Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA.

T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.     

Alpha-interferon in Kikuchi’s disease

In Japan, histiocytic necrotizing lymphadenitis (Kikuchi’s disease) is a relatively common reactive lesion affecting lymph nodes, but the histogenesis and pathogenesis of the disease have not been clarified. Alpha-interferon has a role in the body’s defense against, viral infections. Using a polyclonal antibody to human alpha-interferon, we found numerous cells, mainly histiocytes, containing alpha-interferon in affected foci in the lymph nodes from 24 patients with Kikuchi’s disease. Tubuloreticular structures, thought by some authors to be associated with the production of interferon, were detected by electron microscopy in histiocytes, activated lymphocytes and vascular endothelial cells in the affected foci. These results suggested that the formation of tubuloreticular structures is a secondary phenomenon following stimulation by alpha-interferon. Further, the activity of 2′–5′ oligoadenylate synthetase, which is induced by alpha-interferon and enhanced during the early or active stage of viral infection, showed increased levels of activity in the active stage of Kikuchi’s disease and decreased to normal levels in the convalescent stage 2 weeks later. These results suggested the possibility of a viral etiology for Kikuchi’s disease.     

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

Phylogeny of gymnosperms inferred fromrbcL gene sequences

Partial nucleotide sequences of the large subunit of ribulose-1,5-bisphosphate carboxylase (rubisco) gene (1333 base pairs: about 90% of the gene) from several seed plants were determined. Phylogenetic trees based on amino acid sequences were inferred by using the neighbor joining and maximum likelihood methods. The results indicate (1) monophyly of gnetum group (Ephedra, Gnetum, Welwitschia), (2) monophyly of extant gymnosperms containing gnetum group, which contradicts the results of morphological data.     

Diploid and triploid offspring of triploid agamosporous fernDryopteris pacifica

A cytological and reproductive study of the diploid and triploid agamosporousDryopteris pacifica was made to elucidate the origin of its infraspecific cytotypes. Some triploids produced 16 spore mother cells (SMCs) sometimes with n=41II+41I chromosomes, in addition to eight SMCs with n=123II, in each sporangium. In the former case the 16 SMCs usually underwent abnormal meiosis to give rise to some 50 spores, some of which were regular-shaped; in the latter the eight SMCs multiplied into 32 spores by normal meiosis. We found that spores from one of the triploid plants developed into either diploid or triploid gametophytes, which further apogamously produced diploid or triploid sporophytes, respectively. This novel mechanism of ploidy reduction is discussed in relation to the origin of diploid agamosporous ferns, the taxonomic complexity of the species, and the correlation of agamospory with polyploidy. The mechanism is also compared to that operating in agamospermous angiosperms.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

External characteristics and associated developmental changes in two species of Sulawesi macaques,Macaca tonkeana andM. hecki,with special reference to hybrids and the borderland between the species

The degree of intergradation between two species of Sulawesi macaques,Macaca tonkeana andM. hecki, was studied by examining the diagnostic external characteristics of more than 100 monkeys kept as pets by natives. Two possible hybrid monkeys were found and both originated from the borderland between the two species, located in the most proximal region of the northern peninsula of Sulawesi. The previously postulated wide area of integradation between the two species at the possible contact zone was, however, not recognized, and typical examples oftonkeana orhecki were found to be present on the two sides of a narrow “hybrid” zone which was defined by direct observations. Furthermore, despite considerable individual variations, we were able to allocate most monkeys to one or other of the species. Each of ten external characteristics of the members of both species more or less encompassed the individual variations, but may undergo changes with the development of the monkeys. The mechanisms of reproduction of hybrid monkeys and the maintenance of differences between the species are discussed.