Glycosylated fibronectin point-of-care test for diagnosis of pre-eclampsia in a low-resource setting: a prospective Southeast Asian population study.

OBJECTIVE: To determine the performance of a glycosylated fibronectin (GlyFn) point-of-care (POC) test for pre-eclampsia (PE) in a large Southeast Asian cohort (India) in comparison to previously described biomarkers. DESIGN: A total of 798 pregnant women at ≥20 weeks of gestation were enrolled in a prospective case-control study. Study participants included 469 normotensive women with urinary mg protein/mmol creatinine ratio <0.3, 135 with PE (hypertension with urinary mg protein/mmol creatinine ratio ≥0.3) and 194 with gestational hypertension (hypertension with urinary mg protein/mmol creatinine ratio <0.3). METHODS: GlyFn levels were determined using a POC device and PIGF, sFlt-1 and PAPPA2 levels were determined by immunoassay. Performance was assessed using logistic regression modelling and receiver-operating characteristic (ROC) curves. Classification performance and positive and negative predictive values are reported at specific thresholds. RESULTS: Increased levels of GlyFn, soluble fms-like tyrosine kinase-1 (sFlt-1) and pregnancy-associated placental protein A2 (PAPPA2), and decreased levels of placental growth factor (PlGF) were significantly associated (P < 0.01) with clinically defined PE. Area under the ROC (AUROC) values with 95% confidence intervals were: GlyFn, 0.99 (0.98-0.99); PlGF, 0.96 (0.94-0.98); sFlt-1, 0.86 (0.83-0.89); and PAPPA2, 0.96 (0.94-0.97). Of subjects with GH, 48% were positive for more than two PE biomarkers, and 70% of these delivered preterm. CONCLUSIONS: The Lumella™ GlyFn POC test has been validated in a low/middle-income country setting for PE diagnosis and may be a useful adjunctive tool for early identification, appropriate triage, and improved outcomes. TWEETABLE ABSTRACT: The Lumella™ point-of-care test had excellent performance in diagnosing PE in a large Southeast Asian cohort.

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.

Proteomic analysis of the genotoxicant methylazoxymethanol (MAM)-induced changes in the developing cerebellum.

The genotoxicant methylazoxymethanol (MAM) is a widely used developmental neurotoxin, and its glucoside is an etiological factor for western Pacific amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS/PDC). Identification of global protein expression changes that occur in response to MAM in the developing cerebellum could provide valuable insight into the potential mechanisms involved in the neurodegeneration process. We have utilized fluorescence 2-dimensional differential gel electrophoresis (2D-DIGE), to determine the protein expression changes that occur during normal cerebellar development and in response to MAM. Three day-old postnatal C57BL/6 mice (PND3) received a single injection of MAM, and the cerebella of postnatal day 4 (PND4) and day 22 (PND22) were analyzed. Approximately, 1400 unique spots were matched and quantified in all samples. Comparison of PND4 and PND22 developing cerebellum showed that a significant fraction of the proteome (approximately 68%) changes at this stage. The immediate response of the developing cerebellum to MAM was minimal (approximately 10%). However, significant differences (27%) were noted 14 days after MAM exposure. In contrast, the transcriptome changes were more pronounced at 24 h compared to 14 days. MAM targeted several proteins networks including transport (e.g., alpha-synuclein), cytoskeletal (e.g., beta-tubulin, vimentin), and mitochondrial (e.g., Atp5b) proteins. Immunochemistry confirmed several of the changes in protein expression (alpha-synuclein). Comparison with gene expression changes revealed that the temporal changes observed in the transcriptome and proteome are not correlative. These studies demonstrate for the first time the potential networks involved during neuronal development and neurodegenerative processes that are perturbed by MAM.

Peripheral genotype-phenotype correlations in Asian Indians with type 2 diabetes mellitus.

