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Pluripotent stem cells offer an unparalleled opportunity to investigate cardiac physiology, pharmacology, toxicology and pathophysiology. In this paper we describe the use of both mouse (Nkx2-5(eGFP/w)) and human (NKX2-5(eGFP/w)) pluripotent stem cell reporter lines, differentiated toward cardiac lineage, for live single cell high acquisition rate calcium imaging. We also assess the potential of NKX2-5(eGFP/w) cardiac lineage cells for use toxicological screening as well as establish their sensitivity to a shift between low and high oxygen environments. Differentiated mouse Nkx2-5(eGFP/w) cells demonstrated a wide range of spontaneous oscillation rates that could be reduced by ryanodine (10μM), thapsigargin (1μM) and ZD7288 (10μM). In contrast human NKX2-5(eGFP/w) cell activity was only reduced by thapsigargin (1μM). Human cell survival was sensitive to the addition of trastuzumab and doxorubicin, while the switch from a low to a high oxygen environment affected oscillation frequency. We suggest that the human NKX2-5(eGFP/w) cells are less suitable for studies of compounds affecting cardiac pacemaker activity than mouse Nkx2-5(eGFP/w) cells, but are very suitable for cardiac toxicity studies.

To investigate the optimal anticoagulation methods and monitoring strategy for Chinese patients undergoing heart valve replacement, which is potentially quite different from western populations.In this multicenter prospective cohort study, the anticoagulation and monitoring strategy data was acquired from 25 773 in-hospital patients in 35 medical centers and 20 519 patients in outpatient clinic in 11 medical centers from January 1st, 2011 to December 31th, 2015.As for in-hospital patients, mean age of study population was (48.6±11.2) years old; main etiology of valve pathology was rheumatic (87.5%) origin among study cohort; 94.8% of study population received mechanical valve implantation; international normalized ratio (INR) monitoring (in all the study centers) and low-intensity anticoagulation strategy (31 hospitals chose target INR range of 1.5-2.5, and actual values of INR among 89.2% of 100 069 in-hospital monitoring samples were 1.5-2.5), with mean actual INR values of 1.84±0.53, and warfarin dosage of (2.82±0.93) mg/d were widely adopted among the study centers; strategies of in-hospital warfarin administration were similar in all the study centers; complication rates of low-intensity anticoagulation strategy were low in severe hemorrhage (0.02%), thrombosis (0.05%), and thromboembolism (0.05%) events, without anticoagulation-related death.As for 18 974 outpatient clinic patients, the follow-up rate was 92.47%, with a total of 30 012 patient-years (Pty). Anticoagulation-related morbidity and mortality rates were 0.67% and 0.15% Pty; major hemorrhage morbidity and mortality rates were 0.25% and 0.13% Pty; thromboembolism morbidity and mortality rates were 0.45% and 0.03% Pty.The mean dosage of warfarin daily dosage was (2.85±1.23) mg/d and INR value was 1.82±0.57.No significant regional difference in the intensity of anticoagulation therapy was noted during the study.INR can be used as a normalized indicator for intensity of anticoagulation therapy in China.The optimal anticoagulation intensity with INR range from 1.5 to 2.5 is safe and effective for Chinese patients with heart valve replacement, and there is no significant regional difference in the intensity of anticoagulation therapy.

