CAMK4 gene variation is associated with hypertension in a Uygur population.

Considering that calcium/calmodulin-dependent kinase 4 (CAMK4) plays a pivotal role in blood pressure regulation, we investigated the association between a CAMK4 polymorphism (rs10491334) and hypertension in the Han, Kazak, and Uygur ethnic groups. We studied 1224 patients with hypertension and 967 normotensive controls classified into three ethnic groups (Han, Kazak, and Uygur). The rs10491334 polymorphism was genotyped using a TaqMan® 5'-nuclease assay. In the Uygur group, the T-allele frequency in patients with hypertension was twice that of the controls (12.5 vs 6.38%), and T-allele carriers had a significantly increased risk of hypertension compared with non-carriers (odds ratio = 2.200; 95% confidence interval = 1.473-3.285, P < 0.001). However, no significant correlation was found in the Han and Kazak groups. The T-allele of rs10491334 in CAMK4 was associated with hypertension in the Uygur group.

Activity of corilagin on post-parasiticide liver fibrosis in Schistosomiasis animal model.

This study investigates the effects and possible molecular mechanisms of corilagin extraction on prevention of Schistosoma japonicum ova-induced granulomas and liver fibrosis. As a result, under a light microscope, when compared to a model group, the corilagin group showed smaller granulomas, less liver cell denaturation and less inflammatory cell infiltration, and the connective tissues were significantly decreased. By Masson staining, the liver sections from the corilagin group showed less collagen distributed around granulomas, decreased liver fibrosis in the portal tracts and less formed interlobular tissue. The expression of hydroxyproline, IL-13 in liver and GATA3 in spleen in the model group was significantly higher than that in the normal group (P less than 0.05 or 0.01), while the level of hydroxyproline, IL-13 and GATA3 in the corilagin group were significantly lower than that in the model group (P less than 0.05). In conclusion, corilagin extraction can decrease the level of Th2-associated profibrotic cytokine IL-13, and down-regulate the transcription of GATA3 mRNA in spleen cells, which alleviate the hepatic fibrosis caused by egg granuloma in Schistosoma japonicum infection.

Stretched inverse opal colloid crystal substrates-induced orientation of fibroblast.

Recently, there has been increasing interest in studying the interaction between mammalian cells and nanometer-sized structures. However, the effect of nanostructures on cell behavior, such as cell morphology and alignment, is still largely unknown. Inverse opal colloid crystal substrates, which can be stretched to produce nano-scale pore structures of different degrees of orientation, serve as a convenient model system to study the effect of nanotopography on cell morphology and cell alignment. In this work, we fabricated inverse opal colloidal crystal films that were either unstretched or stretched to three, four or six times their original length, producing pore structures of increasing degree of orientation. Human dermal fibroblast-fetal (HDF-f) cells were seeded and cultured on these four types of substrates. The results from fluorescence microscopy and scanning electron microscopy indicated that cells showed the highest degree of alignment when cultured on inverse opal colloid crystal films that were stretched the most (six times original length). The results also demonstrated that the orientation of nanostructures could affect both the morphology and growth direction of fibroblasts. The ability to control the direction of cell growth through the engineering of nanostructures could have important applications in tissue engineering, especially for tissues with anisotropic structures, such as cardiac muscle, blood vessel, tendon and ligament.

Pharmacokinetics and metabolism of 1,5-dicaffeoylquinic acid in rats following a single intravenous administration.