OBJECTIVE: A genome-wide scan of gene expression in leucocytes in Asian Indians with type 2 diabetes was performed and correlated with their known phenotype. METHODS: Microarray gene profiling of 13,474 sequence-verified, non-redundant human cDNAs was done to study leukocyte gene expression in Asian Indians with type 2 diabetes (DM: n=3) and matched controls (n=3). RESULTS: Significant differential expression (fold change <0.3 or >3) was noted for 897 genes in DM vs. controls. The 147 known genes in this category belonged to following broad functional groups (%): enzyme (32), nucleic acid binding (22), ligand binding or carrier (10), signal transducer (9), transporter (7), structural protein (6), cell adhesion (3), tumor suppressor (3), transcription factor binding (2), enzyme inhibitor (2), chaperone (2), cell cycle regulator (1), and defense/immunity protein (1). The 20 genes with at least a 3-fold change, annotated with known phenotypic associations in the current gene databank (phenotype association, fold change) were aspartoacylase (Canavan disease, 9.96), growth hormone receptor (Laron dwarfism, idiopathic short stature, 8.25), lipoprotein lipase (familial chylomicronemia syndrome, lipoprotein lipase deficiency, 8.00), vitamin D (1,25- dihydroxyvitamin D3) receptor (involutional osteoporosis, vitamin D resistant rickets, 7.94), intercellular adhesion molecule 1 human rhinovirus receptor (cerebral malaria susceptibility, 7.16), peroxisomal membrane protein 3 35-kDa (Refsum disease, infantile form, Zellweger syndrome-3, 6.00), Bardet-Biedl syndrome 2 (Bardet-Biedl syndrome, 5.87), ribosomal protein S19 (Diamond Blackfan anemia, 5.85), apolipoprotein C-III (hypertriglyceridemia, 5.44), argininosuccinate lyase (argininosuccinicaciduria, 5.22), myosin VA (Griscelli syndrome-type pigmentary dilution with mental retardation, 4.92), lysozyme (renal amyloidosis, 4.17), SAM domain, SH3 domain and nuclear localisation signals 1 (Cherubism, 4.12 ), von Hippel-Lindau syndrome (hemangioblastoma, cerebellar, somatic, von Hippel-Lindau syndrome, 3.94), early-onset breast cancer 1 (BRCA1, papillary serous carcinoma of the peritoneum, 3.73), UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (inclusion body myopathy, autosomal recessive, sialuria, 3.53), apolipoprotein A-I (amyloidosis, 3 or more types, hypoalphalipoproteinemia, 3.29), midline 1 Opitz/BBB syndrome (Opitz G syndrome, type I, 3.28), ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide (familial hemiplegic migraine, 3.05). Canavan disease, Zellweger syndrome, infantile Refsum disease, Griscelli syndrome, cherubism, breast cancer, peritoneal papillary serous carcinoma, Opitz G/BBB syndrome, and familial hemiplegic migraine (FHM) are phenotypes not previously reported in association with type 2 DM, but whose underlying genes were up-regulated in this peripheral genome scan of Asian Indians. CONCLUSION: Rare and/or previously unknown phenotypes linked to known genes with significant differential expression in type 2 DM are reported. Further testing of heterogeneity in diabetes phenotype syndromes may reveal common pathogenic mechanisms and potential candidate genes responsible for type 2 DM.

Molecular networks perturbed in a developmental animal model of brain injury.

