Molecular cloning of the human transmembrane secretory component (poly-Ig receptor) and its mRNA expression in human tissues

A 2.5 kilobase (kb) cDNA clone containing 92% of the coding region for human transmembrane secretory component (SC) or poly-Ig receptor, was isolated from a mammary gland cDNA library. The cDNA clone encoded a protein of 693 amino acids which showed 99% homology with the primary amino acid sequence of human free SC as reported by Eiffert et al. (1), and 54% homology with the deduced amino acid sequence of rabbit transmembrane SC for which cDNA was cloned by Mostov et al. (2). Northern blot analysis showed mRNA expression in various human exocrine tissues in good agreement with our previous immunohistochemical studies of SC.     

Retinol and retinyl esters in parenchymal and nonparenchymal rat liver cell fractions after long-term administration of ethanol

Chronic ethanol consumption reduces the liver retinoid store in man and rat. We have studied the effect of ethanol on some aspects of retinoid metabolism in parenchymal and nonparenchymal liver cells. Rats fed 36% of total energy intake as ethanol for 5-6 weeks had the liver retinoid concentration reduced to about one-third, as compared to pair-fed controls. The reduction in liver retinoid affected both the parenchymal and the nonparenchymal cell fractions. Plasma retinol level was normal. Liver uptake of injected chylomicron [3H]retinyl ester was similar in the experimental and control group. The transport of retinoid from the parenchymal to the nonparenchymal cells was not found to be significantly retarded in the ethanol-fed rats. Despite the reduction in total retinoid level in liver, the concentrations of unesterified retinol and retinyl oleate were increased in the ethanol fed rats. Hepatic retinol esterification was not significantly affected in the ethanol-fed rats. Since our study has demonstrated that liver uptake of chylomicron retinyl ester is not impaired in the ethanol-fed rat, we suggest that liver retinoid metabolism may be increased.     

Differences in reactivity of confluent and nonconfluent cultures of human endothelial cells toward thrombin-stimulated platelets or heparinized salt solution

Summary This study examined whether nonconfluent endothelial cell cultures reacted differently than confluent ones toward thrombin-stimulated platelets or a heparinized salt solution. The adherence to the endothelial cell cultures of⁵¹Cr-labeled human platelets stimulated at different thrombin concentrations was studied. There was significantly higher adherence of stimulated platelets to nonconfluent cultures compared with confluent ones. This was confirmed by scanning electron microscopy, which also revealed a tendency for the platelets to adhere at the cell periphery. Electron microscopy also showed that thrombin-stimulated platelets induced endothelial cell contraction. Part of the peripheral endothelial cell surface toward the bottom of the culture dish was inverted, facing the lumen of the dish. This phenomenon was particularly seen in nonconfluent cultures. When⁵¹Cr-labeled endothelial cultures were incubated with a mildly injurious fluid as heparinized sodium acetate and 20% serum, at 20° C for 30 min, the nonconfluent cultures showed significantly more cell detachment and release of⁵¹Cr than the confluent ones. We conclude that under the conditions of the present experiments there are differences in the reactivity of confluent and nonconfluent endothelial cell cultures. These differences probably reflect biological dissimilarities. In experiments where properties of cultured endothelium are studied, care should be taken that the degree of confluency is standardized.     

Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens

Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C. The enterotoxin was detected by serological and biological assays. Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage. The culture maintained enterotoxigenicity throughout cultivation in a continuous system. The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium. No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.     

Phagocytosis of bacteria by human leukocytes measured by flow cytometry

A new method has been developed for the evaluation of the phagocytic activity of human leukocytes using fluorescently labeled bacteria and flow cytometry. By simultaneous measurement of cellular light scatter and fluorescence, extracellular bacteria, phagocytes, and nonphagocytes could be discriminated and quantified. All leukocytes assumed to be capable of phagocytosis were phagocytosing, and about 90% of these cells were polymorphonuclear neutrophilic granulocytes. Within 15 min 85% of the bacteria were phagocytosed and each phagocyte contained an average of 15-20 bacteria. The phagocytic capacity of the leukocytes from healthy individuals showed minor interindividual and day-to-day variations. This method facilitates a rapid and accurate in vitro evaluation of the phagocytic activity of human leukocytes.     

The Gene Encoding the Catalytic Subunit Cα of cAMP-Dependent Protein Kinase (Locus PRKACA) Localizes to Human Chromosome Region 19p13.1

We have determined the chromosomal localization of the gene for the catalytic subunit Cα of cAMP-dependent protein kinase (locus PRKACA) to human chromosome 19 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. In addition, PCR analysis of a chromosome 19 mapping panel revealed the presence of a human Cα-specific amplification product only in cell lines containing the region 19p13.1 to 19q12. Finally, two-color fluorescencein situhybridization to metaphase chromosomes using the human Cα cDNA and human chromosome 19 inter-Alu-PCR product as probes localized the human Cα gene to chromosome region 19p13.1.     

