Characterization of Staphylococcus aureus from Humans and a Comparison with ?solates of Animal Origin,in North Dakota,United States

Different clones of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus have been found in humans as well as in animals and retail meat. However, more information about the genetic characteristics and similarities between strains is needed. The aim of this study was to identify and characterize Staphylococcus aureus from humans, and to compare their characteristics with isolates of animal origin. A total of 550 nasal swabs were taken from healthy humans, and S. aureus was isolated and identified. Positive S. aureus isolates were subjected to molecular typing and susceptibility testing. In addition, 108 MRSA isolates recovered from clinical patients in the state of North Dakota and 133 S. aureus isolates from animals and meat previously analyzed were included. The nasal carriage of S. aureus in healthy people was 7.6% and, in general, clones were genetically diverse. None of the S. aureus strains obtained from healthy people were mecA- or PVL-positive. A total of 105 (97.2%) MRSA isolates from clinical cases harbored the mecA gene and 11 (10.2%) isolated from blood stream infections harbored the PVL gene. The most common resistance profile among S. aureus from healthy people was penicillin, and from clinical cases were erythromycin-penicillin-ciprofloxacin. The rate of multidrug resistance (MDR) was 70% in humans. Most of the S. aureus harboring mecA and PVL genes were identified as ST5 and ST8, and exhibited MDR. However, S. aureus isolates of animal origin used for comparison exhibited a lower rate of MDR. The most common resistance profiles in isolates of animal origin were penicillin-tetracycline and penicillin-tetracycline-erythromycin, in animals and raw meat, respectively. The ST5 was also found in animals and meat, with ST9 and ST398 being the major clones. The genetic similarity between clones from humans and meat suggests the risk of spread of S. aureus in the food chain.     

Metabolic Needs and Capabilities of Toxoplasma gondii through Combined Computational and Experimental Analysis

Toxoplasma gondii is a human pathogen prevalent worldwide that poses a challenging and unmet need for novel treatment of toxoplasmosis. Using a semi-automated reconstruction algorithm, we reconstructed a genome-scale metabolic model, ToxoNet1. The reconstruction process and flux-balance analysis of the model offer a systematic overview of the metabolic capabilities of this parasite. Using ToxoNet1 we have identified significant gaps in the current knowledge of Toxoplasma metabolic pathways and have clarified its minimal nutritional requirements for replication. By probing the model via metabolic tasks, we have further defined sets of alternative precursors necessary for parasite growth. Within a human host cell environment, ToxoNet1 predicts a minimal set of 53 enzyme-coding genes and 76 reactions to be essential for parasite replication. Double-gene-essentiality analysis identified 20 pairs of genes for which simultaneous deletion is deleterious. To validate several predictions of ToxoNet1 we have performed experimental analyses of cytosolic acetyl-CoA biosynthesis. ATP-citrate lyase and acetyl-CoA synthase were localised and their corresponding genes disrupted, establishing that each of these enzymes is dispensable for the growth of T. gondii, however together they make a synthetic lethal pair.     

Quantitation of growth factors in ossein-mineral-compound.

Study was undertaken to identify polypeptide factors in the commercially available ossein-mineral-compound and to see if they are present in a biologically relevant quantity. Using the guanidine-EDTA extraction, 35.7 +/- 0.1 mg proteins were obtained from 1 g of the ossein-mineral-compound. At concentration 1 micrograms/ml, guanidine-EDTA-extractable proteins stimulated the incorporation of thymidine into DNA by human bone cells to 581 +/- 122% (p less than 0.001) of that by bovine serum albumin-treated control cells, decreasing thereafter. Similarly, it stimulated the activity of alkaline phosphatase in the human bone cells. Growth factors IGF-I, IGF-II, and TGF-beta were identified in the ossein-mineral-compound. This leads to speculation regarding possible role of growth factors in explaining the beneficial effects of the compound in retarding bone loss in patients with osteoporosis.     

Beshornin and beshornoside,steroidal saponins of Beshorneria yuccoides

Two new saponins beshornin and beshornoside have been isolated from the methanolic extract of Beshorneria yuccoides leaves and their structures elucidated. Beshornin is 3-O-[α-l-rhamnopyranosyl-(1 → 4)-β-d-glucopyranosyl- (1 → 2)-[α-l-rhamnopyranosyl-(1 -+ 4)-P-D-glucopyranosyl-(1 → 3)]-β-d-glucopyranosyl-(1 → 4)-β-d- galactopyranosyl-(25R)-5α-spirostan-3β-ol, whereas beshornoside is 3-O-[α-l-rhamnopyranosyl-(1 → 4)- β-d)-glycopyranosyl-(1 → 2)]-[α-l-rhamnopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 3)]-β-d-glucopyranosyl- (1 → 4)-β-d-galactopyranosyl 26-O-[β-d]-glucopyranosyl-(25R)-5α-furostan-3β,22α,26-triol.     

Differences in platelet activation by prolactin and leptin.

