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Biomedical subjects

Bin Lu

Publications and source records attributed to Bin Lu.

At least 19 recordsLinked to original sources

Selective solid-phase extraction of tebuconazole in biological and environmental samples using molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) were prepared by precipitation polymerization using tebuconazole (TBZ) as a template. Frontal chromatography and selectivity experiments were used to determine the binding capabilities and binding specificities of different MIPs. The polymer that had the highest binding selectivity and capability was used as the solid-phase extraction (SPE) sorbent for the direct extraction of TBZ from different biological and environmental samples (cabbage, pannage, shrimp, orange juice and tap water). The extraction protocol was optimized and the optimum conditions were: conditioning with 5 mL methanol:acetic acid (9:1), 5 mL methanol and 5 mL water respectively, loading with 5 mL aqueous samples, washing with 1.2 mL acetonitrile (ACN):phosphate buffer (5:5, pH3), and eluting with 3 mL methanol. The MIPs were able to selectively recognize, effectively trap and preconcentrate TBZ over a concentration range of 0.5-15 micromol/L. The intraday and interday RSDs were less than 9.7% and 8.6%, respectively. The limit of quantification was 0.1 micromol/L. Under optimum conditions, the MISPE recoveries of spiked cabbage, pannage, shrimp, orange juice and tap water were 62.3%, 75.8%, 71.6%, 89% and 93.9%, respectively. MISPE gave better HPLC separation efficiencies and higher recoveries than C18 SPE and strong cation exchange (SCX) SPE.

Acetic Acid↗

TIP30/CC3 expression in breast carcinoma: relation to metastasis, clinicopathologic parameters, and P53 expression.

Metastasis is the most frequent cause of death in patients with breast cancer. Tip30/CC3 gene is a putative metastasis suppressor gene, which was first identified by a differential display analysis of messenger RNA from the highly metastatic human variant small cell lung carcinoma (SCLC) versus less metastatic classic SCLC cell lines. The aim of this study was to analyze the relationship between expression of TIP30/CC3 and clinical prognosis in 87 patients with surgically removed breast carcinoma. Tumor tissues were stained immunohistochemically with anti-TIP30/CC3 antibody. We demonstrated that the expression of TIP30/CC3 was inversely associated with axillary lymph node metastasis (P = .0008) and vascular invasion (P = .0016). Expression of TIP30/CC3 was not correlated with tumor grade, estrogen, progesterone, and P53 expression. Inhibition of TIP30/CC3 expression by RNA-mediated interference greatly enhanced breast cancer cell invasion through the extracellular matrix, whereas overexpression of TIP30/CC3 by adenovirus vector suppressed invasion through the extracellular matrix. These data supported the theory that the expression of TIP30/CC3 had a suppressive function on tumor metastasis. In summary, the decrease in expression of TIP30/CC3 is related to metastasis and may represent a new prognosticator in breast carcinoma.

Acetyltransferases↗

Diazoxide prevents diabetes through inhibiting pancreatic beta-cells from apoptosis via Bcl-2/Bax rate and p38-beta mitogen-activated protein kinase.

Increased apoptosis of pancreatic beta-cells plays an important role in the occurrence and development of type 2 diabetes. We examined the effect of diazoxide on pancreatic beta-cell apoptosis and its potential mechanism in Otsuka Long Evans Tokushima Fatty (OLETF) rats, an established animal model of human type 2 diabetes, at the prediabetic and diabetic stages. We found a significant increase with age in the frequency of apoptosis, the sequential enlargement of islets, and the proliferation of the connective tissue surrounding islets, accompanied with defective insulin secretory capacity and increased blood glucose in untreated OLETF rats. In contrast, diazoxide treatment (25 mg.kg(-1).d(-1), administered ip) inhibited beta-cell apoptosis, ameliorated changes of islet morphology and insulin secretory function, and increased insulin stores significantly in islet beta-cells whether diazoxide was used at the prediabetic or diabetic stage. Linear regression showed the close correlation between the frequency of apoptosis and hyperglycemia (r = 0.913; P < 0.0001). Further study demonstrated that diazoxide up-regulated Bcl-2 expression and p38beta MAPK, which expressed at very low levels due to the high glucose, but not c-jun N-terminal kinase and ERK. Hence, diazoxide may play a critical role in protection from apoptosis. In this study, we demonstrate that diazoxide prevents the onset and development of diabetes in OLETF rats by inhibiting beta-cell apoptosis via increasing p38beta MAPK, elevating Bcl-2/Bax ratio, and ameliorating insulin secretory capacity and action.

