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Lactacystin, originally isolated from a microbe as an inducer of neuritogenesis, targets the catalytic beta-subunit of the proteasome, and arrests the cell cycle. Here we report for the first time that lactacystin induces apoptotic cell death in human monoblastic U937 cells. When U937 cells were cultured with lactacystin, their nuclei were shrunken, a morphological change typical of apoptosis, and cell viability was decreased. Electrophoretic analysis revealed that chromosomal DNAs from lactacystin-treated cells were cleaved in an internucleosomal ladder-like pattern, indicating that cell death occurs through an apoptotic process, which was also confirmed by DNA fragmentation analysis using flow cytometry. These findings suggest that inhibition of the proteasome during proliferation results in apoptotic cell death, and that the proteasome is a key enzyme in the course of the cell cycle that destines the cell to proliferate, differentiate or die.

The activation of protein kinase C (PKC) is irreversibly regulated by limited proteolysis catalyzed by a calcium-activated neutral cysteine protease, calpain. Calpain cleaves PKC alpha at specific sites in the hinge region between the catalytic and the regulatory domains of this kinase. Here we show a novel method for production of antibodies that bind specifically to the catalytic fragment of PKC alpha but not to the unproteolyzed protein. To detect proteolyzed PKC alpha 'cleavage site-directed antibodies,' which recognize amino-terminal regions in the nascent catalytic fragments and do not cross-react with the unproteolyzed enzymes, were raised using synthetic peptides corresponding to the amino-terminal sequences. The synthetic peptides used in this study were the sequences of human PKC alpha at the cleavage sites by m- and mu-types of calpains (LGPAGNKV and VISPSEDRKQPSNNLDRVKLT, respectively) and they are designated as CF alpha 2, CF alpha 4, in this order. Each synthetic peptide was injected into rabbit after conjugation with a carrier protein. The antibodies thus obtained (anti-CF alpha 2 or -CF alpha 4) specifically reacted with either the 46- or 45-kDa catalytic fragment of PKC alpha, respectively, whereas they did not cross-react with other fragments. Furthermore, the antibodies did not bind to the unproteolyzed enzyme nor fragments of PKC alpha obtained by treatment with other proteinases unless the fragment carried the same amino-terminal sequence. When human platelets were treated with calcium ionophore, the catalytic fragments of PKC alpha (45- and 46-kDa) were detected in the cytosol by immunoblotting with the antibodies. However, these antibodies did not bind unproteolyzed 80-kDa PKC alpha, although this form was dominant in the cytosol of the calcium ionophore-treated human platelets. In addition, the 45-kDa catalytic fragment of PKC alpha was detected in an apoptotic human fibroblast TIG-3 cells cultured in serum-free medium. Our method is applicable for analysis of proteolysis in various cellular states.

The Fas antigen (CD95) is a receptor for apoptotic cell death signals. Human T Jurkat cells that express the Fas molecule on the cell surface are sensitive to cytotoxic anti-Fas monoclonal antibodies (mAbs). We report here that fetal calf serum (FCS) contains a novel factor (SAF: Serum Anti-Apoptosis Factor) that suppresses Fas-mediated apoptosis of Jurkat cells. The apoptotic cell death of Jurkat cells induced by an anti-Fas mAb was accelerated when FCS was removed from the medium. Depletion of serum itself did not induce apoptosis without the mAb. The apoptosis-suppressing activity was not seen in neonatal calf or adult bovine serum, suggesting that the SAF activity was developmentally regulated. The anti-apoptosis activity was not detected in any fraction of FCS obtained on gel filtration chromatography. However, the activity was recovered in a certain fraction on the addition of calf serum, suggesting that the anti-apoptotic activity of SAF requires other serum factor(s). We purified SAF from FCS using an assay system reconstituted with calf serum, and raised antibodies (Abs) to it in rabbits. The anti-SAF Ab inhibited the apoptosis-suppressing activity, indicating that SAF is a key factor for the anti-apoptotic activity of FCS.

