Membrane guanylyl cyclase receptors: an update.

Recent studies have demonstrated key roles for several membrane guanylyl cyclase receptors in the regulation of cell hyperplasia, hypertrophy, migration and extracellular matrix production, all of which having an impact on clinically relevant diseases, including tissue remodeling after injury. Additionally, cell differentiation, and even tumor progression, can be profoundly influenced by one or more of these receptors. Some of these receptors also mediate important communication between the heart and intestine, and the kidney to regulate blood volume and Na+ balance.     

Gene conversion in a cytochrome P-450 gene family.

The mRNAs encoding the two major phenobarbital-inducible forms of cytochrome P-450 of rat liver, P-450b and P-450e, are remarkably similar (98% homologous) in nucleotide sequence, but the distribution of differences within them is not random. While the 5' halves (approximately equal to 1 kilobase) appear to be identical, there are 36 divergent residues in the remaining sequences of the two mRNAs, with 14 differences residing in two short highly divergent segments, which in the P-450e gene are located within exon 7. DNA sequence analysis of portions of a number of P-450b/e-related genes provides strong evidence that at least one of the short divergent segments is the result of a recent gene conversion event between an ancestor to the cytochrome P-450e gene and a related donor P-450 gene of unknown function. The sequence data also suggest that extensive gene conversion has occurred within all the members of this gene family in the region including exons 7 and 8 and the intron between them, with a resultant homogenization of those sequences relative to other portions of the genes. Genomic Southern blotting analysis demonstrates that the presence of an apparent "constant" region in the 5' halves of the P-450b and P-450e mRNAs does not reflect a rearrangement in somatic cells of a germ-line DNA configuration. It is therefore proposed that it, too, is a consequence of a very recent gene conversion event between ancestors of the genes encoding both proteins or of an unequal crossing-over between them. On the basis of these and other data we propose that gene conversion represents an important evolutionary mechanism for the generation of related cytochrome P-450 isozymes in which regions of extraordinary sequence similarity and dissimilarity are intermixed. The gene conversion mechanism would account for some of the overlaps in substrate specificities of distantly related P-450s as well as for substantial differences in catalytic properties between closely related members of the same P-450 protein family.     

Guanylin: an endogenous activator of intestinal guanylate cyclase.

Intestinal guanylate cyclase mediates the action of the heat-stable enterotoxin to cause a decrease in intestinal fluid absorption and to increase chloride secretion, ultimately causing diarrhea. An endogenous ligand that acts on this guanylate cyclase has not previously been found. To search for a potential endogenous ligand, we utilized T84 cells, a human colon carcinoma-derived cell line, in culture as a bioassay. This cell line selectively responds to the toxin in a very sensitive manner with an increase in intracellular cyclic GMP. In the present study, we describe the purification and structure of a peptide from rat jejunum that activates this enzyme. This peptide, which we have termed guanylin, is composed of 15 amino acids and has the following amino acid sequence, PNTCEICAYAACTGC, as determined by automated Edman degradation sequence analysis and electrospray mass spectrometry. Analysis of the amino acid sequence of this peptide reveals a high degree of homology with heat-stable enterotoxins. Solid-phase synthesis of this peptide confirmed that it stimulates increases in T84 cyclic GMP levels. Guanylin required oxidation for expression of bioactivity and subsequent reduction of the oxidized peptide eliminated the effect on cyclic GMP, indicating a requirement for cysteine disulfide bond formation. Synthetic guanylin also displaces heat-stable enterotoxin binding to cultured T84 cells. Based on these data, we propose that guanylin is an activator of intestinal guanylate cyclase and that it stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins.     

Hereditary chondrocalcinosis in an Ashkenazi Jewish family.

A hereditary chondrocalcinosis is described for the first time in an Ashkenazi Jewish kindred. Of 34 family members in five generations, seven had medical history suggesting the disease. Five of 25 members of generations III-V had direct evidence for their disease. Characteristically, symptoms started at a fairly early age (third decade) while radiological evidence of chondrocalcinosis was delayed to the fourth decade. Joints commonly affected were knees, wrists, and elbows. The course was chronic with acute, exercise induced exacerbations.     

Cloning and characterization of a sixth adenylyl cyclase isoform: types V and VI constitute a subgroup within the mammalian adenylyl cyclase family.