Animals↗

Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease.

Progressive photoreceptor degeneration resulting from genetic and other factors is a leading and largely untreatable cause of blindness worldwide. The object of this study was to find a cell type that is effective in slowing the progress of such degeneration in an animal model of human retinal disease, is safe, and could be generated in sufficient numbers for clinical application. We have compared efficacy of four human-derived cell types in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the Royal College of Surgeons rat early in the progress of degeneration. Umbilical tissue-derived cells, placenta-derived cells, and mesenchymal stem cells were studied; dermal fibroblasts served as cell controls. At various ages up to 100 days, electroretinogram responses, spatial acuity, and luminance threshold were measured. Both umbilical-derived and mesenchymal cells significantly reduced the degree of functional deterioration in each test. The effect of placental cells was not much better than controls. Umbilical tissue-derived cells gave large areas of photoreceptor rescue; mesenchymal stem cells gave only localized rescue. Fibroblasts gave sham levels of rescue. Donor cells were confined to the subretinal space. There was no evidence of cell differentiation into neurons, of tumor formation or other untoward pathology. Since the umbilical tissue-derived cells demonstrated the best photoreceptor rescue and, unlike mesenchymal stem cells, were capable of sustained population doublings without karyotypic changes, it is proposed that they may provide utility as a cell source for the treatment of retinal degenerative diseases such as retinitis pigmentosa.

Animals↗

Sustained-release tablets of indomethacin-loaded microcapsules: preparation, in vitro and in vivo characterization.

Indomethacin (IDM) was encapsulated in gelatin-cellulose acetate phthalate (CAP) microcapsules (A) by complex coacervation method and in CAP microcapsules (B) by simple coacervation method. Microcapsules A and B, having mean diameters of 38.24 and 35.74 microm, respectively, were used to prepare sustained-release tablets A and B. The activation energy of thermal degradation for tablets A and B was calculated based on differential scanning calorimetry (DSC) to be 258.9 and 284.8 kcal/mol, respectively. In vitro release profiles showed no burst effect and release t(1/2) of the two sustained-release tablets were found to be 41.30+/-1.86 and 33.25+/-2.84 min, respectively, while that of IDM plain tablets C was 6.30+/-0.39 min (P<0.01). In vitro release of IDM from tablets A and B could be described by Higuchi equation and zero-order kinetics, respectively. After per os (po) administration with physiological saline, their irritation to rat stomach was obviously reduced in comparison with tablets C. Pharmacokinetic study in rabbits showed that t(max) was delayed and C(max) lowered compared with tablets C and the values of AUC(0-24 h) of the three tablets were very close.

Animals↗

Plus- and minus-stranded foot-and-mouth disease virus RNA quantified simultaneously using a novel real-time RT-PCR.

