The flanking amino acids of the human follitropin beta-subunit 33-53 region are involved in assembly of the follitropin heterodimer.

Previous analyses of the topology of human follitropin (hFSH) with monoclonal antibodies and antipeptide antibodies have led to a current operating hypothesis that some amino acids within the hFSH beta 33-53 region are surface oriented, and others participate in subunit contact. Protein structural analysis predicts beta-turns within this region, and the immunochemical studies indicate that the ends may be involved in subunit contact. In this study, hFSH beta was mutagenized to change 34TRDL37 to 34AAAA37 or 48QKTCT52 to 48AAACA52, allowing us to study the ends of the hFSH beta 33-53 sequence contiguous with the hFSH beta sequence. Wild-type and mutant cDNAs were coexpressed with alpha-subunit cDNA in CHOPro-5 cells. Wild-type hFSH was secreted from cells cotransfected with wild-type hFSH alpha and hFSH beta cDNAs, as expected. However, heterodimeric hFSH was minimally detected in the medium from cells transfected with the 34TRDL37 mutant and was not detected in the case of the 48QKTCT52 mutant. Analysis of cell lysates (intracellular FSH) by immunoprecipitation and polyacrylamide gel electrophoresis showed that wild-type and mutant beta-subunits were indistinguishable and recoverable intact from each cell line. Additionally, analysis of lysates with a conformation-specific monoclonal antibody 3G3 revealed that similar levels of properly folded beta-subunit were produced in cells expressing wild-type or either mutated beta-subunit. These data indicate that the flanking amino acids of the hFSH beta 33-53 region, in particular 48QKTCT52, are critical for assembly of hFSH heterodimer.

Expression of human catalase in acatalasemic murine SV-B2 cells confers protection from oxidative damage.

Reactive oxygen species have been implicated in aerobic organisms as causative agents in damage to DNA, proteins, and lipids. Catalase is a major enzyme in the defense against such oxidant damage. To determine whether increased catalase expression confers greater resistance to oxidant stress, a eukaryotic expression vector harboring a human catalase cDNA clone was constructed. Acatalasemic murine fibroblasts were then co-transfected with that catalase expression vector and pSV2-neo, and successfully transfected cells were identified by their ability to grow in the presence of geneticin. Clones that contained integrated copies of the catalase expression vector were identified by Polymerase Chain Reaction (PCR) analysis. Stably transfected geneticin-resistant cell lines that overexpressed catalase in potentially positive cell lines were confirmed by catalase enzyme assays. To examine the physiological relevance of catalase overexpression, cells were exposed to oxidant stresses (hydrogen peroxide and hyperoxia), and survival rates were determined. Results demonstrated a significant resistance to oxidative stress in cells overexpressing catalase when compared to controls. These transfected cell lines will provide important models for further evaluation of the role of catalase in protecting cells against the toxic effects of oxygen-derived free radicals and their derivatives.

Expression of bovine and mouse endothelial cell antioxidant enzymes following TNF-alpha exposure.

Endothelial cells are primary targets for injury by reactive oxygen species. Endothelial catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganous superoxide dismutase (MnSOD) provide potential antioxidant enzymatic defenses against oxidant-induced cellular damage. Previous studies in vivo and in vitro have demonstrated that in certain cell types exposure to oxidants may increase the expression of one or more of these antioxidant enzymes, thus providing greater intracellular potential to withstand oxidant-induced cell stress. To test whether endothelial antioxidant enzyme expression is influenced by similar oxidant-induced stresses in vitro, we have exposed endothelial cells to tumor necrosis factor-alpha (TNF-alpha) and have measured levels of catalase, CuZnSOD and MnSOD mRNA, and protein. Our results demonstrate a selective increase of MnSOD mRNA, with coordinate increases of both MnSOD protein and enzyme activity in endothelial cells treated for 24/h with TNF-alpha. In contrast, levels of catalase and CuZnSOD mRNA and protein remained unchanged in these cells after TNF-alpha treatment. These observations were made in microvessel endothelial cells derived from murine and bovine sources. Our results indicate that TNF-alpha can act specifically to increase enzymatic antioxidant potential in endothelial cells by induction of a particular antioxidant enzyme encoding mRNA species. These data demonstrate the capacity of endothelial cells to mount an antioxidant defense in response to exposure to an inducer of oxidative damage.