Objective: The purpose of this study was to evaluate the safety and efficacy of hydroxyurea (HU) in spinal muscular atrophy (SMA) in a randomized, double-blind, placebo-controlled trial. Methods: Twenty-eight patients with type 2 SMA and 29 patients with type 3 SMA were randomly assigned (2:1) to receive HU or matching placebo for 18 months. HU was initiated at 10 mg/kg/day with an 8-week titration to 20 mg/kg/day. Subjects were assessed at baseline (T0) and monthly for the first 2 months (T1–T2) and then every 2 months throughout treatment (T3–T10) and posttreatment periods (T11–T13). The primary outcome measures were the Gross Motor Function Measure (GMFM), Manual Muscle Test (MMT), and serum full-length survivor motor neuron (flSMN) mRNA. The secondary outcome measures were Modified Hammersmith Functional Motor Scale and forced vital capacity (FVC). Results: Fifty-five patients completed this trial, which lasted from March 2007 to June 2009. Except for neutropenia, we found no differences in adverse events between the 2 groups. Compared with the placebo group, the HU group had −1.88 for GMFM (p = 0.11), −0.55 for MMT (p = 0.49), and 2.17 for flSMN mRNA (p = 0.13). Similarly, we found no difference in mean improvement of the secondary endpoints. Both groups had a trend toward a decline in FVC with little change in strength and motor function. Conclusion: Under the current regimen and schedule, HU brought about no improvement in patients with type 2 and 3 SMA, and its main side effect was neutropenia. Classification of evidence: This trial provides Class I evidence that HU 20 mg/kg/day does not effectively treat SMA.

Tenofovir disoproxil fumarate (TDF) has demonstrated long-term efficacy and a high barrier to resistance in multiple chronic hepatitis B (CHB) populations outside of China. This study aimed to evaluate the efficacy and safety of TDF compared with adefovir dipivoxil (ADV) in Chinese patients with CHB during 48 weeks of treatment (ClinicalTrial.gov number, NCT01300234). A Phase 3, multicentred, randomized, double-blind, controlled trial compared the efficacy and safety of TDF with ADV in Chinese patients with CHB. The primary endpoint was the proportion of patients with HBV DNA400 copies/mL in each treatment group at Week 48, using an unpooled Z-test for superiority. Secondary endpoints included viral suppression, serologic response, histological improvement, normalization of alanine aminotransferase (ALT) levels and the emergence of resistance mutations. A total of 509 patients, 202 hepatitis B e antigen (HBeAg)-positive and 307 HBeAg-negative, with HBV DNA ≥10(5) copies/mL received either TDF 300 mg od or ADV 10 mg od. At Week 48, TDF demonstrated superior viral suppression compared with ADV in both HBeAg-positive (76.7% vs 18.2%, P0.0001) and HBeAg-negative (96.8% vs 71.2%, P0.0001) patients. The majority of patients in both treatment arms achieved ALT normalization (85%). No resistance to TDF was observed. The frequency of adverse events was comparable between treatment arms (TDF 3.9% vs ADV 4.8%). In this double-blind, randomized, clinical trial, TDF demonstrated superiority over ADV with respect to viral suppression in Chinese patients with CHB at 48 weeks of treatment and without the development of resistance.

Lamivudine (LAM) and adefovir (ADV) are widely used in most Asian countries, though monotherapy is associated with the occurrence of resistance.To evaluate the efficiency of LAM and ADV combined treatment of chronic hepatitis B patients with compensated cirrhosis.206 eligible Chinese patients were randomly assigned in a 1:1 ratio to receive either LAM or ADV for the first 24 weeks. According to virologic response at 24 weeks, the patients either continued to monotherapy or switched to combined therapy for 48 weeks. After 48 weeks, all patients received LAM and ADV combined therapy for 96 weeks.Serum HBV DNA levels significantly decreased in patients with ADV or LAM monotherapy and continuously reduced after the combined therapy. Serum ALT normalized rate were 88.24% and 81.37% at week 48, and 95.74% and 87.36% at week 96 in ADV and LAM group respectively, comparing to 60.78% and 56.73% in ADV and LAM groups at baseline. The accumulated virological breakthrough rate at week 48 and 96 was significantly higher in LAM group.Both combination strategies were resulted in the long term virological, biochemical improvement in Chinese chronic hepatitis B patients with compensated cirrhosis.

