Zingiberis Siccatum Rhizoma, the active component of the Kampo formula Daikenchuto, induces anti-inflammatory actions through α7 nicotinic acetylcholine receptor activation.

BACKGROUND: We previously reported that Daikenchuto (DKT), a gastrointestinal prokinetic Japanese herbal (Kampo) medicine used for the treatment of postoperative ileus (POI), has characteristic potent anti-inflammatory activity. This effect may be partly mediated by the activation of α7 nicotinic acetylcholine receptor (nAChR). In this study, we identified the specific herbs in DKT that induce anti-inflammatory action. METHODS: The herbal components of DKT were individually administered orally to each mouse four times before and after intestinal manipulation (IM) was carried out on the distal ileum. The anti-inflammatory activity of each crude drug was subsequently evaluated using immunohistochemical analyses of relevant molecules. KEY RESULTS: Treatment with Zingiberis Siccatum Rhizoma (ZSR) but not the other components inhibited the infiltration of cluster of differentiation 68 (CD68)-positive macrophages as effectively as DKT treatment. Selective α7nAChR antagonists, such as methyllycaconitine citrate, or transient receptor potential ankyrin 1 (TRPA1) antagonists, such as HC-030031, significantly inhibited the amelioration of macrophage infiltration by ZSR. The inhibition of macrophage infiltration by ZSR was abolished in both α7nAChR and 5-hydroxytryptamine 4 receptor (5-HT4 R) knockout mice. CONCLUSIONS & INFERENCES: Daikenchuto-induced anti-inflammatory activity, which was mediated by inhibiting macrophage infiltration in POI, is dependent on the effects of ZSR. Zingiberis Siccatum Rhizoma activates TRPA1 channels possibly in enterochromaffin (EC) cells to release 5-HT, which stimulates 5-HT4 R in the myenteric plexus neurons to release ACh, which in turn activates α7nAChR on macrophages to inhibit inflammation in POI.

Cycloartenyl ferulate, a component of rice bran oil-derived gamma-oryzanol, attenuates mast cell degranulation.

IgE-targeting therapy could provide significant progress in the treatment of allergic inflammation. In this study, we examined the effect of cycloartenyl ferulate (cycloartenol ferulic acid ester; CAF), a natural product from rice bran oil-derived gamma-oryzanol, on allergic reaction. When CAF and gamma-oryzanol were injected intradermally with anti-DNP IgE into the dorsal skin of rats, the passive cutaneous anaphylaxis reaction induced by DNP-HSA was attenuated. CAF and gamma-oryzanol also inhibited the degranulation of DNP-IgE sensitized RBL-2H3 mast cells stimulated with anti-DNP-HSA. IgE conjugated with CAF could not be detected by anti-IgE antibody in the ELISA analysis. Although incubation of IgE with CAF did not decrease the amount of IgE, it was possible to precipitate IgE by centrifugation. These results demonstrate that CAF captures IgE, prevents it from binding to FcepsilonRI, and attenuates mast cell degranulation.

Attenuation by dietary taurine of dextran sulfate sodium-induced colitis in mice and of THP-1-induced damage to intestinal Caco-2 cell monolayers.

The effects of dietary taurine on the experimental colitis induced by dextran sulfate sodium (DSS) in mice were evaluated. C57BL/6 female mice were given 3% DSS in drinking water for 5 d to induce acute colitis. Taurine at 2% was added to the drinking water 5 d before and during the DSS-treatment to investigate its preventive effect. Taurine supplementation significantly attenuated the weight decrease, diarrhea severity, colon shortening, and the increase in the colonic tissue myeloperoxidase activity induced by DSS. Taurine also significantly inhibited the increase in the expression of a pro-inflammatory chemokine, macrophage inflammatory protein 2 (MIP-2), but not of interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha mRNA. Furthermore, taurine significantly protected the intestinal Caco-2 cell monolayers from the damage by macrophage-like THP-1 cells in an in vitro coculture system. These results suggest that taurine prevented DSS-induced colitis partly in association with (1) its inhibitory effects on the secretion of MIP-2 from the intestinal epithelial cells and on the infiltration of such inflammatory cells as neutrophils and (2) its cytoprotective functions on the epithelial barrier from the direct toxicity of DSS and from the inflammatory cell-induced injury.

