Molecular cloning and functional expression of the first two specific insect myosuppressin receptors

The Drosophila Genome Project database contains the sequences of two genes, CG8985 and CG13803, which are predicted to code for G protein-coupled receptors. We cloned the cDNAs corresponding to these genes and found that their gene structures had not been correctly annotated. We subsequently expressed the coding regions of the two corrected receptor genes in Chinese hamster ovary cells and found that each of them coded for a receptor that could be activated by low concentrations of Drosophila myosuppressin (EC50,4 x 10(-8) M). The insect myosuppressins are decapeptides that generally inhibit insect visceral muscles. Other tested Drosophila neuropeptides did not activate the two receptors. In addition to the two Drosophila myosuppressin receptors, we identified a sequence in the genomic database from the malaria mosquito Anopheles gambiae that also very likely codes for a myosuppressin receptor. To our knowledge, this paper is the first report on the molecular identification of specific insect myosuppressin receptors.     

Chemokine expression by systemic sclerosis fibroblasts: abnormal regulation of monocyte chemoattractant protein 1 expression.

OBJECTIVE: Chemokines are important mediators in the chemoattraction of leukocytes to sites of inflammation. This study investigated the potential contribution of systemic sclerosis (SSc) fibroblasts to chemokine production and its potential relevance to the pathogenesis of SSc. METHODS: The expression of messenger RNA (mRNA) for different C-C and C-X-C chemokines by SSc and normal fibroblasts was studied by RNase protection assay. Monocyte chemoattractant protein 1 (MCP-1) protein production was analyzed by enzyme-linked immunosorbent assay. The chemotactic effect of fibroblast-derived MCP-1 on monocytic cells was analyzed in a transmigration assay. Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) activation in fibroblasts was studied by electromobility shift analysis. MCP-1 expression in SSc skin sections was studied by immunohistochemistry. RESULTS: Among all chemokine genes studied, only MCP-1 and interleukin-8 mRNA were expressed by nonstimulated normal and SSc fibroblasts. SSc fibroblasts displayed increased constitutive expression of MCP-1 mRNA and protein and showed a blunted response to oxidative stress. Increased MCP-1 production was associated with higher chemotactic activity for monocytic cells. Increased NF-kappaB or AP-1 activation was not responsible for the constitutive overexpression of MCP-1 by SSc fibroblasts. In SSc skin sections, MCP-1 expression was detected in fibroblasts, keratinocytes, and mononuclear cells, whereas it was undetectable in normal skin. CONCLUSION: SSc fibroblasts display a specific pattern of chemokine gene expression that is characterized by constitutively increased and abnormally regulated expression of MCP-1 in vitro. MCP-1 is also expressed in lesional skin and can participate in the pathogenesis of SSc.     

Molecular cloning and expression of two type one somatostatin receptors in goldfish brain.

Somatostatin (SRIF or SS) exerts diverse inhibitory actions through binding to specific receptors. In this study, two SRIF receptor complementary DNAs (cDNAs) were cloned and sequenced from goldfish brain using PCR and cDNA library screening. The two cDNAs share 92% similarity in nucleotide sequence and 98% similarity in the deduced amino acid sequences and are presumably derived from duplicate genes, as goldfish are tetraploid. Two cDNAs encode two 367-amino acid goldfish type one SRIF receptors (designated as sst1A and sst1B, respectively), with seven putative transmembrane domains (TMD) and YANSCANP motif in the 7th TMD, a signature sequence for mammalian SRIF receptor (sst) family. In addition, the amino acid sequences of two receptors have 76% and 75% similarity to human or rat sst1, respectively, and 39-55% similarities to other mammalian sst subtypes (sst2-5), suggesting that the two receptors could be the goldfish homologs of mammalian sst1. The difference between goldfish and mammalian sst1 is mainly reflected by the extreme divergence in their extracellular N termini. Both SRIF-14 and [Pro2]SRIF-14, two of the native goldfish SRIF forms, significantly inhibited forskolin-stimulated cAMP release in COS-7 cells transiently expressing goldfish sSt1A or sst1B, suggesting functional coupling of the two receptors to adenylate cyclase. Northern blot and RT-PCR showed that messenger RNAs (mRNAs) for both receptors are widely distributed throughout goldfish brain, whereas only one receptor mRNA is expressed in the pituitary. RT-PCR analysis also detected sst1 receptor mRNAs in several peripheral tissues. These findings provide fundamental information for studying the mechanism of SRIF actions in vertebrates and structural analysis of mammalian sst receptors.     

Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors.

