Lung epithelial cells are essential effectors of inducible resistance to pneumonia.

Infectious pneumonias are the leading cause of death worldwide, particularly among immunocompromised patients. Therapeutic stimulation of the lungs' intrinsic defenses with a unique combination of inhaled Toll-like receptor (TLR) agonists broadly protects mice against otherwise lethal pneumonias. As the survival benefit persists despite cytotoxic chemotherapy-related neutropenia, the cells required for protection were investigated. The inducibility of resistance was tested in mice with deficiencies of leukocyte lineages due to genetic deletions and in wild-type mice with leukocyte populations significantly reduced by antibodies or toxins. Surprisingly, these serial reductions in leukocyte lineages did not appreciably impair inducible resistance, but targeted disruption of TLR signaling in the lung epithelium resulted in complete abrogation of the protective effect. Isolated lung epithelial cells were also induced to kill pathogens in the absence of leukocytes. Proteomic and gene expression analyses of isolated epithelial cells and whole lungs revealed highly congruent antimicrobial responses. Taken together, these data indicate that lung epithelial cells are necessary and sufficient effectors of inducible resistance. These findings challenge conventional paradigms about the role of epithelia in antimicrobial defense and offer a novel potential intervention to protect patients with impaired leukocyte-mediated immunity from fatal pneumonias.

Sensitization to acid-hydrolyzed wheat protein by transdermal administration to BALB/c mice, and comparison with gluten.

BACKGROUND: An increasing number of studies have shown that hydrolyzed wheat protein (HWP) can induce IgE-mediated hypersensitivity by skin contact and/or food ingestion. However, there has been no study of the sensitizing potential of HWP. In this study, the possibility of transdermal pathway for sensitization to acid-HWP (HWP1) was investigated using BALB/c mice, and compared with that of gluten. METHODS: HWP1 or gluten (500 µg/mouse) was transdermally administered using patches. After three or four cycles of sensitization for 3 days/week, active systemic anaphylaxis (ASA) was induced by intraperitoneal injection of the antigen, and rectal temperatures, scores of anaphylactic responses, and plasma histamine levels were determined. Because HWP1 was included in facial soap in Japan, the effect of detergent on the sensitizing potential was also investigated. RESULTS: Transdermal administration of HWP1 induced dose-dependent production of IgE and IgG1. After sensitization for 3 or 4 weeks, intraperitoneal injection of HWP1 caused ASA, leading to decreased rectal temperatures, increased anaphylaxis scores, and increased plasma histamine levels. In addition, splenocytes harvested after ASA produced IL-4, IL-5, and IL-10 by re-stimulation with HWP1. Transdermal exposure to gluten also induced IgE and IgG1 production, and intraperitoneal injection of gluten also induced ASA only in mice sensitized in the presence of sodium dodecyl sulfate. CONCLUSIONS: Transdermal exposure to HWP1 is sufficient to activate key immune pathways necessary for sensitizing mice for immediate hypersensitivity reactions. This study shows that HWP has a sensitizing potential as well as gluten, whereas its allergenicity may be different from that of gluten.

Inhaled corticosteroids stabilize constrictive bronchiolitis after hematopoietic stem cell transplantation.

Post transplantation constrictive bronchiolitis (PTCB) is the most common pulmonary complication among long-term survivors of allogeneic hematopoietic stem cell transplantation (HSCT). It is a late manifestation of GVHD. Its treatment with high-dose systemic corticosteroids and other immunosuppressive regimens is associated with multiple side effects. Topical corticosteroids are used for the treatment of other manifestations of GVHD to minimize these side effects. We conducted a retrospective analysis of a series of adult patients to evaluate the efficacy of high-dose inhaled corticosteroids in the treatment of PTCB. Seventeen patients with new-onset airflow obstruction were diagnosed with PTCB. Their forced expiratory volume in 1 s (FEV1) declined from a median of 84% (range, 56-119) before HSCT to 53% (26-82) after HSCT. All patients received inhaled fluticasone propionate 500-940 microg two times daily. Symptoms of airway obstruction improved and FEV1 stabilized 3-6 months after treatment. We conclude that high-dose inhaled corticosteroids may be effective in the treatment of PTCB and propose a plausible mechanism of its action. A prospective evaluation of its efficacy is warranted.

Description of International Caenorhabditis elegans Experiment first flight (ICE-FIRST).

