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The glycoprotein-gene (G gene) -deleted rabies virus (RV) vector is a powerful tool to examine the function and structure of neural circuits. We previously reported that the deletion of the G gene enhances the transgene expression level of the RV vector. However, the mechanism of this enhancement remains to be clarified. We presume that there are two possible factors for this enhancement. The first factor is the glycoprotein of RV, which shows cytotoxicity; thus, may cause a dysfunction in the translation process of infected cells. The second possible factor is the enhanced expression of the L gene, which encodes viral RNA polymerase. In the RV, it is known that the gene expression level is altered depending on the position of the gene. Since G-gene deletion displaces the L gene in the genome, the expression of the L gene and viral transcription may be enhanced. In this study, we compared the transgene expression level and viral transcription of three recombinant RV vectors. The effect of glycoprotein was examined by comparing the viral gene expression of G-gene-intact RV and G-gene-replaced RV. Despite the fact that the L-gene transcription level of these two RV vectors was similar, the G-gene-replaced RV vector showed higher viral transcription and transgene expression level than the G-gene-intact RV vector. To examine the effect of the position of the L gene, we compared the viral gene expression of the G-gene-deleted RV and G-gene-replaced RV. The G-gene-deleted RV vector showed higher L-gene transcription, viral transcription, and transgene expression level than the G-gene-replaced RV vector. These results indicate that G-gene deletion enhances the transgene expression level through at least two factors, the absence of glycoprotein and enhancement of L-gene expression. These findings enable investigators to design a useful viral vector that shows a controlled desirable transgene expression level in applications.

The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level.

Changes in gastric mucus glycoprotein (mucin) isolated from pirenzepine-treated rats with or without ethanol (50%)-induced gastric damage were studied. The prior administration of pirenzepine inhibited significantly and dose-dependently the occurrence of macroscopically observable hemorrhagic lesions induced by treatment with ethanol. The gastric mucosa was separated into the surface mucosa, including the mucus gel layer, and the deeper mucosa by mechanical scraping, and the mucin in each was isolated. In ethanol-treated animals the mucin content of the deep corpus mucosa was significantly reduced to 68% the control value. This reduction was inhibited by pretreatment with pirenzepine. In the surface mucosa mucin content was also reduced to 48% of control value by ethanol treatment, but pirenzepine pretreatment increased mucin content to 235% the control value. Total mucin content in the entire stomach essentially resumed the control level by pretreatment with 100 mg/kg of pirenzepine. A single oral administration of pirenzepine (100 mg/kg) caused no change in total mucin content, but mucin in the deep corpus mucosa selectively and significantly increased to 124% the control. These and the results of carbohydrate analysis of purified mucin indicate that pirenzepine administration possibly accelerates the secretion of accumulated deep corpus mucus, retains this deep mucus on surface mucosal mucus, and protects the gastric mucosa in ethanol-induced gastric damage. This may be related to the antiulcerogenic effects of pirenzepine.

A method was established for recovering the mucus gel layer of rat gastric mucosa without damage to underlying surface epithelium. The mucus gel was solubilized by stirring the gastric mucosa in a solution of N-acetylcysteine (NAC), a mucolytic agent. Optimal mucus gel solubilization was possible by treatment with 2% NAC for 5 minutes at room temperature. Mucus glycoprotein was quantitatively extracted and measured from the mucus gel sample obtained by the NAC treatment. This treatment caused no damage to surface epithelial cells, as observed by a light microscope. Besides NAC, pronase solution was also adequate for solubilizing the mucus gel layer without any damage to the surface epithelium. However, extraction and measurement of mucus glycoprotein from the pronase-treated mucus gel sample was not possible due to contamination by high molecular hexose-containing substances which were eluted along with the mucus glycoprotein from the column of Bio-Gel A-1.5m. This NAC method was used to examine changes in mucus glycoprotein content in the mucus gel at one hour following the oral administration of 16,16-dimethyl prostaglandin E2. A significant increase in mucus glycoprotein of the gel was brought about by the prostaglandin treatment. Thus, the present method was suitable for estimating the amount of mucus secreted in to the mucus gel layer.

