Adeno-associated viral serotypes differentially transduce inhibitory neurons within the rat amygdala.

Recombinant adeno-associated viruses (AAV) are frequently used to make localized genetic manipulations within the rodent brain. It is accepted that the different viral serotypes possess differing affinities for particular cell types, but it is not clear how these properties affect their ability to transduce specific neuronal cell sub-types. Here, we examined ten AAV serotypes for their ability to transduce neurons within the rat basal and lateral nuclei of the amygdala (BLA) and the central nucleus of the amygdala (CeA). AAV2 based viral genomes designed to express either green fluorescent protein (GFP) from a glutamate decarboxylase (GAD65) promoter or the far-red fluorescent protein (E2-Crimson) from a phosphate-activated glutaminase (PAG) promoter were created and pseudotyped as AAV2/1, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV 2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8. These viruses were infused into the BLA and CeA at equal titers and twenty-one days later tissue within the amygdala was examined for viral transduction efficiency. These serotypes transduced neurons with similar efficiency, except for AAV4 and AAV5, which exhibited significantly less efficient neuronal transduction. Notably, AAV4 and AAV5 possess the most divergent capsid protein sequences compared to the other commonly available serotypes. We found that the Gad65-GFP virus did not exclusively express GFP within inhibitory neurons, as assessed by fluorescent in situ hybridization (FISH), but when this virus was used to transduce CeA neurons, the majority of the neurons that expressed GFP were in fact inhibitory neurons and this was likely due to the fact that this nucleus contains a very high percentage of inhibitory neurons.

Autologous olfactory [corrected] mucosal transplant in chronic spinal cord injury: an Indian Pilot Study.

STUDY DESIGN: Prospective Pilot Study. OBJECTIVES: To determine the safety and feasibility of autologous olfactory mucosal transplantation into the spinal cord in chronic spinal cord injured using the technique developed by Carlos Lima et al. SETTING: Spinal Injury Center, New Delhi. METHODS: Five chronic, motor complete, traumatic spinal cord injury (SCI) patients with neurological level C5-T12 underwent the procedure. Participants were assessed at baseline and at 6 monthly intervals. Safety and tolerability were evaluated through monitoring for any adverse events and tests including magnetic resonance imaging (MRI) evaluation. Efficacy assessment was done through neurological, functional and psychological evaluation, electrophysiological studies and urodynamics. RESULTS: Surgery was tolerated well by all American Spinal Injury Association (ASIA) Impairment Scale (AIS) A participants. The only AIS B participant lost sensory scores significantly after surgery but is gradually regaining it. MRI evaluation revealed a syrinx in one participant and increase in length of myelomalacia in four participants. There were no other adverse findings on MRI evaluation. There was no significant improvement in any of the neurological, electrophysiological or urodynamic efficacy variables. Statistically significant improvement was seen in functional scores as evaluated by Spinal Cord Independence Measure, Beck Depression Inventory scores and life impact scores on International Spinal Cord Injury Scale. CONCLUSIONS: The procedure is relatively safe and feasible in AIS A participants with thoracic level injuries at 18 month follow-up. No efficacy could be demonstrated which could be attributed to the procedure.

Rapamycin induces transactivation of the EGFR and increases cell survival.

The mammalian target of rapamycin (mTOR) signaling network regulates cell growth, proliferation and cell survival. Deregulated activation of this pathway is a common event in diverse human diseases such as cancers, cardiac hypertrophy, vascular restenosis and nephrotic hypertrophy. Although mTOR inhibitor, rapamycin, has been widely used to inhibit the aberrant signaling due to mTOR activation that plays a major role in hyperproliferative diseases, in some cases rapamycin does not attenuate the cell proliferation and survival. Thus, we studied the mechanism(s) by which cells may confer resistance to rapamycin. Our data show that in a variety of cell types the mTOR inhibitor rapamycin activates extracellularly regulated kinases (Erk1/2) signaling. Rapamycin-mediated activation of the Erk1/2 signaling requires (a) the epidermal growth factor receptor (EGFR), (b) its tyrosine kinase activity and (c) intact autophosphorylation sites on the receptor. Rapamycin treatment increases tyrosine phosphorylation of EGFR without the addition of growth factor and this transactivation of receptor involves activation of c-Src. We also show that rapamycin treatment triggers activation of cell survival signaling pathway by activating the prosurvival kinases Erk1/2 and p90RSK. These studies provide a novel paradigm by which cells escape the apoptotic actions of rapamycin and its derivatives that inhibit the mTOR pathway.

Dynamic model of HIV/AIDS population of Agra region.

