Biomedical subjects
Publications and source records attributed to K Ma.
The chemical components of essential oil from herba of Achillea wilsoniana is identified by GC-MS techniques, and 35 compounds are identified, mainly containing beta-pinene, limonene, t-beta-farnesene, delta-cadinene, alpha-cadinol, 1,4-dimethy-7-ethylazulene.
The NADP(+)-dependent glutamate dehydrogenase from Thermococcus litoralis has been crystallized by the hanging-drop method of vapour diffusion using an ammonium sulfate and PEG mixture as the precipitant. The crystals belong to the monoclinic system and are in space group C2 with unit-cell dimensions a = 142.7, b = 202.0, c = 125.8 A with beta = 113.1 degrees with a hexamer in the asymmetric unit. T. Litoralis, a hyperthermophilic organism, belongs to the family of Archaea and has a maximum growth temperature of about 370 K. The glutamate dehydrogenase isolated from this organism has a half-life of 2 h at 373 K and a comparison of this structure with that of other GluDH's from hyperthermophilic organisms and from mesophiles will contribute to an understanding of the molecular mechanisms which underlie thermostability.
In order to search for mutations in the multicopy RBM genes that might be associated with male infertility, we have used sequence data from the reported cDNA clone to determine the intron exon boundaries of the YRRM 1 gene. This gene has 12 exons, three of which encode the putative RNA binding domain of the protein. Different copies of the gene contain sequence variations and, additionally, give rise to transcripts with different numbers of copies of the repeated SRGY motif. Since mutations in the RNA binding domain would seem likely to have an effect on the activity of the protein, we have scanned these exons for mutations by SSCP on DNA from normal and infertile men. Sequence differences in the exon encoding the N-terminal part of the RNA binding domain account for at least four different classes of the gene and give rise to different SSCP conformers. Sequence analysis shows that one of these classes is a pseudogene and that the members of another class are nonfunctional. RT-PCR shows that all classes are transcribed and that the A class is most abundant. We have found a point mutation that alters the highly conserved RNP2 motif in one infertile patient. This mutation is also found in his father. We have used PCR followed by SSCP analysis to map RBM on a Y Chromosome (Chr) YAC contig and have demonstrated a distribution that spans a major part of this chromosome's euchromatin.
From ethanol extracts of the roots of Clematis chinensis, a new saponin, named clematichinenoside C, was isolated and its structure was established as 3-O-beta-D-glucopyranosyl-(1-4)-beta-D-ribopyranosyl-(1-3)-alpha-L - rhamnopyranosyl-(1-2)-alpha-L-arabinopyranosyl oleanolic acid 28-O-alpha-L-rhamnopyranosyl-(1-4)-beta-D-glucopyranosyl- (1-6)-beta-D-glucopyranosyl ester by spectroscopic and chemical means. In addition, one known saponin, huzhongoside B, was identified in the plant.
In vivo neutron activation (IVNA) analysis has the capacity to measure several total body elements in human subjects. Although it has been considered a criterion method for the past 3 decades, the reliability of IVNA analysis has been tested only in phantom calibrations. In 5 male weight-stable patients with AIDS, total body N, Ca, Cl, Na, P, and C were measured three times in 16 weeks at Brookhaven National Laboratory. With tracer dilution methods for total body water (TBW) by 3H2O and for extracellular water (ECW) by 35SO4 and NaBr, and dual-energy x-ray absorptiometry (DXA), total body calcium (TBCa) and fat percentage were measured within 2 weeks of IVNA measurements. For comparison, tracer dilution for TBW by D2O and ECW by NaBr, plus DXA measurements, were performed three times in 5 weight-stable healthy volunteers. The reliability of the IVNA technique was very high in patients with AIDS; it ranged from 0.99 for total body chloride (TBCI) to 0.84 for total body phosphorus (TBP), and it agreed with phantom calibration results in the literature. The reliability for measuring fat percentage and TBCa by DXA was similar in patients with AIDS and in healthy volunteers. Tracer dilution for measuring TBW by 3H2O in patients with AIDS and by D2O in healthy volunteers had a reliability score similar to those found with IVNA and DXA. The reliability scores for measuring ECW in patients with AIDS by 35SO4 and NaBr, 0.66 and 0.68, respectively, were the lowest among all measurements, whereas the reliability score for NaBr in healthy volunteers was 0.96, as with the other measurements.
We have isolated a murine homologue of the human Y-linked RBM genes (previously termed YRRM), a gene family implicated in spermatogenesis and which encodes proteins containing an RNA recognition motif. A number of very similar copies of this gene (called Rbm) are present in the mouse. These mouse homologues are also Y-encoded, mapping on the short arm of the chromosome, proximal to Sry. Expression is confined to the testis, specifically the germ line on the basis of lack of expression in the germ-line negative testes of adult sex-reversed mice. The timing of Rbm transcription is regulated, with fetal message levels reaching a peak at 15 d.p.c. Transcripts are clearly detectable by 4 days after birth and reach their highest level at 14 d.p.p. which is the time at which the Y chromosome condenses during meiotic prophase. These results suggest that Rbm is functionally involved in germline RNA metabolism.