By UV-irradiation, cells are subjected to DNA damage followed by mutation, cell death and/or carcinogenesis. DNA repair systems such as nucleotide excision repair (NER) and translesion DNA synthesis (TLS) protect cells against UV-irradiation. To understand the role of histone acetyltransferase GCN5 in regulation of DNA repair, we studied the sensitivity of GCN5-deficient DT40, GCN5(-/-), to various DNA-damaging agents including UV-irradiation, and effects of GCN5-deficiency on the expression of NER- and TLS-related genes. After UV-irradiation, cell death and DNA fragmentation of GCN5(-/-) were appreciably accelerated as compared with those of DT40. Interestingly, GCN5(-/-) showed a remarkable sensitivity to only UV-irradiation but not to other DNA-damaging agents tested. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically down-regulated in GCN5(-/-) (to ∼25%). In addition, ectopic expression of human POLH in GCN5(-/-) dramatically reversed the sensitivity to UV-irradiation of GCN5(-/-) to almost the same level of wild type DT40. Moreover, chromatin immunoprecipitation assay revealed that GCN5 binds to the chicken POLH gene 5'-flanking region that contains a typical CpG island and acetylates Lys-9 of histone H3, but not Lys-14 in vivo. These data suggest that GCN5 takes part in transcription regulation of POLH gene through alterations in the chromatin structure by direct interaction with its 5'-flanking region, and protects vertebrate cells against UV-induced DNA damage via controlling POLH gene expression.

A number of potential variables are associated with the diagnostic accuracy of endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA). The aim of this study was to evaluate factors affecting the diagnostic accuracy of EUS-FNA for upper gastrointestinal submucosal or extraluminal solid lesions.Patients with such lesions who underwent EUS-FNA between January 2009 and December 2010 were studied retrospectively. Needles of 22, 25 and 19 gauge were used. The associations between the EUS-FNA results and factors such as mass location, mass size, needle size, number of needle passes, combined histologic-cytologic analysis and final diagnosis were analyzed.A total of 170 EUS-FNA procedures were performed in 158 patients with upper gastrointestinal submucosal or extraluminal solid lesions. The overall accuracy of EUS-FNA was 86.5% (147/170). The diagnostic accuracy with three or more needle passes was higher than with less than 3.0 needle passes (90.0%, 108/120 vs 78.0%, 39/50; P0.05). Mass location, mass size, and final diagnosis were not associated with EUS-FNA accuracy. Combined cytologic-histologic analysis had significantly higher diagnostic accuracy than either cytologic or histologic analysis alone (P0.001). In a subgroup of 90 patients, both 22 and 25 gauge needles were used for EUS-FNA. The overall diagnostic accuracy was similar for 25 gauge needles and 22 gauge needles (80.0% vs 78.9% P = 1.000) in this subgroup.Overall, 25 and 22 gauge needles have a similar diagnostic accuracy. Our results suggest that 3.0 or more needle passes and combined cytologic-histologic analysis enhance the diagnostic accuracy of EUS-FNA.

The transcription factor, early B cell factor 1 (EBF1) with an atypical zinc-finger and helix-loop-helix motif, is essential for development and differentiation of lymphocytes. In mice, EBF1 is involved in the generation of pre-pro B cells (the first specified progenitors of B cells) from common lymphoid progenitors (CLPs) and transcription regulations of various genes involved in B cell-development, for instance, mb-1 and Pax5. During B lymphopoiesis, interestingly, EBF1 is detected throughout from CLPs to mature B cells. However, in immature B cells, the physiological role of EBF1 remains to be elucidated. Here, by analyzing EBF1-deficient DT40 cells, EBF1(-/-), generated by us, we show that EBF1-deficiency caused significant increases (to ∼800%) in both mRNA and protein levels of B lymphocyte-induced maturation protein-1 (Blimp-1), the master gene for plasma cell differentiation. In addition, both transcription and protein synthesis of Blimp-1 were remarkably down-regulated (to ∼20%) by re-expression (over-expression) of EBF1. Chromatin immunoprecipitation assay revealed that EBF1 binds to proximal 5'-upstream regions around two putative EBF1 binding motifs of the gene in vivo. These results suggest that EBF1 takes part in transcriptional regulations of the Blimp-1 gene in immature B cells, and may play a key role in B cell differentiation. This is the first report on a novel EBF1 function in immature B cells as a powerful repressor of Blimp-1 gene expression.