In our previous reports, low dose acid secretagogues caused increase in corpus mucus glycoprotein content in rats. This increase was not due to the direct action of protons. The present study was conducted to determine whether this increase has a mucosal function or not Rats were given histamine (0.8 mg/kg) intraperitoneally or tetragastrin (12 micrograms/kg) subcutaneously. One hour following the acid secretagogues administration, the animals were given 1 ml of 30, 40 or 50% ethanol orally and then sacrificed after one hour. Gastric mucus glycoproteins were isolated from the corpus region of stomach. The following results were obtained: 1) Histamine prevented the gastric mucosal damage and the decrease of corpus mucus glycoprotein content induced by 40% ethanol ingestion. 2) Tetragastrin prevented the gastric mucosal damage induced by 30 approximately 50% ethanol ingestion and the decrease of corpus mucus glycoprotein content induced by 40 approximately 50% ethanol ingestion. Based on these results, the increase of corpus mucus glycoprotein content induced by acid secretagogues has a mucosal function. Moreover, tetragastrin strengthened the mucosal defence together with acid secretion.

Radiolabeled glycoconjugates were prepared by incorporation of [35S]sulfate and [3H]glucosamine into the gastric mucosa of human gastric resection specimens, using organ culture techniques. Specimens which incorporated higher amounts of [35S]sulfate into high molecular glycoconjugates (Fr-1) were designated as group I. The average specific activity in Fr-1 of group I was 30 times that of group II, but the glucosamine uptake remained about the same in all cases. All specimens in group I were positive to intestinal metaplasia in their adjacent mucosal area. The sulfated glycoproteins in Fr-1 of group I were found to incorporate 80% of the 35S and the remaining 20% by sulfated glycosaminoglycans. But the glycoconjugates constituting all the Fr-1 of group II were identified as glycoproteins and incorporating 35S only to a small extent were identified as sulfated glycoproteins. The susceptibility of the glycoproteins from group I mucosa to pronase digestion was different from that of group II. The glycoproteins synthesized in group I mucosa were as sulfated glycoproteins as well as nonsulfated glycoproteins and glycosaminoglycans, while the group II glycoproteins consisted primarily of nonsulfated glycoproteins.

Quantitative changes of gastric mucosal glycoproteins with the gastric damage induced by acetylsalicylic acid (aspirin) in rat have been studied. Gastric injury was easily observed macroscopically within one hour after the oral administration of aspirin. The most striking changes occurred at five hours, and the injury was overcome within nine hours after dosing. The glycoproteins extracted from rat stomack with Tris buffer containing Triton X-100 were fractionated on Bio-Gel A-1.5 m column chromatography and divided into three fractions. The first peak, corresponding to gastric mucus macromolecular neutral and acidic glycoproteins with or without sulphate (Fr.I), was diminished after aspirin administration. A considerable alteration of Fr.I (49% of control) appeared at three hours, and a gradual return to the control value was observed subsequently. The changes in the amount of the glycoproteins were detected before the macroscopical changes of the mucosa. These results suggest that gastric ulceration induced by aspirin may be caused by a deficiency of gastric mucus macromolecular glycoproteins of gastric mucus.

Gastric mucus glycoproteins were extracted with 2% Triton X-100 from rat gastric corpus and antrum and purified by CsCl equilibrium centrifugation. Corpus mucus glycoproteins were degraded into what appeared to be two "subunits" (Mw 4.4 x 10(5) and 6 x 10(6)) by the reduction of disulfide bonds. Papain digestion of the latter produced glycopeptides with a molecular weight of approximately 4.4 x 10(5). This type of subunit had carbohydrate chains with about 9 sugars attached to every 2 amino acid residues. Papain digestion of the former type of subunit revealed no change in the elution profile on Bio-Gel A-15m. This type of subunit had carbohydrate chains with 17-19 sugars attached to every 3 amino acid residues. The subunit of antral mucus glycoproteins was essentially the same as the former type of corpus subunits in molecular weight (Mw 4.4 x 10(5)) and average oligosaccharide chain length. These results suggest that there are two distinct types of mucus glycoprotein subunits in rat stomach.

1. The mucosal macromolecular glycoproteins were extracted from forestomach, corpus and antrum region of rat whose weight ratio was 2:5:1, respectively. 2. The glycoproteins were fractionated on Bio-Gel A-1.5 m. 96% of the glycoproteins was localized in glandular stomach. 3. The carbohydrate of the glycoproteins composed of N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and sialic acid in the proportions 1.0:2.7:3.2:1.0:0.14 for corpus, while 1.0:1.4:1.5:0.7:0.04 for antrum.

Gastric lesions induced by aspirin increased the ulcer index and incidence with prolongation of fasting time. Aspirin decreased gastric mucus glycoprotein in both the corpus and antrum. However, the rate of decrease in mucus glycoprotein induced by aspirin differed according to feeding habits and the gastric region. Qualitative change in corpus mucus glycoprotein was induced by aspirin.

