Biomedical subjects

C Shih

Publications and source records attributed to C Shih.

At least 91 records · Page 5Linked to original sources

PubMed1993

Specific and sensitive high-performance liquid chromatographic method with fluorescence detection for measurement of lometrexol and its polyglutamates in biologic samples.

A reversed-phase high-performance liquid chromatographic (HPLC) assay is described for the quantitative determination of lometrexol in biological samples; the assay is rapid, simple, specific, and highly sensitive. The method requires the dissociation of lometrexol from folate-binding proteins present in blood and formation of a fluorescent oxidized derivative of the compound. The dissociation of lometrexol from folate-binding proteins was achieved by acidification to pH 3.5 using ammonium formate, followed by serum protein precipitation with perchloric acid. The protein-free lometrexol was subsequently oxidized by MnO2 at 90 degrees C for 10 min. Chromatographic separation of lometrexol without interference was achieved on a C18 reversed-phase column with a convex gradient, using acetonitrile-0.1% ammonium formate, pH 7.0, as the mobile phase. In human serum and urine the calibration curve was linear between 5 and 300 nM. The lower limit of quantification was 5 nM. The method has been applied successfully to measure serum and urinary levels of lometrexol in patients.

Chromatography, High Pressure Liquid↗

PubMed1993

Infrequent mutation of p53 gene in hepatitis B virus positive primary hepatocellular carcinomas.

Somatic mutation of the p53 oncogene/anti-oncogene allele has been shown to be involved in many different human solid tumors. Recently, there have been reports that p53 mutations are found to occur at high frequency (50%) in aflatoxin-related human primary hepatocellular carcinomas (HCC) (Hsu et al., 1991 Nature, vol 350, p. 427; Bressac et al., 1991 Nature, vol 350, p. 429). Most strikingly, a hotspot G to T mutation at amino acid position 249 was identified. These reports appear to contradict our earlier publications that although p53 mutation is found frequently in human HCC cell lines, it is rarely found in primary tumors. In this paper, we have further examined 20 different primary HCC samples (17 were hepatitis B surface antigen positive) and their adjacent nontumourous tissues, using restriction fragment length polymorphism (RFLP) analyses. Clear loss of heterozygosity (LOH) was found in only 3 out of 20 samples. All three samples were also found to carry a point mutation within the remaining p53 allele. None of these mutations was found to be at the proposed aflatoxin hotspot of amino acid 249. All three point mutations are of somatic origin. Ten samples, randomly chosen from the remaining 17 LOH negative HCC tumors, were analyzed further by DNA sequencing and Western blot analyses. No point mutations of p53 were found. Taken together with our previous report (Hosono et al., 1991, Oncogene vol 6, p. 237-243), we conclude that p53 mutation occurs infrequently, only approximately 18%, in HBV-positive primary hepatomas from Taiwan. Furthermore, p53 mutation appears to be acquired later in tumor development at least in some HCC samples.

Adult↗

PubMed1993

Articular manifestations of renal osteodystrophy.

The relationship between hyperparathyroid bone disorders and chronic renal diseases has been studied extensively for a long period of time. Zvaifler and Bywaters are the first to emphasize articular manifestations in hyperparathyroidism. Sundaram has found 30% in 80 hemodialysis patients presenting with articular disorders in hands. We reviewed routine radiographs of 258 uremia patients retrospectively and found that 36 patients had typical X-ray presentations of renal osteodystrophy. Sixteen among them (44.44%) were noted with articular involvement. Fourteen patients involved axial joints including sacroiliac, acromio-clavicular, sternoclavicular and symphysis pubis in decreasing order. Appendicular joints involvement were found in 6 patients, patellofemoral joints and small joints of hands involvement in 4 patients respectively and hip and shoulder joints in each one patient. None of the 16 patients complained joint pain. Diagnosis is easy because of the uncommon presentations of joint disorders in uremic patients. However, more knowledge about articular manifestations of renal osteodystrophy may eliminate the unnecessary investigations.

Adolescent↗

PubMed1992

A dideazatetrahydrofolate analogue lacking a chiral center at C-6, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid, is an inhibitor of thymidylate synthase.

N-[4-[2-(2-Amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl]benzoyl]-L-glutamic acid (15), prepared in five steps from 2-pivaloyl-7-deazaguanine, has been found to be an antitumor agent with its primary site of action at thymidylate synthase rather than purine synthesis. This compound appears to be a promising candidate for clinical evaluation.

Animals↗

PubMed1992

Synthesis and biological activity of acyclic analogues of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid.

