Mu-opioid-receptor-mediated inhibition of the N-type calcium-channel current.

The predominant consequences of mu-opioid-receptor activation are depression of both neuronal activity and transmitter release. Mu-Opioid agonists have previously been observed to increase a potassium conductance and to inhibit adenylate cyclase. We now report that activation of mu-opioid receptors directly decreases the N-type calcium-channel current in a differentiated, human neuroblastoma cell line (SH-SY5Y). The coupling between the mu-opioid receptor and the calcium channel involves a pertussis toxin-sensitive G protein and is independent of changes in adenylate cyclase activity. The inhibition of the calcium-channel current is voltage dependent because it is largely overcome by strong membrane depolarization. It is not associated with changes in the kinetics of current inactivation. Therefore, the mu-receptor belongs to the superfamily of G-protein-coupled, inhibitory neurotransmitter receptors which modulate the activity of calcium and potassium channels and adenylate cyclase.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

Genes newly identified as regulated by glucocorticoids in murine thymocytes

Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.     

Pyridostigmine enhances even if it does not normalize the growth hormone responses to growth hormone-releasing hormone in patients with Cushing's disease.

Subjects with Cushing's disease have diminished growth hormone (GH) response to growth hormone-releasing hormone (GHRH). The aim of our study was to investigate the underlying mechanism of this diminished GH response in these patients using pyridostigmine (PD), an acetylcholinesterase inhibitor, which is reported to increase GH secretion by reducing somatostatin tone. Eight subjects with untreated Cushing's disease (caused by a pituitary adenoma) and 6 control subjects received GHRH 100 micrograms in 1 ml of saline, as intravenous bolus injection 60 min after (1) placebo (2 tablets, p.o.) or (2) PD (120 mg, p.o.). After GHRH plus placebo, the GH peak (mean +/- SEM) was significantly lower in subjects with Cushing's disease (2.4 +/- 0.5 micrograms/l) compared to control subjects (25.1 +/- 1.8 micrograms/l, p less than 0.05). After GHRH plus PD, the GH peak was significantly enhanced both in subjects with Cushing's disease (7.1 +/- 2.3 micrograms/l, p less than 0.05) and in control subjects (42.3 +/- 4.3 micrograms/l, p less than 0.05). In patients with Cushing's disease, the GH response to GHRH plus PD was lower with respect to the GH response to GHRH alone in normal subjects. We conclude that hypercortisolism may cause a decrease in central cholinergic tone which is in turn hypothesized to be responsible of an enhanced somatostatin release from the hypothalamus. However, other metabolic or central nervous system alterations may act synergistically with hypercortisolism in causing GH inhibition in patients with Cushing's disease.     

Induction of DNA bending by bifunctional intercalating agents of the 7H-pyridocarbazole family

The structures and binding energetics of selected complexes formed between the deoxynucleotides d(CpGpGpCpG).d(CpGpCpCpG), d(CpGpApTpCpG)2, d(GpCpGpCpCpG).d(CpGpGpCpGpC), and d(CpGpCpCpCpG)2 with the DNA bifunctional intercalating agent ditercalinium and three of its rigid linking chain derivatives have been investigated theoretically by means of a molecular mechanics approach that takes into account nucleic acid flexibility, ligand flexibility and solvent dielectric effects (R. Lavery, in: Unusual DNA structures, eds S. Harvey and R. Wells (Pergamon, New York, 1988) p. 189; R. Lavery, in: DNA bending and curvature, eds W.K. Olson et al. (Adenine Press, New York, 1988) p. 191). The piperidinium chains of the bis-intercalating ligands are always located in the major groove of DNA. For the energy-minimized complexes the ligand proceeds to bind following preferentially the 5'-pyrimidine-purine-3' alternating sequence, thus dictating the number of internal exclusion sites. The complexes with three exclusion sites will present (i) a bending of the structure towards the major groove, and (ii) a non-ideal distribution of unwinding angles; complexes with less than three exclusion sites will remain essentially linear. The absence of a bend does not preclude other types of local deformations of the base-pairs such as inclination, buckle and tip. The proposed structures of the d(CpGpApTpCpG)2 complexes are in agreement with NMR structural results. The possible relevance of these findings to a previously proposed mode of interaction for ditercalinium-like DNA ligands is discussed.     

Maternal serum alpha-fetoprotein screening activities of state health agencies: a survey.

Maternal serum alpha-fetoprotein (MSAFP) screening has been demonstrated to be cost-effective on a population basis and is becoming standard practice. The American Society of Human Genetics has twice published policy statements to define the essential elements of a quality screening program. The present study reviewed the impact of these policy statements on state public-health agencies with respect to regulation or provision of MSAFP screening in their jurisdictions. With a few exceptions, states have not elected to play a major role in provision or regulation of this test. There is need to address issues of funding, standards, and data collection in a collaborative effort, if policy statements on genetic services are to be translated into effective state population screening.     

Ubiquity of Cysteine- and Metalloproteinase Activities in a Wide Range of Trypanosomatids

We have analysed the proteinase profiles of 11 species from 7 different genera of trypanosomatids by in situ detection of enzyme activities on SDS-PAGE gels containing co-polymerized gelatin as substrate, and the use of specific proteinase inhibitors. Our survey indicates that while cysteine- and metalloproteinases are distributed ubiquitously among trypanosomatids, there are marked differences between the enzyme profiles from the monogenetic (Crithidia, Herpetomonas, Leptomonas) and digenetic (Trypanosoma, Endotrypanum, Phytomonas, Leishmania) species. The detected metalloproteinase activities, ranging in size from 50–100 kDa, partitioned into the detergent-phase after Triton X-114 extraction, while most of cysteine proteinases, of three distinct molecular mass ranges (30–50 kDa, 80–100 kDa and 116–205 kDa), partitioned into the aqueous phase. Thus, within this group of organisms, the metalloproteinase activities seem to be predominantly membrane-associated proteins. We also show that the plant parasites of the genus Phytomonas exhibit a distinctive cysteine proteinase profile that might be exploited further as a criterion for taxonomy of the genus.