Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Anti-tumor therapy with macroencapsulated endostatin producer cells

Background  

Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors.     

Preimplantation exposure of mouse embryos to palmitic acid results in fetal growth restriction followed by catch-up growth in the offspring

Free fatty acids (FFAs) are energy substrates for many cell types, but in excess, some FFAs can accumulate in nonadipose cells, inducing apoptosis. Also known as lipotoxicity, this phenomenon may play a role in the development of obesity-related disease. Obesity is common among reproductive age women and is associated with adverse pregnancy and fetal outcomes; however, little is known about the effects of excess FFAs on embryos and subsequent fetal development. To address this knowledge gap, murine blastocysts were cultured in excess palmitic acid (PA), the most abundant saturated FFA in human serum, and ovarian follicular fluid. Targets susceptible to aberrations in maternal physiology, including embryonic IGF1 receptor (IGF1R) expression, glutamic pyruvate transaminase (GPT2) activity, and nuclei count, were measured. PA-exposed blastocysts demonstrated altered IGF1R expression, increased GPT2 activity, and decreased nuclei count. Trophoblast stem cells derived from preimplantation embryos were also cultured in PA. Cells exposed to increasing doses of PA demonstrated increased apoptosis and decreased proliferation. To demonstrate long-term effects of brief PA exposure, blastocysts cultured for 30 h in PA were transferred into foster mice, and pregnancies followed through Embryonic Day (ED)14.5 or delivery. Fetuses resulting from PA-exposed blastocysts were smaller than controls at ED14.5. Delivered pups were also smaller but demonstrated catch-up growth and ultimately surpassed control pups in weight. Altogether, our data suggest brief PA exposure results in altered embryonic metabolism and growth, with lasting adverse effects on offspring, providing further insight into the pathophysiology of maternal obesity.     

Cyclohexyl-linked tricyclic isoxazoles are potent and selective modulators of the multidrug resistance protein (MRP1)

Structure–activity relationship (SAR) studies on the tricyclic isoxazole series of MRP1 modulators have resulted in the identification of potent and selective inhibitors containing cyclohexyl-based linkers. These studies ultimately identified compound 21b, which reverses drug resistance to MRP1 substrates, such as doxorubicin, in HeLa-T5 cells (EC₅₀ = 0.093 μM), while showing no inherent cytotoxicity. Additionally, 21b inhibits ATP-dependent, MRP1-mediated LTC₄ uptake into membrane vesicles prepared from the MRP1-overexpressing HeLa-T5 cells (EC₅₀ = 0.064 μM) and shows selectivity (1115-fold) against the related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells. Finally, when dosed in combination with the oncolytic MRP1 substrate vincristine, 21b showed tumor regression and growth delay in MRP1-overexpressing tumors in vivo.     

Single-nucleotide polymorphism,linkage disequilibrium and geographic structure in the malaria parasite <Emphasis Type="Italic">Plasmodium vivax</Emphasis>: prospects for genome-wide association studies

Background

The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized.

Results

We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.

Conclusion

These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.See commentary: http://www.biomedcentral.com/1741-7007/8/90

     

Novel lipoglycopeptides as inhibitors of bacterial signal peptidase I

Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria. Because of its unique physiological and biochemical properties, it serves as a potential target for development of novel antibacterial agents. In this study, we report the production, isolation, and structure determination of a family of structurally related novel lipoglycopeptides from a Streptomyces sp. as inhibitors of SPase I. Detailed spectroscopic analyses, including MS and NMR, revealed that these lipoglycopeptides share a common 14-membered cyclic peptide core, an acyclic tripeptide chain, and a deoxy-alpha-mannose sugar, but differ in the degree of oxidation of the N-methylphenylglycine residue and the length and branching of the fatty acyl chain. Biochemical analysis demonstrated that these peptides are potent and competitive inhibitors of SPase I with K(i) 50 to 158 nm. In addition, they showed modest antibacterial activity against a panel of pathogenic Gram-positive and Gram-negative bacteria with minimal inhibitory concentration of 8-64 microm against Streptococcus pneumonniae and 4-8 microm against Escherichia coli. Notably, they mechanistically blocked the protein secretion in whole cells as demonstrated by inhibiting beta-lactamase release from Staphylococcus aureus. Taken together, the present discovery of a family of novel lipoglycopeptides as potent inhibitors of bacterial SPase I may lead to the development of a novel class of broad-spectrum antibiotics.     

Structure activity studies of the serine-AIB dipeptide domain in 2,3-dihydroisothiazole based growth hormone secretagogues

A series of growth hormone secretagogues (GHSs) based on 2,3-dihydroisothiazole has been synthesized in the search for a potential treatment of growth hormone deficiency or frailty in the elderly. This paper describes the evaluation of the SAR of the benzyl-d-Ser-aminoisobutyric acid dipeptide fragment. Introduction of substituents in the peptide backbone and in the phenyl ring has been investigated, as well as replacements for the benzyl group and for the AIB residue. A number of modifications resulted in enhanced potency over the parent benzyl-d-Ser-AIB derivative.     

Preparation and characterization of a beta-lactamase-Fab' conjugate for the site-specific activation of oncolytic agents.

Antibody-directed catalysis (ADC) is a two-step method for the targeted delivery of chemotherapeutic agents in which enzyme-antibody conjugates, prelocalized to antigen-bearing cells, activate prodrugs designed to be substrates for the enzyme. An enzyme-Fab' conjugate exhibiting both native beta-lactamase activity and immunoreactivity toward carcinoembryonic antigen (CEA) was constructed. Treatment of CEA-expressing LS174T cells with this conjugate imparted beta-lactamase activity to the cells; beta-lactamase activity was not imparted by treatment with unconjugated beta-lactamase and not to CEA negative cells treated with conjugate. Cephalosporin-based prodrugs, and other substrates synthesized as model compounds, were found to have wide variations in their kinetic parameters toward the conjugate, with kcat values ranging from 16 to 3300 s-1 and KM values ranging from 5 to 160 microM. The prodrug derived from desacetylvinblastine-3-carboxylic acid hydrazide (DAVLBHYD) was studied in vitro and found to be 5-fold less cytotoxic to LS174T cells than the parent DAVLBHYD. For antigen-positive cells preincubated with conjugate, however, the prodrug showed the same potency as the parent drug. Thus, the combination of conjugate and prodrug appears to provide antigen-dependent toxicity to tumor cells.