A sixth member of the mammalian adenylyl cyclase family has been isolated from a canine cardiac cDNA library. This isoform is more highly homologous to type V than to the other adenylyl cyclase types; sequence similarity is apparent even in the transmembrane regions where the greatest divergence among the types exists. Type VI mRNA expression is most abundant in heart and brain; however, unlike type V, a low level of expression is also observed in a variety of other tissues examined. Type VI adenylyl cyclase can be stimulated by NaF, guanosine 5'-[gamma-thio]triphosphate, and forskolin but not by Ca2+/calmodulin, whereas it is inhibited by adenosine and its analogues. Comparison of both their structural and biochemical properties suggests that types V and VI constitute a distinct subgroup of the mammalian adenylyl cyclase family.     

Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma

Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41–2272 and BAY 60–2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41–2272 and BAY 60–2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.Asthma is an inflammatory disease that causes airway hyperreactivity (AHR) and bronchoconstriction, which impedes daily life activities and, when severe, can cause death. It is the most common chronic disease of childhood, accounts for one in three emergency department visits daily, and asthma diagnoses are increasing worldwide (1). The leading treatment for relief and acute care is bronchodilation, which relies heavily on the β-adrenergic receptor-cAMP pathway. Nearly 70% of patients, however, develop resistance or tachyphylaxis to the existing β-agonist therapy (2), underscoring a need for new bronchodilators that can act through a different pharmacologic principle.The nitric oxide-soluble guanylate cyclase-cGMP pathway (NO-sGC-cGMP) is the primary signal transduction pathway for relaxing vascular smooth muscle (3). In contrast, a role for the NO-sGC-cGMP pathway in relaxing airway smooth muscle is less clear (4, 5), and bronchodilation was instead suggested to depend on glutathione nitrosothiol levels in the lung (6, 7). However, recent studies have shown that inflammation can desensitize sGC toward its natural activator, NO (8), and new drugs have become available that directly activate sGC, independent of NO (9). These developments encouraged us to re-examine the NO-sGC-cGMP pathway regarding its role in bronchodilation, its becoming damaged in inflammatory asthma, and its potential for alternative bronchodilator development under this circumstance.     

Anticipation in an Indo-Canadian family with Crohn's disease.

Genetic anticipation, associated elsewhere with monogenic neurological disorders, has been hypothesized to be present in familial forms of Crohn's disease. Usually, with studies of parent-child pairs, the parent who is initially diagnosed is older at the onset of disease than the child. With each successive generation, an apparent increase in disease severity or behaviour occurs. This phenomenon is believed to have a molecular basis. In the present report, an Indo-Canadian family with Crohn's disease is described. In all members of the family, disease was diagnosed only after prolonged residence in Canada, supporting the view that Crohn's disease arises in individuals with a genetic predisposition following exposure to some, as yet unknown, common environmental factor. Three siblings with Crohn's disease, first diagnosed between ages 15 and 27 years, or six to 11 years after arrival in Canada, had phenotypically concordant disease localized in the ileum and colon, with fistulizing complications, including perianal sepsis. Crohn's disease was only diagnosed in the father at the age of 76 years, almost three decades after his arrival in Canada. His disease was localized to the ileum and had a fibrostenosing behaviour. This is the first reported instance of familial Crohn's disease in an immigrant population, illustrating potential biases in genetically based studies of Crohn's disease that rely solely on phenotypic expression.     

A tobacco gene family for flower cell wall proteins with a proline-rich domain and a cysteine-rich domain.

Flowering is known to be associated with the induction of many cell wall proteins. We report here five members of a tobacco gene family (CELP, Cys-rich extensin-like protein) whose mRNAs are found predominantly in flowers and encode extensin-like Pro-rich proteins. CELP mRNAs accumulate most abundantly in vascular and epidermal tissues of floral organs. In the pistil, CELP mRNAs also accumulate in a thin layer of cells between the transmitting tissue and the cortex of the style and in a surface layer of cells of the placenta in the ovary. This unique accumulation pattern of CELP mRNAs in the pistil suggests a possible role in pollination and fertilization processes. CELP genes encode a class of plant extracellular matrix proteins that have several distinct structural features: a Pro-rich extensin-like domain with Xaa-Pro3-7 motifs and Xaa-Pro doublets, a Cys-rich region, and a highly charged C terminus. The extensin-like domains in these proteins differ significantly in their length and these differences appear to be results of both long and short deletions within the coding regions of their genes. Furthermore, the number of charged amino acid residues in the C-terminal region varies among the CELPs. These structural differences may contribute to functional versatility in the CELPs. On the other hand, the Cys-rich domain is highly conserved among CELPs and the positions of the Cys residues are conserved, suggesting that this region may have a common functional role. The presence of a Pro-rich domain and a Cys-rich domain in these CELPs is reminiscent of a class of hydroxyproline-rich glycoproteins, solanaceous lectins, that are believed to be important in cell-cell recognition. The structure of these CELPs indicates that they may be multifunctional and that their genes may have arisen from recombinational events.     

