Srinivasa Nagalla

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.

Proteomic Identification of Salivary Biomarkers of Type-2 Diabetes

The identification of biomarkers to noninvasively detect prediabetes/diabetes will facilitate interventions designed to prevent or delay progression to frank diabetes and its attendant complications. The purpose of this study was to characterize the human salivary proteome in type-2 diabetes to identify potential biomarkers of diabetes. Whole saliva from control and type-2 diabetic individuals was characterized by multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS). Label-free quantification was used to identify differentially abundant protein biomarkers. Selected potential biomarkers were then independently validated in saliva from control, diabetic, and prediabetic subjects by Western immunoblotting and ELISA. Characterization of the salivary proteome identified a total of 487 unique proteins. Approximately 33% of these have not been previously reported in human saliva. Of these, 65 demonstrated a greater than 2-fold difference in abundance between control and type-2 diabetes samples. A majority of the differentially abundant proteins belong to pathways regulating metabolism and immune response. Independent validation of a subset of potential biomarkers utilizing immunodetection confirmed their differential expression in type-2 diabetes, and analysis of prediabetic samples demonstrated a trend of relative increase in their abundance with progression from the prediabetic to the diabetic state. This comprehensive proteomic analysis of the human salivary proteome in type-2 diabetes provides the first global view of potential mechanisms perturbed in diabetic saliva and their utility in detection and monitoring of diabetes. Further characterization of these markers in a larger cohort of subjects may provide the basis for new, noninvasive tests for diabetes screening, detection, and monitoring.

Bombesin-like peptides: Of ligands and receptors

Publisher Summary The bombesin-like peptides comprise a large family of peptides common to frogs and man. Bombesin when injected into rats proved to have a wide variety of pharmacologic effects, including effects on body temperature, cardiac output, blood pressure, and the release of gastrointestinal hormones. These many effects prompted a search for mammalian homologs of the amphibian bombesin-like peptides. The bombesin-like peptides have been divided into three subfamilies, based on their penultimate and adjacent amino acids. The bombesin subfamily contains Leu as its penultimate amino acid, the ranatensin subfamily contains Phe as its penultimate amino acid, and the phyllolitorins occur in both a Leu (bombesin-like) and Phe (ranatensin-like) form but substitute a serine adjacent to the penultimate residue. At present, there are two mammalian bombesin-like peptides, GRP and NMB, and two known receptors, the GRP-preferring subtype and the NMB-preferring subtype. Given the potential of crosstalk between these receptors and ligands, there is much complexity still to be unraveled to understand fully the physiologic and pathologic roles of the bombesin-like peptides.

Suppression subtractive hybridization and microarray identification of estrogen-regulated hypothalamic genes.

The gonadal steroid estrogen is a pleiotropic hormone that has multiple effects on numerous cellular functions. One of estrogens major targets is the brain, where the steroid not only affects growth, differentiation, and survival of neurons, but also regulates cell excitability. Because estrogen modulates multiple, overlapping signaling pathways, it has been difficult to scrutinize the transcriptional activity of the steroid. Therefore, we still lack a global picture of how different genes interact and are regulated by estrogen. Herein we report the use of suppression subtractive hybridization followed by custom microarray analysis of thousands of genes that are differentially expressed during the negative feedback phase of the female reproductive cycle. We have found a number of key transcripts that are regulated by estrogen and contribute to the alteration in synaptic transmission and hence excitability of hypothalamic neurons (e.g., GABA neurons). These include gec-1, GABABR2, PI3 kinase subunit p55γ, and a number of proteins containing pleckstrin homology domains that are critical for plasma membrane targeting. Studies are underway to refine our analysis to individual nuclei and individual cells. However, what has emerged from this highly sensitive microarray analysis is that estrogen affects neuronal plasticity in hypothalamic neurons not only by transcription of new membrane proteins (e.g., receptors and channels), but also by altering expression of downstream signaling molecules and proteins involved in neurosecretory pathways.

Comprehensive maternal serum proteomic profiles of preclinical and clinical preeclampsia

We systematically characterized maternal serum proteome in women with clinical preeclampsia (PE) and asymptomatic women in early pregnancy that subsequently developed PE. Clinical PE cohort comprised 30 patients with mild PE, 30 with severe PE, and 58 normotensive women. Preclinical PE cohort included 149 women whose serum samples were collected at 8-14 gestational weeks and in whom 30 women later developed mild and 40 severe PE. Serum proteome was analyzed and enzyme-linked immunosorbent assays were used for protein quantification. In Clinical PE, fibronectin, pappalysin-2, choriogonadotropin-beta, apolipoprotein C-III, cystatin-C, vascular endothelial growth factor receptor-1, and endoglin were more abundant compared to normotensive women. In preclinical PE, differently expressed proteins included placental, vascular, transport, matrix, and acute phase proteins. Angiogenic and antiangiogenic proteins were not significant. We conclude that placental and antiangiogenic proteins are abundant in clinical PE. In preclinical PE, proteomic profile is distinct and different from that in clinical PE.

