PubMed Health⌕ Search

Biomedical subjects

H Cheng

Publications and source records attributed to H Cheng.

At least 55 records · Page 3Linked to original sources
PubMed1999

Development of rabbit embryos during a 96-h period of in vitro culture after superovulatory treatment under conditions of elevated ambient temperature.

The effects of elevated ambient temperature on the response to exogenous gonadotropins were evaluated in female New Zealand White rabbits exposed to 33+/-1 degrees C (mean +/- SE) and 10-30% relative humidity (8 h/day) during a 5-day period. Does were treated with pFSH (0.3 mg/0.3 ml Standard Armour) twice daily during three consecutive days with a minimum interval of 8 h between injections. Six hours after the last FSH injection all does were removed from the experimental chamber, given hCG (25 IU/kg) and paired overnight. Nineteen hours after pairing, embryos were flushed from the reproductive tracts, evaluated, and subjected to in vitro culture during a 96-h period. The ovulatory responses to exogenous gonadotropins and fertilization rates did not differ significantly under conditions of elevated ambient temperature, whereas fewer blastocysts and increased number of degenerate embryos were observed after culture. We conclude that although hyperthermia was induced during exposure to elevated ambient temperature, it did not alter the ovulatory responses to gonadotropin treatment and plasma concentrations of FSH and LH compared with does in a thermoneutral environment. Exposure of donor rabbits to elevated ambient temperature before mating, however, increased embryonic degeneration.

Animals↗
PubMed1999

Cardiac-specific overexpression of the alpha(1) subunit of the L-type voltage-dependent Ca(2+) channel in transgenic mice. Loss of isoproterenol-induced contraction.

The L-type voltage-dependent calcium channel (L-VDCC) regulates calcium influx in cardiac myocytes. Activation of the beta-adrenergic receptor (betaAR) pathway causes phosphorylation of the L-VDCC and that in turn increases Ca(2+) influx. Targeted expression of the L-VDCC alpha(1) subunit in transgenic (Tg) mouse ventricles resulted in marked blunting of the betaAR pathway. Inotropic and lusitropic responses to isoproterenol and forskolin in Tg hearts were significantly reduced. Likewise, Ca(2+) current augmentation induced by iso- proterenol and forskolin was markedly depressed in Tg cardiomyocytes. Despite no change in betaAR number, isoproterenol-stimulated adenylyl cyclase activity was absent in Tg membranes and NaF and forskolin responses were reduced. We postulate an important pathway for regulation of the betaAR by Ca(2+) channels.

Adenylyl Cyclases↗
PubMed1999

G(i) protein-mediated functional compartmentalization of cardiac beta(2)-adrenergic signaling.

In contrast to beta(1)-adrenoreceptor (beta(1)-AR) signaling, beta(2)-AR stimulation in cardiomyocytes augments L-type Ca(2+) current in a cAMP-dependent protein kinase (PKA)-dependent manner but fails to phosphorylate phospholamban, indicating that the beta(2)-AR-induced cAMP/PKA signaling is highly localized. Here we show that inhibition of G(i) proteins with pertussis toxin (PTX) permits a full phospholamban phosphorylation and a de novo relaxant effect following beta(2)-AR stimulation, converting the localized beta(2)-AR signaling to a global signaling mode similar to that of beta(1)-AR. Thus, beta(2)-AR-mediated G(i) activation constricts the cAMP signaling to the sarcolemma. PTX treatment did not significantly affect the beta(2)-AR-stimulated PKA activation. Similar to G(i) inhibition, a protein phosphatase inhibitor, calyculin A (3 x 10(-8) M), selectively enhanced the beta(2)-AR but not beta(1)-AR-mediated contractile response. Furthermore, PTX and calyculin A treatment had a non-additive potentiating effect on the beta(2)-AR-mediated positive inotropic response. These results suggest that the interaction of the beta(2)-AR-coupled G(i) and G(s) signaling affects the local balance of protein kinase and phosphatase activities. Thus, the additional coupling of beta(2)-AR to G(i) proteins is a key factor causing the compartmentalization of beta(2)-AR-induced cAMP signaling.

Adrenergic beta-2 Receptor Agonists↗
PubMed1999

Solution structure and dynamics of a de novo designed three-helix bundle protein.

Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25- to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-helix bundle protein, alpha3D, is reported. The structure of alpha3D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.

Amino Acid Sequence↗
PubMed1999

A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection.

A human mitochondrial DNA (mtDNA) standard reference material (SRM 2392) will provide quality control when mtDNA is sequenced for forensic identifications, medical diagnosis, or mutation detection. SRM 2392 includes DNA from two lymphoblast cell cultures (CHR and 9947A) and cloned DNA from the CHR HV1 region, which contains a C stretch and is difficult to sequence. The mtDNA sequence (but not the DNA) of a third human template GM03798 is provided for comparison. Fifty-eight unique primer sets allow any area or the entire mtDNA (16,569 bp) to be amplified and sequenced. While none of the differences in these three templates correspond to published mutations associated with specific diseases, some of these differences did result in animo acid changes compared with that published by S. Anderson et al. (1981, Nature 290: 457-465). An interlaboratory evaluation of the amplification, sequencing, and data analysis of the CHR template was conducted by four laboratories. Corroboration of the SRM results will provide quality assurance that any unknown mtDNA is also being amplified and sequenced correctly.

