Passive immunization against oral AIDS virus transmission: An approach to prevent mother‐to‐infant HIV‐1 transmission?

To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.     

Effect of thiopental, propofol, and etomidate on vincristine toxicity in PC12 cells

Neurotoxicity is the dose-limiting side-effect of vincristine in cancer therapy. Using the nerve growth factor (NGF)-dependent neurite outgrowth and cell proliferation of the PC12 pheochromocytoma cell line as an in vitroassay, the protective effect of different intravenous anesthetics was assessed. Vincristine (1 nmol/L) significantly decreased the percentage of neurite-forming cells from 68%±9% to 27%±7% within a 3-day incubation period. The longer neurites (>2× cell body) in particular proved to be extremely sensitive to vincristine (from 17%±4% to 0% of total neurite-expressing cells). Flow cytometry results revealed an S-phase percentage of 15.85%±3.25% after NGF induction, with vincristine reducing this percentage to 0.68%±0.38%. Reversal of the inhibitory effect of vincristine was noted in the cells treated with thiopental or propofol but not etomidate. Bicuculline partially antagonized the protective effect of thiopental and propofol in both studies. We conclude that thiopental and propofol, but not etomidate, have a protective effect in vincristine-induced neurotoxicity. The protective effect produced by thiopental and propofol is probably secondary to activation of GABA_Areceptors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Neutral fat hydrolysis and long-chain fatty acid oxidation during anaerobic digestion of slaughterhouse wastewater

Neutral fat hydrolysis and long-chain fatty acid (LCFA) oxidation rates were determined during the digestion of slaughterhouse wastewater in anaerobic sequencing batch reactors operated at 25 degrees C. The experimental substrate consisted of filtered slaughterhouse wastewater supplemented with pork fat particles at various average initial sizes (D(in)) ranging from 60 to 450 microm. At the D(in) tested, there was no significant particle size effect on the first-order hydrolysis rate. The neutral fat hydrolysis rate averaged 0.63 +/- 0.07 d(-1). LCFA oxidation rate was modelled using a Monod-type equation. The maximum substrate utilization rate (kmax) and the half-saturation concentration (Ks) averaged 164 +/- 37 mg LCFA/L/d and 35 +/- 31 mg LCFA/L, respectively. Pork fat particle degradation was mainly controlled by LCFA oxidation rate and, to a lesser extent, by neutral fat hydrolysis rate. Hydrolysis pretreatment of fat-containing wastewaters and sludges should not substantially accelerate their anaerobic treatment. At a D(in) of 450 microm, fat particles were found to inhibit methane production during the initial 20 h of digestion. Inhibition of methane production in the early phase of digestion was the only significant effect of fat particle size on anaerobic digestion of pork slaughterhouse wastewater. Soluble COD could not be used to determine the rate of lipid hydrolysis due to LCFA adsorption on the biomass.     

Germanium oxide inhibits the transition from G2 to M phase of CHO cells

We report here for the first time that germanium oxide (GeO(2)) blocks cell progression. GeO(2) is not genotoxic to Chinese hamster ovary (CHO) cells and has limited cytotoxicity. However, GeO(2) arrests cells at G2/M phase. The proportion of cells stopped at G2/M phase increased dose-dependently up to 5 mM GeO(2) when treated for 12 h, but decreased at GeO(2) concentration was greater than 5 mM. Analysis of 5-bromodeoxyuridine-labeled cells indicated that GeO(2) delayed S phase progression in a dose-dependent manner, and blocked cells at G2/M phase. Microscopic examination confirmed that GeO(2) treatment arrested cells at G2 phase. Similar to several other events that cause G2 block, the GeO(2)-induced G2 block can also be ameliorated by caffeine in a dose- and time-dependent manner. To explore the mechanism of G2 arrest by GeO(2), cyclin content and cyclin-dependent kinase activity were examined. Cyclin B1 level was not affected after GeO(2) treatment in CHO cells. However, GeO(2) decreased p34(cdc2) kinase (Cdk1) activity. The kinase activity recovered within 9 h after GeO(2) removal and correlated with the transition of G2/M-G1 phase of the cells. This result suggests that GeO(2) treatment reduces Cdk1 activity and causing the G2 arrest in CHO cells.     

Endocytosis of epidermal growth factor receptor regulated by Grb2-mediated recruitment of the Rab5 GTPase-activating protein RN-tre

The Grb2 adaptor protein is best known for its role in signaling to the small GTPase p21(ras), mediated through its interaction with the SOS guanine nucleotide exchange factor. Here, we demonstrate that Grb2 also signals to Rab5, a small GTPase that plays a key role in early endocytic trafficking. Grb2 functions through association with RN-tre, a GTPase-activating protein for Rab5. Grb2 and RN-tre associate both in vitro and in vivo, with interaction mediated by both SH3 domains of Grb2 and extended proline-rich sequences in RN-tre. Association between Grb2 and RN-tre is constitutive and occurs independently of Eps8, a previously identified binding partner of RN-tre. Epidermal growth factor (EGF) stimulates recruitment of RN-tre to the EGF receptor (EGFR) in a Grb2-dependent manner. Grb2 and the EGFR are internalized and co-localized in endocytic vesicles in response to EGF. Overexpression of RN-tre blocks the internalization of both proteins, consistent with its function as a negative regulator of Rab5 and endocytosis. Strikingly, RN-tre does not block EGF-induced internalization of a Grb2 mutant deficient in RN-tre binding. These results 1) suggest that the ability of RN-tre to inhibit internalization of the EGFR requires Grb2-mediated binding to the receptor and 2) identify Grb2 as a critical regulator of Rab5 and EGFR endocytosis.     

