Calcitonin gene-related peptide protects rats from cerebral ischemia/reperfusion injury via a mechanism of action in the MAPK pathway.

The aim of the present study was to investigate the protective function and underlying mechanism of calcitonin gene-related peptide (CGRP) on cerebral ischemia/reperfusion damage in rats. Adult male Wistar rats were selected for the establishment of an ischemia/reperfusion injury model through the application of a middle cerebral artery occlusion. Animals were randomly divided into 6 groups of 24 animals. Drugs were administered according to the design of each group; animals were administered CGRP, CGRP8-37, PD98059 and SB20358. The neurobehavioral scores of the rat cerebral ischemia model in each group were calculated. The infarction range of the rat brain tissues was observed by 2,3,5-triphenyltetrazolium chloride staining. The expression levels of three proteins, phosphorylated c-Jun N-terminal kinase (JNK)/JNK, phosphorylated extracellular signal-regulated protein kinase (ERK)/ERK and p-p38/p38, in the mitogen-activated protein kinase (MAPK) pathway in the brain tissues was detected by western blotting. The results showed that CGRP could improve the neurobehavioral function of the ischemic rats and reduce the infarction range. Western blotting results confirmed that the function of the CGRP was mediated mainly through the reduction of the JNK and p38 phosphorylation and the promotion of ERK phosphorylation. Therefore, the present study confirmed that an increase in the exogenous CRGP could effectively improve ischemia/reperfusion injury of the brain tissue. The mechanisms of action were achieved through a reduction in JNK and p38 phosphorylation and an increase in ERL phosphorylation in the MAPK pathway. These mechanisms were interdependent.

Atopic dermatitis phenotype with early onset and high serum IL-13 is linked to the new development of bronchial hyperresponsiveness in school children.

BACKGROUND: Atopic dermatitis (AD) is characterized by a heterogeneous clinical spectrum, and some forms of AD are associated with the initial steps of allergic march. The aims of this study were to determine AD phenotypes in school-age children and investigate their associations with the allergic march in each cluster. METHODS: We included 242 children (6-8 years) with current AD from the Children's HEalth and Environmental Research study, a 4-year prospective follow-up study with 2-year survey intervals. Latent class analysis was used. Serum IL-13 and thymic stromal lymphopoietin (TSLP) levels at the time of enrollment were measured using ELISA. RESULTS: We identified four current AD phenotypes in children, characterized as 'early onset with low atopy' (26.4% of the sample; group 1), 'early onset with high atopy and high eosinophil percentages' (48.3%; group 2), 'late onset with low atopy' (9.9%; group 3), and 'late onset with high atopy and normal eosinophils' (15.3%; group 4). Although groups 2 and 4 demonstrated high atopic burden, children in group 2 showed the persistence of AD and eosinophilia associated with a high prevalence of new cases of bronchial hyper-responsiveness and asthma symptoms during follow-up. The serum IL-13 level was significantly increased in the early-onset AD groups, but there was no significant difference in the serum TSLP levels across all four groups. CONCLUSION: An allergic march-associated AD phenotype exists that is characterized by early onset of AD with its persistence, increased serum IL-13 levels, high atopy, and a persistently increased blood eosinophil percentage.

Lateral spread response monitoring during microvascular decompression for hemifacial spasm. Comparison of two targets of partial neuromuscular blockade.

AIM: The aim of the present study was to determine (1) whether successful intraoperative electromyography monitoring for lateral spread response (LSR) is possible with partial neuromuscular blockade (NMB) in subjects undergoing microvascular decompression (MVD) for hemifacial spasm and (2) the adequate level of NMB to achieve that goal. MATERIAL AND METHODS: A total of 61 patients in whom LSR was monitored during MVD were enrolled in the study. Patients were randomly allocated to two groups: group TOF in which the NMB target was maintenance of two train-of-four (TOF) counts and group T1 in which the NMB target was maintenance of a T1/Tc ratio of 50 % (T1: first twitch height of TOF and Tc: control twitch height). The adductor pollicis brevis muscle was used to monitor TOF responses. The frequency of successful LSR monitoring, defined as successful baseline establishment and maintenance of LSR until surgical decompression, was compared between the two groups. RESULTS: Of the 61 patients 2 were excluded from the study so that 30 patients in group TOF and 29 patients in group T1 were analyzed. The success rate of LSR monitoring was clinically acceptable and significantly higher in group T1 than in group TOF, i.e. n = 15 (50.0 %) in group TOF versus n = 24 (82.8 %) in group T1 (P = 0.008), corresponding to a 32.8 % higher success rate in group T1 than group TOF (95 % CI: 13.9-51.7 %). Mean vecuronium infusion dose was smaller and mean TOF count was higher in group T1 than group TOF with a TOF count = 2 (1) in group TOF versus 3 (1) in group T1 (P = 0.003). Mean sevoflurane and remifentanil infusion doses were not different between groups. There was no incidence of spontaneous movement during microscopy in either group. CONCLUSION: Maintenance of partial NMB with a target T1/Tc ratio of 50 % resulted in a clinically acceptable success rate of LSR monitoring and surgical condition during MVD. Maintenance of partial NMB with a target T1/Tc ratio of 50 % rather than TOF count of two during LSR monitoring for MVD can therefore be recommended.