Thyroid hormone, 3, 3', 5-triiodo-L-thyronine (T(3)), mediates cell growth, development and differentiation by binding to its nuclear receptors (TRs). The role of TRs in cancer is still undefined. Notably, hyperthyroxinemia has been reported to influence the rate of colon cancer in an experimental model of carcinogenesis in rats. Previous microarray analysis revealed that cathepsin H (CTSH) is upregulated by T(3) in HepG2-TR cells. We verified that mRNA and protein expression of CTSH are induced by T(3) in HepG2-TR cells and in thyroidectomized rats following administration of T(3). The possible thyroid hormone-responsive elements of the CTSH promoter localized to the nucleotides -2038 to -1966 and -1565 to -1501 regions. An in vitro functional assay showed that CTSH can increase metastasis. J7 cells overexpressing CTSH were inoculated into severe combined immune-deficient mice and these J7-CTSH mice displayed a greater metastatic potential than did J7-control mice. The clinicopathologic significance of CTSH expression in hepatocellular carcinoma (HCC) was also investigated. The CTSH overexpressing in HCC was associated with the presence of microvascular invasion (P=0.037). The microvascular invasion characteristic is closely related to our in vitro characterization of CTSH function. Our results show that T(3)-mediated upregulation of CTSH led to matrix metallopeptidase or extracellular signal-regulated kinase activation and increased cell migration. This study demonstrated that CTSH overexpression in a subset hepatoma may be TR dependent and suggests that this overexpression has an important role in hepatoma progression.

Objective:To investigate the efficacy of intratympanic steroid therapy in adults with sudden sensorineural hearing loss, and to analyse the factors associated with treatment outcome.Design:Retrospective study of patients undergoing intratympanic steroid injection for sudden sensorineural hearing loss between 1 January 2006 and 30 June 2007 at a teaching hospital in Taipei, Taiwan.Results:Patients who received intratympanic steroid therapy within seven days of disease onset achieved a significantly better response rate (76.1 per cent), compared with the delayed treatment group (50 per cent). The total response rate, after four steroid injections, was 68.9 per cent. Patients with low and mid-frequency hearing loss were more responsive to steroid treatment. Vertigo was a negative prognostic factor for recovery. There were no long-term sequelae of intratympanic steroid treatment.Conclusion:Intratympanic steroid injection may be a simple and effective treatment for patients with sudden sensorineural hearing loss.

In an effort to develop an alternative formulation of podophyllotoxin suitable for drug release and delivery, podophyllotoxin-loaded solid lipid nanoparticles (PPT-SLNs) were constructed, characterized and examined for in vitro cytotoxicity and tumor inhibition. The SLNs were prepared by using a solvent emulsification–evaporation method, and their size was around 50 nm. TEM detection showed that the SLNs were homogeneous and spherical in shape, and differential scanning calorimetry (DSC) measurement revealed a new conformation of PPT-SLNs. An in vitro drug release study showed that PPT was released from the SLNs in a slow but time-dependent manner. Furthermore, the treatment of 293T and HeLa cells with PPT-SLNs demonstrated that PPT-SLNs were less toxic to normal cells and more effective in anti-tumor potency compared with unconjugated PPT. A colony forming efficiency assay showed an effective long-term cancer growth suppression of PPT-SLNs; in addition, they can also enhance the apoptotic and cellular uptake processes on tumor cells compared with PPT. These results collectively demonstrated that this SLN formulation has a potential application as an alternative delivery system for anti-tumor drugs.

Nine eumenorrheic women were studied at five separate times each month for a minimum of 2 months. Fasting bloods and 1-day food records were obtained from all women at these times to describe the pattern and magnitude of within-person variation in selected nutrient and lipid indicators attributable to menstrual cyclicity. Serum cholesterol, beta-carotene, 17 beta-estradiol (E2), and dietary selenium intakes exhibited significant periodic regressions against time. For the group as a whole, however, only serum cholesterol and E2 measures exhibited significantly strong consistent periodicities. Future studies with a larger sample of women are warranted to confirm these findings. Unless strict blood sampling protocols are followed, variation attributable to menstrual cyclicity may mitigate the reliability of the serum cholesterol screenings advocated in the past few years.

