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Biomedical subjects

M Wang

Publications and source records attributed to M Wang.

At least 613 records · Page 34Linked to original sources
PubMed1997

Accumulation and distribution of free folic acid content in red beet (Beta vulgaris L.).

Among vegetable plants, red beet contains a relatively high level of the B vitamin folic acid. Although many leafy green vegetables contain high levels of folic acid, red beet is consumed primarily as a root vegetable. Folic acid levels have been quantified in various vegetable plants, but little information exists regarding the accumulation and distribution of this vitamin in plant tissues. The objective of this study was to characterize free folic acid content (FFAC) in shoot and root tissue during growth of two red beet inbreds. Experiments were conducted in a greenhouse during 1993, 1994 and 1995. Two inbreds, W384 and W357, were planted in randomized complete blocks and shoot and root tissues were separately harvested at 60, 80, and 100 days after planting (DAP). Significant differences between years, tissue portions, and among harvest dates were detected, however, similar patterns in FFAC accumulation and distribution were observed between inbreds and years. FFAC in shoot tissue was significantly greater than root tissue for both inbreds. Accumulation of FFAC was linear for both inbreds across harvest dates for root tissue but not for shoot tissue. FFAC accumulation in shoot tissue increased sharply from 60 to 80 DAP but decreased sharply from 80 to 100 DAP. These results demonstrate that FFAC accumulates differentially in root and shoot tissue in a red beet plant. Maximum folic acid levels in shoot tissue are achieved prior to those in root tissue.

Folic Acid↗
PubMed1997

Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene.

The mouse anaphylatoxin C3a receptor (mC3aR) gene was isolated using a human C3aR cDNA probe. The genomic fragment contains an open reading frame of 1431 base pairs that encodes a peptide of 477 amino acids. A cDNA with identical sequence was subsequently obtained from the mouse pre-B cell line 70Z/3 by reverse transcriptase polymerase chain reaction based on sequence of the mC3aR gene. Northern blot analysis suggested expression of the mC3aR in lung and heart, and to a lesser extent, in brain, liver, muscle, kidney, and testis. The deduced amino acid sequence of the mouse C3aR is 65% identical to that of the human C3aR. Like the human receptor, mouse C3aR contains a predicted large extracellular loop of approximately 165 amino acids (residues 161-325) between the fourth and fifth transmembrane domains. This loop, however, is the least conserved structure (45% identical sequence) of all the extracellular and intracellular domains between the mouse and human C3aRs. The mouse gene product bound 125I-labeled human C3a with a Kd of 2.54 nM when expressed in the stably transfected rat basophilic leukemic cell line RBL-2H3. Bound C3a could be effectively displaced by excess quantities of unlabeled C3a, but not by C4a or C5a. C3a induced dose-dependent calcium mobilization in the transfected cells, which could be blocked by pertussis toxin treatment. These results confirm that the cloned gene encodes a functional C3aR capable of coupling to a pertussis toxin-sensitive G protein. The sequence divergence of the large extracellular loop does not appear to affect C3a binding and transmembrane signaling.

Amino Acid Sequence↗
PubMed1997

Expression of rat steroid 5 alpha-reductase (isozyme-1) in Spodoptera frugiperda, SF21, insect cells: expression of rat steroid 5 alpha-reductase.

The enzyme steroid 5 alpha-reductase (5 alpha R) catalyzes the reduction of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5 alpha R type I isozyme (r5 alpha R 1). The full length of r5 alpha R1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda, Sf 21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K(m) values for NADPH and T were 17 microM and 2.7 microM, respectively. Inhibition of the recombinant r5 alpha R1 by N,N diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5 alpha R1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5 alpha R1. Photoaffinity labeling by [2'-32P]-2-azido-NAD P+ ([2'-32P]2N3-NAD P+) and [1,2(3)H] N-(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1997

Purification and sequence determination of a new neutral mammalian neurotoxin from the scorpion Buthus martensii Karsch.