Mn and Cu/Zn SOD expression in cells from LPS-sensitive and LPS-resistant mice.

We examined the effect of lipopolysaccharide (LPS) treatment on the expression of manganese and copper/zinc superoxide dismutase (MnSOD and Cu/ZnSOD) mRNA and protein in resident peritoneal macrophages and lung endothelial cells derived from LPS-sensitive (LPS-s) and LPS-resistant (LPS-r) mice. Macrophages from both LPS-s and LPS-r mice treated with LPS for 24 h produced increased levels of MnSOD mRNA and protein. In contrast, levels of lung endothelial cell MnSOD mRNA and protein from LPS-s mice were increased by LPS treatment, while no increases in these parameters were observed in endothelial cells from LPS-r mice. Tumor necrosis factor-alpha (TNF alpha) treatment, however, did increase levels of MnSOD mRNA in both LPS-s and LPS-r endothelial cells to an equal extent. Both macrophage and endothelial cell Cu/ZnSOD mRNA and protein levels were not significantly affected by LPS treatment. These results demonstrate that the mutation that affects susceptibility to LPS in LPS-r mice exerts a differential influence on MnSOD inducibility in a cell specific manner.

Transfection of a manganese-containing superoxide dismutase gene into hamster tracheal epithelial cells ameliorates asbestos-mediated cytotoxicity.

To determine if overexpression of manganese-containing SOD (MnSOD) alters cell sensitivity to asbestos, an expression cassette containing murine MnSOD cDNA was cotransfected with pSV2neo, a plasmid conferring resistance to the antibiotic G418, into a diploid cell line of hamster tracheal epithelial (HTE) cells. Pools of G418-resistant transfectants were characterized by Southern and Northern blot analyses and enzyme activity assays. Although increases in MnSOD gene copies in individual cell pools ranged from approximately 7- to 86-fold in comparison to cells transfected with pSV2neo alone, steady-state levels of MnSOD mRNA were increased only by 1.4-to 2.3-fold. Despite modest increases in MnSOD mRNA, significant elevations in MnSOD enzyme activity were observed in pools of G418-resistant cells. MnSOD-transfected cell lines were more resistant to the cytotoxic effects of crocidolite asbestos using a sensitive colony-forming efficiency (CFE) assay. These data show that MnSOD has a direct role in cell defense against asbestos-induced cytotoxicity, an oxidant-dependent process.

A rapid and convenient procedure for identifying recombinant plasmids.

We describe an improved method for identifying plasmids that contain recombinant DNA inserts. The protocol is inexpensive and easy to perform and allows for the examination of small colonies that might normally be excluded from this type of analysis.

Examination of osteoblast-orthopaedic biomaterial interactions using molecular techniques.

Molecular techniques can be used to elucidate the effects of extended periods of cell-biomaterial interactions on the time-course and level of expression of particular genes which determine cellular phenotype. We used the polymerase chain reaction to demonstrate the expression of genes for the bone-related proteins osteocalcin, osteonectin and osteopontin by neonatal rat calvarial osteoblasts. In addition, Northern blotting was subsequently used to show that messenger RNAs encoding osteonectin and osteopontin were consistently expressed during a 5 wk period of interaction of osteoblasts with Ti-6Al-4V, a commercial brand of hydroxyapatite, and tissue culture polystyrene.

Regulation of antioxidant enzyme expression in LPS-treated bovine retinal pigment epithelial and corneal endothelial cells.