Synthesis and in vitro cytotoxicity of 4-alkyl- or 4-arylaminosubstituted cyclopenta[c]quinoline derivatives.

Twelve 4-substituted cyclopenta[c]quinoline derivatives were synthesized and evaluated in vitro cytotoxicity against four human cancer cell lines (HOP62, SK-OV-3, MD-MB-468 and T-47D). The compounds 6c and 6e bearing p-anisidine and pyrrolidine side chain were more active than the others.

Overexpression of Akt inhibits NGF-induced growth arrest and neuronal differentiation of PC12 cells.

To investigate the role of Akt in nerve growth factor (NGF)-induced neuronal differentiation, PC12 cells ectopically expressing wild-type or dominant-inhibitory forms of Akt were analyzed. NGF-induced neurite outgrowth was greatly accelerated in cells expressing dominant-inhibitory Akt, compared to parental PC12 cells, but was almost completely blocked in cells expressing wild-type Akt. Since neuronal differentiation requires an arrest of cell growth, several aspects of cell growth of the different cell lines were compared. Cells expressing wild-type Akt were not susceptible to the growth-arresting effect of NGF, whereas parental PC12 cells and notably cells expressing mutant Akt were so affected. Accompanying this, the expressions of CDKs and p21(WAF1) were down- and up-regulated, respectively, in both parental PC12 cells and cells expressing mutant Akt. When treated with some growth arrest-inducing agents such as sodium nitroprusside, forskolin and butyrolactone I, cells expressing wild-type Akt regained their responsiveness to the effects of NGF on differentiation. In summary, our results indicate that Akt overrides the growth-arresting effect of NGF and thereby, negatively regulates neuronal differentiation.

Synthesis and in vitro cytotoxicity of 2-alkylaminosubstituted quinoline derivatives.

Eight 2-alkylaminosubstituted 5,8-dimethoxy-4-methylquinolines and nine 2-alkylaminosubstituted or 2,6-disubstituted 4-methylquinoline-5,8-diones were synthesized and evaluated in vitro cytotoxicity against four human cancer cell lines (HOP62, SK-OV-3, HCT15 and SF295).

Heat shock-induced, caspase-3-independent rapid breakdown of Akt and consequent alteration of its total phosphorylation/activity level.

The immediate effect of a 15-min heat shock was examined on the level and the activity of Akt. Following heat shock, the Akt level decreased by 15-70% in a temperature-dependent and phosphorylation status-independent manner. This decrease of Akt level was not prevented by caspase inhibitors. At 48 degrees C, the extent of the breakdown was so immense that the total phosphorylation/activity level of Akt was not increased over the control level, implying that the total cellular activity of Akt governed by the level and the molar activity does not necessarily undergo the ensuing change.

Experience with continuous infusion of recombinant activated factor VII in the Asia-Pacific region.

There is increasing interest in continuous infusion of recombinant activated factor VII (rFVIIa) as a convenient and safe alternative to intermittent bolus therapy. In the Australian patients reported in this paper, cost savings of up to 25% in the first 12 h of treatment with continuous infusion of rFVIIa have been achieved safely, suggesting that substantial overall savings are possible. However, in the Thai patient reported, a dose reduction of 35% in the first 12 h was associated with poor haemostatic control, suggesting that a dose reduction of >25% may be inadvisable. The indications for treatment in the five Australian patients were: retroperitoneal haemorrhage (n = 3); right forearm compartment syndrome (n = 1); wrist haemarthrosis and median nerve compression (n = 2); sublingual haematoma (n = 1); and cerebral (mid-brain) haemorrhage (n = 1). Treatment was effective in four out of five patients (six bleeding episodes) and there was one treatment failure where treatment had been substantially delayed. The Thai patient was treated as part of a prospective, uncontrolled, observational study of 34 bleeding episodes in 22 patients in the Asia-Pacific region. Treatment was judged ineffective after 24 h, but full haemostatic control was subsequently achieved with intermittent rFVIIa therapy.

Regulatory mechanism of polarized membrane transport by glucocorticoid in renal proximal tubule cells: involvement of [Ca2+]i.