The synthesis and biological evaluation of a number of analogues of N-[4-[4-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidyl) butyl]benzoyl]-L-glutamic acid (2) (7-DM-DDATHF), an acyclic modification of the novel folate antimetabolite 5,10-dideazatetrahydrofolic acid (DDATHF), are described. The synthetic procedure utilized previously for the synthesis of 2, 15, and 16 was extended to the preparation of analogues modified in the benzoyl region with thiophene and methylene groups replacing the benzene ring (compounds 27a-c) and in the glutamate region with aspartic acid and phenylalanine replacing L-glutamic acid (compounds 36, 37). The 2-amino-4,6-dioxo derivative 33 was obtained from intermediate 30 via a palladium-catalyzed carbon-carbon coupling reaction with diethyl (4-iodobenzoyl)-L-glutamate, followed by reduction and removal of protecting groups with base. Cell culture cytotoxicity studies of all of the above acyclic analogues of DDATHF against CCRF-CEM human lymphoblastic leukemic cells gave IC50s ranging from 0.042 greater than 48 microM. Inhibition and cell culture reversal studies against isolated enzymes suggest the mode of action of these compounds. Compound 2 was only 3-fold less inhibitory toward glycinamide ribonucleotide formyltransferase (GARFT, isolated from L1210 leukemic cells) than DDATHF itself. These acyclic analogues were less efficient substrates for the enzyme folylpolyglutamate synthetase (FPGS) compared with their bicyclic counterparts. Moderate antitumor activity was observed for compound 2 against 6C3HED lymphosarcoma and C3H mammary adenocarcinoma in vivo.

Adenocarcinoma↗

PubMed1992

Morphometric variations of the lumbar vertebrae between Chinese and Indian adults.

A morphometric study of the lumbar vertebrae of 126 adult skeletons, 90 Chinese and 36 Indian, of both sexes without marginal osteophytes were performed. In each lumbar vertebra, the cephalad and caudad midsagittal diameters, the interpedicular diameter of the spinal canal as well as the midsagittal and transverse diameters and the height of the vertebral body were measured. The results showed that the midsagittal and transverse diameters, the heights of the lumbar vertebral bodies and the interpedicular diameters of the lumbar spinal canals increased progressively from L1 to L5, while the midsagittal diameters of the lumbar spinal canals decreased progressively from L1 to L5 in both Chinese and Indian adult skeletons. The lowest mean values of the cephalad and caudad midsagittal and the interpedicular diameters of the spinal canals in Chinese were found to be 5.04 +/- 0.15 mm at L5, 4.67 +/- 0.09 mm at L5 and 25.92 +/- 0.20 mm at L2, respectively, while in Indians they were found to be 4.54 +/- 0.18 mm at L5, 4.25 +/- 0.10 mm at L5 and 25.42 +/- 0.22 mm at L1, respectively. In addition, the mean diameters of the spinal canal and the vertebral body (except the height of the vertebral body) were significantly greater in the Chinese than in the Indian skeletons. The above findings indicate that the mean diameters of both the lumbar spinal canal and the vertebral body vary greatly between Chinese and Indian adults, i.e. there are no mean values of the vertebral dimensions that are valid for all populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

Structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate that determine inhibition of mammalian glycinamide ribonucleotide formyltransferase.

We have investigated the structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) that determine the activity of this compound as an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) purified from mouse L1210 cells. 5-Deazatetrahydrofolate was as good an inhibitor of GARFT as DDATHF, indicating that isosteric replacement of nitrogen by carbon at the 5-position of tetrahydrofolate is sufficient for inhibition of GARFT. 5,10-Dideazafolic acid, 5,8,10-trideazatetrahydrofolate, and 2-desamino-5,10-dideazatetrahydrofolate were poor inhibitors of GARFT, indicating that a reduced pyridopyrimidine ring, N-8, and the 2-amino group of DDATHF, respectively, play an important role in the binding of tetrahydrofolate analogues to this enzyme. DDATHF analogues in which the phenyl ring was replaced either by a cyclohexyl ring or by methylene groups retained activity as inhibitors. 5,10-Dideazatetrahydrohomofolate was about 6 times more potent as an inhibitor of GARFT than DDATHF, but 5,10-dideazatetrahydronorfolate had about one-fifth of the activity of DDATHF. An analogue of DDATHF in which the glutamic acid side chain was replaced by aspartic acid (which was not a substrate for polyglutamation and was only weakly cytotoxic) was equiactive with DDATHF as an inhibitor of purified GARFT. Surprisingly, 5,10-dideazatetrahydropteroic acid was about as active as DDATHF as an inhibitor of GARFT, an indication that the glutamic acid in the side chain of DDATHF does not play a role in this ligand-enzyme interaction. The polyglutamate derivatives of DDATHF bound up to 100 times tighter to GARFT than DDATHF itself; longer chain polyglutamates conformed to Goldstein's zone B behavior under experimental conditions and were projected to be in zone C, i.e., stoichiometric inhibition, in vivo. We conclude that the presence of carbon at the 5-position of tetrahydrofolate analogues is sufficient for inhibition of GARFT, that N-8 and the 2-amino group are involved in binding of DDATHF to GARFT, probably through hydrogen bonds, and that the structures of the phenyl ring and amino acid side chain of DDATHF analogues are not primary determinants of GARFT inhibition by monoglutamate forms of these compounds. We also conclude that polyglutamation plays a major role in the potent cytotoxicity of DDATHF.