Methylazoxymethanol (MAM) is widely used as a developmental neurotoxin and exposure to its glucoside (i.e., cycasin) is associated with the prototypical neurological disorder western Pacific ALS/PDC. However, the specific molecular targets that play a key role in MAM-induced brain injury remain unclear. To reveal potential molecular networks targeted by MAM in the developing nervous system, we examined characteristic phenotypic changes (DNA damage, cytoarchitecture) induced by MAM and their correlation with gene expression differences using microarray assays (27,648 genes). Three day-old postnatal C57BL/6 mice (PND3) received a single injection of MAM and the cerebellum and cerebral cortex of PND4, 8, 15, and 22 mice were analyzed. DNA damage was detected in both the cerebellum (N7-mGua, TUNEL labeling) and cerebral cortex (N7-mGua) of PND4 mice, but progressive disruption of the cytoarchitecture was restricted to the cerebellum. A majority (>75%) of the genes affected (cerebellum 636 genes, cortex 1080 genes) by MAM were developmentally regulated, with a predominant response early (PND4) in the cerebellum and delayed (PND8 and 15) in the cerebral cortex. The genes and pathways (e.g., proteasome) affected by MAM in the cerebellum are distinct from cortex. The genes perturbed in the cerebellum reflect critical cellular processes such as development (17%), cell cycle (7%), protein metabolism (12%), and transcriptional regulation (9%) that could contribute to the observed cytoarchitectural disruption of the cerebellum. This study demonstrates for the first time that specific genes and molecular networks are affected by MAM during CNS development. Further investigation of these targets will help to understand how disruption of these developmental programs could contribute to chronic brain injury or neurodegenerative disease.

Morphine regulation of a novel uridine diphosphate glucuronosyl-transferase in guinea pig pups following in utero exposure.

The uridine diphosphate glucuronosyltransferases (UGTs) catalyze conjugation reactions between various substrates and glucuronic acid, UDPGA (uridine diphosphate glucuronic acid), within the endoplasmic reticulum. Conjugation with UDPGA (glucuronidation) is an important pathway in the elimination, detoxification, and activation of compounds including steroid hormones, xenobiotics, and quaternary ammonium substrates. The guinea pig, which has a placental structure and a glucuronidation profile for morphine that are similar to the human, serves as a good small animal model to study the ontogeny of UGTs and the effect of in utero exposure to morphine on UGTs. We examined type 2 UGTs expressed in the guinea pig using amplification and cloning of partial cDNAs from liver RNA. Sequence analysis revealed a novel UGT2 (subsequently named UGT2A3),(2) that has a 64% amino acid sequence similarity to a known UGT2.(3) Full-length cDNAs were isolated from a guinea pig liver cDNA library. Tissue distribution of UGT2A3 using Northern blot analysis showed expression of three distinct size UGT2A3 mRNAs with unique expression in liver and small intestine. UGT2A3 mRNA is expressed at high levels in liver and lower levels in kidney and small intestine. In utero exposure to chronic intermittent morphine resulted in the up regulation of mRNA in 7-day-old female pups' liver and kidney as determined by quantitative RT-PCR analysis. The conjugation profile for UGT2A3 using stable expression in CHO cells and thin-layer chromatography demonstrated active conjugation of phenolic substrates. Regulation of UGTs by in utero morphine exposure may play an important role in fetal development.

Cloning and characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. CDH contains one heme b and one FAD per molecule and oxidizes cellobiose to cellobionolactone in the presence of cytochrome c. In this report, a thermostable CDH from the thermophilic ascomycete Sporotrichum thermophile has been purified, cloned, and characterized. The temperature optimum for this CDH reaction was 60 degrees C, and the activation energy for the reaction was 26.3 kJ/mol. The Km and kcat were temperature-dependent and increased as reaction temperature increased. These kinetic properties prove that this CDH is truly thermophilic. A 2.8-kb cDNA was isolated by screening an expression library of S. thermophile with a polyclonal antisera raised against Phanerochaete chrysosporium CDH. The cDNA encoded an 807-amino-acid protein with a predicted mass of 86,332 Da. S. thermophile CDH is organized into three domains, an N-terminal flavin domain, a middle heme domain, and a C-terminal cellulose-binding domain, which shows sequence similarity with the cellulose-binding domains of endoglucanases and cellobiohydrolases from Trichoderma reesei. Comparison with the CDH sequences of P. chrysosporium and Trametes versicolor identified Met 95 and His 143 as potential heme coordinations. EFIG, LGGPM, and VNSTH motifs in the heme domain and the XRXPXTDXPSXDGXRY motif in the flavin domain were identified as CDH-specific motifs. With regard to the amino acid composition, S. thermophile CDH has more disulfide linkages and acidic and basic amino acids compared to CDHs from P. chrysosporium and T. versicolor.

Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): characterization of connective tissue growth factor as a member of the IGFBP superfamily.

The insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine settings. Additionally, some IGFBPs appear to exhibit biological effects that are IGF independent. The six high-affinity IGFBPs that have been characterized to date exhibit 40-60% amino acid sequence identity overall, with the most conserved sequences in their NH2 and COOH termini. We have recently demonstrated that the product of the mac25/IGFBP-7 gene, which shows significant conservation in the NH2 terminus, including an "IGFBP motif' (GCGCCXXC), exhibits low-affinity IGF binding. The closely related mammalian genes connective tissue growth factor (CTGF) gene, nov, and cyr61 encode secreted proteins that also contain the conserved sequences and IGFBP motifs in their NH2 termini. To ascertain if these genes, along with mac25/IGFBP-7, encode a family of low-affinity IGFBPs, we assessed the IGF binding characteristics of recombinant human CTGF (rhCTGF). The ability of baculovirus-synthesized rhCTGF to bind IGFs was demonstrated by Western ligand blotting, affinity cross-linking, and competitive affinity binding assays using 125I-labeled IGF-I or IGF-II and unlabeled IGFs. CTGF, like mac25/IGFBP-7, specifically binds IGFs, although with relatively low affinity. On the basis of these data, we propose that CTGF represents another member of the IGFBP family (IGFBP-8) and that the CTGF gene, mac25/IGFBP-7, nov, and cyr61 are members of a family of low-affinity IGFBP genes. These genes, along with those encoding the high-affinity IGFBPs 1-6, together constitute an IGFBP superfamily whose products function in IGF-dependent or IGF-independent modes to regulate normal and neoplastic cell growth.

Cellobiose dehydrogenase from Phanerochaete chrysosporium is encoded by two allelic variants.

The complete nucleotide sequences of two alleles of cellobiose dehydrogenase, cdh-1 (3,627 bp) and cdh-2 (3,623 bp), from Phanerochaete chrysosporium OGC101 are reported. The nucleotide sequences of cdh-1 and cdh-2 exhibit 97% similarity. A total of eighty-six point mutations between cdh-1 and cdh-2 are observed. Both alleles have 14 exons, and the introns are located at exactly the same positions. The translation products of these alleles have identical amino acid sequences. Restriction fragment length polymorphism analyses of homokaryotic derivatives show segregation of the CDH alleles.

Synthesis and characterization of insulin-like growth factor-binding protein (IGFBP)-7. Recombinant human mac25 protein specifically binds IGF-I and -II.

The mac25 cDNA was originally cloned from leptomeningial cells and subsequently reisolated through differential display as a sequence preferentially expressed in senescent human mammary epithelial cells. The deduced amino acid sequence of the human mac25 propeptide shares a 20-25% identity to human insulin-like growth factor-binding proteins (IGFBPs), suggesting that mac25 could be another member of the IGFBP family. In the present study, we have generated recombinant human mac25 (rh-mac25) in a baculovirus expression system and assessed its affinity for IGFs and have evaluated the pattern of expression of the mac25 gene in human tissues. Binding of 125I-IGF-I and 125I-IGF-II to rh-mac25 was demonstrated by Western ligand blotting after nondenaturing polyacrylamide gel electrophoresis and by affinity cross-linking with as little as 2 nM rh-mac25. Specificity of rh-mac25 binding to 125I-IGFs was demonstrated by competition for rh-mac25 binding with unlabeled IGFs, but not with [QAYLL]IGF-II analog, which has 100-fold less affinity for IGFBPs. In comparison with IGFBP-3, rh-mac25 has at least a 5-6-fold lower affinity for IGF-I and 20-25-fold lower affinity for IGF-II. mac25 mRNA was detectable in a wide range of normal human tissues, with decreased expression in breast, prostate, colon, and lung cancer cell lines. In conclusion, mac25 specifically binds IGFs and constitutes a new member of the IGFBP family, IGFBP-7. Its wider distribution in normal tissue and lower expression in several cancer cells indicate that IGFBP-7 may function as a growth-suppressing factor, as well as an IGF-binding protein.