The Human Immunodeficiency Virus / Acquired Immunodeficiency syndrome (HIV/AIDS) is spreading rapidly in all regions of the world. But in India it is only 20 years old. Within this short period it has emerged as one of the most serious public health problems in the country, which greatly affect the socio-economical growth. The HIV problem is very complex and ill defined from the modeling point of view. Keeping in the view the complexities of the HIV infection and its transmission, it is difficult to make exact estimates of HIV prevalence. It is more so in the Indian context, with its typical and varied cultural characteristics, and its traditions and values with special reference to sex related risk behaviors. Therefore, it is necessary to develop a good model which will help in making exact estimates of HIV prevalence that may be used for planning HIV / AIDS prevention and control programs. In this paper Neuro-Fuzzy approach has been used to develop dynamic model of HIV population of Agra region.

O K and Cu LIII edge study of itinerant holes in I2-, Hgl2- and HgBr2- intercalated BSCCO(2212) single crystals.

Intercalation effect on BSCCO (2212) system, although it increases the interlayer distance and the c-axis remarkably, produces only a small change in the transition temperature. Thus, amongst other things, intercalation provides an effective method to investigate the influence of the interblock coupling. Electrons are transferred from the host Cu-O2 layers to the guest molecules I2, HgBr2, HgI2 leading to evolution of the Tc. For this we have made high resolution XANES study on the O K and Cu L3 edges to estimate the density of the doping holes. We attempt on basis of our and earlier results the evolution of Tc in these as also the much larger decrease produced in Tc for I2-intercalation for which the increase in basal spacing is the smallest of the three halides.

Simultaneous measurement of XANES in halide-intercalated BSCCO(2212) using electron and fluorescence yield to compare their performance.

Total Yield with an escape depth of approximately 100-200 A is known to be rather surface sensitive. Fluorescence Yield, on the other hand, with an escape depth of approximately 1000-2000 A is relatively less prone to surface effects but necessitates some corrections to obtain the true signal. Both have their plus and minus points and, if used with care, yield reliable data. In the present experiment both the techniques have been simultaneously employed for measuring orientation dependent O K and the Cu L3 edges from an uncleaved surface of I(2)BSCCO(2212) single crystal to compare the performance of the two modes of detection. Despite glaring differences in intensities the results from the two appear to show reasonable agreement in respect of relative intensities of the spectral features.

Polarization-dependent XANES study of Bi2Sr2Ca(1-x)Pr(x)Cu2O8-delta insulating single crystal.

Pr doping has been extensively studied in cuprate superconductors to understand the mechanism of quenching of superconductivity in cuprate perovskites. Experience has revealed that it acts differently in different cuprate perovskites. We have made high resolution polarization XANES measurements on a Pr-doped single crystal within the ab-plane on the Cu K and Pr L III absorption edges to ascertain the valence state of Pr cation and thereby try learning how it is quenching superconductivity in the present case. Our results show that besides the Pr quenching holes the possibility of its localizing some holes through hybridization with O 2p and Cu 3d cannot be ruled out.

Identification of a strong promoter of bacteriophage MB78 that interacts with a host coded factor and regulates the expression of a structural protein.

A strong promoter of bacteriophage MB78 which controls the expression of a small structural protein of the phage has been identified and characterized. Analysis of its nucloetide sequence upstream of the translational start site revealed the presence of conserved -10 (TAATAT) and -35 (TTCTCCT) regions. It was observed that transcription initiates with thymidine residue, 86 nt upstream of the translational start site. Transcription seems to undergo alternate up and down regulation. This promoter is efficiently recognized by sigma 70 RNA polymerase and a host factor also binds to it. Binding of RNA polymerase is independent of binding of the host factor.

Bradykinin analogs as inhibitors of angiotensin-converting enzyme.

A series of peptide analogs and fragments of bradykinin were designed and synthesized on solid supports using Boc and Fmoc strategies, and on polyethylene pins using Fmoc strategy. The peptides were purified, characterized and tested for their inhibitory effects on angiotensin-converting enzyme. The inhibition of the converting enzyme. The inhibition of the enzyme was measured spectrophotometrically using Furylacryloyl-Phe-Gly-Gly as the substrate. Apparent Ki's were determined for the substrates, which exhibited significant inhibition in the initial screening assay using 10 microM of the peptide inhibitor. Short peptides corresponding to the carboxyl terminus of bradykinin were found to be poor inhibitors of angiotensin-converting enzyme. However, bradykinin-like peptides with modifications at their amino terminus are effective inhibitors. The best inhibitor found in this study, Ala2,6-des-Pro3-bradykinin, has an apparent Ki of 30.2 nM, compared to an apparent Ki of 94 nM for des-Pro3-bradykinin, which was reported to be a better inhibitor of angiotensin-converting enzyme than captopril.