The objective of the present study was to determine the efficacy of prophylactic administration of gabexate for the prevention of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis, hyperamylasemia and pancreatic pain. Patients scheduled for ERCP were randomized into two groups in a double-blind manner: the patients in the gabexate group were treated with continuous intravenous infusion of 300 mg gabexate dissolved in 500 mL Ringer's solution at 111 mL/h, starting 30 min before the endoscopic maneuvers and continuing up to 4 h after them; placebo group patients were treated only with Ringer's solution also starting 30 min before the endoscopic maneuvers and continuing up to 4 h. Data for 193 patients were analyzed. The incidence of post-ERCP pancreatitis was 3 patients (3.1%) in the gabexate group and 10 (10.5%) in the placebo group (P = 0.040). The incidence of hyperamylasemia was 33 patients (33.7%) in the gabexate group and 42 (43.7%) in the placebo group (P = 0.133). The incidence of pancreatic pain was 15 patients (15.3%) in the gabexate group and 28 (29.5%) in the placebo group (P = 0.018). The results suggest that a 4.5-h infusion of gabexate (for a total of 300 mg) could prevent post-ERCP pancreatitis and pancreatic pain.

Summary Background : Recent studies suggest that the Helicobacter pylori eradication rate in patients with non-ulcer dyspepsia is lower when compared to patients with peptic ulcer diseases. Aim : The aim of this study was to study the efficacy of triple therapy for H. pylori infection in patients with duodenal ulcer vs. patients with non-ulcer dyspepsia. Methods : A total of 582 Chinese patients with proven H. pylori infection were recruited to receive: omeprazole 20 mg, amoxicillin 1000 mg and clarithromycin 500 mg all given twice daily for 7 days (OCA regime). Endoscopy with rapid urease test, histology and culture were performed before treatment. Post-treatment H. pylori status was determined by 13C-urea breath test. Metronidazole, clarithromycin and amoxicillin resistance was defined as minimum inhibitory concentration (MIC) of >8 μg/mL, >1 μg/mL and >1 μg/mL, respectively. Results : A significantly higher (intention-to-treat/per-protocol) eradication rate was found in patients with duodenal ulcer than those with non-ulcer dyspepsia (91/94% vs. 84/88% respectively, P = 0.011 and P = 0.016). Clarithromycin resistance rate was higher in patients with non-ulcer dyspepsia than those with duodenal ulcer (14% vs. 6%, P = 0.015). Clarithromycin resistance (40% vs. 5%, P

In this study, low concentrations of histamine2-receptor (H2-)antagonists were effected across a water plug, with separation taking place in a binary buffer comprising ethylene glycol and NaH2PO4 (pH 5.0), and detection at 214 nm. Liquid-liquid extraction with ethyl acetate- isopropanol is shown to provide extracts that are sufficiently clean. The calibration curves were linear over a concentration range of 0.1-2.00 microg/mL cimetidine, 0.2-5.0 microg/mL ranitidine-HCl, 0.3-5.0 microg/mL nizatidine, and 0.1-3.0 microg/mL famotidine. Mean recoveries were82%, while the intra- and interday relative standard deviations (RSDs) and relative errors (REs) were all13%. The method is sensitive with a detection limit of 3 ng/mL cimetidine, 30 ng/mL ranitidine HCl, 50 ng/mL nizatidine and 10 ng/mL famotidine (S/N = 3, electric-driven injection 90 s). This newly developed capillary electrophoresis (CE) method was applied for the determination of analytes extracted from plasma taken from a volunteer dosing a cimetidine, ranitidine, and nizatidine tablet simultaneously. These three H2-antagonists can be detected in real samples by this method, excluding the low dosing of famotidine tablet.

gamma-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K-dependent posttranslational modification of glutamate to gamma-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

Four polymorphisms were identified in the coding exons of the human luteinizing hormone/chorionic gonadotropin receptor (hLHR) gene. A CTGCAG insertion occurred after nucleotide 54 in 8 of 34 independent chromosomes examined. The heterozygosity frequency was 0.353. This Leu-Gln dipeptide insertion in the first Leucine repeat of the hLHR extracellular domain did not affect the ligand binding affinity of the receptor. Among the 54 chromosomes analyzed, 64.8% was A and 35.2% was G at nucleotide 872 in exon 10. The heterozygosity frequency was 0.115. The A/G substitution led to the replacement of Asn by Ser in the G allele and the abolition of a potential N-glycosylation site. Another polymorphism occurred at nucleotide 935. Fifty nine percent of chromosomes examined were A and 41% were G at this site with the encoded amino acid being Ser in the former and Asn in the latter. The heterozygosity frequency was 0.192. This polymorphism did not have biological consequence. Both of the exon 10 polymorphisms showed ethnic prevalence with the 872 G allele and 935 A allele predominantly in non-Caucasians. The fourth polymorphism was neutral and occurred at nucleotide 1065 in exon 11, with C in 60% and T in 40% of the 50 chromosomes examined. These polymorphisms are useful for tracking the inheritance of specific hLHR allele.