Cloning of a cDNA encoding cellobiose dehydrogenase, a hemoflavoenzyme from Phanerochaete chrysosporium.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by cellulose-degrading cultures of the wood-degrading basidiomycete Phanerochaete chrysosporium. CDH contains one flavin adenine dinucleotide (FAD) and one heme b per molecule, and it oxidizes cellobiose to cellobionolactone. In this report, a 2.4-kb cDNA encoding CDH was isolated by screening an expression library of P. chrysosporium OGC101 with a CDH-specific polyclonal antibody. The cDNA encodes a 755-amino-acid protein with a predicted mass of 80,115 Da. Sequence analysis suggests that the heme domain is located at the N terminus and that the falvin domain is located at the C terminus. The flavin domain shows a beta 1-alpha A-beta 2 motif for FAD binding and has high sequence similarity to several FAD-dependent enzymes. Little sequence similarity to hemoflavoenzymes is found. CDH binds to cellulose similarly to cellulases. However, little sequence similarity is observed with the conserved cellulose-binding sequences of cellulases. This suggests that CDH might possess a specific sequence for cellulose binding which is different from that of cellulases. Northern (RNA) blot analysis of total RNA from cellulose-, glucose-, and cellobiose-grown P. chrysosporium indicated that CDH mRNA is produced only in cellulose-grown cells. This suggests that CDH expression is regulated at the transcriptional level by either cellulose or one of its degradation products. Southern blot analysis suggests the presence of only a single gene for CDH in P. chrysosporium OGC101.

There are three distinct forms of bombesin. Identification of [Leu13]bombesin, [Phe13]bombesin, and [Ser3,Arg10,Phe13]bombesin in the frog Bombina orientalis.

Amphibian bombesin is the prototypic peptide that defines the bombesin-like peptide family. In this paper we show that in the frog Bombina orientalis, there are actually 3 distinct forms of bombesin, and each of these peptides is an agonist with differing affinities for the known bombesin receptors. Oligonucleotides complementary to the 5'- and 3'-untranslated regions of the bombesin mRNA were used to amplify bombesin-related cDNAs from the skin, brain, and gut of B. orientalis. Three classes of cDNAs were found. One class encoded the previously characterized form of bombesin which has a Leu at position 13 ([Leu13]bombesin). The other two classes, respectively, encoded new bombesin-like peptides which we have designated as [Phe13]bombesin and [Ser3,Arg10,Phe13]bombesin ([SAP]bombesin). The existence of [SAP]bombesin in skin was confirmed by tandem mass spectrometry. Polymerase chain reaction analysis of genomic DNA showed the mRNAs for [Leu13]bombesin, [Phe13]bombesin, and [SAP]bombesin most likely arise from separate genes. Polymerase chain reaction analysis showed different patterns of tissue-specific expression for each form. [Leu13]Bombesin and [SAP]bombesin were predominantly expressed in skin, brain, and gut; [Phe13]bombesin was expressed only in brain, and [Leu13]bombesin predominated in oocytes. [SAP]Bombesin contained a cleavage site between residues 4 and 5, which if used would yield the peptide [SAP]bombesin(5-14) which has the sequence [Gln3,Arg6]neuromedin B. Thus a frog homolog of NMB could derive from the [SAP]bombesin prohormone. [Phe13]Bombesin, [SAP]bombesin, and [SAP]bombesin(5-14) were synthesized and their affinities for the mammalian bombesin-like peptide (GRP and NMB) receptors determined. These peptides acted as agonists for the GRP and NMB receptors, with relative potencies for the GRP receptor of [Leu13]bombesin > [Phe13]bombesin > [SAP]bombesin(5-14) > [SAP]bombesin and for the NMB receptor of [Phe13]bombesin > [SAP]bombesin(5-14) > [Leu13]bombesin > [SAP]bombesin. None of these peptides demonstrated high affinity binding for the BRS-3 receptor. The different receptor affinities and tissue distribution of these peptides suggests distinct physiologic roles and raises the possibility of as yet uncharacterized mammalian homologs of these new amphibian peptides.

Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4).

Bombesin is a tetradecapeptide originally isolated from frog skin and demonstrated to have a wide range of actions in mammals. Based on structural homology and similar biological activities, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of bombesin. We previously reported that frogs have both GRP and bombesin, which therefore are distinct peptides. We now report the cloning of a bombesin receptor subtype (BB4) that has higher affinity for bombesin than GRP. PCR was used to amplify cDNAs related to the known bombesin receptors from frog brain. Sequence analysis of the amplified cDNAs revealed 3 classes of receptor subtypes. Based on amino acid homology, two classes were clearly the amphibian homologs of the GRP and neuromedin B receptors. The third class was unusual and a full-length clone was isolated from a Bombina orientalis brain cDNA library. Expression of the receptor in Xenopus oocytes demonstrated that the receptor responded to picomolar concentrations of [Phe13]-bombesin, the form of bombesin most prevalent in frog brain. The relative rank potency of bombesin-like peptides for this receptor was [Phe13]bombesin > [Leu13]bombesin > GRP > neuromedin B. In contrast, the rank potency for the GRP receptor is GRP > [Leu13]bombesin > [Phe13]bombesin > neuromedin B. Transient expression in CHOP cells gave a Ki for [Phe13]bombesin of 0.2 nM versus a Ki of 2.1 nM for GRP. Distribution analysis showed that this receptor was expressed only in brain, consistent with the distribution of [Phe13]-bombesin. Thus, based on distribution and affinity, this bombesin receptor is the receptor for [Phe13]bombesin. Phylogenetic analysis suggests that this receptor separated prior to separation of the GRP and neuromedin B receptors; thus, BB4 receptors and their cognate ligands may also exist in mammals.

A rhesus monkey model to characterize the role of gastrin-releasing peptide (GRP) in lung development. Evidence for stimulation of airway growth.

Gastrin-releasing peptide (GRP) is developmentally expressed in human fetal lung and is a growth factor for normal and neoplastic lung but its role in normal lung development has yet to be clearly defined. In this study we have characterized the expression of GRP and its receptor in fetal rhesus monkey lung and determined the effects of bombesin on fetal lung development in vitro. By RNA blot analysis, GRP mRNA was first detectable in fetal monkey lung at 63 days gestation, reached highest levels at 80 days gestation, and then declined to near adult levels by 120 days gestation; a pattern closely paralleling GRP expression in human fetal lung. As in human lung, in situ hybridization localized GRP mRNA to neuroendocrine cells though during the canalicular phase of development (between 63-80 days gestation) GRP mRNA was present not only in classic pulmonary neuroendocrine cells, but also in cells of budding airways. Immunohistochemistry showed that bombesin-like immunoreactivity was present in neuroendocrine cells, but not in budding airways, suggesting that in budding airways either the GRP mRNA is not translated, is rapidly secreted, or a related, but different RNA is present. RNase protection analysis using a probe to the monkey GRP receptor demonstrated that the time course of receptor RNA expression closely paralleled the time course of GRP RNA expression. In situ hybridization showed that GRP receptors were primarily expressed in epithelial cells of the developing airways. Thus GRP would appear to be secreted from neuroendocrine cells to act on target cells in developing airways. This hypothesis was confirmed by organ culture of fetal monkey lung in the presence of bombesin and bombesin antagonists. Bombesin treatment at 1 and 10 nM significantly increased DNA synthesis in airway epithelial cells and significantly increased the number and size of airways in cultured fetal lung. In fact, culturing 60 d fetal lung for 5 d with 10 nM bombesin increased airway size and number nearly to that observed in cultured 80 d fetal lung. The effects of bombesin could be blocked by specific GRP receptor antagonists. Thus this study demonstrates that GRP receptors are expressed on airway epithelial cells in developing fetal lung and that the interaction of GRP with the GRP receptor stimulates airway development.