A search for the plant ingredients that protect cells from air pollutants and benz[a]pyrene phototoxicity.

A diversity of antioxidants and plant ingredients were examined for their protective effect in cultured Balb/c 3T3 cells against ultraviolet A (UVA)-induced cytotoxicities of extracted air pollutants and benz[a]pyrene (B[a]P) in an effort to find effective protectors against the phototoxicity of air pollutants and B[a]P. As has been observed for B[a]P phototoxicity, air pollutants themselves and those previously exposed to UVA light in the absence of cells exhibited faintly weak cytotoxicity, but the toxicity was markedly elevated when they were exposed to UVA light concomitantly with cells. The B[a]P phototoxicity was not eliminated by well-known antioxidants but was markedly diminished by diversity of plant ingredients. Among the plant ingredients tested in the current study, morin, naringin, and quercetin were found to be desirable protectors against B[a]P phototoxicity.

Long-term administration of amlodipine prevents decompensation to diastolic heart failure in hypertensive rats.

UNLABELLED: OBJECTIVES; We assessed the effects of long-term amlodipine administration in a diastolic heart failure (DHF) rat model with preserved systolic function as well as the relationship between changes in left ventricular (LV) myocardial stiffening and alterations in extracellular matrix. BACKGROUND: Although the effect of long-term administration of amlodipine has been shown to be disappointing in patients with systolic failure, the effect is unknown in those with DHF. METHODS: Dahl salt-sensitive rats fed a high-salt diet for seven weeks were divided into three groups: eight untreated rats (DHF group), eight rats given high-dose amlodipine (10 mg/kg/day; HDA group) and seven rats given low-dose amlodipine (1 mg/kg/day; LDA group). RESULTS: High-dose administration of amlodipine decreased systolic blood pressure and controlled excessive hypertrophy, without a decrease in the collagen content, and prevented the elevation of LV end-diastolic pressure at 19 weeks. Low-dose administration of amlodipine with subdeppressive effects did not control either hypertrophy or fibrosis; however, it prevented myocardial stiffening and, hence, the elevation of LV end-diastolic pressure. The ratio of type I to type III collagen messenger ribonucleic acid levels was significantly lower in both the HDA and LDA groups than in the DHF group. CONCLUSIONS: Long-term administration of amlodipine prevented the transition to DHF both at the depressor and subdepressor doses. Amlodipine did not decrease the collagen content, but attenuated myocardial stiffness, with inhibition of the phenotype shift from type III to type I collagen. Thus, amlodipine may exert beneficial effects through amelioration of collagen remodeling in the treatment of DHF.

Chronic treatment with FK506 increases p70 S6 kinase activity associated with reduced nitric oxide synthase activity in rabbit hearts.

FK506, an immunosuppressant, modulates phosphorylation of nitric oxide (NO) synthase, and induces cardiac hypertrophy in clinical settings. Having recently reported that chronic treatment with an inhibitor of NO synthase induces cardiac hypertrophy associated with the activation of 70-kD S6 kinase (p70S6K), which plays an important role in cardiac hypertrophy by regulating protein synthesis, we investigated the effects of chronic administration of FK506 on NO synthase and p70S6K activities in hearts. Twenty rabbits were divided into four groups: untreated rabbits, those treated with low-dose FK506 (0.10 mg/kg), those treated with medium-dose FK506 (0.20 mg/kg), and those treated with high-dose FK506 (0.40 mg/kg). FK506 was administered intravenously twice a day. After 4 weeks of treatment with FK506, calcium-dependent NO synthase activity in myocardium in the high-dose FK506 group was lower (P < 0.05) than in the untreated group. p70S6K activity in myocardium in the high-dose group was higher (P < 0.05) than in the untreated group. There was a significant (P < 0.05) inverse correlation between NO synthase and p70S6K activities in myocardium. However, the endothelial-dependent vasodilation of aortic rings or plasma levels of NO metabolites during experimental protocols did not differ among the groups studied. These findings suggest that chronic treatment of FK506 activates p70S6K and reduces NO synthase activity in rabbit hearts. Reduced NO synthase and/or activated p70S6K activities in hearts might contribute to the cardiac hypertrophy observed in some patients receiving FK506.