Even though tagged RT-PCR and rTth RT-PCR have been developed to improve strand-specific detection of RNA virus, these assays are not quantitative. In this study, a novel real-time RT-PCR assay, which combines the benefits of both the tagged and rTth RT-PCR has been developed to quantify the strand-specific RNA of foot-and-mouth disease virus (FMDV). The tagged-primers plus a TaqMan probe located within the highly conserved viral 3D region were used. The in vitro synthesized minus-and plus-stranded RNA templates were used as a dual control along with the copy number of viral RNA molecules, which is more reliable than reported RT-PCR employing a DNA-based standard. This assay was used to quantify FMDV RNA from 10(9) to 10(1) copies with a maximum sensitivity of between ten and five copies and was shown to be highly reproducible with low intra-and inter-assay variation. Coefficients of variation (CV) values were 0.70-1.39% and 0.98-2.1%, respectively. Importantly, the method was applied to simultaneously quantify both plus-stranded and minus-stranded FMDV RNA using tagged-RT and tagged-FP primer during a high-temperature reverse transcription. Highly sensitive and strand-specific real-time RT-PCR assay has been established. We tested the ratio of viral plus-stranded to minus-stranded RNA in acutely infected and persistently infected BHK-21 cells, for which the values ranged from 22/1 to 143/1 and from 287/1 to 334/1, respectively, suggesting different replication patterns of plus-and minus-stranded RNA in acutely infected and persistently infected cells. This value ranged from 83/1 to 93/1 in enriched FMDV virions, indicating that FMDV encapsidation is highly specific for plus-stranded RNAs. In addition, the method was applied to surveille the FMDV replication at animal level.

Animals↗

Selective solid-phase extraction of cholesterol using molecularly imprinted polymers and its application in different biological samples.

Non-covalent molecularly imprinted polymers (MIPs) of cholesterol were prepared by UV initiated polymerization. A polymer that had the highest binding selectivity and capability was used as solid-phase extraction (SPE) sorbents for direct extraction of cholesterol from different biological samples (human serum, cow milk, yolk, shrimp, pork and beef). The extraction conditions of molecularly imprinted SPE (MISPE) were optimized and the optimum protocol was: conditioning MISPE cartridges with n-hexane, loading with n-hexane, washing with n-hexane and n-hexane:toluene=9:1, respectively, then eluting with chloroform:ethanol:acetic acid=3:1:1. Cholesterol MISPE selectively recognized, effectively trapped and pre-concentrated cholesterol over a concentration range of 10-80 microg/mL. Recoveries ranged from 80.6% to 92.7%, with R.S.D. lower than 9.8%. Under the optimal condition, MISPE recoveries of spiked human serum, yolk, cow milk, shrimp, pork and beef were 91.1%, 80.4%, 86.6%, 78.2%, 81.4% and 80.1%, respectively. Compared with C18 SPE, almost all of the matrix interferences were removed after MISPE, and better baselines and higher selectivity were achieved.

Animals↗

Apolipoprotein B is associated with metabolic syndrome in Chinese families with familial combined hyperlipidemia, familial hypertriglyceridemia and familial hypercholesterolemia.

There is a paucity of data concerning the metabolic syndrome (MetS) in families with familial combined hyperlipidemia (FCHL), familial hypertriglyceridemia (FHTG), familial hypercholesterolemia (FH) and normolipidemic families in China. This study investigated the prevalence of MetS in these families and explored potential factors relevant to MetS. We recruited 70 families with 560 individuals > or = 20 years of age, including 43 FCHL families with 379 individuals, 3 FHTG families with 30 individuals, 16 FH families with 102 individuals and 8 normolipidemic families with 49 individuals. The definition of MetS is determined using modified criteria of National Cholesterol Education Program substituting body mass index for waist circumference. MetS is identified in 60.7% of FCHL patients and 71.4% of FHTG patients. The prevalence of MetS in family members is 36.7% for FCHL, 33.3% for FHTG, 17.6% for FH and 16.3% for normolipidemic families, with an odds ratio (OR) of 2.97 (95% CI 1.29-7.07, P=0.007) in FCHL families compared with normolipidemic families. Apolipoprotein B (apoB) is associated with MetS by multiple logistic analysis with an OR of 1.05 (1.03-1.07, P<0.001) in FCHL families, OR of 1.26 (1.03-1.55, P=0.026) in FHTG and OR of 1.07 (1.01-1.12, P=0.014) in FH families, independent of variables including age, gender, apolipoprotein A1, and low density lipoprotein cholesterol. Apolipoprotein A1 provided an OR of 0.95 (0.94-0.97, P<0.001) in FCHL families and OR of 0.94 (0.90-0.97, P=0.011) in FH families, but neither in FHTG nor in normolipidemic families (both P>0.05). Thus, apoB may be regarded as a relevant factor in the assessment of MetS in FCHL, FHTG and FH families. However, this finding needs to be verified by prospective studies in diverse ethnicities and warrants additional studies to elucidate possible mechanisms linking apoB to MetS.