Cytochrome b558 composed of large and small subunits, and cytosolic 47- and 65-kDa proteins are important constituents of the superoxide (O2-) generating system in phagocytes and B lymphocytes. In this paper, we describe changes in O2(-)-generating activity and expression of O2(-)-generating components during differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. Undifferentiated U937 cells generated no O2- in response to a stimulation, although they expressed the three components other than the cytochrome b558 large subunit. When U937 cells were cultured with agents that induced the cell differentiation, such as vitamin D3, retinoic acid, interferon-gamma, and tumor necrosis factor, O2(-)-generating activity increased 5- to 200-fold depending on the agent used. Immunoblotting analysis revealed that the amounts of the four protein components essential for O2- generation increased, although their induction levels were significantly different between inducers. Among the four protein components, the cytochrome subunits were induced in low levels by all agents tested, which may explain why the O2(-)-generating activity of differentiated U937 cells was much lower than that of neutrophils from peripheral blood.

Recent progress in chemotherapy has prolonged the survival of patients with malignant biliary strictures, leading to increased rates of stent occlusion. Even we employed metallic stents which contributed to higher rates and longer durations of patency, and occlusion of covered metallic stents now occurs in about half of all patients during their survival. We investigated the complication and patency rate for the removal of covered metallic stents, and found that the durations were similar for initial stent placement and re-intervention. In order to preserve patient quality of life, we currently recommend the use of covered metallic stents for patients with malignant biliary obstruction because of their removability and longest patency duration, even though uncovered metallic stents have similar patency durations.

Background and study aims: Recent progress in chemotherapy has prolonged the survival of patients with malignant biliary strictures, leading to increased rates of stent occlusion. Occlusion of covered metallic stents now occurs in about half of all patients with malignant biliary strictures. The removal of metallic stents followed by placement of a second stent has been attempted, but outcomes remain controversial. The aim of the current study was to evaluate the effectiveness and safety of the primary placement and secondary placement (re-intervention) of covered metallic stents and to assess the feasibility and safety of stent removal. Patients and methods: The study included 186 patients with unresectable malignant biliary strictures who underwent primary stent placement between October 2001 and March 2010. Covered biliary self-expandable metal stents (SEMSs) were removed in 39 of these patients, and 36 underwent re-intervention. The patency times, occlusion rates of the first stent and re-intervention, success rates of stent removal, and complications were investigated. Results: Covered SEMSs were placed in 186 patients. The median patency time of the first stent was 352 days. Stent occlusion occurred in 48.9 % of the patients and was mainly caused by debris or food residue (37 %), dislocation (19 %), and migration with hyperplasia (19 %). Stent removal was attempted in 50 patients and was successful without complication in 39 (78 %). Most of the patients in whom stent removal was unsuccessful had migration with hyperplasia. The median patency time of the second stent was 263 days. The stent patency time did not significantly differ between the first and the second stent. Conclusions: Covered SEMSs could be safely removed at the time of stent occlusion. Patency rates were similar for initial stent placement and re-intervention.