Screening for point mutations in exon 10 of the low density lipoprotein receptor gene by analysis of single-strand conformation polymorphisms: detection of a nonsense mutation — FH469→Stop

DNA from 40 unrelated familial hypercholesterolemia (FH) heterozygotes were subjected to analyses of single-strand conformation polymorphisms (SSCPs) of exon 10 of the low density lipoprotein receptor (LDLR) gene. Four different SSCP patterns were observed. The underlying mutations were characterized by DNA sequencing. Three of the patterns represented the three genotypes of a recently described sense mutation in codon 450. A method based upon the polymerase chain reaction (PCR) was developed to analyze this mutation. The frequencies of the wild-type (G at nucleotide 1413) and mutant (A at nucleotide 1413) alleles were 0.56 and 0.44, respectively. The fourth pattern was found in only one FH heterozygote and was caused by heterozygosity at nucleotide 1469 (G/A). Nucleotide 1469 is the second base of codon 469_Trp(TGG). The GA mutation changes this codon into the amber stop codon, and is referred to as FH_469Stop. The mutant receptor consists of the amino terminal 468 amino acids. Because the truncated receptor has lost the membrane-spanning domain, it will not be anchored in the cell membrane. FH_469Stop destroys an AvaII restriction site, and this characteristic was used to develop a PCR method to establish its frequency in Norwegian FH subjects. Two out of 204 (1%) unrelated FH heterozygotes possessed the mutation.     

Effect of penicillin on the morphology of a Neisseria meningitidis strain liberating free endotoxin. An electron microscope study

An endotoxin-liberating strain of Neisseria meningitidis plasmolysed extensively after 2 h of exposure to 100 times MIC values of benzypenicillin. The peptidoglycan layer could be demonstrated after 2 h of treatment in places where the cytoplasm still was close to the cell wall. After 20 h, however, this layer was complete undetectable. In untreated cells the peptidoglycan layer could more easily be found in older cultures than in very young cultures. An increased adhesiveness and aggregation to other bacterial cells and to cell wall material could be observed after 2 h of penicillin treatment, and more pronouncedly after 20 h. A high yield of free cell wall material could be demonstrated after 2 h of penicillin treatment. This corresponded well to an increased content of free endotoxin in the filtrates from the cell cultures treated with penicillin, compared to untreated controls. After 20 h of treatment, free cell wall material had formed large aggregates or was adherent to the cell walls of ghost cells. The corresponding endotoxin analysis showed a reduced content of filtrable endotoxin. Possible implications of the structural changes in relation to penicillin treatment of patients are discussed.     

Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator.

Cell-binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase-type plasminogen activator (u-PA). In contrast to the human system, chemical cross-linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand-blotting analysis, two mouse u-PA-binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u-PA receptor (u-PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u-PAR due to cross-hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Dan?, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand-blotting analysis. Binding of mouse u-PA to its receptor showed species specificity in ligand-blotting analysis, since mouse u-PA did not bind to human u-PAR and human u-PA did not bind to mouse u-PAR. The apparent M(r) of mouse u-PAR varied between different mouse cell lines and ranged over M(r) 45,000-60,000. In four of the cell lines, mouse u-PA bound to two mouse u-PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u-PA-binding protein. In the monocyte macrophage cell line P388D.1, trypsin-treatment of intact cells could remove only the large mouse u-PAR variant (M(r) 60,000) indicating that only this type was a cell-surface-exposed molecule. The smaller mouse u-PAR variant (M(r) 45,000), was deglycosylated by the enzyme endo-beta-N-acetylglucosaminidase H and is probably an intracellular precursor form carrying only high-mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u-PAR variant proteins by diisopropylfluorophosphate-inactivated mouse u-PA-Sepharose affinity chromatography yielded two silver-stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Finite element analysis of different loading conditions for implant-supported overdentures supported by conventional or mini implants

The effect of implants’ number on overdenture stability and stress distribution in edentulous mandible, implants and overdenture was numerically investigated for implant-supported overdentures. Three models were constructed. Overdentures were connected to implants by means of ball head abutments and rubber ring. In model 1, the overdenture was retained by two conventional implants; in model 2, by four conventional implants; and in model 3, by five mini implants. The overdenture was subjected to a symmetrical load at an angle of 20 degrees to the overdenture at the canine regions and vertically at the first molars. Four different loading conditions with two total forces (120, 300 N) were considered for the numerical analysis. The overdenture displacement was about 2.2 times higher when five mini implants were used rather than four conventional implants. The lowest stress in bone bed was observed with four conventional implants. Stresses in bone were reduced by 61% in model 2 and by 6% in model 3 in comparison to model 1. The highest stress was observed with five mini implants. Stresses in implants were reduced by 76% in model 2 and 89% increased in model 3 compared to model 1. The highest implant displacement was observed with five mini implants. Implant displacements were reduced by 29% in model 2, and increased by 273% in model 3 compared to model 1. Conventional implants proved better stability for overdenture than mini implants. Regardless the type and number of implants, the stress within the bone and implants are below the critical limits.