Retinal pigment epithelial (RPE) and corneal endothelial (CE) cells, because of their locations and functions, are continuously exposed to toxic oxidants. Protection from these toxic materials may be due, in part, to the action of endogenous antioxidant enzymes. We have established the presence of mRNAs that encode antioxidant enzymes in bovine RPE and CE cells and have determined the effect of bacterial lipopolysaccharide (LPS) on their expression. The most striking change in antioxidant enzyme expression is an increase in the level of mitochondrial manganous superoxide dismutase (MnSOD) mRNA in the LPS-treated RPE and CE cells. This increase in mRNA expression is accompanied by a slight increase in MnSOD activity as determined by SOD activity gels.

Suspected lead poisoning in a public school.

Reports of lead exposure can generate considerable public concern, particularly when children are involved. In December, 1996, a public elementary school in rural Utah was found to have elevated concentrations of lead in its drinking water. The local public health department responded by instituting remediation of the water supply and by warning parents of the possible danger to their children. Subsequent blood lead testing in 116 of the approximately 300 children involved showed an average lead concentration in the range expected for the U.S. population at large. One of the 116 specimens was marginally elevated and was probably unrelated to the school drinking water. Reducing lead exposure is an important public health concern which sometimes generates a response out of proportion to the danger involved.

Assessing the efficacy of three dentifrices in the treatment of dentinal hypersensitivity.

A multicenter clinical trial conducted by the authors compared the desensitizing efficacy of a new 5 percent potassium nitrate: 0.243 percent sodium fluoride dentifrice along with two clinically proven, commercially available desensitizing dentifrices to a placebo dentifrice. Sensitivity to cold air and tactile stimulation, along with patients' subjective assessments, were evaluated to assess the dentinal desensitizing efficacy of the test dentifrices. Results demonstrated that after four weeks, participants who used the new dentifrice formulation experienced significant decreases in dentinal sensitivity compared to the placebo group for all measured indexes.

A comparison of intraoral antimicrobial effects of stabilized stannous fluoride dentifrice, baking soda/peroxide dentifrice, conventional NaF dentifrice and essential oil mouthrinse.

The intraoral antimicrobial activity of four commercial oral products-conventional NaF dentifrice (Crest), baking soda/peroxide/NaF dentifrice (Mentadent), essential oil mouthrinse (Listerine) and SnF2 dentifrice (Crest Plus Gum Care)-have been compared in three test regimens. Formulations were compared for their ability to suppress the regrowth and apical extension of dental plaque following toothbrushing during thirty hours of non-brushing where products were used as oral rinses (30-hour plaque regrowth model). Formulations were also compared for their ability to suppress the colony-forming units (cfu) of facultative anaerobic bacteria sampled from buccal gingival surfaces following use (Gingival Surface Microbial Index-GSMI model). Lastly, formulations were compared for effects in suppressing the glycolytic metabolic activity and regrowth activity of in vivo-treated dental plaques sampled at various periods following topical use and incubated under controlled ex vivo conditions (Plaque Glycolysis and Regrowth-PGRM model). In thirty-hour plaque regrowth testing, the rank ordered antimicrobial efficacy of formulations followed SnF2 > essential oils > NaF = water = baking soda/peroxide. In GSMI testing, all formulations were shown to suppress the cfu of facultative anaerobic bacteria relative to baseline, although SnF2 treatment was observed to reduce bacterial levels to a significantly greater degree than NaF dentifrice or baking soda/peroxide dentifrice up to two hours following brushing. In PGRM testing, the SnF2 dentifrice provided significant inhibition of bacterial metabolism and regrowth following topical application when compared with the NaF dentifrice as control. The baking soda/peroxide dentifrice provided no reduction in either bacterial metabolism or regrowth in PGRM. Previous studies had demonstrated modest effects for essential oil rinse in reducing PGRM plaque regrowth, with no effects for this treatment on plaque metabolism. Overall, these results demonstrate that SnF2 dentifrice provides substantial intraoral antimicrobial effects. The essential oil mouthrinse also exhibits significant intraoral antimicrobial effects, albeit apparently less than SnF2 dentifrice. The baking soda/peroxide dentifrice did not produce any antimicrobial effects following in vivo use compared with conventional dentifrice. These results provide mechanistic rationale for the chemotherapeutic efficacy of SnF2 and essential oil formulations in reducing gingivitis, while providing no support for the expectation of clinical efficacy for formulations containing baking soda and peroxide.

