Pharmacokinetics of saquinavir after intravenous and oral dosing of saquinavir: hydroxybutenyl-beta-cyclodextrin formulations

The current research evaluated the ability of hydroxybutenyl-beta-cyclodextrin (HBenBCD) to enhance saquinavir in vitro solubility and in vivo oral bioavailability; both the base and mesylate salt forms of saquinavir were investigated. HBenBCD was effective and significantly improved saquinavir solubility in aqueous media. In the presence of 10 wt % HBenBCD, saquinavir base solubility in water was increased to ca. 5.5 +/- 0.4 mg/mL and represents a 27-fold increase from that observed in water (207 +/- 5 microg/mL) in the absence of HBenBCD. Saquinavir-HBenBCD formulations were found to have rapid dissolution over a wide pH range (1.2-6.8), and saquinavir solubility in these media was maintained throughout the experiments. When saquinavir-HBenBCD formulations were administered to Wistar-Hannover rats, saquinavir was rapidly absorbed and rapidly eliminated. Rapid saquinavir elimination was particularly pronounced when saquinavir-HBenBCD formulations were given as an oral aqueous gavage. Saquinavir oral bioavailability in rats obtained from saquinavir mesylate capsules (2.0% +/- 0.7%) was increased (9 +/- 4)-fold (18.6% +/- 7.3%) when dosed with saquinavir base-HBenBCD capsules. Clearly, HBenBCD can significantly improve the solubility and oral bioavailability of saquinavir; however, further formulation studies are required to optimize saquinavir oral delivery using this technology.     

Functional roles for C5a receptors in sepsis

The function of the C5a receptors, C5ar (encoded by C5ar) and C5l2 (encoded by Gpr77), especially of C5l2, which was originally termed a 'default receptor', remains a controversial topic. Here we investigated the role of each receptor in the setting of cecal ligation and puncture-induced sepsis by using antibody-induced blockade of C5a receptors and knockout mice. In 'mid-grade' sepsis (30-40% survival), blockade or absence of either C5ar or C5l2 greatly improved survival and attenuated the buildup of proinflammatory mediators in plasma. In vivo appearance or in vitro release of high mobility group box 1 protein (HMGB1) required C5l2 but not C5ar. In 'high-grade' sepsis (100% lethality), the only protective condition was the combined blockade of C5l2 and C5ar. These data suggest that C5ar and C5l2 contribute synergistically to the harmful consequences in sepsis and that C5l2 is required for the release of HMGB1. Thus, contrary to earlier speculation, C5l2 is a functional receptor rather than merely a default receptor.     

Species of Agave with antimicrobial activity against selected pathogenic bacteria and fungi

The in vitro antimicrobial activity against pathogenic bacteria, yeast, and molds were examined in extracts of the Agave species A. lecheguilla, A. picta, A. scabra and A. lophanta using an agar diffusion technique. The extracts of A. picta produced zones of inhibition of 9–13 mm for E. coli, L. monocytogenes, S. aureus, and V. cholerae, while B. cereus and Y. enterocolitica were not inhibited. The other Agave species did not show any detectable inhibitory activity against the bacteria tested; however, all four Agave sp. were inhibitory against all yeast and molds analyzed as evident by 9–20 mm zones of inhibition. The minimum microbicidal concentration (MMC) of the active extract ranged from 1.8 to 7.0 mg/ml for the sensitive bacteria, and 2.0–3.0 mg/ml for yeast. In the case of molds, the minimum inhibitory concentration (MIC) of the active extracts ranged from 3.0 to 6.0 mg/ml. Together, these data suggest that the Agave sp. analyzed are potential antimicrobial candidates with a broad range of activity.     

Optimized DNA vaccines to specifically induce therapeutic CD8 T cell responses against autochthonous breast tumors

BACKGROUND: Vaccines capable of inducing CD8 T cell responses to antigens expressed by tumor cells are considered as attractive choices for the treatment and prevention of malignant diseases. Our group has previously reported that immunization with synthetic peptide corresponding to a CD8 T cell epitope derived from the rat neu (rNEU) oncogene administered together with a Toll-like receptor agonist as adjuvant, induced immune responses that translated into prophylactic and therapeutic benefit against autochthonous tumors in an animal model of breast cancer (BALB-neuT mice). DNA-based vaccines offer some advantages over peptide vaccines, such as the possibility of including multiple CD8 T cell epitopes in a single construct. MATERIALS AND METHODS: Plasmids encoding a fragment of rNEU were designed to elicit CD8 T cell responses but no antibody responses. We evaluated the use of the modified plasmids as DNA vaccines for their ability to generate effective CD8 T cell responses against breast tumors expressing rNEU. RESULTS: DNA-based vaccines using modified plasmids were very effective in specifically stimulating tumor-reactive CD8 T cell responses. Moreover, vaccination with the modified DNA plasmids resulted in significant anti-tumor effects that were mediated by CD8 T cells without the requirement of generating antibodies to the product of rNEU. CONCLUSIONS: DNA vaccination is a viable alternative to peptide vaccination to induce potent anti-tumor CD8 T cell responses that provide effective therapeutic benefit. These results bear importance for the design of DNA vaccines for the treatment and prevention of cancer.     

