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Objective: To evaluate the effect and clinical significance of adrenomedulin (ADM) and urotensin-II (UII) on pulmonary hypertension (PH), by detecting their levels of patients with congenital heart disease and pulmonary hypertension., Methods: 52 patients with congenital heart disease who had left-to-right shunt were selected randomly. 52 patients were divided three groups according to pulmonary artery systolic pressure (PASP): group A was not pulmonary hypertension (PASP < 30 mm Hg, n = 17); group B was mild pulmonary hypertension (PASP30-49 mm Hg, n = 18); group C was moderate and severe pulmonary hypertension (PASP > or = 50 mm Hg, n = 17). The plasma levels of ADM and UII were detected at each period by radioimmunoassay (RIA) method. It was analyzed the changes of their levels within pre-operation, 20 mins and 7 days post-operation and the interrelation between them and PASP., Results: (1) Following the severity degree of pulmonary hypertension, the plasma levels of ADM increase. There is positive correlation between PAP and plasma level of ADM (pre-operation r = 0.8012, P < 0.01; 20 min post-operation r = 0.6325, P < 0.01; 7 d post-operation r = 0.7126, P < 0.01). (2) Following the severity degree of pulmonary hypertension, the plasma levels of UII don't change obviously. There is no correlation between PAP and plasma level of UII (P > 0.05). (3) The plasma levels of ADM: group A (pre-operation: 33 pg/ml +/- 5 pg/ml, 20 mins post-operation: 29 pg/ml +/- 4 pg/ml, 7 d post-operation: 20 pg/ml +/- 3 pg/ml); group B (pre-operation: 44 pg/ml +/- 8 pg/ml, 20 mins post-operation: 40 pg/ml +/- 6 pg/ml, 7 d post-operation: 34 pg/ml +/- 4 pg/ml); group C (pre-operation: 60 pg/ml +/- 10 pg/ml, 20 mins post-operation: 58 pg/ml +/- 8 pg/ml, 7d post-operation: 38 pg/ml +/- 4 pg/ml). Plasma level of ADM of each group after CPB is lower than that of each group before operation. It is statistical difference only 7 days post-operation (group A q = 5.41, P < 0.01; group B q = 4.76, P < 0.01; group C q = 6.32, P < 0.01). (4) The plasma levels of UII: group A (pre-operation: 2.2 pmol/L +/- 0.5 pmol/L, 20 mins post-operation: 2.2 pmol/L +/- 0.44 pmol/L, 7 d post-operation: 2.2 pmol/L +/- 0.6 pmol/L); group B (pre-operation: 2.7 pmol/L +/- 0.6 pmol/L, 20 mins post-operation: 2.6 pmol/L +/- 0.6 pmol/L, 7 d post-operation: 2.6 pmol/L +/- 0.5 pmol/L); group C (pre-operation: 2.9 pmol/L +/- 0.6 pmol/L, 20 mins post-operation: 2.6 pmol/L +/- 0.7 pmol/L, 7 d post-operation: 2.8 pmol/L +/- 0.4 pmol/L). Compared with that of each group before operation, Plasma level of UII of each group after operation is no obvious difference (P > 0.05)., Conclusion: (1) Following the severity degree of pulmonary hypertension, the plasma levels of ADM increase. ADM plays an important role in the formation of pulmonary hypertension and restructure. (2) Following the severity degree of pulmonary hypertension, the plasma levels of UII don't change obviously. There is no correlation between PAP and plasma level of UII, but UII may be play an important role in the formation of pulmonary hypertension and restructure. (3) Measuring the levels of ADM may be a reliable method to follow the change of pulmonary pressure and worsening of pulmonary hypertension.

Background: The influences of genomic background are confirmed in more diseases. Immunologic tolerance after intrauterine infection of hepatitis B virus is considered to occur in T cells. Cytokines work effectively in eliminating virus by immune system after hepatitis B virus infection. To explore the relationship between cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin-4 and interleukin-10), which expressed abnormal quantity in the peripheral blood to intrauterine hepatitis B virus infectious children, gene single nucleotide polymorphism (SNP) and susceptibility to hepatitis B virus intrauterine infection., Methods: This is a cross sectional study of molecular clinical epidemiology. The subjects in this study were selected from outpatients of hepatitis B vaccine follow-up special clinics of our hospital in the period. According to intrant criteria, the high risk children of hepatitis B virus (HBV) intrauterine infection were divided into immune failure group (group I); and immune effective group (group II) and non high risk children belonged to the control group. Four gene SNP sites of TNF-alpha -238, IFN-gamma +874, IL-4 -590 and IL-10 -1082 were determined by real-time quantitative fluorescent polymerase chain reaction (PCR)., Results: The significant differences of TNF-alpha -238 A allele frequency were found between group I and group II (chi(2) = 6.797, P < 0.05) and between group I and the control group (chi(2) = 9.513, P < 0.05). No evident differences of TNF-alpha -238 A were found between group II and control group (chi(2) = 0.047, P > 0.05); the significant differences of IFN-gamma +874 A allele frequency were found between group I and group II (chi(2) = 7.238, P < 0.05), and between group I and the control group (chi(2) = 5.199, P < 0.05). No evident differences were found between group II and the control group (chi(2) = 0.602, P > 0.05); the significant differences of IL-4 -590 C/T allele frequency were not found between group I and group II (chi(2) = 0.632, P > 0.05), also group I and the control group (chi(2) = 0.584, P > 0.05), and the group II and the control group (chi(2) = 0.004, P > 0.05) respectively; The significant differences of IL-10 -1082 G allele frequency were found between group II and group I (chi(2) = 10.359, P < 0.001), and between group II and the controls (chi(2) = 35.418, P < 0.001), but the significant differences were not found between group I and the control group (chi(2) = 1.759, P > 0.05)., Conclusions: This study suggested the possibility that the TNF-alpha -238 A allele and IFN-gamma +874 A allele were associated with HBV intrauterine infection. There was no evident relationship between IL-4 -590 C/T allele SNP and susceptibility to HBV intrauterine infection, but the IL-10 -1082 G allele was associated with preventive efficacy to HBV intrauterine infection.

Two experiments were conducted to investigate the effect of RH (35, 60, and 85%) on thermoregulation of broiler chickens at high (35 degrees C) and mild (30 degrees C) temperatures at the age of 4 wk. The effects of humidity on rectal temperature (RT) and plumage temperature at back (PBAT) and skin temperature at breast (SBRT) were determined at 1, 4, 8, 16, and 24 h after exposure. The RT, PBAT, and SBRT were all significantly increased by high temperature (35 degrees C). Humidity had a significant influence on RT at 35 degrees C but not at 30 degrees C. The peripheral temperatures (PBAT and SBRT) were significantly affected by humidity but responded differently at high (35 degrees C) compared with mild temperature (30 degrees C). In conclusion, high humidity above 60% impaired the heat transmission from body core to the periphery at 35 degrees C but facilitated it at 30 degrees C in 4-wk-old broiler chickens. The effect of humidity on nonevaporative heat loss was depended on air temperature, as nonevaporative heat loss was suppressed by high humidity (>60% RH) at high temperature but enhanced at the mild temperature. The effect of humidity on the relationship between peripheral and core temperature depends on ambient temperature as well as on the age of the broiler chicken. The disturbance of thermal balance could not be determined only by changes in RT or peripheral temperature at a single time point but could be determined by mean body temperature within a certain time frame.

