[Development of gene therapy for pancreatic cancer].

In order to develop the new therapeutic intervention for pancreatic cancer, we have examined the effect of gene therapy for this miserable pancreatic disease. The transfection of UPRT, a 5-FU converting enzyme, gene resulted in the significant change in sensitivity of pancreatic cancer cells against 5-FU. Anti-angiogenesis gene therapy has been also demonstrated to be a promising strategy for pancreatic cancer. It has been revealed that replication-competent adenoviruses are not only the strong weapon themselves but also useful carriers of genes possessing anti-tumor activities as virus vectors specific to tumors without normal p53 function nor intact Rb pathway. Whether these experimental results are universally true require the clinical trials in future.

Synthesis of 2-O-(4-coumaroyl)-3-(4-hydroxyphenyl)lactic acid, an important intermediate of rosmarinic acid biosynthesis.

A simple method to synthesize (+/-)-2-O-(4-coumaroyl)-3-(4-hydroxyphenyl)lactic acid (1), a key intermediate in rosmarinic acid biosynthesis in higher plant cells, was established by condensation of protected 4-coumaric acid and (+/-)-3-(4-hydroxyphenyl)lactic acid followed by deprotection. A stable supply of 1 thus attained will lead to biochemical and molecular biological characterization of later steps of rosmarinic acid biosynthesis.

Meat allergy: investigation of potential allergenic proteins in beef.

The potential allergenic proteins in beef were investigated. The sera of ten beef-allergic patients suffering from atopic dermatitis and having a positive RAST score to beef, aged 3-18 years, were obtained from Yoshida Hospital in Japan, and five non-allergic individuals were subjected to this study. The sera of the ten patients reacted strongly to a beef extract, but not to pork and chicken extracts by both ELISA and immunoblotting. The sera of the five control subjects did not react to any of these meat extracts. Three bands having molecular masses of approximately 200 kDa, approximately 67 kDa and approximately 60 kDa were observed by immunoblotting after SDS-PAGE. Two fractions of the beef extract from a Sephadex-gel (G-200) filtration column strongly reacted with the sera of the beef-allergic patients by ELISA and immunoblotting: one fraction had the approximately 67 kDa component and the other had the approximately 200 kDa and approximately 60 kDa components. One of them (approximately 67 kDa) was confirmed to be bovine serum albumin (BSA) by an analysis of the N-terminal amino acid sequence. We could not identify the others by sequencing, but the approximately 200 kDa and approximately 60 kDa components were presumed to be glycoproteins. Bovine gamma (BGG:globulin M.W. approximately 160 kDa) is a glycoprotein and has several subunits. The beef-allergic patients showed strong reactivity to the approximately 200 kDa and approximately 60 kDa components of pure BGG by immunoblotting. Inhibition-ELISA showed that pure BGG preparations strongly inhibited the binding of sera from the beef-allergic patients to the beef extract. These results suggest that the approximately 200 kDa, approximately 67 kDa and approximately 60 kDa components in the beef extract had strong allergenicity: approximately 67 kDa was BSA, and approximately 200 kDa and approximately 60 kDa were presumably aggregated BGG and it's heavy chain, respectively.

In vitro analysis of distraction osteogenesis.

Distraction osteogenesis has been widely used for lengthening craniomaxillofacial bone. The healing process of distracted bone has been studied mainly by histological observation in vivo. To analyze the cellular response to the mechanical stress of distraction, we have established and evaluated an in vitro model of distraction osteogenesis using an organ culture technique. Five-week-old male Wistar rats were used for the experiments. The tibial bone was fractured by hand and fixed for 1 week by acrylic resin. After the initial healing period, the tibial bone was harvested and used for this study. A distraction instrument was devised to control the strain on the cultured bone using a micrometer. After distraction, the samples were histologically evaluated for ossification by hematoxylin and eosin stain and Alcian blue stain. As a result, the histological finding for the bone region at a slow rate of distraction (0.5 mm/day) was different from that at a rapid rate of distraction (1.0 mm/day). The proliferation of cartilage was inhibited at the rapid distraction rate. Thus, we hypothesized that mechanical stress regulated cartilaginous growth in tissue cultivation. Judging from this experiment, the model was useful for investigation of the mechanism of bone formation in distraction osteogenesis, because it was simple and served to isolate many factors.

Genotypes of alcohol-metabolizing enzymes in relation to alcoholic chronic pancreatitis in Japan.

