Immunoelectron microscopic study of the luminal release of chromogranin A from rat enterochromaffin cells

 Recently we found that raising the intraluminal pressure caused an increase in the luminal release of serotonin from enterochromaffin (EC) cells and serotonin immunoreactivity normally restricted within the secretory granules was diffusely scattered over the extragranular matrix. In the present study we investigated the intracellular localization of chromogranin A, a protein co-stored with serotonin in the EC cells, after stimulating the luminal release of serotonin. In situ vascularly and luminally perfused rat duodenum was exposed to intraluminal pressure and fixed for immunoelectron microscopic study. For immunoelectron microscopy, the pre-embedding DAB reaction for serotonin combined with the postembedding immunogold reaction for chromogranin A was used. Results showed that a number of secretory granules labeled with immunogold chromogranin A immunoreactivity located close to the apical plasma membrane. Some EC cells showed that one part of the apical cytoplasm was protruded into the lumen and a number of secretory granules with immunogold labeling were included in the protruded cytoplasm. These results suggest that EC cells may release chromogranin A into the intestinal lumen together with serotonin, by means of a different manner of secretion from that in serotonin. Received / Accepted: 9 December 1998     

α-Galactosidase gene mutations in Fabry disease: heterogeneous expressions of mutant enzyme proteins

Five point mutations were identified in unrelated Japanese Fabry disease hemizygotes: three new missense mutations, C142Y (⁴²⁵ G A), A156V (⁴⁶⁷ C T), and L166V (⁴⁹⁶ C G) in exon 3; one new splice site mutation at the 3 end of the consensus sequence in exon 4; one previously reported nonsense mutation, W44X (¹³¹ G A). C142Y expressed 50% of the normal enzyme protein in COS-1 cells, but catalytic activity was not detected. Both A156V and L166V expressed significant amounts of residual enzyme activity (6.7% and 9.8%) and enzyme proteins (10% each), the latter were more thermolabile at neutral pH than at acid pH, in vitro.     

Distribution of serotonin-immunoreactive nerve cells and fibers in the rat gastrointestinal tract

 The distribution of serotonin-immunoreactive (5HT-IR) nerve cells and fibers was thoroughly investigated immunohistochemically in the rat stomach, duodenum, jejunum, ileum, and colon. The immunoreactivity of the 5HT neurons was compared between non-treated controls and animals treated with colchicine, colchicine plus 5-hydroxytryptophan (5HTP), colchicine plus pargyline, and reserpine. The intensity of immunoreactivity in nerve fibers as well as nerve cell bodies was enhanced mostly in colchicine plus pargyline treated animals, therefore these animals were used for an observation of precise localization of 5HT in the rat gastrointestinal (GI) tract. Immunoreactivity in the nerve cell bodies and fibers was completely abolished in the GI tract of reserpine treated animals. The pattern of localization and projection of 5HT-IR neurons was similar in all segments of the rat GI tract. 5HT-IR nerve cell bodies were located in the myenteric plexus and showed the distinctive features of Dogiel type I neurons. Prominent bundles of varicose fibers traversed the myenteric ganglia and some of them surrounded the cell bodies of immunopositive and immunonegative neurons. 5HT-IR nerve fibers were located in the submucous plexus, densely entwined about the submucosal blood vessels. Most characteristically, 5HT-IR nerve fibers invaded the lamina propria of mucosa where they underlay the crypt epithelium. In conclusion, the present study showed that 5HT-IR neurons located in the myenteric plexus projected fibers widely in the rat GI tract. The localization of fibers in the lamina propria of mucosa implies that this neuron may exert an important role in the epithelial function of the GI tract. Accepted: 8 October 1996     

The mechanical binding strengths of Helicobacter pylori BabA and SabA adhesins using an adhesion binding assay–ELISA,and its clinical relevance in Japan

To elucidate a potential role for H. pylori BabA and SabA adhesins in the pathogenesis of gastric mucosal lesions, the MBS of BabA and SabA was examined using an in‐house ABA‐ELISA. Ninety isolates from Japanese patients with gastric cancer (n= 43) and non‐cancerous (n= 47) lesions were subjected to an ABA‐ELISA which had been developed in‐house, and sequential analysis of the babA2 middle region. The BabA‐MBS was significantly higher in the cancer than the non‐cancer group (P= 0.019), but there was no significant difference for SabA‐MBS. A weak correlation between BabA‐MBS and SabA‐MBS (r= 0.418) was observed, the positive correlation being higher in the cancer than the non‐cancer group (r= 0.598 and 0.288, respectively). The isolates were classified into two groups: a BabA‐high‐binding and a BabA‐low‐binding group (in comparison to the average for BabA‐MBS). The average SabA‐MBS in the BabA‐high‐binding group was significantly higher than in the BabA‐low‐binding group (P < 0.0001). Analysis of babA2 middle region diversity (AD1–5) revealed that AD2‐type was predominant in isolates irrespective of BabA‐MBS. H. pylori BabA‐MBS might have an effect on SabA‐MBS and relate to the severity of gastric disorders, including gastric cancer. Evaluation of MBS of the combined two adhesins would be helpful for predicting damage in the H. pylori infected stomach.     

Contribution of Disulfide Bonds to the Characteristics of Casein Micelles

Drainage in foam was analyzed by the capillary model proposed by Haas and Johnson. Foam was produced by introducing air into an ovalbumin solution through a spinneret. The electric conductivity at selected positions in the foam and the drained liquid height below the foam were measured at constant intervals. The measured electric conductivity was converted to the liquid volume fraction by Prager’s equation. The capillary model described well not only the liquid leakage rate from foam but also the liquid volume fraction in foam. The ratio of the liquid volume fraction in foam to the volume fraction of the Plateau border was much larger than the value estimated from Haas and Johnson’s result. It was confirmed that a time constant, T, involved in the model was suitable for estimating foam stability only by one parameter. However, T is affected not only by material characteristics but also by the foam height. On the other hand, since the ratio of the liquid volume fraction in foam to the volume fraction of the Plateau border reflects the material characteristics, it may be used for comparing the foam stability between materials with the same viscosity and density.     