Three trials were conducted to investigate the effect of RH (35, 60, and 85%) on thermoregulation of 1-wk-old broiler chickens at different temperatures (35, 30, and 25 degrees C). The response to humidity in rectal temperature and plumage temperature at the back and breast within 24 h after exposure were recorded at 5 time points (1,4,8,16, and 24 h). Humidity affected the thermoregulation of 1-wk-old broiler chickens by redistributing heat within the body at high, low, and thermoneutral temperatures. The redistribution of heat resulted in decreased rectal temperature and increased peripheral temperature, which were, respectively, beneficial and unfavorable at high and low temperatures. These results suggested that feedback effects of surface temperature on core temperature also exist in poultry, as already observed in mammals, and could be induced not only by changed ambient temperature but also by the changes in humidity at high temperature. The disturbance of thermal equilibrium could not be established solely by changes in RT, but rather core and surface temperatures had to be considered. The daily rhythms in rectal and surface temperatures were affected by humidity.

Objective: To investigate the selectivity and individualization of transpedicular balloon kyphoplasty for aged osteoporotic spinal fracture., Methods: Twenty-two consecutive procedures were performed in 17 aged patients with osteoporotic spinal compression fractures from April 2002 to June 2004. The signal changes in different sequences were confirmed by magnetic resonance imaging before the procedures. This operation involved the percutaneous insertion of two inflatable bone tamps into a fractured vertebral body transpedicularly under fluoroscopic guidance. Every patient was treated individually, according to the results of radiography and CT scan before operation. Preoperative and postoperative complications, visual analogue scale, and radiographic findings such as vertebral height and Cobb angle were recorded and analyzed., Results: All patients tolerated the procedure well with immediate relief of their back pain in 24 hours. There was no leakage of cement into the epidura. The mean loss percent of the anterior and middle vertebral heights were (35.32 +/- 13.15)% and (27.53 +/- 12.61)% before operation, and (14.21 +/- 12.43)% and (16.2 +/- 7.5)% after operation. The height restoration of vertebra was confirmed by X-ray after the procedure (P < 0.01). The mean kyphosis was improved from (25.3 +/- 4.2) degrees to (8.6 +/- 5.1) degrees. No complications occurred. No patient had nerve injury. The patients were allowed to walk next day after the procedure., Conclusion: The selectivity and individualization of transpedicular balloon kyphoplasty for aged osteoporotic spinal fractures has satisfactory short-term clinical efficacy. It is also an effective way to prevent complications.

Objective: To contrast single and double balloon-inflated kyphoplasty for vertebral compression fractures (VCFs) and evaluate its clinical efficacy., Methods: From May 2000 to May 2004, 90 consecutive procedures were performed in 58 patients who suffered from painful vertebral compression fractures, transferring tumour and angioma. Ninety vertebrae were inflated while 62 as A group were double balloon and 28 as B group were single balloon, fracture reduction and bone cement augmentation. Preoperative and postoperative symptom levels, variables, complications were recorded and the vertebral height and Cobb angle were measured and analyzed., Results: All patients' pain was alleviated or disappeared without syndrome, and the vertebral height and Cobb angle of both groups were improved. The average recovery rate was 72.6% (22.9% approximately 100%), Cobb angle from 17.9 degrees (3.1 degrees approximately 31.6 degrees ) were corrected to 9.6 degrees (0.6 degrees approximately 28.2 degrees ), the average angle was 8.7 degrees (0.3 degrees approximately 27.2 degrees ), and the contrast between preoperative and postoperative showed obvious differences (P <0.001). The average recovery rate of A group was 77.6% (55.3% approximately 100%), B group was 64.3% (22.9% approximately 100%). The average postoperative Cobb angle of A group was 9.9 degrees (0.3 degrees approximately 27.2 degrees ), B group was 8.6 degrees (0.6 degrees approximately 19.8 degrees ) (P >0.05)., Conclusions: As a promising minimally invasive surgery, balloon kyphoplasty can provide early relief of pain and improve the function as well as spinal alignment in treatment of painful compression fracture owing to recovering the vertebral height and Cobb angle of the vertebral body. Single balloon-inflated kyphoplasty can improve VCFs as double balloon.

In an attempt to obtain the para-f isomer, rac-(1R,4aR,9aR)-2-methyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2,3-c]pyridin-6-ol, via mesylation of an intermediate 9[small alpha]-hydroxyphenylmorphan, we obtained, instead, a rearranged chloro compound with a 5-membered nitrogen ring, 7-chloro-3a-(2,5-dimethoxyphenyl)-1-methyl-octahydroindole. This indole underwent a second rearrangement to give us the desired para-f isomer. The structures of the intermediate indole and the final product were unequivocally established by X-ray crystallography. A resynthesis of the known rac-(1R,4aR,9aR)-2-methyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2,3-c]pyridin-8-ol, the ortho-f isomer, was achieved using the reaction conditions for the para-f isomer, as well as under Mitsunobu reaction conditions where, unusually, the oxide-bridge ring in the 5-phenylmorphan was closed to obtain the desired product. The synthesis of the para-f isomer adds an additional compound to those oxide-bridged phenylmorphans that were initially visualized and synthesized; the establishment of the structure and configuration of 8 of the theoretically possible 12 racemates has now been achieved. The X-ray crystallographic structure analysis of the para-f isomer provides essential data that will be needed to establish the configuration of a ligand necessary to interact with an opioid receptor.

A synthetic procedure for the preparation of [18F]FPCBT, an imaging agent for the dopamine transporter (DAT), has been developed. The radiosynthesis was carried out in a two step procedure. Even though the yield was low, we were able to prepare 20 to 30 mCi of the product, which was enough for two or three studies. The radiochemical purity was greater than 96%. The in vivo properties of this radiotracer were evaluated using baboon and it showed highest uptake in the striatum. The studies also revealed that the maximum uptake was reached within 7 to 10 minutes post injection. Plasma metabolite analysis indicated that there is only one metabolite and it is less lipophilic than the parent compound. [18F]FPCBT displayed good brain uptake and its high target to non target ratio indicate that it is a potential candidate for DAT imaging.

A novel series of morphinans were synthesized, and their binding affinity at and functional selectivity for micro, delta, and kappa opioid receptors were evaluated. These dimeric ligands can be viewed as dimeric morphinans, which were formed by coupling two identical morphinan pharmacophores (cyclorphan (1) or MCL 101 (2)) with varying connecting spacers. Ligands 6 and 7 with alkyl spacers on the nitrogen position and ligands 8 and 9 in which the two morphinan pharmacophores were coupled by ether moieties at the 3-hydroxyl positions showed significant decrease in affinity at all three opioid receptors. An improvement in the affinity was achieved by introducing an ester moiety as the spacer in the dimeric morphinans. It was observed that the affinity of these ligands was sensitive to the character and length of the spacer. Compound 13 (MCL-139) with a 4-carbon ester spacer, compound 17 (MCL-144) containing a 10-carbon spacer, and compound 19 (MCL-145) with the conformationally constrained fumaryl spacer were the most potent ligands in this series, displaying excellent affinities at micro and kappa receptors (K(i) = 0.09-0.2 nM at micro and K(i) = 0.078-0.049 nM at kappa), which were comparable to the parent compound 2. Ligand 12, a compound containing only one morphinan pharmacophore and a long-chain ester group, had affinity at both micro and kappa receptors almost identical to that of the parent ligand 2. In the [(35)S]GTPgammaS binding assay, ligands 13, 17, and 19 and their parent morphinans 1 and 2 stimulated [(35)S]GTPgammaS binding mediated by the micro and kappa receptors. Compounds 13 and 17 were full kappa agonists and partial micro agonists, while compound 19 was a partial agonist at both micro and kappa receptors. These novel ligands, as well as their interesting pharmacological properties, will serve as the basis for our continuing investigation of the dimeric ligands as potential probes for the pharmacotherapy of cocaine abuse and may also open new avenues for the characterization of opioid receptors.

