Streptococcus pneumoniae DNA polymerase I lacks 3''-to-5'' exonuclease activity: localization of the 5''-to-3'' exonucleolytic domain.

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and single-stranded DNAs. It degraded oligonucleotide substrates to a decameric final product.     

Altered metabolic and adhesive properties and increased tumorigenesis associated with increased expression of transforming growth factor beta 1

Transforming growth factor-beta (TGF-beta) is a potent mediator of cell proliferation and extracellular matrix formation, depending on the cell type and the physiological conditions. TGF-beta is usually secreted in a "latent" complex that needs activation before it can exert its effects. Several observations correlate increased expression of TGF-beta 1 with tumorigenesis. To evaluate the physiological relevance of increased TGF-beta 1 synthesis in tumor cells we established cell clones overexpressing TGF-beta 1 and observed the resulting physiological changes in TGF-beta overproducing cells in vitro and in vivo. As a model system we used the human E1A-transformed 293 tumor cells, which are insensitive to the direct growth modulatory effects of TGF-beta. The selection of this cell line allows an assessment of physiological alterations independent of TGF-beta induced proliferative changes. The use of two TGF-beta 1 expression vectors containing either the natural or a modified TGF-beta 1 precursor cDNA permitted the establishment of separate 293 cell lines overexpressing latent or active TGF-beta. Comparison of the resulting changes in glycolytic rate, adhesiveness and integrin and plasminogen activator expression established that, in vitro, both types of clones behaved similarly, indicating that expression of latent TGF-beta induces autocrine changes in the tumor cells and thus suggesting that some level of cell-associated activation occurs. TGF-beta overexpression resulted in an increased metabolic rate due to enhanced glycolysis, a property long associated with tumor cells. This increased glycolysis was not associated with altered proliferation. Cells overexpressing TGF-beta also displayed enhanced fibronectin mRNA and plasminogen activator synthesis and increased adhesiveness in vitro. They showed enhanced survival when plated sparsely on plastic in the absence of serum, and attached more readily to laminin. In addition, synthesis of several beta 1 integrins, in particular the alpha 1/beta 1, alpha 2/beta 1, and alpha 3/beta 1, all of which recognize laminin, were enhanced. Finally, cells overexpressing active TGF-beta, but not latent TGF-beta, also showed increased tumorigenicity in nude mice. Thus, an increase in endogenous TGF-beta synthesis confers several proliferation-independent phenotypic changes which may be of significance for the survival of the tumor cell inoculum or its subsequent growth, and for tumor formation and development. In the case of cells expressing active TGF-beta, the release of active TGF-beta into the vicinity of the tumor cells may also result in a more hospitable environment for tumor growth.     

Insulin and insulin-like growth factor I in follicular fluid after induction of ovulation in women undergoing in vitro fertilization.

This study was undertaken to evaluate the relationship between concentrations of insulin and insulin-like growth factor I (IGF-I) in follicular fluid and fertilization and cleavage of human oocytes fertilized in vitro. The concentration of oestradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, testosterone, insulin and IGF-I was determined in 36 follicular fluids, free of visible blood contamination and containing mature oocyte-corona-cumulus complexes, obtained from 12 women undergoing in vitro fertilization. Follicular development was induced by clomiphene citrate and human menopausal gonadotrophin, and follicular aspiration was performed 35 h after an ovulatory dose of human chorionic gonadotrophin. Concentrations of IGF-I were significantly higher in follicular fluids associated with mature oocytes that fertilized and cleaved, than in follicular fluid associated with mature oocytes that did not fertilize (P < 0.001). There was no difference in the concentration of insulin between follicular fluids from which fertilized oocytes were obtained and those with oocytes that remained unfertilized. No significant correlations were found between rates of embryo cleavage, concentrations of insulin and IGF-I. Multiple linear regression analysis demonstrated that the concentrations of IGF-I in follicular fluid were predicted statistically by a negative regression coefficient for the concentration of testosterone, and by a positive regression coefficient for the concentration of progesterone in follicular fluid. No candidate variable was included in the model to predict concentrations of insulin. These data suggest an important role for IGF-I in the mature follicle.     

Demonstration of the capacity of nacre to induce bone formation by human osteoblasts maintained in vitro.

Nacre implanted in vivo in bone is osteogenic suggesting that it may possess factor(s) which stimulate bone formation. The present study was undertaken to test the hypothesis that nacre can induce mineralization by human osteoblasts in vitro. Nacre chips were placed on a layer of first passage human osteoblasts. None of the chemical inducers generally required to obtain bone formation in vitro was added to the cultures. Osteoblasts proliferated and were clearly attracted by nacre chips to which they attached. Induction of mineralization appeared preferentially in bundles of osteoblasts surrounding the nacre chips. Three-dimensional nodules were formed by a dense osteoid matrix with cuboidal osteoblasts at the periphery and osteocytic-like cells in the center. These nodules contained foci with features of mineralized structures and bone-like structures, both radiodense to X-ray. Active osteoblasts (e.m.) with abundant rough endoplasmic reticulum, extrusion of collagen fibrils and budding of vesicles were observed. Matrix vesicles induced mineral deposition. Extracellular collagen fibrils appeared cross-banded and electrodense indicating mineralization. These results demonstrate that a complete sequence of bone formation is reproduced when human osteoblasts are cultured in the presence of nacre. This model provides a new approach to study the steps of osteoblastic differentiation and the mechanisms of induction of mineralization.     

