Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos.

This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm-40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.

Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 µs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 µs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.

The study of spermidine-stimulated polypeptide synthesis in cell-free translation of mRNA from lactating mouse mammary gland.

The stimulatory effect of spermidine on the translation of poly(A)+ mRNA from lactating mouse mammary glands in a wheat germ system was studied. Spermidine stimulated total polypeptide synthesis about 2.5-fold relative to that occurring in the presence of an optimal concentration of Mg2+ alone. The size and the number of polysomes were about 1.6-times larger in the presence of spermidine than in its absence. A similar magnitude of increase in peptide chain initiation, 1.4-fold, was found when the extent of peptide chain initiation was measured by determining the residual polypeptide synthesis subsequent to the addition of inhibitor(s) of peptide chain initiation to the in vitro translation system with or without spermidine at various times of the incubation. Time-course study of the release of polypeptide from polysomes showed that spermidine stimulated this process to a much greater extent than peptide chain initiation, indicating that the polyamine also increases the rate of peptide chain elongation. The extent of stimulation of peptide chain elongation by spermidine was estimated to be about 1.5-fold when the disappearance of isotope-labeled nascent peptides from polysomes was measured by pulse-chase experiments. These results indicate that spermidine stimulates the cell-free translation of mammary mRNA by increasing the rates of both initiation and elongation of polypeptide synthesis to almost the same extent. The polyamine also reduced the relative amount of incomplete polypeptides, thereby increasing the yield of full-length translational products.

Structure determination of glycosphingolipids of cultured human keratinocytes.

From cultured human keratinocytes, seven glycolipid fractions were isolated by DEAE and silica-gel column chromatographies, and further by HPLC on a silica-gel column. By means of 1H-NMR spectroscopy, fast atom bombardment mass spectrometry and GLC-mass spectrometry, one fraction was determined to contain acylglucosylceramides, which consist of amide linked omega-hydroxy fatty acids (C30:0, C30:1, C32:1 and C34:1), fatty acids linked to the omega-hydroxy fatty acids through ester linkages (C14:1, C16:1, C18:1 and C18:2), a long-chain base (d18-sphingenine), and beta-glucose. Five of the other fractions contained glucosylceramides, and the seventh fraction contained a mixture of glucosylceramides and galactosylceramides. Glucosylceramides containing long-chain omega-hydroxy fatty acids, which are assumed to be immediate precursors of the acylglucosylceramides, were hardly detected in these glycolipid fractions. Six glucosylceramide fractions were separated due to differences in their fatty acids and sphingosines. On comparison with the results reported in our previous paper, the acylglucosylceramide content of the cultured human keratinocytes was about half that of human epidermis. Under the culture conditions used, the human keratinocytes did not differentiate into granular or horny cells. Taken together, the results suggest that the synthesis of acylglucosylceramides is not activated much in the cultured keratinocytes, but would be more activated in differentiated cells.

Falls among community-dwelling elderly in Japan.

Japanese have a lower incidence of hip fracture than Caucasians despite having lower bone mass. Hip fractures usually occur after a fall, and differing incidence rates of falls might explain the observed differences in hip fracture rates. To explore this hypothesis, we studied falls and related conditions among 1534 (624 men, 910 women) community-dwelling people aged 65 years and over in Japan and compared the prevalence of falls to Japanese-Americans living in Hawaii and to published studies of Caucasians. In Japan, 9% of the men and 19% of the women reported one or more falls during the past year. The prevalence of falls increased with age in both genders and was greater among women compared with men. In logistic regression models, having musculoskeletal disease, physical disability or limited activity increased the risk of falls by two to four times in both genders. Most fallers (92%) reported fear of future falls, and about one third of fallers reported that they went out less often as a result of their falls. Compared with native Japanese, the age-standardized prevalence of falls among Japanese-Americans was similar but about twice as high for Caucasians, which may explain the lower hip fracture risk of Japanese.

Characterization of the human dihydropyrimidinase-related protein 2 (DRP-2) gene.

