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The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

Feeding of sorghum with a high level of tannin (high-tannin sorghum) to rats caused changes in gene expression in parotid glands similar to isoproterenol treatment. Within 3 days the parotid glands were enlarged about 3-fold and a series of proline-rich proteins were increased about 12-fold. Unlike isoproterenol treatment, no changes were observed in the submandibular glands, and a Mr 220,000 glycoprotein in parotid glands was not induced. Amino acid analyses, electrophoretic patterns, and cell-free translations of mRNAs all confirmed that the proline-rich proteins induced by feeding high-tannin sorghum were identical to those induced by isoproterenol treatment. Binding curves for proline-rich proteins to tannins showed affinities 10-fold greater than bovine serum albumin and tannins.A series of unusual proteins containing 25-45% proline have been isolated from human and rat salivary glands and the secretions of salivary glands (1-6). Two families of either acidic or basic proline-rich proteins (PRPs) are commonly found, with variations in glycosylation and phosphorylation. The phosphorylated PRPs are postulated to be involved in dental repair (7) because these substances have a very high affinity for hydroxyapatite. The functions of the basic PRPs, which have isoelectric points of >10 (8), are unknown.The condensed tannins (proanthocyanidins, oligomers of flavan-3-ols) present in many plant tissues characteristically bind and precipitate proteins (9). This astringency of the tannins, which are rich in phenolic groups, apparently protects the plant tissue against ingestion by some insects (10) and seed-eating birds (11). Seeds of bird-resistant cultivars of sorghum, a major cereal of the semi-arid tropics, contain a high level of tannin (high-tannin sorghum), which diminishes the nutritional value of the grain by inhibiting protein digestion (12) and possibly by other mechanisms. In studies designed to define the interaction of tannins and proteins, Hagerman and Butler (13) were able to demonstrate that tannins have an unusually high affinity for proteins rich in proline. Because the gastrointestinal tractspecifically the oral cavity-is a source of PRPs, Hagerman and Butler (13) proposed that salivary PRPs could possibly interact with tannins and serve to protect dietary proteins and digestive enzymes.In this study we report on the dramatic effects on rat parotid glands of feeding high-tannin sorghum. High-tannin sorghum ingestion mimics some of the phenotypic changes observed after treatment of rats with the a-agonist isoproterenol (6,8 Feeding Trials. Sprague-Dawley male rats (70 g) (Murphy Breeding Laboratories, Plainfield, IN) were maintained on Purina Lab Chow for 3-6 days before initiating the feeding trials. Sorghum grains were ground and incorporated into diets of the following composition (% of diet): sorghum, 92.6; corn oil, 2.0; minerals (AIN-76), 3.5; vitamins (AIN-76), 1.0; lysine HCI, 0.75; choline chloride, 0.14; and butylated hydroxytoluene, 0.01. Feed and water were provided ad lib. I...

The small, proline-rich (SPR) genes consist of three subclasses closely linked on human chromosome 1, a region referred to as the epidermal differentiation complex. SPR genes consist of two exons, with the second exon containing the entire open reading frame. SPRs are expressed in all squamous tissues of the skin, scalp, footpad, vaginal epithelia, and most of the epithelial lining of the digestive tract, including the lip, tongue, esophagus, and forestomach. Although SPR1 is absent in normal mucociliary epithelium of the respiratory tract, epithelia that undergo squamous differentiation in response to vitamin-A deficiency or to injury owing to exposure to environmental toxicants express SPR1. High levels of SPR1 are detected in various diseases and cancers of the skin or respiratory epithelia and in nonkeratinizing papillary adenocarcinomas. SPR expression can be regulated by transcriptional factors, by posttranscriptional factors, or by factors that affect SPR1 mRNA translation or protein turnover. Furthermore, regulation can be affected by the state of cell proliferation. The presence of SPR1 in most of these epithelia, and the absence of SPR3 in normal skin, suggest that these subclasses have distinct functions. Various approaches to the study of the cross-linked envelope (CE) components in identifying SPR1 and SPR2 and in suggesting that SPRs are one of the precursor proteins of the CE. However, expression of SPR1 in nonsquamous tissues and cell lines indicates a function not associated with squamous differentiation. Several studies have demonstrated that SPR1 antibodies react with nuclear proteins and that SPR1 is expressed in cells before entering the G0 phase of the cell cycle. Future studies should clarify the role of SPRs by modifying their contents in CE, and should identify SPR-associated proteins to clarify the cell growth-related role of SPR1.

No abstract

Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.

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A B S T R A C T Addition of increasing amounts of JIlabeled desialylated thyroxine-binding globulin (DTBG) ceruloplasmin.An inhibition assay system using hepatic cell membranes showed that desialylated orosomucoid had a greater affinity for membrane binding sites than did DTBG but desialylated fetuin and ceruloplasmin bound less avidly than DTBG.Serum from normal persons and patients with a variety of illnesses was tested for its ability to inhibit ["JI]DTBG binding. The inhibitory activity of 1 ml of normal serum was equivalent to that of 0.2-2 i'g DTBG. Patients with Laennec's cirrhosis, biliary cirrhosis, and hepatic metastases had greatly increased inhibitory activity in their serum. Patients with jaundice due to extrahepatic obstruction had inhibitory activity not significantly different from that found in normal serum.Column chromatography of normal serum on Sephadex G-200 resulted in inhibitory activity throughout the range of protein molecular weight. Desialylation of normal serum with neuraminidase enhanced the inhibitory activity but did not change the distribution of the activity. Gel chromatography of cirrhotic serum showed markedly increased inhibitory activity associated with the macroglobulins and the 4.5S peak and a new peak of