Developmental expression of a mucinlike glycoprotein (MUCLIN) in pancreas and small intestine of CF mice.

The mucinlike glycoprotein MUCLIN, one of two protein products of the CRP-ductin gene, was used to study changes in the expression of sulfated glycoconjugates during the pathogenesis of cystic fibrosis, using the cystic fibrosis transmembrane conductance regulator (CFTR) knockout mouse (CF mouse). We assessed the appearance of dilated lumina containing protein or mucus plugs in pancreatic acini and crypts of the small intestine and quantified MUCLIN protein and CRP-ductin mRNA during postnatal development. In CF mice, the pancreatic acinar lumen was dilated by postnatal day 16 (P16), but MUCLIN protein was first significantly increased by P23 and remained elevated through adulthood compared with normal mice. Similarly, intestinal crypts had CF-like mucus plugs by P16, but MUCLIN protein was first elevated by P23 and remained elevated through adulthood compared with normal mice. In both organs, MUCLIN labeling of the luminal surface was increased concomitantly with dilation and protein or mucus plugging but before upregulation of expression. The morphological changes were then followed by upregulation of MUCLIN protein and CRP-ductin mRNA expression. This is the first direct study of CF pathogenesis and the resultant increase in glycoconjugate gene expression. The data are consistent with CF pathogenesis progressing from an initial alteration in protein secretory dynamics (increased luminal MUCLIN and protein/mucus plugs) to an upregulation of glycoprotein/mucin gene expression, which is expected to exacerbate obstruction of the luminal spaces.

MUCLIN expression in the cystic fibrosis transmembrane conductance regulator knockout mouse.

BACKGROUND & AIMS: Cystic fibrosis is characterized by increased secretion of glycoconjugates with altered carbohydrate composition, but no specific gene products that show these changes have been identified. The aim of this study was to use a recently described sulfated mucin-like glycoprotein (MUCLIN: formerly called gp300) as a model glycoconjugate to study such changes in the gastrointestinal system in the cystic fibrosis transmembrane conductance regulator (CFTR) knockout mouse (cftrm1Unc). METHODS: Western and Northern blots were used to determine the tissue levels of MUCLIN and its messenger RNA (mRNA) in normal and CFTR knockout mice. Immunocytochemistry was used to determine the localization of MUCLIN. RESULTS: MUCLIN is expressed in the normal mouse intestinal tract, pancreas, and gallbladder. In CFTR knockout mice, MUCLIN shows increased expression at both mRNA and protein levels in pancreas and duodenum, but not in the gallbladder. In the duodenum, MUCLIN was localized intracellularly in crypt enterocytes and on the luminal surface, and luminal surface labeling was dramatically increased in the CFTR knockout mouse. In the CFTR knockout mouse duodenum and gallbladder, MUCLIN showed retarded electrophoretic migration indicating altered posttranslational processing. CONCLUSIONS: MUCLIN shows increased expression and possibly altered posttranslational processing in the CFTR knockout mouse and will serve as a good model for understanding changes in the composition of mucous secretions in patients with this disease.

Murine granulocytic cell adhesion to bone marrow hemonectin is mediated by mannose and galactose.

Hemonectin (HN) is a bone marrow (BM) protein that promotes specific attachment of immature granulocytes and their precursors within the BM. We report that HN is a glycoprotein containing both mannose and galactose residues, and provide evidence that these carbohydrates mediate granulocytic cell adhesion to HN. Carbohydrate structure was determined by digoxigenin-conjugated lectin binding to HN and indicated the presence of mannose, galactose, sialic acid, and the absence of fucose-linked oligosaccharides. The role of carbohydrates in mediating cell adhesion was examined by chemical and enzymatic deglycosylation. Deglycosylation of HN with trifluoromethanesulfonic acid, which cleaves N- and O-linked oligosaccharides, inhibits 66% of cell attachment to HN, and results in an apparent decrease in molecular weight from 60 to 50 kD. Enzymatic deglycosylation with endo-B-N-acetylglucosaminidase H, which hydrolyzes specific N-linked mannose residues, inhibits 30% of cell adhesion to HN. Finally, the role of these specific sugars in hemonectin-mediated cell adhesion was confirmed with neoglycoprotein blocking. Preincubation of BM cells with mannosyl- and galactosyl-BSA probes produces a dose-dependent inhibition of cell attachment to HN, whereas fucosyl-BSA does not inhibit cell adhesion to HN. These results show that mannose and galactose partially mediate adhesion of BM granulocytes to HN.