Inhibition by L-arginine of the endothelin-mediated increase in cytosolic calcium in human neutrophils

The effect of endothelin (ET) on the cytosolic-free calcium [(Ca2+]i) changes in polymorphonuclear leukocytes (PMN) from normal humans and Wistar rats was investigated. ET induced a dose-related [Ca2+]i peak. This [Ca2+]i transient was blunted by TMB-8 (10(-5)M) and by Ca(2+)-free EGTA medium, therefore suggesting a role of both intracellular Ca2+ release and Ca2+ influx in the generation of the [Ca2+]i peak. Preincubation of PMN with the nitric oxide (NO)-donor L-arginine (L-Arg) markedly blocked the ET-induced [Ca2+]i transient in an enantiomerically-specific manner. A similar blunting effect of L-Arg on the fMLP (10(-7)M)-induced [Ca2+]i transient was detected. The L-Arg antagonist, NG-monomethyl-L-arginine (L-NMMA), reverted the L-Arg blocking effect on both ET- and fMLP-induced [Ca2+]i transients. These data suggest that ET has a potential role in activating Ca2+ mobilization in PMN, an effect that can be inhibited by L-Arg.     

Persistence of Cell Division Synchrony in Spirogyra Insignis (Gamophyceae): Membrane Proteoglycans Transmitting Synchronizing Information Throughout Generations

Spirogyra insignis shows a long-term persistence of cell division synchrony in the absence of the synchronizing Zeitgeber, so that at least six generations are involved in the process. This tentatively suggests that a mechanism of transmission throughout generations of synchronizing information could maintain this synchrony. Apparently, a vital part of the molecular basis of this mechanism is a membrane proteoglycan complex. This complex could obtain temporal information from a synchronizing Zeitgeber and be transmitted to the progeny by distribution of plasma membrane between daughter cells.     

Sequence of the Streptococcus pneumoniae bacteriophage HB-3 amidase reveals high homology with the major host autolysin.

We have sequenced a DNA fragment containing the pneumococcal bacteriophage HB-3 hbl gene, which codes for the phage lytic amidase. A remarkable nucleotide similarity (87.1%) between the lytA gene, coding for the pneumococcal amidase, the major autolysin of Streptococcus pneumoniae, and the hbl gene was found. This similarity completely disappeared outside the open reading frames coding for both amidases. The hbl gene transformed amidase-deficient strains of S. pneumoniae to the wild-type phenotype, and Southern blotting experiments provided evidence for recombination between donor and recipient genes. A comprehensive evaluation of these and previous results on the peptidoglycan hydrolases of S. pneumoniae and its bacteriophages suggested that recombination mechanisms participate in the evolution of the genes coding for these enzymes.     

Computer simulation of hydrodynamic properties of semiflexible macromolecules: randomly broken chains, wormlike chains, and analysis of properties of DNA

The translational and rotational diffusion coefficients and the intrinsic viscosity of semiflexible, randomly broken, and wormlike chains have been obtained by Monte Carlo simulation in the context of the rigid-body treatment. Both approximate and rigorous rigid-body hydrodynamics are used, so that the error introduced by the approximate methods can be evaluated. A randomly broken chain and a wormlike chain having the same contour length and persistence length have the same radius of gyration but different values for any of the hydrodynamic properties. The two types of chains are compared in this regard. Considering that the cross section of the chain is represented by a cylinder better than by a string of spheres, we devise a cylindrical correction to be applied to the results simulated for chains of beads. Application is made to the analysis of experimental data for the translational and rotational coefficients of DNA fragments with up to 10(3) base pairs, obtaining the persistence length for each model. The values for the wormlike chain agree well with model-independent values obtained from radii of gyration and with other literature data at varying ionic strength. The randomly broken chain is equally able to reproduce the experimental length dependence of the properties, but the resulting persistence length may be too high.     

Growth of rural and urban children in the Valley of Oaxaca,Mexico

Weight, stature, arm circumference, and the triceps skinfold were measured in 1,410 school children, 6 through 14 years of age, from two urban colonias in the city of Oaxaca de Juarez (n = 479), and from two rural Ladino (n = 467) and two rural Zapotec (n = 464) communities in the Valley of Oaxaca in southern Mexico. Children from rural Ladino communities and urban colonias are significantly taller, heavier, and more muscular than children in rural Zapotec communities. The differences between rural Ladino and urban colonia children favor the former, particularly for weight and stature. These observations thus suggest (1) that children in the rural, indigenous communities in the Valley of Oaxaca are relatively undernourished compared to children in Ladinoized and urban communities, and (2) that rural-to-urban migration does not necessarily result in improved growth status.     

Growth of rural and urban children in the valley of Oaxaca,Mexico

Weight, stature, arm circumference, and the triceps skinfold were measured in 1,410 school children, 6 through 14 years of age, from two urban colonias in the city of Oaxaca de Juarez (n = 479), and from two rural Ladino (n = 467) and two rural Zapotec (n = 464) communities in the Valley of Oaxaca in southern Mexico. Children from rural Ladino communities and urban colonias are significantly taller, heavier, and more muscular than children in rural Zapotec communities. The differences between rural Ladino and urban colonia children favor the former, particularly for weight and stature. These observations thus suggest (1) that children in the rural, indigenous communities in the Valley of Oaxaca are relatively undernourished compared to children in Ladinoized and urban communities, and (2) that rural-to-urban migration does not necessarily result in improved growth status.