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Biomedical subjects

J Tong

Publications and source records attributed to J Tong.

At least 37 records · Page 2Linked to original sources

Software scripts for quality checking of high-throughput nucleic acid sequencers.

We have developed a graphical interface to allow the researcher to view and assess the quality of sequencing results using a series of program scripts developed to process data generated by automated sequencers. The scripts are written in Perl programming language and are executable under the cgibin directory of a Web server environment. The scripts direct nucleic acid sequencing trace file data output from automated sequencers to be analyzed by the phred molecular biology program and are displayed as graphical hypertext mark-up language (HTML) pages. The scripts are mainly designed to handle 96-well microtiter dish samples, but the scripts are also able to read data from 384-well microtiter dishes 96 samples at a time. The scripts may be customized for different laboratory environments and computer configurations. Web links to the sources and discussion page are provided.

Base Sequence↗

[Selective isolation of anethole from volatile oil of Foeniculum vulgare Mill by inclusion crystalline with chela-shape host].

AIM: To isolate the components from the volatile oil of Foeniculum vulgare Mill. METHODS: According to the function of molecular recognition of supramolecular chemistry, chela shape molecule, trans-1, 2-biphenyl-1, 2-acenaphthendiol was used as host molecule and the volatile oil of Foeniculum vulgare Mill as guest molecule. Trans-1, 2-biphenyl-1, 2-acenaphthendiol can recognize the components that endowed with interactional complementarity and form inclusion compound as crystals. RESULTS: The anethole in the volatile oil was selectively included as trans-1,2-biphenyl-1,2-acenaphthendiol which was obtained in pure state from the inclusion compound by Kugelrohr vacuum technology. The formation of inclusion compound was confirmed by means of IR and powder XRD. The structure of the selectively isolated component was elucidated as trans-anethole by means of IR, 1HMMR and MS. CONCLUSION: The experimental results showed that the method is simple, rapid and selective for isolation anethole from volatile oil of Foeniculum vulgare Mill.

Allylbenzene Derivatives↗

Preparation of Monodispersed Solid Lipid Microspheres Using a Microchannel Emulsification Technique.

Monodispersed solid lipid microspheres consisting of high melting point edible oil were prepared as a novel sophisticated material. To prepare the monodispersed solid lipid microspheres a temperature-controlled microchannel (MC) emulsification process was devised. The prepared microspheres had diameters of approximately 20 µm or more and the standard deviation of the diameters was less than 1 µm. The effects of different surfactants, the pressure and the shape of the MC on the MC emulsification, and the diameter of the prepared droplet were studied. The experimental results are discussed using the new mechanism of droplets formation which is caused by the interfacial tension. Copyright 2000 Academic Press.

Journal Article↗

Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus.

An NAD(+)-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg(2+)or Mn(2+)as the metal cofactor. Ca(2+)and Ni(2+)mainly support formation of DNA-adenylate intermediates. The catalytic cycle is characterized by a low k (cat)value of 2 min(-1)with concomitant accumulation of the DNA - adenylate intermediate when Mg(2+)is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A < or = G/C < C/G < A/T on both the 3'- and 5'-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3'-side of the nick junction. The requirement of 3' complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3'- or 5'-side of the nick. Furthermore, most of the unligatable 3' mismatched base pairs prohibit formation of the DNA-adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5'-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.

Base Pair Mismatch↗

A neurofibromatosis-1-regulated pathway is required for learning in Drosophila.

The tumour-suppressor gene Neurofibromatosis 1 (Nf1) encodes a Ras-specific GTPase activating protein (Ras-GAP). In addition to being involved in tumour formation, NF1 has been reported to cause learning defects in humans and Nf1 knockout mice. However, it remains to be determined whether the observed learning defect is secondary to abnormal development. The Drosophila NF1 protein is highly conserved, showing 60% identity of its 2,803 amino acids with human NF1 (ref. 12). Previous studies have suggested that Drosophila NF1 acts not only as a Ras-GAP but also as a possible regulator of the cAMP pathway that involves the rutabaga (rut)-encoded adenylyl cyclase. Because rut was isolated as a learning and short-term memory mutant, we have pursued the hypothesis that NF1 may affect learning through its control of the Rut-adenylyl cyclase/cAMP pathway. Here we show that NF1 affects learning and short-term memory independently of its developmental effects. We show that G-protein-activated adenylyl cyclase activity consists of NF1-independent and NF1-dependent components, and that the mechanism of the NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory.

Adenylyl Cyclases↗

Prevalence of methylenetetrahydrofolate reductase C677T and its association with arterial and venous thrombosis in the Chinese population.