Functional analysis of the 5'-flanking region of the human gastrin-releasing peptide gene in small cell lung carcinoma cell lines.

The mammalian bombesin-like peptide, gastrin-releasing peptide (GRP), is expressed by many small cell lung carcinomas (SCLC), stimulates the growth of some SCLC and thus is an autocrine growth factor for a subset of SCLC. The subset of SCLC that expresses GRP typically shows neuroendocrine differentiation and has been designated as "classic" SCLC. To begin to characterize the mechanisms responsible for the expression of GRP in classic SCLC, the 5'-flanking region of the human GRP gene was sequenced and analyzed for cis-acting elements. Constructs containing up to 6.0 kilobases of 5'-flanking region were transiently expressed in SCLC and in control cell lines. Deletion analysis demonstrated that a sequence of 178 bases upstream of the transcriptional start was sufficient for basal expression in SCLC cell lines. A negative regulatory element was located between -5.5 and -2.6 kilobases; a general enhancer element was located between -1409 and -1197 kilobases, and a tissue-specific element was located between -1128 and -793 kilobases. Mobility shift analysis indicated that proteins from nuclear extracts of classic SCLC but not variant SCLC bound to this tissue-specific regulatory element. Thus the regulation of GRP gene expression in SCLC cell lines is a balance between a negative element, a general enhancer, and a tissue-specific element that appears active only in classic SCLC cell lines.

Cloning of complementary DNAs encoding the amphibian bombesin-like peptides Phe8 and Leu8 phyllolitorin from Phyllomedusa sauvagei: potential role of U to C RNA editing in generating neuropeptide diversity.

The bombesin-like peptides were originally characterized in frog skin, then later found to have a wide distribution and range of actions in mammals. The bombesin-like peptides have classically been divided into three subfamilies, the bombesin subfamily, of which gastrin-releasing peptide (GRP) is the mammalian form; the ranatensin subfamily, of which neuromedin-B (NMB) is the mammalian form; and the phyllolitorin subfamily, which to date has only been characterized in amphibians. As a first step in characterizing mammalian phyllolitorin-like peptides, we have cloned complementary DNAs (cDNAs) encoding Leu8 and Phe8 phyllolitorin from Phyllomedusa sauvagei. Sequence analysis revealed that the amphibian phyllolitorin messenger RNA (mRNA) encodes a precursor of 90 amino acids containing a signal peptide sequence, an amino-terminal extension peptide, the phyllolitorin peptide of nine amino acids, and a carboxy-terminal extension peptide. Northern blot, reverse transcriptase-polymerase chain reaction (PCR), and in situ hybridization analysis showed that the mRNA was present at highest levels in skin, at lower levels in brain, and at lowest levels in gut. Phylogenetic analysis of bombesin-like peptide prohormone sequences showed that the phyllolitorin prohormones are much more closely related to the bombesin and ranatensin prohormones than to the GRP and NMB prohormones. This analysis suggests that the bombesin-like peptides should be reclassified into the GRP subfamily, the NMB subfamily, and the skin peptide subfamily. Surprisingly, the cDNAs encoding Phe8 and Leu8 phyllolitorins were identical except for a single T to C difference in the codon coding for the Phe or Leu residue of phyllolitorin.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone receptor, but not estradiol receptor, messenger ribonucleic acid is expressed in luteinizing granulosa cells and the corpus luteum in rhesus monkeys.