The genes within the dihydropyrimidinase-related protein (DRP) family, were originally identified in humans by their homology to dihydropyrimidinase (DHP). Four members of this gene family, DRP-1, -2, -3 and -4, are expressed mainly in the fetal and neonatal brains of mammals and chickens, and have been implicated as intracellular signal transducers in the development of the nervous system. We isolated the human DRP-2 gene, and determined its transcriptional start site and exon/intron organization. The gene spanned more than 62 kb, and contained 14 exons with lengths ranging from 62 bp to 2606 bp. The transcriptional start site was determined by an RNase protection assay and 5' rapid amplification of cDNA ends (RACE), and a highly GC-rich promoter was identified that contained possible regulatory elements such as a TATA box, CAAT box and three GC boxes. Comparison of the phase and position of intron insertions within the human DRP-2 gene with those within DRP-1, DHP and two Caenorhabditis elegans DRP/DHP homologs, indicated that DRPs are more conserved in their exon/intron organization than DHP.

Cloning and characterization of the Caenorhabditis elegans CeCRMP/DHP-1 and -2; common ancestors of CRMP and dihydropyrimidinase?

The vertebrate CRMP (collapsin-response-mediator protein) gene family comprises at least four members. These CRMPs exhibit about 60% amino acid identity with vertebrate dihydropyrimidinase (DHP), an amidohydrolase involved in the pyrimidine degradation pathway. CRMP is also referred to as DRP (DHP-related protein), TOAD-64 (turned on after division, 64 kDa) and Ulip (Unc-33-like phosphoprotein). These vertebrate CRMPs are expressed mainly in early neuronal differentiation, which suggests that they play a role in neuronal development. In this study we isolated two cDNA clones from nematode C. elegans based on their sequence homology to vertebrate CRMPs and DHP. These two molecules, termed CeCRMP/DHP-1 and -2, turned out to be Ulip-B and -A, respectively, which were previously identified in the C. elegans genomic database by Byk et al. (1998). These newly isolated molecules were believed to represent a common ancestral state before the gene duplication between CRMPs and DHP. CeCRMP/DHP-1 and -2 protein retained all putative zinc-binding residues thought to be essential for the amidohydrolase activity of DHP and exhibited a weak amidohydrolase activity when 5-bromo-dihydrouracil was used as a substrate. Whole-mount in situ hybridization and expression analysis using GFP fusions revealed that CeCRMP/DHP-1 was transiently expressed in the hypodermis of C. elegans during the early larva stage. CeCRMP/DHP-1 was also expressed in a single nerve cell between the pharynx and ring neuropil. On the other hand, expression of CeCRMP/DHP-2 was observed in the body wall muscle throughout the lifespan of C. elegans. These results indicate that a major site of CeCRMP/DHP-1 and -2 expression is non-neuronal. Targeted gene disruption of CeCRMP/DHP-2 caused no particular difference in appearance or movement phenotype.

Different mechanisms of thioredoxin in its reduced and oxidized forms in defense against hydrogen peroxide in Escherichia coli.

The present experiments were done to elucidate the roles of thioredoxin and thioredoxin reductase system in defense against hydrogen peroxide (H2O2) in Escherichia coli. The thioredoxin-deficient mutant (trxA) was more sensitive to H2O2 than was the wild-type strain, when challenged in the stationary and exponentially growing phase. Thioredoxin reductase-deficient mutant (trxB) in the stationary phase also exhibited increased sensitivity, compared with the wild-type strain. These results indicated that reduced form of thioredoxin is required for defense against H2O2, possibly by scavenging radicals generated in the cells. In contrast, the trxB mutant in the growing phase had higher survival after exposure to H2O2 than the wild-type strain. The acquirement of resistance related to increased capacity for removing H2O2 in the trxB mutant and was not observed in a catalase-negative background. Furthermore, enhanced expression of the katG :: lacZ gene occurred in the mutant. Therefore, it was concluded that oxidized form of thioredoxin confers H2O2 resistance on E. coli cells by increasing activity to remove H2O2, which was brought about by enhanced induction of the katG-coded catalase/hydroperoxidase I at the transcriptional level. In addition, this resistance to H2O2 correlated well with reduced amount of DNA damage caused by H2O2, determined by the induction level of the recA :: lacZ fusion gene after treatment with H2O2.