Aim: To appraise the effect of sea buckthorn (Hippophae rhamnoides) on cirrhotic patients., Methods: Fifty cirrhotic patients of Child-Pugh grade A and B were randomly divided into two groups: Group A as the treated group (n=30), taking orally the sea buckthorn extract, 15 g 3 times a day for 6 months. Group B as the control group (n=18), taking vitamin B complex one tablet, 3 times a day for 6 months. The following tests were performed before and after the treatment in both groups to determine LN, HA, collagens types III and IV, cytokines IL-6 and TNFalpha, liver serum albumin, total bile acid, ALT, AST and prothrombin time., Results: The serum levels of TNFalpha, IL-6, laminin and type IV collagen in group A were significantly higher than those in the control group. After a course of sea buckthorn treatment, the serum levels of LN, HA, collagen types III and IV, total bile acid (TBA) decreased significantly as compared with those before and after treatment in the control group. The sea buckthorn notably shortened the duration for normalization of aminotransferases., Conclusion: Sea buckthorn may be a hopeful drug for prevention and treatment of liver fibrosis.

Purpose: This study evaluated the marginal discrepancies and leakage of all-ceramic crowns cemented with different luting agents after fatigue tests., Materials and Methods: Forty-eight all-ceramic crowns were cemented onto natural molars. Zinc-phosphate cement, compomer cement, and an adhesive composite resin luting system were used in 16 specimens each. Sixteen metal-ceramic crowns were cemented with zinc-phosphate cement as a control. Half of the specimens in each group were fatigued in a chewing simulator for 600,000 loading cycles with 3,500 thermocycles. The others received the 3,500 thermocycles only. An impression-replica technique and SEM were used for evaluation of the marginal discrepancies. Leakage of the specimens was microscopically assessed., Results: There were no significant differences between marginal discrepancies of the groups, with the exception of porcelain shoulder margins in the metal-ceramic group, which had significantly larger discrepancies (P < .01). There was no significant effect of the fatigue tests on marginal discrepancies. However, significant differences of leakage were found between the groups cemented with the different luting agents after both fatigue tests. The adhesive composite resin luting system demonstrated the least leakage. The compomer cement showed an intermediate level of leakage, and the zinc-phosphate cement showed severe leakage that extended through the dentinal tubules to the pulp chambers. No significantly different effect of the two fatigue tests on the leakage was found., Conclusion: The adhesive composite resin luting system showed clinically acceptable marginal discrepancies and an excellent ability to minimize leakage of all-ceramic crowns.

OBJECTIVE: To evaluate the marginal microleakage of IPS-Empress 2 all-ceramic crowns using three different cements. METHODS: Twenty-four IPS-Empress 2 all-ceramic crowns were built and luted onto standardly prepared human upper third molars using three different cements. All samples were thermocycled then submerged in 2% fuchsin for 24 hours. The marginal microleakage at tooth-cement and ceramic crown-cement interfaces was observed using light stereomicroscopy. RESULTS: The Panavia F adhesive luting system demonstrated the least microleakage(0.90+/-0.75, 0.46+/-0.37). Dyract compomer cement showed an intermediate level of microleakage (4.71+/-1.68, 5.28+/-1.48). Harvard zinc phosphate cement was associated with severe microleakage in all specimens( 6.99+/-0.04, 6.99+/-0.04). THe differences in microleakage among the three cements was highly significant(P<0.01). CONCLUSION: Panavia F adhesive luting system was the most effective dental cement to prevent microleakage. It can be used for IPS-Empress 2 posterior all-ceramic crowns.

A series of novel fluoroalkyl-containing tropane derivatives was synthesized, and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) were determined via competitive binding assays. Among these derivatives, the fluoropropyl ester of beta-CIT (19), the fluoroethyl ester of beta-CIT (20), the N-fluoropropyl derivative of beta-CBT (12), and the fluoropropyl ester of beta-CMT (18) displayed higher affinity and greater selectivity for the DAT versus SERT and NET than FP-CIT, which indicates that they are attractive candidates for the development of (18)F-labeled PET imaging agents for the DAT.

A series of new N-substituted derivatives of morphinan was synthesized and their binding affinity for the three opioid receptors (mu, delta, and kappa) was determined. A paradoxical effect of N-propargyl (MCL-117) and N-(3-iodoprop-(2E)-enyl) (MCL-118) substituents on the binding affinities for the mu and kappa opioid receptors was observed. All of these novel derivatives showed a preference for the mu and kappa versus delta binding.

A series of methoxy-containing derivatives of indatraline 13a-f and 17 were synthesized, and their binding affinities for the dopamine, serotonin, and norepinephrine transporter binding sites were determined. Introduction of a methoxy group to indatraline affected its affinity and selectivity greatly. Except for the 4-methoxy derivative 13a,which had the same high affinity at the dopamine transporter binding site as indatraline, the other methoxy-containing analogues (13b-f and 17) exhibited lower affinity than indatraline for the three transporter binding sites. However, some of the analogues were more selective than indatraline, and the 6-methoxy derivative 13c displayed the highest affinity for both the serotonin and norepinephrine transporters. This compound retained reasonable affinity for the dopamine transporter and is a promising template for the development of a long-acting inhibitor of monoamine transporters. Such inhibitors have potential as medications for treatment, as a substitution medication, or for prevention of the abuse of methamphetamine-like stimulants.

2,4-Bis(3'-indolyl)thiazoles, 3,5-bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis(3'-indolyl)pyrazine were synthesized and evaluated for cytotoxic activity against diverse human cancer cell lines by the National Cancer Institute. These compounds demonstrated significant inhibitory effects in the growth of a range of cancer cell lines. 2,4-Bis(3'-indolyl)thiazole displayed selective cytotoxicity against certain leukemia cell lines with GI50 values in the low micromolar range while the substituted derivatives showed a broad spectrum of cytotoxic activity. 3,5-Bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis[3'-(N-methyl-indolyl)]pyrazine possessed strong inhibitory activity against a wide range of human tumor cell lines. The mechanism of action remained unknown. The results suggested that 2,4-bis(3'-indolyl)thiazoles, 3,5-bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis[3'-(N-methyl-indolyl)] pyrazine offer potential as lead compounds for the discovery of anticancer agents.

