Prostaglandin protects isolated guinea pig chief cells against ethanol injury via an increase in diacylglycerol.

We studied cellular processes activated by prostaglandins (PG) that are involved in the protection of gastric chief cell injury estimated in terms of dye exclusion test, release of lactate dehydrogenase (LDH), or 51Cr from prelabeled chief cells. Pretreatment of chief cells with 3 x 10(-6) M PGE2 or PGE1 at 37 degrees C and pH 7.4 for 15 min maximally reduced not only ethanol- but also taurocholic acid-caused LDH release from chief cells. PGs equipotently stimulated increases in the accumulation of diacylglycerol and cyclic AMP without elevating intracellular Ca2+ concentrations in gastric chief cells. The rank order of the potency was equal to that of PGs to reduce the injury. Pretreatment of chief cells with synthetic 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 12-o-tetradecanoyl phorbol 13-acetate (TPA) reduced the injury of chief cells, while 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, failed to reduce the injury and 1-(5-isouinolinylsulfonyl)-2-methylpiperazine (H7) blocked the protective action of PGE2. On the other hand, forskolin and dbcAMP had no effect on ethanol-caused LDH release and diacylglycerol formation in chief cells. These results suggest that PGE2 and PGE1 possess the direct protective action against ethanol- or taurocholic acid-caused injury in chief cells, presumably through the activation of the diacylglycerol/protein kinase C signaling pathway.

p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells.

To further investigate the role of p53 gene inactivation in gastric tumorigenesis, the mutational status of the p53 gene in primary human gastric cancer samples was examined. Reverse transcriptase polymerase chain reaction and subsequent direct sequencing of the p53 gene from gastric cancer samples revealed frequent point mutations of the p53 gene: some of these coincided with those previously identified in gastric cancer cell lines. In addition, both allelic deletion analysis using pYNZ 22 and polymerase chain reaction-restriction fragment length polymorphism analysis demonstrated an allelic deletion of the p53 gene in cancer tissue which contained a point mutation of the p53 gene in the remaining allele. Transfection of the wild-type or mutant p53 genes into gastric cancer cells showed that the wild-type but none of the mutated p53 genes suppressed the colony formation of gastric cancer cells. Furthermore, the incorporation of thymidine into DNA was reduced in cancer cells expressing the wild-type p53 gene. The glutathione S-transferase-wild type p53 fusion protein bound to simian virus 40 large T antigen in COS-1 cell lysate. None of the p53 fusion proteins containing mutations at codons 143, 175, 248, or 273 bound to simian virus 40 large T antigen. By contrast, two different mutant p53 fusion proteins containing mutations specifically observed in gastric cancer bound to simian virus 40 large T antigen. These results indicate that inactivation of the p53 gene through mutations and the allelic deletion may play an important role in gastric tumorigenesis. These mutations may cause a conformational change in the p53 protein resulting in the loss of the suppression by p53 of the growth of gastric cells, partly through disruption of the association of p53 protein with a cellular component.

Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumor immunity.

Tumor-infiltrating lymphocytes, particularly CD8(+) T cells, could be a manifestation of antitumor immunity. We clinicopathologically analyzed the biological significance of tumor-infiltrating lymphocytes in 221 patients with renal cell carcinoma without preoperative treatments. More abundant infiltration of tumor tissue not only by CD8(+) but also CD4(+) T cells was associated with shorter survival of the patients, because of the positive correlation between the number of lymphocytes and representative tumor grade factors. This suggests that immune cell reactions are more pronounced as the tumor grade/biological malignancy progresses, probably because of increased antigenicity of tumor cells. We next analyzed the proliferative activity of CD8(+) T cells that infiltrated in tumor cell nests, which could also reflect antitumor immunity. Higher labeling index of Ki-67, a proliferation-associated antigen, among CD8(+) T cells in contact to tumor cells was associated with a longer survival by both uni- and multivariate analyses. Our data in human renal cell carcinoma suggest that infiltration of tumor tissue by T cells itself does not denote the efficacy of antitumor immunity because of its dependence on the biological malignancy of tumor cells, but infiltration of tumor tissue by CD8(+) T cells bearing more pronounced proliferative activity could reflect effective antitumor immunity. This concept would be important for future immunotherapy of human cancer.

Diagonal earlobe crease as a marker of the presence and extent of coronary atherosclerosis.