A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCR analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists with the following specificity sequence: domoate greater than kainate much greater than quisqualate greater than or equal to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid greater than or equal to L-glutamate much greater than N-methyl-D-aspartate. The kainate-elicited currents were specifically blocked by 6-cyano-7-nitroquinoxaline-2,3-dione but were insensitive to 2-amino-5-phosphonovalerate and kynurenic acid. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.     

Chemokine expression by systemic sclerosis fibroblasts: Abnormal regulation of monocyte chemoattractant protein 1 expression

Objective

Chemokines are important mediators in the chemoattraction of leukocytes to sites of inflammation. This study investigated the potential contribution of systemic sclerosis (SSc) fibroblasts to chemokine production and its potential relevance to the pathogenesis of SSc.

Methods

The expression of messenger RNA (mRNA) for different C‐C and C‐X‐C chemokines by SSc and normal fibroblasts was studied by RNase protection assay. Monocyte chemoattractant protein 1 (MCP‐1) protein production was analyzed by enzyme‐linked immunosorbent assay. The chemotactic effect of fibroblast‐derived MCP‐1 on monocytic cells was analyzed in a transmigration assay. Nuclear factor κB (NF‐κB) and activator protein 1 (AP‐1) activation in fibroblasts was studied by electromobility shift analysis. MCP‐1 expression in SSc skin sections was studied by immunohistochemistry.

Results

Among all chemokine genes studied, only MCP‐1 and interleukin‐8 mRNA were expressed by nonstimulated normal and SSc fibroblasts. SSc fibroblasts displayed increased constitutive expression of MCP‐1 mRNA and protein and showed a blunted response to oxidative stress. Increased MCP‐1 production was associated with higher chemotactic activity for monocytic cells. Increased NF‐κB or AP‐1 activation was not responsible for the constitutive overexpression of MCP‐1 by SSc fibroblasts. In SSc skin sections, MCP‐1 expression was detected in fibroblasts, keratinocytes, and mononuclear cells, whereas it was undetectable in normal skin.

Conclusion

SSc fibroblasts display a specific pattern of chemokine gene expression that is characterized by constitutively increased and abnormally regulated expression of MCP‐1 in vitro. MCP‐1 is also expressed in lesional skin and can participate in the pathogenesis of SSc.

     

Molecular characterization of two sea bass gonadotropin receptors: cDNA cloning, expression analysis, and functional activity

The follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) play central roles in vertebrate reproduction. They act through their cognate receptors to stimulate testicular and ovarian functions. The present study reports the cloning and characterization of two sea bass (Dicentrarchus labrax) cDNAs encoding a FSH receptor (sbsFSHR) and a LH receptor (sbsLHR). The mature proteins display typical features of the glycoprotein hormone receptor family members, but the sbsFSHR also contains some remarkable differences when compared with other fish or mammalian FSHRs. Among them, a distinct extracellular N-terminal cysteine domain as regards to its length and cysteine number, and the presence of an extra leucine-rich repeat. Expression analysis revealed that the sbsFSHR is exclusively expressed in gonadal tissues, specifically in the follicular wall of previtellogenic and early-vitellogenic follicles. On the contrary, sbsLHR mRNA was found to be widely distributed in sea bass somatic tissues. When stably expressed in mammalian cell lines, sbsFSHR was specifically stimulated by bovine FSH, while sbsLHR was activated by both bovine LH and FSH. Nevertheless, specific stimulation of the sbsLHR was observed when recombinant sea bass gonadotropins were used. The isolation of a FSHR and a LHR in sea bass opens new ways to study gonadotropin action in this species.     

Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hypercholesterolemic primates.

Atherosclerosis is marked by an overt inflammatory infiltrate, with enhanced recruitment of monocytes/macrophages observed in both human and experimental atherosclerosis. We previously determined that monocyte chemoattractant protein 1 (MCP-1) accounts for virtually all of the chemotactic activity produced by vascular (aortic) smooth muscle cells in culture. We now report that arteries from a primate model of atherosclerosis with dietary-induced hypercholesterolemia exhibit increased levels of MCP-1 mRNA expression in vivo, whereas their normal counterparts demonstrate minimal MCP-1 expression. Furthermore, immunohistochemistry and in situ hybridization clearly indicate that the expression of MCP-1 protein and mRNA is in the smooth muscle cells of the medial layer of the artery and in monocyte-like and smooth muscle-like cells found in the overlying intimal lesion. These studies indicate that one of the responses to dietary hypercholesterolemia is the expression of MCP-1 by vascular smooth muscle cells. This expression, when augmented with other cellular and molecular factors, could significantly contribute to the recruitment of monocytes/macrophages to the vessel wall.     

