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Multiplex PCR-direct sequencing method was established to detect 9 different SNPs in exon 6 and exon 7 of ABO genes and could identify at least 28 different ABO genotypes. Population study was carried out in a sample of 80 unrelated Chinese Tibetan minority individual dwelled in Qinghai Province. The method was also applied to forensic cases. A variety of degeneration forensic samples, including blood stain, hair root, swab, bone and mixed stain were successfully identified by this efficient method and in conformance with serological typing. There were no significant deviations from Hardy-Weinberg equilibrium in ABO genotypes of Tibetan population. The heterozygosity, polymorphic information content, discrimination power, paternity of exclusion, and probability of genetic identity were 0.675, 0.672, 0.874, 0.391, and 0.126 respectively. The gene frequency of ABO was O>B>A. The multiplex PCR-directed sequencing method can accurately and reliably detect ABO genotypes in many kinds of samples, and it improves personal identification efficiency. The ABO genotype is high variance in Qinghai Tibetan minority group, and it can be applied in forensic medicine and population genetic study.

Objective: To optimize the formulation of ligustrazine phosphate thermosensitive gel by the central composite design-response surface methodology (RSM plus CCD)., Methods: In the formulation design using RSM plus CCD, independent variables were the amounts of poloxamer 407 and poloxamer 188, and gel temperature was dependent variable. Multilinear and quadratic models were used to estimate the relationship between the dependent and the independent variables, select the optimal formulations and validate., Results: The quantitative relationships between two factors and evaluation index were characterized, quadratic model had better prediction capability than multilinear model., Conclusion: Quadratic model is performed in the optimization of formulation due to the statistical confidence. The optimization of ligustrazine phosphate thermosensitive gel formulation can be achieved by the central composite design and response surface methodology.

Background & Objective: Unexpected splenectomy is sometimes performed simultaneously with radical esophagectomy for esophageal carcinoma because of spleen injury or anatomical abnormity. This study was to investigate the influence of unexpected simultaneous splenectomy on postoperative complications and prognosis of patients undergoing radical esophagectomy for esophageal carcinoma., Methods: Clinical data of 843 esophageal carcinoma patients, underwent esophagectomy (R0 resection) at Cancer Center of Sun Yat-sen University from Aug. 1999 to Jul. 2002, were analyzed. Of these patients, 39 (4.6%) underwent splenectomy. The clinicopathologic parameters and prognosis of the patients in splenectomy group and non-splenectomy group were compared., Results: The amount of intraoperative blood loss was significantly higher in splenectomy group than in non-splenectomy group [(380+/-113) ml vs. (305+/-85) ml, P<0.001]. However, there were no significant differences in clinicopathologic characteristics, intraoperative or postoperative complications between the 2 groups (P>0.05). The occurrence rate of pulmonary complications was higher in splenectomy group than in non-splenectomy group (17.9% vs. 8.5%, P>0.05). The median survival time was shorter in splenectomy group than in non-splenectomy group (18.4 months vs. 21 months, P>0.05)., Conclusion: Unexpected simultaneous splenectomy had no effect on the long-term survival of patients who underwent radical esophagectomy for esophageal carcinoma, but it may result in more intraoperative blood loss and pulmonary complications.

Background & Objective: Mucin-1 (MUC1), a tumor-associated antigen, is an optional molecular target for antitumor immunotherapy protocols. This study was to establish a subcutaneous human esophageal cancer transplantation model with MUC1 high expression in nude mice that closely resembles the biological features of human esophageal cancer, and provide a in vivo model for MUC1-targeting immunotherapy of esophageal cancer., Methods: Human esophageal carcinoma cell line EC-109 with MUC1 high expression was cultured, and subcutaneously transplanted into BALB/c athymic nude mice (4-5 weeks old). The growth status of transplanted tumor was observed. These subcutaneous tumors were examined histologically. The expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. Cell cycle and MUC1 expression of the transplanted tumor cells were analyzed by flow cytometry., Results: Human esophageal carcinoma transplantation models with MUC1 high expression were successfully established in 6 of the 7 nude mice. The histological and biological characteristics of subcutaneous transplanted tumors were similar to those of human esophageal carcinoma. The mean PCNA label index of subcutaneous transplanted tumor cells was (63.5+/-3.6)%. The mean S-phase fraction of cell cycle was (37.6+/-3.7)%. The positive rate of MUC1 in subcutaneous transplanted tumor cells was 97.5%., Conclusion: Human esophageal carcinoma transplantation model with MUC1 high expression in nude mice is similar to human malignant tumors in biological characteristics, and can be used to investigate the biological characteristics of esophageal carcinoma, as well as provides an applicable animal model for research on MUC1-targeting immunotherapy of esophageal cancer.

The aim of this study was to explore the degradation kinetics of water-insoluble lauroyl-indapamide in solutions and predict the stabilities of lauroyl-indapamide encapsulated in liposomes. Buffer-acetone (9:1) was used as the reaction solution and the reaction temperature was maintained at 60 degrees C. The correlation of the apparent degradation constants (k(obs)) of lauroyl-indapamide in liposomes and in buffer-acetone solutions at different pH has been explored. The degradation of lauroyl-indapamide in solutions was found to follow pseudo-first-order kinetics and was significantly dependent on the pH values. Lauroyl-indapamide was the most stable at pH 6.8, increasing or decreasing the pH of the solutions would decrease its stabilities. Buffer concentration had some effects on the stabilities of lauroyl-indapamide. The degradation active energies Ea were 68.19 kJ x mol(-1), 131.75 kJ x mol(-1) and 107.72 kJ x mol(-1) at pH3.6, 6.8 and 12 respectively in acetone-free buffer solutions (0.05M) calculated according to the Arrhenius equation with the extrapolation method. The apparent degradation constants (kobs) of lauroyl-indapamide in liposome and in buffer-acetone (9:1) solutions showed a good correlation at different pH levels, which indicates that the stabilities of the drug that dissolved in acetone-buffer mixture solutions can be used to predict the stabilities of the drug in liposomes as well.