This study evaluates the association between the presence of diagonal earlobe creases (ELC) and coronary artery disease (CAD). One thousand four hundred twenty-four patients (760 men and 664 women, aged 30 to 80 years) were examined for the presence of ELC and classified into 2 groups: group I control--1,086 consecutive patients who denied symptoms of myocardial ischemia and were admitted to a general hospital for other reasons; group II CAD--338 patients with documented CAD (presence of > or = 70% coronary diameter stenosis at angiography). ELC was present in 304 patients (28%) in group I and 220 (65%) in group II (p < 0.0001). The patients were stratified in age groups to isolate the influence of age because the prevalence of ELC and CAD increased with advancing age (p < 0.0001 for both). This association remained statistically significant in all decades, except for patients aged > 70 years. To further remove the confounding effect of different age and sex distributions between the groups, a direct adjustment of the ELC prevalence was performed. When adjusted for age and sex, the prevalence of creases was still 58% higher in patients with CAD than in control subjects (p < 0.001). The presence of ELC was also related to the extent of CAD as measured by the number of major arteries narrowed (p = 0.015). The observed sensitivity of the sign for the diagnosis of CAD was 65%, the specificity 72%, the positive predictive value 42% and the negative predictive value 87%.

EGF stimulates both cyclooxygenase activity and cell proliferation of cultured guinea pig gastric mucous cells.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin all dose-dependently stimulated [3H]-thymidine incorporation into guinea pig gastric mucous cells cultured in vitro. On the other hand, other growth factors, e.g., platelet-derived growth factor (PDGF) and gastrointestinal hormones, such as gastrin, had no effect on DNA synthesis in these cells. Exposure of the cells to EGF at concentrations which stimulated DNA synthesis caused increases in both prostaglandin (PG) E2 release and cyclooxygenase (COX) enzyme protein synthesis, as evaluated by Western blot analysis. These results suggest that such increases in DNA synthesis and PGE2 release may be involved, at least in part, in the mechanism of EGF-induced local regulation of gastric mucosal integrity.

Effects of growth factors and gut hormones on proliferation of primary cultured gastric mucous cells of guinea pig.

Almost completely homogenous gastric mucous epithelial cells of guinea pigs were grown to confluence in the presence of 10% fetal calf serum (FCS). FCS, epidermal growth factor (EGF), and insulin significantly increased 5-bromo-2'-deoxyuridine (BrdU) uptake by the cells and EGF together with insulin increased the cells' [3H] thymidine uptake. Basic fibroblast growth factor (bFGF) enhanced EGF-induced DNA synthesis by the cells, but vasoactive intestinal peptide (VIP), secretin, prostaglandin E2 (PGE2), and dibutyryl cyclic AMP (dbcAMP) neither induced DNA synthesis nor enhanced the effect of EGF on DNA synthesis by the cells. Gastrin, cholecystokinin-octapeptide (CCK8), and carbamylcholine chloride (CCh) also did not enhance the effect of EGF on DNA synthesis. 125I-EGF, 125I-bFGF, and 125I-gastrin binding to the gastric mucous cells revealed the presence of high-affinity receptors for EGF and bFGF, but not for gastrin. Northern blot analysis showed the expression of EGF receptor mRNA, but not gastrin receptor mRNA. These results suggest that EGF, insulin, and bFGF may cooperatively regulate gastric mucous cell growth, but that gastrin and other gastrointestinal hormones do not have a direct stimulatory effect on mucous cell growth in the guinea pig.

Difference in effects of sodium fluoride and cholecystokinin on diacylglycerol accumulation and calcium increase in guinea pig gastric chief cells.

In isolated guinea pig gastric chief cells, sodium fluoride (NaF) stimulated a monophasic increase in diacylglycerol accumulation, while cholecystokinin (CCK) strongly stimulated its biphasic accumulation. NaF evoked an increase in initial Ca2+ influx rate with a slow increase in intracellular free Ca2+ concentration [( Ca2+]i), while CCK stimulated a rapid increase in [Ca2+]i followed by a late sustained phase of the [Ca2+]i increase. Lanthanum chloride (La3+) effectively blocked NaF-stimulated increase in [Ca2+]i, but it blocked only CCK-stimulated late sustained phase of [Ca2+]i increase. The effect of NaF on pepsinogen secretion was enhanced in the presence of 100 microM AlCl3. Furthermore, pertussis toxin did not affect NaF-evoked diacylglycerol accumulation at all. These results suggest that NaF may activate a pertussis-toxin insensitive guanine nucleotide regulatory protein (G protein) coupled to a signal transducing mechanism which seems to be distinct from that activated by CCK, thereby inducing increases in diacylglycerol accumulation, Ca2+ influx and pepsinogen secretion in guinea pig gastric chief cells.