Traveling, living and working in space is now a reality. The number of people and length of time in space is increasing. With new horizons for exploration it becomes more important to fully understand and provide countermeasures to the effects of the space environment on the human body. In addition, space provides a unique laboratory to study how life and physiologic functions adapt from the cellular level to that of the entire organism. Caenorhabditis elegans is a genetic model organism used to study physiology on Earth. Here we provide a description of the rationale, design, methods, and space culture validation of the ICE-FIRST payload, which engaged C. elegans researchers from four nations. Here we also show C. elegans growth and development proceeds essentially normally in a chemically defined liquid medium on board the International Space Station (10.9 day round trip). By setting flight constraints first and bringing together established C. elegans researchers second, we were able to use minimal stowage space to successfully return a total of 53 independent samples, each containing more than a hundred individual animals, to investigators within one year of experiment concept. We believe that in the future, bringing together individuals with knowledge of flight experiment operations, flight hardware, space biology, and genetic model organisms should yield similarly successful payloads.

Genetic analysis of ryanodine receptor function in Caenorhabditis elegans based on unc-68 revertants.

The Caenorhabditis elegans ryanodine receptor is encoded by the unc-68 gene, and functions as a Ca2+-induced Ca2+ release channel during muscle contraction. To investigate the factors that suppress calcium release and identify molecules that interact with the ryanodine receptor, we isolated revertants from two unc-68 mutants. Three of the revertants obtained from the null allele unc-68(e540), which displayed normal motility, had intragenic mutations that resulted in failure to splice out intron 21. The other two, kh53 and kh55, had amino acid insertions in the third of the four RyR domains. The brood size and the egg laying rate remain abnormal in these revertants. This suggests the third RyR domain may be required for egg laying and embryogenesis, although we can not determine a molecular mechanism. Five ketamine sensitive revertants recovered from the missense mutant unc-68(kh30) showed altered responses to caffeine, ryanodine, levamisole and ouabain relative to those of the unc-68(kh30) animals. These may carry second-site suppressor mutations, which may define genes for proteins that regulate the Ca2+ concentration in body-wall muscle. One of these mutants, kh52, shows lower motility and higher sensitivity to drugs, and this mutation was mapped to chromosome X. These observations provide a basis for the study of ryanodine receptor functions in embryogenesis and in calcium-mediated regulation of muscle contraction in C. elegans. This is the first study to show that the conserved RyR domain of the receptor acts in egg laying and embryogenesis.

[Multi-institutional cooperative study on combination chemotherapy with THP, CDDP and 5-FU for squamous cell carcinoma of the head and neck].

Combination chemotherapy with THP, CDDP and 5-FU for squamous cell carcinoma of the head and neck was conducted in 13 institutions in Hyogo Prefecture as a multi-institutional cooperative study. In the initial study (Nov. 1990-Nov. 1993), THP was administered intravenously at 20 mg/m2 on day 1, CDDP at 80 mg/m2 on day 2, and 5-FU at 1,000 mg/body/day in a continuous drip infusion for 120 hours from day 2 to day 6. In the second study (May, 1996-Mar. 1998), THP was administered at 20 mg/m2 on day 1, 5-FU at 10 mg/kg/day from day 1 to day 5, and CDDP at 70 mg/m2 on day 6 in the same way as the initial study. Forty-nine patients (Stage I in 3, Stage II in 12 including 2 recurrent cases, Stage III in 6, Stage IV in 28 including 3 recurrent cases; 1 course chemotherapy in 13 and 2 or more courses in 36) were subjected as complete cases in the initial study, and 36 patients (Stage I in 5 including one recurrent case, Stage II in 11 including 1 recurrent case, Stage III in 9 including 2 recurrent cases, Stage IV in 11 including one recurrent case; 1 course in 18 and 2 or more courses in 18) in the second. The overall response rate was 65.3% (CR in 3 cases) in the initial study and 63.9% (CR in 5 cases) in the second. Primary cases showed a response rate of 65.9% (29/44) in the initial study and 71.0% (22/31) in the second, whereas recurrent cases showed a 60.0% (3/5) response rate in the initial study and a 20.0% (1/5) rate in the second. Treatment-naive patients showed a response rate of 72.7% (24/33) in the initial study and 71.0% (22/31) in the second, whereas previously treated patients showed a 50.0% (8/16) response rate in the initial study and a 20.0% (1/5) rate in the second. Adverse reactions of more than Grade 3 in the initial study were leukopenia in 18.4%, thrombocytopenia in 8.2%, decrease of hemoglobin in 6.1%, loss of hair in 6.1%, anorexia in 36.7%, nausea and vomiting in 26.5%, and diarrhea in 4.1%, whereas those of Grade 3 in the second study were decrease of hemoglobin in 2.8%, anorexia in 22.2% and nausea and vomiting in 8.3%. From these results, it is suggested that the regimen in the second study was more useful than that in the initial study.

