In vivo 31P and multilabel 13C NMR measurements for evaluation of plant metabolic pathways

Reliable measurements of intracellular metabolites are useful for effective plant metabolic engineering. This study explored the application of in situ 31P and 13C NMR spectroscopy for long-term measurements of intracellular pH and concentrations of several metabolites in glycolysis, glucan synthesis, and central carbon metabolic pathways in plant tissues. An NMR perfusion reactor system was designed to allow Catharanthus roseus hairy root cultures to grow for 3-6 weeks, during which time NMR spectroscopy was performed. Constant cytoplasmic pH (7.40+/-0.06), observed during the entire experiment, indicated adequate oxygenation. 13C NMR spectroscopy was performed on hairy root cultures grown in solutions containing 1-13C-, 2-13C-, and 3-13C-labeled glucose in separate experiments and the flow of label was monitored. Activities of pentose phosphate pathways, nonphotosynthetic CO2 fixation, and glucan synthesis pathways were evident from the experimental results. Scrambling of label in glucans also indicated recycling of triose phosphate and their subsequent conversion to hexose phosphates.     

Synthesis of N,N'-disubstituted 3-aminobenzo[c] and [d]azepin-2-ones as potent and specific farnesyl transferase inhibitors

A structure-activity study was performed by synthesis on N,N'-disubstitution of 3-aminobenzo[c] and [d]azepin-2-one 2 and 3 to afford potent and specific farnesyl transferase inhibitors with low nM enzymatic and cellular activities.     

Movement of entomopathogenic nematodes in soils of Fragaria spp

The mobility of three entomopathogenic nematodes (Steinernema carpocapsae UK and All Strain, and S. glaseri (Nematoda: Steinernematidae) was observed in strawberry fields in Vancouver, WA. Nematodes were sprinkled over the soil surface using a mason jar. Soil and soil-moisture content, soil composition, soil pH and conductivity was reported over the evaluation period. 12 cm deep soil samples were taken after surface application at 9 and 25 days, 7, 26 and 45 and 6, 22, 37 and 50 days at the three different test sites within the surface application site and at 5 cm distance. Soil samples taken were divided into three layers (0-4, 5-8 and 9-12 cm) and exposed once to five Galleria larvae in the laboratory to evaluate nematode presence and movement. Results after surface application demonstrated more vertical movement of S. glaseri into deeper soil layers in comparison to both S. carpocapsae strains.     

Parallel liquid synthesis of N,N'-Disubstituted 3-amino azepin-2-ones as potent and specific farnesyl transferase inhibitors

A rapid structure-activity study was performed by parallel liquid synthesis on N,N'-disubstitution of 3-amino azepin-2-one to afford potent and specific farnesyl transferase inhibitors with low nM enzymatic and cellular activities. The activities of the selected compounds were validated in vivo, and compounds 41a and 44a presented significant antitumour activity.     

Anatomical and functional evidence for a stress-responsive,monoamine-accumulating area in the dorsomedial hypothalamus of adult rat brain

The dorsomedial hypothalamus (DMH) plays an important role in relaying information to neural pathways mediating neuroendocrine, autonomic, and behavioral responses to stress. Evidence suggests that the DMH is a structurally and functionally diverse integrative structure that contributes to both facilitation and inhibition of the hypothalamo-pituitary-adrenal axis, depending on the nature of the stimulus and the specific neural circuits involved. Previous studies have determined that stress or stress-related stimuli elevate tissue concentrations of serotonin (5-hydroxytryptamine; 5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine, and noradrenaline selectively within the DMH. In order to determine the specific region of the rat DMH involved, we used high-performance liquid chromatography with electrochemical detection to measure tissue concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline within five different subregions of the DMH in adult female Lewis and Fischer rats immediately or 4 h following a 30-min period of restraint stress. Compared to unrestrained control rats, restrained rats had elevated concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline immediately after a 30-min period of restraint and had elevated concentrations of 5-HT 4 h following the onset of a 30-min period of restraint stress. These effects were confined to a specific region that included medial portions of the dorsal hypothalamic area and dorsal ependymal, subependymal, and neuronal components of the periventricular nucleus. Furthermore, these effects were observed in Lewis rats, but not Fischer rats, two closely related rat strains with well-documented differences in neurochemical, neuroendocrine, autonomic, and behavioral responses to stress. These data provide support for the existence of a stress-responsive, amine-accumulating area in the DMH that may play an important role in the differential stress responsiveness of Lewis and Fischer rats.     

