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Biomedical subjects

G A Silverman

Publications and source records attributed to G A Silverman.

At least 19 recordsLinked to original sources
PubMed1999

Zebrafish YAC, BAC, and PAC genomic libraries.

Numerous positional cloning projects directed at isolating genes responsible for the myriads of observed developmental defects in the zebrafish are anticipated in the very near future. In this chapter, we have reviewed the YAC, BAC, and PAC large-insert genomic resources available to the zebrafish community. We have discussed how these resources are screened and used in a positional cloning scheme and have pointed out frequently formidable logistical considerations in the approach. Despite being extremely tedious, positional cloning projects in the zebrafish will be comparatively easier to accomplish than in human and mouse, because of unique biological advantages of the zebrafish system. Moreover, the ease and speed at which genes are identified and cloned should rapidly increase as more mapping reagents and information become available, thereby paving the way for meaningful biological studies.

Animals↗
PubMed1999

An 18q- syndrome breakpoint resides between the duplicated serpins SCCA1 and SCCA2 and arises via a cryptic rearrangement with satellite III DNA.

The 18q-syndrome is representative of a group of terminal deficiency or macrodeletion syndromes characterized by mental retardation and congenital malformations. To gain insight into the mechanism of chromosomal loss and stabilization in these disorders, we cloned a putative terminal deletion breakpoint from an 18q-syndrome patient. The 18q21.3 breakpoint occurred between two nearly identical serine protease inhibitor (serpin) genes, SCCA1 and SCCA2. Although cytogenetic studies suggested that this chromosomal aberration was formed by a simple terminal deletion, DNA sequence analysis, pulsed-field gel electrophoresis and fluorescence in situ hybridization showed that the breakpoint was contiguous with a 35 bp filler sequence followed by a satellite III DNA-containing telomeric fragment of 475-1000 kb. This type of satellite III DNA sequence was not detected on the normal chromosome 18, but was highly homologous with types of satellite III DNA sequences normally located on the short arms (p11) of the acrocentric chromosomes and other heterochromatic regions. This DNA sequence analysis suggested that the terminal deficiency in this 18q-syndrome patient arose via illegitimate (non-homologous) recombination. Moreover, these data raise the possibility that a subset of chromosomal aberrations appearing cytogenetically and molecularly as simple terminal truncations or deletions are caused by small (<1000 kb) cryptic rearrangements.

Abnormalities, Multiple↗
PubMed1998

A murine ortholog of the human serpin SCCA2 maps to chromosome 1 and inhibits chymotrypsin-like serine proteinases.

Squamous cell carcinoma antigens (SCCA) 1 and 2 are inhibitory members of the high-molecular-weight serine proteinase inhibitor (serpin) family. The biological functions of SCCA1 and 2 are unknown. One approach to determining the function of human proteins is to study orthologs in other species, such as the mouse. The purpose of this study was to determine whether orthologs to human SCCA1 or 2 exist in the mouse. We report the identification and characterization of a novel serpin, sqn5 (now designated Scca2). Comparative amino acid sequence analysis suggests that Scca2 is a member of the ov-serpin subfamily of serpins with highest homology to SCCA1 and SCCA2. Fluorescence in situ hybridization revealed that the Scca2 mapped near Bcl2 on mouse chromosome 1. This region is syntenic with the human locus for SCCA1 and SCCA2 on 18q21.3. The tissue expression patterns as determined by RT-PCR showed a restricted distribution. Scca2 was detected in the lung, thymus, skin, and uterus, as are SCCA1 and SCCA2. Unlike the SCCAs, however, Scca2 was detected also in the gastrointestinal tract. Enzyme-inhibition assays using a GST-SCCA2 fusion protein revealed that SCCA2 inhibited chymotrypsin-like serine proteinases, but not papain-like cysteine proteinases. SCCA2 inhibited CTSG at 1:1 stoichiometry and with a second-order rate constant of kass = 1.7 x 10(5) M-1 s-1. SCCA2 also inhibited human mast cell chymase but the stoichiometry was 2:1, and the second-order rate constant was kass = 0.9 x 10(4) M-1 s-1. This inhibitory profile is identical to that observed for human SCCA2. Based on these findings, Scca2 appears to be the murine ortholog of human SCCA2.

