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Biomedical subjects

P S Nelson

Publications and source records attributed to P S Nelson.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase.

hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln-. A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases. ch-hK4 was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified with an overall yield of 25%. The zymogen was readily self-activated during the refolding process to generate an active form (21 kDa) of hK4 (rhK4). rhK4 cleaved the chromogenic substrates Val-Leu-Arg-pNA (S-2266), Pro-Phe-Arg-pNA (S-2302), Ile-Glu-Gly-Arg-pNA (S-2222), and Val-Leu-Lys-pNA (S-2251), indicating that rhK4 has a trypsin-type substrate specificity. The rhK4 was inhibited by aprotinin (6 kDa), forming an equimolar 27 kDa complex. rhK4 readily activated both the precursor of PSA (pro-PSA) and single chain urokinase-type plasminogen activator (scuPA, pro-uPA). rhK4 also completely degraded prostatic acid phosphatase but failed to cleave serum albumin, another protein purified from human seminal plasma. These results indicate that hK4 may have a role in the physiologic processing of seminal plasma proteins such as pro-PSA, as well as in the pathogenesis of prostate cancer through its activation of pro-uPA.

Acid Phosphatase↗
PubMed2001

Project normal: defining normal variance in mouse gene expression.

The mouse has become an indispensable and versatile model organism for the study of development, genetics, behavior, and disease. The application of comprehensive gene expression profiling technologies to compare normal and diseased tissues or to assess molecular alterations resulting from various experimental interventions has the potential to provide highly detailed qualitative and quantitative descriptions of these processes. Ideally, to interpret experimental data, the magnitude and diversity of gene expression for the system under study should be well characterized, yet little is known about the normal variation of mouse gene expression in vivo. To assess natural differences in murine gene expression, we used a 5406-clone spotted cDNA microarray to quantitate transcript levels in the kidney, liver, and testis from each of 6 normal male C57BL6 mice. We used ANOVA to compare the variance across the six mice to the variance among four replicate experiments performed for each mouse tissue. For the 6 kidney samples, 102 of 3,088 genes (3.3%) exhibited a statistically significant mouse variance at a level of 0.05. In the testis, 62 of 3,252 genes (1.9%) showed statistically significant variance, and in the liver, there were 21 of 2,514 (0.8%) genes with significantly variable expression. Immune-modulated, stress-induced, and hormonally regulated genes were highly represented among the transcripts that were most variable. The expression levels of several genes varied significantly in more than one tissue. These studies help to define the baseline level of variability in mouse gene expression and emphasize the importance of replicate microarray experiments.

Animals↗
PubMed2001

A novel mechanism for chaperone-mediated telomerase regulation during prostate cancer progression.

Telomerase activity has been detected in >85% of all malignant human cancers, including 90% of prostate carcinomas. Using a well-characterized experimental prostate cancer system, we have found that telomerase activity is notably increased (>10-fold) during tumorigenic conversion. Expression profiles of the telomerase components (hTR and hTERT) revealed no substantive changes, which suggests a nontranscriptional mechanism for increased activity. Because the hsp90 chaperone complex functionally associates with telomerase, we investigated that relationship and found that along with telomerase activity, a number of hsp90-related chaperones are markedly elevated during transformation, as well as in advanced prostate carcinomas. Using the nontumorigenic cell protein extract as the source of telomerase, addition of purified chaperone components enhanced reconstitution of telomerase activity, which suggests a novel mechanism of increased telomerase assembly via a hsp90 chaperoning process during prostate cancer progression.

Animals↗
PubMed2001

Prostate short-chain dehydrogenase reductase 1 (PSDR1): a new member of the short-chain steroid dehydrogenase/reductase family highly expressed in normal and neoplastic prostate epithelium.

Genes regulated by androgenic hormones are of critical importance for the normal physiological function of the human prostate gland, and they contribute to the development and progression of prostate carcinoma. We used cDNA microarrays comprised of prostate-derived cDNAs to profile transcripts regulated by androgens in prostate cancer cells. This study identified a novel gene that we have designated prostate short-chain dehydrogenase/reductase 1 (PSDR1), that exhibits increased expression on exposure to androgens in the LNCaP prostate cancer cell line. Northern analysis demonstrated that PSDR1 is highly expressed in the prostate gland relative to other normal human tissues. The PSDR1 cDNA and putative protein exhibit homology to the family of short-chain dehydrogenase/reductase enzymes and thus identify a new member of this family. Cloning and analysis of the putative PSDR1 promoter region identified a potential androgen-response element. We used a radiation-hybrid panel to map the PSDR1 gene to chromosome 14q23-24.3. In situ hybridization localizes PSDR1 expression to normal and neoplastic prostate epithelium. These results identify a new gene involved in the androgen receptor-regulated gene network of the human prostate that may play a role in the pathogenesis of prostate carcinoma.

Amino Acid Sequence↗
PubMed2001

Prostate cancer genomics.

