Notes: Background. Helicobacter pylori adhering to the human gastric epithelium causes gastric diseases such as ulcer, carcinoma and lymphoma. It is thus important to observe in detail both the surface of the epithelial cells and the H. pylori that adhered to it for the elucidation of H. pylori-induced diseases by scanning electron microscopy (SEM). Since the thick mucus layer blocks the observation of the cell surface and the bacteria, it is generally eliminated during the processing for SEM by roughly mechanical methods, but these treatments also demolish the ultrastructure of the cells. We studied the nonmechanical method for removal of mucus layer of gastric epithelium using pronase.Materials and Methods. To determine the optimal concentration of pronase, mucin was used as a substrate for inhibition of the viscosity. Pronase was added in 2% mucin at the concentration of 10, 50, 100, 500, 1000, 2000 or 5000 unit/ml and the flowing time of the mixture was measured. Based on the digestion experiment, biopsied specimens from 24 patients with dyspepsic symptoms were fixed in glutaraldehyde and then washed in rolling with different concentration of pronase. After the pretreatment by pronase, the specimens were treated according to the standard process for SEM.Results. We succeeded in removing the mucus layer on the surface of epithelial cells from the biopsied specimens fixed in glutaraldehyde by rinsing with 2000 unit/ml pronase for 24 hours.Conclusions. Using our digestive method without destroying the ultrastructure, the earliest stage which H. pylori has adhered onto the human gastric epithelium can be observed for the investigation of H. pylori-induced gastric disorders by SEM.

Notes: This study evaluated the relationship between Helicobacter pylori infection, atrophic gastritis and intestinal metaplasia in Japan.〈section xml:id="abs1-3"〉〈title type="main"〉Materials and Methods.This was a multicenter study performed in 21 centers in Japan. A total of 2455 individuals were enrolled. H. pylori status was determined by validated ELISAs. Atrophic gastritis was diagnosed by histology, endoscopy with Congo Red dye scattering or the Kimura-Takemoto endoscopic classification.〈section xml:id="abs1-4"〉〈title type="main"〉Results.Atrophic gastritis increased from 9.4% in those less than 20 years of age to 〉 70% in those aged 60 or older and was strongly associated with H. pylori infection. The overall prevalence of atrophic gastritis in H. pylori infection was 82.9% (1272/1534) compared with 9.8% (90/921) among uninfected (OR = 44.8; 95% CI = 34.7–57.8). Intestinal metaplasia was present in 43.1% (542/1258) of H. pylori positive persons compared with 6.2% (51/823) among the uninfected (OR = 11.5; 95% CI = 8.5–15.5). Atrophic gastritis in H. pylori positive Japanese was very high in the younger generation (38.5% in those aged 20 or less and 58.5% in those 21–30).〈section xml:id="abs1-5"〉〈title type="main"〉Conclusions.Atrophic gastritis and intestinal metaplasia were strongly associated with H. pylori and not with aging. The fall in prevalence of H. pylori in Japan has not been associated with a corresponding fall in the prevalence of atrophic gastritis among those with H. pylori infection. The high prevalence of the precursor lesion, atrophic gastritis with intestinal metaplasia, among those with H. pylori infection suggests that the risk of development of early gastric cancer will continue to remain high in Japan.

Notes: In this study we examined the functional expression of the extraneuronal monoamine transporter (EMT) in normal human astrocytes (NHA). RT-PCR with EMT-specific primers demonstrated the presence of EMT mRNA in NHA. The RT-PCR products were subjected to restriction-site analysis using three different enzymes (HinfI, SacI and BclI). The restriction patterns with the three enzymes were identical and were exactly as expected from the known restriction map of human EMT cDNA. DNA sequencing was performed for the RT-PCR products from NHA. Sequence analysis demonstrated that the sequences of RT-PCR products were identical to that of EMT. The extract of NHA was immunoblotted with anti-EMT polyclonal antibody raised against EMT polypeptides. Western blotting indicated that anti-EMT polyclonal antibody recognized a band of 63 kDa. Immunocytochemical staining using anti-EMT polyclonal antibody in NHA revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or Golgi membranes, showed a considerable level of immunoreactivity. We examined the time course of temperature-dependent [3H]MPP+ uptake in NHA for 60 min. Temperature-dependent [3H]MPP+ uptake increased in a time-dependent manner for the initial 45 min and almost reached a plateau level (8.70 ± 0.59 pmol/mg protein) at 60 min. In the presence of 3 µm decynium22 (D22) (the most potent EMT inhibitor), temperature-dependent [3H]MPP+ uptake was strongly reduced by 61% (3.39 ± 0.76 pmol/mg protein at 60 min). D22-sensitive [3H]MPP+ uptake was saturable over a MPP+ concentration of 6.25–200 µm. Km for this process was 78.01 ± 7.64 µm and Vmax was 295.4 ± 12.8 pmol/mg protein/min. D22-sensitive [3H]MPP+ uptake was reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding Ba2+ to the uptake buffer. These results provide evidence that the MPP+ transport activity in NHA is potential-sensitive. Moreover, D22-sensitive [3H]MPP+ uptake was independent of extracellular Na+. D22-sensitive [3H]MPP+ uptake was inhibited by D22, various organic cations, steroids and monoamine neurotransmitters. Our results showed that the EMT is functionally expressed in NHA and may also play a key role in the disposition of cationic drugs, neurosteroids, the neurotoxin MPP+ and monoamine neurotransmitters in the brain.

Notes: In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis–Menten constant (Km) of 35.7 ± 4.1 µm and a maximal velocity (Vmax) of 49.1 ± 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA 〉 TBA 〉 TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17β-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.

Notes: We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.

Notes: Background.The current study was designed to evaluate the effect of aging and Helicobacter pylori infection on the gastric mucosa in asymptomatic Japanese adults. Materials and Methods.Eighty-five asymptomatic healthy adults were recruited from a health-screening center in Sapporo. All subjects underwent endoscopy and gastric biopsy, and serum was obtained for IgG antibodies to H. pylori, serum gastrin, and pepsinogen levels. Results.The prevalence of atrophic change of the gastric mucosa assessed by pathological findings increased with age (49% in the 30- to 39-year-old group compared to 89% in those 60 years and older, p 〈 .001). The frequency of intestinal metaplasia also increased with age (38% in the 30- to 39-year-old group compared to 82% in those 60 years and older, p 〈 .001). In contrast, the frequency of atrophic gastritis and intestinal metaplasia was extremely low in the H. pylori seronegative group regardless of age. Mean serum gastrin level in H. pylori-positive adults was significantly greater than in those who were H. pylori-negative (114.3 ± 11.2 compared to 65.8 ± 6.5 pg/ml, p 〈 .03). The serum pepsinogen I-II ratio was significantly lower in those with H. pylori infection than in those without (3.1 compared to 6.6, p 〈 .0001). Conclusions.These results suggest that the chronological changes in the gastric mucosa in Japanese individuals are either entirely related to H. pylori infection or the process is greatly accelerated by H. pylori infection.