Long-term consumption of large amounts of alcohol is the main cause of chronic pancreatitis. All heavy drinkers, however, do not contract chronic pancreatitis. Although genetic predisposition to alcoholism and alcoholic liver disease has been reported, genetic susceptibility to alcoholic pancreatitis is still a matter of debate. To determine the relation between genotypes of alcohol-metabolizing enzymes and chronic alcoholic pancreatitis, we examined genotype patterns of aldehyde dehydrogenase 2 (ALDH 2), alcohol dehydrogenase 2 (ADH 2) and cytochrome P-4502E1 (CYP2E1) in 54 patients with chronic alcoholic pancreatitis who were diagnosed in general hospitals in all over Japan and compared with those in 30 patients with chronic nonalcoholic pancreatitis or in 46 alcoholics with normal pancreatic function. There were no significant differences in the distribution of genotypes of ALDH 2 and CYP2E1 among those three groups. As for the ADH 2 genotype, distribution of 2(1)/2(1), 2(1)/2(2), and 2(2)/2(2) was 35%, 30%, and 35% in alcoholics with normal pancreatic function; 4%, 39%, and 57% in the chronic alcoholic pancreatitis group; and 0%, 50%, and 50% in the chronic nonalcoholic pancreatitis group, respectively. The frequency of ADH 2(2) allele was significantly higher in the chronic alcoholic pancreatitis group, compared with alcoholics with normal pancreatic function; but, it was not significantly different from that in the chronic nonalcoholic pancreatitis group. We also examined the relation between pancreatic fibrosis or pancreatitis histologically diagnosed and genotypes of alcohol-metabolizing enzymes in alcoholic autopsy cases. Twenty of 31 cases showed moderate or severe pancreatic fibrosis and showed intralobular + interlobular fibrosis, which is characteristic in alcoholic pancreatitis or intralobular fibrosis. ADH 2(2) allele tended to show a high frequency in the intralobular + interlobular fibrosis group, compared with that in the intralobular fibrosis group (75.0% vs. 41.7%, p < 0.1). The chronic pancreatitis group had a significantly higher frequency of the ADH 2(2) allele than that in cases without such findings (87.5% vs. 58.7%, p < 0.05). However, the ALDH 2 and CYP2E1 genotypes showed no significant relation to the findings of pancreatic fibrosis or histological pancreatitis. These data suggest that the risk of chronic alcoholic pancreatitis diagnosed clinically and pathologically seems to be associated with the ADH 2(2) allele in the genotypes of alcohol-metabolizing enzymes.

Development of grating acuity in infants with Retinopathy of Prematurity.

PURPOSE: To evaluate the visual development in infants with stage 1 approximately 3 ROP and compare their visual results with healthy preterm infants. PATIENTS AND METHODS: One hundred forty-four premature infants were recruited and were divided into 3 groups according to the stage of ROP. Randomly selected preterm subjects with no ROP were taken as controls. Ophthalmic examinations started 4 to 7 weeks after birth and were repeated as needed until the retina was fully vascularized or until any ROP that developed had resolved. Grating acuity was measured by acuity cards between 35-45 weeks of corrected age and by PL method at 12, 18 and 24 months of age. RESULTS: Infants with stage 3 ROP had slightly lower visual acuity scores compared to other infants at most of the testing points throughout the 35-45 week period, which did not show statistical significance at any week. Infants with stage 2 and 3 ROP had similar visual acuity values but slightly lower acuity scores than infants with stage I or no ROP at the 12 month follow-up. The differences were not statistically significant. Stage 3 ROP infants had significantly lower acuity scores compared to infants with stage 1-2 or no ROP at the 18 and 24 month follow-up visits (p<0.0001). CONCLUSION: We stress periodic monitoring of early visual acuity in infants with ROP because of the possibility of impaired visual development.

[The frequency and development of tissue iron deficiency in 6 iron deficiency anemia patients with plummer-vinson syndrome].

The physical signs of tissue iron deficiency include smooth and red tongue, angular stomatitis, koilonychia, and pica. The incidence of these conditions is unknown in Japan. We evaluated the frequency and development of tissue iron deficiency in 353 patients with iron deficiency anemia. The frequency of tissue iron deficiency was 6.8%; papillary atrophy of the tongue, 5.4%; abnormal nails, 5.4%; angular stomatitis, 1.1%; Plummer-Vinson syndrome, 1.7%; and pica, 0.06%. These findings were compared with the date collected by Wintrobe and Beveridge. The development and incidence of tissue iron deficiency correlated significantly with the severity of iron deficiency anemia.

Further evidence for the involvement of human chromosome 6p24 in the aetiology of orofacial clefting.

