Biomedical subjects

Srinivasa R Nagalla

Publications and source records attributed to Srinivasa R Nagalla.

10 recordsLinked to original sources

PubMed2007

Proteomic analysis of cervical-vaginal fluid: identification of novel biomarkers for detection of intra-amniotic infection.

Intra-amniotic infection (IAI) is associated with preterm birth and perinatal mortality. To identify potential biomarkers, we performed a comprehensive survey of the cervical-vaginal fluid (CVF) proteome from a primate IAI model utilizing multidimensional protein identification technology (LC/LC-MS/MS) and MALDI-TOF-MS analyses. Analyses of CVF proteome identified 205 unique proteins and differential expression of 27 proteins in controls and IAI samples. Protein expression signatures and immunodetection of specific biomarkers identified can be employed for noninvasive detection of IAI.

Amniotic Fluid↗

PubMed2006

A novel EWS-WT1 gene fusion product in desmoplastic small round cell tumor is a potent transactivator of the insulin-like growth factor-I receptor (IGF-IR) gene.

Desmoplastic small round cell tumor (DSRCT) is a primitive sarcoma characterized by a recurrent chromosomal translocation, t(11;22)(p13;q12), which fuses the 5' exons of the EWS gene to the 3' exons of the WT1 gene. EWS-WT1 chimeras are heterogeneous as a result of fusions of different regions of the EWS gene to the WT1 gene. We report here a rare and novel EWS-WT1 variant, EWS-WT1 5/10, in a 6-year-old boy diagnosed with DSRCT and analyze the potential transactivation effect of the fusion oncoprotein. The predicted product is comprised of the N-terminal transactivation domain of EWS and lacks any sequence derived from the WT1 gene product. Nonetheless, the truncated protein was able to stimulate expression of the insulin-like growth factor-I receptor gene, a potent antiapoptotic receptor tyrosine kinase with potentially important roles in DSRCT etiology. These findings raise the possibility that the oncogenic potential of EWS-WT1 fusions is not necessarily a consequence of the fusion protein product per se.

Child↗

PubMed2005

Standardizing global gene expression analysis between laboratories and across platforms.

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.

Gene Expression Profiling↗

PubMed2004

GABAergic system gene expression predicts clinical outcome in patients with neuroblastoma.

PURPOSE: Neuroblastoma (NB) is a common childhood malignancy characterized by heterogeneous clinical behavior. The purpose of this study was to identify potential NB biomarkers that may improve outcome prediction. PATIENTS AND METHODS: The suppression subtractive hybridization (SSH) technique was used to identify the genes differentially expressed between NB and control tissue. RNA isolated from 235 primary NB tumor samples obtained from the Children's Cancer Group was evaluated for expression of the candidate markers using quantitative reverse transcriptase polymerase chain reaction (Taqman assays). The association between the mRNA expression levels in the identified candidate genes and clinical outcome was evaluated. RESULTS: SSH analysis identified differential expression of members of the GABAergic gene family in NB. Lower levels of gamma-aminobutyric acid (GABA) receptor-associated protein (GABARAP) gene expression predict decreased survival among all patients. GABA(A) delta receptor subunit gene expression was predictive of a poor outcome among Evans stage IV-S patients. An index of five coexpressed GABA(A) receptor subunits was identified (GABA(A) profile [GAP score]). Patients with a higher GAP score (> -1) had a survival advantage. Multivariate analysis showed that GABARAP and GABA(A) alpha2 receptor subunit gene expression levels and GAP score remained predictors of clinical outcome after accounting for current prognostic indicators. CONCLUSION: Dysregulation of the GABAergic system may constitute a fundamental event in the development of NB, and assessment of GABAergic system gene expression could provide improved patient stratification and potential new therapies.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Diagnosis of intra-amniotic infection by proteomic profiling and identification of novel biomarkers.

CONTEXT: Intra-amniotic infection (IAI) is commonly associated with preterm birth and adverse neonatal sequelae. Early diagnosis of IAI, however, has been hindered by insensitive or nonspecific tests. OBJECTIVE: To identify unique protein signatures in rhesus monkeys with experimental IAI, a proteomics-based analysis of amniotic fluid was used to develop diagnostic biomarkers for subclinical IAI in amniotic fluid and blood of women with preterm labor. DESIGN, SETTING, AND PARTICIPANTS: Surface-enhanced laser desorption-ionization/time-of-flight mass spectrometry, gel electrophoresis, and tandem mass spectrometry were used to characterize amniotic fluid peptides in 19 chronically instrumented pregnant rhesus monkeys before and after experimental IAI. Candidate biomarkers were determined by liquid chromatography-tandem mass spectrometry. Polyclonal antibodies were generated from synthetic peptides for validation of biomarkers of IAI. Amniotic fluid peptide profiles identified in experimental IAI were subsequently tested in a cohort of 33 women admitted to Seattle, Wash, hospitals between June 25, 1991, and June 30, 1997, with preterm delivery at 35 weeks or earlier associated with subclinical IAI (n = 11), preterm delivery at 35 weeks or earlier without IAI (n = 11), and preterm contractions with subsequent term delivery at later than 35 weeks (n = 11). MAIN OUTCOME MEASURES: Identification of peptide biomarkers for occult IAI. RESULTS: Protein expression profiles in amniotic fluid showed unique signatures of overexpression of polypeptides in the 3- to 5-kDa and 10- to 12-kDa molecular weight ranges in all animals after infection and in no animal prior to infection. In women, the 10- to 12-kDa signature was identified in all 11 patients with subclinical IAI, in 2 of 11 with preterm delivery without IAI, and in 0 of 11 with preterm labor and term delivery without infection (P<.001). Peptide fragment analysis of the diagnostic peak in amniotic fluid identified calgranulin B and a unique fragment of insulinlike growth factor binding protein 1, which were also expressed in maternal serum. Mapping of other amniotic fluid proteins differentially expressed in IAI identified several immunoregulators not previously described in amniotic fluid. CONCLUSIONS: This proteomics-based characterization of the differential expression of amniotic fluid proteins in IAI identified a distinct proteomic profile in an experimental primate chorioamnionitis model that detected subclinical IAI in a human cohort with preterm labor. These diagnostic protein expression signatures, complemented by immunodetection of specific biomarkers in amniotic fluid and in maternal serum, might have application in the early detection of IAI.