Translational bypassing without peptidyl-tRNA anticodon scanning of coding gap mRNA

Half the ribosomes translating the mRNA for phage T4 gene 60 topoisomerase subunit bypass a 50 nucleotide coding gap between codons 46 and 47. The pairing of codon 46 with its cognate peptidyl-tRNA anticodon dissociates, and following mRNA slippage, peptidyl-tRNA re-pairs to mRNA at a matched triplet 5' adjacent to codon 47, where translation resumes. Here, in studies with gene 60 cassettes, it is shown that the peptidyl-tRNA anticodon does not scan the intervening sequence for potential complementarity. However, certain coding gap mutants allow peptidyl-tRNA to scan sequences in the bypassed segment. A model is proposed in which the coding gap mRNA enters the ribosomal A-site and forms a structure that precludes peptidyl-tRNA scanning of its sequence. Dissipation of this RNA structure, together with the contribution of 16S rRNA anti-Shine-Dalgarno sequence pairing with GAG, facilitates peptidyl-tRNA re-pairing to mRNA.     

VuNIP1 (NOD26-like) and VuHSP17.7 gene expression are regulated in response to heat stress in cowpea nodule

In tropical areas, high soil temperatures frequently limit biological nitrogen fixation (BNF) activity in a number of legumes species. In contrast with other legumes growing under BNF, cowpea (Vigna unguiculata) nodules are very resistant to high-temperature stress. However, the molecular basis of nodule heat tolerance remains unclear. In order to identify genes involved in the heat stress response in cowpea nodules a cDNA library was screened with cDNA-AFLP derived probes. Two full-length cDNAs corresponding to a small heat shock protein (VuHSP17.7) and to a Nodulin 26 (VuNIP1) were isolated and characterized. VuHSP17.7 encodes a sHSP family class I protein, and VuNIP1 corresponds to a NOD26-like protein, member of NIP subfamily of plant membrane intrinsic protein (MIP). VuHSP17.7 was highly induced by high-temperature stress in nodule, leaves, flower, and bud flower, and VuNIP1 was repressed in nodule after heat stress. Moreover, the spatial expression pattern of VuNIP1 and VuHSP17.7 differs significantly indicating that distinct signaling pathways under heat stress may regulate the expression of these genes.     

Nuclear Ca2+ regulates cardiomyocyte function.

In the heart, cytosolic Ca(2+) signals are well-characterized events that participate in the activation of cell contraction. In contrast, nuclear Ca(2+) contribution to cardiomyocyte function remains elusive. Here, we examined functional consequences of buffering nuclear Ca(2+) in neonatal cardiomyocytes. We report that cardiomyocytes contain a nucleoplasmic reticulum, which expresses both ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (InsP(3)R), providing a possible way for active regulation of nuclear Ca(2+). Adenovirus constructs encoding the Ca(2+) buffer protein parvalbumin were targeted to the nucleus with a nuclear localization signal (Ad-PV-NLS) or to the cytoplasm with a nuclear exclusion signal (Ad-PV-NES). A decrease in the amplitude of global Ca(2+) transients and RyR-II expression, as well as an increase in cell beating rate were observed in Ad-PV-NES and Ad-PV-NLS cells. When nuclear Ca(2+) buffering was imposed nuclear enlargement, increased calcineurin expression, NFAT translocation to the nucleus and subcellular redistribution of atrial natriuretic peptide were observed. Furthermore, prolongation of action potential duration occurred in adult ventricular myocytes. These results suggest that nuclear Ca(2+) levels underlie the regulation of specific protein targets and thereby modulate cardiomyocyte function. The local nuclear Ca(2+) signaling and the structures that control it constitute a novel regulatory motif in the heart.     

Activation of phosphatidylcholine signalling during oxidative stress in synaptic endings

The purpose of the present study was to investigate the involvement of phosphatidylcholine (PC) signalling in synaptic endings incubated under oxidative stress conditions. Synaptosomes purified from adult rats (4 months old) cerebral cortex were exposed to oxidative insult (FeSO₄, 50 μM) or vehicle, and diacylglycerol (DAG) generation and free fatty acid (FFA) release were subsequently evaluated using exogenous [¹⁴C]PC as substrate. DAG formation increased after 5, 30, and 60 min of Fe²⁺-exposure with respect to the control conditions. The contribution of PC-specific phospholipase C (PC-PLC) and phospholipase D (PLD) pathways to DAG generation was evaluated using ethanol in the enzyme assays. Phosphatidylethanol (PEth) production was measured as a marker of PLD activity. In the presence of ethanol (2%) iron significantly stimulated DAG and PEth production at all times assayed. FFA release from PC, however, was inhibited after 5 and 60 min of iron exposure. Similar results were observed in aged animals (28 months old) when compared with adult animals. DAG generation from PC was also evaluated in the presence of the tyrosine kinase inhibitors genistein and herbimycin A. Inhibition of tyrosine kinase activity did not modify the stimulatory effect exerted by iron on PC-PLC and PLD activities. Moreover, the presence of LY294002 (a specific PI3K inhibitor) did not alter DAG production. Our results demonstrate that oxidative stress induced by free iron stimulates the generation of the lipid messenger DAG from PC in synaptic endings in adult and aged rats.