Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells.

INTRODUCTION: In situ hybridization of whole mouse fetuses and their tibias with a stem cell growth factor (SCGF) antisense probe demonstrated specific expression of SCGF mRNA around skeletal tissues, particularly in bone marrow cells, proliferating chondrocytes, the perichondrium and periosteum, but little expression in resting or hypertrophic chondrocytes. METHODS: Recombinant human (rh) SCGF-alpha was purified from a conditioned medium of SCGF-alpha gene-transfected CHO cells. The molecular mass of rhSCGF-alpha, 45 kDa, was shifted down to 40 kDa by digestion with endo-O-glycosidase and sialidase, suggesting O-glycosylation of rhSCGF-alpha with sialic acids. RESULTS: For human bone marrow CD34+Lin- cells, rhSCGF-alpha alone did not stimulate colony-formation, but small cluster-formation (10.3 +/- 2.5/1 x 10(3) CD34+Lin- cells). It promoted growth of erythroid and granulocyte/macrophage (GM) colonies in the primary culture with erythropoietin and GM colony-stimulating factor (CSF) or G-CSF, respectively, and further supported GM progenitor cells in a short-term liquid culture. In contrast, rhSCGF-alpha suppressed stem cell factor (SCF)-stimulated erythroid bursts, indicating some competitive interaction between SCGF and SCF. rhSCGF-alpha was synergistic with interleukin-3 and the flt3 ligand to enhance GM colony-growth, but not synergistic with those inducing ex vivo expansion of GM progenitor cells. CONCLUSION: SCGF is selectively produced by osseous and hematopoietic stromal cells, and can mediate their proliferative activity on primitive hematopoietic progenitor cells.

Appearance of poor-fermenting variants in brewing yeast culture.

A class of yeast variants appears after cultivation of a bottom-fermenting brewing yeast strain, IFO2003. Although IFO2003 fails to grow well above 33 degrees C, the variants can grow up to 34 degrees C. Temperature-resistance and an acquired phenotype of maltose poor-fermentation ability are strictly correlated in the bottom-fermenting brewing yeast, enabling us to develop easy estimation of the fermentation ability of the variants.

Identification of a glutamic acid and an aspartic acid residue essential for catalytic activity of aspergillopepsin II, a non-pepsin type acid proteinase.

Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive acid proteinase. To identify the catalytic residues of the enzyme, all acidic residues that are conserved in the homologous proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.

Simultaneous transport and metabolism of nicotinic acid derivatives in hairless mouse skin.

In vitro simultaneous transport and metabolism of three ester prodrugs of nicotinic acid (NA), methyl nicotinate (MN), ethyl nicotinate (EN) and butyl nicotinate (BN) were studied using excised skin from hairless mouse. Hydrolysis studies of these esters with and without skin homogenate were also done at 37 degrees C. Both the ester and NA were detected in all receiver solutions in permeation studies, and no chemical hydrolysis of the esters was found, indicating that the esters were hydrolyzed during the skin permeation process. The total (ester+NA) flux from a saturated solution of ester prodrugs was higher than that of NA and was highest for MN, followed by EN and BN, whereas the total permeability coefficient of ester prodrugs increased from MN to BN. A difference in the NA/total flux ratio was found among these prodrugs; thus, esterase activity was also dependent on the alkyl chain length of the esters. The total flux from each ester solution increased linearly with the donor concentration. NA flux from MN and EN solutions increased with an increase in the donor concentration and reached a plateau at the high concentration range, suggesting that metabolic saturation occurred. NA fluxes at the plateau were similar among ester prodrugs and corresponded to the Vmax estimated from the hydrolysis experiment. The order of donor concentration at which NA reached a plateau also corresponded to the order of Km. It was confirmed that a difference in alkyl chain length of the ester prodrugs affected not only permeability but also metabolism in the skin permeation process.

Isolation and characterization of a cDNA for human mouse, and rat full-length stem cell growth factor, a new member of C-type lectin superfamily.

cDNA encoding stem cell growth factor (SCGF; 245 aa), a novel human growth factor for primitive hematopoietic progenitor cells, has been previously reported (Hiraoka, A., Sugimura, A., Seki, T., Nagasawa, T., Ohta, N., Shimonishi, M., Hagiya, M. and Shimizu, S. Proc. Natl. Acad. Sci. USA 94, 7577-7582, 1997). Here we report the cloning and characterization of a full-length SCGF cDNA. This protein consists of 323, 328 and 328 aa in the human, murine and rat forms, the latter two of which share 85.1% and 83.3% aa identity, and 90.4% and 90.4% aa similarity to the human protein, respectively. Because the newly identified human clone encodes the protein longer by 78 aa than that previously identified, we term the longer clone as hSCGF-alpha and the shorter one as hSCGF-beta. The computer-assisted homology search reveals that SCGF is a new member of the C-type lectin superfamily, and that SCGF shows the greatest homology to tetranectin among the members of the family (27.2-33.7% aa identity and 46.0-53.6% aa similarity). SCGF transcripts are detected in spleen, thymus, appendix, bone marrow and fetal liver. Fluorescent in situ hybridization mapping indicates that the SCGF gene is located on chromosome 19 at position q13.3 for human form and on chromosome 7 at position B3-B5 for murine form, which are close to flk-2/flt3 ligand and interleukin-11 genes of both human and murine species.

Self-reported oral hygiene habits, health knowledge, and sources of oral health information in a group of Japanese junior high school students.

The purpose of the present study was to analyze self-reported oral hygiene habits, sources of oral health information, and oral health knowledge in a group of Japanese junior high school students and to determine whether there is a need for improvement in the school's present oral health instruction. A sample of 110 students aged 12-14 in Chiba City were surveyed by means of a questionnaire composed of 24 multiple choice questions. The questions focused on: (1) experience of school-based oral health education, (2) sources of oral health information, (3) knowledge about dental caries, periodontal disease, and the preventive action of fluoride, (4) oral hygiene habits, and (5) dietary behavior. Results showed that more than two-thirds of the students had participated in some kind of school-based oral health education program. Most students (76%) claimed that toothbrushing was the main event attended and 63% claimed that toothbrushing was the main subject they had been taught. The school nurse was identified by 48% of the students as their main source of oral health information in school-based oral health education. When asked to identify their main source of oral health information, most of the students identified "school". Half of the students (48%) identified dental plaque as the main cause of dental caries but only 31%, as the main cause of periodontal disease. Few students (11%) were able to identify the preventive action of fluoride; 58% answered "I don't know"). These results suggest that a meaningful target for the oral health education of children should be the improvement of the school's oral health instruction.