Biomedical subjects

K He

Publications and source records attributed to K He.

At least 55 records · Page 3Linked to original sources

PubMed1998

[Studies on complex chromosomal translocation and their relevance to clinical prognosis in acute promyelocytic leukemia].

OBJECTIVE: To study the relationship between the complex chromosome translocation in acute promyelocytic leukemia(APL, M3) and clinical therapy and prognosis. METHODS: Chromosome translocation and PML-RAR fusion transcript in three APL patients were studied by using karyotypic analysis, fluorescence in situ hybridization and reverse transcriptase/polymerase chain reaction. RESULTS: The findings revealed that all these cases had PML/RAR A gene rearrangement. Apart from the chromosomes 15 and 17 involved in the translocation, other multiple chromosomes including 5, 11, 16, 22 were also implicated in complex translocations, which to some extent seemed to be related with clinical prognosis. CONCLUSION: This study provides additional information for monitoring clinical therapy and prognostic evaluation.

Adult↗

PubMed1997

Cytochrome P450 2C11: Escherichia coli expression, purification, functional characterization, and mechanism-based inactivation of the enzyme.

The male-specific P450 enzyme CYP 2C11, whose expression is developmentally and hormonally regulated, is the major steroid 16alpha-hydroxylase of the untreated rat liver. The enzyme metabolizes a host of substrates, including mechanism-based inactivators, such as 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) and spironolactone (SPL). Structural and functional characterization of the specific mode of such inactivation, however, requires sufficient quantities of the fully purified enzyme. Although several laboratories including our own have isolated and purified the enzyme from male rats, the yields are typically low and of the order of 1%. For these reasons, we chose to heterologously express the enzyme in Escherichia coli. The full-length cDNA was excised from the yeast vector pD2M1 and cloned into the plasmid vector pCW after appropriate modifications for optimal expression in E. coli. The enzyme was isolated and purified from E. coli membranes in relatively high yields (approximately 60%) and relatively high specific content (19 nmol/mg protein). The purified recombinant enzyme had spectral and functional characteristics comparable to those reported for the native rat liver enzyme, including mechanism-based inactivation by DDEP and SPL. Studies with 14C-heme-labeled enzyme indicated that the major mode of DDEP inactivation was via heme-N-ethylation. On the other hand, studies with radiolabeled SPL-SH (the proximal inactivating deacetylated metabolite of SPL) revealed that although both [22-14C]SPL-SH and SPL-35SH inactivated the enzyme, only SPL-35SH was found to irreversibly radiolabel the 2C11 protein. The latter findings thus suggest that during mechanism-based inactivation of 2C11, the thiol moiety of SPL-SH is oxidatively activated to a species that attacks the 2C11 protein during or after cleavage from the thiosteroid. Thus, these modes of mechanism-based 2C11 inactivation by DDEP and SPL-SH considerably differ from the corresponding modes of P450 3A inactivation by these agents, wherein heme modification of the protein predominates.

Amino Acid Sequence↗

PubMed1997

Ras activation in platelets after stimulation of the thrombin receptor, thromboxane A2 receptor or protein kinase C.

Several reports have indicated that the small G-protein Ras is not present immunologically in platelets. However, here we report the identification of Ras in platelets by immunoprecipitation with the Ras-specific monoclonal antibodies Y13-259 or Y13-238, followed by Western blotting. The presence of Ras was not due to contamination of samples with erythrocytes or leucocytes. Immunofluorescence studies indicated that Ras was present in a peripheral rim pattern in fixed, permeabilized platelets, suggesting an intracellular, plasma membrane location. Activation of platelets with the thrombin receptor peptide42-50, the prostaglandin H2/thromboxane A2 mimetic U46619 or phorbol 12-myristate 13-acetate induced a rapid increase in GTP-bound, activated Ras. In each case, this increase was inhibited by the protein kinase C (PKC) inhibitor bisindolylmaleimide GF 109203X, suggesting that Ras is activated downstream of PKC in platelets. Thus the activation of Ras in platelets by agonists will now allow consideration of multiple potential Ras-dependent signal transduction pathways in platelet activation processes.

Blood Platelets↗

PubMed1997

Effect of changing the size of lipid headgroup on peptide insertion into membranes.

Adsorption of amphiphilic peptides to the headgroup region of a lipid bilayer is a common mode of protein-membrane interactions. Previous studies have shown that adsorption causes membrane thinning. The degree of the thinning depends on the degree of the lateral expansion caused by the peptide adsorption. If this simple molecular mechanism is correct, the degree of lateral expansion and consequently the membrane thinning should depend on the size of the headgroup relative to the cross section of the hydrocarbon chains. Previously we have established the connection between the alamethicin insertion transition and the membrane thinning effect. In this paper we use oriented circular dichroism to study the effect of varying the size of the headgroup, while maintaining a constant cross section of the lipid chains, on the insertion transition. A simple quantitative prediction agrees very well with the experiment.