A furazolidone-containing therapeutic regimen for Helicobacter pylori infection has attracted special interest in the face of a rising world-wide metronidazole resistant H. pylori, and the expense of currently used antimicrobial regimens.To evaluate the efficacy of furazolidone-containing regimens in eradicating H. pylori.One-hundred and forty H. pylori positive patients with endoscopically confirmed duodenal ulcer or functional dyspepsia received one of four different regimens to eradicate H. pylori. In the first trial, the patients were randomly assigned to receive a 1-week course of furazolidone 100 mg b.d. and clarithromycin 250 mg b.d., with either tripotassium dicitrato bismuthate (TDB) 240 mg b.d. (FCB group) or lansoprazole 30 mg daily (FCL group). In the second trial, the patients were randomly assigned to receive a 1-week course of clarithromycin 250 mg b.d. and omeprazole 20 mg daily, with either furazolidone 100 mg b.d. (FCO group) or metronidazole 400 mg b.d. (MCO group). Endoscopy was repeated 4 weeks following completion of therapy with re-assessment of H. pylori status on gastric biopsies by histology and culture.Four patients (1 in FCB, 1 in FCO and 2 in MCO groups) dropped out because they refused a follow-up endoscopy. Eradication rates of H. pylori on an intention-to-treat basis in the FCB, FCL, FCO and MCO groups were 91% (32/35, 95% CI: 82-99%), 91% (32/35, CI: 82-99%), 86% (30/35, CI: 74-97%) and 74% (26/35, CI: 60-89%) (all P0.05), respectively. Mild side-effects occurred in 15% of the 140 patients. In MCO group, the eradication rate in the patients infected with metronidazole-sensitive isolates of H. pylori was 86%, but dropped to 67% in those with metronidazole-resistance strains (P = 0.198).One-week regimens containing furazolidone and clarithromycin in combination with TDB or a proton pump inhibitor fulfil the criteria for successful H. pylori therapy.

To identify potential mutations in the gamma-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined in 4 patients from a consanguineous kindred with combined deficiency of all vitamin K-dependent procoagulants and anticoagulants and results were compared with normal genomic sequence. All 4 patients were homozygous for a point mutation in exon 9 that resulted in the conversion of an arginine codon (CTG) to leucine codon (CGG) at residue 394. Screening of this mutation based on introduction of Alu I site in amplified fragment from normal allele but not from the mutated allele showed that 13 asymptomatic members of the kindred were heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation which is the first reported in the gamma-glutamyl carboxylase gene fits perfectly with phenotype of the disorder and confirms the suggested autosomal recessive pattern of inheritance of combined deficiency of all vitamin K-dependent procoagulants and anticoagulants in this kindred. The mutated carboxylase protein expressed in Drosophila cells was stable but demonstrated threefold reduced activity compared with WT carboxylase, confirming that the L394R mutation results in a defective carboxylase.

Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.