Acyltransferases↗

PubMed1991

Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus.

We characterized 11 DNA polymerase mutants of human hepatitis B virus (HBV) which contain single missense or nonsense mutations in the various domains within this gene. Except for mutant 738, a tight association between DNA replication and RNA packaging of these missense pol mutants was observed. Further analysis of HBV core particle-associated RNA indicated that only the 3.5-kb core-specific RNA, but not the precore-specific RNA, is selectively packaged in this tissue culture system. Previously, we have demonstrated that only the 3.5-kb core-specific RNA can serve as an efficient template for pol translation. Taken together, our results suggest that selectivity of HBV RNA packaging occurs as a result of selective translation of pol-containing mRNAs. Furthermore, our data suggest that the RNA encapsidation domain of pol overlaps with all of the domains of pol involved in the synthesis of terminal protein, as well as DNA replication. Finally, on the basis of gradient centrifugation analysis, a pol defect appeared to have no negative effect on the assembly or stability of core particles. A new method to assay RNA encapsidation, as well as potential RNase H activity, is reported.

Base Sequence↗

PubMed1991

Surgical anatomy of the mandibular ramus of the facial nerve in Chinese adults.

One hundred and twenty Chinese adults' facial halves were selected for this study. The description of the mandibular ramus of the facial nerve was presented under 4 items: (1) the relationship with the lower border of the mandible, (2) the number of its rami, (3) the relationship with the retromandibular vein, the facial vein or the facial artery, and (4) its anastomoses with the buccal ramus and/or the cervical ramus. In these specimens, anterior to the facial artery, 90% of the mandibular rami ran above the lower border of the mandible, and 10% of the mandibular rami ran below the lower border of the mandible. Posterior to the facial artery, 67% of the mandibular rami ran above the lower border of the mandible, and 33% of the lowest mandibular rami passed in an arc with an average of 0.95 cm (the lowest point being 3 cm or less) below the lower border of the mandible. In 32% of the specimens, the mandibular ramus had no branch and in 68% of the specimens, the mandibular ramus had two or more rami. There were 100% of the mandibular rami lying superficially to the retromandibular vein and the facial vein, while in 5% of the specimens, some other mandibular rami ran deeply to the facial vein. In 83% of the specimens, the mandibular rami lay only superficially to the facial artery and in only 2% deeply to the facial artery. The remaining 15% ran both deeply and superficially to the facial artery. There were 60% of the specimens in which the mandibular rami had one or more anastomoses with the buccal rami.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1990

Hepatitis B virus propagated in a rat hepatoma cell line is infectious in a primate model.

The human hepatitis B virus (HBV) produced by a rat hepatoma cell line through transfection with HBV DNA is infectious in the human primate model--chimpanzee. Since hepadnaviruses are known to have an extremely narrow host range, our results support the idea that the host species barrier of HBV infection resides on the penetration/adsorption step rather than any postpenetration intracellular event during the virus life cycle.

Animals↗

PubMed1990

cis rescue of a mutated reverse transcriptase gene of human hepatitis B virus by creation of an internal ATG.

Using mutational analysis, we have investigated the translation strategy of the reverse transcriptase gene (pol) of human hepatitis B virus. It has been proposed that this pol gene product is synthesized as a core-pol fusion protein from a polycistronic mRNA template via ribosomal frameshifting, a mechanism often seen in retroelements. Our data indicate that creation of a novel ATG initiation codon near the original ATG can compensate for a lethal missense mutation at the first ATG position of the pol open reading frame. Genetic analysis has rigorously ruled out the possibilities of frameshifting, non-ATG initiation, or RNA editing. These results are discussed in the context of a 5'-end entry model versus a novel model of direct internal entry of ribosomes.