Adsorption↗

PubMed1997

Additional bioactive annonaceous acetogenins from Asimina triloba (Annonaceae).

Trilobalicin (1), a new nonadjacent bis-THF ring annonaceous acetogenin, 2,4-cis- (2) and 2,4-trans-trilobacinone (3), the ketolactones of trilobacin, an adjacent bis-THF ring acetogenin, were isolated from the stem bark of Asimina triloba (L.) Dunal (Annonaceae). Their structures were established based on chemical and spectral evidence. The relative stereochemistry of 1 was determined as trans/threo/threo/trans/erythro from C-10 to C-22 by comparisons of NMR data with those of model compounds. Compound 1 is the first example of a nonadjacent bis-THF acetogenin being isolated from the title species and represents a new type of these compounds. Bioactivities of these new structures against brine shrimp larvae and six human solid tumor cell lines were determined, and cytotoxic selectivities were shown for the lung (A-549) and breast (MCF-7) cell lines with up to a million times the potency of adriamycin.

4-Butyrolactone↗

PubMed1997

Bioactive compounds from Taiwania cryptomerioides.

Two new sesquiterpenes, 10 alpha-hydroxyamorphan-4-en-3-one (1) and 4 alpha-methylcadinane-4 alpha-methyl-1 alpha,2 alpha,10 alpha-triol (2), together with four known compounds, sesquiterpenes 10 alpha-hydroxycadinan-4-en-3-one (3) and alpha-cadinol (4), diterpene ferruginol and lignan helioxanthin, were isolated from the whole plant of Taiwania cryptomerioides under bioassay-guided fractionations. The structures of 1 and 2 were elucidated mainly by the NMR spectroscopic analyses. Bioactivities of the isolated compounds against brine shrimp, yellow fever mosquito larvae, and human tumor cells are reported; compound 4 was the most bioactive, showing selectivity for the human colon tumor cell line (HT-29).

Aedes↗

PubMed1997

Konishiol, a new sesquiterpene, and bioactive components from Cunninghamia konishii.

Three sesquiterpenes, konishiol (1), cadalenol (2), 3-cedranol (3), one diterpene, manool (4), and one lignan, (+)-tsugacetal (5), have been isolated, for the first time, from the whole plant of Cunninghamia konishii by using bioactivity-directed fractionation. Compound 1 is new to the literature, and its chemical structure was determined by various spectroscopic analyses including EIMS, HREIMS, NOE, NOESY, and by preparing its di-acetyl derivative (1a). Compounds 2-5 showed moderate to weak bioactivities in brine shrimp (BST) and mosquito larvae (YFM) bioassays as well as cytotoxicities against three human solid tumor cell lines.

Antineoplastic Agents, Phytogenic↗

PubMed1997

Characterization of the erythrocyte superoxide dismutase allozymes in the deer Cervus elaphus.

The cDNA sequences for Cu,Zn superoxide dismutase from two Cervus elaphus subspecies, North American wapiti and European red deer, were determined. The derived amino acid sequences showed two differences: residue 8 was Leu in wapiti and Met in red deer and residue 25 was His in wapiti and Asn in red deer. The extra positive charge at position 25 in the wapiti isoform accounted for its greater mobility towards the cathode during non-denaturing electrophoresis, a procedure widely used in the genetic analysis of deer. There was no difference in specific activity between the two Cu,Zn superoxide dismutase isoforms, but the wapiti isoform was slightly more susceptible to heat denaturation.

Amino Acid Sequence↗

PubMed1997

[Detection of bcr gene rearrangement in chronic myeloid leukemia by fluorescence in situ hybridization].

OBJECTIVE: To detect bcr gene rearrangement in chronic myeloid leukemia (CML). METHODS: Bcr rearrangement was detected by fluorescence in situ hybridization (FISH), using 765E3, a yeast artificial chromosome (YAC)-derived probe flanking the breakpoint within bcr gene. RESULTS: Nine patients with CML were examined, bcr gene rearrangement was revealed in 5 cases in chronic phase, 2 in blastic phase, and one after interferon-alpha therapy. The karyotype of 1 case after autologous bone marrow transplantation (ABMT) was chimera with normal and bcr gene rearrangement chromosomes. CONCLUSION: YAC765E3 is a useful probe for detecting bcr gene rearrangement. FISH technique is likely an important tool for monitoring of treatment and revealing minimal residual disease in CML.