Traffic control: Rab GTPases and the regulation of interorganellar transport.

Membrane proteins, membrane lipids, and luminal contents are exchanged among the intracellular compartments of eukaryotic cells by vesicular transport. This process must be highly ordered to maintain cellular architecture in the face of rapid membrane turnover. The Ras-related Rab GTPases play multiple roles in regulating this traffic.

Calmodulin binding to the C-terminus of the small-conductance Ca2+-activated K+ channel hSK1 is affected by alternative splicing.

We identified three splice variants of hSK1 whose C-terminal structures are determined by the independent deletion of two contiguous nucleotide sequences. The upstream sequence extends 25 bases in length, is initiated by a donor splice site within exon 8, and terminates at the end of the exon. The downstream sequence consists of nine bases that compose exon 9. When the upstream sequence (hSK1(-)(25b)) or both sequences (hSK1(-)(34b)) are deleted, truncated proteins are encoded in which the terminal 118 amino acids are absent. The binding of calmodulin to these variants is diminished, particularly in the absence of Ca2+ ions. The first 20 amino acids of the segment deleted from hSK1(-)(25b) and hSK1(-)(34b) contain a 1-8-14 Ca2+ calmodulin binding motif, and synthetic oligopeptides based on this region bind calmodulin better in the presence than absence of Ca2+ ions. When the downstream sequence (hSK1(-)(9b)) alone is deleted, only the three amino acids A452, Q453, and K454 are removed, and calmodulin binding is not reduced. On the basis of the relative abundance of mRNA encoding each of the four isoforms, the full-length variant appears to account for most hSK1 in the human hippocampus, while hSK1(-)(34b) predominates in reticulocytes, and hSK1(-)(9b) is especially abundant in human erythroleukemia cells in culture. We conclude that the binding of calmodulin by hSK1 can be modulated through alternative splicing.

U73122 inhibits the dephosphorylation and translocation of cofilin in activated macrophage-like U937 cells.

Cofilin, an actin-binding protein, plays an important role in the migration, phagocytosis, and superoxide production of activated phagocytes through cytoskeletal reorganization. In unstimulated phagocytes, cofilin is a major phosphoprotein. However, upon activation, the phosphoprotein is dephosphorylated and translocated from cytosol to plasma membranes. Only the unphosphorylated form of cofilin is an active form that binds actin, whereas the regulatory mechanisms of cofilin have not been elucidated. We found that 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phospholipase C (PLC), suppressed both opsonized zymosan (OZ)-induced dephosphorylation and translocation of cofilin in macrophage-like U937 cells at 4 microM concentration. OZ triggered an increase in inositol 1,4,5-trisphosphate (IP3), and U73122 inhibited it. 1-[6-[[17beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-pyrrodione-dione (U73343), which was employed as an inactive analogue, had no such inhibitory activities as did U73122. Furthermore, herbimycin A, an inhibitor of src-type tyrosine kinase, also inhibited OZ-triggered IP3 formation. These results suggest that the activity and localization of cofilin are regulated by PLC at the downstream of src-family tyrosine kinase.

Incident-angle dependence of the sensitivity of second-harmonic generation of an oxacyanine dye in an LB film.

The second-harmonic generation of a cyanine dye in an LB film was investigated by varying the incident angle of the excitation laser. There was a clear dependence on the incident angle, which was simulated by a simple model. Thus, a highly sensitive determination should be carried out at such an angle where the efficiency of the second-harmonic generation shows its maximum.

The Canadian SCORE questionnaire: optimizing the use of technology for low bone density assessment. Simple Calculated Osteoporosis Risk Estimate.