Objective: To observe the effect of Tiam-l gene silencing on the metastasis of human colorectal carcinoma cell line SW480 in nude mice by real-time whole-body fluorescence imaging., Methods: Enhanced green fluorescence protein (EGFP)-labeled human colorectal carcinoma cells, SW480/EGFP(+)/Tiam-1(-) and SW480/EGFP(+), were implanted into nude mice via tail vein injection or orthotopic colonal inoculation, and real-time whole-body fluorescence imaging was performed to compare the difference in tumor progression and metastasis between the two cells., Results: Both SW480/EGFP(+) and SW480/ EGFP(+)/Tiam-1(-) cells stably expressed EGFP, and Tiam1 gene expression was reduced in SW480/EGFP(+)Tiam-1(-) to 30% of the expression level in SW480/EGFP(+) cells. The growth rate of the two cell lines had no significant difference in vitro (P>0.05), but SW480/EGFP(+)/Tiam1(-) cell proliferation and metastasis were depressed obviously in comparison with SW480/EGFP(+) in vivo (P<0.05)., Conclusion: Tiam-1 gene may play an important role in invasion and metastasis of human colorectal cancer.

In order to minimize the residual tert-butyl alcohol (TBA) level in cyclodextrin complex prepared by freeze drying TBA/water cosolvent system, the formulation and lyophilization procedure that may influence the residual TBA was studied. Residual TBA in freeze dried cyclodextrin complex was determined by gas chromatography. The significant formulation and processing factors that influence residual TBA were identified by adjusting the initial TBA concentration in cosolvent, selecting cyclodextrin type (beta-cyclodextrin or hydroxypropyl beta-cyclodextrin), changing sample volume in flasket, altering freezing mode (fast freezing or slow freezing) and modifying the duration of secondary drying. The results show that the amorphous cyclodextrin material (hydroxypropyl beta-cyclodextrin), initial low TBA concentration in cosolvent and fast freezing would lead to high TBA residue in cyclodextrin complex, annealing was effective in reducing the residual TBA. The duration of secondary drying had no distinct effect on residual TBA. It is concluded that in order to reduce residual TBA in cyclodextrin complex prepared by lyophilization monophase solution, the initial TBA concentration in cosolvent should be higher than the crystal formation concentration, the appropriate cyclodextrin type and freeze drying processing should be choosen.

Allele frequencies for 15 short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) were obtained from 7,636 unrelated individuals of Chinese Han population living in Qinghai and Chongqing, China. Totally 206 alleles were observed, with the corresponding allele frequencies ranging from 0.0001-0.4982. Chi-square test showed that all of the STR loci agreed with the Hardy-Weinberg equilibrium. We also compared our data with previously published population data of other ethnics or areas. The results are valuable for human identification and paternity testing in Chinese Han population.

Background & Objective: Dendritic cells (DCs) are antigen-presenting cells, and DC-based fusion vaccine of DCs with tumor cells can induce specific immune response against tumor cells effectively. This study was to investigate the antitumor immunity efficacy of fusion vaccine of DCs with human esophageal carcinoma EC109 cells in vitro., Methods: Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated, and cultured with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-4 (IL-4) to generate DCs. Fusion cells of DCs with EC109 cells were generated by polyethylene glycol (PEG) protocol. The T-cell proliferation response stimulated by DC/EC109 cells was detected by MTT assay. The killing efficacy of cytotoxic T lymphocytes (CTLs), activated by DC/EC109 cells, on EC109 cells was evaluated by LDH assay in vitro, and compared with the killing efficacy on human gastric carcinoma SGC7901 cells and human breast cancer MCF7 cells., Results: The highest fusion efficiency of DCs with EC109 cells was 22.25%. The stimulating efficacy of DC/EC109 cells on the proliferation of T cells was significantly higher than those of DCs and EC109 cells (P<0.05). DC/EC109 cells induced specific CTLs against EC109 cells, and the killing efficacy of the CTLs was significantly higher for EC109 cells than for SGC7901 cells or MCF7 cells (P<0.05)., Conclusion: C/EC109 fusion vaccine can induce specific antitumor response against EC109 cells effectively.

Objective: To observe the therapeutic efficacy of 131I-labeled granulocyte macrophage colony-stimulating factor (GM-SCF) in SCID mouse-acute myeloid leukemia model, and the relationship between dose and effect., Methods: SCID-mouse acute myeloid leukemia model was established by injecting HL-60 cells through tail vein. GM-CSF was labeled with 131I by the chloramines-T method. SCID mice were randomly divided into 6 groups. Groups I, II and III treatment groups were given 9.25 x 10(5), 22.20 x 10(5) and 37.00 x 10(5) Bq of 131I-GM-CSF, respectively. Group IV was given 131I. Group V was given blending of 131I and GM-CSF. Group VI was control. Changes of HL-60 cells in blood and marrow, as well as white blood cells, red blood cells and platelets in blood were detected. Survival time of the SCID mice was calculated., Result: It was observed that WBC, HL-60 cells in blood and marrow were less in treatment groups than that in control groups, especially in groups II, III. After 2 weeks of treatment, BPC of II, III groups increased remarkably (P < 0.01). Survival time of the SCID mice was prolonged in treatment groups (P < 0.01), especially in group III, the longest survival time of 60 days., Conclusion: 131I-GM-CSF could increase leukemic SCID mice survival rate. The therapeutic efficacy of low dose and mediate dose of 131I-GM-CSF is dose-dependent. 131I-GM-CSF is an effective radiation immunity therapy for leukemic mice.

Objective: To investigate the biodistribution of 131I-GM-CSF in SCID mice bearing human AML in vivo., Methods: The xenograft model of human leukemia was established in SCID mice. In the leukemia mice, the biodistribution of 131I-GM-CSF produced by chlo amine-T method was studied., Results: (1)The inoculated HL-60 cells could grow in SCID mice, which developed leukemia after 4 weeks. (2) 131 I-GM-CSF was concentrated in spleen, bone marrow and tumor tissue of the mice. In spleen and bone marrow, 131 I-GM-CSF was uptaken to peak in 30 minutes after injection, the up taking rate was (442. 9+/-86. 4) % ID/g and (4283. 8+/-252. 8)% ID/g, respectively, and maintained on higher level in 24 hours. The injection of 131I resulted in an even distribution in the whole body., Conclusions: 131 I-GM-CSF is able to concentrate electively in spleen, bone marrow and organs infiltrated by leukemia cells. The biodistribution of 131I-GM-CSF in the leukemia mice is tissue specific.