The mouse ZFH-4 protein contains four homeodomains and twenty-two zinc fingers.

We isolated the mouse zfh-4 cDNA which is 12 kb long and capable of encoding a 3,550-amino acid protein containing four homeodomains and 22 zinc fingers including two pseudo zinc finger motifs. The mouse ZFH-4 is 51% homologous to the mouse ATBF1 and 23% to the Drosophila ZFH-2. The homeodomain and zinc finger regions are highly conserved between ZFH-4 and ATBF1 except that one zinc finger is missing in ZFH-4. Analysis of partial genomic sequences showed that the mouse zfh-4 and ATBF1 genes are similar in exon-intron organization. RT-PCR analysis of zfh-4 transcripts in adult mouse tissues showed that zfh-4 expression was low but reproducibly detectable in brain, heart, lung and muscle. In these mouse tissues, ATBF1 transcripts were poorly amplified by PCR under the conditions where zfh-4 transcripts were amplified, suggesting that the expression of zfh-4 mRNA is higher than that of ATBF1 mRNA. Other comparative analysis suggests functional similarities and dissimilarities between ZFH-4 and ATBF1.

Intermittent administration of human parathyroid Hormone(1-34) prevents immobilization-related bone loss by regulating bone marrow capacity for bone cells in ddY mice.

ddY mice, 6 weeks of age, were neurectomized (Nx) in the right hindlimbs and sham-operated (Sham) in the left limbs for evaluation of the effects of intermittent injections of human parathyroid hormone (hPTH) on trabecular bone turnover and bone marrow cell development in unloaded and loaded limbs. Mice were given subcutaneous injections of hPTH(1-34) five times a week at a dose of 0 (vehicle), 4 (low dose), or 40 (high dose) microg/kg of body weight for 2, 4, or 6 weeks. Histomorphometric analyses of the trabecular bone of the proximal tibiae revealed that high-dose hPTH injections preserved the trabecular bone volume of the Nx limbs, which was reduced after neurectomy, at the same level as that of the contralateral Sham limbs. The mineral apposition rate in the Nx limbs was elevated to values above even that of the Sham limbs by high-dose hPTH injections. The bone formation rate reduced by neurectomy was maintained at the Sham level by low- and high-dose hPTH injections. The neurectomy-induced increase in osteoclast number was suppressed by high-dose hPTH injections. In the bone marrow cells, the numbers of nonadherent and adherent cells per tibia obtained from the Nx and Sham limbs did not change. The hPTH injections decreased the numbers of nonadherent cells and increased those of adherent cells in both the Nx and the Sham limbs, but the effects were less marked in the Nx than in the Sham limbs even at high-dose injections. The formation of osteogenic nodules in the marrow cultures obtained from the Nx limbs was decreased after surgery and was maintained at the level of the Sham limbs by high-dose hPTH injections. The number of osteoclast-like multinucleated cells was reduced in the Sham limbs by high-dose hPTH injections. The value was increased at 2 weeks after neurectomy, but it was maintained at the Sham level by high-dose hPTH injections through the experimental period. The numbers of colony forming units-fibroblastic, which were reduced by neurectomy, and those of colony forming units for granulocytes and macrophages were not altered by hPTH injections. These results demonstrate that intermittent high-dose hPTH administration in the Nx limbs as well as in the contralateral Sham limbs has similar anabolic effects, stimulating osteoblast cell lineage and suppressing osteoclast cell lineage. The anabolic effects at 4 microg were reduced, but the effects at 40 microg seemed to be less affected by unloading due to sciatic neurectomy.

ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells.

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper-interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642-1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin-activated MgATPase activity of myosin II. HeLa ZIPK is the first non-muscle MLCK that phosphorylates MRLC at two sites.

High water intake ameliorates tubulointerstitial injury in rats with subtotal nephrectomy: possible role of TGF-beta.

BACKGROUND: It has been shown that tubulointerstitial injury correlates well with a decline of renal function. In this study, we investigated the effect of high water intake (HWI) on functional and structural parameters in rats with subtotal nephrectomy. METHODS: Two weeks after the ablative procedure, rats were divided into two groups. One group received the treatment with HWI (3% sucrose added to drinking water) for eight weeks. Functional parameters were compared with sham-operated control (CONT) or nephrectomized rats without treatment (NX). Remnant kidneys were then assessed histologically for evidence of interstitial fibrosis and glomerulosclerosis. RESULTS: Creatinine clearance was significantly improved in HWI rats compared with NX rats. Simultaneously, urinary protein was also significantly reduced in HWI rats. HWI predominantly ameliorated interstitial lesions and, to a lesser extent, glomerular lesions. Northern blot analysis demonstrated that transforming growth factor-beta (TGF-beta) mRNA expression was significantly suppressed in HWI rats. In situ hybridization revealed that HWI suppressed TGF-beta mRNA expression mainly in the outer medulla. Fibronectin mRNA was also reduced by the HWI treatment. The changes in TGF-beta and fibronectin mRNA were in parallel with Na+/myo-inositol cotransporter (SMIT) mRNA, which is regulated by extracellular osmolarity. Immunohistochemistry demonstrated that protein expression of TGF-beta and fibronectin coincided with the mRNA expression. CONCLUSION: These results suggest that HWI reduces TGF-beta mRNA expression in medullary interstitium and ameliorates tubulointerstitial injury in rats with reduced renal mass.

Lumped constant for deoxyglucose is decreased when myocardial glucose uptake is enhanced.

Quantification of myocardial glucose uptake by positron emission tomography with [18F]fluorodeoxyglucose (FDG) requires the "lumped constant" (LC), which corrects the difference of affinity between glucose and FDG to glucose transporters and phosphorylating system. Since LC was introduced, it has been considered to be constant. However, this has recently been questioned. To elucidate the constancy of LC by other than radioisotope techniques, the accumulation rate of sugar phosphates (d[SP]/dt) was measured in isolated, perfused rat hearts by 31P NMR spectroscopy with 2-deoxyglucose (DG). We postulate alpha as the affinity of DG to transporters and the phosphorylating system relative to that of glucose. Theoretically, alpha is equivalent to LC. We determined alpha by measuring d[SP]/dt at DG concentration ([DG]) = 10, 7, 5, and 3 mmol/l, keeping the total of glucose concentration ([glucose]) and [DG] to 10 mmol/l. When the glucose uptake was enhanced by insulin (10 mU/ml) or stunning, calculated alpha was reduced (insulin stimulated, 0.15; stunning, 0.19) compared with the control (0.59). These results indicate that LC can be evaluated by methods without radiolabeled tracers and is smaller when glucose uptake is augmented.

Whole-body hyperthermia provides biphasic cardioprotection against ischemia/reperfusion injury in the rat.