Moderate hyperhomocysteinaemia (MHH) is associated with arterial and venous thrombosis. A main genetic defect related to MHH is a C to T substitution at nucleotide 677 of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene. A prothrombin 20210A mutation was recently identified as a risk factor for arterial and venous thrombosis. However, studies on the prevalence of mutant MTHFR C677T and prothrombin G20210A and their association with thrombosis were controversial and seldom reported in the Chinese population. We investigated the prevalence of MTHFR C677T and prothrombin G20210A genotypes by polymerase chain reaction (PCR) followed by restriction enzyme digestion in 420 Chinese subjects: 53 with deep venous thrombosis (DVT); 145 with cerebrovascular disease [115 cerebral infarction, 30 cerebral haemorrhage (CH)]; 100 with coronary artery disease (CAD); and 122 control subjects. The prevalence of the mutated MTHFR 677TT genotype and the 677T allele in normal controls was 12.3% and 30.7% respectively, similar to that in Caucasians and Japanese. The mutant 677T homozygotes and alleles were more frequent in patients with DVT than in controls (18.9% vs. 12.3%, 0.01 < P < 0. 025; 48.1% vs. 30.7%, P < 0.005). The relative risk of DVT among the carriers of 677TT and 677T were significantly increased [odds ratios: 3.4, 95% confidence interval (CI) 1.3-9.5, and 3.6, 95% CI 1. 7-7.7, respectively). The mutant MTHFR heterozygous 677C/T carriers were increased in patients with cerebral infarction compared with controls (53.9% vs. 36.9%, 0.01 < P < 0.025). Relative risk of cerebral infarction was 0.96 (95% CI 0.4-2.3) for 677TT homozygotes and 1.99 (95% CI 1.2-3.4) for 677C/T heterozygotes. However, the distribution of the MTHFR TT genotype was less frequent in patients with CAD with coronary artery stenosis of > 50% than in controls (2. 8% vs. 12.3%, 0.025 < P < 0.05). Relative risk of CAD was not increased among the carriers of 677TT and 677T (odds ratios: 0.2, 95% CI 0-1.1, and 0.97, 95% CI 0.5-1.8, respectively). There were no differences in the distribution of the MTHFR genotypes among CH, CAD with coronary artery stenosis of < 50% and controls. The prothrombin 20210A mutation was not found in any patients or controls. These results demonstrated that MTHFR 677T was associated with DVT and cerebral infarction but was less associated with CAD in the Chinese population.

Adolescent↗

The human nucleus accumbens is highly susceptible to G protein down-regulation by methamphetamine and heroin.

Although the nucleus accumbens is assumed to be a critical brain "pleasure center," its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down-regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of G(alpha)1 and/or G(alpha)2 were reduced by 32-49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug-dependent state (e.g., tolerance) might be related to inhibition of G(alpha)1-linked receptor activity.

Adult↗

Combined free toe and free deep inferior epigastric perforator flap for reconstruction of the thumb and thumb web space.

To repair a complexly injured hand with composite loss of the thumb and the thumb web space in one stage, the combined transfer of a free second toe and a free deep inferior epigastric perforator flap was designed. It was used to simultaneously reconstruct the thumb and thumb web space of the injured hand in five cases. All flaps survived and there were no complications at any donor site after the reconstructions. In follow-up averaging 35.6 months, the final functional and cosmetic outcomes of the reconstructed thumbs and thumb web spaces were satisfactory. Results demonstrated that the combined transfer of a free second toe and a free deep inferior epigastric perforator flap is a valid method for simultaneous reconstruction of the thumb and the thumb web space of the injured hand.

Abdominal Muscles↗

[Mechanism of pineal and suprachiasmatic regulation on circadian rhythm of body temperature in rats].

OBJECTIVE: To investigate the mechanism of circadian rhythm of skin and core temperature as regulated by pineal and suprachiasmatic nucleus (SCN) in rats. METHOD: Pineal destruction and melatonin interference test were employed. RESULT: The two nuclei functioned differently in the maintenance of the temperature rhythm. Changes of the temperature level were nucleus related, i.e., up-regulated by pineal and down-regulated by SCN. In terms of initiation and daily running of the rhythmic oscillation, pineal is probably the central clock of the skin temperature rhythm, while SCN mainly controls the core temperature rhythm. CONCLUSION: The two nuclei interlink by way of neuroendocrine signals to balance over the oscillatory system of the circadian temperature.

Animals↗

Effect of advanced glycosylation end products on activity of protein kinase C in human peripheral blood mononuclear cells.