We examined the effect of glucocorticoids on brush border membrane transporters and, furthermore, the involvement of Ca2+ in its action in the primary cultured rabbit renal proximal tubule cells (PTCs). Dexamethasone (DEX, 10(-9) M) decreased Pi uptake by 17%; whereas DEX affected neither alpha-methyl-glucopyranoside (alpha-MG) uptake nor Na+ uptake. The DEX-induced inhibition of Pi uptake was due to a decrease of V(max). In contrast, other steroid hormones such as progesterone, testosterone, and 17beta-estradiol (10(-9) M) did not induce inhibition of Pi uptake. In order to examine the involvement of Ca2+ in DEX-induced inhibition of Pi uptake, PTCs were treated with A 23187 (10(-6) M, Ca2+ ionophore). A 23187 also inhibited Pi uptake, mimicking DEX action in Pi uptake. Treatments with W-7 (10(-4) M, calmodulin dependent kinase inhibitor), KN-62 (10(-6) M, Ca2+/calmodulin-dependent protein kinase II inhibitor), and BAPTA/AM (10(-6) M) or TMB-8 (10(-4) M) (intracellular Ca2+ mobilization blockers) blocked the DEX-induced inhibition of Pi uptake. However, nifedifine, methoxyverapamil (10(-6) M, L-type Ca2+ channel blockers), and EGTA (1 mM, extracellular Ca2+ chelator) did not block it. In conclusion, DEX inhibited Pi uptake via, in part, Ca2+/calmodulin pathway mediated by intracellular Ca2+ mobilization in the PTCs.

Synthesis and in vitro cytotoxicity of 1-azaanthraquinone-3-carboxamides.

Five 1-azaanthraquinone-3-carboxamides were synthesized and evaluated in vitro cytotoxicity against four human cancer cell lines. The most active compound, 7b, exhibited cytotoxic activity comparable to doxorubicin.

Psammaplin A, a natural bromotyrosine derivative from a sponge, possesses the antibacterial activity against methicillin-resistant Staphylococcus aureus and the DNA gyrase-inhibitory activity.

Psammaplin A, a natural bromotyrosine derivative from an associated form of two sponges (Poecillastra sp. and Jaspis sp.) was found to possess the antimicrobial effect on the Gram-positive bacteria, especially on methicillin-resistant Staphylococcus aureus (MRSA). The minimal inhibitory concentration of psammaplin A against twenty one MRSAs ranged from 0.781 to 6.25 microg/ml, while that of ciprofloxacin was 0.391-3.125 microg/ml. Psammaplin A could not bind to penicillin binding protein, but inhibited the DNA synthesis and the DNA gyrase activity with the respective 50% (DNA synthesis) and 100% (DNA gyrase) inhibitory concentration 2.83 and 100 microg/ml. These results indicate that psammaplin A has a considerable antibacterial activity, although restricted to a somewhat narrow range of bacteria, probably by inhibiting DNA gyrase.

Synthesis and in vitro cytotoxicity of 3-substituted-1,8-diazaanthraquinones produced by Lewis-acid catalyzed hetero Diels-Alder reaction.

A hetero Diels-Alder reaction of quinoline-5,8-dione with 1-(N,N-dimethylamino)-3-methyl-1-aza-1,3-butadiene proceeded to give 3-methyl-1,8-diazaanthraquinone (100% regioselectivity) in the presence of Lewis-acid catalyst (ZnCl2 or ZnBr2). Subsequent functionalizations of the benzylic methyl group resulted in the 1,8-diazaanthraquinone analogues as potential antitumor agents. The most active compound, 8, exhibited in vitro cytotoxic activity comparable to that of doxorubicin.

The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase.

The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.

AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.

Influence of intact and partially hydrolysed guar gum on iron utilization in rats fed on iron-deficient diets.

Effects of partially hydrolysed guar gum (PHGG) or intact guar gum (GG) on iron utilization in rats fed on several iron-deficient diets were examined. Hemoglobin, serum iron and iron storage in liver of rats fed on iron-deficient diets as a control group (without PHGG and GG) significantly decreased, while those of the test group fed together with PHGG or GG were unchanged. In an iron balance test for 3 days, administration of PHGG or GG caused an increase in iron absorption. The results suggested that PHGG or its metabolites increase the bioavailability of dietary iron in deficiency.

Comparison of heterotrimeric protein phosphatase 2A containing different B subunits.