This study examined mechanisms of beta-adrenergic (AR) desensitization in a myocardial infarction (MI) model of heart failure in the rat. Inotropic responses to isoproterenol (non-selective beta-AR agonist) and RO 363 (selective beta1-AR agonist), in left atria and left papillary muscle, were reduced by up to 65%, while chronotropic responses in right atria were unaffected. beta1- and beta2-AR density did not change after MI, suggesting that changes in beta-AR responsiveness are due to changes occurring downstream of the receptor. Inotropic and chronotropic responses to forskolin were not altered in right and left atria and left papillary muscle after MI, suggesting changes at the level of the G-proteins. Pertussis toxin treatment of animals restored inotropic responses to isoproterenol in left atria and left papillary muscle to levels seen in the sham group, indicating that inactivation of Gi-proteins improves inotropic function in MI rats, and that beta-ARs couple to Gi in cardiac failure. Expression of G-protein receptor kinase 2 (GRK2), beta-arrestin1 and the regulatory subunits of cAMPdPK (RI alpha and RII alpha), showed no change after MI. However the expression of Gi alpha2 was significantly increased in left ventricle (sham 0.888+/-0.140, MI 1. 759+/-0.352 P=0.026), right ventricle (sham 0.031+/-0.004, MI 0. 037+/-0.002 P=0.006) and atria (sham 0.107+/-0.006, MI 0.138+/-0.006 P=0.004), with no changes observed in the expression of Gs alpha. These results suggest that increases in Gi play an important role in the decreased beta-AR responsiveness in the rat model of MI., (Copyright 1999 Academic Press.)

Objectives: To assess the eventual presence, tissue localization, molecular forms, amount and activity of cathepsin G in the annulus fibrosus., Methods: Normal non-autolytic disc tissue was collected from cadavers within six hours after death. Degenerate disc samples were collected from low back pain patients undergoing anterior interbody fusion due to severe, discographically verified and painful disc degeneration, and from the posterior parts of intervertebral discs from 10 patients undergoing microscopic discoidectomy because of intervertebral herniation. Avidin-biotinperxidase complex staining of cathepsin G was quantitated by morphometry. Cellular localization was analyzed using double immunofluorescence staining of cathepsin G and CD68, proline 4-hydroxylase or von Willebrand factor. Neutral salt extracts were analyzed by using synthetic cathepsin G substrate in spectrophotometry, dot-immunoblotting and Western blotting., Results: Histological and morphometric image analysis showed increased cellularity, increased numbers of cathepsin G positive cells and neovascularization in degenerated discs compared to control discs. Neutral salt extract of disc tissue, degenerated or normal, in contrast to control material from synovial capsular tissue, did not contain measurable cathepsin G activity, although immunoreactive enzyme was detected in dot-immunoblotting. Western blotting demonstrated that the discal cathepsin G had an apparent molecular weight of 27 kDa., Conclusion: Due to its properties and localization in normal and pathologically altered tissue, cathepsin G probably plays both a direct and an indirect role in extracellular matrix degradation in the annulus fibrosus. Extracted cationic cathepsin G was immunoreactive, but was functionally inhibited by serpins or, more likely, by polyanionic proteoglycans and saccharins derived from the connective tissue matrix of the annulus fibrosus.

The thiazole analogues of the marine bis(indole)alkaloid nortopsentins, 2,4-bis(3-indolyl)thiazoles, were synthesized using Hantzsch reaction between indole-3-thioamides and 3-(alpha-bromoacetyl)indoles as the key step, and these analogues showed potent cytotoxic activities against a variety of human cancer cell lines in vitro.

This study investigated the effects of myocardial infarction (MI)-induced cardiac failure and treatment with an angiotensin-converting enzyme (ACE) inhibitor perindopril (2 mg/kg/day) on rat beta-adrenoceptor (beta-ar) subtypes in anatomically defined regions of infarcted left ventricular (LV) free wall and noninfarcted tissue from right ventricle (RV) by using autoradiography. After 5 weeks of MI, rats with large MI size (>42%) had developed cardiac failure and beta1-ars were significantly decreased (-59%; p < 0.01) in the border region of the infarcted LV and almost abolished in the infarcted area (-90%; p < 0.005) compared with normal LV from sham-operated controls. The beta-ar changes were not found in the noninfarcted area of the same LV or in RV. MI did not significantly alter the number of beta2-ar subtypes in any region of the ventricles. Perindopril treatment for 4 weeks reduced mean cardiac region weights but did not affect beta-ar density in any cardiac region in either sham-operated or MI rats. These results indicate that cardiac failure due to MI causes significant downregulation of beta1-ars only in border and infarcted regions of rat LV and no change in beta2-ar in any area. It also suggests that the improved response of the infarcted rat heart to isoprenaline stimulation after ACE inhibitors does not result from changes in the numbers of cardiac beta-ars.

The aim of this study was to study innervation and angiogenesis in response to grafts of dermis and cultured keratinocytes using immunohistochemical techniques. In a porcine model, fresh autologous de-epidermalized dermis and cultured autologous keratinocytes were combined using a two-stage technique, to produce keratodermal grafts. Wounds were encased within skin graft chambers that prevented the influence of the surrounding skin. As grafts contracted, a peripheral rim of granulation tissue became exposed, allowing us to compare the wound bed beneath grafts with that beneath the raw granulating surface. Grafts were studied for 6 weeks. Angiogenesis was studied using antisera to von Willebrand factor to detect endothelial cells. Nerve growth was studied using antisera to S-100, a Schwann cell marker, and to four axonal markers: protein gene product 9.5, C-flanking peptide of neuropeptide Y, calcitonin gene-related peptide, and vasoactive intestinal peptide. In kerato-dermal grafts (n = 28), organization of blood vessels and nerve growth occurred only beneath areas with epidermal cover as compared with the surrounding granulation tissue. Initially, the immunoreactivity to von Willebrand factor was high, but in areas with epidermal cover it assumed a more orderly pattern with fewer blood vessels. Innervation was first detected by S-100 immunoreactivity seen at 1 to 2 weeks, closely followed by that to protein gene product 9.5 and much later to calcitonin gene-related peptide. C-flanking peptide of neuropeptide Y and vasoactive intestinal peptide immunoreactivity were detected in the wound depth surrounding large blood vessels at 4 to 6 weeks. In control wounds that had been either grafted with de-epidermalized dermis alone (n = 10) or allowed to granulate (n = 10), persistently there was high immunoreactivity to von Willebrand factor but minimal immunoreactivity to the neural markers. In conclusion, kerato-dermal grafts become innervated, and beneath their surface there is also vascular organization to resemble normal skin. Keratinocytes themselves may influence angiogenesis and innervation, as both processes failed to occur beneath granulating areas.

Objective: To compare the distribution and the amount of transforming growth factor beta (TGF beta) in labial salivary glands (LSG) in patients with Sjögren's syndrome (SS) and healthy controls., Methods: LSG from SS patients (n = 10) and healthy controls (n = 6) were labelled with peroxidase-antiperoxidase staining for TGF beta 2, which was quantitated in image analysis using Video Interactive Display System software., Results: In all LSGs in SS and healthy controls, TGF beta 2 was found in endothelial cells of the capillaries and in the capsular and stromal fibroblasts. In LSGs in SS, TGF beta 2 was also found in some lymphocytes in the inflammatory cell foci and in fibroblasts in fibrotic areas. The TGF beta 2 staining index (microns 2/mm2 tissue) was greater in SS than in control LSGs (3670 (SEM 430) v 2061 (176); p < 0.01), with no difference between the primary and secondary forms of SS (p > 0.05)., Conclusion: The localisation and the level of expression of TGF beta 2 indicate its involvement in local tissue fibrosis, and may reflect attempts at immunosuppression.