Chromosomes, Artificial, Yeast↗

PubMed1997

Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents. Decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins.

Grapefruit juice increases the oral availability of a variety of CYP3A4 substrates. It has been shown that recurrent grapefruit juice ingestion results in a loss of CYP3A4 from the small bowel epithelium. We now show that the reduction in intestinal CYP3A4 concentration is rapid; a 47% decrease occurred in a healthy volunteer within 4 hr after consuming grapefruit juice. To identify the specific components of the juice responsible for this effect, we used a recently developed Caco-2 cell culture model of human intestinal epithelium that expresses catalytically active CYP3A4. We found that grapefruit oil and two furanocoumarin constituents (6', 7'-dihydroxybergamottin and a closely related dimer) caused a dose-dependent fall in CYP3A4 catalytic activity and immunoreactive CYP3A4 concentration. The effect was selective in that concentrations of CYP1A1 and CYP2D6 did not fall, consistent with previous results obtained in vivo. Assays of various juices confirmed that 6',7'-dihydroxybergamottin is the major furanocoumarin present and, although its concentration varies significantly among types and brands of grapefruit juice, it is consistently present in concentrations exceeding the IC50 (1 microM) for loss of midazolam 1'-hydroxylase activity determined in the Caco-2 cells. Studies with recombinant CYP3A4 revealed that 6', 7'-dihydroxybergamottin is a mechanism-based inactivator, which supports the idea that loss of CYP3A4 results from accelerated degradation of the enzyme. We conclude that the effect of grapefruit juice on oral availability of CYP3A4 substrates can be largely accounted for by the presence of 6',7'-dihydroxybergamottin although other furanocoumarins probably also contribute.

Biological Availability↗

PubMed1996

Membrane pores induced by magainin.

Magainin, found in the skin of Xenopus laevis, belongs to a broad class of antimicrobial peptides which kill bacteria by permeabilizing the cytoplasmic membrane but do not lyse eukaryotic cells. The 23-residue peptide has been shown to form an amphiphilic helix when associated with membranes. However, its molecular mechanism of action has been controversial. Oriented circular dichroism has detected helical magainin oriented perpendicular to the plane of the membrane at high peptide concentrations, but Raman, fluorescence, differential scanning calorimetry, and NMR all indicate that the peptide is associated with the head groups of the lipid bilayer. Here we show that neutron in-plane scattering detects pores formed by magainin 2 in membranes only when a substantial fraction of the peptide is oriented perpendicular to the membrane. The pores are almost twice as large as the alamethicin pores. On the basis of the in-plane scattering data, we propose a toroidal (or wormhole) model, which differs from the barrel-stave model of alamethicin in that the lipid bends back on itself like the inside of a torus. The bending requires a lateral expansion in the head group region of the bilayer. Magainin monomers play the role of fillers in the expansion region thereby stabilizing the pore. This molecular configuration is consistent with all published magainin data.

Alamethicin↗

PubMed1996

Secobarbital-mediated inactivation of cytochrome P450 2B1 and its active site mutants. Partitioning between heme and protein alkylation and epoxidation.

Secobarbital (SB) is a relatively selective mechanism-based inactivator of cytochrome P450 2B1, that partitions between epoxidation and heme and protein modification during its enzyme inactivation. The SB-2B1 heme adduct formed in situ in a functionally reconstituted system has been spectrally documented and structurally characterized as N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX. The SB-protein modification has been localized to 2B1 peptide 277-323 corresponding to the active site helix I of cytochrome P450 101. The targeting of heme and this active site peptide suggests that the 2B1 active site topology could influence the course of its inactivation. To explore this possibility, the individual SB epoxidation, heme and protein modification, and corresponding molar partition ratios of the wild type and seven structural 2B1 mutants, site-directed at specific substrate recognition sites, and known to influence 2B1 catalysis were examined after Escherichia coli expression. These studies reveal that Thr-302 is critical for SB-mediated heme N-alkylation, whereas Val-367 is a critical determinant of 2B1 protein modification, and Val-363 is important for SB epoxidation. SB docking into a refined 2B1 homology model coupled with molecular dynamics analyses provide a logical rationale for these findings.

Alkylation↗

PubMed1996

Longimicins A-D: novel bioactive acetogenins from Asimina longifolia (annonaceae) and structure-activity relationships of asimicin type of annonaceous acetogenins.