A new neutral mammalian neurotoxin, designated BmK M4, with an isoelectric point (pI) of 7.6 and a relatively low toxicity (LD50 = 4.0 +/- 0.25 microgram/g mice, i.v.) was purified from the venom of scorpion Buthus martensii Karsch (BmK). The complete amino acid sequence of the toxin composed of 64 amino acid residues was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidomethylated protein (up to 30 amino acid residues) and its peptide fragments degraded by lysylendopeptidase or Staphylococcus aureus Va protease. The calculated mol. wt based on the amino acid composition was 7001. By comparison with the sequences of other basic BmK mammalian neurotoxins, it was concluded that the weaker toxicity and lower pI value of BmK M4 might be the result of mutations H10E, R18G and K28D. The sequence comparison of BmK M4 with an acidic toxin, BmK M8, showed that the weakest toxicity and acidic property of BmK M8 may be the consequence of mutations K8D, D53A, V55E and V59E. The substitution of 21 Gly in BmK M4 for Tyrin other BmK toxins may also be of importance. In their tertiary structures, these mutated charged residues are mainly distributed in the surface (face B) that is roughly opposite to the "conserved hydrophobic surface" (face A) proposed by Fontecilla-Camps et al. in 1982. Therefore the toxin-receptor interaction may take a multiposition mode.

Amino Acid Sequence↗
PubMed1997

Nerve growth factor mRNA stability is controlled by a cis-acting instability determinant in the 3'-untranslated region.

Nerve growth factor (NGF) mRNA is rapidly degraded in many non-neuronal cell types with a half-life of between 30 and 60 min. Similar to other short-lived mRNAs the 3'-untranslated region (3'-UTR) of the NGF mRNA contains a short AU nucleotide-rich sequence. To implicate this region as a cis-acting determinant of NGF mRNA instability, expression vectors containing NGF cDNA with and without the 3'-UTR, and vectors containing only the 3'-UTR were constructed and used in cell transfection experiments. Transfection of HEK293 or NIH3T3 cells with these expression vectors followed by measurement of NGF mRNA half-life indicated that NGF mRNA without the AU-rich 3'-UTR was approximately 3-fold more stable than NGF mRNA containing the 3'-UTR. Similar results were seen in a polysome-based cell-free RNA decay assay using NGF mRNA with and without the 3'-UTR prepared from transfected cells. Addition of a short RNA containing the AU-rich 3'-UTR to the cell-free RNA decay system prolonged the half-life of the full-length NGF mRNA, suggesting competition between these two RNA species for polysome-associated factors which degrade the NGF mRNA. Moreover, transfection of HEK293 or astroglial cells with vectors designed to express only the AU-rich region of the 3'-UTR resulted in enhanced expression of NGF mRNA. The results indicate that the 3'-UTR of the NGF mRNA contains a cis-acting instability determinant which, perhaps by interacting with trans-acting RNA-binding proteins, controls the rate of NGF mRNA turnover.

Animals↗
PubMed1997

Cytokine mRNA expression in tolerant heart allografts after immunosuppression with cyclosporine, sirolimus or brequinar.

