Selective recovery of lactate dehydrogenase using affinity foam

Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step.     

Development of "normal" dermatomes and somatotopic maps by "abnormal" populations of cutaneous neurons

During development, motor and sensory axons grow to peripheral targets with remarkable precision. Whereas much has been learned about the development of motoneuron connectivity, less is known about the regulation of cutaneous innervation. In adults, dorsal root ganglia (DRG) innervate characteristic skin regions, termed dermatomes, and their axons project somatotopically in the dorsal horn. Here, we have investigated whether cutaneous neurons are selectively matched with specific skin regions, and whether peripheral target skin influences the central connections of cutaneous neurons. To address these questions, we shifted limb buds rostrally in chick embryos prior to axon outgrowth, causing DRGs to innervate novel skin regions, and mapped the resulting dermatomes and central projections. Following limb shifts, cutaneous innervation arose from more rostral and from fewer DRGs than normal, but the overall dermatome pattern was preserved. Thus, DRGs parcel out innervation of skin in a consistent manner, with no indication of matching between skin and DRGs. Similarly, cutaneous nerves established a "normal" somatotopic map in the dorsal horn, but in more rostral segments than usual. Thus, the peripheral target skin may influence the pattern of CNS projections, but does not direct cutaneous axons to specific populations of neurons in the dorsal horn.     

Recent advances in the search for new anti-coccidial drugs

Coccidia provide a rich hunting ground for drug-designers, as there are significant biochemical differences between the parasites and their hosts. Recent years have brought the discovery of the plastid and its possible metabolic machinery, characterisation of acidocalcisomes, reports on the apparent absence from some coccidia of a typical mitochondrion, and the discovery of the mannitol cycle and shikimate pathway in the parasites. Moreover, modern technologies such as genomics and proteomics are bringing new insights into the biochemistry of coccidia and highlighting possible drug targets in abundance. A major issue for would-be drug discoverers is to decide upon the targets to prioritise. This review provides an update on recent findings on how coccidia differ biochemically from vertebrates. It includes discoveries within coccidian parasites themselves but also uses findings in Plasmodium to provide an overview of biochemical features that may be characteristics of many apicomplexan parasites and so potential targets for broad-spectrum drugs.     

Mgm1p,a dynamin-related GTPase,is essential for fusion of the mitochondrial outer membrane

In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p. We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion. Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division. In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents. Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion. Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes. Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures. However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria. Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane. Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p.     

The Impact of Implementing a Test,Treat and Retain HIV Prevention Strategy in Atlanta among Black Men Who Have Sex with Men with a History of Incarceration: A Mathematical Model

Background

Annually, 10 million adults transition through prisons or jails in the United States (US) and the prevalence of HIV among entrants is three times higher than that for the country as a whole. We assessed the potential impact of increasing HIV Testing/Treatment/Retention (HIV-TTR) in the community and within the criminal justice system (CJS) facilities, coupled with sexual risk behavior change, focusing on black men-who-have-sex-with-men, 15–54 years, in Atlanta, USA.

Methods

We modeled the effect of a HIV-TTR strategy on the estimated cumulative number of new (acquired) infections and mortality, and on the HIV prevalence at the end of ten years. We additionally assessed the effect of increasing condom use in all settings.

Results

In the Status Quo scenario, at the end of 10 years, the cumulative number of new infections in the community, jail and prison was, respectively, 9246, 77 and 154 cases; HIV prevalence was 10815, 69 and 152 cases, respectively; and the cumulative number of deaths was 2585, 18 and 34 cases, respectively. By increasing HIV-TTR coverage, the cumulative number of new infections could decrease by 15% in the community, 19% in jail, and 8% in prison; HIV prevalence could decrease by 8%, 9% and 7%, respectively; mortality could decrease by 20%, 39% and 18%, respectively. Based on the model results, we have shown that limited use and access to condoms have contributed to the HIV incidence and prevalence in all settings.

Conclusions

Aggressive implementation of a CJS-focused HIV-TTR strategy has the potential to interrupt HIV transmission and reduce mortality, with benefit to the community at large. To maximize the impact of these interventions, retention in treatment, including during the period after jail and prison release, and increased condom use was vital for decreasing the burden of the HIV epidemic in all settings.     

Enteric absorption and pharmacokinetics of oseltamivir in critically ill patients with pandemic (H1N1) influenza

Background

Whether the enteric absorption of the neuraminidase inhibitor oseltamivir is impaired in critically ill patients is unknown. We documented the pharmacokinetic profile of oseltamivir in patients admitted to intensive care units (ICUs) with suspected or confirmed pandemic (H1N1) influenza.

Methods

We included 41 patients 18 years of age and older with suspected or confirmed pandemic (H1N1) influenza who were admitted for ventilatory support to nine ICUs in three cities in Canada and Spain. Using tandem mass spectrometry, we assessed plasma levels of oseltamivir free base and its active metabolite carboxylate at baseline (before gastric administration of the drug) and at 2, 4, 6, 9 and 12 hours after the fourth or later dose.

Results

Among the 36 patients who did not require dialysis, the median concentration of oseltamivir free base was 10.4 (interquartile range [IQR] 4.8–14.9) μg/L; the median concentration of the carboxylate metabolite was 404 (IQR 257–900) μg/L. The volume of distribution of the carboxylate metabolite did not increase with increasing body weight (R² = 0.00, p = 0.87). The rate of elimination of oseltamivir carboxylate was modestly correlated with estimations of creatinine clearance (R² = 0.27, p < 0.001). Drug clearance in the five patients who required continuous renal replacement therapy was about one-sixth that in the 36 patients with relatively normal renal function.

Interpretation

Oseltamivir was well absorbed enterically in critically ill patients admitted to the ICU with suspected or confirmed pandemic (H1N1) influenza. The dosage of 75 mg twice daily achieved plasma levels that were comparable to those in ambulatory patients and were far in excess of concentrations required to maximally inhibit neuraminidase activity of the virus. Adjustment of the dosage in patients with renal dysfunction requiring continuous renal replacement therapy is appropriate; adjustment for obesity does not appear to be necessary.A substantial number of cases of pandemic (H1N1) influenza have involved young adults and adolescents without serious comorbidities who present with severe viral pneumonia complicated by acute respiratory distress syndrome, rhabdomyolysis, renal failure and, occasionally, shock.^1,2 Antiviral therapy in such critically ill patients typically requires oral or nasogastric administration of the neuraminidase inhibitor oseltamivir. Current guidelines from the World Health Organization for the pharmacologic management of progressive or severe pandemic (H1N1) influenza recommend the consideration of high-dose therapy (≥ 150 mg twice daily).^3,4 Critically ill patients exhibit defects in gastrointestinal absorption because of impaired gut perfusion, edema of the bowel wall and ileus as a consequence of critical illness and shock.⁵ Whether the enteric absorption of oseltamivir is impaired in such patients is unknown.We undertook this study to document the pharmacokinetic profile of oseltamivir administered orally or by nasogastric tube in patients admitted to intensive care units (ICUs) with respiratory failure due to suspected or confirmed pandemic (H1N1) influenza.