The effects of socioeconomic globalization on health and aging in highlanders compared to lowlanders in Yunnan, China, and Kochi, Japan

In highland areas worldwide, socioeconomic globalization is progressing urbanization and environmental destruction. Urbanization is caused by socioeconomic globalization of development of transportation, movement or immigration of people, and prevailing market economy. Lifestyle-related diseases, such as diabetes and hypertension, are increasing worldwide with greater longevity and changes in lifestyles. Highland areas may also be affected by globalization and the people living there may be especially vulnerable. Our objective was to disclose the features of lifestyle-related diseases and the human aging phenomena of highland people affected by their increasingly urbanized lifestyles by undertaking a detailed geriatric assessment. Our assessment included firstly comparing the prevalence of hypertension and neurobehavioral functions in community-dwelling Tibetan elderly in Shangri-la, Yunnan, China (altitude 3,300 m) with Thai elderly in the city of Jing Hong, Yunnan, China (altitude 500 m) and Japanese in Tosa, Kochi, Japan (altitude 300 m). Secondly, differences in the prevalence of hypertension, obesity, and neurobehavioral function were analyzed between people in an urban area, Jiang Tang, and rural areas and in association with their economic status in Nish in Shangri-la. High prevalences of lifestyle-related diseases such as hypertension and obesity were shown in highlanders, especially those in an urban area. Geriatric functional ability was associated with economic status. Notwithstanding a higher prevalence of physical disorders and lowered functional abilities, a higher quantitative quality of life was found in Shangri-la than in Tosa. We concluded that highland-dwelling people were vulnerable and susceptible to lifestyle-related diseases resulting from socioeconomic globalization.     

Fabry disease: correlation between structural changes in α-galactosidase, and clinical and biochemical phenotypes

Fabry disease comprises classic and variant phenotypes. The former needs early enzyme replacement therapy, and galactose infusion is effective for some variant cases. Attempts of early diagnosis before manifestations appear will begin in the near future. However, it is difficult to predict the phenotype, to determine the therapeutic approach, only from genetic information. Thus we attempted structural analysis from a novel viewpoint. We built structural models of mutant -galactosidases resulting from 161 missense mutations (147 classic and 14 variant), and evaluated the influence of each replacement on the structure by calculating the numbers of atoms affected. Among them, 11 mutants, biochemically characterized, were further investigated by color imaging of the influenced atoms. In the variant group, the number of atoms influenced by amino-acid replacement was small, especially in the main chain. In 85% of the cases, less than three atoms in the main chain are influenced. In this group, small structural changes, located apart from the active site, result in destabilization of the mutant enzymes, but galactose can stabilize them. Structural changes caused by classic Fabry mutations are generally large or are located in functionally important regions. In 82% of the cases, three atoms or more in the main chain are affected. The classic group comprises dysfunctional and unstable types, and galactose is not expected to stabilize the mutant enzymes. This study demonstrated the correlation of structural changes, and clinical and biochemical phenotypes. Structural investigation is useful for elucidating the bases of Fabry disease and clinical treatment.     

Immunoelectron microscopic study of the luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure

 Since definitive morphological studies showing the luminal release of serotonin have not been reported, we used a perfused system which allows physiological monitoring and biochemical as well as morphological evidence indicating release of serotonin from enterochromaffin cells. Isolated vascularly and luminally perfused rat duodenums exposed to 5–35 cmH₂O of luminal pressure were measured for release of serotonin into the blood vessels and intestinal lumen. Immediately after raising the luminal pressure, the duodenum was fixed for immunoelectron microscopic localization of serotonin. Peristaltic contraction and serotonin content of the perfusates were continuously measured. The luminal release of serotonin increased with elevated intraluminal pressure, but the vascular release of serotonin was not altered. Tetrodotoxin had no effect on the pressure-stimulated luminal serotonin release. Enterochromaffin cells in control animals without increased luminal pressure contained immunogold-labeled secretory granules in the apical and basal cytoplasm. After intraluminal pressure increased, many apical secretory granules were no longer dense and immunogold particles were localized over the cytoplasmic matrix and microvilli. These findings indicate that luminal serotonin release is increased after raising the intraluminal pressure and serotonin, normally stored in the secretory granules of enterochromaffin cells, appears to be released into the cytoplasmic matrix and then diffuses or is transported into the intestinal lumen. Accepted: 15 January 1997     

Effect of reserpine on 5-hydroxytryptophan (5HTP)-immunoreactive neurons in the rat brain

By immunohistochemistry of rat brain in conjunction with a specific antibody against 5-hydroxytryptophan (5HTP), we examined immunoreactivity to 5HTP in neurons, from which 5-hydroxytryptamine (5HT; serotonin) was depleted by reserpine treatment. The distribution patterns of 5HTP-positive neurons overlapped with those of 5HT neurons. Treatment with reserpine (5 mg/kg, 90 min before death) caused a complete suppression of 5HT-positive staining, but 5HTP-immunostaining remained in perikarya of the nuclei raphe dorsalis, centralis superior and obscurus. Treatment with reserpine (25 mg/kg, 90 min before death) suppressed the 5HTP-immunoreaction in certain perikarya (e.g. of the nucleus raphe dorsalis) and fibres; however, 5HTP-immunostaining remained in perikarya of the nuclei centralis superior and raphe obscurus. This suggests that these neurons synthesize more 5HTP by a process which appears to be stimulated by reserpine.