The Simple Calculated Osteoporosis Risk Estimation (SCORE) questionnaire is a tool to assist physicians to identify women who might require bone densitometry. The purpose of this study was to develop a Canadian SCORE and to assess validity and reliability. Twenty sites enrolled 307 postmenopausal women ages 50-70 yr. SCORE results were compared to hip and lumbar spine bone density assessed by dual X-ray absorptiometry. Sensitivity and specificity of a range of SCORE cut-points were assessed in a receiver operating characteristics analysis to determine the optimal cut-point for SCORE. With low bone density defined as a T-score < or = -2.0, a SCORE cut-point of 6 in women ages 50-59 yr displayed a sensitivity of 0. 96, 95% confidence interval (CI) (0.89, 1.00), a specificity of 0.51, 95% CI (0.43, 0.58). In women ages 60-70 yr, a SCORE cut-point of 8 displayed a sensitivity of 0.90, 95% CI (0.80, 0.97) and a specificity of 0.20, 95% CI (0.11, 0.29). The test-retest reliability (intraclass correlation coefficient) was 0.95. SCORE performed better in women in their fifties than women in ther sixties. Older women require higher SCORE cut-points. The use of SCORE as an initial measure for identifying those at risk for osteoporosis may reduce costs by limiting unnecessary tests.

Nitric oxide induces chemotaxis of neutrophil-like HL-60 cells and translocation of cofilin to plasma membranes.

Nitric oxide (NO) plays various important roles in the physiological system. With regard to chemotaxis of neutrophils, there are reports that endogenous NO is a mediator of chemotaxis, and others that exogenous NO inhibits chemotaxis. It is also reported that NO itself expressed chemotactic activity. On the other hand, we have recently proposed the importance of cofilin, an actin-binding phosphoprotein, in phagocyte functions through dephosphorylation and translocation to the plasma membrane regions. Because chemotaxis is a phenomenon of dynamic cell movement, cofilin, a regulator of the cytoskeletal system, may be involved in its mechanisms. To clarify further the effect of NO on functions of leukocytes and to examine the effect of NO on cofilin, we investigated the chemotaxis of neutrophil-like HL-60 cells induced by NO, as well as the influence of NO on the phosphorylation and intracellular distribution of cofilin. Two NO donors, 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamin e (NOC5) and S-nitroso-N-acetylpenicillamine (SNAP), were shown to cause chemotaxis, and, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO), a NO-specific scavenger, inhibited the chemotaxis induced by NO-donors, suggesting that NO itself released from the NO donors has chemotactic activity. LY-83583 and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), inhibitors of soluble guanylate cyclase, inhibited the chemotaxis to NO donors, which implies that soluble guanylate cyclase is involved in the signaling pathway of this NO action. We also found that NO caused translocation of cofilin to the cell periphery, though dephosphorylation of cofilin was not detected. These results demonstrate that NO has chemotactic activity for neutrophils and caused the translocation of cofilin to the plasma membrane regions without its dephosphorylation.

Gene structure and promoter function of murine Munc18-2, a nonneuronal exocytic Sec1 homolog.

Sec1 family proteins are regulators of diverse exocytic processes, from yeast to man. Three mammalian homologues, Munc18-1, -2, and -3 have been described. We have studied the structure and expression of the mouse Munc18-2 gene. The Munc18-2 gene comprises 19 exons whose sizes range from 50 to 158 bp, with a total gene size of approximately 11 kb. A single transcript of 2.1 kb is expressed in multiple non-neuronal murine tissues. Munc18-2 has a striking resemblance to Munc18-1 in structure despite only 60% sequence identity, suggesting a recent gene duplication event. Analysis of the region upstream of the transcription start site shows that Munc18-2 has a TATA-less promoter, with a consensus initiator (Inr) sequence at the start of transcription, several Sp1 binding sites, and strong promoter activity in RBL-2H3 mast cells. The region from +5 to -430 is more active than +5 to -800, suggesting upstream repressor elements.

Genomic organization, chromosomal localization, and expression of the murine RAB3D gene.

Rab proteins, members of the Ras superfamily of small GTPases, play regulatory roles in intercompartmental vesicular transport. Each step of traffic seems to require the participation of at least one distinct Rab, with the Rab3 subfamily involved in stimulated exocytosis. We report our studies on the murine rab3D gene, one of the four mammalian Rab3 isoforms. We located this gene on chromosome 13, region A(2-3). The rab3D gene consists of 5 exons spanning 10.6 kb, and the structural gene is contained in exons 2 through 5 with one canonical GTP-binding motif in each exon. Organization of the rab3D gene is identical to that of rab3A but different from other rab genes. Alternative poly-A(+) signals in the 3' untranslated region account for the identities of multiple transcripts detected by Northern blot analysis. Rab3D is expressed in all tissues studied, predominantly in heart, lung, and liver, and binding sites for multiple transcription factors are found in the TATA-less promoter region.

Bisphosphonates for steroid induced osteoporosis.