BACKGROUND: Hyperthermia increases cardiac tolerance to ischemia/reperfusion injury 24 hours after the heat stress. Free radicals and redox mechanisms have been implicated in such tolerance. However, the time course and its relation to the induction of antioxidative enzymes in the protection induced by whole-body hyperthermia against ischemia/reperfusion injury are unknown. METHODS AND RESULTS: Hyperthermia was induced in anesthetized rats by placement in a temperature-controlled water bath. After the defined recovery interval(s) at room temperature, ischemia was induced by occlusion of the left coronary artery for 20 minutes, followed by reperfusion for 48 hours. The exposure to hyperthermia led to a recovery interval- dependent, biphasic reduction in the incidence of ventricular fibrillation during ischemia and in the size of the myocardial infarct as determined after 48 hours of reperfusion. The time course of the late-phase (24- to 96-hour recovery interval) but not the early-phase (0.5 hour) cardioprotection depended on the degree of hyperthermia. The time course of the increase in myocardial manganese superoxide dismutase (Mn-SOD) activity corresponded to that of the cardioprotective effects, although an increase in the content of Mn-SOD and of heat shock protein 72 corresponded only to the late-phase effects. Administration of an antioxidant before hyperthermia abolished the early- and late-phase cardioprotection and the increase in Mn-SOD activity. CONCLUSIONS: THe activation of Mn-SOD mediated by free radical production during hyperthermia is important in the acquisition of early-phase and late-phase cardioprotection against ischemia/reperfusion injury in rats.

Synthesis and antiallergic activity of dimethyl-2-(phenylcarbamoyl)ethylsulfonium p-toluenesulfonate derivatives.

The derivatives of dimethyl-2-(phenylcarbamoyl)ethylsulfonium p-toluenesulfonates were synthesized and evaluated for antiallergic activity. The 2,3-dihydroxyethoxy group was introduced to the phenyl ring from the standpoint of lipophilicity and electronic effects of substituent. The IgE-induced rat passive cutaneous anaphylaxis (PCA) was inhibited by oral administration of several substituted 2-[(4-propoxyphenyl)carbamoyl]ethyldimethylsulfonium p-toluenesulfonate derivatives. Among them (+/-)-2-[N-[4-(3-ethoxy-2-hydroxypropoxy)phenyl]carbamoyl]ethyldimeth ylsulfonium p-toluenesulfonate (1a, IPD-1151T) was found to possess considerable activity in the PCA test, and it was launched as Suplatast tosilate in Japan.

Inhibition of myo-inositol transport causes acute renal failure with selective medullary injury in the rat.

Myo-inositol is a major compatible osmolyte in the renal medulla that is accumulated under hypertonic conditions via the Na+/myo-inositol cotransporter (SMIT). We have recently reported that SMIT is predominantly present in the thick ascending limb of Henle (TAL) and is strongly induced by acute NaCl loading, suggesting an important role of myo-inositol in this nephron segment. In the present study, we sought to examine in vivo effects of inhibition of myo-inositol transport using a transport inhibitor, 2-O, C-methylene-myo-inositol (MMI). Intraperitoneal injection of MMI caused acute renal failure in the rats. Serum creatinine and urea nitrogen were significantly increased 12 hours after MMI injection. Morphologic study revealed that the tubular cells were extensively injured in the outer medulla. A considerable number of the tubular cells were injured in the cortex as well. Immunohistochemical study for Tamm-Horsfall protein (THP), which was used for identification of the TAL cells, showed that THP-positive cells were predominantly injured. The tubular injury apparently appeared to worsen when high concentration of NaCl was injected with MMI. Administration of myo-inositol prevented acute renal failure and improved the tubular injury after MMI injection. Furthermore, supplementation of betaine, another osmolyte in the TAL cells, partially prevented the toxic effects of MMI. These results suggest that myo-inositol play a crucial role in the TAL regarding osmoregulation of the cells.

Nisoldipine selectively induces coronary vasodilation and improves mild myocardial ischemia in dogs: a potential role of cellular acidosis.