Chromosomal translocations affecting the 6p24 region have been associated with orofacial clefting. Here we present a female patient with cleft palate, severe growth retardation, developmental delay, frontal bossing, hypertelorism, antimongoloid slant, bilateral ptosis, flat nasal bridge, hypoplastic nasal alae, protruding upper lip, microretrognathia, bilateral, low set, and posteriorly rotated ears, bilateral microtia, narrow ear canals, short neck, and a karyotype of 46,XX,t(6;9)(p24;p23). The translocation chromosomes were analysed in detail by FISH and the 6p24 breakpoint was mapped within 50-500 kb of other breakpoints associated with orofacial clefting, in agreement with the assignment of such a locus in 6p24. The chromosome 9 translocation breakpoint was identified to be between D9S156 and D9S157 in 9p23-p22, a region implicated in the 9p deletion syndrome.

Morphological variation of the frontal sinus in Melanesian (Fiji) and Polynesian (Western Samoa) populations.

The aims of this study were to investigate gender differences in frontal sinus morphology and to estimate differences between Fijian and Western Samoan populations. Cephalograms of 118 Fijians (59 male, 59 female), 80 W. Samoans (33 male, 47 female) and 89 Japanese (60 male, 29 female) were used in this study. The thickness of the frontal sinus and the convexity of glabella in males were found to be larger than those of females in each population. The angle of Sg-N-G in the Fijians was larger than that of the Western Samoans and Japanese. In order to compare the frontal sinus morphology among populations, Mahalanobis' generalized distances were calculated on the basis of the five distance diameters. The results indicate that gender is separated by the first axis which indicates the overall size. Japanese samples were separated from South Pacific samples by the second axis which indicates the shape factor. In populational discrimination, there was a higher percentage of correct discriminations of the females of the males. In conclusion, gender difference was recognized in the size of the frontal sinus, and the populational differences were shown in the shape factor between Melanesian and Polynesian populations, and also between South Pacific and Japanese populations.

Orientations of tyrosines 21 and 24 in coat subunits of Ff filamentous virus: determination by Raman linear intensity difference spectroscopy and implications for subunit packing.

Virions of the Ff group of bacteriophages (fd, f1, M13) are morphologically identical filaments (approximately 6-nm diameter x approximately 880-nm length) in which a covalently closed, single-stranded DNA genome is sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit (pVIII). Orientations of pVIII tyrosines (Tyr21 and Tyr24) with respect to the filament axis have been determined by Raman linear intensity difference (RLID) spectroscopy of flow-oriented mutant virions in which the tyrosines were independently mutated to methionine. The results show that the twofold axis of the phenolic ring (C1-C4 line) of Tyr21 is inclined at 39.5 +/- 1.4 degrees from the virion axis, and that of Tyr24 is inclined at 43.7 +/- 0.6 degrees. The orientation determined for the Tyr21 phenol ring is close to that of a structural model previously proposed on the basis of fiber x-ray diffraction results (Protein Data Bank, identification code 1IFJ). On the other hand, the orientation determined for the Tyr24 phenol ring differs from the diffraction-based model by a 40 degrees rotation about the Calpha-Cbeta bond. The RLID results also indicate that each tyrosine mutation does not greatly affect the orientation of either the remaining tyrosine or single tryptophan (Trp26) of pVIII. On the basis of these results, a refined model is proposed for the coat protein structure in Ff.

[Fatal pulmonary thromboembolism in a patient with paroxysmal nocturnal hemoglobinuria].

A 38 year old woman with paroxysmal nocturnal hemoglobinuria was admitted to our hospital because of hemoptysis. Pulmonary infarction was diagnosed by the perfusion lung scan. In spite of the administration of prednisolone, dextran, low molecular weight heparin, and warfarin, she died of pulmonary infarction and secondary pulmonary hypertension. Autopsy revealed thromboembolism of both pulmonary arteries and hepatic central vein thrombosis. Recent understanding of the pathogenesis and incidence of thromboembolism in paroxysmal nocturnal hemoglobinuria was discussed.

Immunohistochemical and urinary markers of podocyte injury.