OBJECTIVES: To assess the effects of bisphosphonates for the prevention and treatment of corticosteroid-induced osteoporosis. SEARCH STRATEGY: We searched the Cochrane Musculoskeletal Group trials register, Medline up to 1997 and Embase1988-1997), and selected hand searching of reference lists was conducted. Hand searching of scientific abstracts from relevant meetings for the last five years was also done. An electronic search in Current Contents was done for the last six months. The Cochrane Controlled Trials Register (CCTR) will be searched for future updates. All languages were included in the search. For practical reasons only those in English were included, but all languages will be retrieved and translated for future updates. SELECTION CRITERIA: All controlled clinical trials (CCTs) dealing with prevention or treatment of corticosteroid-induced osteoporosis with bisphosphonates of any type and reporting the outcomes of interest were assessed. Trials had to involve adults only, and subjects had to be taking a mean steroid dose of 7.5 mg/day or more. DATA COLLECTION AND ANALYSIS: All data extraction was performed by two independent reviewers. Outcomes of interest included change in bone mineral density (BMD) at the lumbar spine and femoral neck at six and 12 months. If present, data on number of new fractures and withdrawals due to adverse effects were also extracted. All data extraction was performed by two independent reviewers. Both continuous and dichotomous data were analyzed using fixed effects models. When significant heterogeneity was present, a random effects model was used. MAIN RESULTS: A total of 13 trials, including 842 patients are included in this meta-analysis. Results are reported as a weighted mean difference of the percent change in BMD between the treatment and placebo groups, with trials being weighted by the inverse of their variance. The 95% confidence intervals (95% CI) are presented. At the lumbar spine, the weighted mean difference of BMD between the treatment and placebo groups was 4.3% (95% CI 2.7, 5.9). At the femoral neck, the weighted mean difference was 2.1% (95%CI 0. 01, 3.8). Although there was a 24% reduction in odds of spinal fractures [OR 0.76 (95%CI 0.37, 1.53)], this result was not statistically significant. REVIEWER'S CONCLUSIONS: Bisphosphonates are effective at preventing and treating corticosteroid-induced bone loss at the lumbar spine and femoral neck. Efficacy regarding fracture prevention cannot be concluded from this analysis, although bone density changes are correlated with fracture risk.

Herbimycin A inhibits both dephosphorylation and translocation of cofilin induced by opsonized zymosan in macrophagelike U937 cells.

We previously reported that a 21-kDa phosphoprotein may play an important role in superoxide production through dephosphorylation by neutrophillike differentiated HL-60 cells (Suzuki et al., 1995, Biochim Biophys Acta 1266: 261-267). The phosphoprotein was identified as cofilin, an actin-binding protein, and the activation-induced changes in its intracellular distribution have been described elsewhere (Suzuki et al., 1995, J Biol Chem 270:19551-19556). However, the physiologic roles of cofilin in phagocytes remain to be established, and the regulatory mechanisms for dephosphorylation and translocation of cofilin are unknown. In the present study, we investigated the roles of cofilin in the opsonized zymosan (OZ)-activated macrophagelike U937 cells by using herbimycin A, an inhibitor for protein tyrosine kinase. In the individual adherent phagocytes, OZ induced many events: 1) production of superoxide, 2) phagocytosis of the insoluble particles OZ, 3) dephosphorylation of cofilin, 4) translocation of cofilin from cytosol to plasma membrane regions, 5) decrease in intracellular pH from 7.4 to aprroximately 6.8, and 6) rapid and transient increase in filamentous actin at the cell periphery. All of these events were inhibited or reduced significantly by herbimycin A. OZ increased phosphorylation of tyrosine in 110-, 50-, 34-, and 29-kDa proteins, whereas herbimycin A inhibited it. These results suggest that tyrosine kinase plays an essential role upstream of these events through phosphorylation of such proteins. Furthermore, microinjection of anti-cofilin antibody to the differentiated U937 cells caused inhibition of the phagocytosis. These results suggest that cofilin plays critical roles in phagocytic functions through changes in cytoskeletal organization.

Synaptotagmin II negatively regulates Ca2+-triggered exocytosis of lysosomes in mast cells.