1,5-Dicaffeoylquinic acid (1,5-DCQA) is a potentially important HIV-1 integrase inhibitor widely distributed in many plants. To characterize the pharmacokinetic and metabolic properties of 1,5-DCQA in rats following single intravenous administration (160 mg/kg), the plasma concentrations of 1,5-DCQA were measured by high-performance liquid chromatography (HPLC) and the metabolites formed in urine were identified by liquid chromatography-mass spectrometry (LC-MS) in parallel to diode-array detection (DAD). The results showed that the concentrations of 1,5-DCQA in plasma declined rapidly in a biphasic manner with a mean terminal half-life (t(1/2)) of 1.40 h. The mean clearance (CL) and the apparent volume of distribution (Vd(B)) of 1,5-DCQA were 0.44l/h/kg and 0.89l/kg, respectively. A total of 15 metabolites in rat urine were identified, including four isomeric O-mono-methylated (M1-M4), six isomeric O-di-methylated (M5-M10), one isomeric O-mono-methyl-glucuronidated (M11) and four isomeric O-di-methyl-glucuronidated (M12-M15) metabolites. The O-methylation positions of three important metabolites (M1, M2 and M5) were determined (3''-, 3'-, and 3',3''-) by comparing with synthesized standards. These results suggested that the disappearance of 1,5-DCQA from plasma was rapid, and that its quick urinary excretion and extensive metabolism, including methylation and glucuronidation, were two factors causing its rapid elimination from the circulation.

An effective method for quantitative evaluation of proteins adsorbed on biomaterial surfaces.

An effective method for the quantitative evaluation of proteins adsorbed on biomaterial surfaces has been developed. First, the kinetic behavior of a range of human fibrinogen (Fib) adsorbed onto polystyrene (PS) films was investigated by using a reflectometry interference spectroscopy setup. The specific molecular number of adsorbed proteins, N(p,) was then defined. According to the definition, the numbers of Fib molecules adsorbed on PS films were calculated. An atomic force microscope (AFM) was used to scan the lateral distribution of the Fib molecules adsorbed on the PS films. From the AFM images, the practical specific molecular numbers were obtained by direct counting of the molecules. In order that the adsorbed number of Fib molecules on a unit area of the PS films could be counted easily, the solution concentration of proteins was reduced to 5 ag/mL (10(-18)g/mL). There was good consistency between the numbers calculated with the formula defined by us and the numbers counted from AFM images. Therefore, the results of the present study prove the validity of our definition of the specific molecular number of adsorbed proteins and the effectiveness of the reflectometry interference spectroscopy-based method for quantitative evaluation of adsorptive proteins.

Neoadjuvant chemotherapy in inoperable, locally advanced, and inflammatory breast carcinoma: a pilot study of MTT assay in vitro and outcome analysis of 10 patients.

Patients with inoperable, locally advanced, and inflammatory breast carcinoma (LAIBC), whether with supraclavicular lymph nodes (SLN) or not (stage IIIB and IV), usually carry an overall poor prognosis. The current treatment for these patients is by means of combined modality, including preoperative chemotherapy. This strategy has led to a substantial improvement in clinical response, making some patients operable, and even making breast conservative surgery possible. However, the long-term results still are not promising. The aim of this pilot study was to determine the efficacy of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in vitro in directing chemotherapy (including preoperative adjuvant chemotherapy and postoperative adjuvant chemotherapy) for these patients. Between June 1994 and March 1997, 10 patients with inoperable LAIBC, whether with SLN or not, were enrolled. During the period of the combined therapy modalities, the neoadjuvant chemotherapy was adopted for three cycles according to the results of chemosensitivity in vitro by MTT assay. Then a modified radical or radical mastectomy was performed, which was followed by radiotherapy and further postoperative adjuvant chemotherapy with the same regimen as that of neoadjuvant chemotherapy. All patients had been followed up from the beginning of neoadjuvant chemotherapy to the end of October 1999. Two patients had clinical complete response (CRs), with one having pathologic CR in both breast tumor and axillary lymph node, and the other having pathologic CR in axillary lymph node. The other eight patients had partial response. By the time of analysis, six patients had been dead of relapse or progression. Among the four patients who were still alive, one had local relapse, one had distant metastatic disease, and the other two had no evident disease. By retrieving from MEDLINE before 1999, the authors learned that this is the first pilot study of neoadjuvant chemotherapy for inoperable LAIBC using MTT assay to predict the chemosensitivity in vitro. Compared with conventional chemotherapy, the clinical response and long-term results seem to be more encouraging.

["Target secondary structural motif" in the action of antisense oligodeoxynucleotides].