Objective: To explore the effects of di(2-ethylhexyl)phthalate (DEHP) on neonatal mice's testes and Leydig cells in vivo., Methods: Pregnant mice were exposed to DEHP at the dose of 100 mg/kg, 200 mg/kg or 500 mg/kg (body weight) per day by gavage from gestation day 12 (GD 12) through postnatal day 3 (PND 3), respectively. The testis and body weights, testicular histopathology and the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) of the neonatal mice were investigated., Results: The body and testis weights of the male mice's offspring were significantly reduced following DEHP exposure. Leydig cell morphology was affected significantly by DEHP as compared with the controls. Leydig cells obviously increased in the neonatal mice's testes on PND 15 and PND 30 when exposed to DEHP (500 mg/[kg x d]). Activities and positive area of the steroidogenic enzymes 3beta-HSD immunoexpression decreased markedly when exposed to DEHP (100 mg/[kg x d] or 200 mg/[kg x d]). Image analysis showed a decrease in the activities of 3beta-HSD in the animals exposed to DEHP (500 mg/[kg x d]), but an increase in the positive area of 3beta-HSD immunoexpression as compared with the control animals on PND 15 (P < 0.01)., Conclusion: DEHP affects the Leydig cell morphology, the activity of 3beta-HSD, the testis and body weights and the testicular histopathology of neonatal mice, and it may function as an antiandrogenic agent.

The degradation kinetics of fluorouracil-acetic-acid-dextran conjugate (FUAC-dextran) was investigated in various buffer solutions with different pH value and physiological saline solution at 60 degrees C and 37 degrees C, respectively. The hydrolytic reaction displayed pseudo-first-order degradation kinetics. Hydrolytic rate constant obtained was the function of pH value and independent of species of buffering agents. The smallest rate constant was observed at pH round 3.00. The activation energy of the hydrolytic reaction was estimated from Arrhenius equation as 88.73 +/- 6.00 kJ.mol-1. The special base catalytic degradation of the conjugate was observed from acidic to slight alkaline condition and the special base catalytic rate constants were calculated. The conjugate was more stable in physiological saline than that in buffer solution at pH 7.00 or 9.00 at 37 degrees C. The results revealed that the conjugate was stable in acidic condition and will degrade in alkaline condition.

Organophosphate pesticides are used widely all over the world and play an important role in plant pest control. However these pesticides are considered as pollutants and harmful to human health. To search for microorganisms that can degrade organophosphate pesticides with high efficiency, a bacterial strain, coded as JS018, was isolated and screened from the soil in the vicinity of Shanming Pesticides Factory, Shanming, Fujian. Laboratory tests showed that the bacterium could degrade several kinds of organophosphate pesticides, such as Parathion-methyl and phoxin. The strain's degrading rates on phoxin, Parathion-methyl, hostathion and dichlorvos in LB liquid fermentation medium for 36 h were 99%, 96%, 80.4% and 69.0% respectively. The bacterial colonies on LB plate appeared shiny and pale-pink in color. The bacteria were Gram-negative coccoids, 0.5 - 0.7 microm in diameter. They grew well at 30 - 38 degrees C and pH 7.0 - 9.0. The optimal temperature and pH for cell growth was 32 degrees C and pH 7.5 - 8.0, respectively. They did not grow in medium containing 6% or more NaCl. The antibiotic susceptibility tests showed that the strain was resistant to ampicillin, penicillin and lincomycin. It was sensitive to kanamycin, tetracycline and gentamicin. Laboratory tests also showed that the strain could ferment D-glucose, trehalose, melezitose and ethanol. It was negative in the production of indole and hydrogen sulfide. It could not liquefy gelatin, utilize citrate, nor ferment L-arabinose, sucrose, D-mannitol, D-xylose, fructose, D-galactose, maltose or lactose. The catalase, urease and nitrate reduction were positive. Based on its morphological, physiological and biochemical properties as well as the 16S rDNA sequence analysis result, the strain was tentatively identified as Roseomonas sp.

Objective: To investigate the frequencies distribution of human leukocyte antigen (HLA)-B27 subtypes in unrelated healthy Chinese., Method: Polymerase chain reaction sequence-based typing (PCRSBT) was used to determine HLA high-resolution genotypes of 825 unrelated healthy Chinese., Results: A total of 25 HLA-B27-positive individuals and 8 HLA-B27 subtypes were detected. These subtypes and their corresponding frequencies were B * 2704 (30.77%) , B * 2705 (23.08%), B * 2707 (19.23%), B * 2711 (7.69%), B * 2712 (7.69%), B * 2701 (3.85%), B * 2713 (3.85%) and B * 2721 (3.85%)., Conclusion: The data obtained through PCR-SBT method may serve as important reference for the research of relationship between HLA-B27 subtypes and some diseases such as ankylosing spondylitis.

Aim: The liposome/water partition coefficients of salmeterol and budesonide between aqueous phase and liposomes were determined and the factors that influence their partition coefficients were studied, the mechanism of interaction between the two drugs and phospholipid bilayer was elucidated., Methods: The liposome/water partition coefficients of the two drugs were determined by equilibrium dialysis technique. The change of the partition coefficients of the two drugs along with liposome composition and medium was also studied., Results: The partition coefficients of the two drugs decreased with the increase of cholesterol content and saturation of phospholipid used. The liposome/water partition coefficient of salmeterol increased with the increase of liposome surface negative charge, medium pH and ionic strength, while the liposome surface charge, medium pH and ionic strength had no distinct effect on the liposome/water partition coefficient of budesonide., Conclusion: The liposome/water partition coefficient of drug was affected by the type, saturation of phospholipid used in liposome preparation, the cholesterol content and surface charge of liposome, as well as the pH and ionic strength of medium also have effect on the liposome/water partition coefficient of drug. Accordingly, in order to reflect the actual partition of drug in biological membrane, the determination condition including liposome composition and medium should be similar to the biological membrane.