The extent of gastric damage induced by aspirin was found to differ according to rat strain. The occurrence of ulcers varied, from high to low, in the following strain order: Donryu, Sprague-Dawley (SD) and Wistar. The content of corpus mucus glycoprotein was essentially the same in all the strains: about 6 mg as hexose of dry tissue. Antral mucus glycoprotein content increased in the order Wistar, SD and Donryu: 7.1, 8.3 and 9.1 mg, respectively. Gastric mucus glycoprotein carbohydrate composition was essentially the same in all three strains. The relatively low proportions of N-acetylglucosamine, galactose and sialic acid from the antrum was a characteristic feature in contrast to mucus glycoprotein from the corpus which contained a high proportion of these sugars.

The effects of two synthetic prostaglandin E derivatives on mucus glycoproteins in the stomachs of rats were evaluated. Neither derivative caused change in mucus glycoprotein content in the gastric corpus or antrum, but both increased the biosynthetic activity of mucus glycoproteins in these regions (about 15-30%). Furthermore, the effects of the pretreatment of these derivatives on conserving mucus glycoproteins in ethanol-induced gastric lesions were examined. Treatment with these derivatives 60 min prior to 70% ethanol administration markedly inhibits the decrease in gastric mucus glycoprotein content caused by 70% ethanol. But the glycoprotein content under these pretreatment conditions was significantly less (12-19%) than that in the control group without any drug treatment.

The effects of cimetidine on gastric mucus glycoprotein biosynthesis in rat gastric mucosa were investigated using organ culture technique. 2 h following oral administration of cimetidine (40 mg/kg body weight), corpus tissue was placed in the organ culture. Mucus glycoprotein synthesis was accelerated to 137% of control (p less than 0.01). In order to investigate the direct effect of cimetidine on mucus glycoprotein biosynthesis, corpus tissue was cultured in the medium containing cimetidine. 100 microM cimetidine enhanced mucus glycoprotein biosynthesis to 174% (p less than 0.01). Cimetidine was found to stimulate gastric mucus glycoprotein biosynthesis both in vivo and in vitro.

Sulfated mucus glycoprotein biosynthesis by human gastric mucosal biopsies was studied. Human gastric healthy specimens and cancer specimens were obtained from antral and corpus normal mucosal areas by biopsy. A comparison was made of the biosynthesized sulfated mucus glycoproteins assessed by the incorporation of 35S-sulfate and 14C-glucosamine into the specimens using an organ culture technique. A significant uptake of 35S-sulfate or 14C-glucosamine into the mucus glycoproteins was demonstrated in all specimens. 14C-glucosamine incorporation into the glycoproteins was almost the same for all biopsies. The synthesis of sulfated glycoprotein in healthy antral mucosa was about four times that in corpus mucosa. Significantly raised levels in sulfated glycoprotein biosynthesis were found in cancer specimens, being about 30 and 13 times higher in antrum and corpus mucosa than that of healthy specimens, respectively.

Biosynthetic activity of gastrin mucus glycoprotein in rats after fasting for 24 and 72 hr was studied by the organ culture technique. Fasting produced a slight reduction in gastric mucus glycoprotein biosynthesis in the corpus and antrum (about 70-90% of fed rats). Sulfation of gastric mucus glycoprotein was restrained in the corpus (18% in control for 72 hr).

The efficacy of two anti-ulcer drugs, Cimetidine and Cetraxate, on the mucus glycoproteins of gastric mucosa in the aspirin-induced gastric damage was studied in rats. Simultaneous or previous oral administration of Cimetidine or Cetraxate with aspirin reduced the diminution of the mucus glycoproteins which was occurred by aspirin administration. The recovery of the content of mucus glycoprotein in drug dosed rats occurred within 3 h after aspirin dosing and was nearly 90% of control at 5 h in all cases. Single administration of Cetraxate or Cimetidine produced an increase in the mucus glycoprotein content greater than that of the untreated control. Although a macroscopical method for the measurement of gastric damage was applied to this work, neither erosions nor linear ulcers were observed in all cases except the single administration of aspirin. The biochemical method used should be able to assess the efficacy of drugs on the mucosal lesion which cannot be expressed as the ulcer index.

The qualitative changes of gastric mucus glycoproteins occurring after aspirin dosing in rats were further investigated. Mucus glycoprotein contents of test animals were 55 and 53% of the control value at 3 and 7 h after dosing, respectively. The glycoproteins were further fractionated by CM-Sepharose CL-6B chromatography. 5 fractions were separated, 1 unadsorbed (fraction 1) and 4 adsorbed (fractions 2-5). Ratios of the major fractions 1:2:4 were 36:47:17 for control, 40:35:25 3 h after aspirin dosing, and 47:33:20 7 h after aspirin administration. Fractions 1, 2, and 4 corresponded to sulfomucin and sialomucin (relatively high content of sialic acid), sialomucin, and neutral mucin, respectively. While the absolute amount of each mucus glycoprotein fraction decreased with aspirin administration, the diminution of fraction 2 was distinct.