Adult↗

Pathway analysis using random forests classification and regression.

MOTIVATION: Although numerous methods have been developed to better capture biological information from microarray data, commonly used single gene-based methods neglect interactions among genes and leave room for other novel approaches. For example, most classification and regression methods for microarray data are based on the whole set of genes and have not made use of pathway information. Pathway-based analysis in microarray studies may lead to more informative and relevant knowledge for biological researchers. RESULTS: In this paper, we describe a pathway-based classification and regression method using Random Forests to analyze gene expression data. The proposed methods allow researchers to rank important pathways from externally available databases, discover important genes, find pathway-based outlying cases and make full use of a continuous outcome variable in the regression setting. We also compared Random Forests with other machine learning methods using several datasets and found that Random Forests classification error rates were either the lowest or the second-lowest. By combining pathway information and novel statistical methods, this procedure represents a promising computational strategy in dissecting pathways and can provide biological insight into the study of microarray data. AVAILABILITY: Source code written in R is available from http://bioinformatics.med.yale.edu/pathway-analysis/rf.htm.

Algorithms↗

Single amino acid substitutions in the hemagglutinin of influenza A/Singapore/21/04 (H3N2) increase virus growth in embryonated chicken eggs.

Most of the recently circulating H3N2 influenza A strains do not replicate well in embryonated chicken eggs and had to be isolated by cell culture, which presents a great challenge for influenza vaccine production using embryonated chicken eggs. We previously reported that a human H3N2 virus, A/Fujian/411/02, which replicates poorly in eggs, could be improved by changing a minimum of two HA residues (G186V/V226I or H183L/V226A). Here, we extended our work to the A/Singapore/21/04 strain that was also unable to grow in eggs. We showed that a single amino acid substitution of either G186V or A196T in the HA resulted in significantly increased virus replication in eggs without affecting virus antigenicity.

Amino Acid Substitution↗

Phosphatidylinositol 3-kinase/Akt positively regulates Fas (CD95)-mediated apoptosis in epidermal Cl41 cells.

Fas (CD95)-mediated apoptosis is an essential mechanism for the maintenance of homeostasis, and disruption of this death pathway contributes to many human diseases. The cell survival protein kinase Akt/protein kinase B (PKB) is a known regulator of apoptosis, but its role in Fas-mediated cell death and its regulatory mechanisms are unclear. In this study, we show that stimulation of the Fas receptor by its ligand (FasL) induces rapid phosphorylation of Akt/PKB and a parallel increase in cell apoptosis in epidermal Cl41 cells. Inhibition of PI3K/Akt by dominant-negative overexpression of PI3K (Deltap85) and Akt (Akt-T308A/S473A) protects the cells from apoptosis, indicating an unexpected proapoptotic role of PI3K/Akt in the Fas signaling process. Treatment of the cells with pharmacological inhibitors of PI3K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-1 (LY294002), similarly inhibits FasL-induced apoptosis and Akt/PKB phosphorylation, indicating that PI3K is an upstream mediator of Akt/PKB and is involved in Fas-mediated cell death. Electron spin resonance studies show that FasL treatment induces rapid generation of reactive oxygen species, and inhibition of ROS by antioxidants effectively inhibits Akt/PKB signaling, suggesting that FasL activation of Akt/PKB is redox sensitive. In cells transfected with dominant-negative PI3K/Akt, Fas expression is down-regulated, but FLIP expression is unaffected. Reporter gene and mRNA expression assays show that FasL activates fas transcriptional activity and this effect is inhibited by PI3K/Akt suppression. Together, our results indicate that the PI3K/Akt, in addition to its normal prosurvival role, also plays an apoptotic role in Fas-mediated cell death through a mechanism that involves transcriptional activation of Fas receptor.

