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.     

A serpin gene cluster on human chromosome 6p25 contains PI6, PI9 and ELANH2 which have a common structure almost identical to the 18q21 ovalbumin serpin genes

The human genes encoding the "ovalbumin" subgroup of closely related serine proteinase inhibitors (serpins) are located at 18q21.3 and 6p25. Those at 6p25 include proteinase inhibitor 6 (PI-6; gene symbol PI6), proteinase inhibitor 9 (PI-9; gene symbol PI9) and monocyte neutrophil elastase inhibitor (M/NEI; gene symbol ELANH2). Here we describe the fine mapping of these genes to a 200-kb region of chromosome 6 that includes the markers WI-8835 and D6S1338, and the establishment of the gene order: tel-PI6-PI9-ELANH2-cen. PI6 and ELANH2 are transcribed towards the telomere, and structural analysis shows that PI6 and PI9 are organized identically, having seven exons and six introns. PI6 and PI9 are almost identical in structure to the ovalbumin serpin genes at 18q21.3. The 18q21.3 genes have an extra exon and intron, otherwise all the other exon/intron boundaries are conserved between the two groups. These results represent the first detailed map of the chromosome 6 serpin gene cluster, and demonstrate that although they are very closely related, the 6p25 and 18q21-->q23 ovalbumin serpin genes form two structurally distinct groups. These findings do not support a previously proposed model for evolution of the clusters which invoked an inter-chromosomal duplication of the entire 6p25 group to 18q21.3.     

Aberrant hypermethylation at the bcl-2 locus at 18q21 in human lung cancers

Accumulating evidence suggests that altered DNA methylation may play a role in the oncogenesis of human neoplasms, including lung cancer. The presence of aberrant hypermthylations at 3p, 9p, 11p, ad 17p, which are known to be hot spots for allele loss in lung cancers, is suggested to be a reflection of the existence of tumor suppressor genes in these chromosomal regions. In the present study, we investigated the methylation status of the Rb locus at 13q14 as well as that of the bcl-2 locus at 18q21 in 134 lung cancer specimens, representing all major histological subtypes. As a result, 18q21 was identified to be the fifth chromosomal region affected by frequent tumor-specific aberrant hypermethylation in lung cancers. The occurrence of aberrant hypermethylation at the bcl-2 locus at 18q21 was restricted to non-small cell lung cancers, and among non-small cell lung cancers, such epigenetic aberrations were observed most frequently in adenocarcinomas without any association with bcl-2 expression. Interestingly, allelic loss at the bcl-2 locus was also seen in 40% (7 of 17 informative cases) of adenocarcinomas; this frequency was also the highest among values for the various histological subtypes of lung cancers. These results suggest that aberrant hypermethylation at the bcl-2 locus may be a reflection of a putative tumor suppressor gene residing at 18q21, and aberrant hypermethylation might play a role in its inactivation. In contrast, altered methylation status of the Rb locus appears to be quite rare in lung cancers, if present at all.     

Specificity of serine proteinase/serpin complex binding to very-low-density lipoprotein receptor and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein

Very-low-density lipoprotein receptor (VLDLR) and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein (alpha2MR/LRP) are multifunctional endocytosis receptors of the low-density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type-1 plasminogen-activator inhibitor (PAI-1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and alpha2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinase-type and tissue-type plasminogen activators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, alpha2-antiplasmin, alpha1-proteinase inhibitor, alpha1-antichymotrypsin, protease nexin-1, heparin cofactor II, and antithrombin III. Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor-associated protein (RAP)-sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS-1 cells, which have a high endogenous expression of alpha2MR/LRP. We found that the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrombin with PAI-1, protease nexin-1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/alpha2-antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and alpha2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and alpha2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.     

MADR2 maps to 18q21 and encodes a TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma

The MAD-related (MADR) family of proteins are essential components in the signaling pathways of serine/threonine kinase receptors for the transforming growth factor beta (TGFbeta) superfamily. We demonstrate that MADR2 is specifically regulated by TGFbeta and not bone morphogenetic proteins. The gene for MADR2 was found to reside on chromosome 18q21, near DPC4, another MADR protein implicated in pancreatic cancer. Mutational analysis of MADR2 in sporadic tumors identified four missense mutations in colorectal carcinomas, two of which display a loss of heterozygosity. Biochemical and functional analysis of three of these demonstrates that the mutations are inactivating. These findings suggest that MADR2 is a tumor suppressor and that mutations acquired in colorectal carcinomas may function to disrupt TGFbeta signaling.     

Molecular markers for the bovine fibrillin 1 gene (FBN1) map to 10q26

A 2-year-old girl lost both lips after an aggressive attack of noma (gangrenous stomatitis). After an unsuccessful attempt to reconstruct her upper lip, a bipedicled chin flap was used to reconstruct both lips. The cosmetic result was good and the functional result was acceptable.     

Localization of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3

The three bona fide genes coding for calmodulin CALM1, CALM2, and CALM3, were assigned to human chromosomes 14, 2, and 19, respectively, by polymerase chain reaction-based amplification of calmodulin gene-specific sequences using DNA from human-hamster cell hybrids as template. Employing calmodulin gene-specific DNA probes of mainly intronic or flanking parts of the individual genes, regional sublocalization was performed by in situ hybridization on metaphase spreads of human lymphocytes. The three calmodulin genes map to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3, respectively. These results represent the first complete chromosomal localization study on a mammalian calmodulin gene family and indicate that these structurally closely related genes were most likely dispersed throughout the genome concomitantly with their generation from an ancestral precursor gene.     

The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster

Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.     