Overall, approximately 11% of men attending infertility clinics suffer unexplained oligo- or azoospermia. Cytogenetic observations of loss of the distal portion of the Y chromosome long arm (Yq) were found to be associated with disrupted spermatogenesis. The existence of a gene locus involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was thus postulated. It is suggested that microdeletions, or mutations, at the AZF locus could result in impaired spermatogenesis in chromosomally normal men. In order to test this hypothesis we have carried out Y chromosome genetic screening of 100 oligo- or azoospermic 46XY patients. We have also assessed phenotype/genotype relationships in those patients whose infertility has an underlying genetic aetiology. Patients were screened by polymerase chain reaction (PCR) with a set of Y chromosome-specific sequence tagged sites (STS) for submicroscopic deletions of their Y chromosome. Our results show that as many as 8% of cases of unexplained male infertility may have an underlying genetic aetiology related to microdeletions in two specific regions of the Y chromosome. Positive results from such a screen will be important when deciding the suitability of a patient for assisted conception schemes such as intracytoplasmic sperm injection.
The oxidized and reduced forms of the [4Fe-4S]-containing ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, Pf, have been investigated by 1H nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and thiol titrations. We have identified and isolated at Ambient temperature four distinct redox states for the [4Fe-4S] form of the ferredoxin. These states differ in the redox state of the cluster, which is coordinated by Cys 11, Asp 14, Cys 17, and Cys 56, and of a disulfide bridge between Cys 21 and Cys 48. The protein, as isolated under anaerobic conditions, designated 4Fe FdBred, contains the reduced cluster and two free thiols. The cluster, but not the thiols, is readily oxidized by brief exposure to O2 to yield 4Fe FdBOX. Prolonged O2 treatment (> 24 h at 30 degrees C) is required to generate the protein with a disulfide (4Fe FdAOX) while this fully oxidized form is readily converted by brief reduction with sodium dithionite to the protein with a reduced cluster and a disulfide (4Fe FdAred). Analyses of the magnitude and the number of hyperfine-shifted resonances in each of the four redox states are discussed.
Using chromosomal in situ hybridization it has been demonstrated that specific members of the YRRM and the TSPY families are multicopy and Y chromosome specific in hominoids. After hybridization with the YRRM-related cosmid A5F and the TSPY-related cosmids cos36 and cY91, a reverse and complementary pattern of main and secondary signals is detected on the Y chromosomes of the human, the pygmy chimpanzee and the gorilla, while the location of signals coincides on the Y chromosomes of the chimpanzee, both orang-utan subspecies and the white hand gibbon. This complementary distribution of YRRM and TSPY sequences on the hominoid Y chromosomes possibly originates from a similar sequence motif that is shared by and evolutionarily conserved between certain members of both gene families and/or repeated elements flanking those genes. Otherwise this complementary distribution could go back to a common organization of these genes next to each other on an ancient Y chromosome which was disrupted by chromosomal rearrangements and amplification of one or other of the genes at each of the locations.
From the roots of Clematis chinensis, two new triterpenoid saponins, named clematichinenoside A and B, were isolated and their structures were elucidated as 3-O-beta-D-ribopyranosyl-(1-3)-alpha-L-rhamnopyranosyl-(1-2)-alpha -L- arabinopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester and 3-O-beta-D-glucopyranosyl-(1-4)-beta-D-ribopyranosyl-(1-3)-alpha-L - rhamnopyranosyl-(1-2)-alpha-L-arabinopyranosyl hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-6)- beta-D-glucopyranosyl ester, respectively. In addition oleanolic acid, hederagenin and seven known saponins were obtained.
Two new medicagenic acid saponins, named asterbatanoside J and K, were isolated from the roots of Aster batangensis. On the basis of chemical and spectral studies especially 2D NMR including COSY, HETCOR, HMQC, HOHAHA, TOCSY, ROESY and HMBC techniques, their structures were established as 3-O-beta-D-glucopyranosyl-(1--> 6)-beta-D-glucopyranosyl-2 beta, 3 beta-dihydroxy-olean-12-en-23 alpha, 28-dioic acid- 28-O-alpha-L-arabinopyranosyl-(1-->3)-alpha-L- rhamnopyranosyl-(1-->2)-beta-D-fucopyranoside and 3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl- medicagenic acid-28-O-beta-D-xylopyranosyl-(-->4)-[alpha-L- arabinopyranosyl-(1 -->3)-alpha-L-rhamnopyranosyl-(1-->2) -beta-D-fucopyranside, respectively.
Two new triterpenoid saponins named asterbatanoside D and E have been isolated from Aster batangensis and their structures elucidated as 3-O-beta- D-glucopyranosyl-bayogenin-28-O-beta-D-glucopyranosyl-(1-->6)-beta -D- glucopyranoside and 3-O-6'-acetyl-beta-D-glucopyranosyl-bayogenin-28-O-beta-D- glucopyranosyl-(1-->6)-beta-D-glucopyranoside by means of MS, 1D and 2D NMR techniques (COSY, TOCSY, ROESY, HMQC, and HMBC), and chemical reactions.