AKAP350 at the Golgi apparatus. II. Association of AKAP350 with a novel chloride intracellular channel (CLIC) family member

AKAP350 can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. We performed a yeast two-hybrid screen of a rabbit parietal cell library with a 3.2-kb segment of AKAP350 (nucleotides 3611-6813). This screen yielded a full-length clone of rabbit chloride intracellular channel 1 (CLIC1). CLIC1 belongs to a family of proteins, all of which contain a high degree of homology in their carboxyl termini. All CLIC family members were able to bind a 133-amino acid domain within AKAP350 through the last 120 amino acids in the conserved CLIC carboxyl termini. Antibodies developed against a bovine CLIC, p64, immunoprecipitated AKAP350 from HCA-7 colonic adenocarcinoma cell extracts. Antibodies against CLIC proteins recognized at least five CLIC species including a novel 46-kDa CLIC protein. We isolated the human homologue of bovine p64, CLIC5B, from HCA-7 cell cDNA. A splice variant of CLIC5, the predicted molecular mass of CLIC5B corresponds to the molecular mass of the 46-kDa CLIC immunoreactive protein in HCA-7 cells. Antibodies against CLIC5B colocalized with AKAP350 at the Golgi apparatus with minor staining of the centrosomes. AKAP350 and CLIC5B association with Golgi elements was lost following brefeldin A treatment. Furthermore, GFP-CLIC5B-(178-410) targeted to the Golgi apparatus in HCA-7 cells. The results suggest that AKAP350 associates with CLIC proteins and specifically that CLIC5B interacts with AKAP350 at the Golgi apparatus in HCA-7 cells.     

Metastatic sweat gland adenocarcinoma: A clinico-pathological dilemma

Background  

Sweat gland adenocarcinoma is a rare malignancy with high metastatic potential seen more commonly in later years of life. Scalp is the most common site of occurrence and it usually spreads to lymph nodes. Liver, lung and bones are the distant sites of metastasis with fatal results. The differentiation between apocrine and eccrine metastatic sweat gland carcinoma is often difficult. The criteria's are inadequate to be of any practical utility.     

Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.The ability to discriminate between bovine and other sources of fecal contamination is necessary for the accurate evaluation of human health risks associated with agricultural runoff and focused water quality management to make waters safe for human use. Many methods have been proposed to identify bovine fecal pollution using a variety of different microbiology and molecular techniques. One of the most widely used approaches utilizes a PCR to amplify a gene target that is specifically found in a host population. Currently, there are numerous PCR-based assays for the detection and/or quantitative assessment of bovine fecal pollution available for microbial source-tracking (MST) applications (1, 5-7, 11, 14, 17, 18, 21, 23). These assays target genes ranging from mitochondrial DNA to ribosomal rRNA to other functional genes involved in microorganism-host interactions.The majority of the reported bovine-associated PCR assays target 16S rRNA genes from the order Bacteroidales. This bacterial group constitutes a large proportion of the normal gut microbiota of most animals, including bovines (28), and contains subpopulations closely associated with other animal hosts such as swine, horse, and human (1, 3, 6, 18, 24). Host-associated PCR-based assays targeting Bacteroidales genetic markers have been used to investigate the sources and levels of fecal pollution at a number of beaches and inland watersheds, with variable levels of success (10, 13, 22, 27). Researchers have postulated that differences in host animal age, health, diet, and geographic location may influence bacterial community structures in the bovine gastrointestinal tract (2, 9, 26). Without a priori knowledge of the potential representational bias introduced by such factors, it may be difficult to use these assays with confidence as indicators of bovine fecal pollution.Assay specificity and sensitivity and the prevalence and abundance of genetic marker determinations are typically estimated from the systematic testing of a collection of reference fecal sources collected from known animal sources. However, the characterization of assay performance has been limited, in most cases, to animal sources originating from a particular geographic region or industry, such as dairy or beef. The determination of assay performance across a range of different host populations is essential as the field moves toward the implementation of PCR-based host-associated fecal pollution assessment approaches.We report a performance study of seven PCR and quantitative PCR (qPCR) assays targeting Bacteroidales genes reported to be associated with either ruminant (e.g., bovine, goat, sheep, deer, and others) or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations. Assay specificity was determined by testing 175 fecal DNA extracts from 24 different animal species. For qPCR assays, the abundance of each genetic marker was measured within each bovine population and compared to quantities of Bacteroidales 16S rRNA genetic markers. These analyses indicated large discrepancies in assay performance across different bovine populations.