A simplified method for detection of visual abnormality in rats on reproduction study: the critical intensity of illumination and the effect of dark-adaptation

A newly designed Y-shaped box was previously reported by authors to be useful for a screening test of visual abnormality in rats because of easy numerization and statistical analysis of the results. In the present paper, the relationship between the intensity of illumination and the negative phototaxic response were examined. The total selection rate for the dark area (total time of selecting dark area/total trials) of non-treated rats under 30 lux illumination was 98% and identical wit the result of those under 1600 lux. The selecting rates of non-treated rats under the illumination of 15, 10, 7.5, 5 and 1.25 lux were 95, 93, 89, 82 and 67%, respectively. On the other hand those of dark-adapted rats one hour under 15, 7.5 and 5 lux showed 98, 95 and 92%, respectively. From these results, the critical intensities of illumination-unaffected selection rate for darkness in non-treated and dark-adapted rats were estimated at 30 and 15 lux, respectively. There was an obvious effect of dark-adaptation on the visual ability of rats. The selection rate of eyelid-sutured rats under 30 lux was 52%, an approximate theoretical value of true blindness, but it was 85% under 1600 lux. It is suggested that a more exact detection of visual abnormality would be possible under the critical intensity of illumination-unaffected selection rate for darkness.     

Formation and removal of benzo[a]pyrene metabolites--DNA adducts in cultured normal human fibroblasts using a microsome-activating system

The rate of removal of DNA adducts of several benzo[a]pyrene metabolites from nuclear DNA was compared by introducing a microsome-activating system in human fibroblast cells. Confluent human fibroblasts were exposed to benzo[a]pyrene in the presence of a microsomal activating system and DNA adducts were formed in the nuclear DNA. The adducts present in DNA were determined after 1 h of incubation and 48 h later. There was no difference in the rate of removal between 7S- and 7R -N2-[10-(7 beta, 8 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene)yl]deoxyguanosine, 7R -N2-[10(7beta, 8 alpha, 9 beta-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]deoxyguanosine and the covalent adduct of 9-hydroxybenzo[a]pyrene-4,5-epoxide to guanosine. This finding does not agree with the idea that metabolites forming 'persistent DNA adducts' are always responsible for the carcinogenicity of their parent compound.     

Development of human pancreas

The developmental sequence of human pancreatic secretory proteins has not previously been studied in detail. We applied immunohistochemistry to study 20 fetal and neonatal pancreas' (8th to 39th gestational weeks) using antisera against the following pancreatic secretory proteins: pancreatic secretory trypsin inhibitor (PSTI), serine proteinases (trypsin, chymotrypsin, and elastase I), and amylase. PSTI was first detected in developing buds of the pancreas during the 8th gestational week, and proteinases were observed in acinar cells during the 14th week of gestation. Immunoreactivity for both PSTI and proteinases was found in most acinar cells soon after their appearance. Immunoreactivity for amylase could not be detected in fetal or neonatal pancreas tissue. PSTI was also found in developing islets during the 14th gestational week, but the number of immunoreactive cells had decreased by term. Cells positive for serine proteinases were occasionally in contact with islets in second-trimester fetuses. In discussing these results, we give particular attention to the nonparallel appearance of secretory products in the fetal pancreas, and the significance of cells immunoreactive for secretory proteins in endocrine islets.     

Freeze-fracture studies of the sinoatrial and atrioventricular nodes of the caprine heart,with special reference to the nexus

Summary The caprine sinoatrial node (SAN) and atrioventricular node (AVN) were studied by freeze-fracture techniques, and their nexus or gap junction structure were compared with that of ordinary atrial and ventricular muscle cells. The general features of the nexus in both the SAN and AVN were essentially identical. Approximately two-thirds of the nexuses observed in the nodal cells consisted of typical macular arrangements of nexal particles, and the remaining third, of atypical configurations of either circular arrangements or linear arrays of particles in continuity with the macular nexuses. Such atypical nexuses were never observed in the ordinary adult myocardial cells. Quantitative analysis revealed that all of the nexuses in the nodal cells measured, were less than 0.1 m², whereas the majority of the nexuses in ordinary myocardial cells (64% in the atrium and 76% in the ventricle) were larger than 0.1 m². No significant differences in diameter and center-to-center distance of nexal particle were found between the nodal cells and ordinary myocardial cells.     

A new class of site-specific endodeoxyribonucleases. Endo.Sce I isolated from a eukaryote, Saccharomyces cerevisiae

We had found that yeasts had intracellular endodeoxyribonucleases that cut phage DNA into a set of double-stranded fragments with discrete chain lengths. We purified one of them to apparent homogeneity from Saccharomyces cerevisiae and designated it Endo.Sce I. Sequence analysis around 5 cleavage sites in plasmid DNA and phage DNA revealed that Endo.Sce I cuts a defined phosphodiester bond in each strand of double helix at the cleavage sites and produces free cohesive ends consisting of 4 nucleotides protruding at 3'-termini. However, unlike in the case of prokaryotic type II-restriction endonucleases, (i) Endo.Sce I seems to consist of two nonidentical subunits, (ii) no common palindrome or consensus sequence including more than 5 base pairs is detected at or near these cleavage sites, and (iii) Endo.Sce I can cut the DNA isolated from the cells that produced Endo.Sce I. All of the 5 cleavage sites are included in inverted repeats, but these inverted repeats are variable in size, nucleotide sequence, and distance between repeating units. An inverted repeat itself is not a structure recognized by Endo.Sce I. This study shows that Endo.Sce I is the first example of eukaryotic site-specific endonuclease and has properties, as described above, which distinguish it from prokaryotic restriction endonucleases.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Active Oxygen Contributes to the Major Part of Chromosomal Aberrations in V79 Chinese Hamster Cells Exposed to N-Hydroxy-2-Naphthylamine

N-hydroxy-2-naphthylamine (NOH-2NA). an active metabolite of human occupational bladder carcinogens, induced, in V79 Chinese hamster cells. chromosomal aberrations which were suppressed in the presence of catalase and/or superoxide dismutase. The induction of the aberrations was more efficient in a more basic pH in parallel with the generation of hydrogen peroxide from NOH-2NA. The possible role of the oxidation product of NOH-2NA in the induction of the aberrations is discussed.     

Construction of a chimeric series of Bacillus cyclomaltodextrin glucanotransferases and analysis of the thermal stabilities and pH optima of the enzymes

The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from the alkalophilic Bacillus sp. strain no. 17-1 was cloned in Escherichia coli. The cloned CGTase gene consisted of a single open reading frame which would encode a polypeptide of 713 amino acids, and the first 27 amino acid residues comprised a signal peptide. The nucleotide sequence and the amino acid sequence of this CGTase (CGTase 17-1) gene had strong homology with those of the CGTase (CGTase 38-2) gene previously cloned in our laboratory from the alkalophilic Bacillus sp. strain no. 38-2, although the enzymic properties of the CGTase 17-1 were distinct from those of the CGTase 38-2. To analyse those enzymic properties further, we constructed 12 chimeric CGTases using three restriction nuclease sites and compared the enzymic properties of the chimeric CGTases. The N-terminal part of the enzyme was important for heat stability, and the pH-activity profile was influenced by both the N- and the C-terminal parts. A third segment was less important for enzymic properties.