Influence of culture method on the proliferation and osteogenic differentiation of human adipo-stromal cells in nonwoven fabrics

The initial attachment, proliferation, and osteogenic differentiation of stromal cells from human fat tissue were investigated in three-dimensional nonwoven fabrics prepared from polyethylene terephthalate (PET) fiber with different diameters. The largest number of cells initially attached was observed in the nonwoven fabrics prepared from PET fiber with a diameter of 22.0 microm, irrespective of fabric porosity. The number of cells attached was larger and the cells were distributed more homogeneously in the fabrics by the agitated seeding method than by the static seeding method. The culture method depended on the time profile of cell proliferation. Cell proliferation improved in the following order: stirred (spinner flask) culture method > agitated culture method > static culture method. In addition, cells proliferated homogeneously in fabrics by the stirred culture method. When evaluated as a measurement of cell osteogenic differentiation, the activity of alkaline phosphatase (ALP) was not influenced by the diameter of fabrics. The static culture method tended to enable cells to enhance ALP activity, in contrast with the stirred and agitated culture methods. It is concluded that fabric fiber diameter and culture method greatly affected the proliferation and differentiation of cells in nonwoven fabrics.     

Conjugation of acetaldehyde with cysteinylglycine,the first metabolite in glutathione breakdown byγ-glutamyltranspeptidase

Glutathione and its metabolites were examined for reactivity to acetaldehyde. When acetaldehyde was incubated with glutathione alone, there was only a slight decrease of acetaldehyde, while an apparently equimolar reaction between acetaldehyde and free sulfhydryl was observed with the addition of -glutamyltranspeptidase. Cysteinylglycine, the first metabolite in the glutathione breakdown by -glutamyltranspeptidase, showed a rapid and equimolar reactivity to acetaldehyde and such was comparable to the reaction seen withl-cysteine ord-penicillamine. In light of the chemical structure, cysteinylglycine probably conjugates with acetaldehyde to form thiazolidinecarboxylic acid derivatives, 2-methyl-thiazolidine-4-carbonyl-glycine, and if so, the alteration of glutathione metabolism by acetaldehyde during ethanol intoxication warrants further attention.     

Relationship between muscle fatigue and oxygen uptake during cycle ergometer exercise with different ramp slope increments

Summary The surface electromyogram (EMG) from active muscle and oxygen uptake ( ) were studied simultaneously to examine changes of motor unit (MU) activity during exercise tests with different ramp increments. Six male subjects performed four exhausting cycle exercises with different ramp slopes of 10, 20, 30 and 40 W · min^–1 on different days. The EMG signals taken from the vastus lateralis muscle were stored on a digital data recorder and converted to obtain the integrated EMG (iEMG). The was measured, with 20-s intervals, by the mixing chamber method. A non-linear increase in iEMG against work load was observed for each exercise in all subjects. The break point of the linear relationship of iEMG was determined by the crossing point of the two regression lines (iEMG_bp). Significant differences were obtained in the exercise intensities corresponding to maximal oxygen uptake ( ) and the iEMG_bp between 10 and 30, and 10 and 40 W · min –1 ramp exercises (P < 0.05). However, no significant differences were obtained in and corresponding to the iEMG_bp during the four ramp exercises. With respect to the relationship between and exercise intensity during the ramp increments, the -exercise intensity slope showed significant differences only for the upper half (i.e. above iEMG_bp). These results demonstrated that the and at which a nonlinear increase in iEMG was observed were not varied by the change of ramp slopes but by the exercise intensity corresponding to and the iEMG_bp was varied by the change of ramp slopes. In addition, the significant differences in the exercise intensity slopes for the upper half of the tests would suggest that the recruitment patterns of MU and/or muscle metabolic state might be considerably altered depending upon the ramp slope increments.     

J chain synthesis in human myeloma cells: light and electron microscopic studies

The presence of J chain in human immunocytes containing various immunoglobulin classes has already been reported and in this paper we investigated the presence of J chain in human myeloma cells using light and electron microscopy. Myeloma cells were positive for cytoplasmic J chain in thirteen cases: six of fifteen IgG myeloma, two of seven IgA myeloma, two of nine B-J myeloma and three of three primary macroglobulinaemia. None of the four with benign monoclonal gammopathy was positive for J chain. The ultrastructural localization of J chain in IgG myeloma cells was similar to that of gamma chain, i.e. around the nuclear membrane, in the endoplasmic reticulum (ER) and in the Golgi apparatus. The latter finding is compatible with the presence of J chain in the urine of the same patient. We examined the urine from nine patients with J chain-positive cells and secretion of J chain was confirmed in three patients: two with IgG myeloma and one with B-J myeloma. In noe of twelve in whom the immunocytochemical findings were negative for J chain was there evidence of urinary J chain.     

Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.     