Cloning and sequence analysis of hydroxyquinol 1,2-dioxygenase gene in 2,4,6-trichlorophenol-degrading Ralstonia pickettii DTP0602 and characterization of its product.

A gene encoding hydroxyquinol 1,2-dioxygenase was cloned from 2,4,6-trichlorophenol-degrading Ralstonia (Pseudomonas) pickettii strain DTP0602. Cell-free extracts of Escherichia coli containing a cloned 1.4-kb StuI-XhoI DNA fragment of R. pickettii DTP0602 hydroxyquinol 1,2-dioxygenase converted hydroxyquinol into maleylacetate and also degraded 6-chlorohydroxyquinol. The 1.4-kb DNA fragment contained one open reading frame (designated hadC) composed of 948 nucleotides. The molecular mass of 34,591 deduced from the gene product (HadC) was in agreement with the size (35 kDa) of the purified HadC protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of HadC exhibited high homology to that of the hydroxyquinol 1,2-dioxygenase of 2,4,5-trichlorophenoxyacetic acid-degrading Burkholderia cepacia AC1100 (Daubaras, D. L. et al., Appl. Environ. Microbiol., 61, 1279-1289, 1995). The active enzyme had a molecular mass of 68 kDa, suggesting that it is functional as a homodimer. The enzyme also catalyzed the oxidation of pyrogallol and 3-methylcatechol, possible intermediates in the degradation of 2,4,6-trichlorophenol, in addition to 6-chlorohydroxyquinol and hydroxyquinol. The dioxygenase catalyzed both ortho- and meta-cleavage of 3-methylcatechol.

Combination chemotherapy for malignant paraganglioma.

Treatment with a combination chemotherapeutic regimen consisting of cyclophosphamide, vincristine, and dacarbazine for malignant paraganglioma with hepatic metastasis is reported. A 51-year-old male presented with tumors in the retroperitoneal space and liver. The patient was diagnosed as having paraganglioma based on elevated levels of serum neuron-specific enolase, urinary catecholamine and vanillylmandelic acid, and on histological findings of the liver specimen. The patient was treated with this combination chemotherapy in repeated 21-day cycles. Temporary improvement in laboratory findings and a 20% reduction in the size of the hepatic masses were observed without severe adverse effects.

Combination of ascorbic acid and methylprednisolone pulse therapy in the treatment of idiopathic thrombocytopenic purpura.

Ascorbic acid, reported in 1988 to be effective for idiopathic thrombocytopenic purpura (ITP), is an attractive drug because of its lack of toxicity. Further studies are necessary in order to improve its effectiveness without increasing secondary effects. We present a chronic ITP patient treated with a combination of ascorbic acid and methylprednisolone pulse (MP) therapy who was previously treated with MP therapy alone. The effect of this combination therapy seems to be better than MP therapy alone. This therapy is worth further examination as another therapeutic choice due to its fewer secondary effects than the usual regimen of corticosteroids, splenectomy, and other immunosuppressive drugs.

Pigmentary retinopathy with nephrotic syndrome, Ménétrier's disease, and diabetes mellitus.

A patient with pigmentary retinopathy, nephrotic syndrome, Ménétrier's disease, and diabetes mellitus is presented. Other complications were congestive heart failure, hypothyroidism, hypertension, and hypertriglyceridemia. Hypogenitalism was also suspected. Pigmentary retinopathy is known to associate with many systemic diseases, which are classified into several syndromes. This case superficially resembles Alström's disease due to the common characteristics of pigmentary retinopathy, diabetes mellitus, renal disease, and hypogenitalism. But clinically and histologically, there are distinct differences. To our knowledge, this association has never been reported.

[Salvage coronary angioplasty in a young patient with Takayasu arteritis and myocardial infarction]. / Angioplastia coronária de salvamento em jovem com arterite de Takayasu e infarto do miocárdio.

