Studying rat brain neurochemistry using nanoprobe NMR spectroscopy: a metabonomics approach

In the present experiments, in vivo microdialysis techniques together with nanoprobe NMR spectroscopy were used to evaluate the neurochemical environment of the rat frontal cortex. Metabonomics techniques of data reduction and pattern recognition were used to examine whether collected neurochemicals were sensitive to tetrodotoxin (TTX), a neurotoxin that when infused into discrete brain regions can help distinguish between the neuronal versus glial origin of neurochemicals in cerebrospinal fluid microdialysate. (1)H NMR spectra recorded on samples collected from the rat frontal cortex before and after an intracortical TTX infusion (10 microM for 60 min) were subjected to multivariate statistical analysis. Glutamate, isoleucine, valine, alanine, and alpha- and beta-hydroxybutyrate were found to have decreased concentrations after the addition of TTX, suggesting that their release is likely from cortical neurons. In contrast, lactate, formate, acetate, glucose, creatinine, pyruvate, and other neurochemicals remained unchanged following local application of TTX. The present findings extend our previous work combining the analytical technology of small-volume nanoprobe NMR spectroscopy with in vivo microdialysis in freely moving animals and show that it is possible to apply metabonomics methodology to this important class of biofluid to monitor changes in neurochemical composition of the rat brain.     

Production of active recombinant human chymase from a construct containing the enterokinase cleavage site of trypsinogen in place of the native propeptide sequence

Human chymase, a chymotrypsin-like proteinase found in mast cells, was produced in an enzymatically active recombinant form. The protein was expressed in Escherichia coli as part of an insoluble fusion protein which was solubilized and renatured. The structure of the fusion protein was NH2-ubiquitin-enterokinase cleavage site-chymase-COOH. The enterokinase cleavage site of trypsinogen replaced the native propeptide sequence of chymase, allowing for activation by a readily available proteinase (enterokinase) of known specificity. Characterization of refolded-activated recombinant chymase with substrates and inhibitors demonstrated properties identical to that of the native proteinase isolated from skin.     

Isradipine produces neither a conditioned place preference nor aversion

Isradipine (ISR) has been reported to block cocaine-induced conditioned place preference. Using this procedure, the pairing of this L-type calcium blocker, at doses of 2.5, 5.0 and 10 mg/kg, with a preferred (cue-distinct) environment was investigated. In a separate experiment, ISR injection (10 mg/kg) was paired with the less-preferred environment to determine whether ISR produces a place preference. Testing in the non-drugged state revealed that ISR conditioning failed to affect side preference in both experiments. The neutral affective properties of ISR may be relevant to the development of cocaine use/abuse treatment regimens.     

Detection of time-resolved photoionization currents from pesticide-contaminated vegetation

Multiphoton-ionization fast-conductance (MPI-FC) was directly applied for detection of photoionization currents originated from pesticide traces irradiated by UV-laser. The principal methodology required for direct screening of pesticide traces deposited on various vegetational specimens was addressed. This approach was exemplified with 1,2,3,4,5,6-hexachlorocyclohexane and 3-amino-1,2,4-triazole, which are frequently found in agriculture-related industries. The range of pesticide concentrations covered was from 0.001 ng/mL to 1 mg/mL, and all measurements were carried out under ambient conditions. Our target vegetation substrates were sampled from the outdoors: grass, plant, and/or tree leaves. Neither material purification nor laborious extraction routines were utilized; thus employing MPI-FC for in situ/on-line monitoring of pesticide-contaminated vegetation is feasible.     

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.     

Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 microg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-alpha1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.     

A pilot study of hydroxyurea among patients with advanced human immunodeficiency virus (HIV) disease receiving chronic didanosine therapy: Canadian HIV trials network protocol 080

To assess the in vivo short-term antiretroviral effect of hydroxyurea in human immunodeficiency virus (HIV)-infected persons chronically treated with didanosine (ddI), 26 patients with CD4 cell counts between 100 and 350 were enrolled in a 12-week, open-label pilot study and randomly assigned to receive 500 or 1000 mg/day hydroxyurea. Clinical status, laboratory toxicities, CD4 lymphocyte count, and HIV RNA plasma virus load were assessed weekly. Median declines from baseline of 0.02 and 0.63 log10 HIV-1 RNA copies/mL of plasma were observed for the 500- and 1000-mg/day groups, respectively (P = .02). CD4 cell counts did not change significantly with the addition of hydroxyurea; however, a small but statistically significant decrease in counts was observed during the washout phase. Both doses of hydroxyurea were well-tolerated. These results demonstrate a substantial decrease in plasma virus load when 1000 mg of hydroxyurea is administered over 1 month as adjunctive therapy to ddI among HIV-infected persons with 100-350 CD4 cells/mm3.     

Needle exchange is not enough: lessons from the Vancouver injecting drug use study

OBJECTIVE: To describe prevalence and incidence of HIV-1, hepatitis C virus (HCV) and risk behaviours in a prospective cohort of injecting drugs users (IDU). SETTING: Vancouver, which introduced a needle exchange programme (NEP) in 1988, and currently exchanges over 2 million needles per year. DESIGN: IDU who had injected illicit drugs within the previous month were recruited through street outreach. At baseline and semi-annually, subjects underwent serology for HIV-1 and HCV, and questionnaires on demographics, behaviours and NEP attendance were completed. Logistic regression analysis was used to identify determinants of HIV prevalence. RESULTS: Of 1006 IDU, 65% were men, and either white (65%) or Native (27%). Prevalence rates of HIV-1 and HCV were 23 and 88%, respectively. The majority (92%) had attended Vancouver's NEP, which was the most important syringe source for 78%. Identical proportions of known HIV-positive and HIV-negative IDU reported lending used syringes (40%). Of HIV-negative IDU, 39% borrowed used needles within the previous 6 months. Relative to HIV-negative IDU, HIV-positive IDU were more likely to frequently inject cocaine (72 versus 62%; P < 0.001). Independent predictors of HIV-positive serostatus were low education, unstable housing, commercial sex, borrowing needles, being an established IDU, injecting with others, and frequent NEP attendance. Based on 24 seroconversions among 257 follow-up visits, estimated HIV incidence was 18.6 per 100 person-years (95% confidence interval, 11.1-26.0). CONCLUSIONS: Despite having the largest NEP in North America, Vancouver has been experiencing an ongoing HIV epidemic. Whereas NEP are crucial for sterile syringe provision, they should be considered one component of a comprehensive programme including counselling, support and education.     

Solutions and debugging for data consistency in multiprocessors with noncoherent caches

We analyze two important problems that arise in shared-memory multiprocessor systems. Thestale data problem involves ensuring that data items in local memory of individual processors are current, independent of writes done by other processors.False sharing occurs when two processors have copies of the same shared data block but update different portions of the block. The false sharing problem involves guaranteeing that subsequent writes are properly combined. In modern architectures these problems are usually solved in hardware, by exploiting mechanisms for hardware controlled cache consistency. This leads to more expensive and nonscalable designs. Therefore, we are concentrating on software methods for ensuring cache consistency that would allow for affordable and scalable multiprocessing systems. Unfortunately, providing software control is nontrivial, both for the compiler writer and for the application programmer. For this reason we are developing a debugging environment that will facilitate the development of compiler-based techniques and will help the programmer to tune his or her application using explicit cache management mechanisms. We extend the notion of a race condition for IBM Shared Memory System POWER/4, taking into consideration its noncoherent caches, and propose techniques for detection of false sharing problems. Identification of the stale data problem is discussed as well, and solutions are suggested.