ASCORBIC ACID DEFICIENCY IN CULTURED HUMAN FIBROBLASTS

Fibroblasts grown in medium containing less than 1 µg of ascorbic acid per milliliter showed evidence of ascorbic acid deficiency when compared with cells grown in medium containing 50 µg of ascorbic acid per milliliter. This was manifested morphologically by dilated endoplasmic reticulum, a decrease in number, size, and intensity of staining of the mitochondria, by defective intercellular fibril formation, and by easy disaggregation of the cells from the intercellular matrix after treatment with pronase. When 50 µg per milliliter of ascorbic acid was incorporated into the medium, the altered morphology was corrected, banded fibrils were produced which were organized into bundles, and the cells were tightly bound in a matrix which was resistant to disaggregation with a variety of proteolytic enzymes. Collagen and sulfated glycosaminoglycan synthesis were less in the control than in the ascorbic acid supplemented cells. Similar morphological and chemical changes have been reported in the connective tissue of scorbutic animals. The effects of low ascorbic acid concentration on fibroblasts in culture indicate that these cells require ascorbic acid to maintain connective tissue functions.     

Heterotrophic nitrification and aerobic denitrification inAlcaligenes faecalis strain TUD

Heterotrophic nitrification and aerobic and anaerobic denitrification byAlcaligenes faecalis strain TUD were studied in continuous cultures under various environmental conditions. Both nitrification and denitrification activities increased with the dilution rate. At dissolved oxygen concentrations above 46% air saturation, hydroxylamine, nitrite and nitrate accumulated, indicating that both the nitrification and denitrification were less efficient. The overall nitrification activity was, however, essentially unaffected by the oxygen concentration. The nitrification rate increased with increasing ammonia concentration, but was lower in the presence of nitrate or nitrite. When present, hydroxylamine, was nitrified preferentially. Relatively low concentrations of acetate caused substrate inhibition (K_I=109 M acetate). Denitrifying or assimilatory nitrate reductases were not detected, and the copper nitrite reductase, rather than cytochrome cd, was present. Thiosulphate (a potential inhibitor of heterotrophic nitrification) was oxidized byA. faecalis strain TUD, with a maximum oxygen uptake rate of 140–170nmol O₂·min^-1·mg prot^-1. Comparison of the behaviour ofA. faecalis TUD with that of other bacteria capable of heterotrophic nitrification and aerobic denitrification established that the response of these organisms to environmental parameters is not uniform. Similarities were found in their responses to dissolved oxygen concentrations, growth rate and ammonia concentration. However, they differed in their responses to externally supplied nitrite and nitrate.     

Differentiation inhibiting activity (DIA/LIF) and mouse development.

Analysis of the differentiation in culture of murine embryonic stem (ES) cells has resulted in the identification and characterization of the regulatory factor differentiation inhibiting activity (DIA). DIA specifically suppresses differentiation of the pluripotential ES cells without compromise of their developmental potential. DIA is identical to the pleiotropic cytokine leukaemia inhibitory factor (LIF) which has a broad range of biological activities in vitro and in vivo. It is produced in both diffusible and matrix-localised forms whose expression is differentially regulated. The compartmentalization of DIA/LIF and the modulation of its expression during stem cell differentiation and by other cytokines may be significant elements in the control of early embryo development. These features may also indicate general principles of the regulatory networks which govern stem cell renewal and differentiation in later development.     

Identification and characterization of a novel high-molecular-weight form of the integrin alpha 3 subunit.

The integrin alpha 3 beta 1 is a multiligand extracellular matrix receptor found on many cell types. Immunoprecipitations of 125I-surface-labeled prostate carcinoma cell lines, DU145 and PC-3, with the anti-alpha 3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected alpha 3 beta 1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa alpha 3 and 115-kDa beta 1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti-alpha 3 antibody-Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa alpha 3 subunit generated 130- and 25-kDa polypeptides. The 225-kDa protein did not generate a 25-kDa polypeptide. Deglycosylation and reduction of the 225-kDa protein resulted in the generation of 110- and 95-kDa polypeptides, while deglycosylation and reduction of the 155-kDa alpha 3 resulted in a 110-kDa polypeptide identical in size to the 110-kDa polypeptide generated from the 225-kDa protein. Peptide maps generated from the 110-kDa components of the 225-kDa polypeptide and the 155-kDa alpha 3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the alpha 3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa alpha 3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human alpha 3 cDNA probe identified an mRNA species of 6.2 kb in addition to a major mRNA species of 4.3 kb. The larger mRNA species, which is of an appropriate size for encoding a polypeptide of approximately 220-kDa, was not detectable in cells which did not express the 225-kDa protein. These data demonstrate that the 225-kDa polypeptide represents a novel integrin alpha 3 subunit consisting of the alpha 3 integrin heavy chain disulfide-bonded to a 95-kDa polypeptide which may represent an alternative "light" chain to the 25-kDa light chain of the alpha 3 subunit.     

Identification and characterization of the familial adenomatous polyposis coli gene.

DNA from 61 unrelated patients with adenomatous polyposis coli (APC) was examined for mutations in three genes (DP1, SRP19, and DP2.5) located within a 100 kb region deleted in two of the patients. The intron-exon boundary sequences were defined for each of these genes, and single-strand conformation polymorphism analysis of exons from DP2.5 identified four mutations specific to APC patients. Each of two aberrant alleles contained a base substitution changing an amino acid to a stop codon in the predicted peptide; the other mutations were small deletions leading to frameshifts. Analysis of DNA from parents of one of these patients showed that his 2 bp deletion is a new mutation; furthermore, the mutation was transmitted to two of his children. These data have established that DP2.5 is the APC gene.     

Effect of temperature on the toxicities of ten pyrethroids to German cockroach (Dictyoptera: Blattellidae).

Of 10 pyrethroids tested by topical application of male German cockroaches, Blattella germanica (L.), d-phenothrin was the least toxic at 19, 26, or 31 degrees C. lambda-cyhalothrin was most toxic. Pyrethroids with the alpha-cyano moiety (fluvalinate, fenvalerate, cypermethrin, esfenvalerate, tralomethrin, cyfluthrin, and lambda-cyhalothrin) were more toxic than those without this functional group (d-phenothrin, resmethrin, and permethrin). At LC50, toxicity was negatively related to temperature. Temperature-toxicity responses of five of seven alpha-cyano pyrethroids were parallel, possibly indicating qualitatively identical but quantitatively different levels of detoxification enzymes.