We attempted to characterize the genes expression of CD4+ T lymphocytes for the pathogenesis of systemic lupus erythematosus (SLE). Genomewide gene expression profiles of CD4+ T cells, which were isolated from the disease severe activity (T4-1s) and nonactivity (T4-2s) with an SLE patient by using long serial analysis of gene expression (LongSAGE). We picked out 289 genes matching to Unigene cluster with different expression more than four copies between T4-1s and T4-2s libraries and analyzed their roles from the collectedly published articles of PubMed by genes functional clustering. The genes functions were related to a diverse cellular process including: (1) most of these genes were associated with CD4+ T cells functions, particularly related to cellular developments; (2) Ras pathway genes as RANBP10, GMIP, RASGRP2 and ARL5 might be responsible for the abnormal development of CD4+ T cells of SLE; (3) HIG2, TCF7, KHSRP, WWP1, SMAD3, TLK2, AES, CCNI and PIM2 belong to Wnt/beta-catenin way, they could play roles in modulating proliferation and differentiation of T lymphocytes; (4) uncertain viral infections may initiate autoimmunity because high levels expression genes were detected in T4-1s such as TRIM22, IER2, ABCE1, DUT, G1P2, G1P3, HNRPUL1, EVER2, IFNAR1, TNFSF14, TMP21 and PVRL2; and (5) apoptosis relating genes as EIF3S8, SH3BGRL3, GPX4, TOSO, PFDN5, BIN1, XIAPAF1, TEGT and CUGBP2 may contribute to over uploading of selfantigens in SLE cells. Abnormalities findings of multiple genes expression involving with a variety of CD4+ T cells process might be meaningful to understanding the pathogenesis of SLE, and immature CD4+ T cells may be responsible for SLE.

Objective: To study the pharmacokinetics of PVP costed beta-elemente liposmes in rats., Methods: Gas chromatography was established to determine the concentration of beta-elemene in plasma of rats after administered through i.g., Results: The pharmacokinetics parameters was: T(1/2) = 95.07 +/- 20.46 min, AUC = 348.72 +/- 32.49 microg x min/ml, Cmax = 4.39 +/- 0.33 microg/ml, Tmax = 60 min., Conclusion: The bioavailability of PVP coated beta-elemene liposomes is 140.2 +/- 7.5% compared with conventional liposomes.

Objective: To investigate the gene polymorphism of human leukocyte antigen (HLA)-A, B, DRB1 loci in the population of Beijing region, and research on the application feasibility of polymerase chain reaction sequence-based typing (PCR-SBT) method., Methods: PCR-SBT method was applied to determine HLA- A, B, DRB1 genotypes of 618 unrelated healthy individuals of Beijing region., Results: A total of 84 different alleles and 199 genotypes of HLA-A, 143 alleles and 366 genotypes of HLA-B, 122 alleles and 286 genotypes of HLA-DRB1 were detected., Conclusion: The results showed the characteristics of HLA-A, B, DRB1 distributions, and provided more comprehensive and accurate gene data that may serve as normal reference values for all of Beijing people.

DNA typing for human leukocyte antigen (HLA)-A, -B and -DRB1 was performed using polymerase chain reaction-sequence-based typing method on 618 randomly selected healthy individuals of the Han population in Northern China. Allele frequencies and haplotypes were statistically analyzed. A total of 84 HLA-A alleles, 143 B alleles, and 122 DRB1 alleles were detected, and 853 A-B-DRB1 haplotypes, 473 A-B haplotypes, and 551 B-DRB1 haplotypes were statistically inferred. Statistical analysis of three-locus haplotypes showed that A*0207-B*4601-DRB1*0901 (3.06%) was the most predominant. Gene frequencies and haplotypic associations within HLA-A, -B, and -DRB1 loci were determined at a high-resolution (four digit) allelic level and should provide useful information in anthropology, bone marrow donor registry, legal medicine, and disease association studies.

Objective: To explore the methods of isolation, cultivation, purification, identification of the fetal mice testis Leydig cell and to observe its biological characteristics in vitro., Methods: Leydig cells were isolated by 0.03% collagenase (type I) from fetal mice testis and cultured in DMEM/F12 medium. The identity and purity of Leydig cell were assessed by 3beta-hydroxysteroid dehydrogenase delta4-delta5 isomerase (3beta-HSD). Cell viability was measured by trypan blue. Testosterone level in the medium of cultured Leydig cells was measured in various culture phases and cell density by radioimmunoassay., Results: The purity of Leydig cell was (45.10 +/- 1.66)% before culture, and (81.17 +/- 2. 32)% 72 h after culture. The level of testosterone secreted by Leydig cells could be detected in the medium and its level was associated with the density and time of cultured Leydig cells. The secretion capacity of testosterone by single Leydig cell decreased gradually during the culturing period., Conclusion: The fetal Leydig cells isolated from fetal mice testis have high purity. It can be cultured and kept the secretion ability of testosterone for a few days in vitro. This system can provide a valuable model for further study on the cellular function of the Leydig cells of fetal mice.

Small unilamellar liposomes (SUV) of different phospholipid/polymer composition were labeled with NBD-PC, which served as a bilayersituated fluorescence marker. Neonatal cardiomyocytes were incubated with liposomes and then the cell-associated fluorescence was measured. The factors influencing the liposome uptake by cardiomyocytes such as concentration of lipid, time of incubation, membrane fluidity of liposomes, charge lipid/polymer modification of liposomes and anoxia of cultured cardiomyocytes were investigated. The liposome uptake by cardiomyocytes increased dose-dependently and time-dependently. Liposome uptake was strongly influenced by the electrical charge and modified polymer. After 2 h incubation, the uptake of positively charged liposomes was 1.7-fold higher than that of negatively charged one and both higher than that of the neutral one. The presence of PE-PEG2000 distinctly reduced the liposome uptake and the difference between the uptake of charged and neutral liposome. Anoxia increased the uptake of liposome at the first hour (increased 20%), but after 2 h incubation the liposome uptake by hypoxia cellswas less than that of normoxia cells (decreased 18%). Mechanisms involved are also discussed.

