Regulation of Osteoblast Gene Expression in Intratypic Osteosarcoma Hybrid Cells

Intratypic osteosarcoma hybrids were constructed by fusing the human osteoblast-like osteosarcoma SaOS-2 with the rat osteoblast-like osteosarcoma UMR-106. Both of these osteosarcomas express liver/bone/kidney alkaline phosphatase (ALPL), but only the UMR-106 cell line expresses osteopontin (OPN), a gene expressed during later stages of osteoblast differentiation. Analysis of osteoblast gene expression in these hybrids demonstrated that ALPL continued to be expressed; however, OPN steady-state mRNA levels were dramatically reduced in four hybrids. Quantitative measurements indicated theft OPN steady-state mRNA levels were extinguished by a factor of 20- to 1000-fold. Since SaOS-2 chromosomes are preferentially lost from these hybrids, subclones of extinguished hybrids were isolated that reexpressed OPN mRNA at levels similar to the UMR-106 parental line. These data indicate that trans-acting negative regulatory factors, expressed from the SaOS-2 genome, are responsible for OPN extinction. This report provides the first demonstration of the negative regulation of OPN gene expression and also provides additional evidence that extinction plays a role in the regulation of osteoblast gene expression.     

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The V_H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG^TM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V_H-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the V_H molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the V_H isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the V_H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its¹H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V_H molecule, prevented dimerization of the V_H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.     

Male-specific cell migration into the developing gonad

Background: The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing β-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for β-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad.Results: Migration of mesonephric cells occurred into XY but not XX gonads from 11.5–16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells.Conclusions: Migration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.     

A temperate bacteriophage specific for strains ofAeromonas salmonicida possessing A-layer,a cell surface virulence factor

A temperate bacteriophage designated TP446 was isolated from culture supernatants ofAeromonas salmonicida strain A446. Phage TP446 adsorbed to all of the typical and atypical strains ofA. salmonicida tested that possessed A-layer, the surface protein array that represents the primary virulence factor of this fish pathogen. In contrast, TP446 failed to adsorb to mutants lacking A-layer. These results indicate that the A-layer is a component of the receptor for phage TP446.     

Cyanophycin granule polypeptide protease in a unicellular cyanobacterium

An assay was developed to measure the proteolysis of cyanophycin granule polypeptide in crude extracts of a unicellular cyanobacterium. The substrate was radioactively labeled cyanophycin granule polypeptide formed by an unicellular cyanobacterium grown in the presence of chloramphenicol. Substrate polypeptide displayed identical chemical properties with polypeptide isolated from non-chloramphenicol-treated cells. Solubilization of radioactivity as arginine indicated hydrolysis of polypeptide. Radioactively labeled aspartate and arginine from hydrolyzed polypeptide was related to nmol amino acid using a combination of paper chromatography, liquid scintillation analysis, and ninhydrin quantitation. Protease activity was found in extracts of nitrogen-limited cells harvested 16–24 h after a nitrogen source was added back. Optimal pH for protease activity was 8.0 and optimum temperature was 35°C. Protease activity in crude extracts followed Michaelis-Menten kinetics with a V _max of 92 nmol arginine per 15 min/mg protein and a K _m of 2.1×10³ nmol arginine. Protease activity was inhibited by arginine and by high concentrations of aspartate.     

Determination of the Carbon-Bound Electron Composition of Microbial Cells and Metabolites by Dichromate Oxidation

The applicability of the silver sulfate-acid dichromate oxidation (chemical oxygen demand) method for determining the carbon-bound electron compositions of microbial cells, substrates, and metabolic by-products was evaluated. An approach for approximating the carbon-bound electron composition of microbial cells from CHN data is also presented. Ten aliphatic and aromatic carboxylic acids, 17 amino acids, and 8 sugars generally gave 96 to 101% (mainly ≥98%) recovery with 0.0625 N dichromate (digestion mixture of 10 ml of sample-10 ml of 0.25 N dichromate-20 ml of Ag₂SO₄-amended concentrated H₂SO₄). Recoveries of nicotinic acid (5%) and methionine (65%) were incomplete; arginine (125%) and two purine and three pyrimidine bases (105 to 120%) were overestimated. The validity of 0.0625 N dichromate for determining the carbon-bound electron composition of bacterial cells was supported by theoretical analysis of the carbon-bound electron composition of hypothetical bacterial cell material (defined monomer composition) and by the compatibility of elemental and dichromate oxidation-derived carbon-bound electron compositions of typical bacterial cells.     

Préparation et forme sérique de la protéine réactive C de lapin

The preparation of rabbit C-reactive protein (CRP) involves a single step affinity chromatography. This preparation takes advantage of the calcium-dependent affinity of CRP for an agarose gel bearing 2-aminoethanol dihydrogen-phosphate as a ligand. A prior chromatography on agarose gel without the ligand allows the uptake of the serum amyloid P-component (SAP).The CRP prepared according to this method is able to form precipitating complexes in agarose with rabbit lipoproteins. The specificity of these interactions is studied. It is demonstrated that CRP-High Density Lipoproteins (HDL) association produces a second precipitation arc when the pure CRP is revealed by a specific antiserum in agarose. Moreover, CRP in the serum is shown to be in the bound form only, and the binding involves Low Density Lipoproteins (LDL) exclusively.