Expression of enzymes and oncogene induced after radiotherapy and/or chemotherapy in patients with brain tumors.

The relationship between the degree of the expression of Cu/Zn SOD, GST-pi and bcl-2 in the initial and recurrent tumor tissue after radiotherapy and/or chemotherapy and the cellular heterogeneity obtained from DNA content by image cytometry was investigated. Subjects were 7 patients who had glial tumors which were surgically removed at onset and removed a second time at recurrence. Radiotherapy and chemotherapy were also administered after initial resection. Immunoreactivity for copper/zinc super oxide dismutase (Cu/Zn SOD), GST (glutathione-S-transferase)-pi, and bcl-2 were evaluated from routinely prepared tissue blocks. Tumors were classified into two groups by cytometric analysis of DNA ploidy in the G2M cell cycle phase. One tumor group consisted of single clonal cells in both the initial and recurrent tumors and the other group consisted of tumors with polyclonal cells in the initial and recurrent tumor. In this study, one patient (case 3) with single clonal cell glioblastoma at recurrence did not show high Cu/Zn SOD activity after radiotherapy and chemotherapy but showed a short survival time after recurrence. In three patients (cases 1, 2, 3) with single clonal-cell glioblastoma, the recurrent tumor cells showed high GST-pi immunoreactivity and survival time was short after recurrence. Tumor cells in two patients (cases 5, 7) with single clonal cell anaplastic glioma at recurrence, showed high GST-pi immunoreactivity and had a short survival time after recurrence. In three single clonal glioblastomas (cases 1, 2, 3), the recurrent tumor showed the increased bcl-2 immunoreactivity and showed a short survival time after recurrence. In two patients (case 5, 7) with single clonal cell anaplastic glioma at recurrence, tumor cells showed a high bcl-2 immunoreactivity and these patients showed a short survival time after recurrence. Although the number of subjects is very small, our study shows that the immunoreactivity of bcl-2 and GST-pi in malignant gliomas may be very important factors in radio- and chemosensitivity, and shows that GST-pi is induced by radiation and anti-cancer drugs.     

Reduction by phytate-reduced soybean beta-conglycinin of plasma triglyceride level of young and adult rats.

This study examined the effects of soybean beta-conglycinin, from which phytate was mostly removed, on the plasma lipids in young and adult rats. Male Wistar young (6 week-old) and adult (21 week-old) rats were fed high cholesterol diets containing 20% casein, soy protein isolate (SPI), or soybean beta-conglycinin for 10 days. In young rats, although the food intake of the beta-conglycinin group was higher than those of the casein and SPI groups, the weight gain was significantly lower than those of the other groups. However, in adult rats, the weight gain was not different among the groups. In young and adult rats, relative liver weights of SPI and beta-conglycinin groups were significantly lower than that of the casein group, and the degree of the reduction was more marked in the beta-conglycinin group than in the SPI group. In young rats, the plasma triglyceride level was significantly lower in the SPI and beta-conglycinin groups than that in the casein group. In addition, the plasma triglyceride level of the beta-conglycinin group was significantly lower than that of the SPI group. Plasma total cholesterol levels of the SPI and beta-conglycinin groups were significantly lower than that of the casein group. However, there was little difference in the lowering effect between SPI and beta-conglycinin. These results indicate that soybean beta-conglycinin may have lowering functions not only on plasma total cholesterol level, but also on plasma triglyceride level.     

Computational analysis of full-length mouse cDNAs compared with human genome sequences

Although the sequencing of the human genome is complete, identification of encoded genes and determination of their structures remain a major challenge. In this report, we introduce a method that effectively uses full-length mouse cDNAs to complement efforts in carrying out these difficult tasks. A total of 61,227 RIKEN mouse cDNAs (21,076 full-length and 40,151 EST sequences containing certain redundancies) were aligned with the draft human sequences. We found 35,141 non-redundant genomic regions that showed a significant alignment with the mouse cDNAs. We analyzed the structures and compositional properties of the regions detected by the full-length cDNAs, including cross-species comparisons, and noted a systematic bias of GENSCAN against exons of small size and/or low GC-content. Of the cDNAs locating the 35,141 genomic regions, 3,217 did not match any sequences of the known human genes or ESTs. Among those 3,217 cDNAs, 1,141 did not show any significant similarity to any protein sequence in the GenBank non-redundant protein database and thus are candidates for novel genes. Received: 18 January 2001 / Accepted: 17 May 2001     

The receptor tyrosine phosphatase Dlar and integrins organize actin filaments in the Drosophila follicular epithelium.