BACKGROUND: Various factors, such as the optimal number of passes, aspiration pressure, and the use of 19-gauge and Trucut biopsy needles, have been studied to improve the diagnostic accuracy of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). We retrospectively compared the diagnostic accuracy of EUS-FNA between 25- and 22-gauge needles, which have been widely used recently. SUBJECTS AND METHODS: The study group comprised 47 consecutive patients who underwent EUS-FNA with both 22- and 25-gauge needles from October 2007 through March 2010. Their underlying diseases were pancreatic cancer in 24 patients, submucosal tumors in 11, other pancreatic tumors in 4, chronic pancreatitis in 4, enlarged lymph nodes in 3, and gall bladder cancer in 1. Tissue specimens, which were pushed out of the puncture needle, were placed into physiological saline solution. Gray-whitish, worm-like specimens were used for histologic diagnosis. The remaining specimen was centrifuged, and the sediment was plated on slides and examined by a cytopathologist to obtain the cytologic diagnosis. RESULTS: A total of 75 punctures (mean, 1.6) were performed with 25-gauge needles, and 69 punctures (mean, 1.4) were performed with 22-gauge needles. The overall tissue-sampling rate for cytology was 100% (47/47), which was significantly (p=0.01) superior to 83% (39/47) for histology. The overall diagnostic accuracy on the cytologic and histologic examinations was 79% (37/47) and 85% (33/39) (p=0.48). According to needle type, the tissue-sampling rate for cytology and histology on each puncture was 97% (73/75) and 56% (42/75) with 25-guage needles, and was 97% (67/69) and 58% (40/69) with 22-guage needles, the accuracy of cytologic diagnosis on each puncture was 73% (53/73) with 25-gauge needles and 66% (44/67) with 22-gauge needles (p=0.37); the accuracy of histologic diagnosis on each puncture was 60% (25/42) and 75% (30/40) (p=0.14), respectively. No patient had complications. CONCLUSIONS: The tissue-sampling rate and diagnostic accuracy did not differ significantly between 22- and 25-gauge needles in patients with pancreatic or gastrointestinal diseases who underwent EUS-FNA.

Histone acetyltransferase(s) (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. General control non-depressible 5 (GCN5) was first identified as a global coactivator and transcription-related HAT. Here we report that GCN5 regulates the activation of phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) survival pathway in B cells exposed to oxidative stress via controlling gene expressions of spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk). The GCN5-deficiency remarkably caused apoptotic cell death by treatment with exogenous hydrogen peroxide (H(2)O(2)) in chicken DT40 cells. In GCN5-deficient DT40 cells, gene expressions of Syk and Btk, which are involved in activation of PI3K/Akt survival pathway in DT40 cells exposed to exogenous H(2)O(2), were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H(2)O(2)-treated GCN5-deficient cells was remarkably suppressed as compared to that of DT40. Chromatin immunoprecipitation assay revealed that GCN5 binds to proximal 5'-upstream regions of Syk and Btk genes in vivo. These results suggest that GCN5 takes part in transcriptional regulations of the Syk and Btk genes, and plays a key role in epigenetic regulation of PI3K/Akt survival pathway in B cells exposed to reactive oxygen species such as H(2)O(2).

Endoscopic ultrasonography (EUS) has widened its applications and been become an indispensable examination. Recent advances is as follows: Electronic radial scanning EUS, three-dimensional EUS (3D-EUS), enhancement, elastography, and EUS-FNA. Because of electronic radial scanning EUS, we can employ Doppler function. 3D-EUS have introduced not only preciseness for preoperative evaluation for EMR and ESD but also surface rendering similar to endoscopic image. Enhancement and elastography have a possibility of tissue chracterization. First EUS-FNA, in which Vilmann reported fine needle aspiration cytology of pancreas cancer and Grimm treated a case with pseudocyst, were made in 1992. Then EUS-FNA has become popular in the clinical fields and the accuracy of diagnostic EUS-FNA has been reported 70 to 100%. Furthermore EUS-FNA has also widened its applications to therapeutic techniques such as pseudocyst drainage, biliary drainage, pancreatic duct drainage, celiac plexus neurolysis (CPN), ethanol injection therapy, radiofrequency ablation (RFA), immunotherapy, and gene therapy etc. Finally, EUS-FNA is the future promising technique which has potential for developing new treatments.