AIM: To optimize the antisense drug design based on the methods of secondary structure prediction of target mRNA by computer and the quantitative structure-activity relationship analysis. METHODS: The secondary structures of mRNA were predicted by the software RNAstructure, then the antisense phosphorothioate oligodeoxynucleotides (AS-ODN) were designed against the secondary structural elements. The in vitro anti-tumor bioactivity of AS-ODN was evaluated by A549 lung carcinoma cell line. The multiple regression was performed with the computer program SPSS. RESULTS: AS-ODN with high bioactivity concentrated on some local secondary structural motifs that were composed of several secondary structural elements, designated here as "target secondary structural motif" (target motif). The target motifs were relatively stable in the whole mRNA structures, but there were one or more unstable secondary structural elements (free energy > 0) such as internal loops, knots, and hairpins, especially the bulge loops in the motif. Bioactivities of AS-ODN targeting different "target motifs" were statistically different (P < 0.01) while AS-ODN against the same "target motif" were with similar effects. CONCLUSION: The concept of the "target motif" was helpful for optimizing the antisense drug design and might be useful for discovering the local function of mRNA and designing oligonucleotide probes and primers.

Application of secondary structure prediction in antisense drug design targeting protein kinase C-alpha mRNA and QSAR analysis.

AIM: To optimize the design of antisense drug targeting protein kinase C-alpha (PKC-alpha) mRNA and obtain better antisense drugs than ISIS3521 that is undergoing clinical trials. METHODS: RNAstructure (version 3.21, 1999) was utilized to predict the optimal and suboptimal secondary structures of human PKC alpha mRNA (GenBank, X52479), and 29 antisense phosphorothioate oligodeoxynucleotides (S-ODN) targeting the secondary structural elements, 3 partly matched S-ODN and 1 scrambled 3521 were designed. ISIS3521 was set as positive control. Mean (n = 3-5) 50% inhibitory effects on proliferation of A549 cells (IC50) of S-ODN were evaluated. Free energies (delta G degree 37) relating to the target secondary structural elements were calculated according to the nearest neighbor model. The quantitative structure-activity relationship (QSAR) analysis through multiple regression was obtained by SPSS. RESULTS: Three S-ODN; (5'-AGCCCA-GCCGCTTGGCTGGG-3', 5'-AGGAGTGCAGCTGC-GTCAAG-3', 5'-TCAGAGGG-ACTGATGACTTT-3') had lower IC50[(48 +/- 7), (50 +/- 4), (64 +/- 2.7) nmol.L-1, respectively] than that of ISIS3521 [(81 +/- 25) nmol.L-1]. The number of bases comprising the target secondary structural element bulge loop, internal loop, and knot, the free energy of S-ODN (delta G degree 37S), and reaction (delta G degree 37R) were important parameters in QSAR equation. In the multiple regression, R was 0.68, P = 0.0193. Not tally with the equation, two S-ODN (5'-TCAAATGGAGG-CTGCCCGGC-3', 5'-AAAACGTCAGCCATGGTCCC-3') with favorable target structures and delta G degree 37 did not behave good activities. CONCLUSION: Computer aided design was helpful to obtain S-ODN with better in vitro effect than current positive drug. The degree of instability of secondary structural elements and delta G degree 37 were important factors for drug activity. Other important factors needed for further investigation.

Pharmacokinetics and peripheral platelet counts after a single dose of recombinant human thrombopoietin in rhesus monkeys.

AIM: To study the pharmacokinetics and the change of peripheral platelet counts after a single dose of recombinant human thrombopoietin (rhTpo). METHODS: After i.v. or s.c. injections of rhTpo in 12 rhesus monkeys, rhTpo concentration in serum was determined by ELISA. Platelets were counted by automatic microcell counter. RESULTS: The terminal half-lives of rhTpo were 12-18 h. AUC following s.c. were linearly increased with dose, while Cls were 0.061, 0.08, and 0.07 L.kg-1.h-1 in s.c. 0.5, 2, and 8 micrograms.kg-1 groups, respectively. Bioavailability was 0.50 +/- 0.18 after s.c. Single dose of rhTpo was associated with an increase in platelets (55.9%-107.4%, P < 0.05) in a dose-related manner. The peak response and the sustained days of platelet increase were dose-related. The degree of platelet increase (% x time) correlated with the systemic exposure to rhTpo (C x time). CONCLUSION: rhTpo behaved as a linear pharmacokinetics in the monkey within dose range of 0.5-8 micrograms.kg-1.