The catalytic subunit of human DNA polymerase (pol) delta was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells. The recombinant enzyme was separated from endogenous DNA polymerases by phosphocellulose, Mono Q-Sepharose, and single-stranded DNA-cellulose chromatography. Recombinant DNA pol delta was also purified by immunoaffinity chromatography. The enzymatic properties of the purified catalytic subunit were characterized. The enzyme was active and possessed both DNA polymerase and associated 3' to 5' exonuclease activities. NH2-terminal deletion mutants retained polymerase activity, whereas the core and COOH-terminal deletion mutants were devoid of any measurable activities. Coinfection of Sf9 cells with recombinant baculovirus vectors for pol delta and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with 32Pi showed that the recombinant catalytic subunit of pol delta could be hyperphosphorylated by G1 phase-specific cdk-cyclins. When cdk2 was coexpressed with pol delta in Sf9 cells, pol delta was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol delta showed that the NH2-terminal region was essential for this interaction. Coimmunoprecipitation and Western blot experiments in Molt 4 cells confirmed the interaction in vivo. Preliminary experiments showed that phosphorylation of the catalytic subunit of pol delta by cdk2-cyclins had little or no effect on the specific activity of the enzyme.

In order to establish the rat testis as a model system for studying the human pregnancy-specific beta1-glycoprotein (PSG), expression and cellular distribution of PSG in rat testis were examined. Three partial PSG cDNAs, namely, rnCGM6, rnGCM7, and rnCGM8 were obtained when rat testis cDNA libraries were screened with a human placental PSG cDNA probe. Unlike the human PSGs, the rat PSGs show less nucleotide and amino acid sequence homology among family members. The rat PSGs also have multiple truncated leader sequences followed by immunoglobulin variable-like N domains while human PSGs have a single N domain. Examination of the testis, intestine, kidney, liver, lung, and muscle of male rats by reverse transcription-polymerase chain reaction (RT-PCR) with nested gene-specific primers showed that rnCGM6 was present only in the testis, while rnCGM8 was present in the testis, intestine and lung. On the other hand rnCMG7 was found in all tissues examined. Furthermore, rnCGM7 transcript was present in all somatic cells examined whereas rnCGM6 was predominantly in myoid cells and rnCMG8 in Leydig cells. These results suggest that there is cell-specificity in the expression of PSGs in the rat testis and that the rat testis is a good model for studying the biological activities of the PSGs.

Clotting mechanisms, the coagulation cascade, platelet function, and platelet-leukocyte-endothelial cell interactions are all very similar in humans and pigs. Because of these similarities, the authors concluded that the pig would be an ideal model for the study of thromboembolism resulting from prosthetic heart valves. To date, they have successfully recovered a total of 11 pigs (52.9 +/- 8.1 kg), 3 with bioprosthetic valves and 8 with mechanical valves, all in the mitral position (25 mm od). The normal presence of high numbers of pulmonary endothelial macrophages and other unique aspects of porcine cardiovascular and pulmonary function dictate somewhat different surgical protocols than those normally used for human patients and ruminant species. Some of these special procedures include 1) crystalloid prime without the use of plasma volume expanders, especially those with a starch base; 2) pharmacologic protection against arrhythmias (lidocaine, 4 mg/kg); 3) special attention to adequate hypothermic cardioprotection during the time of cross-clamp; 4) the use of shock doses of corticosteroid (prednisolone sodium succinate, 0.5 mg/kg) before removal of the aortic cross-clamp; and 5) positive inotropic support (dopamine, 0.008 mg/kg) while weaning from cardiopulmonary bypass. Gamma camera images of 111In tagged autologous platelets 24 hours after surgery show most thrombi located on the sewing ring with fewer on the pledgets and anchor sutures. The latter observations were confirmed by quantification of platelet deposition using a gamma counter.

The human gene for gamma-glutamyl carboxylase is 13 kb in length and contains 15 exons. Transcription starts at a cytosine 217 base pair upstream of the first codon. There are two major transcripts in all tissues examined. They are distinguished by the presence of an Alu sequence in the 3' nontranslated end of the longer species. Relative mRNA levels for 12 bovine tissues are presented.