Molecular analysis of an acatalasemic mouse mutant.

The Csb acatalasemia mouse mutant differentially expresses reduced levels of catalase activity in a tissue specific manner. In order to pinpoint the molecular lesion that imparts the acatalasemia phenotype in Csb mice we have utilized the polymerase chain reaction technique to isolate catalase cDNA clones from control and Csb mouse strains. Sequence analyses of these cDNA clones have revealed a single nucleotide difference within the coding region of catalase between control and Csb mice. This nucleotide transversion (G----T) is located in the third position of amino acid 11 in the catalase monomer. In control mouse strains glutamine (CAG) is encoded at amino acid 11, while in Csb mice this codon (CAT) encodes histidine. This amino acid is located within a region that forms the first major alpha-helix in the amino-terminal arm of the catalase subunit and, as such, may render the catalase molecule unstable under certain physiological conditions.

Turnover of cytoplasmic and mitochondrial aspartate aminotransferase isozymes in mouse liver and transplantable hepatomas.

Activities of the cytoplasmic and mitochondrial isozymes of aspartate aminotransferase (aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1, AAT) in transplantable mouse hepatomas BW7756 and H-4 are reduced when compared to normal adult liver. Both proteins have been purified to homogeneity from a single preparation of mouse liver and monospecific antibodies raised to each isozyme. By quantitative immunotitration analysis, the activity of each isozyme in liver and hepatoma has been shown to correlate with levels of immunoprecipitable protein. Furthermore, for each isozyme, the liver versus hepatoma species is indistinguishable by heat inactivation kinetics, Km's for substrates, and molecular weights. Thus, the reduction of mitochondrial and cytoplasmic AAT activities in hepatoma tissue is due not to alterations in the catalytic activity of the enzyme molecules, but to a decrease in the number of enzyme molecules present. Turnover of the isozymes was studied in liver and hepatoma tissue using in vivo radiolabeling and specific immunoprecipitation techniques. The cytoplasmic isozyme has a similar rate of degradation in liver and hepatoma, while the rate of synthesis of this isozyme in hepatoma is approximately tenfold less than in liver. The mitochondrial isozyme is also degraded at a similar rate in both tissues, but the rate of synthesis is sixfold greater in normal liver tissue than in hepatoma. It is concluded that decreased amounts of both isozymes in hepatoma as compared to liver are the result of a reduction in the rate of synthesis of each isozyme without any change in the rate of degradation.

Genetic determination of sn-glycerol-3-phosphate dehydrogenase synthesis in Drosophila melanogaster. A cis-acting controlling element.

A variant that uniformly reduces the activity of larval and adult isozymes of sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) throughout development has been isolated and analyzed. This low activity line (BI 114), which bears the fast electrophoretic structural allele, has been analyzed in comparison with two high activity control lines, WGM 74 (fast electrophoretic variant) and RI09 (slow electrophoretic variant). The enzyme has been purified from each line and all three variants have similar kinetic, immunological, and physicochemical parameters, except for pI differences associated with the electrophoretic differences observed. Enzyme-specific cross-reacting material analysis corroborates the activity data indicating differential rates of enzyme accumulation between lines throughout development. Protein turnover studies indicate that the rate of intracellular degradation between lines is the same, and that the activity and enzyme-specific cross-reacting material differences observed are due to differential rates of synthesis of the enzyme. Genetic analysis indicates that the variation in the rate of glycerol phosphate dehydrogenase synthesis segregates as a single gene with additive inheritance and is closely linked to the structural gene. Zymogram analysis of F1 heterozygotes has indicated that the variant acts as a cis-regulator and affects both isozymes equally throughout development. We have therefore designated this variant as a systemic regulator controlling the rate of glycerol phosphate dehydrogenase synthesis.