Novel potassium-channel openers: preparation and pharmacological evaluation of racemic and optically active N-(6-amino-3-pyridyl)-N'-bicycloalkyl-N"-cyanoguanidine derivatives.

The previous paper reported on the synthesis and pharmacological evaluation of N-(6-amino-3-pyridyl)-N'-bicycloalkyl-N"-cyanoguanidine derivatives, from among which three compounds were selected as potent potassium-channel openers. In the present study, selected compounds were tested for antagonism of potassium-induced contraction of rat aorta, hypotensive activity in normotensive rats, and diuretic activity in spontaneously hypertensive rats. This led to further evaluation of compound (+/-)-10 and selection of (+)-N-(6-amino-3-pyridyl)-N'- [(1S,2R,4R)-bicyclo- [2.2.1]hept-2-yl]-N"-cyanoguanidine ((+)-10) (AL0670) for development as an antihypertensive agent. Although AL0670 is regarded as a pinacidil-type K(+)-channel opener, it showed different pharmacological and conformational profiles from pinacidil.

Novel potassium channel openers: synthesis and pharmacological evaluation of new N-(substituted-3-pyridyl)-N'-alkylthioureas and related compounds.

This report describes the synthesis and pharmacological evaluation of a series of novel potassium channel openers related to the pinacidil-type compounds. Thioureas, cyanoguanidines, and pyridine N-oxides were systematically evaluated for their effects on both the inhibition of spontaneous mechanical activity in rat portal vein (in vitro) and their antihypertensive activity (in vivo), and the structure-activity relationship for this series of compounds was discussed. Good correlation between in vitro and iv antihypertensive activity was observed for these compounds. Among them, cyanoguanidines bearing a conformationally rigid unit such as a norbornyl group generally possessed potent activity in both in vitro and in vivo studies. Especially, N-(6-amino-3-pyridyl)-N'-cyano-N"-(1-methyl-2-norbornyl)guanidine (23d) was identified as a more potent potassium channel opener in vitro (EC100 = 3 x 10(-8) M) than pinacidil (EC100 = 10(-7) M).

Effects of glutamic acid analogues on identifiable giant neurones, sensitive to beta-hydroxy-L-glutamic acid, of an African giant snail (Achatina fulica Férussac).

The effects of the seven glutamic acid analogues, alpha-kainic acid, alpha-allo-kainic acid, domoic acid, erythro-L-tricholomic acid, DL-ibotenic acid, L-quisqualic acid and allo-gamma-hydroxy-L-glutamic acid were examined on six identifiable giant neurones of an African giant snail (Achatina fulica Férussac). The neurones studied were: PON (periodically oscillating neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone), RAPN (right anterior pallial neurone), FAN (frequently autoactive neurone) and v-RCDN (ventral-right cerebral distinct neurone). Of these, d-RPLN and RAPN were excited by the two isomers (erythro- and threo-) of beta-hydroxy-L-glutamic acid (L-BHGA), whereas PON, VIN, FAN and v-RCDN were inhibited. L-Glutamic acid (L-Glu) had virtually no effect on these neurones. alpha-Kainic acid and domoic acid showed marked excitatory effects, similar to those of L-BHGA, on d-RPLN and RAPN. Their effective potency quotients (EPQs), relative to the more effective isomer of L-BHGA were: 0.3 for both substances on d-RPLN, and 1 for alpha-kainic acid and 3-1 for domoic acid on RAPN. alpha-Kainic acid also had excitatory effects on FAN and v-RCDN (EPQ for both: 0.3), which were inhibited by L-BHGA but excited by gamma-aminobutyric acid (GABA). Erythro-L-tricholomic acid showed marked effects, similar to those of L-BHGA, on VIN (EPQ: 0.3) and RAPN (EPQ: 3-1), but produced weaker effects on PON and d-RPLN (EPQ: 0.1). DL-Ibotenic acid produced marked effects, similar to those of L-BHGA, on PON, VIN (EPQ for both: 1) and RAPN (EPQ: 1-0.3), but had weak effects on d-RPLN (EPQ: less than 0.1) and FAN (EPQ: 0.1). It had excitatory effects on v-RCDN (EPQ: 0.1). This neurone was inhibited by L-BHGA but excited by GABA. L-Quisqualic acid showed the same effects as L-BHGA on all of the neurones examined (EPQ range 30-0.1). It was the most potent of the compounds tested on RAPN (EPQ: 30-10), FAN (EPQ: 30) and v-RCDN (EPQ: 3). alpha-Allo-kainic acid and allo-gamma-hydroxy-L-glutamic acid had no obvious effect on any of the neurones examined. As described above, the responses of the neurones examined to these substances varied widely. However, L-quisqualic acid generally had effects on the neurones similar to those of L-BHGA; the L-BHGA-excited neurones were also excited by alpha-kainic acid and domoic acid.