OBJECTIVES: To investigate the effect of advanced glycosylation end products (AGEs) on the activity of protein kinase C (PKC) in human peripheral blood mononuclear cells (PBMC) and to observe whether aminoguanidine (AG) can influence the effect of AGEs. METHODS: After PBMC were isolated from human peripheral blood and incubated with different concentrations of AGEs-BSA for various periods, total PKC activity in PBMC was determined by measuring the incorporation of 32P from [gamma-32 P] ATP into a special substrate using Promega PKC assay kit. RESULTS: AGEs-BSA increased the total PKC activity in PBMC from 83.43 +/- 6.57 pmol/min/mg protein to 116.8 +/- 13.82 pmol/min/mg protein with a peak at 15 min. AGEs-BSA also increased the total PKC activity in a concentration-dependent manner from 83.1 +/- 6.4 pmol/min/mg protein (control) to 119.1 +/- 13.3 pmol/min/mg protein (control vs AGEs-BSA 400 mg/L, P < 0.01). Furthermore, AGEs-BSA induced an elevation of PKC activity in a glycosylating time-related manner, from 80.9 +/- 8.2 (control) to 118.3 +/- 11.5 pmol/min/mg protein (glycosylation for 12 wk, P < 0.01). The total PKC activity stimulated by AGEs-BSA pretreated with AG (100, 200 mg/L) was markedly lower than that of AGEs-BSA group not pretreated with AG (P < 0.05, P < 0.01). CONCLUSIONS: AGEs-BSA increased the total PKC activity in PBMC in a concentration and incubation time dependent manner. The ability of AGEs-BSA to stimulate PKC activity was markedly decreased by pretreatment of AGEs-BSA with AG.

Cells, Cultured↗

[Studies on cell transformation and cell cycle in human embryo lungs induced by nickel compounds].

OBJECTIVE: To elucidate the association between nickel compounds and occupational lung cancer. METHODS: Human embryo lung cells (MRC-9 and IMR-90) were transformed by water-insoluble nickel oxide and nickel subsulfide, and changes in cell cycle of these transformed cells were analyzed by flow cytometric technique. RESULTS: Morphological transformation in MRC-9 and IMR-90 cells were induced after exposure to nickel compounds for 3 - 5 months, including formation of transforming foci, overlaying growth and epithelium-like changes in cells, etc. Some transformed cells could form colonies in soft agar media. Changes in cell cycle of the transformed cells were observed with flow cytometry. Proportions of cells at G(1) phase and G(2)/M phase were 62.61% and 16.37%, respectively, in the untreated MRC-9 cells and hyperploid cells were hardly seen in cell cycle plots. But, in the transformed MRC-9 cells, proportion of cells at G(1) phase reduced to about 50%, and that at G(2)/M phase increased to over 20% and that of hyperploid cells to over 10%. The same phenomenon was observed in the IMR-90 cells. CONCLUSIONS: Water-insoluble nickel compounds can induce morphological transformation of human embryo lung cells and those transformed cells appeared certain biological characteristics of tumor cells.

Cell Cycle↗

[Clinical application of artificial blood vessel graft for arteriovenous fistulization].

OBJECTIVE: To investigate the clinical application of artificial blood vessel graft for arteriovenous fistulization. METHODS: From October 1995 to August 1998, 23 cases with renal failure received PTEF artificial vessels grafting for arteriovenous fistulization in the forearm. The PTFE artificial vessel was 6 mm in diameter, and 40 cm in length. Artificial vessel "U"-shaped loop was formed from elbow incision to wrist incision, and perfused by 20 ml heparin saline. The two ends of artificial vessel were end-to-side anastomosed with superficial cubital vein and cubital artery respectively. RESULTS: All of arteriovenous fistulas were successfully formed, and could be performed hemodialysis periodically. The artificial vessels could be punctured repeatedly, and had sufficient volume of blood flow. It had no rejection, no formation of false aneurysm, and no ischemia in arm or exacerbated reflux to heart. CONCLUSION: The artificial vessel grafting for arteriovenous fistulization is a safe and convenient technique in clinical practice, especially when there is no autogenous vessels for arteriovenous fistula.

Aged↗

HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine.

Because HEK-293 cells are widely used for the functional expression of channels, exchangers and transporters involved in Ca(2+) homoeostasis, the properties of intracellular Ca(2+) stores and the methods used for measuring intracellular Ca(2+) release in HEK-293 cells were evaluated. Ca(2+) imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca(2+) release in HEK-293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbachol- and thapsigargin-induced Ca(2+) release in untransfected HEK-293 cells. Intracellular Ca(2+) release in untransfected HEK-293 cells was also observed if medium changes were performed by aspirating and replacing fresh medium (stop-flow), but not if medium changes were performed by a continuous over-flow procedure. Stop-flow medium-change-induced Ca(2+) release in HEK-293 cells was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the observation that the level of expression of endogenous RyR proteins was below the limits of detection by Western blotting or [(3)H]ryanodine binding. Thus the level of endogenous expression of RyR is so low in HEK-293 cells as to provide a negligible background in relation to functional analysis of recombinant RyR molecules. These results are inconsistent with those of Querfurth et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Biochem. J. 334, 79-86], who reported higher levels of endogenous RyR expression in untransfected HEK-293 cells.

