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The proteases of human leukocytes were cytochemically studied by use of new a-naphthyl esters, tosyl-l-lysine-a-naphthyl ester (TLNE) and acetyl-l-tyrosine-a-naphthyl ester (ATNE). The hydrolytic activities were strong only in neutrophils, with both substrates. They were inhibited completely by DFP and chymostatin, but not by leupeptin and iodoacetate. These results indicate that chymotrypsin-like enzyme(s), capable of hydrolyzing both substrates, exist in neutrophils.

Somatic cell hybrids were obtained by cell fusions between Lck-positive EL4 mouse T lymphoma cells and Lck-negative B82 mouse fibroblasts or S194 mouse plasmacytoma cells to examine negative control of lck gene expression in the resulting hybrids. Western blot analysis using a monoclonal antibody against the Lck protein showed a marked decrease in p56lck expression in B82 × EL4 (BEL) hybrids. In contrast to BEL hybrids, the level of p56lck was not changed significantly in S194 × EL4 (SEL) hybrids and was approximately one-half of that seen in EL4 cells. Diminished expression of the Lck protein in BEL hybrids paralleled downregulation of lck mRNA, which was exclusively transcribed from the distal promoter in EL4 cells. It is unlikely that the suppression was simply a consequence of chromosome segregation critical for lck gene expression, since BEL hybrids retained the EL4-derived lck gene and most of the chromosomes from both parental cells. The results from treatment of BEL hybrids with actinomycin D or cycloheximide suggested that suppression of lck gene expression in the hybrids might not be due to posttranscriptional control. DNA methylation status in the lck distal promoter and the coding regions did not appear to correlate with the expression of the gene. Our results suggest that negative control of lck gene expression differs between fibroblasts and B cells, in that lck gene expression in T cells can be shut down by transfer of a putative repressor factor or factors in fibroblasts but not in B cells.

The first intron (intron I) of the human factor IX gene, which has been previously suggested of having an expression-augmenting activity, was systematically studied for its potential enhancer activity. When tested with the chloramphenicol acetyltransferase expression vector with a minimal factor IX promoter, subregions of intron I showed only marginal enhancing activities (1.7-1.9-fold enhancement at the highest). Smaller subregions encompassing nucleotides 5660-6350 of the intron sequence even showed some weak negative regulatory activities (approximately 50% suppression at the highest), while a cytomegalovirus enhancer sequence, which was used as the positive control, had a 7-fold enhancement. A set of three factor IX minigene expression vectors with the same factor IX promoter were then constructed: p-416FIXc which contained the factor IX cDNA, p-416FIXm1 which contained the factor IX cDNA with a largely truncated intron I, and p-416FIXm2 which contained the factor IX cDNA with the intron I sequence further truncated. The p-416FIXm1 and p-416FIXm2 constructs showed 7-9-fold higher expression activities than p-416FIXc. The elevated factor IX antigen levels agreed well with the grossly elevated factor IX clotting activity and mRNA levels. These results indicate that the expression enhancing activity of intron I is not due to specific enhancer elements present in the intron subsequences, but is due to functional splicing sequences present in the precursor mRNAs produced from the minigene constructs containing intron I. By being efficiently assembled into spliceosome complexes, transcripts with splicing sequences may be better protected in the nucleus from random degradations than those without such sequences.

Laminin-rich extracellular matrix, EHS-gel, has been demonstrated to keep a high level of liver-specific gene expression in cultured rat hepatocytes. To obtain information about the effect of EHS-gel on liver-specific functions, gene expression of liver-enriched transcription factors in rat hepatocytes was investigated. The apolipoprotein A-I and albumin mRNA levels were higher in hepatocytes cultured on EHS-gel than in those cultured on type I collagen (TIC). The levels of mRNA for HNF-4, C/EBPα and C/EBPβ were also higher on EHS-gel than on TIC. The level of HNF-4 mRNA in hepatocytes on EHS-gel was almost comparable to that in liver. The HNF-3α mRNA level was lower on EHS-gel than on TIC. C/EBPβ mRNA was induced by dexamethasone in both EHS-gel and TIC. The induction of C/EBPα and HNF-4 by dexamethasone was observed only on TIC. These data suggest that EHS-gel leads hepatocytes to keep the phenotypic expression through high expression of liver-enriched transcription factors, such as HNF-4.

To investigate the presence of a negative regulatory factor(s) suppressing T-cell receptor alpha-chain (TCR alpha) gene expression in non-T cells, 10 independent cell hybrid clones were generated between mouse T-cell lymphoma EL4 cells (TCR alpha+/beta+) and mouse fibroblast B82 cells. These cell hybrids showed a typical fibroblastic morphology and retained an approximate sum of chromosome numbers derived from both parental cells. No transcripts of the TCR alpha gene were detected in the cell hybrids, although the presence of the rearranged TCR alpha allele from EL4 cells was confirmed. The possibility of involvement of nuclear proteins responsible for the activity of the TCR alpha gene enhancer in the extinction of TCR alpha gene expression in the cell hybrids was examined. Nuclear proteins which bind to the lymphoid enhancer-binding factor 1 (LEF-1) binding motif present in EL4 cells disappeared in the hybrid clones, whereas no significant change was observed in DNA-binding activity of nuclear proteins to a consensus cyclic AMP response element (CRE) and the Ets-1 binding motif between the parental cells and the cell hybrids. No transcripts of the LEF-1 gene were detected in the cell hybrids, despite the retention of the LEF-1 gene and murine chromosomes 3, on which the LEF-1 allele is located, from both parental cells. These results suggest that a trans-acting negative regulatory factor(s) present in fibroblasts suppresses LEF-1 gene expression and that suppression of LEF-1 may lead to the extinction of TCR alpha gene expression in the cell hybrids.

d-Mannono-1,4-lactone was efficiently converted into l-ribose in eight steps. A key step of this synthesis is the cyclization of a γ-hydroxyalkoxamate under Mitsunobu conditions. It is noteworthy that the O-alkylation product was obtained in 94% yield and that none of the N-alkylation product was detected in this cyclization.

For the first time, we report highly stable CdS quantum dots sensitized TiO2sandwich type photoelectrochemical solar cells. A unique combination of a sensitized electrode and a polysulfide electrolyte has provided the most stable solar cell with the highest IPCE of approximately 70%.