Incorporation of dl-[2-14C]ornithine and dl-[5-14C]arginine in milk constituents by the isolated lactating sheep udder

1. Lactating mammary glands of sheep were perfused for several hours in the presence of dl-[2-(14)C]ornithine or dl-[5-(14)C]arginine and received adequate quantities of acetate, glucose and amino acids. 2. In the [(14)C]ornithine experiment 1.4% of the casein and 1% of the expired carbon dioxide came from added ornithine; 96% of the total radioactivity in casein was recovered in proline; 13% of the proline of casein originated from plasma ornithine. 3. In this experiment the results of chemical degradation of proline of casein as well as relative specific activities in the isolated products are consistent with the view that ornithine is metabolized, by way of glutamic gamma-semialdehyde, to proline or glutamic acid. 4. In the [(14)C]arginine experiments 3% of the casein and 1% of the expired carbon dioxide came from arginine; 84% of the arginine and 9% of the proline of casein originated from plasma arginine. 5. In these experiments the relative specific activities of arginine, ornithine and proline in plasma are in agreement with the view that arginine is metabolized by way of ornithine to proline. The conversion of arginine into ornithine is probably catalysed by arginase, so that arginase in mammary tissue may be involved in the process of milk synthesis.     

Kinetin inhibits protein oxidation and glycoxidation in vitro

We tested the ability of N(6)-furfuryladenine (kinetin) to protect against oxidative and glycoxidative protein damage generated in vitro by sugars and by an iron/ascorbate system. At 50 microM, kinetin was more efficient (82% inhibition) than adenine (49% inhibition) to inhibit the bovine serum albumin (BSA)-pentosidine formation in slow and fast glycation/glycoxidation models. Kinetin also inhibited the formation of BSA-carbonyls after oxidation significantly more than adenine did. However both compounds inhibited the advanced glycation end product (AGE) formation to the same extent (59-68% inhibition). At 200 microM, kinetin but not adenine, limited the aggregation of BSA during glycation. These data suggest that kinetin is a strong inhibitor of oxidative and glycoxidative protein-damage generated in vitro.     

Study of recombinant antibody fragments and PAI-1 complexes combining protein-protein docking and results from site-directed mutagenesis

Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) have been correlated with cardiovascular diseases such as myocardial infarction and venous thrombosis. PAI-1 has also been shown to play an important role in tumor development, diabetes, and obesitas. Monoclonal antibodies MA-8H9D4 and MA-56A7C10, and their single-chain variable fragments (scFv), exhibit PAI-1-neutralizing properties. In this study, a rigid-body docking approach is used to predict the binding geometry of two distinct conformations of PAI-1 (active and latent) in complex with these antibody fragments. Resulting models were initially refined by using the dead-end elimination algorithm. Different filtering criteria based on the mutagenesis studies and structural considerations were applied to select the final models. These were refined by using the slow-cooling torsion-angle dynamic annealing protocol. The docked structures reveal the respective epitopes and paratopes and their potential interactions. This study provides crucial information that is necessary for the rational development of low-molecular weight PAI-1 inhibitors.     

Comparative Analysis of Carbohydrate Active Enzymes in Clostridium termitidis CT1112 Reveals Complex Carbohydrate Degradation Ability

Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.     

Targeted Delivery of Amoxicillin to C. trachomatis by the Transferrin Iron Acquisition Pathway

Weak intracellular penetration of antibiotics makes some infections difficult to treat. The Trojan horse strategy for targeted drug delivery is among the interesting routes being explored to overcome this therapeutic difficulty. Chlamydia trachomatis, as an obligate intracellular human pathogen, is responsible for both trachoma and sexually transmitted diseases. Chlamydia develops in a vacuole and is therefore protected by four membranes (plasma membrane, bacterial inclusion membrane, and bacterial membranes). In this work, the iron-transport protein, human serum-transferrin, was used as a Trojan horse for antibiotic delivery into the bacterial vacuole. Amoxicillin was grafted onto transferrin. The transferrin-amoxicillin construct was characterized by mass spectrometry and absorption spectroscopy. Its affinity for transferrin receptor 1, determined by fluorescence emission titration [K_affTf-amox = (1.3 ± 1.0) x 10⁸], is very close to that of transferrin [4.3 x 10⁸]. Transmission electron and confocal microscopies showed a co-localization of transferrin with the bacteria in the vacuole and were also used to evaluate the antibiotic capability of the construct. It is significantly more effective than amoxicillin alone. These promising results demonstrate targeted delivery of amoxicillin to suppress Chlamydia and are of interest for Chlamydiaceae and maybe other intracellular bacteria therapies.     

Immunohistochemical Typing of Adenocarcinomas of the Pancreatobiliary System Improves Diagnosis and Prognostic Stratification

MethodsThis was a retrospective observational cohort study on patients with adenocarcinoma of the pancreatobiliary system who underwent diagnostic core needle biopsy or surgical resection at a tertiary referral center. 409 tumor samples were analyzed with up to 27 conventional antibodies used in diagnostic pathology. Immunohistochemical scoring system was the percentage of stained tumor cells. Bioinformatic analysis, internal validation, and survival analysis were performed.ResultsHierarchical clustering and differential expression analysis identified three immunohistochemical tumor types (extrahepatic pancreatobiliary, intestinal, and intrahepatic cholangiocarcinoma) and the discriminant markers between them. Among patients who underwent surgical resection of their primary tumor with curative intent, the intestinal type showed an adjusted hazard ratio of 0.19 for overall survival (95% confidence interval 0.05–0.72; p value = 0.014) compared to the extrahepatic pancreatobiliary type.ConclusionsIntegrative immunohistochemical classification of adenocarcinomas of the pancreatobiliary system results in a characteristic immunohistochemical profile for intrahepatic cholangiocarcinoma and intestinal type adenocarcinoma, which helps in distinguishing them from metastatic and pancreatobiliary type adenocarcinoma, respectively. A diagnostic immunohistochemical panel and additional extended panels of discriminant markers are proposed as guidance for their pathological diagnosis.     

The Milk Protein Trial: Influence Analysis of the Dropout Process

Diggle and Kenward (1994) proposed a selection model for continuous longitudinal data subject to possible non‐random dropout. Their method in general, and the milk protein example in particular, has provoked a large debate about the role of such models. The original enthusiasm was followed by skepticism about the strong but untestable assumption upon which this type of models invariably rests. Concern was raised about the very nature of incompleteness which is arguably more due to design reasons (the experiment was stopped due to insufficient feed supply), than to genuine dropout. In the meantime, the view has emerged that these models should ideally be made part of a sensitivity analysis. This paper presents a formal and flexible approach to such a sensitivity assessment, based on both global influence (Chatterjee and Hadi , 1988) as well as local influence (Cook , 1986). It will be argued that local influence is more apt to zoom in on a particular source of influence, such as the assumed non‐response mechanism. The method is applied to a set of data on milk protein contents in dairy cattle. The same data were used in the original paper by Diggle and Kenward (1994), who concluded that the dropout process was non‐random.