Renal podocytes play an important role in glomerular filtration. We estimated podocyte injury by light microscopic examination of kidney specimens or by urinary excretion of substances related to podocytes. Fresh kidney sections from 69 patients and urinary sediments from 84 patients with various renal diseases were examined immunohistochemically using fluorescent labelling. Four kinds of monoclonal antibodies which recognize podocytes were used: (1) anti-podocalyxin (anti-PCX) antibody, (2) anti-C3b receptor (anti-C3bR) antibody, (3) anti-podocyte protein, 44 kilodalton, (anti-pp44) antibody, and (4) anti-alpha 3 integrin (anti-Int alpha3) antibody. Labelling of kidney sections by anti-C3bR (k-C3bR) was reduced in cases of severe glomerular injury, although there were no changes in k-PCX, k-pp44, or k-Int alpha3 labelling. PCX labelling of urinary sediments (u-PCX) was detected in nearly all cases of glomerulonephritis, u-C3bR was seen in some cases of glomerular injury, u-Int alpha3 was seen in only a small number of cases of severe glomerular injury, and u-pp44 was not detected in any urinary samples. u-PCX, u-C3bR, u-Int alpha3, and k-C3bR correlated with the degree of pathological change, the degree of proteinuria and hematuria. These results suggest that immunostaining kidney sections with anti-C3bR antibody and urinary sediments with anti-PCX, anti-C3bR, and anti-Int alpha3 antibodies might be useful in detecting podocyte injury.

Urinary excretion of podocytes reflects disease activity in children with glomerulonephritis.

The significance of the presence of podocytes in the urine was studied in various renal diseases in children. The podocytes were detected by immunofluorescence using monoclonal antibodies against the podocalyxin that is present on the surface of podocytes which serves as a glycocalyx. They were scored according to the numbers per partitioned area on cytospun urine sediments. Urine podocytes were absent in normal control, nonglomerular diseases such as urinary tract infection and nonglomerular hematuria, and glomerular, non-inflammatory diseases such as minimal change nephrotic syndrome and membranous nephropathy. Conversely, the excretion of podocytes in the urine were detected in various glomerular, inflammatory diseases. A significantly higher level of the podocyte score was found in the acute state of glomerular diseases which was defined as within 6 months after disease onset. Positive correlations were obtained between the presence of urinary podocytes and the histological features of active extracapillary changes and mesangial proliferation. Urinary podocytes were examined monthly for 12 months in 7 cases with IgA nephropathy and 2 cases with Henoch-Schönlein purpura nephritis, and a consistently higher urinary podocyte score was observed in the patients with histological progression. The scoring of urinary podocytes was found to be useful clinically, as a diagnostic tool for glomerular or nonglomerular diseases, inflammatory or noninflammatory diseases, a marker for the estimation of the severity of active glomerular injury and also as a predictor of disease progression.

Involvement of protein kinase C activation in endothelin-1-induced secretion of interleukin-6 in osteoblast-like cells.

We previously reported that endothelin-1 (ET)-1 stimulates phospholipase D (PLD) independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of ET-1 on the secretion of interleukin-6 (IL-6) and the involvement of protein kinase C (PKC) activation in the IL-6 secretion in these cells. ET-1 significantly stimulated IL-6 secretion time-dependently up to 72 h. The stimulative effect was dose-dependent in the range between 1 nM and 1 microM. BQ123, a selective antagonist of endothelinA (ETA) receptor, inhibited the ET-1-induced IL-6 secretion. On the contrary, BQ788, a selective antagonist of endothelinB (ETB) receptor, had no effect. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, significantly stimulated IL-6 secretion. However, 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, did not affect IL-6 secretion. The effect of a combination of ET-1 and TPA on IL-6 secretion was not additive. Calphostin C, a specific PKC inhibitor, significantly inhibited the ET-1-induced IL-6 secretion. Both ET-1- and TPA-induced IL-6 secretion were reduced in PKC downregulated MC3T3-E1 cells. These results strongly suggest that ET-1 stimulates IL-6 secretion via ETA receptor in osteoblast-like cells and that PKC activation is involved in the ET-1-induced IL-6 secretion.

An acromegalic patient with pulsatile secretion of growth hormone (GH) coincident with the slow-wave sleep.

A 43-year-old woman was admitted to our hospital for further treatment of acromegaly with high plasma GH and IGF-I levels after transsphenoidal adenomectomy and subsequent treatment with bromocriptine. Physical examination and magnetic resonance imaging (MRI) showed an active acromegalic appearance with residual pituitary macroadenoma. Laboratory findings revealed an increase in basal levels of plasma GH (21.3 microg/L) and plasma IGF-I (470 ng/ml). Plasma GH levels were suppressed from 21.3 microg/L to 9.9 microg/L following oral administration of 75 glucose and did not respond to either TRH or LHRH injection. When plasma GH levels were measured after repeated blood sampling every 20 min for 24 h and sleep stages were analyzed, there were three GH peaks during the night which corresponded to the slow-wave sleep. Mean plasma GH levels which corresponded to the slow-wave sleep stages were much greater than those of other sleep stages during the night. After the patient was treated with intermittent sc injections of octreotide (40 microg/every 2 h, 480 microg/day) in combination with oral administration of bromocriptine (15 mg/day, t.i.d.), episodic GH release was somewhat suppressed but plasma GH levels were slightly increased, corresponding to the slow-wave sleep stage during the night. Mean plasma GH levels were much higher during the sleeping period than the waking period for 24 h before and after the treatment. These findings suggest that GH secretion is correlated to the slow-wave sleep in this particular patient with pituitary GH producing adenoma.

