Lysosome is a primary organelle in B cell receptor-mediated apoptosis: an indispensable role of Syk in lysosomal function

To investigate the mechanism of B cell receptor (BCR)-mediated apoptosis, we utilized immature B cell lines, DT40 and WEHI-231. In both cell lines, BCR-crosslinking caused the increase in lysosomal pH with early apoptotic changes characterized by chromatin condensation and phosphatidylserine exposure. This increase was detected in c-Abl-deficient DT40 cells but not in Syk-deficient cells, which corresponded to the fact that the former cells but not the latter revealed BCR-induced apoptosis. In contrast, BCR-crosslinking caused no apparent change in mitochondrial transmembrane potential. Therefore, the lysosomal change might be a primary event in BCR-induced apoptosis in DT40 cells. The increased activity of cathepsin B and apoptosis-preventing effect of a cathepsin inhibitor suggested a significant role of lysosomal enzymes in this apoptosis. By microscopic studies, lysosomes of wild-type DT40 cells fused to BCR-carrying endosomes became enlarged and accumulated one another. In contrast, these changes of lysosomal dynamics did not occur in Syk-deficient cells but transfer of wild-type Syk restored the lysosomal changes and apoptosis. These results demonstrated that the lysosomal change accompanied with the activation of lysosomal enzymes is a primary step in BCR-crosslinking-mediated apoptosis and Syk is responsible for this step through the fusion of BCR-carrying endosomes to lysosomes.     

Role of protein-tyrosine kinase syk in oxidative stress signaling in B cells

Oxidative stress induces the activation of multiple signaling pathways related to various cellular responses. In B cells, Syk has a crucial role in intracellular signal transduction induced by oxidative stress as well as antigen receptor engagement. Treatment of B cells with hydrogen peroxide (H(2)O(2)) induces enzymatic activation of Syk. Syk is essential for Ca(2+) release from intracellular pools through phospholipase C-gamma2 and the activation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase-Akt survival pathway following H(2)O(2) stimulation. Oxidative stress-induced cellular responses in B cells follow different patterns, such as necrosis, apoptosis, and mitotic arrest, according to the intensity of H(2)O(2) stimulation. Syk is involved in the protection of cells from apoptosis and induction of G2/M arrest. Syk leads to the activation of the phosphatidylinositol 3-kinase-Akt survival pathway, thereby enhancing cellular resistance to oxidative stress-induced apoptosis. On the other hand, Syk-dependent phospholipase C-gamma2 activation is required for acceleration toward apoptosis following oxidative stress. These findings suggest that oxidative stress-induced Syk activation triggers the activation of several pathways, such as proapoptotic and survival pathways, and the balance among these various pathways is a key factor in determining the fate of a cell exposed to oxidative stress.     

Functional evaluation of lung by Xe-133 lung ventilation scintigraphy before and after lung volume reduction surgery (LVRS) in patients with pulmonary emphysema

We evaluated the respiratory functions of patients with pulmonary emphysema who underwent lung volume reduction surgery (LVRS) by the mean transit time (MTT) with Xe-133 lung ventilation scintigraphy, forced expiration volume in 1 sec (FEV1.0), residual volume (RV), distance walked in 6 min (6-min walk), and the Hugh-Jones classification (H-J classification) before and after LVRS. In 69 patients with pulmonary emphysema (62 men, 7 women; age range, 47-75 years; mean age, 65.4 years +/- 6.1, preoperative H-J classification, III (two were II)-V) who underwent LVRS, all preoperative and postoperative parameters (MTT 3 weeks after LVRS and the others 3 months after LVRS) were judged statistically by the Wilcoxon signed-ranks test and Odds ratio. Every postoperative parameter was improved with a significant difference (P < 0.05) compared to preoperative parameters. MTT at 3 weeks after LVRS was not associated with %FEV1.0 and the H-J classification at 3 months after LVRS, but was associated with RV and a 6-min walk at 3 months after LVRS. MTT was useful for the clinical evalution of aerobic capability after LVRS.     

Raft.1, a monoclonal antibody raised against the raft microdomain,recognizes G-protein beta1 and 2, which assemble near nucleus after shiga toxin binding to human renal cell line

SUMMARY: Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein beta subunits 1 and 2 (Gbeta1 and 2). That Raft.1 recognized Gbeta1 and 2 was further confirmed by the reactivity to recombinant Gbeta1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gbeta1 and 2. Because Gbeta1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.     

The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei

Trypanosoma brucei evades the immune system by switching between Variant Surface Glycoprotein (VSG) genes. The active VSG gene is transcribed in one of approximately 20 telomeric expression sites (ESs). It has been postulated that ES polymorphism plays a role in host adaptation. To gain more insight into ES architecture, we have determined the complete sequence of Bacterial Artificial Chromosomes (BACs) containing DNA from three ESs and their flanking regions. There was variation in the order and number of ES-associated genes (ESAGs). ESAGs 6 and 7, encoding transferrin receptor subunits, are the only ESAGs with functional copies in every ES that has been sequenced until now. A BAC clone containing the VO2 ES sequences comprised approximately half of a 330 kb 'intermediate' chromosome. The extensive similarity between this intermediate chromosome and the left telomere of T. brucei 927 chromosome I, suggests that this previously uncharacterised intermediate size class of chromosomes could have arisen from breakage of megabase chromosomes. Unexpected conservation of sequences, including pseudogenes, indicates that the multiple ESs could have arisen through a relatively recent amplification of a single ES.     