A 16 year old boy with Takayasu's disease was admitted with myocardial infarction. Thrombolytic therapy with intravenous streptokinase showed no signs of reperfusion. Rescue angioplasty of the left descending coronary artery was performed with success.

[Cerebellar infarction due to vertebral artery dissection in a girl].

We report here a case of vertebral artery dissection, which is rare in childhood. A 12-year-old, previous healthy girl was admitted to our hospital with symptoms of vertigo, tinnitus, hearing loss, nausea and vomiting. Although there was neither higher cortical dysfunction, motor weakness, sensory disturbance nor slurred speech. She could not stand up because of severe vertigo. Cranial magnetic resonance imaging (MRI) revealed a subacute cerebellar infarct. A left vertebral artery angiogram on the hospital day 3 demonstrated a sharp narrowing at the C1-C2 level. After an anticoagulant therapy for about 2 weeks, all the symptoms disappeared except for mild tinnitus. Two months later, a left vertebral artery angiogram showed an abrupt occlusion at the C1 level. MRI T1-weighted images demonstrated a thrombus within the false lumen of the dissected vessels. A flow void revealed the patency of the residual true lumen. From these findings, we made a diagnosis of vertebral artery dissection, which was considered to have caused cerebellar infarction. The patient was mostly normal at discharge, and 100 mg/day of aspirin has been given until present.

[Flow cytometric analysis of DNA ploidy in colorectal adenoma].

Flow cytometric measurement of nuclear DNA content in 159 colorectal adenomas was carried out to investigate the relationship between DNA ploidy and the histological findings. DNA aneuploidy was detected in 18 lesions (12.8%). The incidence of DNA aneuploidy was significantly higher in tubulovillous adenomas than in tubular adenomas (30.4% vs. 8.1%; p < 0.01). DNA aneuploidy was not found in any adenoma with mild dysplasia, but was noted in 19.1% of those with moderate dysplasia and in 33.3% of those with severe dysplasia. The mean size of the lesions was significantly larger in adenomas with aneuploidy than in those without aneuploidy (14.0 mm vs. 7.7 mm; p < 0.01). The DNA index values of 18 adenomas with aneuploidy were divided into two groups: one ranged from 1.07 to 1.23 and the other from 1.66 to 1.85. DNA index values correlated with the size of the lesions (p < 0.05), but not with the histologic type and degree of dysplasia.

[Clinical significance of DNA ploidy heterogeneity in early gastric cancers].

To evaluate the clinical significance of DNA ploidy heterogeneity (DH), four or more fresh tissue specimens were obtained from a tumor in 68 resected early gastric cancers. DNA content was measured by flow cytometry and the presence of DH was prospectively investigated. The incidence of DH correlated to invasion depth (m < sm), lymph vessel invasion (negative < positive) and tumor size (10 mm or less in diameter < more than 10). When the criteria of indication for minimum surgery were determined as the intramucosal cancer without n, ly and v factor, 85% of contraindication cases demonstrated DH. These results indicate that DH is a useful marker of tumor progression in early gastric cancer and will be an aid for determining indications for minimum resection.

[Flow cytometric analysis of DNA ploidy in intramucosal gastric carcinoma].

In order to investigate whether or not DNA ploidy was altered in intramucosal gastric carcinomas, nuclear DNA content of biopsy specimen was measured using flow cytometry in 38 intramucosal carcinomas. DNA aneuploidy was detected in 27 of 38 lesions (71.1%), and noted more frequently in differentiated carcinomas than in undifferentiated ones (83.0% vs. 20.0%, p < 0.01). There was no significant relationship between the frequency of DNA aneuploidy and macroscopical type or tumor size. DNA aneuploidy was even found in two of three minute carcinomas (5 mm or less in diameter). DNA indices showed 1.2 or lower values in 40% of the lesions with DNA aneuploidy. The average value of DNA index was significantly larger in depressed type than in elevated type (p < 0.01). In conclusion, DNA ploidy is altered in most differentiated intramucosal carcinomas. A high resolution method is essential for accurate determination of DNA ploidy in intramucosal carcinomas, especially elevated ones.

[Flow cytometric DNA analysis of colorectal carcinoma in adenoma].