Bioactivity-directed fractionation of the ethanol extract of Asimina longifolia led to the isolation of four novel bioactive annonaceous acetogenins: longimicins A-D (1-4). Compounds 1-4 represent the asimicin type of acetogenins; however, the locations of the adjacent bis-tetrahydrofuran (THF) ring moieties are shifted along the aliphatic chains compared to the known compounds of this type. They are the first examples among this type of acetogenins with the placements of the ring systems altered. Compounds 1-4 showed bioactivities in several bioassays, but they are less active than their structural isomers. Study of their structure-activity relationships (SAR) reveals that the position of the adjacent bis-THF ring moiety is essential for maximization of the bioactivities among these asimicin type annonaceous acetogenins.

Animals↗

PubMed1996

cis-gigantrionenin and 4-acetyl gigantetrocin A, two new bioactive annonaceous acetogenins from Goniothalamus giganteus, and the stereochemistries of acetogenin 1,2,5-triols.

Using activity-directed fractionation, two new bioactive acetogenins, cis-gigantrionenin (1) and 4-acetyl gigantetrocin A (2), have been isolated from the bark of Goniothalamus giganteus (Annonaceae). Compound 1 has a cis-mono-THF ring with one flanking hydroxyl and possesses a cis-double bond at C-21/22 of the aliphatic chain; it represents only the second example of the cis-mono-THF ring annonaceous acetogenins having one flanking hydroxyl. Compound 2 has a trans-mono-THF ring with one flanking hydroxyl, but it possesses a mono-acetyl group at the 4-OH position; it represents only the second natural example of the acetylated annonaceous acetogenins; the first acetogenin reported, uvaricin, was mono-acetylated at the 24-OH. The stereochemistries of 1 and 2 were determined by the advanced Mosher ester method. In addition, the absolute stereochemistries of gigantriocin (3), gigantrionenin (4), and giganenin (5) were determined by the advanced Mosher ester method and by circular dichroism (CD). The stereochemistries of the 7,8-diols in murihexocins A (6) and B (7) were determined to have the S,S-configurations, respectively.

Animals↗

PubMed1996

Applying Mosher's method to acetogenins bearing vicinal diols. The absolute configurations of muricatetrocin C and rollidecins A and B, new bioactive acetogenins from Rollinia mucosa.

Muricatetrocin C (1), rollidecin A (2), and rollidecin B (3), three new bioactive annonaceous acetogenins bearing vicinal diols, were isolated from the leaves of Rollinia mucosa (Annonaceae) using activity-directed fractionation. The total structural elucidations of 1-3, including the absolute stereochemistries of the vicinal diols, were achieved by analyzing their per-Mosher ester derivatives. All three compounds showed potent and selective inhibitory effects against several human cancer cell lines.

4-Butyrolactone↗

PubMed1996

Mechanism of alamethicin insertion into lipid bilayers.

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.

Adsorption↗

PubMed1996

Neutron scattering in the plane of membranes: structure of alamethicin pores.

A technique of neutron in-plane scattering for studying the structures of peptide pores in membranes is described. Alamethicin in the inserted state was prepared and undeuterated and deuterated dilauroyl phosphatidylcholine (DLPC) hydrated with D2O or H2O. Neutron in-plane scattering showed a strong dependence on deuteration, clearly indicating that water is a part of the high-order structure of inserted alamethicin. The data are consistent with the simple barrel-stave model originally proposed by Baumann and Mueller. The theoretical curves computed with this model at four different deuteration conditions agree with the data in all cases. Both the diameter of the water pore and the effective outside diameter of the channel are determined accurately. Alamethicin forms pores in a narrow range of size. In a given sample condition, > 70% of the peptide forms pores of n and n +/- 1 monomers. The pore size varies with hydration and with lipid. In DLPC, the pores are made of n = 8-9 monomers, with a water pore approximately 18 A in diameter and with an effective outside diameter of approximately 40 A. In diphytanoyl phosphatidylcholine, the pores are made of n approximately 11 monomers, with a water pore approximately 26 A in diameter, with an effective outside diameter of approximately 50 A.

Alamethicin↗

PubMed1996

Rollinecins A and B: two new bioactive annonaceous acetogenins from Rollinia mucosa.

Two new mono-THF ring acetogenins, rollinecins A (1) and B (2), were isolated from the partitioned ethanolic extracts of the leaves of Rollinia mucosa (Annonaceae) by activity-directed fractionation. 1 and 2 are epimeric at the C-14 carbinol stereocenter. Their absolute stereochemical structures were solved by preparing their respective per-Mosher ester derivatives. 1 and 2 showed equivalent and selective in vitro activities against several human solid tumor cell lines.

Antineoplastic Agents, Phytogenic↗