Molecular cloning and expression of the gene for a human D1 dopamine receptor

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.     

Hyperpolarization and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium

Stimulation of the endothelial lining of arteries with acetylcholine results in the release of a diffusible substance that relaxes and hyperpolarizes the underlying smooth muscle. Nitric oxide (NO) has been a candidate for this substance, termed endothelium-derived relaxing factor. But there are several observations that argue against the involvement of NO in acetylcholine-induced hyperpolarization. First, exogenous NO has no effect on the membrane potential of canine mesenteric arteries. Second, although haemoglobin (believed to bind and inactivate NO (refs 11-15)) and methylene blue (which prevents the stimulation of guanylate cyclase) inhibit relaxation, neither has an effect on hyperpolarization. Finally, nitroprusside, thought to generate NO in vascular smooth muscle, relaxes rat aorta without increasing rubidium efflux. Nevertheless, nitrovasodilators, nitroprusside and nitroglycerin cause hyperpolarization in some arteries. NO might therefore be responsible for at least part of the hyperpolarization induced by acetylcholine. We now report that hyperpolarization and relaxation evoked by acetylcholine are reduced by NG-monomethyl-L-arginine, an inhibitor of NO biosynthesis from L-arginine. Thus NO derived from the endothelium can cause hyperpolarization of vascular smooth muscle, which might also contribute to relaxation by closing voltage-dependent calcium channels. Our findings raise the possibility that hyperpolarization might be a component of NO signal transduction in neurons or inflammatory cells.     

Cryopreservation of Drosophila melanogaster embryos

There is an urgent need to preserve the ever-increasing number (greater than 30,000) of different genetic strains of D. melanogaster that are maintained in national and international stock centres and in the laboratories of individual investigators. In all cases, the stocks are maintained as adult populations and require transfer to fresh medium every two to four weeks. This is not only costly in terms of materials, labour and space, but unique strains are vulnerable to accidental loss, contamination, and changes in genotype that can occur during continuous culture through mutation, genetic drift or selection. Although cryopreservation of Drosophila germ-plasm would be an enormous advantage, many attempts using conventional procedures have been unsuccessful. D. melanogaster embryos are refractory to conventional cryopreservation procedures because of the contravening conditions required to minimize mortality resulting from both intracellular ice formation and chilling injury at subzero temperatures. To overcome these obstacles, we have developed a vitrification procedure that precludes intracellular ice formation so that the embryos can be cooled and warmed at ultra-rapid rates to minimize chilling injury, and have recovered viable embryos following storage in liquid nitrogen. In a series of 53 experiments, a total of 3,711 larvae emerged from 17,280 eggs that were cooled in liquid nitrogen (18.4 +/- 8.8%). Further, using a subset from this population, approximately 3% of the surviving larvae (24/800) developed into adults. These adults were fertile and produced an F1 generation.     

Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains.

The structure of an N-terminal fragment of CD4 has been determined to 2.4 A resolution. It has two tightly abutting domains connected by a continuous beta strand. Both have the immunoglobulin fold, but domain 2 has a truncated beta barrel and a non-standard disulphide bond. The binding sites for monoclonal antibodies, class II major histocompatibility complex molecules, and human immunodeficiency virus gp120 can be mapped on the molecular surface.     

Comparative platelet anti-aggregant activity of D-cysteinolic acid analogues

D-Cysteinolic acid (1) analogues with an S-C-C-N skeleton showed increased platelet anti-aggregant activity in the following order: 2-aminoethanesulfonic acids, thiazolidines, 2-aminoethanethiols and 2-aminoethyl disulfides. Methyl substitutions at the 2-position potentiated the activity. Of these analogues, bis [(R)-2-aminopropyl] disulfide was the most potent inhibitor of platelet aggregation, with about 600-fold the activity of (1).     

Identification of hemolytic granules isolated from human myocardial cells

Summary Human myocardial cells from fresh autopsy material contained granules which possessed hemolytic activity against guinea pig and rabbit erythrocytes. The hemolytic granules, which had a density of 1.02 and a diameter of 200–300 nm, were recovered as a microsome fraction from subcellular homogenates of human myocardial cells by differential centrifugation in 300 mM sucrose containing 0.1 mM PMSF and 10 mM EDTA. The membrane lesions caused by the granules were ring-like structures with an internal diameter of about 10–17 nm, analogous to that caused by perforin- and complement-induced lysis. However, the requirement for divalent cation differed from that for perforin-induced lysis, since the microsome-mediated lysis occurred in the presence of EDTA.     

Muscle-specific gene expression controlled by a regulatory element lacking a MyoD1-binding site

Muscle-specific expression of the gene encoding the delta subunit of the acetylcholine receptor is controlled by a 54-base-pair region that does not contain a binding site for MyoD1, a protein involved in activation of the myogenic program. A MyoD1-binding site is present in the proximal promoter region of the gene encoding the delta-subunit, but is neither sufficient nor necessary for muscle-specific expression in transfected muscle cells.     

Attachment of transported vesicles to microtubules in axoplasm is facilitated by AMP-PNP

Axoplasm extruded from the squid giant axon has been used to analyse the molecular mechanisms of intracellular vesicle transport. Using video-enhanced light microscopy, vesicle transport can be observed directly on individual microtubules at the edge of the axoplasm. Here we report that AMP-PNP (adenyl-5'-yl imidodiphosphate), a non-hydrolysable analogue of ATP, reversibly inhibited vesicle transport. Moreover, vesicles in solution attach to the microtubules and form relatively stable complexes. AMP-PNP may produce this effect by binding to an ATP-binding site on the transport machinery, thereby stabilizing the motility complex that is normally formed by a transported vesicle, an ATPase and a microtubule. The effects of AMP-PNP on the vesicle transport system indicate that the enzymatic machinery of this system differs significantly from that of the actomyosin system or the dynein-microtubule system.     

The ras oncogene product p21 is not a regulatory component of adenylate cyclase

Harvey (Ha-MSV) and Kirsten (Ki-MSV) murine sarcoma viruses induce tumours in animals and transform various cells in culture because of the expression of the ras oncogene product, p21 (ref. 1). Proto-oncogenes homologous with these genes are highly conserved evolutionarily and activated ras oncogenes have been detected in many human cancers. Whether c-ras oncogenes are directly responsible for human carcinogenesis is uncertain; however, it is clear that p21 mediates virus-induced transformation, although by an unknown mechanism. Epithelial and fibroblast cell lines transformed with Ha-MSV and Ki-MSV express p21 (ref. 8) and exhibit reduced adenylate cyclase activity. Like the guanine nucleotide regulatory proteins, Ns and Ni, which mediate stimulation and inhibition, respectively, of adenylate cyclase, p21 is a membrane-associated GTP binding protein, which exhibits GTPase activity. These similarities suggest that p21 and the adenylate cyclase regulatory proteins are related in cellular function, and that p21 depresses adenylate cyclase by inhibiting the activity of Ns or acting as Ni. We have therefore now examined the structural and functional similarities between p21 and Ns and Ni and find no evidence that p21 regulates adenylate cyclase activity by acting as one of these regulatory proteins.