In attempt to increase the accumulation of topotecan in tumours and improve its anti-cancer activity, PEGylated liposome (H-PEG) containing topotecan was prepared. The in-vitro cytotoxicity, in-vivo biodistribution pattern and anti-tumour effect of H-PEG were studied systemically. Compared with free topotecan or conventional liposome (H-Lip), H-PEG improved the cytotoxic effect of topotecan against human ovarian carcinoma A2780 and human colon carcinoma HCT-8 cells. The IC50 value (concentration leading to 50% cell-killing) of H-PEG decreased 5 fold (P<0.01) and 9 fold (P<0.01) against A2780 and HCT-8 cells compared with H-Lip, respectively. The results of biodistribution studies in sarcoma S(180) tumour-bearing mice showed that liposomal encapsulation increased the concentration of total topotecan and the ratio of lactone form in plasma. H-PEG resulted in a 70-fold and 3.7-fold increase in AUC(0-->24 h) compared with free topotecan and H-Lip, respectively. Moreover, H-PEG increased the accumulation of topotecan in tumours and the relative tumour uptake ratio compared with free topotecan was 5.2, and higher than that of H-Lip. The anti-cancer effect studies in murine heptocarcinoma H(22) tumour-bearing mice showed that H-PEG improved the therapeutic efficiency of topotecan and decreased the toxicity of topotecan to a certain extent compared with H-Lip. These results indicated that PEG-modified liposome might be an efficient carrier of topotecan.

The 2004 Southeast Asia Tsunami killed nearly 5,400 people in Southern Thailand, including foreign tourists and local residents. To recover DNA evidence as much as possible from the seriously decomposed bodies, we explored procedures of sample preparation from both bone and tooth samples as well as both mitochondrial and nuclear markers. Despite having failed to recover enough DNA for nuclear marker typing, we succeeded in obtaining fully informative results for mitochondrial markers (HV1 and HV2) from 258 tooth samples with a success rate of 51% (258/507). Using an organic DNA extraction method coupled with an ultrafiltration step, we obtained 16 STR (including 13 CODIS loci, one sex discrimination locus, and two Identifiler loci) profiles for 834 samples with a success rate of 79% (834/1,062). In addition, by comparing the allelic frequencies between the typed samples as a group and other index populations, we conclude that the Thai tsunami victims are a combined group of several populations. Our results provide valuable evidence and protocols for the future forensic practice.

Divalent or trivalent cations such as Ca2+, Ba2+, Mg2+, Zn2+, Fe2+, Al3+ and Fe3+ can cause a significant increase in the entrapment efficiency of lauroyl-indapamide in liposomes, from about 5% to more than 90%, which suggests that the presence of these ions plays an important role in the encapsulation of lauroyl-indapamide.

This report firstly describes the pharmacokinetic study of liposomal breviscapine (LB) after oral administration in rats. The mean Cmax and AUC(0-->t) of LB were 3.3 and 3.1-fold higher than those of breviscapine solution (BS). The oral absorption of breviscapine was significantly increased after encapsulation in the liposomal formulation.

Aim: To prepare the cardiomyocyte-targeting liposomes and investigate their cardiomyocyte targetability in vitro., Methods: Liposomes modified with compound PAC were prepared (PAC-L); The uptake of PAC-L by cardiomyocytes was studied by incubating fluorescence labeled liposomes with cardiomyocytes in vitro and measuring the association of liposomes by a fluorescence spectrophotometer., Results: A high affinity of PAC-L to the cardiomyocytes was observed, the amount of cell uptake of PAC-L by cardiomyocytes was higher than that by nonmyocyte (P < 0.001); The amount of cardiomyocyte uptake of PAC-L on the normoxia condition 2 h was 2.9-fold higher than that of plain-L, and the increase was 5.2-fold when hypoxia occured; The form of liposome uptake changed, the amount of cardiomyocyte uptake of Plain-L by internalization was only 11%, while that of PAC-L was 56%., Conclusion: It is indicated that PAC-L was a potential drug carrier for targeting to ischemic myocardium.

Liposome as a carrier of topotecan (TPT), a promising anticancer drug, has been reported in attempt to improve the stability and antitumor activity of TPT. However, the biodistribution pattern of TPT liposome in vivo and PEG-modified liposome containing TPT have not been studied systemically. In this paper, the in vitro stability and in vivo biodistribution behavior of several liposomes containing TPT with different lipid compositions and PEG-modification were studied. Compared with the 'fluid' liposome (S-Lip) composed of soybean phosphatidylcholine (SPC), the 'solid' liposome (H-Lip) composed of hydrogenated soybean phosphatidylcholine HSPC decreased the leaking efficiency of TPT from liposome and enhanced the stability of liposome in fetal bovine serum (FBS) or human blood plasma (HBP). The results of biodistribution studies in S180 tumor-bearing mice showed that liposomal encapsulation increased the concentrations of total TPT and the ratio of lactone form in plasma. Compared with free TPT, S-Lip and H-Lip resulted in 5- and 19-fold increase in the area under the curve (AUC(0-->infinity)), respectively. PEG-modified H-Lip (H-PEG) showed 3.7-fold increase in AUC(0-->infinity) compared with H-Lip, but there was no significant increase in t(1/2) and AUC(0-->infinity) for PEG-modified S-Lip (S-PEG) compared with S-Lip. Moreover, the liposomal encapsulation changed the biodistribution behavior, and H-Lip and H-PEG dramatically increased the accumulation of TPT in tumor, and the relative tumor uptake ratios were 3.4 and 4.3 compared with free drug, respectively. There was also a marked increase in the distribution of TPT in lung when the drug was encapsulated into H-Lip and H-PEG. Moreover, H-PEG decreased the accumulation of TPT in bone marrow compared with unmodified H-Lip. All these results indicated that the membrane fluidity of liposome has an important effect on in vitro stability and in vivo biodistribution pattern of liposomes containing TPT, and PEG-modified 'solid' liposome may be an efficient carrier of TPT.

The effect of novel 3-{4-[2-hydroxyl-(1-methyl ethylamine) propyl oxygen]phenyl}propionic acid cetylester (PAC) as a surface modification ligand on the delivery of liposomes into cultured cardiomyocytes was investigated. Small unilamellar liposomes with and without PAC (PAC-liposome and Plain-liposome) were labeled with a fluorescence marker. The cultured neonatal cardiomyocytes were incubated with liposomes under normoxia or hypoxia conditions, and then the cell-associated fluorescence was measured. A high affinity of the PAC-liposomes to cardiomyocytes was observed. The amount of cell uptake of PAC-liposomes under normoxia conditions was 4-fold higher than that of plain-liposome, and the increase was 8.5-fold when hypoxia occured. The results suggested that PAC is a potential surface modification ligand for liposome targeting the ischemic myocardium.

