Striatal dopamine level contributes to hydroxyl radical generation and subsequent neurodegeneration in the striatum in 3-nitropropionic acid-induced Huntington's disease in rats

We tested the hypothesis that dopamine contributes significantly to the hydroxyl radical (OH)-induced striatal neurotoxicity caused by 3-nitropropionic acid (3-NP) in a rat model of Huntington's disease. Dopamine (10–100 μM) or 3-NP (10–1000 μM) individually caused a significant increase in the generation of hydroxyl radical (OH) in the mitochondria, which was synergistically enhanced when the lowest dose of the neurotoxin (10 μM) and dopamine (100 μM) were present together. Similarly, systemic administration of l-DOPA (100–250 mg/kg) and a low dose of 3-NP (10 mg/kg) potentiated OH generation in the striatum, and the rats exhibited significant decrease in stride length, a direct indication of neuropathology. The pathology was also evident in striatal sections subjected to NeuN immunohistochemistry. The significant changes in stride length, the production of striatal OH and neuropathological features due to administration of a toxic dose of 3-NP (20 mg/kg) were significantly attenuated by treating the rats with tyrosine hydroxylase inhibitor α-methyl-p-tyrosine prior to 3-NP administration. These results strongly implicate a major contributory role of striatal dopamine in increased generation of OH, which leads to striatal neurodegeneration and accompanied behavioral changes, in 3-NP model of Huntington's disease.     

Molecular modeling of flexible arm‐mediated interactions between bacterial chemoreceptors and their modification enzyme

Sensory adaptation in bacterial chemotaxis is mediated by methylation and demethylation of specific glutamyl residues in the cytoplasmic domain of chemoreceptors. Methylation is catalyzed by methyltransferase CheR. In E. coli and related organisms, methylation sufficiently rapid to be physiologically effective requires a carboxyl terminal pentapeptide sequence on the receptor being modified or, via adaptational assistance, on a neighboring homodimer in a receptor cluster. Pentapeptide‐enhanced methylation is thought to be mediated by a ～30 residue, potentially disordered sequence that serves as a flexible arm connecting the receptor body and pentapeptide‐bound methyltransferase, thus allowing diffusionally restricted enzyme to reach methyl‐accepting sites. However, it was not known how many or which sites on the same or neighboring receptors were accessible to the tethered enzyme. We investigated using molecular modeling and found that, in a hexagonal array of trimers of receptor dimers, CheR tethered to a dimer of chemoreceptor Tar by its native 30‐residue flexible‐arm sequence could reach all methyl‐accepting sites on the dimer to which it was tethered plus 48 methyl‐accepting sites distributed among nine neighboring dimers, equivalent to the total sites carried by six receptors. This modeling‐determined methylation neighborhood of one enzyme‐binding dimer and six neighbors corresponds precisely with the experimentally identified neighborhood of seven. Thus, the experimentally observed adaptational assistance can occur by docking of pentapeptide‐bound, diffusionally restricted enzyme to methyl‐accepting sites on neighboring receptors. Our analysis introduces the notion that physiologically relevant adaptational assistance could occur even if only a subset of sites on a particular receptor are within reach.     

Nevirapine Resistance and Breast-Milk HIV Transmission: Effects of Single and Extended-Dose Nevirapine Prophylaxis in Subtype C HIV-Infected Infants

Background

Daily nevirapine (NVP) prophylaxis to HIV-exposed infants significantly reduces breast-milk HIV transmission. We assessed NVP-resistance in Indian infants enrolled in the “six-week extended-dose nevirapine” (SWEN) trial who received single-dose NVP (SD-NVP) or SWEN for prevention of breast-milk HIV transmission but who also acquired subtype C HIV infection during the first year of life.

Methods/Findings

Standard population sequencing and cloning for viral subpopulations present at ≥5% frequency were used to determine HIV genotypes from 94% of the 79 infected Indian infants studied. Timing of infection was defined based on when an infant''s blood sample first tested positive for HIV DNA. SWEN-exposed infants diagnosed with HIV by six weeks of age had a significantly higher prevalence of NVP-resistance than those who received SD-NVP, by both standard population sequencing (92% of 12 vs. 38% of 29; p = 0.002) and low frequency clonal analysis (92% of 12 vs. 59% of 29; p = 0.06). Likelihood of infection with NVP-resistant HIV through breast-milk among infants infected after age six weeks was substantial, but prevalence of NVP-resistance did not differ among SWEN or SD-NVP exposed infants by standard population sequencing (15% of 13 vs. 15% of 20; p = 1.00) and clonal analysis (31% of 13 vs. 40% of 20; p = 0.72). Types of NVP-resistance mutations and patterns of persistence at one year of age were similar between the two groups. NVP-resistance mutations did differ by timing of HIV infection; the Y181C variant was predominant among infants diagnosed in the first six weeks of life, compared to Y188C/H during late breast-milk transmission.

Conclusions/Significance

Use of SWEN to prevent breast-milk HIV transmission carries a high likelihood of resistance if infection occurs in the first six weeks of life. Moreover, there was a continued risk of transmission of NVP-resistant HIV through breastfeeding during the first year of life, but did not differ between SD-NVP and SWEN groups. As with SD-NVP, the value of preventing HIV infection in a large number of infants should be considered alongside the high risk of resistance associated with extended NVP prophylaxis.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00061321","term_id":"NCT00061321"}}NCT00061321     

Optimization and scale-up of Wharton's jelly-derived mesenchymal stem cells for clinical applications

MSCs are promising candidates for stem cell therapy and regenerative medicine. Umbilical cord is the easiest obtainable biological source of MSCs and the Wharton's jelly of the umbilical cord is a rich source of fetus-derived stem cells. However, the use of MSCs for therapeutic application is based on their subsequent large-scale in vitro expansion. A fast and efficient protocol for generation of large quantities of MSCs is required to meet the clinical demand and biomedical research needs. Here we have optimized conditions for scaling up of WJ-MSCs. Low seeding density along with basic fibroblast growth factor (bFGF) supplementation in the growth medium, which is DMEM-KO, resulted in propagation of more than 1 x 10(8) cells within a time period of 15 days from a single umbilical cord. The upscaled WJ-MSCs retained their differentiation potential and immunosuppressive capacity. They expressed the typical hMSC surface antigens and the addition of bFGF in the culture medium did not affect the expression levels of HLA-DR and CD 44. A normal karyotype was confirmed in the large-scale expanded WJ-MSCs. Hence, in this study we attempted rapid clinical-scale expansion of WJ-MSCs which would allow these fetus-derived stem cells to be used for various allogeneic cell-based transplantations and tissue engineering.     

1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) induces phosphorylation of eukaryotic elongation factor-2 (eEF2): a cautionary note on the anticancer mechanism of an eEF2 kinase inhibitor

Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.     

In Vivo Substrates of the Lens Molecular Chaperones αA-Crystallin and αB-Crystallin

αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin.     

Preservation of cGMP-induced relaxation of pulmonary veins of fetal lambs exposed to chronic high altitude hypoxia: role of PKG and Rho kinase

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.