Hormones such as prolactin and leptin have recently been recognized as potent platelet aggregation co-activators, and have therefore been postulated as an additional risk factor for both arterial and venous thrombosis. Clinical situations exist that are known to be associated with higher leptin and/or prolactin levels (obesity, pregnancy, prolactinomas and anti-psychotic therapy respectively) and increased venous thrombosis or atherosclerosis risk. Therefore, we compared the impact of both hormones on platelet activation in vitro and in vivo. First, we investigated platelet aggregation and P-selectin expression after stimulation with 1,000 mU/l prolactin or 100 ng/ml leptin in five healthy volunteers in vitro. Prolactin revealed significant higher levels of P-selectin expression and platelet aggregation than leptin in all subjects. We also compared the correlation of prolactin and leptin values with the P-selection expression on platelets. Previously, we detected a significant correlation between prolactin values and ADP-stimulated P-selectin expression on platelets in pregnant women, patients with pituitary tumours, and patients on anti-psychotic therapy. In contrast, leptin did not correlate with P-selectin expression in all subject groups investigated. However, leptin correlated with body mass index in the subjects investigated. Our data indicate that prolactin has a stronger effect on platelet activation as leptin in vitro and in vivo. Moreover, our data suggest that the stronger effect of prolactin on ADP-stimulated platelet aggregation, compared to leptin, depends on higher stimulation of CD62p expression by prolactin.     

Central venous catheters for chemotherapy of solid tumors--our results in the last 5 years

Central venous catheters provide an easy access for intravenous medications. Having a central line in place will relieve a child from the discomfort and danger of multiple regular intravenous lines for chemotherapy. The use of indwelling central venous catheters has become commonplace in the management of children undergoing oncological treatment. There are two types of central lines commonly used. There are Broviac catheters and Port-A-Cath (PAC) catheters. In the last 5 years we inserted 194 catheters in 175 children. We inserted 121 Broviac catheters and 73 PAC catheters. During the follow up of 39382 catheter days 44 complications were observed. In Broviac group the median follow up was 155 days and in PAC group was 230 days. We observed differences in the incidence between two devices. In Broviac group infections were more frequent and in PAC group other complications were more frequent than infections.     

Identification of a 5-HT4 receptor antagonist clinical candidate through side-chain modification

Replacement of the N-butyl side-chain of lead 5-HT4 receptor antagonist 2 with propanesulfonylpiperidinyl, morpholinyl, and piperazinyl groups led to higher affinity analogs 4-6. In vitro drug metabolism screens and cassette pharmacokinetic studies in the dog led to identification of the N-methylpiperazinyl analog (6b), which displayed pharmacokinetic, selectivity, and safety parameters sufficient for advancement to the clinic for the treatment of urinary incontinence.     

Gastrin biosynthesis in canine G cells

Gastrin requires extensive posttranslational processing for full biological activity. It is presumed that progastrin is cleaved at pairs of basic amino acids by a prohormone convertase to form a glycine-extended intermediate (G-Gly) that serves as a substrate for peptidyl-glycine alpha-amidating monooxygenase (PAM), resulting in COOH-terminally amidated gastrin. To confirm the nature of progastrin processing in a primary cell line, we performed [(35)S]methionine-labeled pulse-chase biosynthetic experiments in canine antral G cells. Radiolabeled progastrin reached a peak earlier than observed for G-Gly or amidated gastrin. G-Gly radioactivity accumulated in G cells and preceded the appearance of radioactivity in amidated gastrin. The conversion of G-Gly to amidated gastrin was enhanced by the PAM cofactor ascorbic acid. To determine whether one member of the prohormone convertase family (PC2) was responsible for progastrin cleavage, G cells were incubated with PC2 antisense oligonucleotide probes. Cells treated with antisense probes had reduced PC2 expression, an accumulation of radiolabeled progastrin, and a delay in the formation of amidated gastrin. Progastrin in antral G cells is cleaved via PC2 to form G-Gly that is converted to amidated gastrin via the actions of PAM.     

Disruption of cardiac Ena-VASP protein localization in intercalated disks causes dilated cardiomyopathy

Vasodilator-stimulated phosphoprotein (VASP) and mammalian enabled (Mena) are actin cytoskeleton and signaling modulators. Ena-VASP proteins share an identical domain organization with an NH2-terminal Ena VASP homology (EVH1) domain, which mediates the binding of these proteins to FPPPP-motif containing partners such as zyxin and vinculin. VASP and Mena are abundantly expressed in the heart. However, previous studies showed that disruption by gene targeting of VASP or Mena genes in mice did not reveal any cardiac phenotype, whereas mice lacking both VASP and Mena died during embryonic development. To determine the in vivo function of Ena-VASP proteins in the heart, we used a dominant negative strategy with cardiac-specific expression of the VASP-EVH1 domain. Transgenic mice with cardiac myocyte-restricted, alpha-myosin heavy chain promoter-directed expression of the VASP-EVH1 domain were generated. Overexpression of the EVH1 domain resulted in specific displacement of both VASP and Mena from cardiac intercalated disks. VASP-EVH1 transgenic mice developed dilated cardiomyopathy with myocyte hypertrophy and bradycardia, which resulted in early postnatal lethality in mice with high levels of transgene expression. The results demonstrate that Ena-VASP proteins may play an important role in intercalated disk function at the interface between cardiac myocytes.