A calmodulin-sensitive adenylate cyclase in the prothoracic glands of the tobacco hornworm, Manduca sexta

The Ca2+/calmodulin (CaM) dependence of adenylate cyclase activity in Manduca sexta prothoracic glands was investigated. Membrane fractions from two developmental stages were used, day 3 of the last larval instar and day 0 of the pupal stage, both of which respond to the neuropeptide prothoracicotropic hormone (PTTH) with increased cAMP production dependent on extracellular Ca2+. The data revealed that both larval and pupal prothoracic gland membrane fractions have a Ca2+/CaM-dependent adenylate cyclase which is inhibited by CaM antagonists and EGTA. The larval adenylate cyclase shows a multiphasic response to Ca2+/CaM, with a 2-fold stimulation between 0.02 and 0.01 microM, a further increase in adenylate cyclase activity at concentrations greater than 2 microM and a potentiation of NaF-stimulated activity at doses greater than 0.1 microM Ca2+/CaM. Pupal prothoracic gland membrane fractions exhibit only the second phase of stimulation. Stimulation by the GTP analogs GTP-gamma-S and Gpp(NH)p is dependent on CaM in larval, but not in pupal membrane fractions, suggesting a role for CaM in Gs protein-mediated regulation of adenylate cyclase. However, adenylate cyclase activity in glands from both stages is dependent on CaM, supporting our initial premise that Ca2+ is required for cAMP synthesis in the prothoracic glands.     

Simple model for hormone-activated adenylate cyclase systems.

A simple model is developed to explain the activation of rat liver plasma membrane adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by guanosine nucleotides and glucagon and the dependence of the cATALYTIC RATE ON Mg2+, H+, and substrate concentrations. The basic model proposes that the adenylate cyclase system can exist in two states, A and B; that activating ligands bind preferentially to the B state; and that only the B state is active. Kinetic data are quantitatively fit to this model, and the binding constants for the interaction of the A and B states with glucagon, GTP, and guanyl-5'-ylimidodiphosphate are obtinaed. The substrates ATP and adenyl-5'-ylimidodiphosphate appear to show little preference between the A and B states, and simple Michaelis-Menten kinetics are sufficient to describe the dependence of the catalytic rate on substrate concentration under optimal conditions. The dependence of the rate on pH can be explained by postulating that one ionizable group in its acid form and one ionizable group in its basic form must be present at the active site in order for catalysis to occur. The activation and inhibition of the activity by Mg2+ can be explained by a similar mechanism with Mg2+ binding to activating and inhibiting sites. Glucagon and guanosine nucleotides appear to influence the dependence of the rate on Mg2+ and glucagon. The Mg2+ also may display some preference for the B state. A comparison of this model with others that have been proposed is given. The proposed model appears to provide a simple conceptual frame-work that is applicable to many adenylate cyclase systems.     

Treating tobacco use and dependence: an evidence-based clinical practice guideline for tobacco cessation

The prevention of tobacco-related morbidity and mortality through smoking cessation intervention is among the most vital missions of the chest clinician. This article summarizes the major findings and clinical recommendations of the US Department of Health and Human Services/Public Health Service Guideline, Treating Tobacco Use and Dependence, which is a comprehensive, evidence-based blueprint for smoking cessation. By becoming fluent in the clinical interventions and by implementing the simple institutional changes described in this article and in the guideline, chest clinicians can more effectively intervene with their patients who smoke.     

Gene for an extracellular matrix receptor protein from Pneumocystis carinii.