A population study on risk factors for insomnia among adult Japanese women: a possible effect of road traffic volume.

In an effort to identify risk factors for insomnia and determine the contribution of nightime road traffic volume to insomnia in the general population, a questionnaire survey was carried out among 3,600 adult Japanese women living in eight urban residential areas. The crude prevalence rate of insomnia was 11.2%. Multivariate analysis revealed that aging, living with a child/children aged six or younger, undergoing medical treatment, experiencing major life events, having an irregular bedtime, having a sleep apnealike symptom, and living near a road with a heavy volume of traffic are risk factors for insomnia. Taking into account other risk factors, there was a level-response relationship between the nighttime traffic volume of main roads and the risk of insomnia in the subjects living in the zones 0-20 m from these roads. These results suggest that road traffic noise raises the sound level in bedrooms in such zones, and consequently the prevalence rate of insomnia among the residents, and that noise-induced insomnia is an important public health problem, at least in highly urbanized areas. To confirm this, a further study on noise exposure is needed.

Oligoclonal expansion of cd8+ T-lymphocytes in cultured peripheral lymphocytes derived from asymptomatic HTLV-I carriers.

Peripheral blood mononulear cells (PBMC) derived from 10 asymptomatic human T lymphotropic virus type I (HTLV-I) carriers were cultured for a short term (10-14 days) in the absence of exogenous antigens. In 5 carriers, when compared with 5 HTLV-I non-carriers an apparent increase in the proportion of CD8+ DR+ cells was observed. The clonality of cultured lymphocytes was then examined by analyzing the usage of Vbeta families of T cell receptor genes. In three of the 5 carriers with an increased CD8+ population, two to four Vbeta genes were dominant in the CD8+ population but not in the CD4+ population. No dominance of Vbeta gene usage was observed in lymphocytes derived from the 5 noncarriers. The sequence of cDNA from Vbeta families which were especially dominant revealed their oligoclonal characteristics. These results were quite similar to our previous findings from HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients in whom the same oligoclonal CD8+ cells were expanded cytotoxic T lymphocyte clones for HTLV-I genome products. We posed the question of whether the dominance of TCR Vbeta usage in cultured PBMC was associated with the HTLV-I genome dose in the PBMC or with anti-HTLV-I antibody titers. The three carriers who showed an increased CD8+ population mentioned above all showed a rather high HTLV-I genome dose, which again was similar to HAM/TSP patients. These three carriers however, did not necessarily show high anti-HTLV-I antibody titers in contrast with HAM/TSP patients, who generally do.

Effects of rebamipide, a gastro-protective drug on the Helicobacter pylori status and inflammation in the gastric mucosa of patients with gastric ulcer: a randomized double-blind placebo-controlled multicentre trial.