The membrane bound cytochrome b558 composed of large gp91-phox and small p22-phox subunits, and cytosolic proteins p40-, p47- and p67-phox are important components of superoxide ( O 2 - )-generating system in phagocytes and B lymphocytes. A lack of this system in phagocytes is known to cause serious life-threatening infections. Here, we describe that curcumin, a polyphenol responsible for the yellow color of curry spice turmeric, dramatically activates the O 2 - -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and curcumin, the O 2 - -generating activity increased more than 4-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and curcumin slightly enhanced gene expressions of the five components compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and curcumin caused remarkable accumulation of protein levels of p47-phox (to 7-fold) and p67-phox (to 4-fold) compared with those of the RA-treatment alone. These results suggested that curcumin dramatically enhances RA-induced O 2 - -generating activity via accumulation of cytosolic p47-phox and p67-phox proteins in U937 cells. Therefore, it should have the potential as an effective modifier in therapy of leukemia and/or as an immunopotentiator.

Alterations in the chromatin structure are essential for easy accesses to chromosomal DNA. Such architectural alterations can be achieved by four means: (i) variants of histone subtypes, (ii) chromatin remodeling, (iii) post-translational modification, and (iv) chromatin assembly. This chapter discusses mainly on the first, third and fourth mechanisms, and especially on the acetylation of core histones, one of the third mechanisms. Taking the advantage of the gene targeting technique, we systematically established numerous mutant DT40 cell lines, each lacking particular gene, of interest such that encoding histones, histone deacetylases (HDACs), acetyltransferases (HATs) and chaperones, etc. Every subtype member of the histone gene family is capable of compensating the loss of others to maintain the mRNA level of each histone subtype, and most of histone variants are involved positively or negatively in the transcription regulation of particular genes. Regarding HDACs, HDAC-2 controls the amount of the IgM H-chain at the steps of both transcription and alternative pre-mRNA processing, and HDAC-3 is indispensable for cell viability. Concerning HATs, GCN5 has tremendous impact on growth kinetics by preferentially acting as a supervisor in the normal cell cycle progression. The distinct participatory roles of the N-terminal and C-terminal halves of HIRA, one of histone chaperones, in both cell growth and transcription regulations of cell cycle-related genes, have also been highlighted. Therefore, the gene targeting technique in the DT40 cell line can be used as a powerful tool for the functional analysis of histones, histone modifying enzymes and histone chaperones relevant to chromatin biology.

We previously reported that histone deacetylase-2 (HDAC2) controls the amount of IgM H-chain at the steps of transcription of its gene and alternative processing of its pre-mRNA in DT40 cells. Here, we showed not only that the HDAC2-deficiency caused repressions of gene expressions for HDAC7, EBF1, Pax5, Aiolos and Ikaros, and elevations of gene expressions for HDAC4, HDAC5, PCAF and E2A, but also that it caused altered acetylation levels of several Lys residues of core histones. Using gene targeting techniques, we generated three homozygous DT40 mutants: EBF1(-/-), Aiolos(-/-) and E2A(-/-), devoid of EBF1, Aiolos and E2A genes, respectively. Semiquantitative RT-PCR analysis of the resultant mutants revealed not only that EBF1 and Aiolos down-regulate expressions of IgM H- and L-chain genes, but also that E2A up-regulates expressions of these two genes. These results, together with others, indicate that HDAC2 controls indirectly expressions of IgM H- and L-chain genes through opposite transcriptional regulations of EBF1, Pax5, Aiolos plus Ikaros and E2A genes.