Polymorphism of monocyte chemoattractant protein 1 in Crohn's disease

BACKGROUND AND AIMS: The chemokine MCP-1 is thought to be important for the recruitment of mononuclear cells and the maintenance of inflammation in inflammatory bowel disease. We investigated whether MCP-1 protein expression is correlated with the degree of mucosal inflammation in patients with Crohn's disease. Furthermore, we studied whether a functional single nucleotide polymorphism (G or A) located in the distal regulatory region of the MCP-1 gene is associated with Crohn's disease and/or its phenotype. PATIENTS AND METHODS: MCP-1 concentration in tissue homogenates was analyzed in mucosal biopsy specimens of 31 patients with Crohn's disease and 48 controls by enzyme-linked immunosorbent assay, and the correlation with an endoscopic macroscopic score was analyzed. In 179 patients with Crohn's disease and 189 controls MCP-1 genotyping was carried out by polymerase chain reaction restriction fragment length polymorphism technique. Subgroup phenotypic analysis was performed according to the Vienna classification. RESULTS: MCP-1 tissue concentrations were significantly associated with the macroscopic degree of inflammation. The gene frequency of the different MCP-1 alleles did not differ from healthy controls. However, the G/A and G/G genotype was significantly decreased in patients with a later onset of the disease and both genotypes presented also less frequently with a fistulizing disease behavior. CONCLUSION: The degree of intestinal inflammation in Crohn's disease is associated with MCP-1 tissue levels. Furthermore there is evidence for an association of different disease behavior with different MCP-1 genotypes.     

Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.

By mediating the coupled movement of Na, K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2-kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na-K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.     

Strong association of a functional polymorphism in the monocyte chemoattractant protein 1 promoter gene with lupus nephritis

OBJECTIVE: Lupus nephritis (LN) is a major contributor to morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is evidence that polymorphisms in the genes of inflammatory mediators may predispose to the development of LN in patients with SLE. In this study, we examined the role of a functional monocyte chemoattractant protein 1 (MCP-1) polymorphism in SLE and LN. METHODS: DNA and paired urine and serum samples were obtained from 134 SLE patients (> or =4 American College of Rheumatology criteria for SLE; 49 with and 85 without LN) and 118 controls. MCP-1 genomic variants were detected by polymerase chain reaction followed by restriction enzyme-fragment analysis. Urinary and serum MCP-1 levels and MCP-1 production by peripheral blood macrophages were measured by enzyme-linked immunosorbent assay. RESULTS: The A/A genotype was more common in controls than in SLE patients (P = 0.0002), whereas both the A/G (P = 0.009) and G/G (P = 0.0212) genotypes were more frequent in SLE patients. The A/A genotype was observed in only 23% of the patients with LN compared with 58% of those without LN (P < 0.0001). MCP-1 production by peripheral blood mononuclear cells from patients with the A/G and G/G phenotypes was markedly higher than the production by cells from patients with the A/A genotype. Urinary levels of MCP-1 were significantly higher in patients with LN. CONCLUSION: These results suggest that an A/G or G/G genotype may predispose to the development of SLE and further indicate that SLE patients with these genotypes may be at higher risk of developing LN. Moreover, measurement of urinary levels of MCP-1 may be a useful tool for the detection and management of LN.     

Molecular cloning reveals isoforms of bovine alpha 1-antichymotrypsin.

Comparison of bovine alpha 1-antichymotrypsin (ACT) protease inhibitor with that in human was achieved by cloning a nearly full-length bovine ACT cDNA of 1.5 kb, obtained by screening a bovine liver cDNA library with the human liver ACT cDNA. The deduced primary sequence indicated that the 1456-bp bovine ACT cDNA encodes a protein of 416 amino acids that contains the predicted full-length ACT with a 26-residue NH2-terminal signal sequence. Overall, the primary sequence of bovine ACT possesses a high degree of homology (55%) with human ACT; both bovine and human ACTs share common sequences in the reactive-site domains. Importantly, the reactive site of bovine ACT possesses serine as the predicted P1 position (residue at the NH2-terminal side of the cleaved peptide bond) of the reactive site, whereas human ACT contains leucine in the P1 position. Interestingly, further evidence for heterogeneity in P1 residues was provided by a second partial 0.9-kb bovine liver ACT cDNA clone (pHHK11) that contains isoleucine as P1 residue and shares only partial homology (68%) with the deduced primary sequence of the full-length bovine liver ACT cDNA clone (pHHK12). These findings suggest that isoforms of ACT in bovine liver vary in reactive-site P1 residues; the P1 position of the reactive site is often involved in protease inhibitor specificity. Consistent with the hypothesis of ACT isoforms was the demonstration of multiple copies of the bovine ACT gene by genomic blots.     

