Satsuki Matsushima

Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic β cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A−/−) mice. Synt1A−/− β cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

Abnormalities of epidermal growth factor receptor in lung squamous-cell carcinomas, adenosquamous carcinomas, and large-cell carcinomas: tyrosine kinase domain mutations are not rare in tumors with an adenocarcinoma component.

Tyrosine kinase domain (TKD) gene mutations of the epidermal growth factor receptor gene (EGFR) have proven to be clinically significant in nonsmall‐cell lung cancer (NSCLC), particularly in adenocarcinoma. However, TKD mutations together with deletion mutations in the extracellular domain of EGFR (EGFRvIII) have not been fully investigated in NSCLC except for adenocarcinoma. The present study sought to gain further insight into the significance of EGFR mutations in NSCLC by focusing on nonadenocarcinoma NSCLC.

α-Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Is Expressed in Pancreatic β Cells and Functions in Insulin but Not γ-Aminobutyric Acid Secretion

The function of solubleN-ethylmaleimide-sensitive attachment protein-α (α-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of α-SNAP in the secretion of insulin and γ-aminobutryric acid (GABA) from pancreatic β cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed α-SNAP, but not β-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that α-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. α-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. Anin vitro binding study showed that both wild-type α-SNAP and C-terminal–deleted α-SNAP mutant (1–285) can bind to syntaxin 1A. α-SNAP mutant (1–285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of α-SNAP mutant (1–285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous α-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if α-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic β cells, the functional role of α-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of α-SNAP mutant (1–285) overexpression on GABA release. We conclude that 1) α-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic β cells; and 2) the interaction of α-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process.

Differentiation and morphogenesis in pellet cultures of developing rat retinal cells

We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, 1990, Neuron 4:461–467). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures by using both cell‐type‐specific markers with immunofluorescence and electron microscopy. We demonstrated that, in addition to rods, the other major retinal cell types, including amacrine cells, bipolar cells, Müller cells, and ganglion cells were all present in the pellets, where most were able to develop from dividing precursor cells in vitro. The different cell types in the pellets became organized into two distinct structures: dark rosettes and pale rosettes. The cellular composition of these structures indicated that the dark rosettes correspond to the outer nuclear layer and the pale rosettes to the inner nuclear layer of the normal retina. Ultrastructural studies have indicated that the thin layer of neuronal processes surrounding the dark rosettes correspond to the outer plexiform layer, and the central region of the pale rosettes correspond to the inner plexiform layer of the normal retina. Other features of normal retinal development also occurred in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Thus, pellet cultures provide a convenient way to study different aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate. J. Comp. Neurol. 377:341–350, 1997.

Clinico-Pathological and Biological Significance of Tyrosine Kinase Domain Gene Mutations and Overexpression of Epidermal Growth Factor Receptor for Lung Adenocarcinoma

Introduction: Mutations in the tyrosine kinase domain (TKD) of the epidermal growth factor receptor (EGFR) gene have proven to be clinically significant in non-small cell lung cancer. However, relationships between these mutations and EGFR expression or deletion mutations in the extracellular domain of EGFR (EGFRvIII) remain unclear. The purpose of this study was to gain further insight into the clinical significance of these molecular abnormalities in lung adenocarcinoma. Methods: We investigated EGFR TKD mutations using direct sequencing, EGFR protein expression using Western blotting, and EGFRvIII using reverse transcriptase–polymerase chain reaction in samples from 48 adenocarcinoma patients. Correlations with various clinico-pathological features were analyzed. Results: EGFR TKD mutations were detected in 25 of 48 adenocarcinomas (52.1%), and overexpression of EGFR protein was identified in 19 patients (39.6%). Presence of EGFR TKD mutations was significantly correlated with EGFR overexpression (p = 0.021). EGFR TKD mutations were significantly correlated with never-smoker status (p = 0.043), absence of emphysematous or fibrotic appearance on computed tomography (p = 0.001), papillary subtype (p = 0.041), and bronchioloalveolar carcinoma features (p = 0.045). EGFRvIII was not detected in any adenocarcinomas. Retrospective analysis revealed that patients with EGFR TKD mutations displayed better postoperative prognosis than patients with wild-type EGFR (p = 0.033). Conclusions: These results suggest that EGFR TKD mutation is associated with EGFR overexpression, representing an important factor for consideration when investigating the clinical significance, including susceptibility to chemotherapy, of EGFR TKD mutations in adenocarcinoma. EGFRvIII does not seem to play a major role in the development of lung adenocarcinoma.

A simple and sensitive method for detecting major mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene in non-small-cell lung carcinoma.

The detection of mutations in the epidermal growth factor receptor (EGFR) gene impacts therapeutic decision-making for non–small-cell lung carcinoma (NSCLC). Although direct sequencing has been most frequently used to detect EGFR mutations, this method displays several disadvantages. We set up simple mutation-specific polymerase chain reaction (PCR) for common delE746-A750 and L858R mutations of EGFR using primers specific to these mutations. Both mutation-specific PCR and direct sequencing methods were used to investigate 62 samples of NSCLC, and the results were compared. To evaluate the sensitivity of mutation-specific PCR, DNA mixtures containing various proportions of mutant alleles were analyzed. Mutation-specific PCR revealed delE746-A750 in 8 samples and L858R in 14 samples. All samples with either delE746-A750 or L858R mutation revealed by direct sequencing also displayed positive results for mutation-specific PCR. Conversely, mutations in 3 samples revealed by L858R-specific PCR were barely detectable by direct sequencing. In DNA mixture analysis, DNA mixtures containing 2.5% of delE746-A750 allele or 0.25% of L858R allele yielded positive results with mutation-specific PCR. Our mutation-specific PCR showed satisfactory sensitivity and reliability for detecting major EGFR mutations in clinical NSCLC samples. Given the practical availability, this method could be widely applicable to the treatment of lung cancers.