Role of acetylcholine and gastrin-releasing peptide (GRP) in gastrin secretion.

Using an isolated rat stomach infusion model, we investigated the role of gastrin-releasing peptide (GRP) and acetylcholine in the secretion of gastrin (which plays a major role in gastric acid secretion), and the relationship between gastrin secretion and stomach pH. Bombesin, which has a structure analogous to that of GRP, was used in the experiment. We also investigated whether acetylcholine has muscarine-like or nicotine-like action. Our findings pointed to the presence of an alternative, GRP-mediated, route for stimulating gastric secretion from G cells, other than the acetylcholine-mediated route. We injected bombesin to confirm the presence of such a GRP-mediated route; significantly increased gastrin secretion was observed, even under acidic conditions, in the gastric lumen, which has been considered to show almost no gastric secretion. This secretion was not inhibited by atropine. The results suggested that there are two routes for inducing gastrin secretion from G cells: an acetylcholine-mediated route and a GRP-mediated route (intramural peptide neurons). As GRP induced gastrin secretion, regardless of stomach pH, GRP was considered to be more closely related to gastrin secretion. The results also suggested that a muscarine-like action, particularly in the M3 receptor-mediated route, plays a significant role in acetylcholine-mediated gastrin secretion and that nicotine-like action is not involved in gastrin secretion.

Clinical evaluation of hepatocyte growth factor in patients with gastrointestinal and pancreatic diseases with special reference to inflammatory bowel disease.

Hepatocyte growth factor (HGF) is widely expressed in the gastrointestinal tract and pancreas. However, the clinical significance of HGF in gastrointestinal and pancreatic diseases remains unclear. To clarify its clinical significance in these diseases, we determined serum HGF in patients with gastrointestinal and pancreatic diseases. Serum HGF was measured in 81 patients with gastrointestinal diseases, pancreatic diseases, and 150 healthy individuals, using a highly sensitive immunoradiometric assay (IRMA). The patients included 55 patients with colonic disorders, 20 with gastric disorders and 6 with pancreatic disorders. Serum HGF levels in patients with inflammatory bowel diseases and chronic pancreatitis were higher than those in normal individuals (p < 0.05, each). Symptomatic patients with inflammatory bowel diseases showed higher values of HGF than symptom-free patients (p < 0.05). Patients with moderately severe or severe ulcerative colitis showed higher values of HGF than patients with mild disease (p < 0.05). Serum HGF values were correlated with C-reactive protein (CRP) and serum HGF changed in parallel with clinical courses in patients with ulcerative colitis. The immunohistochemical study showed that HGF was present around the neutrophils infiltrating into the lamina propria, which was biopsied from endoscopically active colonic mucosa in patients with ulcerative colitis, while little HGF was observed in the inactive mucosa. The results of the present study suggest that serum HGF changes in gastrointestinal and pancreatic diseases, especially in inflammatory bowel diseases.

Induction of plasma hepatocyte growth factor in acute colitis of mice.

OBJECTIVE AND DESIGN: The study examined plasma levels of hepatocyte growth factor (HGF) in acute colitis models on mice. MATERIALS: Acute colitis was induced in male FVB mice (about 25 g) by intrarectal administration of 200 microliters of 0.5% acetic acid. METHODS: Plasma was taken at 0, 12, 24, 36, 48 and 72 h after acetic acid treatment. Plasma HGF was measured by an enzyme immunoassay in duplicate. RESULTS: Plasma levels of HGF at 0, 12, 24, 36, 48 and 72 h after acetic acid administration were 0.31 +/- 0.11 ng/ml (n = 4), 0.45 +/- 0.04 ng/ml (n = 4), 0.46 +/- 0.13 ng/ml (n = 4), 0.71 +/- 0.22 ng/ml (n = 4), 0.82 +/- 0.15 ng/ml (n = 4) and 0.44 +/- 0.04 ng/ml (n = 4), respectively. Significant increases in plasma HGF levels were observed at 36 and 48 h after the treatment (p < 0.05, compared to at 0 h). CONCLUSIONS: Plasma HGF was induced by acute inflammation of colonic tissues. These findings suggest that HGF in blood may be one of the non-specific markers of inflammation in mice.