A chemo-pharmacological study of Hypericum perforatum L

Arthritis was produced in adult albino rats weighing 120+-10g through intramuscular injections of 0.5ml of 2 percent w/w formalin beneath the plantar aponeurosis on the first and third day. The anterioposterior diameter of the ankle was recorded and statistically analyzed. It was evident that relatively low doses, 0.25ml/100g body weight, of tincture of Hypericum perforatum produced a significant 48 percent anti-inflammatory effect, whereas higher doses, 0.5ml/100g body weight, of the drug showed 32 percent activity under identical conditions. Parallel studies with 1mg/100g body weight hydrocortisone administration gave similar anti-inflammatory effects. Local application of an alcoholic extract of the drug in very small doses also produced significant skin softness and flexiblility of muscle

Synthesis and biological evaluation of the superagonist [N alpha-chlorotriazinylaminofluorescein-Ser1,Nle4,D-Phe7]-al pha-MSH.

The fluorescein-labeled melanotropin [N alpha-chlorotriazinylaminofluorescein-Ser1,Nle4,D-Phe 7]-alpha-MSH, was prepared by solid-phase techniques of peptide synthesis. The biological actions of this analogue were determined in several melanocyte bioassays and were compared with the parent peptide [Nle4,D-Phe7]-alpha-MSH and the native hormone alpha-MSH. The fluorescein compound was a superpotent agonist with approximately 10 times more activity than alpha-MSH in both the frog and the lizard skin bioassays. Murine S91 melanoma cells assayed in vitro (tyrosinase bioassay) were as responsive to the fluorescein analogue as to alpha-MSH. The analogue exhibited ultraprolonged biological activity and the biological activities were unaffected by treatment of the analogue with alpha-chymotrypsin. The fluorescein-labeled melanotropin should prove useful for melanotropin receptor characterization.

Synthesis and biological actions of highly potent and prolonged acting biotin-labeled melanotropins.

Biocytin derivatives of a superpotent analogue of alpha-melanotropin, [Nle4,D-Phe7]-alpha-MSH, were prepared. [N alpha-Bct-Ser1, Nle4,D-Phe7]-alpha-MSH and [12-Bct-N alpha-dodecanoyl-Ser1,Nle4,D-Phe 7]- alpha-MSH were synthesized by solid-phase techniques, and the coupling of biotin and 12-aminododecanoic acid was achieved through their succinimido esters. These melanotropins possessed almost identical actions to [Nle4,D-Phe 7]- alpha-MSH as determined by several melanocyte bioassays. Both biocytin derivatives were highly potent agonists and exhibited prolonged biological activity as determined in the frog and lizard skin bioassays. Both biotinylated peptides were at least equipotent to alpha-MSH in stimulating Cloudman S91 mouse melanoma tyrosinase activity. The analogues were resistant to inactivation by alpha-chymotrypsin.

Carbon-13 chemical shifts on oxytocin as a consequence of its interaction with neurophysins.

Carbon-13 NMR was used to study the interaction of the hormone oxytocin with neurophysin (NP). Oxytocins specifically enriched to 90% 13C in the alpha-carbons of Leu-3 (in [3-leucine]oxytocin), Gln-4, and Leu-8 and in the carbonyl carbon of Cys-6 were synthesized, so that the effect on these positions of binding to NP could be monitored. The alpha-carbons of residues 3 and 4 experienced shifts of -4.2 and -1.5 ppm (negative shifts are downfield), respectively, upon binding of the hormone to NP. The carbonyl carbon of residue 6 underwent a shift of +0.7 ppm, while the alpha-carbon of residue 8 displayed no shift. For each enriched residue, the hormone diastereoisomer in which this residue had the D configuration was also synthesized. NMR was then used to determine the binding affinity of the various diastereoisomers to NP, as well as to measure the NMR parameters of the bound peptides. When position 3 had the D configuration, the binding affinity for NP was 10-20% that of the native hormone. For positions 4, 6, and 8, the D diastereoisomers bound with the same affinity as oxytocin. The alpha-carbons of D residues of positions 3 and 4 shifted by -2.5 and +0.4 ppm, respectively, the carbonyl carbon of D-Cys-6 shifted by +1.4 ppm, and the alpha-carbon of D-Leu-8 was unshifted on binding to NP. The shift and diastereoisomer binding data, combined with previous results involving enriched carbons and/or diastereoisomers of residues 1, 2, and 9, support the conclusion that residues 1 and 2 are most crucial for binding of oxytocin to NP, residue 3 is less important, and residues 4-9 are of only slight significance.(ABSTRACT TRUNCATED AT 250 WORDS)

A pilot study of role of brahmyadiyoga in chronic unmada(schizophrenia).

Brahmyadiyoga a compound drug was used on fourteen Chronic Unmada patients suffering from 2 years to 8 years between the ago range of 18 to 40 years of either sex. The dose of the drug was 8 gms. to 16 gms. for three months. Assessments were done independently by Ayurvedic physician, Psychiatrist and Clinical Psychologist. Seven out of 10 patients who underwent treatment for three months and all the four patients who took the drug for two months improved.