BACKGROUND: Regulation of actin structures is instrumental in maintaining proper cytoarchitecture in many tissues. In the follicular epithelium of Drosophila ovaries, a system of actin filaments is coordinated across the basal surface of cells encircling the oocyte. These filaments have been postulated to regulate oocyte elongation; however, the molecular components that control this cytoskeletal array are not yet understood. RESULTS: We find that the receptor tyrosine phosphatase (RPTP) Dlar and integrins are involved in organizing basal actin filaments in follicle cells. Mutations in Dlar and the common beta-integrin subunit mys cause a failure in oocyte elongation, which is correlated with a loss of proper actin filament organization. Immunolocalization shows that early in oogenesis Dlar is polarized to membranes where filaments terminate but becomes generally distributed late in development, at which time beta-integrin and Enabled specifically associate with actin filament terminals. Rescue experiments point to the early period of polar Dlar localization as critical for its function. Furthermore, clonal analysis shows that loss of Dlar or mys influences actin filament polarity in wild-type cells that surround mutant tissues, suggesting that communication between neighboring cells regulates cytoskeletal organization. Finally, we find that two integrin alpha subunits encoded by mew and if are required for proper oocyte elongation, implying that multiple components of the ECM are instructive in coordinating actin fiber polarity. CONCLUSIONS: Dlar cooperates with integrins to coordinate actin filaments at the basal surface of the follicular epithelium. To our knowledge, this is the first direct demonstration of an RPTP's influence on the actin cytoskeleton.     

Difference in genesis between antigen-induced IgM antibody-forming cells and B-cell mitogen-induced IgM antibody-forming cells in mouse spleen cell cultures.

To determine the mechanisms in the triggering of thymus-independent lymphocytes (B cells) for development into antibody-forming cells (AFC), genesis of IgM AFC elicited polyclonally by nonspecific stimulation with B-cell mitogen, such as nystatin and bacterial lipopolysaccharide, was compared with that of IgM AFC specifically elicited by antigenic stimulation, using mouse spleen cell cultures as an experimental system and sheep erythrocytes (SRBC) as a test antigen. Considering that differentiation and proliferation are necessary cellular events for precursor B cells to develop into AFC, the effect of different antimetabolic agents on the generation of each type of AFC in spleen cell cultures was examined. The generation of anti-SRBC IgM hemolysin plaque-forming cells (PFC) in B-cell mitogen-stimulated spleen cell cultures was found to be less susceptible to X-irradiation or mitomycin C than that in the SRBC-stimulated cultures. These apparently paradoxical results were affiirmed using colcemid as an inhibitor of cell mitosis and hydroxyurea (HU) as an inhibitor of cellular DNA synthesis. Thus, when spleen cell cultures responding to either SRBC or B-cell mitogen were exposed to colcemid or HU during a period from 2 days to 3 days after the stimulation, the exponential generation of anti-SRBC IgM PFC in the cultures responding to SRBC was completely halted, whereas that in the cultures responding to B-cell mitogen was not. Furthermore, N⁶, O^2′ -dibutyryl adenosine 3′, 5′ -cyclic monophosphoric acid was found to halt the exponential generation of antigen-induced anti-SRBC IgM PFC but not that of the B-cell mitogen-induced anti-SRBC IgM PFC. From these results it was suggested that B-cell mitogen might stimulate precursor Bμ cells at a late stage in the differentiative pathway to develop into AFC without cell division, and that antigenic stimulation might stimulate relatively primitive precursor Bμ cells to proliferate and then differentiate into AFC. Based on this idea, mechanisms in the triggering of B-cell activation are discussed.     

Differences in environment of FAD between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase shown by active site probe study

Rat liver deflavoxanthine dehydrogenase has been prepared by incubating native enzyme with calcium chloride. On reconstitution with FAD, about 85% of the original activity is recovered, all which is the O2-dependent type. In contrast, when dithiothreitol-treated deflavoenzyme is incubated with FAD, the recovery of activity is almost the same as above, but most of the recovered activity is of the NAD-dependent type. Deflavoenzyme with or without previous treatment with dithiothreitol was also reconstituted with two artificial FAD analogues, 8-mercapto-FAD and 6-OH-FAD. The difference spectra between the reconstituted enzymes and the initial deflavoenzyme indicate that, in each case, the FAD analogue is bound in its neutral form in dithiothreitol-treated enzyme, whereas it is bound in the anionic form in enzyme without previous dithiothreitol treatment. Furthermore, the protonated forms can be converted into the anionic forms on storage with a concomitant change of activity from the NAD-dependent to the O2-dependent type. This clearly indicates different environments around FAD in the two types of enzyme protein, which are shown to be interconvertible through oxidation-reduction of enzyme cysteinyl residues.     

Amidolytic activity of porcine trypsin bound to human plasma alpha-1-antiproteinase

Human plasma alpha-1-antiproteinase interacted with porcine trypsin in two different manners. One was a well known interaction, which resulted in inhibition of the proteolytic activity of the trypsin. The other has not been described to date, and resulted in retention of the amidolytic activity of the trypsin towards benzoyl-L-arginine p-nitroanilide in the presence of soybean trypsin inhibitor. The latter, so-called trypsin-protein amidase, activity is essentially the same as that observed with vertebrate alpha-macroglobulin and rodent murinoglobulin under similar conditions. All attempts to separate the two different activities as well as to abolish either activity by means of chemical or physical modifications were unsuccessful. The proteolysis-inhibiting interaction, which was virtually completed within 5 min, was predominant over the amidolysis-retaining interaction, when the inhibitor/trypsin molar ratio was less than 1. On the other hand, the amidolysis-retaining interaction, which proceeded much more slowly, became evident when the molar ratio was greater than 1.