Stimulation of m1 and m3 muscarinic acetylcholine receptors, which are coupled to phosphoinositide hydrolysis and protein kinase C activation, has been shown to increase the release of soluble amyloid precursor protein derivatives (APPs). The effect is mimicked by phorbol esters, which directly activate protein kinase C. Using human embryonic kidney cells expressing individual muscarinic receptor subtypes, we found that stimulation of APPs release by the muscarinic agonist carbachol was only partially reduced by a specific inhibitor of protein kinase C (the bisindolylmaleimide GF 109203X), while the response to phorbol 12-myristate 13-acetate (PMA) was abolished. The increase in APPs release elicited by carbachol and PMA was accompanied by elevated tyrosine phosphorylation of several proteins and reduced by tyrosine kinase inhibitors; GF 109203X significantly reduced the stimulation of tyrosine phosphorylation by carbachol and PMA. Inhibition of protein tyrosine phosphatases by vanadyl hydroperoxide markedly increased cellular tyrosine phosphorylation and enhanced APPs release as effectively as PMA and carbachol. Direct phosphorylation of amyloid precursor protein on tyrosine residues following treatment with carbachol, PMA, or vanadyl hydroperoxide was not observed. The results implicate both tyrosine phosphorylation and protein kinase C-dependent mechanisms in the regulation of APPs release by G protein-coupled receptors, and suggest that carbachol and PMA increase APPs release from human embryonic kidney cells expressing m3 muscarinic receptors via partially divergent pathways that converge at a tyrosine phosphorylation-dependent step.

We have developed a low stringency polymerase chain reaction (LSPCR) to isolate the unknow neighboring region around a known DNA sequence, thus allowing efficient targeted gene walking. The method involves the polymerase chain reaction (PCR) with a single primer under conditions of low stringency for primer annaing (40°C) for the first few cycles followed by more cycles at high strency (55°C). This enables the ampltion a targee DNA fr -nt along with other nontargeted fa ts. High s (55°C) nested PCRs with end-labeled primers are then used to generate a ladder of radioactive bands, which accurately identifies the targeted fragment(s). We performed LSPCR on human placental DNA using a highly conserved sodium l-s primer for 5 cycles at 400C followed by 27 cycles at 55C for primer annealing. Subsequently, using higher stringency (550C) PCR with radiolabeled nested primers for 8 cycles, we have solated a 0.66-kb fra t ofa putative human sodium channel gene. Partial sequence (325 bp) of this fgent revealed a 270-bp region (exon) with homology to the rat brain sodium chane Iea (RBEU) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, we putatively assign this sequence as a part of a gene coding the a-subunit of a human brain type III sodium channel (SCN3A). Using PCR on two human/rodent somatic cell hybrid panels with primers specific to this putative

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Pen culture for in situ raising of stocking material was conducted in Sareni Jheel, located in the Rae Bareli District of Uttar Pradesh. Sareni Jheel, being rich in organic carbon (4.5%) and nutrients, higher values of silt (29%) and clay (15%) were recorded. Rich oxygen levels, alkaline pH, high organic matter, moderate to high chemical parameters suggested moderately productive nature of this wetland. Pen with high-density polyethylene (HDPE) net was installed in an area of 0.1378 ha and stocked with advanced fry of the Indian major carps, rohu Labeo rohita and catla Catla catla in the ratio of 1:1 @ 25 nos. m-2. No significant changes in water quality parameters were noticed within and outside the installed pen. Mean weight at stocking of rohu and catla was 2.88±1.41 g and 2.1±1.16 g, which increased to 57.2±13.84 g and 67.38±25.79 g respectively, in 120 days. The average feed conversion ratio (FCR) recorded was 1.08 with overall survivability of 69.52% in rohu and 74.74% in catla. Benefit-cost ratio and return on investment were calculated as 1.69 and 0.69, respectively. Intervention through pen culture resulted in the increase in fish productivity of Sareni Jheel from 310 to 833 kg ha-1.

We have identified four putative human sodium channel gene sequences, 55 bp each, using the polymerase chain reaction (PCR) on total human placental DNA with primers specific for the cDNA sequence of the rat brain sodium channel Iα (Scn1a) gene. One of these sequences was extended bidirectionally by genomic inverse-PCR to obtain a 1.6-kb fragment. Sequencing of this 1,556-bp fragment showed a 282-bp complete exon, which has 95% and 94% homology at the nucleotide and amino acid levels, respectively, with the rat Scn1a gene. We putatively assign this sequence as belonging to the gene coding the α-subunit of a human brain type I sodium channel (SCN1A). PCR on human × rodent somatic cell hybrids with primers derived from SCN1A localized this gene to chromosome 2. Fluorescence in situ hybridization to human metaphase chromosomes sublocalized the gene to chromosome band 2q24.