An initial and crucial step in the establishment of many microbial infections is the attachment of the pathogen to the host cells. Thus, adherence of Pneumocystis carinii (Pc) to type I pneumocytes is believed to be important in the induction of Pc pneumonia. Little is known about the nature of the attachment of Pc to type I cells, although extracellular matrix (ECM) proteins, such as fibronectin and laminin, have been implicated in the process. We report here the isolation of a Pc gene encoding a receptor protein that binds both fibronectin and laminin in vitro. A cDNA clone encoding the Pc ECM receptor was isolated from a Pc cDNA library and identified on the basis of sequence homology to the human colon carcinoma laminin receptor. Southern blot analysis of Pc genomic DNA confirmed that the cDNA was of Pc origin. Northern blot analysis of Pc total RNA showed a predominant mRNA of approximately 1400 nucleotides that hybridized to the ECM receptor gene. The ECM receptor predicted from the cDNA sequence is 295 amino acid residues long, with a molecular mass of 32.8 kDa. The C-terminal third of the polypeptide is highly negatively charged, whereas the N-terminal two-thirds contains hydrophobic segments that may play a role in membrane association. Sequence analysis and alignment of the N terminus with the laminin receptor cDNA sequence of human colon carcinoma support the conclusion that the Pc ECM receptor cDNA clone is a full-length clone. A Western blot of the overexpressed ECM receptor protein bound both laminin and fibronectin in vitro. Antibodies raised to the overexpressed receptor protein interacted with a 33-kDa protein in total Pc cell lysates. These findings raise the possibility that the Pc ECM receptor protein may mediate the organism's attachment to type I pneumocytes and, thus, may play a crucial role in Pc pathogenesis.     

Active conformation of an insect neuropeptide family.

To understand the structural and chemical basis for insect neuropeptide activity, we have designed, synthesized, and determined the conformation of a biologically active cyclic analog of the pyrokinins, an insect neuropeptide family that mediates myotropic (visceral muscle contractile) activity. Members of this insect neuropeptide family share the common C-terminal pentapeptide sequence Phe-Xaa-Pro-Arg-Leu-NH2 (Xaa = Ser, Thr, or Val). Circular dichroic, nuclear magnetic resonance, and molecular dynamics analyses of the conformationally restricted cyclic pyrokinin analog cyclo(-Asn-Thr-Ser-Phe-Thr-Pro-Arg-Leu-) indicated the presence of a beta-turn in the active core region encompassing residues Thr-Pro-Arg-Leu. The rigid cyclic analog retains biological activity, suggesting that its C-terminal beta-turn is the active pyrokinin conformation recognized by the myotropic receptor. As individual pyrokinins and pyrokinin-like neuropeptides demonstrate both oviduct-contractile and pheromone-biosynthesis activities in various insects, the biologically active beta-turn structure reported here holds broad significance for many biological processes.     

Hereditary pancreatitis. Presentation of an additional family

A West German family with hereditary pancreatitis is described. Four members are definitely known to have had pancreatitis, while three additional members are suspected of having the disease. The mean age of onset of symptoms was 14 years. Known causes of secondary pancreatitis and amino aciduria were ruled out in each case. HLA-segregation was analysed on the A, B, C, and DR loci in all members of the family, but no coupling between distribution of HLA haplotypes and incidence of pancreatitis was detected.     

Identification of an allatostatin from the tobacco hornworm Manduca sexta.

A peptide (Manduca sexta allatostatin) that strongly inhibits juvenile hormone biosynthesis in vitro by the corpora allata from fifth-stadium larvae and adult females has been purified from extracts of heads of pharate adult M. sexta by a nine-step purification procedure. The primary structure of this 15-residue peptide has been determined: pGlu-Val-Arg-Phe-Arg-Gln-Cys- Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-OH, where pGlu is pyroglutamate). To our knowledge, this neuro-hormone has no sequence similarity with any known neuropeptide from other organisms. The synthetic free acid and amide forms showed in vitro activity indistinguishable from that of native M. sexta allatostatin. The ED50 of synthetic M. sexta allatostatin on early fifth stadium larval corpora allata in vitro was approximately 2 nM. This inhibition was reversible. In a cross-species study, M. sexta allatostatin also inhibited the corpora allata of adult female Heliothis virescens but had no effect on the activity of corpora allata of adult females of the beetle Tenebrio molitor, the grasshopper Melanoplus sanguinipes, or the cockroach Periplaneta americana.     

Rheumatic diseases in an Icelandic family. Clinical and immunological survey

We have studied 192 members of a highly inbred Icelandic family with clustering of rheumatic diseases. Twelve consanguineous marriages are known in the family and 54 of 65 surviving offsprings of these (inbred group) were traced. Thirty-nine family members were affected by rheumatic diseases; 18 of them belonged to the inbred group. Eleven of 20 family members with rheumatoid arthritis (RA) came from the same inbred group. Eleven of the inbred group had a positive Rose-Waaler test for rheumatoid factor (RF) and the inbred group had significantly higher serum levels of IgG and IgM than an age and sex matched group from the family. Serum IgM RF was significantly associated with the age of the family members, but IgA RF and IgG RF did not show any such association. The possible role of recessive genes in the rheumatic diseases, as well as the inbreeding effect regarding certain extended HLA-complotypes is discussed.