Androstadienes↗

Diverse morphological lesions and serious arrhythmias with hemodynamic insults occur in the early myocardial contusion due to blunt impact in dogs.

To investigate the morphology and hemodynamics of the early myocardial contusion, an animal model of cardiac contusion was established by impact to the precordial region at sternum at velocity of 10.0m/s with a mechanical elastic-cord propelled impactor in 19 dogs. The electrocardiogram and both the left and right intra-ventricular pressures were recorded continuously throughout the experiment. Histological and immunohistochemical examinations of myoglobin, creatine kinase-MB and fibrinogen were conducted. At the moment of impact, abrupt over-pressures within the left and right ventricles occurred with concomitant serious arrhythmias followed by variety of cardiac conduction disorders and depressed left and right ventricular systolic pressures during the observation times. Histologically, lesions of myocardial contusions were identified at subepicardial, myocardial or subendocardial layer as interstitial hemorrhage, disruption or coagulative necrosis as well as contraction band necrosis of the muscle fibers, which might be categorized into the hemorrhagic, necrotized and mixed forms. The three forms of lesions were found to exist independently, or co-existed in a heart. However, severity of the lesions varied greatly with different parts even within a heart. Intravascular thromboses were occasionally discovered post-impact. Immunohistochemically, loss of myoglobin and creatine kinase-MB from cardiac cells, and accumulation of fibrinogen at the cell membranes were detected 5min post-impact. The intracellular accumulation of fibrinogen increased with extension of post-impact intervals. Our results indicate that diverse morphological lesions concomitant with hemodynamic compromise and serious, even fatal arrhythmias occur in the early myocardial contusion, and intravascular thromboses are occasionally produced, suggesting that traumatic myocardial ischemic lesion may be induced due to blunt impact to the precordial region.

Animals↗

Selective solid-phase extraction of bisphenol A using molecularly imprinted polymers and its application to biological and environmental samples.

Molecularly imprinted polymers (MIPs) were prepared using bisphenol A (BPA) as a template by precipitation polymerization. The polymer that had the highest binding selectivity and ability was used as solid-phase extraction (SPE) sorbents for direct extraction of BPA from different biological and environmental samples (human serum, pig urine, tap water and shrimp). The extraction protocol was optimized and the optimum conditions were as follows: conditioning with 5 mL methanol-acetic acid (3:1), 5 mL methanol, 5 mL acetonitrile and 5 mL water, respectively, loading with 5 mL aqueous samples, washing with 1 mL acetonitrile, and eluting with 3 mL methanol. MIPs can selectively recognize, effectively trap and preconcentrate BPA over a concentration range of 2-20 microM. Recoveries ranged from 94.03 to 105.3%, with a relative standard deviation lower than 7.9%. Under the optimal condition, molecularly imprinted SPE recoveries of spiked human serum, pig urine, tap water and shrimp were 65.80, 82.32, 76.00 and 75.97%, respectively, when aqueous samples were applied directly. Compared with C18 SPE, a better baseline, better high-performance liquid chromatography separation efficiency and higher recoveries were achieved after molecularly imprinted SPE.

Animals↗

Tid1 isoforms are mitochondrial DnaJ-like chaperones with unique carboxyl termini that determine cytosolic fate.

