Tetrahydrobiopterin protects against guanabenz-mediated inhibition of neuronal nitric-oxide synthase in vitro and in vivo.

It is established that guanabenz inhibits neuronal nitric-oxide (NO) synthase (nNOS) and causes the enhanced proteasomal degradation of nNOS in vivo. Although the time- and NADPH-dependent inhibition of nNOS has been reported in studies where guanabenz was incubated with crude cytosolic preparations of nNOS, the exact mechanism for inhibition is not known. Moreover, even less is known about how the inhibition of nNOS triggers its proteasomal degradation. In the current study, we show, with the use of purified nNOS, that guanabenz treatment leads to the oxidation of tetrahydrobiopterin and formation of a pterin-depleted nNOS, which is not able to form NO. With the use of 14C-labeled guanabenz, we were unable to detect any guanabenz metabolites or guanabenz-nNOS adducts, indicating that reactive intermediates of guanabenz probably do not play a role in the inhibition. Superoxide dismutase, however, prevents the guanabenz-mediated oxidation of tetrahydrobiopterin and inhibition of nNOS, suggesting the role of superoxide as an intermediate. Studies in rats show that administration of tetrahydrobiopterin prevents the inhibition and loss of penile nNOS due to guanabenz, indicating that the loss of tetrahydrobiopterin plays a major role in the effects of guanabenz in vivo. Our findings are consistent with the destabilization and enhanced degradation of nNOS found after tetrahydrobiopterin depletion. These studies suggest that drug-mediated destabilization and subsequent enhanced degradation of protein targets will likely be an important toxicological consideration.     

Binding of vascular heparan sulfate proteoglycan to Alzheimer's amyloid precursor protein is mediated in part by the N-terminal region of A4 peptide

The exact mechanisms of deposition and accumulation of amyloid in senile plaques and in blood vessels in Alzheimer's disease remain unknown. Heparan sulfate proteoglycans may play an important role in amyloid deposition in Alzheimer's disease. Previous investigations have demonstrated high affinity binding between heparan sulfate proteoglycans and the amyloid precursor, as well as with the A4 peptide. In the current studies, a specific vascular heparan sulfate proteoglycan found in senile plaques bound with high affinity to two amyloid protein precursors (APP₆₉₅ and APP₇₇₀). Vascular heparan sulfate proteoglycan also bound the Alzheimer's amyloid A4 peptide, and not other amyloid protein precursor regions studied, with high affinity. Both heparan sulfate glycosaminoglycan chains and chemically deglycosylated vascular heparan sulfate proteoglycan protein core bound to A4. High affinity interactions between vascular heparan sulfate proteoglycan and the A4 peptide may play a role in the process of amyloidogenesis in Alzheimer's disease, by localizing the site of deposition of A4, protecting A4 from further proteolysis, or by promoting aggregation and fibril formation.     

Using photography to cross generational, linguistic, and cultural barriers to develop useful survey instruments

Photographs are used as a research tool by anthropologists and as a technique to empower special populations, advocacy groups, and policy makers. This case describes how photography was used to develop a survey to study alcohol expectancies among Thai adolescents. A multi-cultural research team faced generational, linguistic, and cultural barriers in understanding Thai adolescent alcohol use well enough to write useful questions about alcohol expectancies. Asking adolescents to take and then discuss their photographs about alcohol allowed them to express themselves without the imposition of an organizational framework by the investigators. Group discussions of the photographs revealed nuances and subtleties of interpretation of behavior and expectancies involving alcohol that might otherwise have not emerged. The results of this activity were used to refine an expectancy scale used in a large-scale survey of alcohol expectancies and use.     

A novel HIV-1 reporter virus with a membrane-bound Gaussia princeps luciferase

HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1(NL4-3) genome using a picornaviral 2A-like sequence. The resulting HIV-1(NL4-3mGluc) virus expresses G. princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity.     

Autoxidation of brain homogenates from various animals as measured by thiobarbituric acid assay

INTRODUCTION: The TBARS assay has been well recognized for determination of lipid peroxidation and oxidative injury in biological samples including brain homogenates. In general, the homogenates are freshly prepared using rat brains as the tissue sources. In this study, we compared the rates of spontaneous lipid peroxidation in brain homogenates obtained from bovine, canine, hen, rat, and swine. In addition, the influences of lyophilization process and storage time up to six months at -20 degrees C without the freeze-thaw cycle were also determined in the swine brain preparations. METHODS: The standard assay for thiobarbituric acid-reactive substances (TBARS) was performed at 37 degrees C, using spectrophotometry to quantify the malondialdehyde (MDA) content. RESULTS: Rat brain homogenate exhibited the highest autoxidation rate (0.128+/-0.002 microM/min) whereas the bovine brain exhibited the lowest rate (0.032+/-0.001 microM/min). Swine brain homogenate could be kept at -20 degrees C up to 3 months without a significant increase in rate of autoxidation. Lyophilization caused a significant increase in the autoxidation rate of brain homogenate. However, the autoxidation rates of the lyophilized preparation were quite comparable throughout the six-month freezing time. DISCUSSION: Swine brain was a good candidate for tissue source in the TBARS reaction. The homogenate could be kept in the lyophilized form under the storage condition at -20 degrees C without the freeze-thaw cycle in the dark for at least six months.     

Pharmacologic management of the cardiorenal syndrome in heart failure

Cardiorenal syndrome describes the impairment of renal function and associated diuretic resistance in patients with heart failure and clinically manifest volume overload. The pathophysiology of this syndrome is poorly understood, but appears to be caused by impairment of tubuloglomerular feedback, neurohormonal activation, and other factors and therapies used in the management of heart failure. Early diagnosis of the cardiorenal syndrome by way of markers of renal injury and function is critical for timely interventions that may attenuate progression. Many novel therapies have been evaluated in the cardiorenal syndrome setting, including agents that block key local factors (eg, adenosine A_I receptor antagonists), improve diuresis, aquaresis, and natriuresis, and augment natural vasodilator mechanisms to improve renal perfusion. Furthermore, device-based approaches such as ultrafiltration may also play an important therapeutic role.     

Effect of topology of poly(L-lactide-co-ε-caprolactone) scaffolds on the response of cultured human umbilical cord Wharton’s jelly-derived mesenchymal stem cells and neuroblastoma cell lines

In this study, for the first time, a biodegradable poly(L-lactide-co-ε-caprolactone), PLC 67:33 copolymer was developed for use as temporary scaffolds in reconstructive nerve surgery. The effect of the surface topology and pore architecture were studied on the biocompatibility for supporting the growth of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) and human neuroblastoma cells (hNBCs) as cell models. Porous PLC membranes were prepared by electrospinning and phase immersion precipitation with particulate leaching and nonporous PLC membranes were prepared by solvent casting. From the results, the porous PLC membranes can support hWJ-MSCs and hNBCs cells better than the nonporous PLC membrane, and the interconnected pore scaffold prepared by electrospinning exhibited a more significant supporting attachment of the cells than the open pore and nonporous membranes. We can consider that these electrospun PLC membranes with 3-D interconnecting fiber networks and a high porosity warrant a potential use as nerve guides in reconstructive nerve surgery.