In order to explore the transfection and expression of hRI gene on human umbilical blood stem cells, and observe it's effect on the tumor growth. After enriching human umbilical cord blood CD34+ cells with a high-gradient magnetic cell sorting system (MACS), transfected them with supernatant of retrovirus containing human Ribonuclease inhibitor (hRI) cDNA. Hematopoietic progenitor clonogenic assay and PCR were used to evaluate transfection efficiency, and Western-blot and immune fluorescence were used to evaluate the expression quantity of hRI gene after transfection. Observe the effect of RI on the growth of melonoma in B16C57BL mice. The results showed that human umbilical blood CD34+ cells were highly purified by MACS, which made the purity of human umbilical blood CD34+ cells average 96.15%. hRI can be transfected on umbilical blood CD34+ cells, and the transfection efficiency was 35%. The positive expression of hRI gene on transfected CD34+ cells is identified by Western-blot and immune fluorescence assay. Mice injected with transfected CD34+ cells show a significant restraint of the tumor growth, a lower efficiency of tumor formation, a lower weight of the tumor and a longer incubation period of tumor formation with respect to the control groups. The results demonstrated the capacity of RI to inhibited the tumor growth by blocking the vasculature in tumor.

In this paper,the ESTs of pig fat tissue were sequenced and analyzed using the large-scale DNA sequencing method, 7790 high quality ESTs were gained,from which 4354 genes were obtained using cluster analysis with the STACK-PACK software, including 3609 singlet genes and 745 multicopy genes. The 4354 candidate genes were compared with BLASTN to the nr library (e =1e-10), of which 2712 were known genes, containing 1 987 singlet genes and 725 multicopy genes,there are 2 109 unknown genes and new ESTs. Based on the results of BlastN and the index of GenBank Accenssion No., the known gene expression profile of pig fat were constructed. It showed that the genes participating in metabolism held the highest proportion in the 7 sorts, and in some aspects showed the hearty metabolism activity of fat tissue. 257 total reads of the Major Histocompatibility Complex (MHC) genes in the pig fat tissue were found,including 181 singlet genes and 44 multicopy genes. They account for 44.9% of the cell and organism defense, and 5.4% of all known genes,from this we can see that the MHC has a fairly high expression degree. Comparing all ESTs related with MHC (257reads) with known pig BAC we found the partial sequences (approximately 200 bp) of the ESTs distributing to every exon of. We can predict that the ESTs in MHC contain approximately 200 bp highly consistent regions and every exon possesses one of these regions. Although these MHC sequences in different BAC have different protein domains,they are highly consistent and related with immune functions. When the MHC genes transfer to the next generation,they can duplicate repeatly and inherit stably.

Several oil-based solution formulations of insulin were prepared, in which insulin was solubilized in the form of anhydrous reverse micelles. The preparation process involved micellar dissolution of insulin followed by freeze drying, then reconstitution of lyophilized product with an oil phase. These formulations were stable at room temperature for up to 12 months. No significant changes in the appearance were observed and no degradation products of insulin were detected during the course of the stability study. The efficacy of these formulations was evaluated in-vivo using diabetic Wistar rat as an animal model and then a specific formulation was chosen for further study in non-diabetic New Zealand rabbits. It was found that the efficacy of insulin oil solution was dose dependent and insulin oil solution had the same efficacy as insulin emulsion with the same formulation composition. If ethylene-diaminetetraacetic acid (EDTA) was pre-delivered 40 min before the delivery of insulin oil solution, the hypoglycaemic effect of insulin oil solution was greatly enhanced, with an AUC (% glucose reduced) value increase from 28.5 +/- 14.7 to 167.1 +/- 72.3. The improvement of oral absorption induced by predelivery of EDTA might be attributed to enzyme inhibition, reduced gut mobility and the opening of paracellular routes.

Objective: To discuss the reliability of SARS-CoV antibody detection for SARS diagnosis., Methods: Using SARS-CoV ELISA kit to detect relevant antibody in fresh serum of healthy, fever, probable, and suspect cases., Results: The positive rate is 0%, 40%, and 95% respectively in healthy, probable, and suspect cases., Conclusions: It is reliable to detect SARS-CoV antibody in late suspect patients, but there will be high false-positive result in ordinary fever cases.

STR is a universal genetic marker that has changeable polymorphism and stable heredity in human genome. It is a specific DNA segment composed of 2-7 base pairs as its core sequence, and is formed through the repeated connection of the same one. Since it has the characteristics such as numerous allelic genes, highly heterozygosity and easy recognition and short PCR segment, it is employed as an ideal DNA marker in such practical fields as human genetics and forensic medicine. In this study, we investigated the polymorphism of STR of Naci minority with STR genescan marked by fluorescence. Seventy-two alleles of 9 STR in Naci were detected with their frequency 0.0052-0.5208 and 165 genotypes were found out with frequency 0.0104-0.3021. Hi-Square test indicated the distribution of genotypes agreed with Hardy-Weinberg equilibrium (P > 0.05). Statistical analysis showed the followings: the heterozygosity (H) > 0.6 in each locus, the average polymorphism information content (PIC) > 0.7, Mean discrimination power (DP) > 0.8, probability of paternity exclusion (EPP) > 0.5, indicating that the STR markers used in the study were of great value in the researches of minority genetics. This not only founds the base for genetic structures of STR of Chinese but also provides valuable information for anthropology, forensic medicine and ethnology.

Objective: To investigate proliferating cell nuclear antigen labeling indices (PCNA-LI) in craniopharyngiomas in association with tumor recurrence., Methods: Immunohistochemistry was employed to examine the expression of PCNA in 43 craniopharyngioma samples of different pathological types, and the relationship between PCNA-LI and tumor recurrence was evaluated., Results: PCNA-LI was much higher in adamantinous tumors than in squamous papillary tumors that did not give rise to postoperative recurrence. Although PCNA-LI was higher in the recurrent lesions than in the primary tumor in adamantinoma group, the difference was not statistically significant., Conclusions: Craniopharyngioma cells with different pathological features may possess varied proliferating potentials, and active proliferation is the main factor for postoperative recurrence of adamantinoma.