Base Sequence↗

PubMed1990

Preferred translation of human hepatitis B virus polymerase from core protein- but not from precore protein-specific transcript.

In the human hepatitis B virus (HBV) genome, the 5' end of the polymerase coding sequence overlaps with the 3' end of the core protein coding sequence. Recent results obtained from genetic studies have suggested that translation of HBV polymerase initiates from the first ATG codon of the polymerase reading frame and is not a result of frameshift translation from the core protein reading frame, as in the case of retroviruses. By using in vitro-synthesized SP6 RNA transcripts, we now demonstrate that HBV core protein-specific mRNA can direct the synthesis of polymerase from the internal polymerase ATG codon in rabbit reticulocyte lysates and Xenopus oocytes. A related message with an additional 60 nucleotides at the 5' end (pre-core protein mRNA) was not as efficient as the core protein mRNA for translation of polymerase. Furthermore, translation of polymerase from the core protein mRNA was not inhibited by the cap analog m7GpppG. This result, together with the results described above, indicates that translation of HBV polymerase occurs in a novel, cap-independent manner.

Animals↗

PubMed1990

Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium.

The purpose of this investigation was to assess the applicability of two well established procedures: (i) the product isolation assay and (ii) the radiometric 3H2O assay for the determination of very low levels of aromatase activity. The methods were validated and used to assess the capacity of normal and neoplastic human endometrium to synthesize oestrogens from androgens. Using the product isolation assay, various specimens (n = 27) of normal and neoplastic endometrium were incubated with [1,2,6,7-3H]testosterone either by a standard incubation procedure or by a superfusion technique. Following the incubation, carrier oestrone and oestradiol or [14C]oestrone and [14C]oestradiol were added, and the oestrogens were isolated and purified by paper chromatography and high-performance liquid chromatography. The radiochemical purity of oestrone and oestradiol was checked by the isotope dilution technique. In all samples, the 3H associated with oestrone and oestradiol failed to recrystallize as oestrone and oestradiol. No radioactivity was detectable in the oestrone and oestradiol crystals after acetylation. Similarly, 16 endometrial samples were tested for aromatase activity by the 3H2O release assay using [1 beta-3H]androstenedione as substrate. The results indicate that 3H2O was indeed released during these incubations, but this activity could not be inhibited by the aromatase inhibitor 4-hydroxyandrostenedione, by excess substrate or by heat inactivation of the tissue. Furthermore, the release of 3H2O from [1 beta-3H]androstenedione under the incubation conditions used (Dulbecco's modified Eagle's medium or RPMI-1640 containing fetal bovine serum and NADPH) also occurred in the absence of any tissue. This activity was not inhibited by 4-hydroxyandrostenedione nor by excess substrate. The results demonstrate that the human endometrium does not contain detectable levels of aromatase activity and that the radiometric assay can give rise to false-positive results if used for detection of very low levels of aromatase activity.

Androstenedione↗

PubMed1989

The 6S- and 6R-diastereomers of 5, 10-dideaza-5, 6, 7, 8-tetrahydrofolate are equiactive inhibitors of de novo purine synthesis.

The diasteromers of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) differing in chirality about carbon 6 were resolved and studied as inhibitors of folate-dependent processes in mouse leukemia cells. Both diastereomers of DDATHF were found to be potent inhibitors of leukemia cell growth due to effects on de novo purine synthesis. Cell growth inhibition by these compounds was prevented by 5-formyltetrahydrofolate in a dose-dependent manner. This indicated that the effects of the DDATHF diastereomers were due to inhibition of folate-dependent processes. Metabolite reversal experiments indicated that 5'-phosphoribosylglycinamide formyltransferase was the major site of action of these compounds in mouse cells. Another site in de novo purine synthesis was affected at higher concentrations of diastereomer B in L1210 cells. Low concentrations of both diastereomers were found to inhibit pure L1210 5'-phosphoribosylglycinamide formyltransferase competitively with the folate substrate. The two diastereomers were also efficient substrates for mouse liver folylpolyglutamate synthetase. We conclude that the 6R- and 6S-diastereomers of DDATHF are remarkably similar and equiactive antimetabolites inhibitory to de novo purine synthesis and that the biochemical processes involved in their cytotoxicity display little stereochemical specificity.

Acyltransferases↗

PubMed1989

Synthesis and antitumor activity of 5-deaza-5,6,7,8-tetrahydrofolic acid and its N10-substituted analogues.