For assessing the participation of mucus glycoproteins in the cytoprotective process, mucus glycoprotein content in the rat gastric mucosa was measured after treatment with 70% ethanol with or without prostaglandin (PG) E derivatives or 20% ethanol pretreatment. Oral administration of two synthetic PGE derivatives did not cause any significant changes in mucus glycoprotein content. Seventy percent ethanol administration caused marked reduction in mucus glycoprotein content (about 50% of control) as well as severe gastric mucosal damage. Treatment with two PGE derivatives (10-100 micrograms/kg) prior to 70% ethanol administration markedly inhibited the gross mucosal lesion, whereas the glycoprotein content under these conditions was significantly less than that in the untreated control group (ranging from 67-88% of control). Pretreatment with 20% ethanol markedly inhibited the gross mucosal damage caused by 70% ethanol dosing but the inhibition in the reduction of mucus glycoprotein content was restricted to about 80% of the untreated controls. In summary, cytoprotection induced by PG was not accompanied by entire conservation of intramucosal mucus glycoprotein in the gastric mucosa.

Investigations were carried out to determine the effects of omeprazole on mucus glycoprotein in rat gastric mucosa. Quantitative and qualitative changes in mucus glycoprotein, isolated from omeprazole-treated rats with or without acetylsalicylic acid-induced gastric damage, and the degree of gastric mucosal injury were studied. Omeprazole, in a dose-dependent manner, prevented macroscopical mucosal damage induced by acidified acetylsalicylic acid. A considerable decrease in corpus mucus glycoprotein (54%) and antral mucus glycoprotein (72%) was observed in acetylsalicylic acid-treated rats and acetylsalicylic acid reduced the amounts of fucose and galactose of corpus mucus glycoprotein. Pretreatment with omeprazole partially restored a quantitative reduction and a qualitative change of carbohydrate portion in corpus mucus glycoprotein induced by acetylsalicylic acid. A single administration of a high dose of omeprazole (200 mumol/kg) also induced the reduction of corpus mucus glycoprotein content (82%), although no changes in the carbohydrate component were observed. The decrease in corpus mucus glycoproteins induced by omeprazole might be due to a cause different from that of acetylsalicylic acid-induced mucus glycoprotein reduction.

The site of the synthesis of the sulfated glycoproteins in rat gastric mucosa using organ culture system has been studied. About 98% of the 35S radioactivity associated with the glycoproteins was found in corpus region. The sulfated glycoproteins were synthesized mainly in corpus, poorly in antrum and none in forestomach. The synthesized sulfated glycoproteins were heterogeneous by equilibrium centrifugation in CsCl density gradients. The 35S-labeled oligosaccharides released from the glycoproteins under alkaline condition were composed of 4-8 sugar residues.

Expression of members of the epidermal growth factor family, including epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin (AR), and Cripto, as well as their putative receptor, epidermal growth factor receptor (EGFR), was studied immunohistochemically in human gastric mucosa to evaluate their possible roles in cell proliferation of normal and regenerative gastric mucosa. We also examined the correlation betwen cell proliferation and EGFR by double immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and EGFR. In normal gastric mucosa, TGF-alpha, Cripto, and AR immunoreactivities were observed in the surface epithelial and parietal cells of gastric fundic glands, respectively. EGF immunoreactivity was not observed in any of normal mucosa examined. EGFR immunoreactivity was detected on foveolar cells in proliferative zones and in parietal cells. Double immunostaining revealed that EGFR immunoreactivity was distributed much more widely than PCNA immunoreactivity. PCNA positive epithelial cells adjacent to gastric ulcer margin expressed relatively intense EGFR but did not express any of the growth factors examined. On the other hand, relatively intense immunoreactivity of both TGF-alpha and Cripto was detected in PCNA-negative regenerative epithelium located distant from gastric ulcer margin. Relative immunoreactivity of AR in regenerative gastric epithelium associated with ulcer was not different from that in normal gastric mucosa. TGF-alpha, AR, and Cripto are considered to play important roles in normal gastric mucosal proliferation, and TGF-alpha and Cripto may be involved in ulcer healing, possibly via a paracrine mechanism. [ABSTRACT FROM AUTHOR]