Alterations in the chromatin structure are essential for easy accesses to chromosomal DNA. Such architectural alterations can be achieved by four means: (i) variants of histone subtypes, (ii) chromatin remodeling, (iii) post-translational modification, and (iv) chromatin assembly. This chapter discusses mainly on the first, third and fourth mechanisms, and especially on the acetylation of core histones, one of the third mechanisms. Taking the advantage of the gene targeting technique, we systematically established numerous mutant DT40 cell lines, each lacking particular gene, of interest such that encoding histones, histone deacetylases (HDACs), acetyltransferases (HATs) and chaperones, etc. Every subtype member of the histone gene family is capable of compensating the loss of others to maintain the mRNA level of each histone subtype, and most of histone variants are involved positively or negatively in the transcription regulation of particular genes. Regarding HDACs, HDAC-2 controls the amount of the IgM H-chain at the steps of both transcription and alternative pre-mRNA processing, and HDAC-3 is indispensable for cell viability. Concerning HATs, GCN5 has tremendous impact on growth kinetics by preferentially acting as a supervisor in the normal cell cycle progression. The distinct participatory roles of the N-terminal and C-terminal halves of HIRA, one of histone chaperones, in both cell growth and transcription regulations of cell cycle-related genes, have also been highlighted. Therefore, the gene targeting technique in the DT40 cell line can be used as a powerful tool for the functional analysis of histones, histone modifying enzymes and histone chaperones relevant to chromatin biology.

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.

A digital 3D broadcasting system which uses a 525-line progressively scanned digital broadcasting system was developed. Up to now, extensive research has been conducted on 3D systems themselves, but little research has been carried out on the actual broadcasting of 3D signals. One reason has been the inherent difficulty of sending multiple channels using conventional terrestrial analog broadcasting technology. We have recently developed a vertical multiplex method which converts a stereoscopic image consisting of two 525-line interlaced scan video signal (525i), into one 525- line progressive scan video signal (525p). This converted signal is then transmitted using a 525p digital broadcasting system. This 3D system has several merits: (1) It is possible to broadcast 3D video signals using simple equipment attached to a 525p broadcasting system. (2) It is possible to receive left and right eye's information at the same time and no synchronization process is necessary. In order to test this 3D system, we have carried out experiments using a 525p broadcasting system which was constructed for satellite transmission experimentation the year before last. Through these experiments, operating at a bit rate of 9 Mbps, we have confirmed that the 3D system we have constructed does work without any loss of quality of the 3D effect. We are expecting this 3D broadcasting system to be one of the applications of our 525 p experimental broadcasting.© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Interferon-γ (IFN-γ), vitamin D3 (VD), and retinoic acid (RA) induce differentiation of human monoblastic leukemia U937 cells to macrophage-like cells with potential superoxide anion-generating activity upon further stimulation. Here we report that U93 7 cells thus differentiated show various responses to apoptotic induction with a cytotoxic anti-Fas antibody and tumor necrosis factor (TNF). VD-or RA-treated U937 cells acquired resistance against Fas- or TNF receptor (TNFR)-mediated apoptosis, whereas apoptotic cell death was accelerated in IFN-γ-treated cells. By flow cytometric analyses, no decrease in expression of surface Fas antigen or p55 TNFR was observed in differentiated U937 cells. Cell surface expression of CD11b was seen only when differentiation was induced with VD or RA but not with IFN-γ. The growth of VD- or RA-treated cells was retarded but IFN-γ-treated cells were prolific. These findings suggest that the differentiation state differs with the inducer and that the cellular response to apoptotic induction is closely related to the state including the cell cycle.