The aim of this study was to investigate the pattern of both neovascularization and reinnervation, and the relationship between the two processes, in keratodermal grafts, using confocal laser scanning microscopy, at different time points during the healing process. Keratodermal grafts were prepared in pigs by combining autologous dermis with cultured autologous keratinocytes. Immunohistochemistry was carried out on thick cryostat sections (100-150 microns), using antisera to the endothelial marker von Willebrand factor (vWf) and the pan-neuronal marker protein gene product 9.5 (PGP9.5). The results suggest that the neovascularization and reinnervation in the cultured keratodermal graft is almost complete at 6 weeks. Neovascularization precedes innervation, reaching the surface covered by the keratinocytes at 2 weeks, initially with a linear vascular pattern. From 3 weeks, there is a gradual arborization of the vessels to form a typical vascular plexus. The process of reinnervation is similar in pattern to that of neovascularization, although slower in developing a full network of fibres. In conclusion, the use of confocal microscopy allows the precise definition of complex patterns of neovascularization and nerve growth, which are not fully apparent when using conventional microscopy. Because angiogenesis occurs first, it probably plays a leading role in the survival of keratodermal grafts during wound healing. Indeed, new blood vessels form a pathway for the subsequent innervation process, and quickly reach the epidermal layer which, in turn, may play a key role in the tropism of both blood vessels and nerves.

Endothelin-1 (ET-1) is a vasoconstrictor peptide which is produced by endothelial cells. The subcellular distribution of ET-1 in human skin and the variation of immunostaining for ET-1 by light microscopy in skin biopsies of diabetic patients have been analysed using immunohistochemistry and image analysis quantification. Skin biopsies were collected from 17 patients with type 1 diabetes of different durations and with presence or absence of microangiopathy in the retina; skin biopsies of healthy subjects were utilized as controls. The distribution of ET-1 immunoreactivity (IR) at both light and electron microscopy was compared to that of von Willebrand factor (vWf), a general marker of total cutaneous microvessels. Immunohistochemistry revealed that in controls the distribution of immunostaining was similar for ET-1 and vWf, being localized to microvessels in all areas of the skin. However, at the electron microscopical level ET-1-IR was localized in the endothelial cytoplasm rather than in specific organelles, while vWf immunostaining was associated with Weibel-Palade bodies. ET-1-IR was observed in 4/8 (50 per cent) biopsies from healthy subjects; this increased to 81.8 per cent in biopsies of patients affected by diabetes for less than 10 years and decreased to 16.6 per cent in patients with diabetes for more than 10 years. Quantification of ET-1 staining showed a significant decrease of ET-1-IR in patients affected by diabetes for more than 10 years compared with those affected by diabetes for less than 10 years (P < 0.05). Also, the percentage of biopsies showing positive ET-1 staining was lower in patients with retinopathy than in patients without retinopathy. On the contrary, vWf-IR was observed in all skin specimens and its quantification showed no differences between diabetic patients and controls. These changes are not related to variations in the number of blood vessels, and it is suggested that they reflect a possible functional alteration of the endothelial cells during diabetes.

Recombinant DNA Yeast-Derived Hepatitis B Vaccine (RYHB vaccine) is comparable to and can replace Plasma-Derived Hepatitis B Vaccine (PHB vaccine) for the prevention of mother-infant transmission of hepatitis B virus (HBV), but the duration of immune efficacy of RYHB vaccine is not clear. This study indicates the long-term efficacy for the prevention of mother-infant transmission of HBV. One hundred and six neonates born to HBsAg-carrier mothers with HBeAg positive were randomly divided into two groups, one receiving 20 micrograms per dose of RYHB vaccine and the another receiving 20 micrograms per dose of PHB vaccine on the day of birth, at 1 month and at 6 months (three times). Physical examination and blood tests were performed for all infants at 6, 12, 24, 36, 48 and 60 months of age. The results showed that the protective efficacies at 6, 12, 24, 36, 48 and 60 months were 67%, 75%, 63%, 62%, 57% and 56%, respectively for the RYHB vaccine group and 58%, 76%, 51%, 41%, 24% and 18%, respectively for the PHB vaccine group. The protective efficacy was notably significant in the last two years. The study indicates that the duration of protective efficacy is over 5 years with RYHB vaccine, being longer than that of PHB vaccine. These recipients of RYHB vaccine showed no side effects, and the vaccine is regarded as safe and effective.

Suction blister injury is an experimental model for the investigation of the possible derangement of dermal/epidermal interaction in injury. An extensive fibre network can be stained in skin using antisera to the pan-neuronal marker protein gene product 9.5 (PGP) and the sensory neuropeptide calcitonin gene-related peptide (CGRP), while microvessels are identifiable with antisera to the endothelial marker von Willebrand's factor (vWf) and the peptide endothelin (ET). To investigate the possible involvement of superficial cutaneous innervation and microcirculation during the repair process in injury, human skin biopsies taken at different times after suction blister injury were investigated by quantitative immunohistochemistry. Neural and endothelial changes were seen in both edge and blister areas. PGP- and CGRP-immunoreactive nerves showed an increase in both areas compared with control skin up to 6 h after injury, followed by a decrease which lasted until 72 h. This was followed by a gradual increase of both nerve types starting from the blister edge and lasting up to 8 days after injury when the values were similar to controls. Similarly, in the blister area of the skin, vWF-immunoreactive capillaries showed statistically significant increases between 0 and 6 h after injury, followed by a decrease at 12 and 18 h which was maintained up to 72 h. ET-1 immunoreactivity showed a similar, although more variable, pattern of changes. At the blister edge from 23 h onwards, both vWf and ET-1 immunoreactivities showed a second increase from the edge of the blister spreading towards the centre of the blister ahead of the nerve increase. This lasted up to 8 days, when vWf immunoreactivity showed a statistically significant increase compared with control skin. Generally the numerical increase observed at early time points was accompanied by a strong staining intensity, which reverted to a normal staining intensity at later time points. These results suggest that there is a reactive process for all immunoreactive elements in the early stages of injury, followed by changes indicative of neuronal and endothelial damage (depletion phase) and the subsequent repair process. The repair started with re-epidermalization from the blister edge, followed by revascularization and, lastly, by reinnervation of the tissue. These results indicate a close relationship between epidermis, blood vessels, and nerve fibres during the healing process which follows suction blister injury.