This study was designed to determine the cord blood lead (BPb) levels of babies born in one urban area of Shanghai, and to preliminarily identify the demographic, social environment and prenatal factors which have an effect on the cord BPb concentrations. From August to November 1993, umbilical cord blood samples were obtained from 605 live newborns in the Yangpu Maternal and Child Hospital. 257 samples were excluded from measurement because of clotting. In 348 cord samples, the geometric mean of cord BPb levels was 9.2 micrograms/dl, with a 95% confidence interval of the mean 8.86-9.54 (micrograms/dl). 142 babies (40.8%) had cord BPb levels of 10 micrograms/dl or greater. As a result of this high percentage of newborns with BPb levels equal to or greater than 10 micrograms/dl, we estimate that each year in the Shanghai City about 60,000 newborns are at risk for developing neuropsychological deficiencies caused by maternal lead exposure during pregnancy. To investigate the factors affecting cord blood levels, the subjects with levels greater than the 70th percentile (10.7 micrograms/dl) (n = 104) and less than the 30th percentile (7.4 micrograms/dl) (n = 104) were selected to compare the demographic, environment and prenatal medical history. Increased BPb levels at birth were associated with maternal passive smoking, a family member being occupationally exposed to lead, proximity to major traffic way, household coal combustion, neighborhood coal combustion, low level of maternal occupations, and the increasing occurrence of having the high lead foodstuff pidan (preserved duck egg) during pregnancy. We conclude that prenatal lead exposure has become an important health issue for young children in Shanghai.

The effect of positioning on pulmonary function has been previously evaluated, and the prone position has been reported to be preferable for neonates with various respiratory diseases. Studies in healthy neonates have yielded conflicting results. Using a crying pulmonary function test, we examined the effect of positioning on pulmonary function in healthy full-term neonates. Thirty-nine infants with a mean birthweight (+/- SD) of 3,140 +/- 379 g and a mean gestational age (+/- SD) of 39.8 +/- 1.6 weeks were investigated during the first 6 hours of life. Measurements were obtained in both supine and prone positions using a computerized volume-flow system. There were statistically significant decreases in crying vital capacity (CVC) and peak expiratory flow rate (PEF) in the prone compared with the supine position. However, there were no significant differences in forced expiratory flow rate at 75% (V(75)), 50% (V(55)), and 25% (V(25)) of vital capacity between the two positions. These results suggest that prone positioning decreases lung volume and increases resistance of upper airways. We conclude that healthy neonates should be in the supine posture for optimal ventilation.

A monoclonal antibody against human DNA polymerase delta (pol delta) was isolated with properties suitable for its utilization for immunoaffinity chromatography. The antibody was immobilized after periodate oxidation and coupled to a hydrazide-activated support. Starting from a partially purified preparation, calf thymus pol delta was purified about 200-fold in a single step. Further purification on ssDNA-cellulose resulted in isolation of a homogeneous preparation. The amount of enzyme isolated, ca. 0.3 mg of pure pol delta from 0.75 kg of calf thymus, is about 15-fold greater than can be achieved by conventional procedures. This procedure provides a significant advance in the isolation of pol delta in allowing its facile isolation from tissues in good yield. The isolated enzyme consisted of two subunits of 125 and 50 kDa. Characterization of the enzyme showed that these two subunits remained associated on glycerol gradient ultracentrifugation even in the presence of 2.8 M urea.