The BRCA1 and 1A1.3B promoters are parallel elements of a genomic duplication at 17q21

A search for new genes was performed in a 220-kb region around the tumor necrosis factor gene cluster in the human central major histocompatibility complex region using a cDNA hybridization and selection method. In addition to the seven known genes in this region, we identified a new gene that is preferentially expressed in spleen. We also identified two pseudogenes that have high degrees of homology to cytokeratin and cyclophillin, respectively. Expressed sequences for a human homologue of the mouse B144 gene were also found in the current analysis. RT-PCR analysis showed that B144 is expressed in spleen, in thymus, and prominently in the macrophage cell line, U937. We also independently identified the BAT1 gene to be the well-conserved homologue of a previously described rat liver nuclear protein.     

Chromosome 18 breakpoint in t(11;18)(q21;q21) translocation associated with MALT lymphoma is proximal to BCL2 and distal to DCC

OBJECTIVE: To identify independent predictors of intracranial hemorrhage (ICH) during neonatal extracorporeal membrane oxygenation (ECMO). STUDY DESIGN: This retrospective cohort consisted of all neonates who did not have an ICH before treatment with ECMO identified in the Extracorporeal Life Support Organization Registry from 1992 to 1995 (n = 4550). Multiple logistic regression analysis was used to identify factors independently correlated with ICH and to develop a model that could be used to predict the risk of ICH in neonates treated with ECMO. RESULTS: ICH was identified in 9.9% of patients. The factors associated with ICH remaining after adjusting for other significant variables (P <.01) were gestational age (GA) <34 weeks (odds ratio [OR] 12.1, 95% confidence intervals [CI] [6.6, 22]), GA 34 to <36 weeks (OR 4.1, CI [2.9, 5.8]), GA 36 to <38 weeks (OR 2.1, CI [1.6, 2.8]) primary diagnosis of sepsis (OR 1.8, CI [1.4, 2.3]), epinephrine use (OR 1.9, CI [1.5, 2.5]), coagulopathy (OR 1. 6, CI [1.1, 2.2]), arterial pH <7.0 (OR 2.5, CI [1.6, 3.9]), and arterial pH 7.0 to <7.2 (OR 1.8 CI [1.3, 2.5]). ICH rates for neonates receiving venovenous versus venoarterial ECMO and for those treated with or without cephalic jugular venous drainage were not significantly different. CONCLUSIONS: Gestational age, acidosis, sepsis, coagulopathy, and treatment with epinephrine are major independent factors associated with ICH in neonates treated with ECMO. In particular, GA <34 weeks remains a major barrier for use of current ECMO technologies.     

Construction of a gene map of the nephronophthisis type 1 (NPHP1) region on human chromosome 2q12-q13

The dispersed fluorescence spectrum of lines in the 460 nm band system of NiCl2 (nickel dichloride) is presented. The fluorescence was pumped on single rotational transitions of two spin components in a vibrational band observed previously in the laser excitation spectrum. Fluorescent transitions to levels of the &Xtilde;3Sigma-g (ground) state are assigned and discussed. The spectra also show clear evidence for a previously unidentified low-lying electronic state, assumed to be the ? state, which lies about 2000 cm-1 above the ground state. This is likely to be of 3Pig character and is discussed on that basis. The wavenumber of the symmetric stretching vibration of NiCl2 in the ? state is determined to be 367 cm-1, slightly larger than the &Xtilde; state value (360 cm-1). Copyright 1998 Academic Press.     

Strategy to design peptide inhibitors: structure of a complex of proteinase K with a designed octapeptide inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2 at 2.5 A resolution

To assess the relationship between hepatitis C virus infection and Fas antigen expression on hepatocytes, we examined changes in hepatic Fas antigen expression in the presence or absence of active hepatitis C virus infection. Twenty patients with chronic hepatitis C infection were treated with interferon and underwent pre- and posttreatment liver biopsies. Patients were classified according to the absence (group A; n = 9) or the presence (group B; n = 11) of hepatitis C virus RNA (HCV-RNA) in the liver after interferon therapy. An immunohistochemical assay showed Fas antigen staining in hepatocytes membranes and cytoplasm with expression concentrated mainly in periportal areas. The percentage of Fas-positive cells in the liver before treatment was not different between group A (39.5+/-19.1%) and group B (32.5+/-15.6%). Hepatic Fas expression was reduced significantly after treatment (24.3+/-10.6%) compared with the pretreatment values in group A (p < 0.05) but not in group B (25.9+/-16.9%). There was no significant difference between the two groups in the degree of histologic improvement. These results suggest that hepatic Fas expression is associated with persistent infection of hepatitis C virus.     

Inhibition and stimulation of LFA-1 and Mac-1 functions by antibodies against murine CD18. Evidence that the LFA-1 binding sites for ICAM-1, -2, and -3 are distinct

The murine CD18 monoclonal antibody (mAb) M18/2 was reported to inhibit lymphoma metastasis [Zahalka, M. A. et al. (1993) J. Immunol. 150, 4466]. To identify the pathways potentially blocked, we studied the effects of M18/2 compared with two new mAb against murine CD18, GAME-46, and -245. Whereas the GAME mAb blocked most Mac-1-mediated interactions, M18/2 had no effect, or even stimulated. The same was true for adhesion of LFA-1 to ICAM-1. To test effects on interactions with different ICAMs, we used L cells transfected with human ICAM-1, -2, and -3. As previously described, mouse LFA-1 does not bind to human ICAM-1 but we show here that mouse LFA-1 does bind to human ICAM-2 and -3. Again, the GAME mAb blocked completely, but M18/2 did not. These results indicate that the LFA-1 binding sites for ICAM-1 and ICAM-2 and -3, although in close vicinity, are distinct. Furthermore, effects of M18/2 on metastasis cannot be ascribed to blocking of any known beta2-integrin activity.