The molecular processes contributing to cancer of the human prostate gland are under intensive investigation. Methods used for discovering genetic alterations involved in prostate neoplasia include family studies designed to map hereditary disease loci, chromosomal studies to identify aberrations that may locate oncogenes or tumor suppressor genes, and comprehensive gene expression studies. These studies determine how various molecular signaling pathways influence or reflect the process of carcinogenesis. However, a comprehensive overview of the cell is necessary to understand all of the dynamic interactions between genes, their protein products, and the network of cellular processes resulting in tumorigenesis. Unraveling the complexity of these systems in a timely manner involves the integration of computers, miniaturization, and automation into molecular biology. New biotechnologies such as the development of automated DNA sequencing and complementary DNA microarrays allow for a systematic, "discovery-driven" approach. These and other technologies afford a comprehensive view of biology and pathology that have the potential to fully characterize the processes involved in neoplasia and therefore provide potential targets for the therapy of prostate and other cancers.

Genetic Therapy↗
PubMed2001

Sequence databases and microarrays as tools for identifying prostate cancer biomarkers.

Identification, acquisition, and assessment of molecular markers that could be adopted as surrogate endpoints for evaluating a response to prostate cancer intervention strategies is highly desirable. Recent advances in the fields of genomics and biotechnology have dramatically increased the quantity and accessibility of molecular information that is relevant to the study of prostate carcinogenesis. One major advance involves the construction of comprehensive databases that archive gene sequences and gene expression data. This information is in a format suitable for virtual queries designed to distinguish the molecular differences between normal and cancer cells. A second major advance uses robotic tools to construct microarrays comprising thousands of distinct genes expressed in prostate tissues. Such arrays offer a powerful approach for monitoring the expression of thousands of genes simultaneously and provide access for techniques designed to assess patterns or "fingerprints" of gene expression that may ultimately be used as signatures of response to therapeutic intervention.

Base Sequence↗
PubMed2001

From genomics to proteomics: techniques and applications in cancer research.

New technologies designed to facilitate the comprehensive analyses of genomes, transcriptomes and proteomes in health and disease are poised to exert a dramatic change on the pace of cancer research and to impact significantly on the care of cancer patients. These approaches have already demonstrated the power of molecular medicine in discriminating among disease subtypes that are not recognizable using traditional pathological criteria and in identifying specific genetic events involved in cancer progression. This review outlines the current status of these technologies and highlights recent studies in which they have been applied in the context of carcinogenesis.

Affinity Labels↗
PubMed2000

Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells.

Using a culture model of glial tumorigenesis, we identified a novel gene that was up-regulated in malignant mouse astrocytes following the loss of p53. The gene represents the murine homologue of pescadillo, an uncharacterized gene that is essential for embryonic development in zebrafish. Pescadillo is a strongly conserved gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1. Pescadillo displayed a distinct spatial and temporal pattern of gene expression during brain development, being detected in neural progenitor cells and postmitotic neurons. Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is dramatically elevated in malignant human astrocytomas. Yeast strains harboring temperature-sensitive mutations in the pescadillo gene were arrested in either G(1) or G(2) when grown in nonpermissive conditions, demonstrating that pescadillo is an essential gene in yeast and is required for cell cycle progression. Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein. These results suggest that pescadillo plays a crucial role in cell proliferation and may be necessary for oncogenic transformation and tumor progression.

Amino Acid Sequence↗
PubMed2000

PART-1: a novel human prostate-specific, androgen-regulated gene that maps to chromosome 5q12.

Genes regulated by androgenic hormones are of critical importance for the normal physiological function of the human prostate gland, and they contribute to the development and progression of prostate carcinoma. We used cDNA microarrays containing 1500 prostate-derived cDNAs to profile transcripts regulated by androgens in prostate cancer cells. This study identified a novel gene that we have designated PART-1 (prostate androgen-regulated transcript 1), which exhibited increased expression upon exposure to androgens in the LNCaP prostate cancer cell line. Northern analysis demonstrated that PART-1 is highly expressed in the prostate gland relative to other normal human tissues and is expressed as different transcripts using at least three different polyadenylation signals. The PART-1 cDNA and putative protein are not significantly homologous to any sequences in the nonredundant public sequence databases. Cloning and analysis of the putative PART-1 promoter region identified a potential binding site for the homeobox gene PBX-la, but no consensus androgen response element or sterol-regulatory element binding sites were identified. We used a radiation hybrid panel and fluorescence in situ hybridization to map the PART-1 gene to chromosome 5q12, a region that has been suggested to harbor a prostate tumor suppressor gene. These results identify a new gene involved in the androgen receptor-regulated gene network of the human prostate that may play a role in the etiology of prostate carcinogenesis.

Amino Acid Sequence↗
PubMed2000

The prostate expression database (PEDB): status and enhancements in 2000.

The Prostate Expression Database (PEDB) is an online resource designed to access and analyze gene expression information derived from the human prostate. PEDB archives >55 000 expressed sequence tags (ESTs) from 43 cDNA libraries in a curated relational database that provides detailed library information including tissue source, library construction methods, sequence diversity and sequence abundance. The differential expression of each EST species can be viewed across all libraries using a Virtual Expression Analysis Tool (VEAT), a graphical user interface written in Java for intra- and inter-library species comparisons. Recent enhancements to PEDB include: (i) the functional categorization of annotated EST assemblies using a classification scheme developed at The Institute for Genome Research; (ii) catalogs of expressed genes in specific prostate tissue sources designated as transcriptomes; and (iii) the addition of prostate proteome information derived from two-dimensional electrophoreses and mass spectrometry of prostate cancer cell lines. PEDB may be accessed via the WWW at http://www.mbt.washington.edu/PEDB/

Databases, Factual↗
PubMed2000

Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.