To understand the function of B cell antigen receptor (BCR)-related complex on pre-B cells (pre-BCR, Vpre-B/lambda 5/mu heavy chain/Ig-alpha/Ig-beta), we examined pre-BCR- and BCR-mediated signaling events in human and mouse pre-B (Nalm-6, 697, NFS-5), immature B (IgM+ Daudi, WEHI-231) and mature B (IgM+ IgD+ BALL1) cell lines. Anti-mu cross-linking induced tyrosine phosphorylation of the cytoplasmic proteins in each cell type, but did not induce a detectable Ca2+ mobilization response in pre-B cells. While the pre-B cells expressed Syk protein at levels similar to those found in B cell lines, pre-BCR cross-linkage did not induce phosphorylation of Syk tyrosine residues. Different protein kinase C isozymes were expressed by pre-B (PKC-alpha), immature B (PKC-alpha and -beta) and mature B (PKC-beta) cell lines. Anti-mu cross-linking induced PKC translocation from the cytosolic to the membrane compartment in immature and mature B cells, but did not have this effect in a pre-B cell line. Anti-mu cross-linking induced tyrosine phosphorylation of the p85 and p110 subunits of phosphatidylinositol 3-kinase (P13-kinase) in both pre-B and B cell lines, but the pre-BCR induced P13-kinase activation was Syk independent. Ligation of the pre-BCR complex thus triggers a characteristic signaling pattern in pre-B cells.

We show here a novel method for the in situ analysis of proteolyzed proteins in a cell. As a model, we focused on protein kinase C (PKC) beta, which is cleaved at a specific site between the catalytic and regulatory domains by calpain, the intracellular calcium-activated neutral proteinase. To detect proteolyzed PKC beta 'cleavage-site-directed antibodies', which specifically recognize the amino-terminal region of the catalytic fragment but do not cross-react with the unproteolyzed enzymes, were raised using synthetic peptide. The synthetic peptide used in this study was QGTKVPEEKTT, corresponding to the amino-terminal region of the catalytic fragment from human PKC beta generated by calpain. Rabbits were immunized with the synthetic peptide after conjugation with a carrier protein. Antibodies obtained reacted with the 46-kDa catalytic fragment of PKC beta, whereas they did not cross-react with unproteolyzed enzyme nor other fragments with different amino-termini. Thus, our antibody is specific to the amino-terminal sequence QGTKVPEEKTT, but does not recognize the same sequence located internally in native PKC beta. When human monoblast U937 cells were treated with calcium ionophore, the catalytic fragment of PKC beta was detected in the cytosol by immunoblotting with the antibody. However, this antibody did not bind unproteolyzed 80-kDa PKC beta, although this form was dominant in the cytosol of the calcium ionophore-treated cells. We could also detect comparable amounts of catalytic fragment in the calcium ionophore-treated cells by immunocytochemical staining with the same antibody. Our method was applied to examine the proteolysis of PKC beta in neutrophils stimulated with various reagents.

Techniques for Cannulation of the Bile Duct During Endoscopic Retrograde Cholangiopancreatography (ERCP) Hiroshi Imaizumi, Mitsuhiro Kida, Hidehiko Kikuchi, Miyoko Takezawa, Yoshiki Kida, Katsunori Saigenj

A prototype forward-viewing echoendoscope has been developed for therapeutic endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). The hard tip of the forward-viewing echoendoscope, which is shorter than that of the convex type echoendoscope, can be maneuvered flexibly. Using the forward-viewing echoendoscope, the gastrointestinal wall can be vertically punctured along the same axis as the scope, and this process is done more easily than with an oblique-viewing echoendoscope. The diagnostic accuracy of EUS-FNA with the forward-viewing echoendoscope is 97.4%, which is not significantly different to that of the oblique-viewing echoendoscope. The forward-viewing echoendoscope may be useful in situations where the location and procedure are difficult with the oblique-viewing scope, The forward-viewing echoendoscope is able to puncture the gastrointestinal wall vertically with minimal effort, therefore allowing therapeutic EUS procedures such as pseudocyst and abscess drainage, biliary drainage, and pancreatic duct drainage to be performed easily. However, a significant difference between the forward-viewing and oblique-viewing echoendoscopes in pseudocyst drainage has been reported recently. In the future, the forward-viewing and oblique-viewing echoendoscopes will probably be selectively used depending on not only lesion site but also the procedure required in individual patients, thereby facilitating various processes including puncture, tissue collection, and diagnosis, as well as therapeutic procedures.