The nervous system may be actively involved in bone repair and in remodelling of callous tissue in bone fractures, as well as in the regulation of nociceptive impulses from the site of the trauma. The aim of this study was to assess the distribution and nature of the periosteal innervation of normal control bone and during bone healing subsequent to fracture of rat tibiae at seven, 14 and 21 days after experimental fracture using immunocytochemistry and image analysis quantification of the neuronal marker protein gene product 9.5 and sensory neuropeptide calcitonin gene-related peptide. At seven days, periosteal protein gene product 9.5- and calcitonin gene-related peptide-immunoreactive fibres showed dense ramifications and terminal sprouting. In addition to periosteum, the nerve fibres were found in the middle of the callus interspersed with inflammatory cells and penetrating into secondary minor fractures. At days 14 and 21 many tortuous nerves were found in the periosteum but not in mid callus. Image analysis quantification revealed a uniform increased proliferation of nerves after seven days. At 21 days, the intercept countings showed in excess of a three-fold increase of calcitonin gene-related peptide-immunoreactive nerve fibres compared with the normal control group (P > or = 0.0001) and were almost as numerous as protein gene product 9.5-immunoreactive fibres (P < 0.005). It is postulated that calcitonin gene-related peptide-containing sensory innervation may have a potential importance in the fracture vascular control, angiogenesis and osteogenesis in addition to a protective role against excessive fracture movement. The results are consistent with the neural involvement in bone growth and remodelling.

Diabetic neuropathy affects both sensory and autonomic peripheral nerve fibres. Vasoactive intestinal polypeptide (VIP) is present in autonomic fibres which modulate sweat secretion, while calcitonin gene-related peptide (CGRP) is localized to cutaneous sensory fibres. In this study, immunohistochemistry and image analysis were used to assess changes of VIP and CGRP, and of the pan-neuronal marker protein gene-product (PGP)-9.5, in skin biopsies of 18 patients affected by type 1 diabetes (age range 18-46 years) and from seven aged-matched controls. Patients were divided into three groups: group 1 (n = 6), with diabetes for 6 months to 3 years; group 2 (n = 5), with the disease for 5-10 years; and group 3 (n = 7), with diabetes for more than 10 years. VIP immunoreactivity (IR) and PGP-9.5-IR were significantly reduced around sweat glands (P < 0.005) in groups 2 and 3. Epidermal CGRP-IR and PGP-9.5-IR were significantly reduced in group 3 (P < 0.05). Twenty-eight per cent (5/18) of all patients showed high VIP-IR around sweat glands (> 95 per cent confidence limits of controls) and all of these patients had diabetes for less than 3 years. Conversely, 55 per cent (10/18) of patients had low VIP-IR (< 5 per cent confidence limit of controls). The latter, compared with the former, showed a significantly longer duration of diabetes (Fisher exact test P = 0.002), presence of clinical autonomic neuropathy (Fisher exact test P = 0.04), and a reduced sural nerve conduction velocity (Fisher exact test P = 0.04). These results suggest that quantitative immunohistochemical analysis of peptide-containing cutaneous nerves allows an objective evaluation of nerve fibre alterations at early stages of diabetes than is currently possible with neurophysiological functional tests.

In order to investigate a possible role of GABA in the regulation of insulin secretion, we have studied the effect of GABA on insulin secretion from the isolated perfused rat pancreas in vitro and on the changes in the cytoplasmic Ca2+ of Beta-cells from the isolated rat islets. When glucose is present, GABA caused a dose dependent inhibition of the first phase of arginine-induced insulin secretion during the range of 10-1000 microM, but GABA did not affect arginine-induced insulin secretion in the absence of glucose. GABA inhibited not only the first phase but also the second phase of glucose-induced insulin secretion. A GABAB-receptor agonist, baclofen, also inhibited both phases of insulin secretion induced by 16.7 mM glucose. Furthermore, GABA inhibited the rise in cytoplasmic Ca2+ of Beta-cells in response to 16.7 mM glucose. These studies indicate that GABA decreases Beta cell secretory activity mainly in response to glucose. These inhibitory effects of GABA on insulin secretion may be mediated through GABAB-receptor and the inhibition of the rise in cytoplasmic Ca2+.

The binding characteristics of [125I]endothelin-1 (ET-1) to cardiac membranes isolated from rat hearts subjected to Ca(2+)-free perfusion or the Ca2+ paradox were examined. The effect of treatment with 2, 3 butanedione monoxime (BDM), which inhibits the tissue damage associated with the calcium paradox, was also investigated. Membranes from rat hearts perfused under control conditions bound [125I]ET-1 to a single population of sites with a Bmax of 107.7 +/- 3.7 fmol/mg protein and an affinity (KD) of 153 +/- 12 pM. Ten minutes of Ca(2+)-free perfusion resulted in a significant (P < 0.01) increase in Bmax to 167.5 +/- 8.3 fmol/mg protein without change in KD. Ca2+ repletion following Ca(2+)-free perfusion tended to increase further the Bmax (180.6 +/- 10.4 fmol/mg protein) without change in KD. Treatment with BDM attenuated but did not prevent the rise in Bmax following Ca(2+)-free perfusion. Following Ca2+ repletion, however, Bmax returned to control levels in the BDM treated group. These changes were not associated with changes in the ability of ET-1 and ET-3 to inhibit [125I]ET-1 binding. The results demonstrate that Ca(2+)-free perfusion is associated with an increase in the binding site density of [125I]ET-1 which is maintained or further increased upon Ca2+ repletion. If, however, the tissue damage associated with the Ca2+ paradox is prevented with BDM, Ca2+ repletion is associated with a reversal of the increase due to Ca(2+)-free perfusion.

Endothelin-1 (ET-1) may be implicated in the pathophysiology of myocardial ischaemia. To determine whether the long-acting calcium antagonist amlodipine attenuates the ischaemia- and reperfusion-induced increase in cardiac ET-1 binding sites, hearts from rats pretreated with amlodipine (0.25 or 0.5 mg/kg) 2 or 5 h before they were killed were made ischaemic for 20 or 40 min, reperfused, and subfractionated. Twenty- and 40-min ischaemia caused a time-dependent increase in ET-1 binding site density (Bmax) identified with [125I]ET-1. Amlodipine pretreatment attenuated this increase in a time- and dose-dependent manner. 0.25 and 0.5 mg/kg amlodipine also suppressed the reperfusion-induced increase in [125I]ET-1 binding site density, even when the 0.5-mg/kg pretreatment series reperfusion was administered after 40-min ischaemia.