Drosophila sn-glycerol-3-phosphate dehydrogenase isozymes are generated by alternate pathways of RNA processing resulting in different carboxyl-terminal amino acid sequences.

Glycerol-3-phosphate dehydrogenase (GPDH, Ec 1.1.1.8) in Drosophila melanogaster consists of a family of three isozymes designated as GPDH-1, 2, and 3 which exhibit a unique temporal and tissue-specific pattern of expression. While each isozyme is encoded by the same structural gene, they differ by the amino acid sequence at the COOH-terminal end, with GPDH-3 having the sequence Asn-His-Pro-Glu-His-Met-COOH and with GPDH-1 extended by the three amino acid sequence Glu-Asn-Leu-COOH. We have isolated both genomic and cDNA clones in order to examine the structure of the 3'-end of this gene and its transcriptional products. This analysis has demonstrated three classes of transcripts, each differing in the 3'-untranslated region and coding for an enzyme with a different COOH-terminal amino acid sequence. Each transcript is shown to arise through the differential expression of three isotype-specific exons at the 3'-end of the gene. We propose a model where the expression of each isotype-specific transcript is controlled through a developmentally regulated process of 3'-end formation and alternate splicing pathways of the pre-mRNA. Furthermore, since each transcript and its cognant isozyme is tissue-specific in expression, this model suggests a role for tissue-specific trans-acting factors in these processing events.

Isolation of a cDNA clone for murine catalase and analysis of an acatalasemic mutant.

We have investigated the genetic control of murine catalase expression by analyzing catalase transcription and translation products from the tissues of control (Csa) and acatalasemic (Csb) mouse strains. Csb animals possess nearly normal catalase enzyme activity levels in liver, while displaying approximately 20 and 1% of normal activity levels in kidney and red blood cells, respectively. Immunoblot analyses of catalase in these tissues have revealed reduced levels of immunologically reactive catalase protein in Csb kidney and red blood cells, paralleling the reduction of catalase enzyme activity in these tissues. In order to determine the molecular basis for Csb acatalasemia, we have isolated a cDNA clone for murine catalase and have used this probe to analyze Csa and Csb genomic DNA and catalase mRNA. These studies have revealed: 1) no restriction fragment length polymorphisms between Csa and Csb genomic DNAs; 2) no differences in the levels of Csa and Csb catalase mRNA within a single tissue; and 3) no differences in the sizes of Csa and Csb catalase mRNAs. These observations suggest that the genetic defect that produces the tissue-specific reduction of catalase expression in Csb mice is not due to a marked rearrangement of DNA within the Csb catalase structural gene. Furthermore, the Csb mutation does not act at the level of gene transcription or mRNA stability, but rather at the level of mRNA translation and/or catalase protein turnover.

Differential expression and isozymic composition of sn-glycerol-3-phosphate dehydrogenase in tissues from variant lines of Drosophila melanogaster.

The tissue-specific expression and isozymic composition of Drosophila sn-glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8) have been determined for a high-activity control line and two variant lines that alter either the temporal or systemic expression of GPDH through a reduction in rates of polypeptide synthesis. The temporal variant exhibits a reduction in enzyme levels in all larval tissues and in the adult abdomen, while levels of activity in the adult thorax are equal to the control line. Isozymic analyses of these tissues demonstrate that it is the GPDH-3 species that is reduced in a temporal and tissue-specific manner. In contrast, the systemic variant demonstrates a uniform reduction of all isozymic species in each tissue and developmental stage. Analyses of the tissues of F1 hybrid offspring of each variant line and appropriately marked electrophoretic variants demonstrate that the tissue-specific effects observed are due to cis-acting elements that are tightly linked to the structural gene.