Tissue distribution of recombinant human tumor necrosis factor alpha derivative in mice.

AIM: To study the tissue distribution and its mechanism of a new recombinant tumor necrosis factor alpha derivative (rhTNF alpha Da) in mice. METHODS: 125I-rhTNF alpha Da was prepared by Iodogen method. Tissue distribution of 125I-rhTNF alpha Da in mice was studied by determining radioactivity of tetrachloroacetic acid (TCA)- precipitable fraction in tissues. The isolated heart-lung perfusion study using 125I-rhTNF alpha Da perfusate was carried out to study the distribution characteristics of 125I-rhTNF alpha Da in lung. RESULTS: Except for thyroid, AUC of the TCA-precipitable 125I-rhTNF alpha Da in tissues was highest in lung, which was 12.2-fold of that in serum, while concentrations in other tissues were all lower than that in serum. Perfusion study in vitro revealed that the concentration of radio-labeled peptide in lung was higher than that in perfusate. On the contrary, level in heart was much lower than that in perfusate. The overall distribution of 125I-rhTNF alpha Da in lungs showed rapidly equilibratory, dose-dependent, saturable, competitive, and highly affinitive, with Kd 47.6 pmol.L-1 and Bmax 348 fmol.g-1 (lung tissue). CONCLUSION: The specific distribution of rhTNF alpha Da in lungs was its distinctive characteristics.

Predictive chemotherapy of advanced breast cancer directed by MTT assay in vitro.

In order to investigate the predictive value of in vitro MTT assay for directing chemotherapy of breast cancer patients, from 1992 to 1995, 156 advanced breast cancer patients who had evaluable lesions were recruited for a prospective study. Of them 83 had MTT assay before chemotherapy; the 73 patients in the MTT sensitive group received chemotherapy according to the result of the MTT assay. The other 10 patients in the MTT resistant group and 73 patients in the control group were given chemotherapy according to clinicians' discretion. The response rate in the MTT sensitive group was 76.7% (56/73). There was statistically significant difference as compared with 0 (0/10) in the MTT resistant group and 43.8% (32/73) in the control group. Between in vitro and in vivo, the overall coincident rate was 79.5% [(56 + 10)/83]. In the MTT sensitive group, the response rate of the subgroups of lesions and the chemotherapy regiments tended to be higher than that in the control group. Patients in the MTT sensitive group had longer response and survival than those in the control group. However, there was no statistical difference in the median response duration and the median survival between the two groups. Further exploration of in vitro chemosensitivity testing by MTT assay for patients with advanced breast cancer is warranted.

Evaluation of in vitro chemosensitivity of antitumor drugs using the MTT assay in fresh human breast cancer.

Practical criteria were developed in this paper for the purpose of evaluating chemosensitivity of fresh human breast cancer by the MTT assay. The survival rates at maximum inhibition (Imax %) and the concentrations of drugs which caused fifty percent reduction in absorbance compared to baseline values (IC50) of 175 samples of 10 anti-tumor drugs were evaluated by logistic analyses of the dose-response curves. Distributions of Imax% appeared as normal curves, while those of the IC50 significantly deviated from normal distribution (p < 0.0001). We assessed the in vitro chemosensitivity by comparing the Imax % of each drug on individual samples with the mean Imax % + SD which was obtained from the Imax% of 175 samples. If the individual Imax % > mean Imax % + SD. we thought the tumor sample was resistant to this drug. If the Imax % < or = mean Imax % + SD, we would compare its IC50 with Q50 which was used as a cutoff point for in vitro chemosensitivity of anti-tumor drugs. The in vitro chemosensitivity could be graded as sensitive (Q1-Q25), intermediate (Q26-Q75), and resistant (Q76-Q100) by means of percentile method. If the individual IC50 > or = Q76, the tumor sample would be defined as resistant. If the individual IC50 < or = Q25, it would be defined as sensitive. In the range of Q26-Q75, we used Q50 as a cutoff point between relative sensitivity and relative resistance. Preliminary results showed that the in vitro chemosensitivity to different anti-tumor drugs determined by these criteria were consistent with the clinical response in 83 advanced breast cancer patients.