Image-analysis was used to measure nerves immunoreactive to the general neuronal marker protein gene product 9.5 (PGP 9.5-IR) and the neuropeptides calcitonin gene-related peptide and vasoactive intestinal polypeptide in standardised leg skin biopsies of three age-matched groups of young subjects: non-diabetic (n = 14), diabetic patients with normal small fibre function ("non-neuropathic", (n = 11) and diabetic patients with abnormal small fibre function ("neuropathic", n = 11). Depletion of nerves and neuropeptides was most marked in the epidermis, where calcitonin gene-related peptide-immunoreactivity was more frequently absent than PGP 9.5-IR in diabetic patients. Epidermal PGP 9.5-IR nerve area and counts were reduced in neuropathic compared with normal subjects (p less than 0.001), as were epidermal calcitonin gene-related peptide nerve counts (p = 0.003). Sweat gland PGP 9.5 and vasoactive intestinal polypeptide, which may be involved in sweat production, showed no diminution in diabetic patients (area: p = 0.160, p = 0.372 by ANOVA). Two diabetic patients showed elevated sweat gland PGP 9.5-IR and three had increased sweat gland vasoactive intestinal polypeptide; this may represent nerve proliferation. In local sweat tests, acetylcholine-stimulated sweat output was associated with increased immunoreactivity, while the sympathetic skin response showed inverse correlations with immunoreactivity. There were no consistent changes with other commonly-used neurophysiological tests. HbA1 correlated negatively with immunohistochemical measurements. Neuropeptide changes were seen in the absence of macro- and microvascular disease, and epidermal nerve depletion occurred in patients with normal thermal thresholds and cardiac autonomic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical expression of the low-affinity nerve growth factor receptor was studied in human fetal and adult tissues using the monoclonal antibody ME20.4. In dorsal root ganglia, a few immunoreactive neurons were first detected in nine-week-old fetuses and many more were found in the following weeks of gestation. However, none was present in adult ganglia. The ME20.4-positive cells were larger than neurons immunostained by substance P, calcitonin gene-related peptide or galanin antibodies. In the spinal cord, fibres immunostained by ME20.4 appeared in a characteristic pattern that differed from the spatial and temporal distributions of synaptophysin- and neurofilament-immunoreactive fibres. Those expressing the low-affinity nerve growth factor receptor were only detected in regions containing collaterals of primary sensory axons: (i) in the dorsal funiculus between seven and 18 weeks of gestation; (ii) in a ventrodorsal bundle reaching the ventral horn from weeks 12-14; (iii) in the medial region of the dorsal horn between weeks 12 and 20; (iv) in the superficial layers and lateral portion of the dorsal horn after the 14th week of gestation and also in adult spinal cord. During the fetal period, ME20.4 immunoreactivity was also found in motoneurons and peripheral nerve fibres in the skin, myotomes and gut. Sheaths of peripheral nerves and the adventitia of blood vessels were stained both in fetal and adult tissues. Thus, the low-affinity nerve growth factor receptor is: (i) strongly expressed in the developing human nervous system; (ii) transiently associated with a subset of large primary sensory neurons and with motoneurons; (iii) transiently and sequentially expressed by various groups of sensory afferents to the spinal cord; (iv) permanently expressed by fibres in the superficial layers of the dorsal horn, Clarke's column, nerve sheaths and the adventitia of blood vessels.

To establish whether the density, affinity, or selectivity of endothelin-1 (ET-1) binding sites in cardiac ventricular membranes varies with age, membranes were harvested from 5- to 7-day-, 20-day-, and 8- to 9-week-old Sprague Dawley rats and labeled with [125I]ET-1. Selectivity was established by using cold ET-1, ET-2, ET-3, big ET-1, and (+)PN200-110 to inhibit specific binding of [125I]ET-1. Over the age span studied, selectivity and affinity of the [125I]ET-1 binding sites was unchanged, but density (Bmax) decreased from 209.7 +/- 18.4 at 5-7 days to 154.0 +/- 8.9 (p less than 0.02) at 20 days, and to 89.7 +/- 5.2 (p less than 0.01) fmol/mg protein at 8-9 weeks. These age-dependent differences in Bmax were not accompanied by a change in membrane yield and occurred at a time when the specific binding of (+)[3H]PN200-110 increased.

Eight compounds have been isolated from the root bark of Aralia elata. Their structures have been identified by means of physico-chemical and spectral analysis. They are (6'-O-palmitoyl)-beta-sitosterol-3-O-beta-D-glucoside (A5), silphiosideA (A9), chikusetusaponin Ib (A11), araloside A (A12), araloside C (A15), acanthoside D (B1). Compound A10 is a new natural product, named as araloside A methyl-ester. 3-O-beta-D-glucopyranosyl (1----3)[beta-D-glucopyranosy (1----4)]-beta-D-glucopyranosyl-oleanolic acid-28-O-beta-D-glucopyranoside (A16) is a new compound, named as araloside G. Compounds A5, A9, A10, A11, A16, and B1 were isolated for the first time from the plant. 13C-NMR chemical shifts of compounds A9 and A15 were assigned for the first time.

The specific binding of [125I]ET-1 to rat cardiac membrane fragments was inhibited by [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P [substance P(D)], a potent bombesin antagonist. This inhibitory effect required high concentrations (greater than 3X10(-6)M) of substance P(D) and was accompanied by a steep increase in non-specific binding, and not a decrease in total binding. Such results indicate that substance P(D) does not competitively inhibit the specific binding of [125I]ET-1 to rat cardiac membrane fragments.

Amlodipine is a 'second generation', long-acting calcium antagonist. To characterise the binding properties of this drug saturation binding studies were undertaken using (-)[3H]amlodipine and rat cardiac membrane fragments. (-)[3H]Amlodipine bound to a single population of high affinity binding sites with a KD of 1.64 +/- 0.17 nM, a Bmax of 0.45 +/- 0.08 pmol/mg protein and a Hill coefficient approaching unity. Binding was slow and required up to 5 hours to reach asymptote during incubation at 25 degrees C. The specific binding was totally inhibited by (-)amlodipine and (-)D600 and partially inhibited by (+)PN200-110, Bay K8644, (+)D600 and d-cis diltiazem. These results indicate that (-)[3H]amlodipine interacts strongly with the phenylalkylamine as well as the dihydropyridine binding sites. It also interacts with the benzothiazepine binding sites. The inhibition of (-)[3H]amlodipine binding by D600 is stereospecific, (-) greater than (+)D600. Bound (-)[3H]amlodipine dissociated slowly from its binding sites, less than 40% dissociation occurring during 5 hours of incubation (k-1 = 1.53 x 10(-3) min-1). These results indicate that the binding profile of amlodipine differs from that of other dihydropyridine-based Ca2+ antagonists. In addition they explain its slow onset of action, and slowed recovery on withdrawal.

Amlodipine is a newly developed long-acting dihydropyridine-based calcium antagonist. To characterize the binding properties of this compound, saturation binding studies were undertaken, using (-)[3H]amlodipine and rat cardiac membrane fragments. (-)[3H]Amlodipine bound to a single population of high-affinity binding sites with a KD of 1.68 +/- 0.12 nM, a Bmax of 0.34 +/- 0.08 pmol/mg protein, and a Hill coefficient approaching unity. Binding required up to 5 h to reach asymptote, and was pH- and temperature-sensitive. The specific binding was totally inhibited by (-) amlodipine and (-) D600 (IC50 values of 9.20 +/- 5.56 and 6.58 +/- 6.57 nM, respectively) and only partially inhibited by (+) PN 200-110, (-) Bay K 8644, (+) D600, and d-cis diltiazem (IC50 values of 60 +/- 10, 160 +/- 20, 250 +/- 40, and 200 +/- 30 nM, respectively). These results indicate that in addition to its ability to bind to the dihydropyridine and benzothiazepine recognition sites in rat cardiac membrane fragments, (-)[3H]amlodipine also binds strongly to the recognition sites for the phenylalkylamine-based calcium antagonists. The results also show that the inhibition of (-)[3H]amlodipine binding by D600 is stereospecific with (-) greater than (+)D600. Dissociation of bound (-)[3H]amlodipine was slowed under acidotic (pH 6.0) and accelerated under alkalotic (pH 10.0) conditions.

Salmonella typhi (S typhi) infected models were established to evaluate the latex agglutination test (LAT) and staphylococcal coagglutination test (SCT) for the detection of S typhi Vi antigen in blood and urine. Antigens in serum or urine were detected within 7 days after infection with positive cultures, and decreased in 2 weeks. LAT and SCT were positive in 7 mice with no bacteria isolation from blood and urine but their spleen aspirates yielded S typhi. Both tests are rapid and sensitive and may be used for the diagnosis of typhoid infection, especially in partially treated patients when their blood cultures are negative.