We sought to examine the impact of the preferential activation of Th2 cells on the induction and maintenance of a tolerant state in heart allograft rat recipients treated with a short course of cyclosporine (CsA), sirolimus (SRL) or brequinar (BQR). A quantitative polymerase chain reaction (PCR) method was used to measure the levels of cytokine mRNAs, namely interferon (IFN)-gamma and interleukin (IL)-2 in T helper 1 (Th1) cells and IL-4, IL-5 and IL-10 in Th2 cells. Our main findings were that on day 5 postgrafting allografts from untreated recipients had increased levels of IFN-gamma (216 +/- 119 fg), IL-2 (449 +/- 75 fg), IL-4 (6.2 +/- 1.3 fg), IL-5 (34.8 +/- 9.3 fg) and IL-10 (1554 +/- 184 fg) mRNAs compared with normal hearts. CsA reduced the levels of IFN-gamma, IL-2, IL-5 and IL-10, but not IL-4, mRNAs. SRL did not affect the expression of cytokine mRNAs. BQR decreased the levels of IFN-gamma, IL-2 and IL-10, but not IL-5 or IL-4 mRNAs. Compared with grafts from untreated recipients, those from CsA- or BQR-treated tolerant hosts (day 100) displayed undetectable IL-2 mRNA levels, and reduced levels of IFN-gamma, IL-4 and IL-10 mRNAs. In fact, the patterns of cytokine mRNA expression in grafts from CsA- and BQR-treated tolerant hosts were similar to those of normal hearts. Grafts from SRL-treated tolerant hosts merely showed slightly increased Th2 cell activity. In conclusion the selective activation of Th2 cells is not absolutely required for induction or maintenance of tolerance.

Animals↗
PubMed1997

A cyclic N7,C-8 guanine adduct of N-nitrosopyrrolidine (NPYR): formation in nucleic acids and excretion in the urine of NPYR-treated rats.

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen that is present in the diet and tobacco smoke and may form endogenously in humans. Biomarkers to assess NPYR exposure and metabolic activation in humans are needed. The cyclic N7,C-8 guanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purin-4(3H)-one (8), which is formed in tissues of rats treated with NPYR, is one potential candidate for such a biomarker. In this study, we evaluated the formation of this and other NPYR adducts in reactions of alpha-acetoxyNPYR with dGuo, Guo, DNA, and RNA and determined the extent of urinary excretion of adduct 8 in rats treated with NPYR. alpha-AcetoxyNPYR, a stable precursor to the major product of NPYR metabolic activation, was allowed to react with dGuo, Guo, DNA, or RNA at 37 degrees C, pH 7. The most striking observation was that the cyclic N7,C-8 guanine adduct 8 was formed 9 times more extensively in the reaction with Guo than with dGuo. It was also formed 2.5 times more extensively in RNA than in DNA. In rats treated with NPYR, levels of the cyclic N7,C-8 guanine adduct 8 were 2 times as high in RNA than in DNA. Rats treated with [14C]adduct 8 excreted 51% of this adduct unchanged in urine. Rats treated with [3,4-3H]NPYR excreted 0.00004% of the dose as adduct 8. The major differences in product formation in reactions of alpha-acetoxyNPYR with dGuo versus Guo are unusual for alkylating agents; potential mechanisms are discussed. The higher levels of adduct 8 in RNA than in DNA suggest that RNA may be superior as a source of adduct 8 as a biomarker.

Animals↗
PubMed1997

A point mutation in the human estrogen receptor gene is associated with the expression of an abnormal estrogen receptor mRNA containing a 69 novel nucleotide insertion.

A novel ER-like mRNA containing a 69 nucleotide insertion precisely between exon 5 and 6 sequences was previously identified in human breast cancer biopsy samples. Data are presented which suggest that the 69 nucleotide sequence is normally present in intron 5 of the human estrogen receptor gene. The region corresponding to and surrounding this 69 nucleotide sequence was cloned and the nucleotide sequence determined. Cloning and sequencing of the corresponding region in genomic DNA isolated from a breast tumor expressing the 69 nucleotide inserted ER mRNA, revealed an A-->G point mutation immediately 3' to the 69 nucleotide sequence. This point mutation resulted in the generation of a consensus splice donor site. A consensus splice acceptor site sequence is normally present immediately 5' to the 69 nucleotide sequence. These data are consistent with the 69 nucleotide sequence being recognized as an exon by the splicing machinery, and resulting in processing of a mature ER mRNA containing the 69 nucleotide insert.

Base Sequence↗
PubMed1997

In situ hybridization studies of metalloproteinases 2 and 9 and TIMP-1 and TIMP-2 expression in human prostate cancer.