Caffeine↗

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease.

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Amino Acid Sequence↗

Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D.

NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98%. Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+. While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent. Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfectly matched substrate.

Amino Acid Sequence↗

Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels.

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be "leaky." In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca2+ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca2+ release, indicating that maximal drug-induced Ca2+ release depends on the size of the endoplasmic reticulum Ca2+ store. The content of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay, 45Ca2+ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the amplitude of Ca2+ release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca2+-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca2+ concentration.

Animals↗

Effects of cooling germinal vesicle-stage bovine oocytes on meiotic spindle formation following in vitro maturation.

Attempts to cryopreserve bovine oocytes result in low survival because of their sensitivity to temperatures near 0 degrees C. This study evaluates the effects of chilling germinal vesicle-stage (GV) oocytes on their formation of microtubules and the meiotic spindle. In experiment 1, five groups of GV-stage oocytes, each consisting of approximately 90 oocytes, were held at 39 degrees C as controls, or at 31 degrees C, or cooled to 24, 4 or 0 degrees C for 10 min. After being treated, all oocytes were cultured at 39 degrees C for 24 hr. Compared to the controls, holding oocytes for 10 min at 31 or 24 degrees C did not significantly alter the formation of normal spindles, but chilling them to 4 or 0 degrees C did. After 24 hr of maturation, the respective percentages of oocytes containing normal meiotic spindles observed in the controls or those held at 31 or 24 degrees C were 69.8%, 71.9%, or 69.4% (P > 0.05). In contrast, the percentages of oocytes with normal spindles after they had been cooled to 4 or 0 degrees C were 44.0% or 29.1%, respectively. In experiment 2, approximately 90 oocytes/group were cooled to 4 degrees C for various times before being warmed and cultured. Regardless of the time of exposure, cooling oocytes to 4 degrees C reduced the formation of normal spindles. The percentages of oocytes cooled to 4 degrees C for 10, 20, 30, 45, or 60 min with normal spindles were 44.0%, 38.4%, 37.5%, 34.5% and 30.9%, respectively. In experiment 3, approximately 60 oocytes per group that had been held at 31 degrees C or cooled to 24, 4 or 0 degrees C for 10 min were allowed to mature for 24 hr before being subjected to in vitro fertilization. The cleavage rates of oocytes subjected to various chilling treatments exhibited the same pattern as that of oocytes with normal spindles. That is, there were no significant differences in cleavage rates among the control oocytes and those held at 31 or 24 degrees C (70.4%, 71.8%, and 72.4%; P > 0.05). However, only 37. 0% and 30.4% of oocytes chilled to 4 or 0 degrees C cleaved after fertilization. These results suggest that: (1) chilling bovine oocytes no lower than 24 degrees C does not reduce formation of normal meiotic spindles; (2) however, chilling oocytes to 4 degrees C or lower for as little as 10 min drastically reduces the formation of normal meiotic spindles and of fertilization; (3) the rates of fertilization and cleavage of resultant zygotes mimic that of formation of normal spindles.

Animals↗

A bone marrow-derived stroma cell line, ST2, can support the differentiation of fetal thymocytes from the CD4+ CD8+ double negative to the CD4+ CD8+ double positive differentiation stage in vitro.

T-cell precursors differentiate into mature T cells predominantly in the thymus. However, it has also been reported that T-cell precursors mature in extrathymic organs such as the liver, bone marrow, or intestines. In order to investigate the nature of the extrathymic microenvironment that supports T-cell maturation, we examined the effect of a bone marrow-derived stroma cell line, ST2, on T-cell precursors by using a reaggregate thymic organ culture (RTOC) system. We found that ST2 cells supported the differentiation of fetal thymocytes at day 14.5 of gestation from a CD4- CD8- double negative (DN) to a CD4+ CD8+ double positive (DP) differentiation stage in a manner similar to that observed in thymus. Anti-interleukin-7 receptor (IL-7R) and anti-c-kit antibodies blocked the growth of thymocytes in RTOC with ST2 cells, but did not inhibit the generation of DP thymocytes. These data indicate that a bone marrow-derived stroma cell, ST2, which supports B-cell differentiation, is also able to support T-cell development and may constitute one of the microenvironmental components for extrathymic T-cell development.

Animals↗