Characterization of calcitonin- and adrenocorticotropin-producing human cloned cell lines.

We have developed three human cloned cell lines that produce immunoreactive human calcitonin (ihCT) and ACTH (iACTH) as well as exhibit characteristics of cultured neural cells. Clones HMS-41/I, -78/2, and -98/2 were developed from cell lines HeLa AV3, MBA 9812 (bronchogenic carcinoma), and SW 267 (pheochromocytoma), respectively. Karyological analysis of both the parent and the cloned cell lines confirmed the identity of HeLa AV3 and MBA 9812. When grown in serum-free media designed for culturing neural cells, the patterns of production for both ihCT and iACTH varied among the clones. The multiple patterns of hormone production suggest that the mechanisms involved in the biosynthesis, processing, and secretion of these hormones differ among the clones. The clones contain neuron-specific enolase and the putative neurotransmitters beta-alanine and gamma-amino butyric acid, and they respond to cAMP analogs by differentiating, as noted by the extension of neurites (except the HeLa-derived HMS-41/I). The iACTH extracted from cells and synthetic ACTH exhibited equivalent profiles upon isoelectric focusing. The forms of ihCT noted in cell extracts were similar to those observed in extracts of human tumor tissue. Our rabbit antiserum to hCT failed to detect ihCT in those cell extracts prepared for ACTH determination or in extracts of rat pituitaries, but it did detect CT in rat thyroids by both RIA and immunofluorescent procedures. We concluded that our antisera to hCT do not detect the precursor form of ACTH. The availability of these cloned cell lines provides model systems for examining the production of these peptide hormones and the concomitant expression of neural and endocrine characteristics.

Use of the API 20E system to identify non-Enterobacteriaceae from veterinary medical sources.

The capability of the API 20E system to identify gram-negative nonfermenters and nonenteric fermenters was evaluated for 272 isolates from veterinary sources. Two different methods were used for interpreting the carbohydrate fermentation reactions on the strip. In method I, weakly fermented (yellow-green) carbohydrates were considered positive for all oxidase-positive organisms, and in method II, yellow-green carbohydrates were considered positive for all organisms requiring incubation for 48 hours. By both methods, the API system correctly identified 62% of the isolates. With method I, 31% of the isolates were misidentified and 6% were not identified. With method II, 21% of the isolates were misidentified and 17% received no identification. Organisms most affected by these 2 methods of interpretation were Pasteurella and Actinobacillus. Identifications reached by the API system were also compared with identifications made by veterinary diagnostic laboratories. The frequency of identifications agreements was not significantly affected by the method of API carbohydrate fermentation reaction interpretation. Generally, 30% of the identifications agreed (diagnostic laboratories vs API) when using only the API Index, whereas 51% agreed when the entire API computer data base identifications were included. The type of identification disagreements between diagnostic laboratories and the API system, however, was significantly affected by the method of API strip interpretation. With method I, 42% of the identifications were different and 6% were not in the API data base. With method II, 33% of the identifications were different and 17% were not in the API data base. Biotype differences between human and veterinary isolates were also compared. Significant differences between the predicted and actual reactions were noted for Pseudomonas aeruginosa and Bordetella bronchiseptica; however, these differences did not affect their correct identification to the API Index. For Pasteurella multocida, most profile numbers were not listed in the API Index because of differences in the actual vs predicted oxidase and nitrate reduction reactions; however, they were correctly identified with the total computer data base.

Regulation of cementoblast gene expression by inorganic phosphate in vitro.

Examination of mutant and knockout phenotypes with altered phosphate/pyrophosphate distribution has demonstrated that cementum, the mineralized tissue that sheathes the tooth root, is very sensitive to local levels of phosphate and pyrophosphate. The aim of this study was to examine the potential regulation of cementoblast cell behavior by inorganic phosphate (P(i)). Immortalized murine cementoblasts were treated with P(i) in vitro, and effects on gene expression (by quantitative real-time reverse-transcriptase polymerase chain reaction [RT-PCR]) and cell proliferation (by hemacytometer count) were observed. Dose-response (0.1-10 mM) and time-course (1-48 hours) assays were performed, as well as studies including the Na-P(i) uptake inhibitor phosphonoformic acid. Real-time RT-PCR indicated regulation by phosphate of several genes associated with differentiation/mineralization. A dose of 5 mM P(i) upregulated genes including the SIBLING family genes osteopontin (Opn, >300% of control) and dentin matrix protein-1 (Dmp-1, >3,000% of control). Another SIBLING family member, bone sialoprotein (Bsp), was downregulated, as were osteocalcin (Ocn) and type I collagen (Col1). Time-course experiments indicated that these genes responded within 6-24 hours. Time-course experiments also indicated rapid regulation (by 6 hours) of genes concerned with phosphate/pyrophosphate homeostasis, including the mouse progressive ankylosis gene (Ank), plasma cell membrane glycoprotein-1 (Pc-1), tissue nonspecific alkaline phosphatase (Tnap), and the Pit1 Na-P(i) cotransporter. Phosphate effects on cementoblasts were further shown to be uptake-dependent and proliferation-independent. These data suggest regulation by phosphate of multiple genes in cementoblasts in vitro. During formation, phosphate and pyrophosphate may be important regulators of cementoblast functions including maturation and regulation of matrix mineralization.

