Immunocytochemical localization of secretogranin III in the endocrine pancreas of male rats

Secretogranin III (SgIII), a member of the granin protein family, is expressed specifically in neuronal and endocrine cells. To examine the precise localization of SgIII in the endocrine pancreas, pancreatic tissues of rats were analyzed immunocytochemically with a polyclonal anti-serum raised against rat SgIII. By light microscopy of semithin sections, the immunoreactivity for SgIII was readily detected in pancreatic A- and B-cells, faintly so in D-cells, and not at all in the exocrine pancreas. By immunoelectron microscopy, immunogold particles indicative of SgIII were observed in the peripheral regions of secretory granules, and universally in the pancreatic endocrine cells. Morphometrical analyses indicated that SgIII is most preferentially localized in the periphery of the secretory granule among granins. These findings suggest that SgIII is closely associated with the secretory granule membrane, serving to anchor the aggregates of other soluble constituents to the membrane.     

Generation of fractal patterns for probing the visual memory.

The effective use of computer-generated pictures as a trial-unique probe for studying the visual memory is described. The shape of the pattern is determined by means of a fractal algorithm with pseudorandom parameters. This method enables us to easily obtain thousands of moderately complex and sufficiently diversified pictures in series from a given number which serves as the seed of a pseudorandom number generator. We can thereby create a new and unique set of pictures if a new seed is given, as well as retrieve exactly the same pictures in the same sequence as when the original seed is given. These properties eliminate the demand for the massive memory space in a computer otherwise needed to store the entire set of stimulus pictures.     

Mutation analysis of two Japanese patients with Fanconi-Bickel syndrome

Fanconi-Bickel syndrome (FBS), or glycogen storage disease type XI, is a rare autosomal recessive disorder characterized by hepatorenal glycogen accumulation, Fanconi nephropathy, and impaired utilization of glucose and galactose. Recently, this disease was elucidated to link mutations in the glucose transporter 2 (GLUT2) gene. Only three mutations in three FBS families have been reported. Therefore, it is important to elucidate mutations in the GLUT2 gene in FBS by answering the question of whether the syndrome is a single gene disease. In this report, we describe two patients in two unrelated families clinically diagnosed with FBS. No mutation in the entire protein coding region of the GLUT2 gene was detected in patient 1, which suggested that no mutation existed in the GLUT 2 gene, or that some mutations had affected the expression of the GLUT 2 gene. In patient 2, a novel homozygous nonsense mutation (W420X, Trp at codon 420 to stop codon) was detected. These results support the correlation between GLTU2 gene mutation and FBS syndrome. However, many patients must be analyzed to determine whether other genes are involved in FBS. Received: July 16, 1999 / Accepted: September 3, 1999     

MORPHOLOGICAL DIFFERENCES IN HEARTS OF RATS WELL ADAPTED AND POORLY ADAPTED TO CHRONIC HYPOXIA

We carried out an experiment to analyze morphological differences in hearts of rats well adapted and poorly adapted to chronic hypoxia. Male and female Wistar rats, 1 week, 4 weeks and 9 weeks old, were employed on the assumption that adaptive ability was dependent on age and sex. These rats were raised at an altitude of 2,400 m and were kept for 7 to 9 weeks. Control groups were maintained at an altitude of 600 m during the same period of time. Each group consisted of 4 to 6 rats. At the end of the experiment, body weight, heart weight, ratio of heart weight to body weight and hematocrit were measured, and ventricular wall thickness, myocardial fiber diameter, capillary supply and mitochondria were morphometrically studied. Of the 6 experimental groups, the 4-week-old male rats (M2) had the highest body weight, as compared with the other experimental groups. In addition, relative to these other experimental groups, the following features were found for M2. Heart weight was intermediate, heart weight/body weight ratio was low and hematocrit was also low. Ventricular wall thickness was intermediate in the right ventricle (RV) and interventricular septum (IVS) but was thin in the left ventricle (LV). Myocardial fiber diameter was intermediate in the RV, large in the IVS and small in the LV. Capillary supply was intermediate in the RV and dense in the IVS and LV. Mitochondria were small but cristal density and percentage area, estimated from electron micrographs, were found to be high. These data showed that in well developed rats under chronic hypoxia, there is good development of capillary supply with corresponding restriction of cardiac hypertrophy, while hematocrit count and mitochondria are also affected.     

Effects of fluid flow on elution of hydrophilic modifier from dialysis membrane surfaces

When uremic blood flows through dialyzers during hemodialysis, dialysis membrane surfaces are exposed to shear stress and internal filtration, which may affect the surface characteristics of the dialysis membranes. In the present study, we evaluated changes in the characteristics of membrane surfaces caused by shear stress and internal filtration using blood substitutes: water purified by reverse osmosis and 6.7 wt% dextran70 solution. We focused on the levels of a hydrophilic modifier, polyvinylpyrrolidone (PVP), on the membrane surface measured by attenuated total reflectance Fourier transform infrared spectroscopy. Experiments involving 4 h dialysis, 0-144 h shear-stress loading, and 4 h dead-end filtration were performed using polyester-polymer alloy (PEPA) and polysulfone (PS) membranes. After the dialysis experiments with accompanying internal filtration, average PVP retention on the PEPA membrane surface was 93.7% in all areas, whereas that on the PS membrane surface was 98.9% in all areas. After the shear-stress loading experiments, PVP retention on the PEPA membrane surface decreased as shear-stress loading time and the magnitude of shear stress increased. However, with the PS membrane, PVP retention scarcely changed. After the dead-end filtration experiments, PVP retention decreased in all areas for both PEPA and PS membranes, but PVP retention on the PEPA membrane surface was lower than that on the PS membrane surface. PVP on the PEPA membrane surface was eluted by both shear stress and internal filtration, while that on the PS membrane surface was eluted only by internal filtration.     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Cyclic AMP-dependent Cl− secretion induced by thromboxane A2 in isolated human colon