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.

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid

OBJECTIVE We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. STUDY DESIGN Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. RESULTS Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. CONCLUSION Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection.

Genetic basis for chamber-specific ventricular phenotypes in the rat infarct model

BACKGROUND We, and others, have previously reported a strong correlation between increased inter-ventricular dispersion of repolarization and the occurrence of fatal arrhythmia in animal models of CHF. The existence of this and other such distinct electrophysiologic phenotypes in right (RV) vs. left ventricles (LV) could be explained by chamber-specific patterns of gene expression. METHODS We employed microarray gene profiling of 13824 sequence-verified, nonredundant rodent cDNAs to compare myocardial gene expression in RV vs. LV of rats with surgically induced myocardial infarction (MI: n=3) and in sham-operated animals (Sham: n=3). RESULTS Significant LV infarction (32+/-4% LV) and severe CHF were observed in all MI animals at 4 weeks. In Sham animals, 937 genes exhibited significant differential expression in RV vs. LV myocardium. In MI animals, 1158 genes exhibited significant differential expression in RV vs. LV. Of those genes exhibiting significant differential expression, only 241 were common to both Sham and MI animals. Differentially expressed genes included those involved in signal transduction, cell growth and maintenance, and apoptosis. Genes with potential roles in altered dispersion of repolarization included voltage-dependent Ca(2+) channel gamma subunit (MI 8-fold increasing) and K(+) inwardly rectifying channel subfamily J, member 10 (MI 6-fold decreasing). Gap junction membrane channel protein alpha 4 (MI 6-fold decreasing) and cardiac troponin I (MI 8-fold decreasing) were also significantly differentially expressed. Inter-ventricular comparisons revealed significantly greater alterations in gene expression vs. intra-ventricular comparisons. CONCLUSIONS Microarray gene profiling has revealed candidate genes, some of them novel, which may account for chamber-specific ventricular electrophysiologic phenotypes, both in physiologic as well as in arrhythmogenic states such as CHF.

Glycosylated Fibronectin as a First-Trimester Biomarker for Prediction of Gestational Diabetes

OBJECTIVE: To evaluate the potential clinical utility of serum biomarkers for first-trimester prediction of gestational diabetes mellitus (GDM). METHODS: Maternal serum concentrations of glycosylated (Sambucus nigra lectin–reactive) fibronectin, adiponectin, sex hormone–binding globulin, placental lactogen, and high-sensitivity C-reactive protein (CRP) were measured at 5–13 weeks of gestation in a case-control study of 90 pregnant women with subsequent development of GDM and in 92 control group participants. Ability to detect GDM was assessed using logistic regression modeling and receiver operating characteristic (ROC) curves. Classification performance and positive and negative predictive values were reported at specific thresholds. Glycosylated fibronectin variation across trimesters was evaluated using a serial-measures analysis of 35 nondiabetic control group participants. RESULTS: First-trimester serum concentrations of glycosylated fibronectin, adiponectin, high-sensitivity CRP, and placental lactogen were significantly associated (P<.001) with GDM. After adjustment for maternal factors and other biomarkers, glycosylated fibronectin demonstrated an independent association with GDM (P<.001). Adiponectin, high-sensitivity CRP, and placental lactogen demonstrated modest classification performance compared with glycosylated fibronectin (respectively: area under the curve [AUC] 0.63; 95% confidence interval [CI] 0.53–0.71; AUC 0.68; 95% CI 0.60–0.76; and AUC 0.67, 95% CI 0.59–0.75; compared with AUC 0.91; 95% CI 0.87–0.96). Glycosylated fibronectin levels above a threshold of 120 mg/L correctly identified 57 GDM case group participants with a positive predictive value of 63% (95% CI 53–72%) and a negative predictive value of 95% (95% CI 94–95%) at a population prevalence of 12%. There was no association between sex hormone–binding globulin and GDM. CONCLUSION: First-trimester glycosylated fibronectin is a potential pregnancy-specific biomarker for early identification of women at risk for GDM. LEVEL OF EVIDENCE: II

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 acquorinemission 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.