NeuroPAL: A Multicolor Atlas for Whole-Brain Neuronal Identification in C. elegans

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Stereospecific synthesis of α-methylated amino acids

Summary. Both 2,5-trans and 2,5-cis disubstituted 2-tert-butyl-5-(indol-3-yl)methylimidazolidin-4-ones were synthesised and their enolates were prepared using LDA. While the enolate of the 2,5-trans disubstituted derivative could not be methylated, the enolate of the cis-2,5-disubstituted derivative was successfully methylated with methyl iodide to a product which on hydrolysis gave enantiomerically pure α-methyl-L-tryptophan. Received October 31, 1998, Accepted July 23, 1999     

Optimization of pulsed electromagnetic field therapy for management of arthritis in rats

Studies were undertaken to find out the effects of low frequency pulsed electromagnetic field (PEMF) in adjuvant induced arthritis (AIA) in rats, a widely used model for screening potential therapies for rheumatoid arthritis (RA). AIA was induced by an intradermal injection of a suspension of heat killed Mycobacterium tuberculosis (500 mug/0.1 ml) into the right hind paw of male Wistar rats. This resulted in swelling, loss of body weight, increase in paw volume as well as the activity of lysosomal enzymes viz., acid phosphatase, cathepsin D, and beta-glucuronidase and significant radiological and histological changes. PEMF therapy for arthritis involved optimization of three significant factors, viz., frequency, intensity, and duration; and the waveform used is sinusoidal. The use of factorial design in lieu of conventional method resulted in the development of an ideal combination of these factors. PEMF was applied using a Fransleau-Braunbeck coil system. A magnetic field of 5 Hz x 4 muT x 90 min was found to be optimal in lowering the paw edema volume and decreasing the activity of lysosomal enzymes. Soft tissue swelling was shown to be reduced as evidenced by radiology. Histological studies confirmed reduction in inflammatory cells infiltration, hyperplasia, and hypertrophy of cells lining synovial membrane. PEMF was also shown to have a membrane stabilizing action by significantly inhibiting the rate of release of beta-glucuronidase from lysosomal rich and sub-cellular fractions. The results indicated that PEMF could be developed as a potential therapy in the treatment of arthritis in humans.     

Thymidine kinase-independent intracellular delivery of bioactive nucleotides by aryl phosphate derivatives of bromo-methoxy zidovudine (compounds WHI-05 and WHI-07) in normal human female genital tract epithelial cells and sperm

The compounds WHI-05 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-methoxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-bromophenyl] methoxyalaninyl phosphate) are aryl phosphate derivatives of zidovudine (ZDV) with dual-function anti-human immunodeficiency virus and contraceptive activity. These drugs were rationally designed to bypass the thymidine kinase (TK) dependency of ZDV activation as well as to achieve spermicidal activity. We investigated the TK activity and intracellular metabolism of WHI-05 and WHI-07 in normal human vaginal and cervical epithelial cells as well as sperm. The time- and concentration-dependent intracellular formation of ZDV metabolites following addition of WHI-05 and WHI-07 to normal human vaginal, ectocervical, and endocervical epithelial cells as well as motile sperm was studied by analytical HPLC. Thymidine kinase activity in these cells was determined by the flow cytometric method based on intracellular phosphorylation of the fluorescent nucleoside, 5-amino-2-deoxyuridine-dansyl chloride and by the ability of cell-free extracts to convert [(3)H]thymidine to thymidine monophosphate in comparison to NALM-6, a pre-B leukemia cell line. TK activity of genital tract epithelial cells and sperm was found to be relatively low or lacking. Addition of WHI-05 and WHI-07 to vaginal and cervical epithelial cells resulted in their concentration- and time-dependent conversion to alaninyl ZDV monophosphate (Ala-ZDV-MP) and 5'-ZDV monophosphate as the major metabolites. Studies using motile human sperm also demonstrated the conversion of WHI-05 and WHI-07 to Ala-ZDV-MP. These results demonstrate that human female genital tract epithelial cells and sperm efficiently convert WHI-05 and WHI-07 to bioactive ZDV metabolites despite their TK deficiency.     