Percutaneous subclavian implantation of an indwelling central venous catheter is an easy technique and provides convenient venous access for long-term intravenous therapy. Although rarely reported, spontaneous fracture of the catheter is an ominous complication which requires a prompt diagnosis and urgent treatment. We present a case of "pinch-off sign" resulting in a spontaneous fracture of an indwelling central venous catheter. A 49-year-old female breast cancer patient was admitted and Port-A-Cath was implanted for chemotherapy. Immediately after the implantation, fluid infusion and blood withdrawal was smooth until clinical "pinch-off sign" developed 3 weeks later. Chest X ray revealed no abnormal findings. Extravasation of antineoplastic drugs was noted 113 days after operation. Fracture of the indwelling catheter was found at the clavicle-rib junction. The fractured fragment was removed with a transvenous snare under fluoroscope. There was no hemodynamic derangement during the peri-operative period. Microscopy studies suggested that intermittent pressure on the catheter between the clavicle and the first rib may be responsible. The catheter wore on the medial side ue to a tearing and scissoring effect associated with free shoulder joint movement exerted additional forces on this wearing point which led to catheter fracture. The relationship between the spontaneous catheter fracture and "pinch-off sign" is reviewed. Our suggestions are: (1) By avoiding the traditional cannulation of the median subclavian vein, the lateral subclavian vein, infraclavicular axillary vein or internal jugular vein should be better routes for implantation. (2) Chest X ray (anterior-posterior and lateral view) should be examined routinely 3 weeks after the operation.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the expression in Escherichia coli of a fusion protein that contains the propeptide sequence and gamma-carboxyglutamic acid domain (residues -18 to 41) of human factor IX (FIXGla). CNBr was used to release FIXGla from the fusion protein. The 59-amino acid peptide is an efficient substrate for in vitro gamma-carboxylation. Its Km,app (0.55 microM) is several thousand-fold lower than that of the commonly used substrate FLEEL and about 5 times lower than proPT28 or proFIX28, (Hubbard, B. R., Jacobs, M., Ulrich, M. M. W., Walsh, C., Furie, B., and Furie, B. C. (1989) J. Biol. Chem. 264, 14145-14150). In addition, FIXGla is the first peptide substrate that is carboxylated in vitro to more than one gamma-carboxyglutamic acid/molecule (6-11 gamma-carboxyglutamic acids/molecule). We created peptides with mutations identical to FIXSan Dimas or FIXCambridge as well as a peptide with both mutations in the propeptide sequence and examined the effect of the mutations on in vitro carboxylation. Enzyme kinetic studies revealed no significant difference in Vmax/Km values between normal and mutant substrates. Maximum carbon dioxide incorporation was achieved with the double mutant. From these data we conclude the following. 1) FIXGla and its mutants are excellent substrates for studying the mechanism of gamma-carboxylase. 2) Although arginines at positions -4 and -1 are highly conserved in the propeptide sequence of all the vitamin K-dependent proteins, neither is critical for gamma-carboxylation.

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.

We have analysed seven polymorphic restriction sites of the human beta-globin gene cluster of six members of a Chinese family with a beta +-thalassemic sibling. The seven polymorphic sites analysed are the HincII site at the 5'-end of the epsilon-globin gene, the HindIII sites in the two gamma-globin genes, two HincII sites within and at the 3'-end of the psi beta 1 pseudogene, the AvaII site in the beta-globin gene and the BamHI site located at the 3' side of the beta-globin gene. The beta thal chromosome has been identified to have a haplotype of +----++ with respect to these seven polymorphic sites. This is also the most predominant haplotype associated with beta +-thalassemia in Mediterranean and Chinese populations (Chen et al., 1984; Orkin et al., 1982). Of the seven sites analysed in this family, four will be useful in prenatal diagnosis of beta-thalassemia in subsequent pregnancies in the family.

The establishment of the carcinogenic role of aflatoxins has been impeded by the lack of suitable tests to measure individual exposure for long-term studies. It was shown that the use of immuno-concentration followed by high pressure liquid chromatography (HPLC) or immunoassay can regularly detect aflatoxins down to pg/ml in fluids including urine. Aflatoxin M1 (AFM1) in free form was identified as the major metabolite in urine suitable for use as an approximate dosimetric indicator of recent exposure to aflatoxin B1 (AFB1). A panel of monoclonal antibodies of IgG class were developed in the murine system against AFB1 and/or AFM1. Their affinity constants are at the level of 10(8)-10(9) L/mol, and they are suitable for use in either effective immuno-concentration or in high sensitivity immunoassays. It was shown in a high risk area of liver cancer (Qidong of China) that 10% or more of the local inhabitants had a urinary output of AFM1 one or two orders of magnitude higher than that of Beijing people, especially during the wet seasons. The ingestion of AFB1 among these local people was estimated to exceed 1 mg/year. The major source came from contaminated corn and rice, but local alcoholic beverages also contributed. The increased excretion of AFM1 was more pronounced in patients with chronic active hepatitis. This observation of very low AFM1 content in urine of a significant percentage of local people implies their food storage practices may offer a feasible method for such prevention. The value and possible problems in conducting long-term studies on primary prevention are discussed.