellular reaction network, especially in the development of diseases. Unfortunately, appropriately designed drugs often fail to show the expected biological effect, since the multitude of interactions in the biochemical reaction network buffers the individual changes or causes significant side effects. We address this problem through a computational approach, which considers the effect of drug application within a generalized biochemical pathway and by studying the effect of changes regarding the type and strength of inhibitors on the reduction of flux. This allows us to systematically search for the appropriate target and for type and concentration of the optimal inhibitor. We propose the flux selectivity as a measure for the discrimination of the effect on different pathways. Since the calculation of the flux selectivity is based on flux control coefficients that are calculated in the non-affected state, it is also a means for predicting the inhibitor efficacy. Furthermore, we will propose how to increase discriminative inhibition in the case of a parasitic disease by using multi-target drugs.This work is devoted to the memorial of our teacher Reinhart Heinrich, who made important contributions to the investigation of the regulation of metabolic networks, namely by introducing and applying the concept of metabolic control.     

The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).     

Quantification of campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens

Numerous outbreak investigations and case-control studies for campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a risk factor for infection and illness. There is currently extremely limited quantitative data on the levels of Campylobacter cross-contamination in the kitchen, hindering risk assessments for the pathogen commodity combination of Campylobacter and chicken meat. An exposure assessment needs to quantify the transfer of the bacteria from chicken to hands and the kitchen environment and from there onto ready-to-eat foods. We simulated some typical situations in kitchens and quantified the Campylobacter transfer from naturally contaminated chicken parts most commonly used in Germany. One scenario simulated the seasoning of five chicken legs and the reuse of the same plate for cooked meat. In another, five chicken breast filets were cut into small slices on a wooden board where, without intermediate cleaning, a cucumber was sliced. We also investigated the transfer of the pathogen from chicken via hands to a bread roll. The numbers of Campylobacter present on the surfaces of the chicken parts, hands, utensils, and ready-to-eat foods were detected by using Preston enrichment and colony counting after surface plating on Karmali agar. The mean transfer rates from legs and filets to hands were 2.9 and 3.8%. The transfer from legs to the plate (0.3%) was significantly smaller (P < 0.01) than the percentage transferred from filets to the cutting board and knife (1.1%). Average transfer rates from hands or kitchen utensils to ready-to-eat foods ranged from 2.9 to 27.5%.     

Optimal offspring size in a small mammal: an exception to the tradeoff invariant life-history rule

Offspring size and number were examined in a captive population of wild guinea pigs ( Cavia aperea ), and findings were compared with models of optimal offspring size for small litters. Median and modal litter size was two, regardless of maternal size or parity. Females producing their second litter tended to have litters that were larger than average. In contrast, young females that were still growing never had litters that were larger than average. Mean offspring size decreased and variation in offspring size tended to decrease with increasing litter size. Optimal offspring size models, in which offspring survival depended on the amount of resources invested, as well as litter size, predict such a trend. Little support was found for Charnov and Downhower's (1995) tradeoff invariant life-history rule that the range in offspring sizes between litters is inversely proportional to the size of the litter. Cavia aperea may be an exception to this rule because pup mass at birth did not reflect total reproductive investment, because conversion of resources into litter mass may not be linearly related to litter size and because resources were not equally partitioned among offspring within large litters. Experimental data are needed to determine the relevance of these results among mammals in general.     

A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology

Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This makes comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms.     

Transgenic papaya: a useful platform for oral vaccines

Planta - Transgenic papaya callus lines expressing the components of the S3Pvac vaccine constitute a stable platform to produce an oral vaccine against cysticercosis caused by Taenia solium or T....     

Function of the mammalian oviductal sperm reservoir.

Sperm are stored in the isthmic region of the oviduct under conditions that maintain sperm viability and suppress motility. This region is also the site in which essential steps of the capacitation process are coordinated with the appearance of the ovulated egg. The influx of Ca(2+) and phosphorylation of sperm proteins are features of the ongoing capacitation process. Using a cell-culture system of oviductal epithelial cells, it was found that sperm bound to the epithelial cells showed a reduced Ca(2+) uptake and almost no tyrosine phosphorylation as shown by indirect immunofluorescence. Furthermore, sperm viability, measured as membrane integrity with propidium iodide, is significantly prolonged as compared to sperm in suspension. The formation of the sperm reservoir appears to be mediated by carbohydrate-protein interaction. In the pig, it has been found that mannosyl-oligosaccharides exposed by the epithelial cells are high-affinity ligands for sperm-associated lectins. Ovalbumin and mannopentaose are effective inhibitors of sperm binding to explants of oviductal epithelium. Spermadhesins, a new class of animal lectins and the major secretory products of the porcine seminal vesicle, associate with the sperm surface at ejaculation and are candidate molecules for the receptors of the epithelial carbohydrates.     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.     

Acrosin and the acrosome in human spermatogenesis

Using the indirect immunofluorescent staining technique, the developmental patterns of (pro) acrosin and the outer acrosomal membrane were studied in human spermatogenesis. Specific antibodies against purified acrosin and outer acrosomal membranes from boar spermatozoa were raised in the rabbit and were found to crossreact with (pro)acrosin and outer acrosomal membrane from human spermatogenic cells. It was concluded that (pro)acrosin as well as the molecules building up the outer acrosomal membrane have been highly conserved during mammalian evolution. In the course of human spermatogenesis (pro)acrosin as well as the outer acrosomal membrane first appear in the haploid spermatids; the fluorescent areas of the individual cells steadily increase during spermiogenesis. Staining for acrosin and the outer acrosomal membrane, respectively, was found in identical compartments of the spermatogenic cells in juxtaposition to the nucleus. Round-headed spermatozoa from an infertile patient did not stain for (pro)acrosin or outer acrosomal membrane. The lack of the acrosin system was further substantiated by the gelatin substrate film technique demonstrating the absence of a gelatinolytic protease in round-headed spermatozoa. Hence, round-headed spermatozoa lack the acrosome with its constituent membrane proteins and the acrosin system housed by the acrosome of normal spermatozoa.