Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

The ATM protein kinase is activated by intermolecular autophosphorylation in response to DNA damage and initiates cellular signaling pathways that facilitate cell survival and reduce chromosomal breakage. Here, we show that NBS1 and BRCA1 are required for the recruitment of previously activated ATM to the sites of DNA breaks after ionizing irradiation, and that this recruitment is required for the phosphorylation of SMC1 by ATM. To explore the functional importance of SMC1 phosphorylation, murine cells were generated, in which the two damage-induced phosphorylation sites in SMC1 are mutated. Although these cells demonstrate normal phosphorylation and focus formation of ATM, NBS1, and BRCA1 proteins after IR, they exhibit a defective S-phase checkpoint, decreased survival, and increased chromosomal aberrations after DNA damage. These observations suggest that many of the abnormal stress responses seen in cells lacking ATM, NBS1, or BRCA1 result from a failure of ATM migration to sites of DNA breaks and a resultant lack of SMC1 phosphorylation.     

Changes in T,B, and NK Lymphocyte Subsets During and After Normal Pregnancy

PROBLEM: Pregnancy affects the maternal immune system and the clinical course of maternal diseases. Here we report the changes in the detailed lymphocyte subsets of helper T cells, suppressor T cells, CD5⁺ B cells, T cell receptor (TCR) αβ-positive T cells (Tαβ cells), TCRαβ-negative T cell (Tγδ cells), and others during and after pregnancy through to one year postpartum, and discuss the significance of the changes. METHOD: The absolute numbers of helper T cells, suppressor T cells, cytotoxic T cells, TCRαβ-negative T cells (Tγδ cells), CD5^— B cells, CD5⁺ B cells, and NK cell subsets were examined by two-color flow cytometry in peripheral blood from 51 healthy non-pregnant women, 106 healthy pregnant women, and 148 healthy postpartum women. RESULTS: In early pregnancy, the numbers of suppressor T cells and NK cells with strong cytotoxicity (NK⁺⁺⁺ cells) increased, and the number of cytotoxic T cells decreased. In late pregnancy, the helper T cell and NK⁺⁺⁺ cell numbers decreased. Tαβ, CD5^— B and CD5⁺ B cells decreased during pregnancy. After delivery, helper T cells and cytotoxic T cells increased from 1 to 4 months postpartum, and suppressor T cells increased at 7 months postpartum. TCRαβ-negative T cells increased at 4 to 10 months postpartum. Both CD5^— and CD5⁺ B cells decreased further at 1 month postpartum, but CD5⁺ B cells increased markedly at 7 to 10 months postpartum. CONCLUSIONS: These data indicate that 1) early increases of suppressor T cells and NK⁺⁺⁺ cells during pregnancy may be related to the mechanism to accept or reject the fetus in early pregnancy, respectively; 2) late decreases of helper T cells and NK⁺⁺⁺ cells may be related to the maintenance of pregnancy: 3) postpartum increases of helper T cells, cytotoxic T cells, TCRαβ-negative T cells (Tγδ cells), and CD5⁺ B cells may be related to the postpartum aggravation of autoimmune diseases; and 4) the immunological effects of pregnancy remains until about 1 year after delivery.     

Bone formation using novel interconnected porous calcium hydroxyapatite ceramic hybridized with cultured marrow stromal stem cells derived from Green rat

The clinical use of cultured marrow stromal stem cells (MSCs) has recently attracted attention in the field of tissue engineering. For the clinical use of the MSCs, a prominent scaffold is needed. A scaffold hybridized with MSCs is transformed into a "bioactive bone substitute," and this provides good osteoconduction. In this study, a novel calcium hydroxyapatite ceramic with an interconnected porous structure (IP-CHA) was used as a scaffold. MSCs were harvested from Green rats containing Green Fluorescent Protein (GFP), and then these hybrids were implanted into the tibias of Sprague-Dawley rats. The purposes of this study were to examine the osteogenic ability of these hybrids without coculture, and to evaluate whether the resulting bone formation originated from the grafted MSCs or the recipient's cells. The hybridized group showed excellent bone formation compared with the IP-CHA-only implant group. Observation of the implanted MSCs revealed that they survived 8 weeks after surgery, and differentiated into osteoblast-like cells, thus providing bone formation. This implantation of the MSCs/IP-CHA composite provides excellent osteoconduction, and is expected to have extensive clinical applications.     

Investigation of albumin synthesis in rat livers during the perinatal period using immunocytochemistry andin situ hybridization

The immunoreactivity of albumin (ALB) was observed in the hepatocytes of fetal rats on day 18 of gestation, and was especially observable in immature rough endoplasmic reticulum (rER) and Golgi apparatus (GA); by then, a small amount of silver grains of ALB mRNA could already be detected. Just after birth, immunoreactivity of ALB could be observed in fine granules or diffusely in all hepatocytes, and was present in rER and GA. One week after birth immunoreactivity of ALB was observed in all hepatocytes and was visible in developed rER and GA; the grains of ALB mRNA were present in all hepatocytes.     