[Clinical study on relationship between liver-blood stasis and liver fibrosis].

OBJECTIVE: Exploring the relationship between Liver-Blood Stasis (LBS) and liver fibrosis (LF) to lay a theoretical foundation of rational using traditional Chinese medicines against LF. METHODS: Human procollagen peptide III (hPC III), hyaluronic acid (HA), laminin (LN) in the sera of 35 patients with hepatopathy and Blood Stasis Syndrome (HP-BS) and 35 patients with hepatopathy and Non-Blood Stasis Syndrome (HP-NBS) were measured by radioimmunoassay. Thirty healthy subjects were taken as control. Correlation analysis between the degrees of LBS and those of the serum indexes of LF was made. RESULTS: (1) hPC III, HA, LN in the sera of the patients with HP-BS were markedly higher than those in the sera of the patients with HP-NBS, but the latter were markedly higher than healthy subjects. (2) Degrees of LBS correlated closely with those of LF. (3) (Xuefu Zhuyu Decoction XFZYD) not only might improve the degrees of LBS but also decline the serum LF indexes. Nevertheless, the curative effects in early period of taking the herbs only showed serum HA dropped. CONCLUSION: The nature of LF in traditional Chinese medicine concept is mainly LBS. The degrees of LBS might reflect those of LF to a certain extent. XFZYD showed effective in declining serum LF indexes but it needs at least 2 months.

Pharmacokinetics of recombinant human granulocyte colony-stimulating factor in rabbits and mice.

AIM: To study the pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhGCSF) in rabbits and mice. METHODS: 125I-rhGCSF was prepared by iodogen method and determined by size exclusive HPLC (SEHPLC). RESULTS: Concentration-time curves after i.v. 125I-rhGCSF in rabbits were best fitted with 2-compartment open model. The alpha and terminal elimination T1/2 were 0.25-0.33 and 3.2-4.6 h, respectively. AUC increased with doses, and Cls and K10 were similar. Tpeak was 0.59 +/- 0.25 h after s.c., and elimination T1/2 was similar to that after i.v. The bioavailability after sc was 1.0. In mice the highest level was found in renal system, the next was bile-enteric system. Levels in lymph nodes, bone marrow, and spleen were approximately equal to or slightly lower than that in plasma, while the levels in brain, fat, and muscles were the lowest. About 68%-86% were recovered in urine and feces. CONCLUSION: Pharamcokinetics of 125I-rhGCSF in rabbits and mice provided a useful index for clinical trial.

Doxorubicin cellular pharmacokinetics plays no role in chemosensitizing effect of verapamil on Swiss-3T3 cells.

AIM: To find whether or not the doxorubicin (Dox) cellular pharmacokinetics plays a role in chemosensitizing effect of verapamil (Ver) on drug sensitive cells. METHODS: Cytotoxicity and cellular Dox contents (during accumulation and retention periods) were measured in the absence and presence of verapamil in Swiss-3T3 cells and compared with those in multidrug resistant (MDR) MCF-7Adr cells and drug sensitive MCF-7WT cells. mdr-1 mRNA expression in Swiss-3T3 cells was analyzed. RESULT: Dox cytotoxicity was enhanced 2.0-fold in Swiss-3T3 cells by Ver (3 mumol.L-1) and 3.6-fold in MCF-7Adr cells by Ver (6 mumol.L-1), but not in MCF-7WT cells (Ver 6 mumol.L-1). Cellular accumulation of equi-effective concentrations of Dox increased at 6-h incubation in the presence of Ver in Swiss-3T3 (1.5-fold)i and MCF-7WT cells (2.1-fold) but decreased rapidly in MCF-7Adr cells by 20% to 50% compared to that in the absence of Ver. Cellular retention of Dox decreased after 10-min increase in the presence of Ver in Swiss-3T3 cells compared to that in the absence of Ver, that was similar to that in MCF-7WT cells, while the retention was augmented by Ver in MCF-7Adr cells. Slot blot analysis of RNA revealed no mdr-1 gene expression in Swiss-3T3 cells. CONCLUSION: Changes in cellular accumulation and retention of Dox did not account for the chemosensitizing effect of Ver on Swiss-3T3 cells.