AIMS: To investigate the effects of rebamipide on the Helicobacter pylori eradication rate with amoxicillin and omeprazole. The trial also examined its histological effects on gastro-mucosal inflammation after eradication. METHODS: Two hundred and six H. pylori-positive patients with active gastric ulcer underwent 8-week based therapy (OA) consisting of 2-week amoxicillin with omeprazole and subsequent 6-week omeprazole. They randomly received either rebamipide (OA-R) or placebo (OA-P) for 16 weeks: combined with the OA based therapy, and subsequently for another 8 weeks. Besides eradication rate, inflammatory findings of gastric mucosa after eradication were evaluated histologically. RESULTS: Per Protocol Set analysis showed no significant difference in eradication rate between OA-R (64.6%; 95% confidence interval, 54.3-75.0%) and OA-P (67.9%; 95% CI, 57.6-78.3%). Histological findings in the gastric mucosa of the ulcer region, however, indicated a significant improvement (P = 0.017) in inflammation scores in OA-R (1.84 +/- 0.41) compared with that in OA-P (2.02 +/- 0.39) after 16-weeks of treatment. This suppressive effect on inflammation was observed even in the OA-R patients unsuccessfully eradicated. CONCLUSION: Rebamipide demonstrated a suppressive effect on the persistent and possibly chronic inflammation in the gastric mucosa of the ulcer region after eradication, but the drug did not improve the eradication rate.

In vitro investigation of a combination of two drugs, cisplatin and carboplatin, as a function of the area under the c/t curve.

The log/log relationship between the IC50 of cisplatin or carboplatin and the exposure time, determined by human tumor clonogenic assay (HTCA) and MTTAI (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with additional incubation) using PC-14 cells, exhibited a straight line with a slope of -1, indicating that both drugs have AUC-dependent cytotoxicity (AUC, area under the c/t curve). The combined effect of cisplatin and carboplatin was estimated by the median-effect analysis using HTCA, and it was additive when the AUC ratio (AUC of free platinum from carboplatin/that from cisplatin) was low (3.2, 6.5 or 13.1 in each of PC-7, PC-9, PC-14, H-69, and K562). However, it was significantly worse at a higher AUC ratio (19.5 in PC-7, PC-9, and PC-14). The log/log relationship of each drug, determined by MTTAI using human bone marrow cells, showed that each drug exerts an AUC-dependent cytotoxicity on marrow granulocytes. When cisplatin and carboplatin were combined at an AUC ratio of 14, the therapeutic index was significantly better than that of carboplatin alone and less than that of cisplatin alone using K562, PC-9 and PC-14, indicating the usefulness of this combined therapy for tumor cells with high sensitivity to platinum compounds at this AUC ratio.

Molecular cloning and characterization of MACIF, an inhibitor of membrane channel formation of complement.

Human erythrocytes contain a membrane protein, MACIF, which inhibits the formation of a membrane attack complex (MAC) of complement. We have cloned and sequenced the complementary DNA of MACIF messenger RNA. The amino acid sequence predicted from its nucleotide sequence consists of 128 amino acids. The amino-terminal 25 residues may correspond to a signal peptide. The carboxy-terminal sequence confirmed that MACIF is a glycosylphosphatidylinositol (GPI)-anchored protein. The amino acid sequence of MACIF was partially determined by established techniques for protein chemistry and the resultant sequence was consistent with that predicted from the nucleotide sequence. The results of sequence analyses also suggested that asparagine at the 18th position was N-glycosylated. When mRNA obtained from the MACIF cDNA clone with SP6 RNA polymerase was microinjected into Xenopus oocytes, the oocytes synthesized a product which exhibited MACIF activity and reacted with anti-MACIF antibody. Comparison of the predicted sequence revealed significant homology with mouse Ly-6 antigens.

Clinical evaluation of teprenone, a mucosal protective agent, in the treatment of patients with gastric ulcers: a nationwide, multicenter clinical study.

The recurrence rate of gastric ulcers healing to a white scar is low, whereas that of ulcers healing to a red scar is high. Teprenone is a mucosal protective agent that can promote epithelial regeneration by increasing the mucosal hexosamine content. It promotes white scar formation when administered as maintenance therapy for ulcers. A nationwide, multicenter study was performed to determine whether white scar formation was also promoted when teprenone was used during initial therapy. Analysis of the data from 1249 patients showed that teprenone significantly promoted white scar formation. The presence of severe ulcers (large size and active stage), ulcer location at the gastric angulus, and smoking retarded white scar formation irrespective of the use of teprenone.