The Increase of Memory T Cell Subsets in Children with Idiopathic Nephrotic Syndrome

Two-color and three-color flow cytometry was carried out to determine whether the memory T cells (CD45RO+ T cells) play a major role in lymphocyte dysfunction of 26 children with idiopathic nephrotic syndrome (INS). The INS patients were divided into three groups: (1) 10 patients who were not receiving glucocorticoid hormone (GCH) and were suffering from acute nephrotic state were referred to as N1; (2) 8 patients who were in remission maintained by GCH therapy alone were referred to as N2; (3) 8 patients who were free of GCH therapy for at least 4 months were referred to as N3. Group N1 demonstrated a significant increase in the percentage of CD45RO+CD4+ T cells and CD45RO+CD8+ T cells (p < 0.05) compared with 11 controls, and these subsets were noted to have a tendency to decrease to control levels in groups N2 and N3. Furthermore, interleukin-2 receptor-α expressed subsets in CD45RO+CD4+ T cells (CD45RO+CD4+CD25+ T cells) were also increased only in group N1 (p < 0.02). A similar tendency of absolute counts was observed in these subsets. These results suggest that activated memory T cells reflect lymphocyte dysfunction at initial onset or relapse in INS children.

LOCALIZATION AND ONTOGENY OF GLUT3 EXPRESSION IN THE RAT RETINA

This study investigates the presence, localization, and developmental expression of a neuron-specific facilitated-diffusion glucose transporter, GLUT3, in the rat retina so as to elucidate molecular mechanisms regulating glucose homeostasis in support of the visual function. Immunoblot analysis using anti-GLUT3 antibody (ALM3-C) revealed the presence of GLUT3 as a heterogeneously glycosylated protein with an average molecular weight of approximately 44 kDa. Although immunofluorescence staining showed it to be localized primarily in the inner and outer plexiform layers, some of the cell bodies in the inner nuclear layer also showed weak immunoreactivity. Immunoblot analysis of developing rat retinal tissues revealed the presence of the GLUT3 protein as early as embryonic day 15 (E15), and immunofluorescence staining revealed its expression in the inner plexiform layer near the time of birth and in the outer plexiform layer at postnatal day 14 (P14), i.e., when the eyes normally open and retinal activity commences. The proteins abundance remained at a relatively low level during the embryonic stages and up until the end of the first postnatal week (P7), though a transient increase was confirmed to occur at E18. From P13, however, the abundance steadily increased, rapidly reaching the adult level at P24. Based on these observations, we hypothesize that GLUT3 is expressed in some subsets of retinal neurons, being preferentially abundant in their neuronal processes, and that its ontogeny is closely associated with morphological and functional development of the retina. As such, this suggests that GLUT3 plays some important role(s) in the retina where glucose metabolism is essential.

Low concentrations of serum n-3 polyunsaturated fatty acids in non-alcoholic fatty liver disease patients with liver injury

Tomonori Kishino*, Hiroaki Ohnishi, Kouki Ohtsuka, Satsuki Matsushima, Tsuyoshi Urata, Keiko Watanebe, Yukihisa Honda, Yoshitake Mine, Motoko Matsumoto, Kaori Nishikawa, Hideaki Mori, Shin’ichi Takahashi, Hitoshi Ishida and Takashi Watanabe 1 Department of Laboratory Medicine, Kyorin University School of Medicine, Tokyo, Japan 2 Department of Clinical Laboratory, Kyorin University Hospital, Tokyo, Japan 3 The Third Department of Internal Medicine, Kyorin University School of Medicine, Tokyo, Japan

Non-functional role of syntaxin 2 in insulin exocytosis by pancreatic β cells

This study was designed in order to examine the expression and functional role of syntaxin 2/epimorphin in pancreatic β cells. Northern blot analysis revealed that syntaxin 2 mRNA was able to be detected in mouse βTC3 cells, but not in isolated mouse islets. In agreement with this result, immunoblot analysis detected an appreciable amount of syntaxin 2 protein in βTC3 cells, but not in mouse islets. Immunohistochemistry of the mouse pancreas demonstrated that syntaxin 2 was little evident in islet cells of Langerhans, and somewhat predominant in exocrine tissues. In order to examine whether syntaxin 2 is anchored to cell surfaces in βTC3 cells, living cells were incubated with a monoclonal antibody against syntaxin 2 (MC‐1). The antibody bound to their surfaces, indicating that syntaxin 2 was localized on cell surfaces. The addition of MC‐1 to the culture medium of βTC3 cells did not affect insulin release under the presence or absence of 11 mM glucose, indicating that syntaxin 2 is not associated with insulin exocytosis. Thus, the expression of syntaxin 2 in islets of Langerhans is very low and the function of this protein is probably unrelated to the insulin exocytosis pathway.