The expression of MMP-2, MMP-9, TIMP-1, TIMP-2, and the urokinase receptor were examined in fetal and normal prostate tissues, benign prostatic hyperplasia and prostate cancer (n = 117). In situ hybridization with digoxigenin-labeled oligonucleotide probes demonstrated that TIMP-1 and TIMP-2 were expressed at elevated levels in the stroma of Gleason sum 5 tissues, whereas MMP-2 and MMP-9 were expressed at relatively low levels. In higher Gleason sum tissues (GS 8-10), TIMP-1 and TIMP-2 were not expressed, whereas MMP-2 and MMP-9 were intensely expressed. Furthermore, TIMP-1 and TIMP-2 expression was high in organ-confined specimens (OC, n = 43), somewhat lower in specimens with capsular penetration (CP, n = 29), and low or negative in samples with surgical margin/seminal vesicle (M/SV, n = 17) and lymph node (LN, n = 13) involvement. In contrast, MMP-2 and MMP-9 expression was low in the OC tissues; and noticeably higher in CP, M/SV, and LN specimens. Finally, correlation of TIMP and MMP expression with GS and pathological stage versus cure rate further revealed that a high percentage of organ-confined, GS 5 specimens expressing TIMP and little MMP were cured. In comparison, few of the GS 7-10 patients with capsular penetration and expressing MMP and little TIMP were cured. The data suggest that TIMP-1 (and TIMP-2) and MMP-2 (and MMP-9) are independent predictors of outcome.

Collagenases↗
PubMed1997

In vitro mechanical and biological assessment of hydroxyapatite-reinforced polyethylene composite.

In vitro performance of hydroxyapatite (HA)-reinforced polyethylene (PE) composite (HAPEX) has been characterized from both mechanical and biological aspects. The mechanical properties of HAPEX, such as tensile strength and Young's modulus, showed little change after immersion in a physiological solution at 37 and 70 degrees C for various periods. In addition, the biological response of primary human osteoblast-like (HOB) cells in vitro on HAPEX was assessed by measuring DNA synthesis and osteoblast phenotype expression. Cell proliferation rate on HAPEX was demonstrated by an increase in DNA content with time. A high tritiated thymidine ([3H]-TdR) incorporation/DNA rate was observed on day 1 for HAPEX, indicating a stimulatory effect on cell proliferation. The alkaline phosphatase (ALP) activity was expressed earlier on HAPEX than on unfilled PE and increased with time, indicating that HOB cells had commenced differentiation. Furthermore, it was found that the HA particles in the composite provided favourable sites for cell attachment. It appears that the presence of HA particles in HAPEX may have the advantage of acting as microanchors for bone bonding in vivo.

Journal Article↗
PubMed1997

Evaluation of in vitro bioactivity and biocompatibility of Bioglass-reinforced polyethylene composite.

The bioactivity and biocompatibility of Bioglass-reinforced high-density polyethylene composite (Bioglass/HDPE) have been evaluated in simulated body fluid (SBF) and by in vitro cell culture, respectively. The formation of a biologically active hydroxy-carbonate apatite (HCA) layer on the composite surface after immersion in SBF was demonstrated by thin-film X-ray diffraction, infrared spectroscopy and scanning electron microscopy, indicating the in vitro bioactivity of Bioglass/HDPE composites. The HCA layer was formed on the 40 vol% composite surface within 3 days immersion in SBF at a formation rate comparable to those on bioactive glass-ceramics, showing that in vitro bioactivity could be obtained in a composite. Furthermore, the composite was biocompatible to primary human osteoblast-like cells. In comparison with unfilled HDPE and tissue culture plastic control, a significant increase in cellular metabolic activity was found on the composite. Therefore, Bioglass/HDPE composites have a promising biological response as a potential implant material.

Journal Article↗
PubMed1997

Ethnic variation in the prevalence of a common NAD(P)H quinone oxidoreductase polymorphism and its implications for anti-cancer chemotherapy.