Effects of alpha-kainic acid, domoic acid and their derivatives on a molluscan giant neuron sensitive to beta-hydroxy-L-glutamic acid.

No identifiable giant neuron of an African giant snail (Achatina fulica Férussac) was found to be sensitive to L-glutamic acid (L-Glu). However, some of them, including the RAPN (right anterior pallial neuron), were sensitive to beta-hydroxy-L-glutamic acid (L-BHGA). The effects on RAPN of alpha-kainic acid (alpha-KA), domoic acid (DMA) and their derivatives, which are structurally related to L-Glu and L-BHGA, were examined in order to elucidate the chemical structures necessary to produce the effects under study. Erythro- and threo-L-BHGA showed similar excitatory effects on RAPN (the minimal effective concentration (M.E.C.): 10(-4)M). alpha-KA, alpha-KA methylketone and DMA also showed marked excitatory effects on the same neuron (M.E.C.: 3 X 10(-5)-10(-4)M). The two derivatives of alpha-KA, (2S, 3S, 4S)-2-carboxy-4-(1-methyl-5(R)-hydroxymethyl-l(Z),3(Z)-hexadienyl) pyrrolidine-3-acetic acid and (2S,3S,4S)-2-carboxy-4-(1-methyl-5(R)-hydroxymethyl-1(Z),3(E)-hexadie nyl) pyrrolidine-3-acetic acid, also had excitatory effects (M.E.C.: about 3 X 10(-4)M) which were somewhat less potent than those of L-BHGA, alpha-KA etc. However, alpha-allo-KA, alpha-allo-KA methylketone and dihydro-alpha-KA even at a high concentration of 4.7 X 10(-4)M, had no effect on RAPN. From the results obtained, it appears likely that the seven excitatory compounds mentioned above act on the common receptors of the RAPN neuromembrane, due to similarities in their structures and in the type of excitation caused by these compounds.

Effects of various mucosal protective drugs on diethyldithiocarbamate-induced antral ulcer in rats.

This study was designed to determine the effects of exogenous Cu,Zn-superoxide dismutase (SOD) and various mucosal protective agents against antral ulcer induced by diethyldithiocarbamate (DDC), inhibitor of Cu,Zn-SOD. Exogenous Cu,Zn-SOD reduced ulcer formation and prevented a decrease in SOD activity in gastric mucosa. This result indicates that maintenance of mucosal SOD activity is essential to prevent the ulcerogenicity of DDC. Many mucosal protective drugs that increase blood flow, mucus secretion, and endogenous prostaglandin failed to prevent ulcer formation and decrease of mucosal SOD activity. Rebamipide, however, significantly reduced ulcerogenesis and maintained mucosal SOD activity. This suggests that rebamipide has new protective effects on gastric mucosa.

Transforming growth factor-beta1 and basic fibroblast growth factor modulate osteocalcin and osteonectin/SPARC syntheses in vitamin-D-activated pulp cells.

Vitamin D deficiency elicits hypocalcified dentin. However, little is known about the action of vitamin D on the syntheses of dentin matrix proteins. In this study, we examined the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expressions of osteocalcin and osteonectin/secreted protein, acidic and rich in cysteine (SPARC), by human pulp cells in the presence or absence of transforming growth factor-beta1 (TGF-beta1) or basic fibroblast growth factor (bFGF). 1,25(OH)2D3 markedly increased osteocalcin at protein and mRNA levels. The osteocalcin level induced by 1,25(OH)2D3 was decreased and increased by TGF-beta1 and bFGF, respectively. 1,25(OH)2D3 suppressed SPARC synthesis at protein and mRNA levels. TGF-beta1, but not bFGF, increased SPARC synthesis in the presence of 1,25(OH)2D3. SPARC, but not osteocalcin, increased DNA synthesis in pulp cells. These findings suggest that 1,25(OH)2D3 and growth factors interactively regulate the expression of osteocalcin and SPARC in pulp cells, and that SPARC can stimulate DNA synthesis by pulp cells.