Several methods have been developed for the comprehensive analysis of gene expression in complex biological systems. Generally these procedures assess either a portion of the cellular transcriptome or a portion of the cellular proteome. Each approach has distinct conceptual and methodological advantages and disadvantages. We have investigated the application of both methods to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cells. This pathway is of critical importance for the development and progression of prostate cancer. Of clinical importance, modulation of androgens remains the mainstay of treatment for patients with advanced disease. To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate-derived expressed sequence tags (ESTs). A total of 55000 prostate ESTs were assembled into a set of 15953 clusters putatively representing 15953 distinct transcripts. These clusters were used to construct cDNA microarrays suitable for examining the androgen-response pathway at the level of transcription. The expression of 20 genes was found to be induced by androgens. This cohort included known androgen-regulated genes such as prostate-specific antigen (PSA) and several novel complementary DNAs (cDNAs). Protein expression profiles of androgen-stimulated prostate cancer cells were generated by two-dimensional electrophoresis (2-DE). Mass spectrometric analysis of androgen-regulated proteins in these cells identified the metastasis-suppressor gene NDKA/nm23, a finding that may explain a marked reduction in metastatic potential when these cells express a functional androgen receptor pathway.

DNA, Complementary↗
PubMed2000

Toll receptors: an expanding role in our understanding of human disease.

Toll receptor proteins in Drosophila are involved in establishing the dorsal-ventral axis in embryogenesis as well as participating in the innate immune response to invading pathogens. The basic mediators of this response show striking similarities in plants, insects, and vertebrates. The cytoplasmic signaling cascade is exemplified by the human interleukin-1 receptor complex (IL-1R), resulting in transcriptional activation of effector proteins through nuclear factor-kappaB (NF-kappaB). Six mammalian/human Toll-like receptors (TLR) have been described to date. The TLRs share the IL-1R cytoplasmic signaling cascade but are distinguished by their extracellular leucine-rich repeat (LRR) structure. The LRR superfamily comprises a diverse group of proteins, including a cohort involved in transmembrane signaling. Two of the human TLRs (TLR2, TLR4) have been shown to be involved in the innate response to bacterial pathogens and appear to provide a link between the innate and adaptive immune response. A better understanding of this response may provide improved therapeutic modalities in the treatment of bacterial and fungal sepsis, which continues to be a significant source of morbidity and mortality worldwide. In addition, similar to Drosophila, Toll receptors and related proteins in the LRR superfamily may also be involved in human development, as well as in noninfectious human disease.

Animals↗
PubMed1999

Negative selection: a method for obtaining low-abundance cDNAs using high-density cDNA clone arrays.

The identification of the entire complement of genes expressed in a cell, tissue, or organism provides a framework for understanding biological properties and establishes a tool set for subsequent functional studies. The large-scale sequencing of randomly selected clones from cDNA libraries has been successfully employed as a method for identifying a large fraction of these expressed genes. However, this approach is limited by the inherent redundancy of cellular transcripts reflecting widely variant levels of gene transcription. As a result, a high percentage of transcript duplications are encountered as the number of sequenced clones accrues. To address this problem, we have developed a negative hybridization selection method that employs the hybridization of complex cDNA probes to high-density arrays of cDNA clones and the subsequent selection of clones with a null or low hybridization signal. This approach was applied to a cDNA library constructed from normal human prostate tissue and resulted in the reduction of highly expressed prostate cDNAs from 6.8 to 0.57% with an overall decline in clone redundancy from 33 to 11%. The selected clones also reflected a more diverse cDNA population, with 89% of the clones representing distinctly different cDNAs compared with 67% of the randomly selected clones. This method compares favorably with cDNA library re-association normalization approaches and offers several distinct advantages, including the flexibility to use previously prepared libraries, and the ability to employ an iterative screening approach for continued accrual of cDNAs representing rare transcripts.

DNA, Complementary↗
PubMed1999

Comparative hybridization of an array of 21,500 ovarian cDNAs for the discovery of genes overexpressed in ovarian carcinomas.

Comparative hybridization of cDNA arrays is a powerful tool for the measurement of differences in gene expression between two or more tissues. We optimized this technique and employed it to discover genes with potential for the diagnosis of ovarian cancer. This cancer is rarely identified in time for a good prognosis after diagnosis. An array of 21,500 unknown ovarian cDNAs was hybridized with labeled first-strand cDNA from 10 ovarian tumors and six normal tissues. One hundred and thirty-four clones are overexpressed in at least five of the 10 tumors. These cDNAs were sequenced and compared to public sequence databases. One of these, the gene HE4, was found to be expressed primarily in some ovarian cancers, and is thus a potential marker of ovarian carcinoma.

Biomarkers, Tumor↗