A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control

Ghrelin is a 28 a.a. gastric peptide, recently identified as a natural ligand of the growth hormone secretagogue receptor (orphan receptor distinct from the receptor for growth hormone releasing hormone). In the present study, radioimmunoassay demonstrated ghrelin-like material in the rat oxyntic mucosa with moderate amounts also in antrum and duodenum. Small amounts were found in the distal intestines and pancreas. Northern blot analysis revealed abundant ghrelin mRNA in the oxyntic mucosa. Immunocytochemistry demonstrated ghrelin-immunoreactivity in endocrine-like cells in the oxyntic mucosa. Such cells occurred in low numbers also in the antrum and duodenum. The rat oxyntic mucosa is rich in endocrine (chromogranin A/pancreastatin-immunoreactive) cells, such as the histamine-rich ECL cells (65-75% of the endocrine cells), the A-like cells (20-25%) and the D cells (somatostatin cells) (10%). The ghrelin-immunoreactive (IR) cells contained pancreastatin but differed from ECL cells and D cells by being devoid of histamine-forming enzyme (ECL cell constituent) and somatostatin (D cell constituent). Hence, ghrelin seems to occur in the A-like cells. The ghrelin-IR cells in the antrum were distinct from the gastrin cells, the serotonin-containing enterochromaffin cells and the D cells. Conceivably, ghrelin cells in the antrum and distally in the intestines also belong to the A-like cell population. The concentration of ghrelin in the circulation was lowered by about 80% following the surgical removal of the acid-producing part of the stomach in line with the view that the oxyntic mucosa is the major source of ghrelin. The serum ghrelin concentration was higher in fasted rats than in fed rats; it was reduced upon re-feeding and seemed unaffected by 1-week treatment with the proton pump inhibitor omeprazole, resulting in elevated serum gastrin concentration. Infusion of gastrin-17 for 2 days failed to raise the serum ghrelin concentration. Omeprazole treatment for 10 weeks raised the level of HDC mRNA but not that of ghrelin mRNA or somatostatin mRNA in the oxyntic mucosa. Hence, unlike the ECL cells, ghrelin-containing A-like cells do not seem to operate under gastrin control.     

The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin-binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins.     

Structure of the O-glycosidically linked carbohydrate units of rat brain glycoproteins.

The O-glycosidically linked carbohydrate units of rat brain glycopeptides were released as reduced oligosaccharides with NaOH/NaBH4 treatment. Five oligosaccharides were isolated using gel filtration, ion-exchange chromatography and preparative thin-layer chromatography. Studies employing periodate oxidation, methylation analysis, chromium trixide oxidation and gas-liquid chromatography-mass spectrometry indicated the following structures: (I) alpha-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (II) beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (III) N-acetylneuraminyl-[beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol], (IV) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol and (V) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)[N-acetylneuraminyl-(2 leads to 6)]-N-acetylgalactosaminitol.     

Intestinal epithelial cell proliferation and migration in Atlantic salmon, Salmo salar L.: effects of temperature and inflammation

A 28-day feeding trial was carried out to characterise intestinal epithelial cell (IEC) turnover in Atlantic salmon (Salmo salar L.) post-smolts in seawater. Four groups of fish raised at two temperatures of 8°C or 12°C and fed two different diets were investigated. The diets included a reference maize gluten and fishmeal-based diet (FM) and an experimental enteropathy-causing diet containing 20% extracted soybean meal (SBM). IEC proliferation and migration were investigated by labelling cells with the in vivo proliferation marker 5-bromo-2′-deoxyuridine (BrdU). Proliferating cell nuclear antigen (PCNA) labelling was used as a control for identifying proliferating cells. Samples of the proximal (PI), mid (MI) and distal (DI) intestinal regions were collected at five time points (3 h–28 days) over the experimental period. Histologically, FM-fed fish had normal mucosa, whereas the SBM-fed fish developed DI enteropathy. Major zones of cell proliferation were observed in the mucosal fold bases for all intestinal regions. Over time, BrdU-labelled cells migrated up mucosal folds to the tips before being lost. Migration rates were dependent on intestinal region, temperature and diet. Highest migration rates were observed in the PI followed by the MI and DI for FM-fed fish. Diet and temperature barely affected migration in the PI and MI. Migration in the DI was most sensitive to diet and temperature, with both SBM and the higher water temperature increasing proliferation and migration rates. The slow IEC turnover in the DI might help to explain the sensitivity of this region to dietary SBM-induced enteropathy.     