Differential regulation of IL-1beta and TNF-alpha RNA expression by MEK1 inhibitor after focal cerebral ischemia in mice

Activation of the extracellular-signal-responsive kinase (ERK 1/2) by MAP kinase/ERK kinase (MEK1/2) following ischemia/reperfusion in the brain has been associated with cell death since inhibition of MEK1/2 provides neuroprotection in cerebral ischemia injury. Since inflammation has been implicated in ischemic brain injury, the present study investigated whether MEK1/2 modifies expression of two key inflammatory cytokines, IL-1beta and TNFalpha, that have been shown to exacerbate ischemic brain injury. A mouse model of transient cerebral ischemia was deployed to test the effect of selective MEK1/2 inhibitor (SL327) on infarct size and cytokine expression. SL327 (100 mg/kg, i.p.) administered 15 min prior to ischemia resulted in 64% reduction in infarct size over controls (n = 8, P < 0.01). Under the same condition, SL327 significantly reduced peak expression of IL-1beta mRNA (59% reduction compared to vehicle, P < 0.01, n = 4) but not TNF-alpha mRNA. A parallel reduction in IL-1beta protein (67%, P < 0.05, n = 6) was also observed using ELISA analysis. These data suggest that the neuroprotective effect of MEK1/2 inhibition may be mediated by suppression of IL-1beta. The study also demonstrates for the first time that these two cytokines are differentially regulated by kinase mediated signaling pathways.     

Studies on the protective role of vitamin C and E against polychlorinated biphenyl (Aroclor 1254)—induced oxidative damage in Leydig cells

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. Polychlorinated biphenyls (PCBs) are global environmental contaminants that cause disruption of the endocrine system in human and animals. The present study was conducted to elucidate the protective role of vitamin C and E against Aroclor 1254-induced changes in Leydig cell steroidogenesis and antioxidant system. Adult male rats were dosed for 30 days with daily intraperitoneal (ip) injection of 2 mg/kg Aroclor or vehicle (corn oil). One group of rats was treated with vitamin C (100 mg/kg bw/day) while the other group was treated with vitamin E (50 mg/kg bw/day) orally, simultaneously with Aroclor 1254 for 30 days. One day after the last treatment, animals were euthanized and blood was collected for the assay of serum hormones such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), prolactin (PRL), triiodothyronine (T₃), thyroxine (T₄), testosterone and estradiol. Testes were quickly removed and Leydig cells were isolated in aseptic condition. Purity of Leydig cells was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. Purified Leydig cells were used for quantification of cell surface LH receptors and steroidogenic enzymes such as cytochrome P₄₅₀ side chain cleavage enzyme (P₄₅₀scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β- HSD). Leydig cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl transpeptidase (γ-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. Aroclor 1254 treatment significantly reduced the serum LH, TSH, PRL, T₃, T₄, testosterone and estradiol. In addition to this, Leydig cell surface LH receptors, activities of the steroidogenic enzymes such as cytochrome P₄₅₀scc, 3β-HSD, 17β-HSD, antioxidant enzymes SOD, CAT, GPX, GR, γ-GT, GST and non-enzymatic antioxidants such as vitamin C and E were significantly diminished whereas, LPO and ROS were markedly elevated. However, the simultaneous administration of vitamin C and E in Aroclor 1254 exposed rats resulted a significant restoration of all the above-mentioned parameters to the control level. These observations suggest that vitamin C and E have ameliorative role against adverse effects of PCB on Leydig cell steroidogenesis.     

Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases

3′-phosphoadenosine 5′-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5′-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3′-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3′-OH requires Mg₂⁺ and purported to involve residues D₈₇GD₈₉N. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D₈₇ and D₈₉ did not hamper ATPS activity however abolished APSK activity severely. Thus, D₈₇GD₈₉N region is required for stabilization of Mg²⁺-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3′-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gamma³²P-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg²⁺ and APS of wild type and mutants supports the experimental results.     

South American Plasmodium falciparum after the malaria eradication era: clonal population expansion and survival of the fittest hybrids

Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradication's successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999-2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006-2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.