Amlodipine is a long-acting dihydropyridine calcium antagonist with vascular selectivity. Although structurally related to nifedipine, amlodipine differs in several important respects, including its slow rate of onset and slow recovery. These effects probably reflect the relatively slow rate of association and dissociation of amlodipine with its binding site. The interaction of amlodipine with the calcium antagonist binding site associated with the slow Ca2+ channels differs from that of other dihydropyridines in that it involves the binding domains for the phenylalkylamine- and benzothiazepine-based antagonists, as well as for the dihydropyridines. The prolonged duration of action of amlodipine makes it suitable for use in conditions where calcium channel blockade is required on a 24-h basis. To determine whether amlodipine has a vascular protective effect, amlodipine was given orally to either cholesterol-fed rabbits or stroke-prone hypertensive rats. In the cholesterol-fed rabbits amlodipine (1 or 5 mg/kg/day) produced a significant, dose-dependent reduction in the incidence of atheromatous lesions in the thoracic aorta over an 8-week period. In stroke-prone rats the administration of amlodipine at a dose of 5 mg/kg/day reduced the incidence of mortality over a 30-week treatment period. In spontaneously hypertensive rats amlodipine (5 mg/kg/day) caused a fall in systolic blood pressure, accompanied by a significant (P less than 0.01) reduction in cardiac hypertrophy. When administered intravenously (0.25 mg/kg) 5 h before hearts were excised and made globally ischaemic for short periods (the 'stunned' heart) amlodipine pretreatment improved functional recovery associated with reperfusion.

Although calcium antagonists were originally developed for use in the management of patients with angina pectoris, they are now used in the management of other cardiovascular disorders, including hypertension. More recently, the calcium antagonists have been under investigation for their potential protective role in atherosclerosis. Coupled with these new possibilities for therapeutic use are the development of new, long-acting, tissue-specific calcium antagonists. Amlodipine belongs to this group, and although it is a dihydropyridine-based calcium antagonist, its pharmacologic profile differs from that of other dihydropyridine-based calcium antagonists. Differences include: different pH optimum for receptor binding, different rates of association and dissociation, and differences in allosteric interaction with the diltiazem and verapamil binding sites. Amlodipine, when given orally to rabbits receiving a high-cholesterol diet, reduces atheroma formation. Evidence of its ability to protect the vasculature is provided by its ability to significantly increase (p less than 0.001) survival in stroke-prone hypertensive rats.

Standard binding techniques were used to establish whether either hypoxia, reoxygenation, perfusion under acidotic conditions, or "stunning" of the myocardium resembles ischemia and postischemic reperfusion in increasing cardiac membrane [125I]endothelin-1 (ET-1) binding site density (Bmax). Membranes from aerobically perfused rat hearts bound [125I]ET-1 to a single population of sites, with an affinity (KD) of 0.093 +/- 0.004 nM and a Bmax of 98.8 +/- 5.2 fmol/mg of protein. Bmax was increased (p less than 0.01) after 30 min of global ischemia, and further increased upon reperfusion, without changes in KD or selectivity. Neither three 10 min episodes of ischemia separated by 15 min of perfusion, nor perfusion at pH 6.8 instead of 7.4, nor 60 min of hypoxia altered Bmax, KD, or selectivity. Reoxygenation after 60 min of hypoxia increased Bmax (p less than 0.01) and KD (p less than 0.01) without changing selectivity. These results are interpreted to mean that the ischemia-induced increase in Bmax for [125I]ET-1 cannot be explained simply in terms of the ischemia-induced acidosis, or the accompanying reduction in tissue adenosine triphosphate and creatine phosphate.

125I-labelled endothelin-1 was used to identify specific high affinity endothelin-1 binding sites in left ventricular and brain membranes harvested from female age-matched normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Relative to results obtained for brain membranes from normotensive rats, brain membranes from hypertensive rats exhibited an increase in 125I-endothelin-1 binding site density (P less than 0.02). In left ventricular membranes from the hypertensive rats 125I-endothelin-1 binding site density was reduced (P less than 0.02), relative to the density in ventricular membranes of normotensive rats. These results indicate that hypertension may have an organ specific effect on 125I-endothelin-1 binding site density.

We explored the effect of age and of hypertrophy on Ca2+ antagonist binding site density (Bmax), affinity (Kd), and selectivity in cardiac membranes harvested from the hearts of young adult (9-week-old) and older (25-week-old) Sprague Dawley (SD) rats, Wistar Kyoto rats (WKY), and spontaneously hypertensive rats (SHR). Radioligand binding studies with (+)[3H]PN200-110 failed to show a significant difference between the Bmax obtained for the cardiac membranes of 9-week-old SD, WKY, or SHR. Similarly, at 25 weeks, the Bmax values were the same for each group, but in each group the Bmax tended to increase with age. The Kd and selectivity were unchanged. For (-)[3H]D888 binding, the Kd values changed with age, but there was no hypertension or hypertrophy-linked increase in Bmax. In the SD and SHR series, but not in the WKY, there was a tendency for the Bmax to increase with age. We interpreted these results to mean that age may contribute to the different Kd and Bmax values described for cardiac membranes from 25-week-old WKY and SHR.

When used in high concentrations, the immunosuppressive agent cyclosporine A has toxic side effects, including cardiac fibrosis and calcification. Cyclosporine-treated mice were used to investigate whether a cyclosporine dose regime which produces cardiac fibrosis and calcification alters the density of the specific binding sites for the endothelial-derived vasoconstrictor polypeptide, endothelin-1. After twenty-one days of treatment the endothelin-1 binding site density in cardiac cell membranes was increased, without any change in selectivity. This increase in endothelin-1 binding site density could contribute to the cytotoxic effects of cyclosporine.

The [3H]-prazosin binding activity of membranes isolated from aerobically perfused, ischaemic and reperfused cat and rat hearts was investigated. Alpha 1 adrenoceptors in membranes from aerobically perfused cat hearts had a KD of 0.363 +/- 0.067 nM and a Bmax of 68.8 +/- 7.0 fmol/mg protein; 30 min normothermic global ischaemia caused an increase (P less than 0.01) in density (Bmax, 111.4 +/- 9.3 fmol/mg protein), without any change in affinity (KD 0.430 +/- 0.069 nM). Post-ischaemic reperfusion (15 min) caused the Bmax to return to control values, with no change in KD. Membranes isolated from aerobically perfused rat hearts contained a single population of high affinity alpha 1 adrenoceptor binding sites, with a KD of 0.092 +/- 0.02 nM and a Bmax of 43.02 +/- 2.49 fmol/mg protein. Neither global nor low-flow (0.1 ml/min) ischaemia for either 30 or 60 min, nor post-ischaemic reperfusion, caused any change in either the affinity or density of these binding sites. In both species, and under all the above experimental conditions, the selectivity of the alpha 1 adrenoceptor binding sites was maintained, with phentolamine much greater than rauwolscine in displacing bound [3H]-prazosin. These results show that the ability of ischaemia to increase alpha 1 adrenoceptor density in cardiac membranes is species specific, and that it can occur as a direct (as opposed to a reflex) response to ischaemia.