Differences in the abilities of individual fingers during the performance of fast,repetitive tapping movements

Using 12 healthy male subjects, the dynamic motor ability of individual fingers was investigated under four different finger tapping conditions. These were: maximum speed tapping with one finger (single-finger tapping), alternate movement of two fingers (double-finger tapping), double-finger tapping in an unsupported condition, and submaximum constant speed tapping with one finger in a passive manner. Key-contact forces for all fingers and the movement velocity of the tapping finger were monitored. With the exception of the unsupported condition, non-tapping fingers were maintained in contact with designated keys during the tapping tasks. It was found that the index finger attained the fastest cadence and greatest movement velocity, followed by the middle, little and ring fingers, respectively. Subjective assessment of rank order of "difficulty" of tapping by the subjects was highly correlated with tapping cadence. Thus dynamic motor function, as indicated by rapid, repetitive movement, differs among the individual fingers. Parallel changes were observed in the key-contact force of the neighboring non-tapping fingers during tapping. The range of the non-tapping finger forces was largest during tapping by the ring finger. A similar trend was found for passive tapping, during which the magnitude of key-contact force was less than one-third of that observed during active tapping. The lower cadence achieved by the ring finger may be attributed more to a lack of independence at the level of voluntary neuromuscular control, than to innate mechanical interaction with the other fingers. Tapping cadence of each finger was lower for the double-finger mode than for the single-finger mode. The magnitude of the observed decrease in cadence during double-finger tapping was, on the other hand, strongly dependent on finger-combination. The decrease was smallest for the index-middle finger-combination, and greatest for the ring-little finger-combination. Compatibilities with other fingers can play an essential role in the dynamic motor function of individual fingers. During the unsupported task, in which interactions were diminished by allowing all fingers to move freely, tapping cadence increased markedly. Therefore, the lower cadences observed in specific finger-combinations may be partly attributed to anatomical and neural interdigit interactions.     

11q trisomy detected by fluorescence in situ hybridization

Takano T, Yamanouchi Y, Kawashima S, Date M, Hashira S, Kida M, Abe T, Nakahori Y, Nakagome Y. 11q trisomy detected by fluorescence in situ hybridization. Clin Genet 1993: 44: 324–328. © Munksgaard, 1993 A patient with psychomotor developmental delay, multiple minor anomalies, congenital heart disease and left inguinal hernia is reported. His karyotype was 45,X/46,X,+mar (3 : 37 cells), and the marker chromosome was identified as t(Y;11) (q12;q14?) using fluorescence in situ hybridization and fluorescent chromosome painting. He was diagnosed as mosaic for de novo 11q trisomy.     

Hyperplastic foci in chronic liver disease: Their proliferative activity assessed by nucleolar organizing region

In the cirrhotic and precirrhotic liver, there may be small foci with increased cellularity and amphophilic cytoplasm. These are microscopic lesions that do not form macroscopically detectable nodules, which differ from the macroscopically apparent nodules of dysplastic nodules. In the present study, we assessed the proliferating activity of 12 hyperplastic foci in 11 patients with cirrhosis or chronic hepatitis, by staining for agyrophilic nucleolar organizing regions (AgNOR). The mean AgNOR count per nucleus in the hyperplastic foci ranged from 0.96 to 1.36 (mean, 1.13; SD 0.12), and from 0.81 to 1.06 (mean, 0.94; SD 0.08) in the controls. The AgNOR count In the hyperplastic foci was significantly higher than that In the controls (P> 30.01). Small hyperplastic foci show Increased proliferative activity. Further study on these foci is required to clarify their relation to hepatocarcinogenesis.     

No evidence of PEG1/MEST gene mutations in Silver‐Russell syndrome patients

Silver‐Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (α and β) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST α coding region, and there were no significant mutations in the 5′‐flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST α were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS. © 2001 Wiley‐Liss, Inc.     

Alteration of cell cycle regulators correlates with survival in epithelial ovarian cancer patients

The p16-cyclinD1/CDK4-pRb pathway (RB pathway) and p14ARF-MDM2-p53 pathway (p53 pathway) work at the G1-S checkpoint, and the ATM-chk2-CDC25-cyclinB1/cdk1 pathway works at the G2-M checkpoint. The disruption of these pathways is thought to be related to the prognosis of human cancer. In this study, we analyzed the status of these pathways in 107 epithelial ovarian cancer (EOC) patients by immunohistochemistry and evaluated the relationship of these results with chemotherapy response and the prognosis. Altered RB, p53, and G2 pathways were detected in 50.5% (54/107), 51.4% (55/107), and 33.6% (36/107) of cases, respectively. The overall survival (OS) of 77.3% for patients with a normal RB pathway was significantly higher than the OS of 50.0% for patients with an altered RB pathway (by Kaplan-Meier analysis, P = 0.0021). The OS of 66.2% for patients with a normal G2 pathway was significantly higher than the OS of 58.3% for patients with an altered G2 pathway (P = 0.0416). However, the status of the p53 pathway was not related to OS. By univariate and multivariate analyses, advanced stage, high histological grade, altered RB pathway, and altered G2 pathway were significant predictors of poor OS. However, there was no significant relationship between pathway status and chemotherapy response. The status of the RB pathway and of the G2 pathway were independent prognostic factors of EOC.