Defining the roots of cementum formation.

Significant progress has been seen in research aimed at regeneration of the disease-damaged periodontium. Our own strategy has been to approach periodontal tissue development (i.e. root, cementum, periodontal ligament, and bone) as a source for the identification of key regulators of cellular processes that may be applicable to periodontal tissue repair. Specifically, enamel-like molecules, bone morphogenetic proteins (BMPs), and phosphates have been investigated for their role in altering gene expression and cell functions in follicle cells, periodontal ligament cells, and cementoblasts. Amelogenin, leucine-rich amelogenin peptide, and tyrosine-rich amelogenin peptide have been found to similarly affect cementoblast gene expression and cementoblast-mediated mineralization in vitro; however, these enamel-like factors do not increase cell proliferation as has been observed in cells treated with Emdogain (Biora AB, Malmö, Sweden), an enamel matrix derivative. BMP-2 has been found to promote differentiation of follicle cells into a cementoblast/osteoblast phenotype, and BMP-3 is being investigated as a negative regulator of mineralization. The increased ratio of phosphate to pyrophosphate in the local region during root development has been found to significantly enhance the extent of cementum formation in animal models. Furthermore, phosphate has been identified as a regulator of cementoblast SIBLING (small integrin-binding ligand N-linked glycoprotein) gene expression in vitro. These investigations of candidate factors for periodontal regeneration have uncovered mechanisms regulating gene expression and cell function in cells controlling the behavior of periodontal tissues (i.e. follicle cells, periodontal cells, and cementoblasts) and offer new directions to consider for clinical repair of periodontal defects.

Use of the API 20E system to identify veterinary Enterobacteriaceae.

A total of 503 veterinary enteric bacterial pathogens obtained from state veterinary diagnostic laboratories were tested on API 20E strips to determine whether this rapid microidentification system could be utilized for veterinary clinical microbiology. The API 20E strip accurately identified 96% of the veterinary isolates and misidentified 3%. Identifications by the API system and the diagnostic laboratories were in agreement in 85% of the isolates, disagreement on 16% of the isolates, and 1% were not identified by the API strip. Differences in identification occurred primarily in distinguishing between Klebsiella and Enterobacter and between Enterobacter and Escherichia coli. These disagreements were most often due to incorrect identifications by the diagnostic laboratory rather than by the API system. Biotype differences between human and veterinary isolates were compared. Significant differences were noted in several biochemical reactions. The main differences observed for E. coli isolates were in ornithine decarboxylase production and melibiose fermentation. The largest differences for Salmonella occurred in arginine dihydrolase production, citrate utilization, and inositol fermentation, whereas for Klebsiella pneumoniae the main differences were noted in urease production and nitrate reduction. These biotype differences, however, did not affect the accurate identification of organisms on the API strip.

Translocatable elements in Staphylococcus aureus.

The properties of the first translocatable element in Gram-positive bacteria, a 5.2 kb segment encoding erythromycin resistance in S. aureus, are described. This element translocates from plasmid to multiple chromosomal sites and from chromosome to multiple plasmid sites, sometimes causing insertional inactivation and deletion. The genetic control of translocation and its role in natural plasmid evolution are discussed and preliminary evidence for translocation of penicillin and chloramphenicol resistance is presented. In the latter case, translocation involves in intact plasmid.

Genetic translocation in Staphylococcus aureus.

A 5.2-kilobase pair transposon, Tn551, has been found in Staphylococcus aureus, a Gram-positive bacterium. Initially detected on plasmid pI258, it undergoes rec-independent transposition to multiple chromosomal and plasmid sites, sometimes causing insertional inactivation. Unlike most other transposons, Tn551 undergoes apparently precise excision as a rule. The initial observation of Tn551 transition involved UV inactivation of the carrier plasmid; this would appear to be a general means of detecting transposable elements.