Amino Acid Sequence↗
PubMed1998

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition.

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin-serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

Amino Acid Sequence↗
PubMed1998

Delineation of the breakpoint at 18q21.1 in a cell line (Karpas1106) derived from mediastinal B-cell lymphoma by fluorescence in situ hybridization with multiple YAC clones.

The breakpoint of the 18q21 translocation of B-cell-non-Hodgkin's lymphoma (NHL) cell line Karpas1106P was delineated by fluorescence in situ hybridization (FISH). Karpas1106P was derived from mediastinal lymphoblastic B-cell lymphoma and exhibited the immunophenotype characteristic of marginal-zone B-cell lymphoma (MZL): smIg+, pan-B antigen+, CD5-, CD10- and CD23-. The original G-banded karyotype showed a complex translocation containing t(X;18;13)(q28;q21;q12.1). Double-color FISH (DCFISH) with whole-chromosome-painting (WCP) probes for chromosomes X, 13 and 18, and 18q-specific yeast artificial chromosome (YAC) clones defined t(X;18;13) as ider(X)t(X;18; 13)(q28;q 12.3q21.1;q12.1). The immunoglobulin-heavy-chain (IgH) gene was not involved in the chromosomal translocation as detected by DCFISH with VH and Cgamma probes. By using contiguous YAC clones mapped from 18q12.3 to q21.1, we identified a YAC clone y852H2 with its breakpoint at 18q21.1. In Karpas1106P, the distal part of chromosome 18 from the breakpoint (18q21.1-qter) was deleted, showing loss of heterozygosity of this region. In addition, the chromosomal segment 18q21.1 was duplicated and inserted to ider(X)t(X;18; 13) between Xq28 and 13q12.1 with maintaining its original orientation. The DNA sequence of the breakpoint region contained in y852H2 can serve as a candidate locus for further molecular dissection to identify the causative gene of MZL.

Chromosome Mapping↗
PubMed1998

Structure and sequence of human M/NEI (monocyte/neutrophil elastase inhibitor), an Ov-serpin family gene.

Human monocyte/neutrophil Elastase Inhibitor (M/NEI) is a proteinase inhibitor that regulates the activity of the neutrophil proteases: elastase, cathepsin G and proteinase-3. Evidence indicates that M/NEI belongs to the Ov-serpin family (ovalbumin-related serpins), functionally diverse proteins with shared structural features. Recombinant lambda phage clones were isolated that encompass the full-length M/NEI gene plus upstream and downstream regions. The gene, 9.5kb long, consists of 7 exons and 6 introns. The 5' transcription start site identified by primer extension corresponds to a 60bp exon 1; the translation start site is in exon 2. Southern blots established a gene copy number of one. The 3' untranslated region (UTR) contains three AATAAA/AATTAA sites; these were shown to function as alternative polyadenylation signals. A 14-nucleotide upstream motif including the atypical TATA box TATAAGAG otherwise occurs only twice in GenBank, in the genes encoding neutrophil elastase and proteinase-3, target proteases inhibited by M/NEI. Comparison of M/NEI and previously characterized related genes strongly suggests that all Ov-serpins, despite a difference in chromosomal localization and exon number, nonetheless, share a common basic gene structure.

Base Sequence↗
PubMed1998

Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis.

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.

Amino Acid Sequence↗
PubMed1998

Gene expression in activated brain microglia: identification of a proteinase inhibitor that increases microglial cell number.