A case of mixed connective tissue disease developed into overlap syndrome of progressive systemic sclerosis, systemic lupus erythematosus, polymyositis and Sj?gren's syndrome

We encountered a patient who developed an overlap syndrome of progressive systemic sclerosis (PSS), systemic lupus erythematosus (SLE), polymyositis (PM) and Sj?gren's syndrome (SjS) while we were treating her for mixed connective tissue disease (MCTD). This 42-year-old woman had been photosensitive since 18 years of age. In 1986, Raynaud's phenomenon, swollen hands and arthralgia appeared; therefore, we started to treat this patient based on a diagnosis of MCTD. At that time, her anti-RNP antibody titer was 82,920, but she was negative to anti-Sm antibody. In 1988, she was admitted to our hospital with chief complaints of aggravation of polyarthralgia and myalgia. On physical examination, she showed difficulty in opening her mouth, systemic dermal sclerosis, a decrease in muscular strength and rales. In laboratory tests, her myogenic enzyme level was increased, and she was found to be positive to LE cells, antinuclear antibody, anti-DNA antibody, anti-ENA antibody and anti-SSA antibody. Furthermore, histological features clearly corresponding to those of PSS were found by skin biopsy, myogenic changes by electromyography, evidence of chronic inflammation of the salivary glands by lip biopsy, and proliferative changes in the mesangium were detected by renal biopsy. The concept of MCTD, especially the differences from overlap syndrome, is vague. Therefore we need further study about many cases. Since there have been no reports on cases having sufficient evidence of the development of the overlap syndrome of PSS, SLE, PM and SjS during a course of MCTD, our patient would provide very useful data contributing to the study of MCTD.     

Antioxidant enzyme systems in skeletal muscle atrophied by immobilization

To clarify the mechanism of oxidative stress in skeletal muscle atrophied by immobilization, we investigated the change of antioxidant enzyme activities in a typical slow red muscle, the soleus. Atrophied soleus muscles were collected from male Wistar rats (16 weeks old), one ankle joint of which had been immobilized in the fully extended position for 7 days. Also, soleus muscles were collected from intact age-matched rats as control. The activities of Mn-containing superoxide dismutase (Mn-SOD), Cu,Zn-containing superoxide dismutase (Cu,Zn-SOD), Se-dependent glutathione peroxidase (Se-GSHPx), glutathione S-transferase (GST), catalase, and glutathione reductase (GSSGRx) were measured. The activities of Cu,Zn-SOD, GST, and GSSGRx were significantly higher in atrophied muscles, while the others were unchanged. Increased Cu,Zn-SOD and unchanged Mn-SOD levels might reflect increased generation of superoxide anions in the cytoplasm rather than in the mitochondria. Owing to the enhancement of Cu,Zn-SOD and the unaltered Se-GSHPx and catalase activities, hydrogen peroxide is thought to be increased in the cytoplasm. Because there is also an increase of iron in the microsomes of atrophied muscles, the production of hydroxyl radicals, the most aggressive of radicals, might consequently be elevated.     

Inhibition of in vivo tumorigenicity and invasiveness of a human glioblastoma cell line transfected with antisense uPAR vectors

Our previous studies showed that glioblastomas express increased urokinase-type plasminogen activator receptors (uPARs) in comparison to low-grade gliomas (Yamamoto et al., Cancer Res., 54, 5016-5020, 1994). To explore whether downregulation of uPAR inhibits tumor formation and invasiveness, a human glioblastoma cell line was transfected with a cDNA construct corresponding to 300 bp of the human uPAR's 5¢ end in an antisense orientation, resulting in a reduced number of uPA receptors. Co-culture studies with tumor spheroids and fetal rat brain aggregates showed that antisense SNB19-AS1 cells expressing reduced uPAR failed to invade fetal rat brain aggregates. Intracerebral injection of SNB19-AS1 stable transfectants failed to form tumors and were negative for uPAR expression in nude mice. Thus uPAR appears in this model to be essential for tumorigenicity and invasion of glioblastomas in vivo.     

Application of personal computer to an analysis of smallde novo chromosomal insertion: A case ofde novo 3q2 trisomy with ins(8;3)

To identify the origin of a small inserted segment in ade novo 8p+ chromosome, an originally programmed computerized data-base for chromosomal aberration syndromes was utilized. The system selected 3q2 trisomy and 10q2 trisomy as candidates. As a result of a careful comparison of several high-resolution banding patterns among chromosomes 3, 10 and the inserted segment, her karyotype was disignated as: 46,XX,–8,+der(8), inv ins(8;3)(p21.1;q26.32q24)de novo. A small segment from 3q24 to 3q26.32 was trisomic, and invertedly inserted into the short arm of chromosome 8. This computerized database was considered to be useful for analyses of the smallde novo inserted chromosomal segment.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

Bruceine B, A Potent Inhibitor of Leukocyte-Endothelial Cell Adhesion

Leukocyte adhesion to vascular endothelial cells is an essential step in the development of inflammatory diseases. We have searched for inhibitors of leukocyte-endothelial cell adhesion that could be used as anti-inflammatory drugs and found that bruceine B (0.2 g/ml; 0.44 M) inhibited human neutrophil or T cell adhesion to tumor necrosis factor- (TNF) stimulated human umbilical vein endothelial cells (HUVEC). The inhibition of neutrophil adhesion to TNF-stimulated HUVEC by bruceine B was not derived from cytotoxic effects, as determined by measurement of the level of lactate dehydrogenase (LDH) activity in conditioned medium. The effect of bruceine B on neutrophil adhesion to HUVEC was not seen when the neutrophils were preincubated with bruceine B. However, inhibitory effects were evident when the HUVEC were preincubated with bruceine B. Bruceine B also inhibited neutrophil adhesion to lipopolysaccharide-stimulated HUVEC and T cell adhesion to TNF-stimulated HUVEC. These findings suggest that bruceine B may have anti-inflammatory activity.