Chemosensitizing effect of verapamil on Swiss-3T3 cells transfected with human MDR1 gene.

To further understand the characteristics of drug resistance reversal of verapamil, the relationship between the level of doxorubicin resistance and the magnitude of modulation by verapamil was examined in multidrug resistant Swiss-3T3 cells transfected with human MDR1 gene. In independently isolated transfectants doxorubicin cytotoxicity decreased markedly compared with parent cells. Potentiation of doxorubicin toxicity by a noncytotoxic concentration of 3 mumol.L-1 of verapamil was much greater in transfectants than in parent cells, while the magnitude of reversal was inversely dependent on the level of resistance. Southern blot hybridization indicated the MDR1 cDNA integration in genomics of each transfectant. Defect in cellular accumulation of doxorubicin was restored by verapamil in transfected cells. The saturation of active drug transport that may involve the magnitude changes of potentiation by verapamil, and the mode of interaction between P-glycoprotein and drugs, were discussed.

[Therapy of cantharides extract for perennial allergic rhinitis and its effect on total IgE in serum].

The therapy of 10% Cantharides extract in treating 50 cases of perennial allergic rhinitis (PAR) was studied. The extract was plastered and blistered on Dazhui, Neiguan point. It was observed by nasal mucosa provocative test, cells in nasal secretion test and serum total IgE test. The results showed that its effective rate was 88%, the allergic nasal mucosa provocative test of treated group alleviated obviously after the treatment (P < 0.01), the number of eosinophil and basophil in nasal secretion decreased (P < 0.01, P < 0.05); and the serum total IgE also reduced significantly (P < 0.01).

Pharmacokinetics and tissue distribution of human recombinant interleukin-2 in mice.

125I-labeled human recombinant interleukin-2 (125I-rIL-2) was prepared by iodogen method with rIL-2 and Na125I. Product was purified by Sephacryl S-200 gel filtration. Eluate fractions were identified by SDS-PAGE and compared with standard rIL-2. Radioactive 95% purified 125I-rIL-2 fractions were selected for pharmacokinetic study, with a specific activity of 56 PBq.mol-1. Concentration-time curves after iv 75, 530, 603, and 6767 ng of 125I-rIL-2/mouse were fitted to a 3-compartment model: with a fast distribution phase T1/2 of 2 min, a slow distribution phase T1/2 of 30-120 min, and a terminal elimination T1/2 of 6-15 h. AUC was linearly related to the dosage (r = 0.9998). Systematic clearances were independent of the dosages. SDS-PAGE of plasma and urine samples showed that radioactivities due to 125I-rIL-2 were 81 +/- 13% (n = 16) and 91 +/- 8% (n = 3, at 4 h), respectively. Levels of 125I-rIL-2 after im were lower than those after i.v., with bioavailability of 0.57. Time to peak concentration was about 1.1 h. The highest levels were seen at 15 min after i.v. in liver, bile and kidneys, the concentration gradients were blood > adrenals > plasma > lungs > thyroid > spleen > jejunum > mesenteric lymph nodes > jejunum contents > ovaries > heart > bladder > thymus > feces in colon > thigh skeletal muscle > testes > brain > fat. Peak concentration time in most tissues were found at 15 min, but at 4 h in the feces.(ABSTRACT TRUNCATED AT 250 WORDS)