Objective: To investigate the association between porcine gastric epithelial cell proliferation and Helicobacter pylori (Hp) infection., Method: Animal models of gastritis associated with Hp infection were established using "Chinese No.1 pigs". The expressions of proliferating cell nuclear antigen (PCNA) and DNA polyploid content in porcine gastric epithelial cells were quantitatively assayed by means of immunohistochemistry and Feulgen stain respectively., Results: The values of PCNA labeling index (LI) in the porcine gastric epithelial cells of the experimental group were significantly higher than those of the control group (40.95+/-3.60 vs 29.4+/-12.82, P<0.01). The DNA diploid or approximate diploid content in the gastric mucosa of the pigs was significantly lower (70.78% vs 90.65%, P<0.01), but the proliferation polyploid and non-doubleploid of DNA significantly higher in the experimental group than in the control group (P<0.01)., Conclusion: Hp infection may play a role to enhance the proliferation of gastric epithelial cells in pigs.

A comparative study of the third-order nonlinear optical properties, via the newly developed heterodyned optical Kerr effect (OHD-OKE) measurements, of silver phenylacetylide and related compounds is reported. [AgC[triple bond]CC(6)H(5)](n) (1) was found to exhibit efficient third-order nonlinear optical susceptibility chi((3)) of 2.4 x 10(-14) esu, and second hyperpolarizability gamma of 9.07 x 10(-32) esu. These results are compared with those of two related silver phenylacetylide compounds, namely, a double salt, (silver phenylacetylide).(silver tert-butylthiolate) [AgC[triple bond]CC(6)H(5).AgS(t-C(4)H(9))](n) complex (2), and a cluster, triphenylphosphine silver phenylacetylide tetramer, [(C(6)H(5))(3)PAgC[triple bond]CC(6)H(5)](4) (3), as well as that of the related organic polymer polyphenylacetylene (4). These four compounds represent different types of phenylacetylide derivatives: 1 is an organometallic polymer, 2 a polymeric double salt, 3 a discrete metal cluster, and 4 an organic polymer. It was found that the third-order optical nonlinear response was enhanced by the incorporation of silver d electrons into the delocalized conjugated organic pi system, and its magnitude is highly dependent upon the extent of the pi delocalization. Specifically, the relative magnitudes of chi((3)) and gamma follow the order silver phenylacetylide polymer (1) > (silver phenylacetylide).(silver tert-butylthiolate) double salt (2) > polyphenylacetylene polymer (4) > tetrameric (triphenylphosphine silver phenylacetylide)(4) cluster (3). The observed trend may be attributed to the decreasing length of pi conjugation. It is interesting to note that the incorporation of Ag(I) into the polymeric framework of polyphenylacetylene enhances the chi((3)) by 25-fold for the same degree of polymerization (n = 7). The signs of chi((3)) and gamma, which are related to the response mechanisms, were found to be solvent dependent.

Type 1 diabetes mellitus is strongly associated with HLA-DQ8 in humans and I-A(g7) in the NOD mouse. The disease is characterized by loss of tolerance to auto-antigens such as GAD, insulin, and the protein tyrosine phosphatase-like molecule, IA-2. We identified T cell epitopes on the intracytoplasmic region of IA-2 by immunizing DQ8/NOD, DQ8/B10, and NOD mice with overlapping 18 mer peptides in CFA. We identified four peptides presented both by DQ8 and NOD, five DQ8 specific peptides, and six NOD specific peptides. Both mouse lines failed to respond to ten peptides. We demonstrated MHC class II and CD4 restriction of proliferative responses using appropriate blocking antibodies. To understand the role of non-MHC genes in the generation of immune response to the islet auto-antigen, we evaluated cytokine secretion following immunization of DQ8 transgenic mice with strongly immunogenic peptides. The NOD background resulted in increased secretion of cytokines. In conclusion, we have identified IA-2 peptides that induce lymphoproliferative responses in DQ8 transgenic and NOD mice and shown that these peptides stimulate production of Th1 and Th2 cytokines.

Objective: To determine the isotypes and clonality of antibodies to GAD (GADA) and IA-2 (IA-2A) in patients with type 1 and type 2 diabetes., Research Design and Methods: We studied the following consecutive series of patients who attended a diabetes center for antibodies to GADA and IA-2A: 52 newly diagnosed type 1 diabetic patients, 199 type 2 diabetic patients, 200 control patients, and a cohort of 34 nondiabetic identical twins of patients with type 1 diabetes (15 of whom developed diabetes) who were followed prospectively., Results: GADA or IA-2A were detected in 37 (71%) type 1 diabetic patients compared with only 10 (5%) type 2 diabetic patients (P<0.0001). Both GAD and IA-2 antibodies, regardless of the type of diabetes, were usually subclass restricted to IgG1 and were polyclonal. IgM, IgG3, and IgE isotypes were also detected, but all isotypes of GADA and IA-2A were less prevalent than IgG1 (P<0.017 for either antibody). There was no evidence of spreading or switching of isotypes before the onset of type 1 diabetes., Conclusions: These observations suggest that the pathogenesis of antigen-specific antibodies in type 1 and type 2 diabetes is similar and probably involves a chronic nonrandom antigen-driven polyclonal B-cell activation that is consistent with a Th1-type immune response.

Human monoclonal antibody 63 (mAb63) is an IgM/lambda polyreactive antibody that binds to multiple self and non-self antigens. The molecular basis of polyreactivity is still unclear. The present study was initiated to prepare a recombinant Fab of mAb63 and use it to study the determinants involved in polyreactivity. The baculovirus system was employed to express large amounts of mAb63 Fab in Sf9 cells. Our experiments showed that infected Sf9 cells secreted a soluble 50-kD Fab heterodimer that bound to multiple self and non-self antigens. The antigen-binding activity of mAb63 Fab was inhibited by both homologous and heterologous antigens. To study in more detail the molecular determinants involved in polyreactivity, the heavy chain complementarity-determining region 3 (HCDR3), which is known to play a key role in the binding of monoreactive antibodies to antigens, was subjected to site-directed mutagenesis. A single substitution, alanine for arginine, at position 100A resulted in complete loss of antigen-binding activity. The 19 amino acids comprising the HCDR3 of mAb63 were then synthesized and a cyclic peptide prepared. The cyclic peptide showed the same antigen-binding pattern as the parental mAb63 and the recombinant mAb63 Fab. A five amino acid motif (RFLEW), present in the HCDR3 of mAb63, was found by searching the GenBank in three of 50 other human polyreactive antibodies, but in none of nearly 2500 human antibodies thought to be monoreactive. It is concluded that HCDR3 plays a major role in polyreactivity and that in some cases cyclic peptides comprising the HCDR3, by themselves, may be polyreactive.