Protein phosphatase 2A (PP2A) is composed of structural (A), catalytic (C), and regulatory subunits (B). Immunological analyses identified B alpha/PR55 alpha as the major regulatory subunit of brain PP2A while a unique B' subunit was associated with the cardiac enzyme. Recombinant PP2A heterotrimers were purified from insect cells infected with baculoviruses expressing A and C, in combination with viruses expressing B alpha/PR55 alpha, B beta/PR55 beta, or SV40 small tumor antigen (st). Phosphatase activities of rAC-B alpha and rAC-B beta were similar to those for brain AC-B alpha, while rAC-st was 50-80% less active. Heparin had no effect on rAC-st myosin light chain phosphatase activity, while the B subunit-containing forms were stimulated 2-3-fold. Protamine caused a 3-4-fold increase in AC-B alpha and rAC-st activities and a marked activation of rAC-B beta (6-fold) and AC-B' (10.5-fold). When histone H1 was used as substrate, all of the heterotrimers were stimulated approximately 4-fold by heparin. The activity of AC-B' and rAC-B beta were increased 2-fold by Mn2+, while a 6-fold stimulation was observed with rAC-st. Chemical cross-linking of AC-B alpha and AC-B beta generated 200-kDa complexes, while AC-st was present as a 150-kDa complex. These results demonstrate that different regulatory proteins affect enzyme activity and the response to agents that modify PP2A activity in vitro. Different PP2A heterotrimers are likely to have distinct functions in vivo, and changes in subunit composition will have an important impact on signal transduction pathways.

p-Chlorophenylalanine damages pancreatic acinar cell of the chicken.

1. Birds were injected intraperitoneally with DL-p-chlorophenylalanine (p-CP), which is an inhibitor of phenylalanine hydroxylase, or saline one day before the experiment. The weight and cell size of pancreas increased by the p-CP treatment. 2. The application of acetylcholine (5.50 x 10(-9) to 5.50 x 10(-5) M) caused a dose dependent increase in amylase release from the superfused segment of pancreas, although the response significantly decreased in the pancreatic segment treated with p-CP, compared with the control.

Medium-chain triacylglycerols enhance release of cholecystokinin in chicks.

Whether medium-chain triacylglycerols (MCT) affect the plasma concentration of cholecystokinin (CCK) and crop-emptying rate in chicks was investigated after 0, 30, 60, 90, 120 or 180 min of diet intubation. Triacylglycerol sources used were corn oil [containing long-chain triacylglycerols (LCT)], glyceryl tricaprate and glyceryl tricaprylate at a level of 200 g/kg diet. Plasma CCK concentration was significantly enhanced in chicks given the two MCT treatments, but not in those given the LCT treatment, after 30 min feeding relative to the initial level. At all time points, chicks fed the diet containing LCT had significantly lower plasma CCK concentrations than those fed MCT, and chicks fed glyceryl tricaprate had higher concentrations than those fed glyceryl tricaprylate. Dietary MCT sources significantly delayed diet passage from the crop compared with dietary LCT. These results indicate that MCT are more potent stimulators of CCK secretion in chicks than LCT.

Influence of dietary protein concentrations or of duodenal amino acid infusion on cholecystokinin release in goats.

1. Whether dietary protein levels or duodenal infusion of amino acids alters the release of cholecystokinin (CCK) in blood plasma of goats was investigated in three experiments. The CCK determination was done by radioimmunoassay with specific CCK-8 antibody. The male adult Shiba goat, a miniature Japanese native goat, was used. 2. In Experiment 1, four goats were offered a diet containing 4.94 g crude protein (CP)/kg BW0.75 for the first 7 days. They were then given a diet containing 5.86 g CP/kg BW0.75 for 7 days and thereafter 6.79 g CP/kg BW0.75 for the following 7 days. On the last day of each experimental period, blood samples were taken from the jugular vein at zero (before feeding), 30, 60, 120, 240 or 360 min after the feeding of the diet. Plasma CCK levels were not affected by dietary protein levels and time after feeding. 3. Influence of phenylalanine or tryptophan (2 mmol/20 ml) infusion into the duodenum was investigated by a 3 x 3 latin square in Experiment 2. Plasma CCK level was determined at 15 min intervals for 1 hr. Both phenylalanine and tryptophan gradually enhanced plasma CCK concentrations with time after infusion. 4. Branched-chain amino acids such as leucine and isoleucine were supplemented intraduodenally in Experiment 3 as in Experiment 2. No significant change in plasma CCK levels was observed.

Influence of dietary sorbose on lipogenesis in gold thioglucose-injected obese mice.

1. The influence of dietary sorbose on food intake and fatty acid synthesis of the liver and epididymal white adipose tissue (EWAT) was investigated in gold thioglucose (GTG)-injected obese mice from 12 to 14 weeks of age. 2. Sorbose was supplemented to a semi-purified diet at a level of 200 g/kg diet at the expense of sucrose. 3. On the last day of the experiment, fatty acids synthesis in the liver and EWAT was measured using an i.p. injection [1-14C]sodium acetate. 4. The decreases in body weight and food intake by dietary sorbose in GTG-injected obese mice were greater than those in control mice. 5. Lipid content and fatty acid synthesis in the liver and EWAT of control mice were not influenced by dietary sorbose. 6. In GTG-injected obese mice, the reduction of food intake by dietary sorbose suppressed fatty acid synthesis and lipid deposition in both liver and EWAT.