Syntheses of 5-deaza-5,6,7,8-tetrahydrofolic acid (7a) and its 10-formyl (7b), 10-acetyl (7c), and 10-methyl (7d) derivatives are described. These compounds, prepared as analogues of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), the lead compound of a new class of folate antimetabolites, exhibit potent growth inhibition against leukemic cells in culture as well as substantial antitumor activity against transplantable murine solid tumors in vivo.

Animals↗

PubMed1988

Assessment of the relative locations of the greater palatine foramen in adult Chinese skulls.

One hundred dry skulls of adult Chinese of both sexes were studied. They were homogeneous in the form of maxillary arch and having full eruption of the upper third molar, without missing teeth and malposition of teeth. Our findings revealed that the mean distance from the center of the greater palatine foramen (GPF) to the midsagittal plane of the hard palate was 16.00 mm, and to the posterior border of the hard palate, 4.11 mm. The location of the GPF related to the maxillary molars was expressed as percentage in 5 relations. We found that the most common location of the GPF was between the maxillary second and third molars (relation III: 48%), and less common was lingual to the maxillary third molar (relation IV: 33.5%). The usually accepted description of the GPF location was lingual to the second molar (relation II), but in our study this relative position occurred in only 17% of the skulls. The long axis of the greater palatine canal directing to the GPF in the oral cavity was found to be directed anteriorly in 181 openings (90.5%) of the 200 GPF, and only 19 openings (9.5%) directed vertically. The bilateral symmetry of GPF on both sides of each skull was remarkable. The discrepancy of our observations on the Chinese skulls from those on other ethnic groups was discussed. Our findings suggest, therefore, the existence of an ethnic variation and the necessity of a more accurate method of locating the GPF in clinical practice.

Anatomy, Regional↗

PubMed1988

Effects of prostaglandin E2 on alveolar bone resorption during orthodontic tooth movement.

Twenty Sprague-Dawley rats weighing 280-300 g were divided into two groups of ten animals each. They were treated by daily submucosal injections of 50 micrograms prostaglandin E2 (PGE2) per kilogram body weight into the region below the apex of the left first maxillary molar (experimental), or vehicle into the region below the apex of the right first molar (control), for a period of 5 days. The animals of the first group were sacrificed immediately following the treatment period, while those of the second group were sacrificed 5 days after the treatment period. Twenty-two hours prior to sacrifice, a piece of latex orthodontic elastic was secured to the adjacent area between the first and second maxillary molars of both sides of each rat by using two mosquito hemostats. The periodontal ligament (PDL) mesial to the mesiobuccal root of the first maxillary molar was assayed for changes in PDL cell factors. The results showed that immediately following the 5-day treatment period the left PDL had a significant decrease in the total number of fibroblasts and a significant increase in the total number of both osteoclasts and nuclei per osteoclast, while no significant changes in the osteoblasts when compared with those of the right control PDL. The left PDL of animals which were sacrificed 5 days after the treatment period revealed a significant decrease in the number of total fibroblasts and only a slight decrease in both numbers of total osteoclasts and total nuclei per osteoclast, but again no significant changes in osteoblasts when compared with those of the right control PDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Process↗

PubMed1987

Tight clustering of human hepatitis B virus integration sites in hepatomas near a triple-stranded region.

The open circular genome of human hepatitis B virus (HBV) is known to contain a partially double-stranded DNA with a single-stranded gap region of variable length. This circular structure of the genome is maintained by base-pairing of the 5' ends of the two DNA stands, the long or L(-) strand and the short or S(+) strand. By cloning, mapping, and sequencing studies, we have localized three recombinational junctions of the integrated HBV in two hepatoma samples, HT14 and FOCUS. Breakpoints of recombination derived from these results and those of others appear to be clustered and coincidental with the identified 5' or the deduced 3' end of the long-strand DNA, respectively. Statistical analysis of these results supports the hypothesis that integration preferentially occurs in an extremely narrow region on the HBV genome. This site-specific recombinational mechanism appears to be conserved among different HBV subtypes. No extensive sequence homology was found between each pair of the recombining parental molecules; however, at the site of crossover, 2- to 3-base-pair junctional homology was consistently observed. Examination of the patterns of the integrated HBV DNAs allowed us to categorize these various patterns into four different groups according to their end specificity and strand polarity. The molecular form of relaxed circle is proposed to be one major substrate for HBV integration. The effect of free strand in the integration of HBV is emphasized in this model. Unlike any other known DNA animal viruses, the site specificity of HBV integration appears to be similar to that of the retroviruses.

Adult↗