A comparative study of gut‐associated lymphoid tissue in calf and chicken

The calf contains two types of Peyer's patches (PPs): jejunal and ileal. The ileal PP has been thought to be equivalent to the bursa of Fabricius (BF) as a central lymphoid organ. The morphologies of ileal and jejunal PPs in the calf were compared with those of the BF and the caecal tonsil (CT) in the chicken. Immunoglobulin G–positive (IgG⁺) cells appear in the follicles of them all and exhibited a dendritic appearance after birth. We investigated whether the IgG in these follicles was produced in situ. IgG‐producing cells were detected in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. CD4⁺ cells were distributed in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. The data suggest that Ig class switching occurs in both jejunal PP follicles and CT follicles, but does not occur in either the ileal PP follicles or the bursal follicles. Because CD4⁺ T cells would be prerequisite for Ig class switching in these follicles, IgG⁺ cells of the follicular medullas in the ileal PP and the BF would trap immune complexes from the gut lumen. The primary B‐cell repertoire might be selected by gut‐derived antigens in the ileal PP and the BF before seeding the periphery. Anat Rec 266:207–217, 2002. © 2002 Wiley‐Liss, Inc.     

CD44 stimulation down-regulates Fas expression and Fas-mediated apoptosis of lung cancer cells

Cytotoxic T lymphocytes (CTL) play a major role in the rejection of tumor cells, but tumor rejection does not always occur in vivo, indicating that defects in anti-tumor immune responses may be common. We here document a novel function for CD44--using lung cancer cells, we showed that stimulation of CD44 reduced Fas expression and Fas-mediated apoptosis: (i) lung cancer cells expressed high levels of CD44; (ii) engagement of CD44 on the cells by a specific antibody or fragmented hyaluronan reduced Fas expression; (iii) CD44 cross-linking reduced Fas-mediated apoptosis; (iv) stimulation of CD44 on lung cancer cells decreased IFN-gamma production by autologous CTL; and (v) CD44 stimulation prevented killing of lung cancer cells by autologous CTL. Based on these findings, we postulate a new concept--that interaction of CD44 on lung cancer cells with fragments of extracellular hyaluronan present in the surrounding extracellular matrix reduces Fas expression as well as Fas-mediated apoptosis of cancer cells. This leads to reduced susceptibility of the cells to CTL-mediated cytotoxicity through the Fas-Fas ligand pathway.     

Rapid screening of point mutations of the Neisseria gonorrhoeae parC gene associated with resistance to quinolones.

To detect quinolone resistance-associated mutations within the Asp-86, Ser-87, Ser-88, and Glu-91 codons of the Neisseria gonorrhoeae parC gene, we developed a rapid and simple assay based on amplification of the regions of the parC gene containing the mutations sites by PCR and digestion of the PCR products with restriction enzymes. By using the method of primer-specified restriction site modification, artificial SalI, PstI, EcoRI, and HinfI restriction sites were created in the regions containing the Asp-86, Ser-87, Ser-88, and Glu-91 codons, respectively. The mutations generating alterations at Asp-86, Ser-87, Ser-88, and Glu-91 were detected as failures of SalI, PstI, EcoRI, and HinfI to digest the respective PCR products. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the parC gene by this assay. Appropriate mutations at either the Asp-86, Ser-87, Ser-88, or Glu-91 codon were detected in each of 11 strains in which a mutation had previously been observed by DNA sequencing. This rapid and simple assay could be a useful device for screening genetic alterations in the parC gene associated with resistance to quinolones in N. gonorrhoeae.     

Comparison among performances of a ligase chain reaction-based assay and two enzyme immunoassays in detecting Chlamydia trachomatis in urine specimens from men with nongonococcal urethritis.

We evaluated the performances of a ligase chain reaction (LCR)-based assay and two enzyme immunoassays (Chlamydiazyme and IDEIA) in the detection of Chlamydia trachomatis in urine specimens. We compared the results of testing urine specimens by these assays with those of urethral swab culture by examining samples from 131 men with nongonococcal urethritis. Discrepant results were analyzed by testing urethral swab specimens for C. trachomatis by a PCR-based assay. After the resolution of discrepant results, the sensitivity of urethral swab culture was 85.3%, whereas those of the LCR assay, Chlamydiazyme, and IDEIA with urine specimens were 94.1, 82.4, and 94.1%, respectively. The LCR assay and IDEIA were more sensitive than was urethral swab culture. In addition, the LCR assay, with a sensitivity equal to that of IDEIA, was more specific. Overall, the LCR assay proved to be superior to the enzyme immunoassays in detecting C. trachomatis in urine specimens. Testing urine specimens by LCR assay should be a helpful alternative method for diagnosing C. trachomatis urethral infection in men with nongonococcal urethritis.     

The effect of immunoglobulin G1 structure on macrophage binding to supported planar lipid monolayers.

We have studied the antibody-dependent binding of macrophages to supported planar lipid monolayers containing haptenated phospholipids (Tnp-Cap-DPPE). Eight monoclonal anti-TNP IgG1s, which had similar affinities to the TNP residues in solution and in the membranes, were used in the experiment. The results showed that mouse macrophages (P388D1 and J774.1) bound with different affinities to these IgG1-coated lipid monolayers. The monoclonal antibody shown to be deficient in macrophage binding was also relatively ineffective in activating complement. These results indicated that individual monoclonal antibodies of a given subclass may prove deficient in terms of the biological activities associated with the group as a whole.