Occurrence of N-acetylglucosamine 6-phosphate in complex carbohydrates. Characterization of a phosphorylated sialyl oligosaccharide from bovine colostrum

The occurrence of phosphate-containing sialyl oligosaccharides in bovine colostrum was investigated. Two major sialyl oligosaccharide phosphates were identified, one of which was structurally similar to the previously characterized sialyl oligosaccharide 1-phosphates of human urine. The second sialyl oligosaccharide phosphate of bovine colostrum was found to be of a novel type. Structural studies including monosaccharide and phosphate analysis, glycosidase and phosphatase treatments, methylation analysis, and periodate treatment indicated the structure of this compound to be NeuAc alpha 2-6Gal beta 1-4GlcNAc-6-P. This provides the first evidence for the occurrence of N-acetylglucosamine 6-phosphate as an integral component in complex carbohydrates.     

The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes

Two murine mAb have been generated with a reactivity toward a 30,000 m.w. DNA binding protein found on the cell surface of human leukocytes; mAb 12A has an IgG1/k isotype, and mAb 24T has an IgG2b/k isotype. Both react with the DNA binding domain or adjacent region of the putative DNA receptor and inhibit the binding of [3H]DNA to PBMC at concentrations as low as 100 ng/ml. Stoichiometric studies indicate that both mAb react with monocytes and T cells with a kDa of 10(-7) M; about 0.5 x 10(6) molecules bind per cell at saturation. Flow cytometry indicated that 67% of lymphocytes and 98% of monocytes bore the DNA receptor. Dual labeling studies showed that 90% of B cells and 50% of T cells express the receptor; 50% of CD4+ T cells are receptor positive. Immunomatrices constructed with both mAb 12A and 24T allowed the receptor to be purified to a high degree of purity. A single protein of Mr 30,000 was readily observed after SDS-PAGE and silver staining of the gel; after electropheretic transfer of nitrocellulose this protein was shown to be a DNA binding molecule by use of a probe of biotin labeled DNA. These experiments provide further evidence to support the existence of a specific DNA receptor on human leukocytes; the availability of mAb to the receptor should be useful in its further characterization.     

Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.     

Alpha-tocopherol and MRI Outcomes in Multiple Sclerosis – Association and Prediction

Objective

Alpha-tocopherol is the main vitamin E compound in humans, and has important antioxidative and immunomodulatory properties. The aim of this study was to study alpha-tocopherol concentrations and their relationship to disease activity in Norwegian multiple sclerosis (MS) patients.

Methods

Prospective cohort study in 88 relapsing-remitting MS (RRMS) patients, originally included in a randomised placebo-controlled trial of omega-3 fatty acids (the OFAMS study), before and during treatment with interferon beta. The patients were followed for two years with repeated 12 magnetic resonance imaging (MRI) scans and nine serum measurements of alpha-tocopherol.

Results

During interferon beta (IFNB) treatment, each 10 µmol/L increase in alpha-tocopherol reduced the odds (CI 95%) for simultaneous new T2 lesions by 36.8 (0.5–59.8) %, p = 0.048, and for combined unique activity by 35.4 (1.6–57.7) %, p = 0.042, in a hierarchical regression model. These associations were not significant prior to IFNB treatment, and were not noticeably changed by gender, age, body mass index, HLA-DRB1*15, treatment group, compliance, or the concentrations of 25-hydroxyvitamin D, retinol, neutralising antibodies against IFNB, or the omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid. The corresponding odds for having new T1 gadolinium enhancing lesions two months later was reduced by 65.4 (16.5–85.7) %, p = 0.019, and for new T2 lesions by 61.0 (12.4–82.6) %, p = 0.023.

Conclusion

During treatment with IFNB, increasing serum concentrations of alpha-tocopherol were associated with reduced odds for simultaneous and subsequent MRI disease activity in RRMS patients.