Synaptotagmins (Syts) I and II are believed to act as Ca2+ sensors in the control of neurotransmission. Here we demonstrate that mast cells express Syt II in their lysosomal fraction. We further show that activation of mast cells by either aggregation of FcepsilonRI or by Ca2+ ionophores results in exocytosis of lysosomes, in addition to the well documented exocytosis of their secretory granules. Syt II directly regulates lysosomal exocytosis, whereby overexpression of Syt II inhibited Ca2+-triggered release of the lysosomal processed form of cathepsin D, whereas suppression of Syt II expression markedly potentiated this release. These findings provide evidence for a novel function of Syt II in negatively regulating Ca2+-triggered exocytosis of lysosomes, and suggest that Syt II-regulated secretion from lysosomes may play an important role in mast cell biology.

Participation of cofilin in opsonized zymosan-triggered activation of neutrophil-like HL-60 cells through rapid dephosphorylation and translocation to plasma membranes.

We studied the roles of cofilin, an actin-binding phosphoprotein, in superoxide production of neutrophil-like HL-60 cells triggered by opsonized zymosan (OZ). OZ caused dephosphorylation of cofilin as well as a transient increase of F-actin. Both reactions were complete within 30 s. Okadaic acid (OA) magnified the OZ-triggered O2--production 3.3-fold at 1 microM, but inhibited it completely at 5 microM. We used these critical concentrations to study the effects of OA on changes in phosphorylation and intracellular localization of cofilin. The OZ-induced dephosphorylation of cofilin was inhibited by 5 microM OA but not by 1 microM OA. Subcellular fractionation and immunoblotting revealed that 1 microM OA increased cofilin on the phagosomal membranous fraction but 5 microM OA decreased it. At 1 microM, OA increased translocation of p47phox to membranes, which may explain in part the enhancing effect of 1 microM OA. Confocal laser scanning microscopy showed that: (i) Cofilin diffused throughout the cytosol of resting cells, but accumulated at the plasma membranes forming phagocytic vesicles in activated cells. (ii) At 1 microM, OA had little effect on the OZ-evoked translocation of cofilin, whereas 5 microM OA suppressed it completely. (iii) OA alone, which could not trigger the phagocytic respiratory burst, did not cause any change in the distribution of cofilin at such concentrations. Furthermore, in a superoxide-producing cell-free system employing membranous and cytosolic fractions, affinity-purified anti-cofilin antibody showed an enhancing effect. These results suggest that cofilin participates in the superoxide production of the OZ-activated phagocytes through dephosphorylation and translocation. The roles of cofilin in the activated leukocytes will be discussed.

Rab3D, a small GTPase, is localized on mast cell secretory granules and translocates to the plasma membrane upon exocytosis.

Although mast cell secretion has been intensively studied because of its pivotal role in allergic reactions and its advantages as a physiologic model, the molecular composition of the secretory machine is virtually unknown. In view of the guanine-nucleotide dependency of mast cell exocytosis and the participation of Rab3 proteins in synaptic vesicle release, we hypothesized that a Rab3 isoform regulates mast cell secretion. Fragments of Rab3A, 3B, and 3D were cloned from RBL-2H3 mast cells by reverse transcription- polymerase chain reaction (RT-PCR). Northern blot analysis revealed Rab3D transcripts to be relatively abundant, Rab3B substantially less so, and Rab3A and 3C undetectable. By ribonuclease (RNase) protection assay, Rab3D transcripts were at least 10-fold more abundant than those of other isoforms, and by immunoblot analysis, Rab3D protein was at least 60-fold more abundant than that of Rab3B. Rab3D was more abundant in RBL cells than in brain, but the total mass of Rab3 proteins in RBL cells was 10-fold less than in brain. Rab3D only partly colocalized with secretory granules in RBL cells, but fully colocalized in mature peritoneal mast cells. There was a descending concentration gradient of Rab3D from peripheral to central granules, and no cytoplasmic pool was detectable in resting mast cells. Following exocytotic degranulation, Rab3D translocated to the plasma membrane and remained there for at least 15 min. These studies suggest that Rab3D is a component of the regulated exocytotic machine of mast cells, and identify differences between mast cells and neurons in Rab3 expression and trafficking.

Development of the autofluorescence endoscope imaging system.

It is a well known fact that light emitted at a specific wavelength induces fluorescence in the human body. This kind of fluorescence is called autofluorescence. The application of autofluorescence diagnosis, on the other hand, is a more complicated system designed to detect faint autofluorescence inherent in tissues/cells. We have adopted this autofluorescence diagnosis method and developed a new autofluorescence endoscope imaging system called the SAFE-1000. Normal mucosa emitting autofluorescence appears green on the monitor, while abnormal mucosa shows a dark image caused by the lack of autofluorescence.