Estrogens (i.e. estradiol) and progestins (i.e. progesterone) may act as local regulators of ovarian function in various species. This study tested the hypothesis that if progesterone and estradiol act via receptor-mediated pathways in the primate ovary, then receptor messenger RNAs (mRNAs) should be detectable in ovarian cells. The reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect progesterone and estradiol receptor (PR and ER, respectively) mRNAs in the rhesus monkey ovary. Total RNA was isolated from macaque uterine myometrium (positive control), spleen (negative control), whole ovary, germinal (surface) epithelium-enriched cortical and medullary compartments of the ovary, granulosa cells in preovulatory follicles before and after an ovulatory stimulus, and corpora lutea from early (days 3-5), mid (days 7 and 8)-, and late (days 14 and 15) luteal phase of the menstrual cycle. Using primers to the hormone-binding region encoded by the receptor mRNAs, RT-PCR products of the expected sizes were detected for PR and ER from 1 microgram myometrial RNA, whereas products were not obtained from spleen. PR mRNA product was detected in all ovaries, germinal epithelium-enriched cortical and medullary compartments, and corpora lutea from all three stages of the luteal phase (n = 3/stage). PR mRNA product was detected as a strong band in one of three preparations obtained from granulosa cells before an ovulatory stimulus. In contrast, PR mRNA was detected in granulosa cells from all animals after an ovulatory dose of hCG. ER mRNA was detected in whole ovary and in germinal epithelium-enriched cortical compartments, with a barely visible product occasionally observed in medullary compartments of the ovary. In contrast to PR mRNA, ER mRNA was not detected in any corpora lutea throughout the luteal phase or in granulosa cells obtained before or after an ovulatory stimulus. To confirm the specificity of the RT-PCR products, restriction enzymes cleaved the PR product from myometrium, germinal epithelium-enriched cortical compartment, and corpus luteum into the predicted size fragments. Similarly, the ER product from the myometrium and the germinal epithelium-enriched compartment was cleaved into the expected size fragments. Sequence analysis of the PR and ER RT-PCR products revealed 99% homology to the complementary DNA for the hormone-binding region of human PR and ER, respectively. Thus, PR mRNA detection supports the hypothesis of progesterone action via classical receptor-mediated pathways in the luteinizing follicle and corpus luteum of the primate ovary.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular cloning and characterization of receptors for the mammalian bombesin-like peptides.

The bombesin-like peptides comprise a large family of peptides common to both amphibians and mammals that function as growth factors, neurotransmitters, and paracrine hormones. GRP, the mammalian homolog of bombesin and its receptor, as well as NMB, the mammalian homolog of ranatensin, are expressed in human neoplasms and, in particular, in small cell lung carcinomas (SCLC). To better characterize the physiological roles of bombesin-like peptides, our laboratory has cloned the receptors for GRP in murines, rats, and humans. The 3T3 GRP receptor was isolated and characterized using the two-electrode-voltage-clamp analysis and acquorin-emission methods in xenopus oocytes expression system. The rat and human GRP and NMB receptors were cloned by hybridization at low stringency, using the mouse cDNA receptor probe. Sequence analysis of the receptors showed 384 and 390 amino acids for GRP and NMB receptors, respectively. The homology between the two receptors is 60% and between species in the same receptor, 90%. The receptors belong to the 7-membrane spanning domains superfamily. The specific GRP-R antagonist blocked the response to bombesin in oocytes injected with GRP-R, but failed to do so in oocytes injected with NMB-R. The two receptors differ in their distribution of tissue expression. RNA blot and RNase protection analysis showed the same size of mRNA without alteration in the receptors. RT + PCR analysis performed on genomic DNA revealed similarity between normal and cell DNAs, suggesting no major gene deletion or rearrangement. Southern blot analysis indicated the absence of gene amplification. Sequence analysis of the exonic segments of the receptor genes displayed identical amino acids to the respective cDNAs. None of the genes had classic TATAA box. Somatic cell hybrids localized the GRP-R on the X-chromosome and the NMB-R on chromosome 6. The same sequence of normal genes and cDNAs of GRP and NMB receptors, together with the gene characterization, demonstrated that SCLC cell lines do not require a structural change in receptor protein or genomic rearrangement.

Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.