Triplet surveys were conducted in the city of Xi' an and two villages (one in the vicinity and the other at a distance) in Shaanxi Province in China in October-November (when agricultural activities were low), 1997, to elucidate nutrient intakes with a focus on possible urban-rural differences. Total food duplicate samples were collected from non-smoking and non-habitually drinking adult healthy women (about 50 subjects per site and 149 in total). The nutrient intakes were estimated from the weight of food items in reference to national food composition tables. On average, the women took 1873 kcal energy, 54 g protein and 37 g lipid per day, with a lipid energy ratio of 18.4%. Both excess and insufficient energy intake was observed as a result of food intake analysis and body mass index determination. With regard to minor nutrient intakes, insufficiency was serious in the case of calcium, vitamin A and vitamin B2, but not with iron. Whereas dependency on plant foods for sources of energy and protein was common to the three regions, Xi' an people consumed more animal foods than those in the villages. Intake of fish and shellfish was quite low throughout the three regions. Among the four types of cereals, wheat was consumed most substantially in the three regions and in three meals (except for the village where people essentially did not take lunch in reflection of low agricultural activities), whereas rice was consumed more in Xi' an than in the two villages. Maize consumption was higher in the two villages (especially for breakfast) than in the city. In contrast, foxtail millet (although in small amounts) was taken primarily in Xi' an and only at the time of breakfast.

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of 35S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of approximately 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.

The Arg-Gly-Asp (RGD) sequence within the third complementarity-determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin alphaIIbbeta3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (VH domain + Cgamma1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 kappa chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified alphaIIbbeta3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1. 1 Fab molecules bound per platelet was 17% in the presence of 1 m Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 microM Mn2+. The ratio of Fab molecules bound after versus before activation (mean =/- S.D.; n = 3) was: for AP7.3, 9.8 =/- 0.6; for PAC1.1, 8.8 +/- 0.3; and for AP7, 1.4 =/- 0.2. In addition, AP7 bound to the stably expressed integrin mutant alphaIIbbeta3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin alphaIIbbeta3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of alphaIIbbeta3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.

Characterization of the epitopic structures of neuropeptide Y (NPY) has been studied by alanine-scanning mutagenesis, based on our previous investigation of a panel of six murine anti-NPY IgM monoclonal antibodies. To evaluate the structural requirement for these anti-NPY IgM antibodies, recognition variants of the native sequences of the NPY fragment (19-36) were prepared by single alanine substitutions in residues 22 and 25-36. Their binding to these antibodies was examined by competitive inhibition assays. The results demonstrated that the epitopic structures are largely confined to residues 25-36 of NPY and the C-terminal residues of NPY are essential for these anti-NPY IgM antibodies recognition. It emphasizes the notion that even small regions of a protein consisting of as few as 15 residues (22-36) can exhibit multiple epitopic structures. In several anti-NPY IgM antibodies, pairs of residues on opposite faces of the alpha-helix interact with the antibody site, which indicates that the antibody site consists either of a cavity or a deep groove either of which encompasses the alpha-helical segment sufficiently to allow simultaneous contact with most of the residues of this segment.

To assess the value of intravascular ultrasound in detecting carotid atherosclerosis, we compared the ultrasound images of 48 carotid artery segments from autopsies with their histological findings. The results showed that by intravascular ultrasonography one could distinguish between elastic and muscular tissues of arteries, determine the lesions of fibroelastosis and calcified plaques on arterial wall, and precisely measure the wall thickness, inner and outer diameter, luminal area and cross-sectional area of arteries with a high correlation between the data measured from ultrasonography and histological study (r values were 0.98, 0.97, 0.97, 0.96 and 0.96, respectively). This study suggests that intravascular ultrasound might be effectively used for morphological study and detection of atherosclerotic lesions in vivo.

PAC1 is an IgM kappa murine monoclonal antibody that, like the Arg-Gly-Asp-containing ligand fibrinogen, binds to integrin alpha IIb beta 3 only on activated platelets. The unique binding properties of PAC1 may be determined by its large size, its multivalency, and by variable region sequences, including an Arg-Tyr-Asp at residues 100A-C in H-CDR3. To study the molecular determinants of PAC1 function, baculoviruses containing cloned cDNA for the Fd heavy and kappa light chains of PAC1 were used to co-infect Sf9 insect cells. Infected cells secreted a soluble, monovalent, 50-kDa Fab fragment that bound saturably to agonist-stimulated platelets but not to resting cells. Fab binding was inhibited > 85% by 10 mM EDTA, 1 mM RGDS, 1 mM fibrinogen gamma 397-411, or 12 microM fibrinogen, but not by 1 mM RGES. Compared to PAC1 IgM, a 60-fold higher molar concentration of PAC1 Fab was required for half-maximal binding to platelets or for half-maximal inhibition of fibrinogen binding. PAC1 Fab bound to an activated form of alpha IIb beta 3 expressed in Chinese hamster ovary cells, but not to the resting form of the receptor in these cells or to alpha v beta 3 in human endothelial cells. Conversion of Asp100C to Glu by site-directed mutagenesis rendered the antibody inactive, indicating that the Arg-Tyr-Asp sequence in H-CDR3 is essential for PAC1 recognition of alpha IIb beta 3. Binding of fibrinogen or PAC1 IgM to platelets induced tyrosine phosphorylation of a 140-kDa platelet protein, but binding of PAC1 Fab did not. These studies demonstrate that the specificity of PAC1 for activated alpha IIb beta 3 is determined by an integrin recognition sequence within H-CDR3. However, the strength of this binding interaction and the ability of PAC1 to trigger signaling in platelets also depend on antibody valency.