Setting limits on homeotic gene function: restraint of Sex combs reduced activity by teashirt and other homeotic genes.

Each of the homeotic genes of the HOM or HOX complexes is expressed in a limited domain along the anterior-posterior axis. Each homeotic protein directs the formation of characteristic structures, such as wings or ribs. In flies, when a heat shock-inducible homeotic gene is used to produce a homeotic protein in all cells of the embryo, only some cells respond by altering their fates. We have identified genes that limit where the homeotic gene Sex combs reduced (Scr) can affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is prevented from inducing prothoracic structures by the three bithorax complex (BX-C) homeotic genes. However, two of the BX-C homeotic genes, Ultrabithorax (Ubx) and abdominal-A (abd-A), have no effect on the ability of Scr to direct the formation of salivary glands. Instead, salivary gland induction by Scr is limited in the trunk by the homeotic gene teashirt (tsh) and in the last abdominal segment by the third BX-C gene, Abdominal-B (AbdB). Therefore, spatial restrictions on homeotic gene activity differ between tissues and result both from the regulation of homeotic gene transcription and from restraints on where homeotic proteins can function.

Schistosoma mansoni: cell-specific expression and secretion of a serine protease during development of cercariae.

Eukaryotic serine proteases are an important family of enzymes whose functions include fertilization, tissue degradation by neutrophils, and host invasion by parasites. To avoid damaging the cells or organisms that produced them, serine proteases must be tightly regulated and sequestered. This study elucidates how the parasitic blood fluke Schistosoma mansoni synthesizes, stores, and releases a serine protease during differentiation of its invasive larvae. In situ hybridization with a cDNA probe localized the protease mRNA to acetabular cells, the first morphologically distinguishable parasite cells that differentiate from the embryonic cell masses present in the intermediate host snail. The acetabular cells contained vimentin but not cytokeratins, consistent with a mesenchymal, not epithelial, origin. Antiprotease antibodies, localized by immunoperoxidase, showed that the protease progressively accumulated in these cells and was packaged in vesicles of three morphologic types. Extension of cytoplasmic processes containing protease vesicles formed "ducts" which reached the anterior end of fully differentiated larvae. During invasion of human skin, groups of intact vesicles were released through the acetabular cytoplasmic processes and ruptured within the host tissue. Ruptured protease vesicles were noted adjacent to degraded epidermal cells and dermal-epidermal basement membrane, as well as along the surface of the penetrating larvae themselves. These observations are consistent with the proposed dual role for the enzyme in facilitating invasion of host skin by larvae and helping to release the larval surface glycocalyx during metamorphosis to the next stage of the parasite.

Different patterns of transcription from the two Antennapedia promoters during Drosophila embryogenesis.

The homeotic genes of Drosophila control the differentiation of segments during development. Mutations in these genes cause one or more segments to develop structures normally found elsewhere in the organism. Several studies have shown that the spatial patterns of homeotic gene transcription are highly complex, and that these precise patterns of transcription are critical to normal development. The homeotic gene Antennapedia (Antp), a member of the Antennapedia Complex, is required for the correct differentiation of thoracic segments in both embryos and adults. The patterns of total Antp transcript and protein accumulation have been described in detail, but the contribution of each promoter to the overall pattern in embryos has not been reported. We have examined in detail the spatial distribution of transcripts from each of the Antp promoters in both embryo sections and whole embryos by in situ hybridization using promoter-specific probes. We show that the transcripts from each of the two promoters accumulate in distinct, but overlapping patterns during embryogenesis. The results demonstrate that the two Antp promoters are differentially regulated in embryos and provide a basis for examining the regulation of the two promoters and characterizing more fully the function of Antp during embryogenesis. In addition, we have examined the regulation of each of the Antp promoters by genes of the bithorax complex (BX-C). We show that in BX-C- embryos both promoters are derepressed in the abdomen.