Increased release of thromboxane A₂ (TXA₂) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA₂ analogue (STA₂) on the electrical parameters in isolated human colonic mucosa. In the human mucosa set between Ussing chambers, STA₂ stimulated Cl⁻ secretion in a concentration-dependent manner with an EC₅₀ of 0.06 μ m . The STA₂-induced Cl⁻ secretion was significantly inhibited by ONO-3708 (10 μ m ), a specific TXA₂ receptor antagonist. The effect of STA₂ (0.3 μ m ) was independent of the colonic segment from which the tissue was obtained, from caecum to rectum. Chromanol 293B, an inhibitor of the cAMP-dependent KvLQT1 channel, attenuated the STA₂-induced Cl⁻ secretion in the human colonic mucosa (IC₅₀ value 1.18 μ m ). We found that KvLQT1 mRNA and protein were expressed in all the tested segments of the human colon. The STA₂-induced Cl⁻ secretion was significantly inhibited by 8-bromo-2'-monobutyryladenosine-3',5'-cyclic monophosphorothioate (50 μ m ), a membrane-permeant cAMP antagonist. STA₂ (0.3 μ m ) significantly increased the intracellular cAMP levels and the short-circuit current via TXA₂ receptor in a human colonic cell line. These results suggest that the TXA₂-induced Cl⁻ secretion in the colon is mediated via the cAMP pathway in addition to the Ca²⁺–calmodulin pathway which was previously reported.     

Neuronal protein NP25 interacts with F-actin

Neuronal protein NP25 is a neuron-specific protein present in highly differentiated neural cells, but its functional properties have not been well characterized. NP25 shows high amino acid sequence homology with the smooth muscle cell cytoskeleton-associated proteins, SM22, mp20, and calponin. To gain an insight into the biological functions of NP25, we first examined its subcellular localization in the human neuroblastoma cell line, SK-N-SH. NP25 diffusely distributed in the cytoplasm and fiber-like staining was also observed. It showed that NP25 co-localized with F-actin on stress fibers. A co-sedimentation assay demonstrated that NP25 bound to filamentous actin. Further investigations using fluorescence resonance energy transfer (FRET) technique revealed intracellular binding of NP25 and actin. The significance of the interaction between NP25 and F-actin is discussed.     

Effect of influenza virus vaccine on the expression of human immunodeficiency virus co-receptor CCR5.

BACKGROUND: Administration of influenza vaccine to human immunodeficiency virus (HIV)-infected children can lead to increased viral load. CCR5 and CXCR4 are known to play an important role in HIV cell entry and viral replication. OBJECTIVE: To determine the effects of influenza vaccine on chemokine receptors and on viral load in HIV-infected children. METHODS: Eight HIV-infected children receiving stable therapy and 11 healthy adults were enrolled. Chemokine expression and immune activation were determined before and 48 hours after influenza vaccination. CCR5 and beta-chemokine gene expression were analyzed using real-time polymerase chain reaction. Viral load was measured at baseline, 48 hours, and 6 to 12 weeks. RESULTS: Forty-eight hours after influenza vaccination, mean CCR5 expression was significantly decreased on the CD3 (21.1% vs 11.3% in HIV-infected children; P = .02; and 18.3% vs 10.7% in controls; P = .008) and CD4 (13.0% vs 3.6% in the HIV group; P = .04; and 13.6% vs 6.5% in controls; P = .02) lymphocytes. This was observed in conjunction with an increase in HLA-DR expression on T lymphocytes in HIV-infected children (P = .046). No significant changes were observed in HIV viral load, CD3 and CD8 lymphocyte counts, expression of interleukin 2 receptor and CXCR4, or gene expression of CCR5 and beta-chemokines 48 hours after vaccination. CONCLUSIONS: Influenza virus vaccine markedly decreased chemokine receptor CCR5 expression on CD4 T lymphocytes. However, this immunomodulatory effect does not seem to affect overall viral replication in HIV-infected children who received highly active antiretroviral therapy.     

The PPARgamma ligand, 15-Deoxy-Delta12,14-PGJ2, regulates apoptosis-related protein expression in cholangio cell carcinoma cells

PPARgamma is known to induce apoptosis in malignant tumor cells, but the mechanism of this induction is not well understood. We investigated induction of apoptosis with 15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), a PPARgamma ligand, in cholangio cell carcinoma (CCC) cells (RBE, ETK-1 or HuCCT-1). Apoptosis was induced in RBE and ETK-1 cells with 15d-PGJ2, but not in HuCCT-1 cells, although PPARgamma was expressed in all CCC cells. Apoptosis-related proteins were also expressed, including FLIP, bclx, Apaf-1 and XIAP, but expression levels differed among the three cell lines. RBE cells treated with 15d-PGJ2 showed caspase activation, and it appeared that PPARgamma-induced apoptosis was dependent on caspase activation. However, neither ETK-1 nor HuCCT-1 cells showed significant activation of caspase-8 or -3 with 15d-PGJ2 treatment, raising the possibility of a caspase-independent apoptosis induction pathway. XIAP was down-regulated by 15d-PGJ2 in all three CCC cell lines. Therefore, 15d-PGJ2 induces apoptosis in CCC cells via caspase-dependent or independent pathways. 15d-PGJ2 may also induce down-regulation of XIAP and may promote caspase cascade activation through TNF-family receptor signaling pathways.