We evaluated the DNA ploidy in 23 lesions of colorectal carcinoma in adenoma (CIA) and 90 adenomas without carcinomas by flow cytometry using fresh samples. DNA ploidy of carcinoma and adenoma components were assessed, respectively, with 17 paraffin-embedded samples of CIAs. The incidence of DNA aneuploidy (AP) was significantly higher in CIAs than in adenomas (47.8% vs. 12.2%, p < 0.01). Even in adenoma components of CIAs, AP tended to be found more frequent than in adenomas (41.2% vs. 12.2%). The incidence of AP in adenoma components was similar to that in carcinoma components (35.3%) in CIAs. In conclusion, DNA aneuploidy in adenomas may be a marker of malignant potential.

[Diagnostic value of DNA ploidy in epithelial elevated lesions of the stomach].

Using flow cytometry, we evaluated the diagnostic usefulness of DNA aneuploidy in epithelial elevated lesions of the stomach. Four biopsy specimens were obtained from each lesion in 14 elevated early cancers, 21 adenomas, 37 hyperplastic polyps and 11 cases of chronic gastritis with intestinal metaplasia. All samples obtained from benign lesions indicated diploidy. DNA aneuploidy was detected in 12 lesions (85.7%) of early cancers. DNA indices ranged from 1.10 to 1.19 in six of 10 intramucosal cancers with DNA aneuploidy. Meanwhile, a total of two submucosally invasive cancers had stemlines with a DNA index of more than 2.00. Four early cancers, including two submucosally invasive ones, had multiple aneuploid stemlines. DNA aneuploidy is a useful marker of malignancy in epithelial elevated lesions of the stomach. A higher DNA index and multiple aneuploid stemlines may be possible markers of submucosal invasion in early cancer.

[Effect of palmitate and phorbol ester on synthesis of phosphatidylcholine in isolated guinea pig gastric glands].

To better understand the mechanism of phosphatidylcholine synthesis in the stomach, [3H] choline incorporation into phosphatidylcholine in response to agents which have been shown to induce phosphatidylcholine biosynthesis in other tissues was examined using isolated guinea pig gastric glands. Palmitate and 12-O-tetradecanoyl phorbol 13-acetate (TPA) which has been shown to activate protein kinase C directly, stimulated [3H] choline incorporation into phosphatidylcholine in gastric glands, by 189 +/- 12.9%, and 129 +/- 10.4% of control, respectively (n = 4, p less than 0.01, p less than 0.05). On the other hand, dibutyryl cyclic AMP had no effect on the incorporation. When the glands were pulsed with [3H] choline followed by incubation in the presence of palmitate and TPA for 180 min to see the effects of the agents on the limiting step of the phosphatidylcholine synthesis, phosphatidyl-[3H] choline was increased to 167 +/- 7.5% and 142 +/- 7.5% of control respectively (n = 4, p less than 0.01, p less than 0.05). In parallel to the increase in phosphatidylcholine synthesis, phosphoryl-[3H] choline in the glands incubated with palmitate and TPA was decreased as compared with control. These results suggest that palmitate or TPA may stimulate phosphatidylcholine biosynthesis through the activation of cytidylyltransferase in the stomach.

[Protective effect of prostaglandin E2 on ethanol-induced injury of chief cells isolated from guinea pig].

The mechanism by which PGE2 directly protects individual gastric cells from ethanol-induced injury was studied by using isolated gastric chief cells from guinea pig. Ethanol dose-dependently caused chief cell injury which was estimated by the release of lactate dehydrogenase (LDH) from chief cells. Pretreatment of chief cells with PGE2 reduced the cell damage caused by ethanol in time- and dose-dependent manner. The pretreatment at 37 degrees C and pH 7.4 with PGE2 maximally reduced the cell damage. This protective effect was reduced when the pretreatment was performed at either acid or alkaline pH or at reduced temperature. PGE2 did not stimulate any increase in cytosolic free Ca2+ concentration and initial Ca2+ influx rate. On the other hand, PGE2 stimulated an increase of cAMP accumulation in chief cells. However, pretreatment of chief cells with secretion, VIP, dbcAMP or forskolin failed to reduce subsequent injury caused by ethanol. These results suggest that PGE2 may protect chief cells against ethanol-caused injury probably via PGE type receptors coupled to as yet unidentified signal in gastric chief cells.