The NAD(P)H quinone oxidoreductase (NQO1:EC 1.6.99.2) is an important biotransformation enzyme system that is also known to metabolize important novel chemotherapeutic compounds. The gene that codes for this enzyme has recently been found to be polymorphic in humans. Here, we describe the ethnic distribution of the polymorphism and note that this may have implications for anti-tumour drug development and use.

Antineoplastic Agents↗
PubMed1997

Stable in vivo gene transduction via a novel adenoviral/retroviral chimeric vector.

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The available vector systems have not allowed the simultaneous achievement of both goals. We have developed a chimeric viral vector system that incorporates favorable aspects of both adenoviral and retroviral vectors. Adenoviral vectors induce target cells to function as transient retroviral producer cells in vivo. The progeny retroviral vector particles are then able to stably transduce neighboring cells. In this system, the nonintegrative adenoviral vector is rendered functionally integrative via the intermediate generation of a retroviral producer cell. The chimeric vectors may allow realization of the requisite goals for specific gene-therapy applications.

Adenoviridae↗
PubMed1997

Absence of memory for intraoperative information during surgery under adequate general anaesthesia.

Using the isolated forearm technique (IFT), we wished to determine if patients known to be unresponsive to commands during general anaesthesia with nitrous oxide, halothane and neuromuscular blocking agents had any evidence of explicit or implicit recall. Two groups of women, studied in a single-blind sequential block design, heard different tapes, either a command and information tape (n = 34) or radio static (n = 34), throughout surgery. Four women (two radio static, two command) had unequivocal evidence of explicit recall for a period near the beginning or end of the procedure, at a time when the IFT was not being used. With or without hypnosis, category generation, serial position of category exemplars and word association tests did not reveal evidence of priming. We conclude that during light general anaesthesia with nitrous oxide, halothane and atracurium, patients had neither explicit nor implicit memory for information presented during a period when they are known to be unresponsive to commands.

Adolescent↗
PubMed1997

Mevalonate-regulated mechanisms in cell growth control: role of dolichyl phosphate in expression of the insulin-like growth factor-1 receptor (IGF-1R) in comparison to Ras prenylation and expression of c-myc.

One or more mevalonate derivatives of non-sterol type have been proposed to be of indispensable importance for cell growth. Conceivable mevalonate-dependent mechanisms involved in growth control are farnesylation of Ras proteins, regulation of c-myc expression, and N-linked glycosylation of the IGF-1 receptor. The latter mechanism might be rate-limited by dolichyl phosphate, which acts as a donor of oligosaccharides in glycoprotein synthesis in the endoplasmic reticulum. In order to study the significance for cell proliferation of the three aforementioned mevalonate-dependent processings, their inhibitory response due to mevalonate deprivation was explored and compared with the effect on DNA synthesis in the malignant melanoma cell line SK-MEL-2. We found that mevalonate depletion due to treatment with 3 microM lovastatin for 24 h, which efficiently growth-arrested the cells, hardly at all affected the expression of c-myc, and although Ras prenylation was inhibited by 50%, the most pronounced effect of lovastatin was seen on N-linked glycosylation of IGF-1 receptors, which was inhibited by more than 95%. The order and magnitude of the decreased IGF-1 receptor glycosylation, which was followed by a decreased expression of IGF-1 receptors at the cell membrane, correlated well with the inhibition of DNA synthesis. We investigated whether dolichol, and in particular dolichyl phosphate, through its participation in N-linked glycosylation, act as regulators of IGF-1 receptor expression. First, we could confirm that exogenous dolichol became phosphorylated and in this form took part in the glycosylation processing. Secondly, we showed that dolichyl phosphate, in a dose-dependent manner, could increase the number of IGF-1 receptors at the cell membrane, simultaneously as DNA synthesis was stimulated. Taken together, our results provide direct evidence for an important role of dolichyl phosphate as a regulator of cell growth through limiting N-linked glycosylation of the IGF-1 receptor.

Cell Division↗