The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91 degrees C by the fermentation of peptides and reduces elemental sulfur (S(o)) to H2S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S(o) in the medium, and no growth was observed in the absence of S(o). The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S(o) concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S(o) limitation. This was accompanied by increased amounts of H2 and alcohol (ethanol and butanol) from cultures grown with limiting S(o). Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M(r) of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under So limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.
Thermococcus strain ES-1 is a strictly anaerobic, hyperthermophilic archaeon that grows at temperatures up to 91 degrees C by the fermentation of peptides. It is obligately dependent upon elemental sulfur (S(o)) for growth, which it reduces to H2S. Cell extracts contain high aldehyde oxidation activity with viologen dyes as electron acceptors. The enzyme responsible, which we term aldehyde ferredoxin oxidoreductase (AOR), has been purified to electrophoretic homogeneity. AOR is a homodimeric protein with a subunit M(r) of approximately 67,000. It contains molybdopterin and one W, four to five Fe, one Mg, and two P atoms per subunit. Electron paramagnetic resonance analyses of the reduced enzyme indicated the presence of a single [4Fe-4S]+ cluster with an S = 3/2 ground state. While AOR oxidized a wide range of aliphatic and aromatic aldehydes, those with the highest apparent kcat/Km values (> 10 microM-1S-1) were acetaldehyde, isovalerylaldehyde, and phenylacetaldehyde (Km values of < 100 microM). The apparent Km value for Thermococcus strain ES-1 ferredoxin was 10 microM (with crotonaldehyde as the substrate). Thermococcus strain ES-1 AOR also catalyzed the reduction of acetate (apparent Km of 1.8 mM) below pH 6.0 (with reduced methyl viologen as the electron donor) but at much less than 1% of the rate of the oxidative reaction (with benzyl viologen as the electron acceptor at pH 6.0 to 10.0). The properties of Thermococcus strain ES-1 AOR are very similar to those of AOR previously purified from the saccharolytic hyperthermophile Pyrococcus furiosus, in which AOR was proposed to oxidize glyceraldehyde as part of a novel glycolytic pathway (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208-14216, 1991). However, Thermococcus strain ES-1 is not known to metabolize carbohydrates, and glyceraldehyde was a very poor substrate (kcat/Km of < 0.2 microM-1S-1) for its AOR. The most efficient substrates for Thermococcus strain ES-1 AOR were the aldehyde derivatives of transaminated amino acids. This suggests that the enzyme functions to oxidize aldehydes generated during amino acid catabolism, although the possibility that AOR generates aldehydes from organic acids produced by fermentation cannot be ruled out.
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Two continuous cell lines derived from the neoplastic urothelium had been maintained in culture for more than two years. The first cell line derived from the urothelium of a fusion papillocarcinoma on the left lateral wall of the bladder was designated as TBC-1 and grown in vitro for more than 150 generations. The second cell line derived from the urothelium of a papillocarcinoma in the left renal pelvis was designated as TPC-1 and grown in vitro for more than 100 generations. Characterization studies made on both cell lines showed that the cells had a rapid doubling time, exhibited multilayering and produced tumors in sc of BALB/c. Tumor nodules that produced sc of BALB/c kept similar cellular and pathological features to those of the primary biopsy specimens under light and electron microscopes. TPC-1 cell line exhibited a three-dimensional structure of transitional epithelium on the nylon-mesh disk which was coated with a layer of rat tail collagen. Both TBC-1 and TPC-1 cell lines formed colonies in soft agar. Their forming rates were 35% and 28%, respectively. The chromosome number of TBC-1 cells ranged from 17 to 84, with a modal number of 54; whereas that of TPC-1 cells ranged from 28 to 139, with a modal number of 49. The TBC-1 cells showed mutant p53 and ras p21 protein expression and expressed weakly ABH blood group isoantigens. Analysis of lactic dehydrogenase (LDH) isozymes showed the highest levels of LDH isozyme 4 sonicated cell lysates of TBC-1 and TPC-1 respectively.
Burn of knee joint often happens on the extended side surface and hurts the articular capsule and cavity. Five patients with severe burn of knee joint were successfully treated with fasciocutaneous flap of the lower leg. After the operation, all the flaps survived. Three patients' articular cavity got initial sealing-up. Except two patients' joints merging, joint movement was normal or near normal, and with satisfactory appearance. The timing, key points, and advantages of this operation were discussed. The fasciocutaneous flap of the lower leg is a good selection for severe burn of knee joint.
It has been discovered by tissue cultivation and isotope tracing that the injection of Rhizoma Drynariae significantly promotes the calcification of chick embryo bone primordium in vitro, increases the ALP activity of cultivated tissues and accelerates the synthesis of proteoglycan. It has also been proved that the promotion of the synthesis of proteoglycan is an important factor of the promotion of calcification.