Tid1 is a human homolog of bacterial DnaJ and the Drosophila tumor suppressor Tid56 that has two alternatively spliced isoforms, Tid1-long and -short (Tid1-L and -S), which differ only at their carboxyl termini. Although Tid1 proteins localize overwhelmingly to mitochondria, published data demonstrate principally nonmitochondrial protein interactions and activities. This study was undertaken to determine whether Tid1 proteins function as mitochondrial DnaJ-like chaperones and to resolve the paradox of how proteins targeted primarily to mitochondria function in nonmitochondrial pathways. Here we demonstrate that Tid1 isoforms exhibit a conserved mitochondrial DnaJ-like function substituting for the yeast mitochondrial DnaJ-like protein Mdj1p. Like Mdj1p, Tid1 localizes to human mitochondrial nucleoids, which are large protein complexes bound to mitochondrial DNA. Unlike other DnaJs, Tid1-L and -S form heterocomplexes; both unassembled and complexed Tid1 are observed in human cells. Results demonstrate that Tid1-L has a longer residency time in the cytosol prior to mitochondrial import as compared with Tid1-S; Tid1-L is also significantly more stable in the cytosol than Tid1-S, which is rapidly degraded. The longer cytosolic residency time and the half-life of Tid1-L are explained by its interaction with cytosolic Hsc70 and potential protein substrates such as the STAT1 and STAT3 transcription factors. We show that the unique carboxyl terminus of Tid1-L is required for interaction with Hsc70 and STAT1 and -3. We propose that the association of Tid1 with chaperones and/or protein substrates in the cytosol provides a mechanism for the alternate fates and functions of Tid1 in mitochondrial and nonmitochondrial pathways.

Amino Acid Sequence↗

Solid lipid nanoparticles of mitoxantrone for local injection against breast cancer and its lymph node metastases.

In an attempt to improve the therapeutic effect of mitoxantrone (MTO) against breast cancer and its lymph node metastases, solid lipid nanoparticles (SLN) of MTO were prepared, characterized and evaluated on mice. Film dispersion-ultrasonication method was used to prepare MTO-SLN, optimized by central composite design. MTO-SLN were prepared with a mean size of 61 nm, drug content (DC) of 4.18+/-0.10% and encapsulation yield (EY) of 87.23+/-2.16%. MTO-SLN were lyophilized and their mean size became 79 nm without significant change in DC and EY. The in vitro release study revealed a profile of sustained release of MTO from MTO-SLN without burst effect: the cumulative release rate Q24 h = 25.86 +/- 0.82%, t50 = (5.25 +/- 1.10)d and t90 = (28.38 +/- 4.50)d. The drug concentration of MTO-SLN in local lymph nodes was much higher and the drug concentrations in other tissues lower than that of MTO solution (MTO-Soln). Human MCF-7 breast cancer in nude mice and animal model of P388 lymph node metastases in Kunming mice were applied to investigate the therapeutic effects. There was no observed toxicity to the main tissues after local injection of MTO-SLN, but, for MTO-Soln, medium to serious toxicity to liver and lung was produced. The percent inhibition of MTO-SLN against breast cancer was 81.81 +/- 14.03%, while that of MTO-Soln with a double dose was 82.86 +/- 11.13%. The tests for lymph node metastases showed that MTO-SLN gave a mean size of lymph node of 41.85 +/- 27.42 mm3, while that of the MTO-Soln was 119.32 +/- 57.30 mm3 and that of the placebo was 186.83 +/- 77.71 mm3. This study opens a new perspective of active delivery of antitumor drug against breast cancer and its lymph node metastases with inspiring therapeutic effect and little side effects.

Algorithms↗

Direct enrichment and high performance liquid chromatography analysis of ultra-trace Bisphenol A in water samples with narrowly dispersible Bisphenol A imprinted polymeric microspheres column.

Direct injection, enrichment and high performance liquid chromatography (HPLC) analysis of ultra-trace Bisphenol A (BPA) in water samples using one narrowly dispersible BPA imprinted polymeric microspheres (MIPM) column in one analysis process was developed. One BPA imprinted MIPM that had the best globular morphology and imprinted efficiency was used as HPLC stationary phase and applied to direct analysis of ultra-trace BPA in water. The optimum direct analysis conditions were: conditioning the MIPM column with water for 10min, injecting 40mL water sample directly, eluting with 70% methanol for 13min and then 100% methanol for 7min. Under the optimum conditions, the MIPM column can simultaneously extract, enrich, separate and determine ultra-trace BPA in one analysis process with UV detector by injection of large volume water samples (40mL). The calibration graph was linear with R(2)>0.998 in the ranges from 0.1-100nmol/L BPA standard solution. The intra- and inter-day RSD are less than 9.5 and 9.6%, respectively. The limit of quantification was 0.1nmol/L. RSD for spiked tap and lake water was less than 8.9% and the recoveries were 96-101.8%. The enrichment factor for BPA was 10,000 as 40mL water sample was directly injected and analyzed.