Adult↗

PubMed2004

High-throughput identification of proteins and unanticipated sequence modifications using a mass-based alignment algorithm for MS/MS de novo sequencing results.

With the increasing availability of de novo sequencing algorithms for interpreting high-mass accuracy tandem mass spectrometry (MS/MS) data, there is a growing need for programs that accurately identify proteins from de novo sequencing results. De novo sequences derived from tandem mass spectra of peptides often contain ambiguous regions where the exact amino acid order cannot be determined. One problem this poses for sequence alignment algorithms is the difficulty in distinguishing discrepancies due to de novo sequencing errors from actual genomic sequence variation and posttranslational modifications. We present a novel, mass-based approach to sequence alignment, implemented as a program called OpenSea, to resolve these problems. In this approach, de novo and database sequences are interpreted as masses of residues, and the masses, rather than the amino acid codes, are compared. To provide further flexibility, the masses can be aligned in groups, which can resolve many de novo sequencing errors. The performance of OpenSea was tested with three types of data: a mixture of known proteins, a mixture of unknown proteins that commonly contain sequence variations, and a mixture of posttranslationally modified known proteins. In all three cases, we demonstrate that OpenSea can identify more peptides and proteins than commonly used database-searching programs (SEQUEST and ProteinLynx) while accurately locating sequence variation sites and unanticipated posttranslational modifications in a high-throughput environment.

Algorithms↗

PubMed2004

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 estrogen's 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, GABA(B)R2, PI3 kinase subunit p55gamma, 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.

Animals↗

PubMed2003

Cloning and characterization of Xen-dorphin prohormone from Xenopus laevis: a new opioid-like prohormone distinct from proenkephalin and prodynorphin.

Opioid-like peptides mediate analgesia and induce behavioral effects such as tolerance and dependence by ligand-receptor-mediated mechanisms. The classical opioid prohormones can generate several bioactive peptides, and these divergent families of prohormones share a common well conserved ancestral opioid motif (Tyr-Gly-Gly-Phe). Evidence from pharmacological and molecular cloning studies indicates the presence of multiple isoforms of opioid ligands and receptors that are as yet uncharacterized. To identify potential new members we used the opioid motif as an anchor sequence and isolated two distinct isoforms (Xen-dorphins A and B) of an opioid prohormone from Xenopus laevis brain cDNA library. Xen-dorphin prohormones can generate multiple novel opioid ligands distinct from the known members of this family. Both isoforms are present in a wide variety of tissues including the brain. Two potential bioactive peptides, Xen-dorphin-1A and -1B, that were chemically synthesized showed opioid agonist activity in frog and rat brain membranes using a [35S]GTPgammaS assay. Initial radioligand binding experiments demonstrated that Xen-dorphin-1B binds with high affinity to opioid receptor(s) and with potential preference to the kappa-opioid receptor subtype. Cloning of the Xen-dorphin prohormone provides new evidence for the potential presence of other members in the opioid peptide superfamily.

Amino Acid Motifs↗

PubMed2003

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.

Animals↗

PubMedDate not supplied

Identification of protein modifications using MS/MS de novo sequencing and the OpenSea alignment algorithm.

Algorithms that can robustly identify post-translational protein modifications from mass spectrometry data are needed for data-mining and furthering biological interpretations. In this study, we determined that a mass-based alignment algorithm (OpenSea) for de novo sequencing results could identify post-translationally modified peptides in a high-throughput environment. A complex digest of proteins from human cataractous lens, a tissue containing a high abundance of modified proteins, was analyzed using two-dimensional liquid chromatography, and data was collected on both high and low mass accuracy instruments. The data were analyzed using automated de novo sequencing followed by OpenSea mass-based sequence alignment. A total of 80 modifications were detected, 36 of which were previously unreported in the lens. This demonstrates the potential to identify large numbers of known and previously unknown protein modifications in a given tissue using automated data processing algorithms such as OpenSea.

Aged↗