Microglia, the intrinsic immune cells of the central nervous system, are activated in a variety of inflammatory brain diseases in which they play a pathogenetic role. However, mechanisms underlying activation are largely unknown. To begin elucidating molecular mechanisms associated with activation, we characterized the pattern of gene expression in virtually pure dissociated microglial cultures, using RT-PCR differential display. Microglia were activated with bacterial lipopolysaccharide (LPS), a traditional stimulant, and the profile of gene expression was compared to that in basal, control cultures. Activation resulted in altered expression of six genes. The cDNAs were isolated, sequenced and characterized. Homology searches identified three novel genes, and two that exhibited very high sequence similarity to the gene encoding squamous cell carcinoma antigen (SCCA). SCCA (1 and 2) are tandemly arranged genes that encode two serine proteinase inhibitors (serpins). SCCA has been detected exclusively in cancer cells, and is a plasma marker for squamous cell carcinoma. Immunoblot analysis indicated that gene expression was accompanied by a 5-fold increase in the synthesis of SCCA protein in LPS-activated microglia. To assess potential biological actions of the SCCA serpins, SCCA1 protein was added to cultures. SCCA1 altered microglial morphology, and elicited a dramatic, 5-fold increase in cell number within 72 h. The effects appeared to be cell-specific, since the protein had no effect on other cell types: cortical astrocytes and neurons from cortex or basal forebrain were unaffected. We tentatively conclude that SCCA1 may play a cell-specific role in increasing cell number, a critical early step in microglial activation and brain inflammation. More generally, differential display of genes in the microglial model system may help define patterns of expression associated with CNS disease, thereby identifying pathogenetic mechanisms and new therapeutic targets.

Animals↗
PubMed1998

SCCA1 and SCCA2 are proteinase inhibitors that map to the serpin cluster at 18q21.3.

The genes for the squamous cell carcinoma antigen (SCCA) were found flanking a deletion breakpoint from a patient with the 18q-syndrome. The genes are <10 kb apart, tandemly arrayed in a head-to-tail fashion, and approximately 10 kb in size. Both genes also contain 8 exons and identical intron-exon boundaries. The cDNAs encode for proteins that are 92% identical and 95% similar. Amino acid comparisons show that SCCA1 and SCCA2 are members of the high-molecular weight serine proteinase inhibitor (serpin) family. Physical mapping studies show that the genes reside within the 500-kb region of 18q21.3 that contains at least four other serpin genes. The gene order is cen-maspin (PI5), SCCA2, SCCA1, PAI2, bomapin (PI10), PI8-tel. Biochemical analysis of recombinant SCCA1 and SCCA2 proteins shows that SCCA1 is a potent cross-class inhibitor of papain-like cysteine proteinases such as cathepsins L, S and K, whereas SCCA2 is an inhibitor of chymotrypsin-like serine proteinases such as cathepsin G and mast cell chymase. These findings suggest that SCCA1 and SCCA2 are capable of regulating proteolytic events involved in both normal (e.g., tissue remodeling, protein processing) and pathologic processes (e.g., tumor progression).

Amino Acid Sequence↗
PubMed1997

Cytoplasmic antiproteinase 2 (PI8) and bomapin (PI10) map to the serpin cluster at 18q21.3.

High-molecular-weight serine proteinase inhibitors (serpins) regulate a diverse set of intracellular and extracellular processes such as complement activation, fibrinolysis, coagulation, cellular differentiation, tumor suppression, apoptosis, and cell migration. The ov-serpins are a subset of the serpin superfamily and are characterized by their high degree of homology to chicken ovalbumin, the lack of N- and C-terminal extensions, the absence of a signal peptide, and a Ser rather than an Asn residue at the penultimate position. Recently, we mapped four members of the family [SCCA1, SCCA2, PAI2, and PI5 (maspin)] to a 300-kb region within 18q21.3. Using a panel of 18q21.3 YAC clones, PCR, and DNA blotting, we mapped two additional ov-serpins, cytoplasmic antiproteinase 2 [CAP2 (PI8)] and bone marrow-associated serpin [bomapin (PI10)], to the same region. Three of the serpins, PI8, PI10, and PAI2 mapped to the same YACs, yA27D8 and yA24E4. We estimated that the size of the 18q21.3 serpin cluster spanned approximately 500 kb and contained at least six serpin genes. The order was cen-PI5, SCCA2, SCCA1, PAI2, PI10, PI8-tel. The clustering of serpins at 18q21 provides new opportunities to study coordinate gene regulation and the evolution of gene families.