Cloning of the proteinase that facilitates infection by schistosome parasites.

Four cDNA clones encoding a proteinase which facilitates skin invasion by schistosome parasites were isolated by screening a schistosome sporocyst cDNA library, using an oligonucleotide probe containing sequences complementary to predicted 5'-translated regions of its RNA. The amino acid sequence of the enzyme, as deduced from the DNA sequence of the clones, indicates that the enzyme is a serine protease which in many respects is similar to vertebrate pancreatic elastases, although regions outside of the putative active site, binding pocket, and amino-terminal cysteines differ significantly. Regulation of expression of the enzyme occurs at the level of mRNA transcription as well as posttranslationally, the latter involving the processing of a previously unidentified pre-proenzyme (zymogen) sequence. In situ hybridization of the cDNA clones to tissue sections of developing larvae indicates that the enzyme is synthesized within a discrete time frame in specialized cells of the organism.

Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms.

We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.

Preparation of mouse monoclonal antibodies and evidence for a host immune response to the preacetabular gland proteinase of Schistosoma mansoni cercariae.

Forty-six monoclonal antibodies were produced against the preacetabular gland secretions of Schistosoma mansoni cercariae by two different immunization protocols. These antibodies were of both the IgG and the IgM classes. One IgM monoclonal antibody (Ia4D6) was further characterized. It was specific to the cercarial stage by ELISA and showed specific binding to the 30,000 Mr proteinase in crude cercarial secretions by Western blot analysis. Preincubation of this antibody with purified cercarial proteinase resulted in inhibition of proteolytic activity, and it mediated complement-dependent cytotoxicity to cercariae in vitro. Immunoperoxidase staining of cercariae localized this antibody to vesicles visible within the preacetabular glands and their secretory ducts, and to secreted material. ELISA and Western blot analysis also showed that sera from infected mice and patients with schistosomiasis reacted with the cercarial proteinase. These studies demonstrate that a proteinase secreted into the host by invading cercariae is immunogenic and provide a monoclonal antibody probe for further characterization of its structure and function.

The major neutral proteinase of Entamoeba histolytica.

FPLC anion-exchange and chromatofocusing chromatography were used to purify the major neutral proteinase from secretions of axenically cultured Entamoeba histolytica trophozoites. HM-1 strain trophozoites, which were more proteolytically active than the less virulent HK-9 strain, were used for purification of the enzyme. It is a thiol proteinase with a subunit Mr of approximately 56,000, a neutral pH optimum, and a pI of 6. The importance of this enzyme in extraintestinal amoebiasis is suggested by its ability to degrade a model of connective tissue extracellular matrix as well as purified fibronectin, laminin, and type I collagen. The enzyme caused a loss of adhesion of mammalian cells in culture, probably because of its ability to degrade anchoring proteins. Experiments with a peptide substrate and inhibitors indicated that the proteinase preferentially binds peptides with arginine at P-1. It is also a plasminogen activator, and could thus potentiate host proteinase systems.

Hydrofluoric acid burns of the eye.

A case of hydrofluoric acid (HF) burns of the eye is reported and a review is presented of our investigation into the mechanism of HF toxicity in ocular tissues. A number of therapeutic procedures that have been successful in the treatment of HF skin burns were studied in the rabbit for use in the eye. Immediate single irrigation with water, normal saline or isotonic magnesium chloride solution is the most effective therapy for ocular HF burns. Extrapolation of other skin burn treatments to use in the eye is unacceptable due to the toxicity of these agents in normal eyes and the additive damage caused in burned eyes.