Benzhydryl Compounds↗

Toxoplasma gondii: expression pattern and detection of infection using full-length recombinant P35 antigen.

A complete P35 surface antigen of Toxoplasma gondii was sequenced (GenBank ). Immunoblot found that it reacted specially with T. gondii acute infected sera, and the recombinant P35 signal was specific for P35 antigen. The P35-GST protein was used as antigen to detect 125 sera samples by double-sandwich ELISA. P35-IgM positive rate in a chronic infected group, a persistent IgM positive chronic group, a recently seroconvered group and an acute infected group were 4% (1 out of 25), 16% (4 out of 25), 88% (22 out of 25), and 100% (25 out of 25), respectively. The sensitivity and specificity of the recombinant full-length P35 antigen were 100 and 96%, respectively. The detailed expression patterns of P35 antigen were studied in 36 IgM and IgG positive sequential samples from 10 recently seroconvered patients. Results showed that the P35-IgM positive rate decreased as the time after the first seroconversion increased. P35-IgM positive samples in the first, second, third, fourth, and fifth month after the first seroconversion test were 90, 78, 57, 50, and 33%, respectively. P35-IgG positive samples in the first, second, third, fourth, fifth, sixth, and seventh month after the first seroconversion test were 70, 100, 100, 100, 67, 100, and 100%, respectively. All samples were P35-IgM negative after the fifth month, and P35-IgG negative after the seventh month from seroconversion. P35-IgM existed the shortest time and was a more specific marker for T. gondii acute infection than P35-IgG, IgM, and IgG to whole tachyzoites antigens.

Acute Disease↗

cDNA cloning, expression, purification, distribution, and characterization of biologically active canine alanine aminotransferase-1.

Alanine aminotransferase (ALT) is a pyridoxal enzyme found mainly in the liver and kidney, but also in small amounts in the heart, muscle, fat, and brain. Serum aminotransferase activities have been used broadly as surrogate markers for tissue injury and disease in human and veterinary clinical settings and in safety assessment of chemicals and pharmaceuticals. Because of its relative abundance in liver, increased serum ALT activity is generally considered indicative of liver damage. Two ALT isoenzymes, ALT1 and ALT2, are known and have been cloned and sequenced from human, rat, and mouse. In this study, we have cloned the complementary DNA encoding the canine orthologue of ALT1 (cALT1). The complete cDNA sequence comprised 1852 bases and contained a 1485-base open reading frame, which encodes a polypeptide of 494 amino acid residues. Canine ALT1 shares 87.7, 87.2, and 87.0% amino acid identity to its human, mouse, and rat orthologues, respectively. The cDNA was expressed in Escherichia coli, with a N-terminal His (6x) tag, and the recombinant enzyme was purified using immobilized metal-affinity chromatography. The final yield of the purified recombinant cALT1 was greater than 5mg/L culture. The alanine transaminase activity of purified cALT1 was 229.81U/mg protein, which is approximately 38-fold higher than that of total soluble recombinant E. coli cell lysate, confirming that the enzyme is a functional ALT. Evaluation of various canine tissues by RT-PCR revealed that the level of ALT1 expression is in the order of: heart>liver>fat approximately brain approximately gastrocnemius>kidney. The purified cALT1 will be helpful to develop isoenzyme-specific anti-bodies, which could further improve the diagnostic resolution of current ALT assays in drug safety studies.

Alanine Transaminase↗