Molecular cloning and functional expression of a brain-specific somatostatin receptor.

The PCR and conventional library screening were used to clone the brain-specific somatostatin receptor rSSTR-4 from a rat genomic library. The deduced amino acid sequence encodes a protein of 384 amino acids and displays structural and sequence homologies with members of the G protein-receptor superfamily. The amino acid sequence of rSSTR-4 is 60% and 48% identical to that of somatostatin receptors SSTR-1 and SSTR-2, respectively, two recently cloned subtypes. Competition curve analysis of the binding properties of the receptor transiently expressed in COS-1 cells revealed a higher apparent affinity for somatostatin 14 than for somatostatin 28. In contrast, the somatostatin analogs SMS 201-995, IM 4-28, and MK-678 failed to displace specific binding in transfected cells. These characteristics resemble the pharmacological binding properties of the previously described brain-specific somatostatin-receptor subtype. Examination of the tissue distribution of mRNA for rSSTR-4 revealed expression limited to various brain regions with highest levels in the cortex and hippocampus. Thus, based on the pharmacology and tissue localization of this receptor, we conclude that rSSTR-4 represents a brain-specific somatostatin receptor.     

Expression of monocyte chemoattractant protein 1 mRNA in human idiopathic pulmonary fibrosis.

Macrophages are thought to play an important role in the pathologic changes associated with idiopathic pulmonary fibrosis (IPF). The mechanisms for increased monocyte/macrophage recruitment in IPF are unknown. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cell types in culture. We examined the expression of MCP-1 mRNA and its protein product in vivo in IPF and non-IPF lung specimens by in situ hybridization and immunocytochemistry. The cell types expressing MCP-1 in vivo were identified by immunostaining with specific antibodies. We demonstrated the expression of MCP-1 mRNA in pulmonary epithelial cells, in monocytes/macrophages, and in vascular endothelial and smooth muscle cells. Lung epithelial cells in patients with IPF strongly expressed MCP-1 mRNA and its protein product. In contrast, epithelial cells in non-IPF specimens did not express MCP-1 mRNA. Macrophages and vascular endothelial and smooth muscle cells were shown to express MCP-1 in both IPF and non-IPF lung specimens. These findings provide a basis for the understanding of the in vivo physiologic processes that mediate monocyte/macrophage recruitment and infiltration in the lung interstitium and the pathologic state contributing to an increased alveolar monocyte/macrophage population and inflammation in IPF.     

C/EBP-related protein 2 confers lipopolysaccharide-inducible expression of interleukin 6 and monocyte chemoattractant protein 1 to a lymphoblastic cell line.

C/EBP-related proteins 2 and 3 (CRP2 and CRP3) are differentially expressed by P388 lymphoblasts and their derivative P388D1(IL1) macrophages. We have ectopically expressed CRP2, the predominant CRP in macrophages, in P388 lymphoblasts. The expression of CRP2 is sufficient to confer the lipopolysaccharide (LPS)-inducible expression of interleukin 6 and monocyte chemoattractant protein 1 to lymphoblasts, which normally do not display LPS induction of inflammatory cytokines. Consistent with these findings, the expression of CRP2 antisense RNA blocks the LPS induction of IL-6 expression in P388D1(IL1) macrophages. This work clearly establishes the essential role of CRP2 in the induction of cytokine genes by LPS. Additionally, these data add MCP-1 to the list of cytokines showing an involvement of CRP2 in their expression.     

结直肠癌趋化因子单核细胞超化蛋白-1的表达

目的：通过检测结直肠癌组织中单核细胞超化蛋白－1（MPC－1）的表达情况，研究MCP－1的表达与结直肠癌生物学行为的关系。方法：采用RT－PCR方法，检测临床收集的新鲜结肠癌组织标本中MCP－1mRNA的表达；采用免疫组化方法，检测结直肠癌组织中MCP－1蛋白的表达，结果：12例结直肠癌组织均可出现MCP－1mRNA的表达，40例结直肠癌组织MCP－1蛋白表达的阳性率为90％，结直肠癌组织MCP－1蛋白的表达与结直肠癌的转移及Dukes分期有关，表达强者，转移发生率低，Dukes分期早，结论：结直肠组织中MCP－1的表达能影响结直肠癌的生物学行为。