We examined whether nisoldipine, a calcium (Ca) channel blocker, increases coronary blood flow (CBF) without decreasing aortic blood pressure (AoP) with ischemic and nonischemic hearts, and whether the presence of cellular acidosis in ischemic myocardium contributes to the augmentation of coronary vasodilation due to nisoldipine. In 42 dogs, coronary perfusion pressure (CPP) was reduced so that CBF decreased to 60% of the baseline, and CPP was maintained constant thereafter. First, we administered nisoldipine into a systemic vein in the ischemic and nonischemic hearts. Second, nisoldipine was administered into the canine coronary artery of the ischemic myocardium, with and without administration of either sodium bicarbonate (NaHCO3), sodium hydroxide (NaOH), or amiloride. Nisoldipine (0.25-4.0 mg/kg, i.v.) increased CBF by 59% in the ischemic myocardium more than the nonischemic myocardium (by 34%) without reducing AoP. The infusion of nisoldipine (40 ng/kg/min, IC) increased CBF markedly by about 55% in the ischemic myocardium with increases in fractional shortening (FS; 11 +/- 2% to 21 +/- 2%) and lactate extraction ratio (LER; -19 +/- 4% to 15 +/- 2%). Increases in CBF, FS, and LER were markedly attenuated during administration of nisoldipine with concomitant administration of either NaHCO3 or NaOH. Furthermore, the extent of increases in CBF (54 +/- 2 mL/100 g/min), FS (13 +/- 2%), and LER (-17 +/- 4%) were also markedly attenuated due to the concomitant treatment with amiloride. We conclude that myocardial cellular acidosis plays an important role in mediating coronary vasodilation affected by nisoldipine in the ischemic myocardium. H+ may modulate the property of voltage-dependent Ca channels via Na(+)-H+ exchange.

Enhancement of glucose uptake in stunned myocardium: role of glucose transporter.

This study quantifies the myocardial glucose uptake and clarifies the pathway of augmented glucose uptake in myocardium reperfused after a brief period of ischemia (stunned myocardium). The glucose uptake rate was determined from the time course of the sugar phosphate (SP) resonance in rat myocardium (d[SP]/dt) with 31P nuclear magnetic resonance after the substitution of glucose with its analog 2-deoxyglucose. The d[SP]/dt in stunned myocardium [1.03 +/- 0.05 (SE) micromol x g wet wt(-1) x min(-1); n = 8] increased significantly compared with nonischemic control myocardium (0.18 +/- 0.03 micromol x g wet wt(-1) x min(-1); n = 8; P < 0.0001), reaching the maximal stimulatory uptake rate during exposure to insulin (1.05 +/- 0.04 micromol x g wet wt(-1) x min(-1); n = 8). Twenty minutes after reperfusion, the d[SP]/dt was still augmented (0.41 +/- 0.05 micromol x g wet wt(-1) x min(-1); n = 5; P < 0.05 vs. control myocardium). To elucidate further the mechanism of augmented glucose uptake, N6-(L-2-phenylisopropyl)-adenosine (PIA; 100 micromol/l), a potent blocker of the glucose transporter, was administered to stunned hearts and, as a control, to insulin-stimulated hearts. PIA significantly and comparably inhibited the increase in d[SP]/dt in stunned myocardium (0.36 +/- 0.07 micromol x g wet wt(-1) x min(-1); n = 4; P < 0.0001 vs. without PIA) and in insulin-stimulated myocardium (0.38 +/- 0.02 micromol x g wet wt(-1) x min(-1); n = 4; P < 0.0001 vs. without PIA). These results indicate that the augmented glucose uptake in stunned myocardium is maintained by the glucose transporter, the amount of which is almost equal to that which can be maximally recruited by insulin.

Plausible novel therapeutic strategy of uterine endometrial cancer with reduction of basic fibroblast growth factor secretion by progestin and O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM-1470).

