Sultan Ahmad

Cloning and in situ hybridization analysis of the expression of polysialyltransferase mRNA in the developing and adult rat brain

Polysialyltransferase (PST) is an enzyme that catalyzes the addition of polysialic acid (PSA), a homopolymer of alpha-2,8-linked sialic acid residues, onto neural cell adhesion molecule (NCAM). The expression of PSA-NCAM in the brain is developmentally regulated and is of critical importance; however, the temporal and spatial developmental expression of brain PST, a potential key player in the control of PSA-NCAM levels, remains unclear. In the present study, we have cloned the coding region of rat PST cDNA by reverse transcription-polymerase chain reaction, using primers based on the hamster PST-1 cDNA sequence. A 39-mer oligonucleotide complementary to rat PST cDNA was synthesized to investigate the distribution of its mRNA in the developing and adult rat brain by Northern blot and in situ hybridization. In the embryonic rat brain, PST mRNA was detected abundantly throughout the neuroepithelia of most brain regions. At post-natal days 1 and 14, PST was detected throughout the neocortex, in the pyramidal cells (PC) of the hippocampus proper, the granule cell layer (GCL) of the dentate gyrus, the anterior ventral nucleus of the thalamus (AVNT) and the GCL and external germinal layer of the cerebellum. Finally, from PD21 until adulthood, expression of PST mRNA was restricted to the PC layer of the hippocampus proper, the GCL of the dentate gyrus, the AVNT, the GCL of the cerebellum and the dorsal and lateral nucleus of the anterior olfactory bulb. The developmental profile of PST mRNA is paralleled in some structures by that of the PSA-NCAM, there are, however, notable exceptions. Therefore, our results demonstrate that expression of rat PST mRNA is developmentally regulated, is present in the adult rat brain in restricted areas and may be involved in regulating temporal and spatial expression of PSA-NCAM.

Cloning and evaluation of the role of rat GALR-2, a novel subtype of galanin receptor, in the control of pain perception.

Abstract: We have identified a novel subtype of galanin receptor (GALR‐2) in rat dorsal root ganglia and spinal cord. The open reading frame of GALR‐2 is 1116 nucleotides long, encoding a protein of 372 amino acids with a theoretical molecular mass of 40.7 kD. Membranes prepared from stable pools of 293 cells expressing GALR‐2, but not wild‐type 293 cells, demonstrated high affinity galanin binding sites. Rat galanin and galanin‐related peptides M40, C7, M15, and galanin1–16 effectively competed for binding; peptide C7 demonstrated a lower affinity for rGALR‐2, and all these peptides were agonists at rGALR‐2 when assessed on a microphysiometer. Studies on the expression of GALR‐2 in various tissues by Northern and in situ hybridization analyses suggest a low abundance but wide distribution of GALR‐2 mRNA, including several discrete areas in brain and spinal cord and a high abundance in the dorsal root ganglia.

Characterization of beta adrenoceptors on cultured endothelial cells by radioligand binding

The properties of beta-adrenoceptors present on the cultured bovine pulmonary artery endothelial cells were studied by radioligand binding. These cell contain a high density of beta-adrenoceptors (approximately 16,000 receptors/cell) having high affinity (Kd 18 pM) for 125I-iodocyanopindolol (ICYP). Competition binding experiments suggested the presence of more than two subtype of beta-adrenoceptors. 25% of the total population of receptors was found to be of beta 1-type. The remaining 75% represented a mixed population containing what is suggested to be a mixture of beta 2- and atypical beta-adrenoceptors.