Amino Acid Sequence↗
PubMed1997

Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase.

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for more advanced squamous cell tumors. Molecular cloning of the SCCA genomic region revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. Analysis of the primary amino acid sequences shows that both genes are members of the high molecular weight serpin superfamily of serine proteinase inhibitors. Although SCCA1 and SCCA2 are nearly identical in primary structure, the reactive site loop of each inhibitor suggests that they may differ in their specificity for target proteinases. SCCA1 has been shown to be effective against papain-like cysteine proteinases. The purpose of this study was to determine whether SCCA2 inhibited a different family of proteolytic enzymes. Using recombinant DNA techniques, we prepared a fusion protein of glutathione S-transferase and full-length SCCA2 . The recombinant SCCA2 was most effective against two chymotrypsin-like proteinases from inflammatory cells, but was ineffective against papain-like cysteine proteinases. Serpin-like inhibition was observed for both human neutrophil cathepsin G and human mast cell chymase. The second order rate constants for these associations were on the order of approximately 1 x 10(5) M-1 s-1 and approximately 3 x 10(4) M-1 s-1 for cathepsin G and mast cell chymase, respectively. Moreover, SCCA2 formed SDS-stable complexes with these proteinases at a stoichiometry of near 1:1. These data showed that SCCA2 is a novel inhibitor of two physiologically important chymotrypsin-like serine proteinases.

Amino Acid Sequence↗
PubMed1997

Rapid detection of lymphoma-specific translocations in interphase nuclei of non-Hodgkin's lymphoma by fluorescence in situ hybridization.

We have recently developed a method to detect tumor-specific rearrangement of the IgH gene in interphase nuclei by fluorescence in situ hybridization. Tumor-specific IgH gene rearrangement is equivalent to 14q32.33 translocation. Using this approach, we detected 14q32.33 translocation in 29 of 70 patients with B-cell non-Hodgkin's lymphoma (NHL). Chromosome t(3;14) was found in 10 of these 29 patients, and were demonstrated as a fusion signal of BCL6 and VH gene probes in interphase nuclei. Furthermore, in another series of 11 patients and a NHL cell line, we demonstrated t(14;18) and t(11;14) in interphase and metaphase cells with a combination of BCL2 (or PRAD1) with IgH gene probes. Interphase FISH with lymphoma-associated gene probes is a rapid procedure for cytogenetic diagnosis of B-cell NHL.

Cell Nucleus↗
PubMed1995

Human macrophage metalloelastase. Genomic organization, chromosomal location, gene linkage, and tissue-specific expression.

Human macrophage metalloelastase (HME) is a recent addition to the matrix metalloproteinase (MMP) family that was initially found to be expressed in alveolar macrophages of cigarette smokers. To understand more about HME expression, analysis of the structure and location of the gene was performed. The gene for HME is composed of 10 exons and 9 introns, similar to the stromelysins and collagenases, and HME shares the highly conserved exon size and intron-exon borders with other MMPs. The 13-kilobase (kb) HME gene has been localized by fluorescence in situ hybridization to chromosome 11q22.2-22.3, the same location of the interstitial collagenase and stromelysin genes. We determined that HME and stromelysin 1 genes are physically linked within 62 kb utilizing pulse-field gel electrophoresis. The promoter region of the HME gene contains several features common to other MMP genes including a TATA box 29 bp upstream to the transcription initiation site, an AP-1 motif, and a PEA3 element. HME mRNA is not detectable in normal adult tissues but is induced in rapidly remodeling tissues such as the term placenta. In situ hybridization and immunohistochemistry of placental tissue demonstrated HME mRNA and protein expression in macrophages and stromal cells. Cell-specific expression and response to inflammatory stimuli such as endotoxin is conferred within 2.8 kb of the HME 5'-flanking sequence as demonstrated by HME promoter-CAT expression constructs. Knowledge of the genomic organization and chromosomal location of HME may allow us to further define mechanisms responsible for cell- and tissue-specific expression of HME.

Base Sequence↗