RNA directed synthesis of catalytically active Drosophila sn-glycerol-3-phosphate dehydrogenase in Xenopus oocytes.

Xenopus laevis oocytes injected with poly(A)+ RNA isolated from Drosophila melanogaster direct the synthesis of catalytically active glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8). The de novo synthesized enzyme reflects the electrophoretic properties appropriate to the stock of flies from which the injected RNA was isolated and is electrophoretically distinct from endogenous Xenopus activity. Immunoprecipitation of 35S-labeled translation products has demonstrated two immunologically related proteins with molecular masses of 32- and 34kDa which are encoded by two separate mRNA molecules. The 32-kDa protein is identical in size and charge properties to the protein purified from the fly and possesses the catalytic activity observed in the Xenopus translational assay. Poly(A)+ RNA isolated from a strain of flies bearing a CRM- null mutation at the GPDH locus does not contain translatable RNA for the 32-kDa protein. These results suggest that the two immunologically related proteins are the translational products of two separate transcripts derived from either two related loci or from differential transcription and/or processing of the same genetic locus.

Analysis of genetically defined liver enzymes in transplantable hepatomas and developing liver of the mouse.

The activities and isozyme patterns of seven enzymes involved in glycolysis, the malate-aspartate shuttle and the pentose phosphate pathway have been examined in five transplantable murine hepatomas, adult liver and fetal liver. The liver marker enzyme, alcohol dehydrogenase, has also been studied. The developmental expression of the activity and isozyme pattern of these enzymes have been determined. Seven defined loci on six different chromosomes code for these enzymes and isozymes. The tumors express no unique gene product for any of the enzymes examined, but the activities of all the enzymes in the hepatomas are more like fetal liver than adult liver, regardless of whether activities are increased or decreased in hepatomas. The activity of one enzyme is unchanged in hepatomas, and this enzyme has the same activity in fetal and adult liver. The enzyme activities in the five hepatomas vary more than the enzyme activities in liver and fetal liver of the three inbred strains of mice examined.

Site-directed alanine mutagenesis of Phe33, Arg35, and Arg42-Ser43-Lys44 in the human gonadotropin alpha-subunit.

Residues Phe33 and Arg35, individually, and a composite mutation of residues Arg42, Ser43, and Lys44 were changed to alanine in the human glycoprotein hormone common alpha-subunit using site-directed mutagenesis. These specific residues are highly conserved across species and have by chemical modification and synthetic peptide approaches been implicated in the binding of human chorionic gonadotropin (hCG) to leutinizing hormone (LH) receptor. In the present study we tested the hypothesis that specific alpha-subunit amino acid residues which stabilize the hormone receptor interaction for hCG have the same function in human follicle-stimulating hormone (hFSH). Wild type or mutant alpha-subunit cDNAs were coexpressed with wild type hFSH or hCG beta cDNA in sialylation defective Chinese hamster ovary cells. Recombinant hormones were tested in a radioligand receptor competition assay, using rat testis membranes as a source of FSH and LH receptors. Mutant hFSH heterodimers F33A-FSH, R35A-FSH, Arg42-Ser43-Lys44/Ala42-Ala43-Ala44- FSH all displaced 125I-hFSH in a similar fashion, indicating that these residues are not important for binding of hFSH to the rat FSH receptor. On the other hand, F33A-CG evidenced a 5-fold decrease in binding, while R35A-CG had over a 100-fold decrease in binding to the rat LH receptor when compared to the wild type recombinant hCG. These data demonstrate that a receptor-binding site on the common alpha-subunit which is very important for hCG binding to LH receptor is not important for the binding of hFSH to FSH receptor. Our interpretation of these findings is that there are fundamental structural differences in the receptor interface contacts of the common alpha-subunit, which stabilize receptor binding among members of the glycoprotein hormone family.