To know the potential of growth, invasion and metastasis of endometrial cancer associated with neovascularization, the effects of sex steroids and O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM-1470) on basic fibroblast growth factor (FGF) expression and secretion and its mRNA expression were investigated in well-differentiated endometrial cancer cell line Ishikawa and in undifferentiated endometrial cancer cell line AN3 CA. Basic FGF expression and secretion and its mRNA expression in Ishikawa cells, but not in AN3 CA cells, were increased by estrogen, while progesterone diminished the estrogen-induced increases. TNP-470 reduced the levels regardless of estrogen treatment in AN3 CA cells. Therefore, basic FGF secretion may be inhibited by progestin in differentiated cells, and by TNP-470 in undifferentiated cells. Since endometrial cancer consists of differentiated and undifferentiated cells as heterogeneity, a combination therapy for endometrial cancer with progestin and TNP470 might be effective.

Effects of intermittent administration of low dose human PTH(1-34) on cancellous and cortical bone of lumbar vertebral bodies in adult beagles.

This study assessed the effect of low dose human parathyroid hormone [hPTH(1-34)] administration on cancellous and cortical bone of lumbar vertebrae in intact male beagles. 16 19-20-month-old beagle dogs were randomized into four groups: in group 1, the vehicle control group, saline was injected daily; in group 2, the sequential group, 0.375 microg/kg of PTH was injected daily for 4 weeks, then off 8 weeks, and this sequence was once repeated for another 4 and 8 weeks; in group 3, the same dose of PTH was injected once per week for 24 weeks; and, in group 4, PTH was injected three times per week for 24 weeks. Histomorphometric assessment on cancelllous and cortical bone (both ventral and dorsal shell) and two-dimensional node-strut analysis were done on the fifth lumbar vertebral bodies after calcein double bone labeling. In intact adult beagles, on the group treated with 0.375 microg/kg per day three times per week (group 4): (1) had a higher mean value in cancellous bone formation parameters [osteoid surface (+74%), osteoid volume (twofold), mineral apposition rate (+21%), and bone formation rate (twofold)]; (2) exhibited no effect on cortical thickness and porosity in both the ventral and dorsal shell; and (3) showed a lower mean value of node to termini (0.11 +/- 0.02 vs. 0.22 +/- 0.09) and a higher mean value of cortex to node (0.18 +/- 0.06 vs. 0.08 +/- 0.02), but not in trabeculae to trabeculae node, than age-related controls. In conclusion, we found that a low dose of PTH administration: (1) stimulated cancellous bone formation; (2) improved connectivity of trabeculae joined to the cortex; (3) did not decrease cortical thickness; and (4) did not increase cortical porosity in both ventral and dorsal cortexal shell of the lumbar vertebrae during this dosage and period in intact male beagles.

Modulation of picryl chloride-induced contact hypersensitivity reaction in mice by nitric oxide.

We have studied the possible involvement of nitric oxide (NO) in the contact hypersensitivity reaction. A biphasic response of ear swelling was observed at 2 h (early phase) and 24 h (late phase) after application of the antigen to picryl chloride (PC1)-sensitized CBA/J mice. Intravenous injection of NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), at the time of PC1 challenge, inhibited in a concentration-dependent fashion the antigen-induced contact hypersensitivity reaction. Low-dose (1 mg/kg) L-NAME inhibited the early-phase reaction but not the late-phase reaction. High-dose (250 mg/kg) L-NAME inhibited both early- and late-phase reactions. D-NAME (enantiomer of L-NAME) did not inhibit the antigen-induced ear swelling. High-dose (250 mg/kg) L-arginine increased both early and late phase reactions. D-Arginine (enantiomer of L-arginine) did no increase the antigen-induced ear swelling. L-NAME injection, however, did not suppress phenol-induce irritant inflammation. Treatment of mice undergoing PC1-induced contact hypersensitivity reaction with L-NAME reduced the production of interleukin-2 and interferon-gamma by draining lymph node cells. Treatment with L-arginine, on the other hand increased the production of interleukin-2 and interferon-gamma. These results suggest that NO plays a modulating role in contact hypersensitivity reaction.

Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides.

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T-antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [32P]phosphorous-containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS-dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [gamma-32P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T-antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T-antigen, and the C-terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.