HNK-1 sulfotransferase null mice express glucuronyl glycoconjugates and show normal cerebellar granule neuron migration in vivo and in vitro

Sulfoglucuronyl carbohydrate (SGC), reactive with antibody against human natural killer cell antigen, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system and has been implicated in cell-cell recognition, neurite outgrowth and neuronal migration during development, through its interaction with SGC-binding protein (SBP) 1. However, sulfotransferase (ST) null mutant mice, which lack SGC, were shown to have normal development with usual gross anatomy of the nervous system and other organs. Failure to observe a severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoproteins of the ST mutant nervous system. In the ST null mice, SGC-containing molecules were absent; instead the precursor glucuronyl carbohydrate (GC)-containing molecules accumulated. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites in the nervous system as the SGC molecules in the wild type. In vitro binding studies showed that, similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP-1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected in the mutant, possibly owing to interaction of SBP-1 with GC molecules. The results suggested that in vivo SBP-1-GC interaction was sufficient to allow normal neurite outgrowth and cell migration in the mutant, giving rise to a wild-type phenotype. However, the role of other compensatory molecules involved in these processes cannot be completely ruled out.     

Monitoring the correction of glycogen storage disease type 1a in a mouse model using [(18)F]FDG and a dedicated animal scanner

Monitoring gene therapy of glycogen storage disease type 1a in a mouse model was achieved using [(18)F]FDG and a dedicated animal scanner. The G6Pase knockout (KO) mice were compared to the same mice after infusion with a recombinant adenovirus containing the murine G6Pase gene (Ad-mG6Pase). Serial images of the same mouse before and after therapy were obtained and compared with wild-type (WT) mice of the same strain to determine the uptake and retention of [(18)F]FDG in the liver. Image data were acquired from heart, blood pool and liver for twenty minutes after injection of [(18)F]FDG. The retention of [(18)F]FDG was lower for the WT mice compared to the KO mice. The mice treated with adenovirus-mediated gene therapy had retention similar to that found in age-matched WT mice. These studies show that FDG can be used to monitor the G6Pase concentration in liver of WT mice as compared to G6Pase KO mice. In these mice, gene therapy returned the liver function to that found in age matched WT controls as measured by the FDG kinetics in the liver compared to that found in age matched wild type controls.     

The distally based lesser saphenous venofasciocutaneous flap for ankle and heel reconstruction

Finding an appropriate soft-tissue grafting material to close a wound located over the ankle and heel can be a difficult task. The distally based lesser saphenous venofasciocutaneous flap mobilized from the posterior aspect of the upper leg, used as an island pedicle skin flap, can be useful for this purpose. The vascular supply to the flap is derived from the retrograde perfusion of the accompanying arteries of the lesser saphenous vein. These arteries descend along both sides of the lesser saphenous vein to the distal third of the leg, either terminating or anastomosing with the septocutaneous perforators of the peroneal artery. Between February of 1999 and March of 2001, four variants of this flap were applied in 21 individuals, including 11 fasciocutaneous, five fascial, three sensory, and two fasciomyocutaneous flaps. Skin defects among all patients were combined with bone, joint, and/or tendon exposure. The authors found that the flap was reliable and technically simple to design and execute. This one-stage procedure not only preserves the major arteries and the sural nerve of the injured leg, but it also has proved valuable for covering a weight-bearing heel and filling a deep defect, because it potentially provides protective sensation and a well-vascularized muscle fragment. When conventional local flaps are inadequate, this flap should be considered for its reliability and low associated morbidity.     

Design and synthesis of aminophenol-based factor Xa inhibitors

A novel potent and selective aminophenol scaffold for fXa inhibitors was developed from a previously reported benzimidazole-based naphthylamidine template. The aminophenol template is more synthetically accessible than the benzimidazole template, which simplified the introduction of carboxylic acid groups. Substitution of a propenyl-para-hydroxy-benzamidine group on the aminophenol template produced selective, sub-nanomolar fXa inhibitors. The potency of the inhibitors is partially explained with the aid of a trypsin complex crystal structure.     

Strain improvement to enhance the production of recombinant penicillin acylase in high-cell-density Escherichia coli cultures

Using fed-batch operation for high-cell-density cultivation, efforts are frequently made for optimization of culture parameters, particularly feeding strategy. The current study also emphasized the importance of selecting strains for the production of recombinant proteins in high-cell-density cultures. With Escherichia coli penicillin acylase (PAC) as a target protein, the host/vector system of MDdeltaP7 harboring pTrcKnPAC2902 and pKS12 was designed for optimization of fed-batch cultivation for recombinant protein production. The host, MDdeltaP7, potentially had a high translational and periplasmic processing efficiency for pac expression. On the other hand, the vector, pTrcKnPAC2902, was genetically constructed for pac overexpression. Coexistence of the other vector, pKS12, significantly enhanced PAC production by improving cell physiology and reducing the amount of inclusion body formation upon pac overexpression. An extremely high volumetric PAC activity at 37,500 U/L was obtained with the use of the developed host/vector system under optimum fed-batch culture conditions.