Cell Line↗
PubMed1999

Protein kinase C isoform expression and activity alter paclitaxel resistance in vitro.

OBJECTIVE: The aim of this study was to assess the relationship of protein kinase C (PKC) isoform expression and functional activity to the development of multidrug resistance in gynecologic malignancies. METHODS: Paclitaxel-resistant subclones (T30 and T30-Res) of the Mes-sa human uterine sarcoma cell line were selected through exposure to paclitaxel in vitro. Indices of relative drug resistance were determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) assay. Differences in the expression pattern of PKC isoforms were assessed by Western blot of cell lysates. Finally, the influence of PKC activity (i.e., translocation to the plasma membrane, confirmed by Western blot of plasma membrane bound protein) on resistance to paclitaxel was examined with the MTT assay in cells preincubated with PMA. RESULTS: The indices of relative paclitaxel resistance of Mes-sa, Mes-sa-T30, and Mes-sa-T30-Res were 1-, 5-, and 11-fold, respectively. Five (alpha, gamma, iota, lambda, and mu) of the 11 known PKC isoforms were detected in all cell lysates. Only PKC-alpha and PKC-gamma expression increased with increasing indices of paclitaxel resistance. Interestingly, PMA induction of PKC activity reversed resistance to paclitaxel in all cell lines by 2- to 3-fold, and this reversal of drug resistance was associated with a time-dependent translocation of PKC-alpha and PKC-gamma to the plasma membrane compartment. CONCLUSIONS: Increased expression of only the PKC-alpha and PKC-gamma isoforms correlates with increasing levels of paclitaxel resistance in Mes-sa cells in this in vitro experimental model. However, increased functional activity of these and other PKC isoforms leads to reversal in paclitaxel resistance. Therefore, PKC activating mechanisms normally present in primary tumor cells may be compromised in drug-resistant clones.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Familial adenomatous polyposis-associated thyroid cancer: a clinical, pathological, and molecular genetics study.

We report two familial adenomatous polyposis (FAP) kindreds with thyroid cancer, harboring two apparently novel germlineAPC mutations. The clinical phenotype in the first kindred was typical of classical adenomatous polyposis, whereas the second kindred exhibited an attenuated adenomatous polyposis phenotype. There was a female predominance with a mean age of 34 years (range, 23-49) at cancer diagnosis. Multiple sections of four thyroid tumors from three FAP patients were analyzed in detail. Histological examination of thyroid tumors showed a range of morphological features. Some tumors exhibited typical papillary architecture and were associated with multifocal carcinoma; in others, there were unusual areas of cribriform morphology, and spindle-cell components with whorled architecture. Immunoreactivity for thyroglobulin and high molecular weight keratins was strong. Somatic APC mutation analysis revealed an insertion of a novel long interspersed nuclear element-1-like sequence in one tumor sample, suggesting disruption of APC. In three FAP patients, ret/PTC-1 and ret/PTC-3 were expressed in thyroid cancers. No positivity was observed for ret/ PTC-2. p53 immunohistochemistry was positive in only one section of a recurrent thyroid tumor sample. Our data suggest that genetic alterations in FAP-associated thyroid cancer involve loss of function of APC along with the gain of function of ret/PTC, while alterations of p53 do not appear to be an early event in thyroid tumorigenesis.

Adenomatous Polyps↗
PubMed1999

Colossal crypts bordering colon adenomas in Apc(Min) mice express full-length Apc.

Enlarged but nondysplastic crypts are frequently observed at the margins of colon tumors, forming what has been called a transitional epithelium. It is now thought that this is a reactive state and not a preneoplastic condition as previously suggested. We have used the mouse familial adenomatous polyposis model, ApcMin, to study these abnormal adenoma-associated crypts. We report that these nondysplastic crypts are enormous (as much as 10 times normal length) and branch more frequently than normal crypts. They express wild-type Apc protein and display the wild-type Apc allele. We conclude that the colossal crypts at adenoma margins have normal Apc gene function, consistent with the suggestion that their phenotype is a reactive state. The cause remains an open question, but the dramatic epithelial response hints at the presence of potent epithelial trophic factors in the vicinity of colon tumors.

Adenoma↗
PubMed1999

Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method.

Determination of the calcium spark amplitude distribution is of critical importance for understanding the nature of elementary calcium release events in striated muscle. In the present study we show, on general theoretical grounds, that calcium sparks, as observed in confocal line scan images, should have a nonmodal, monotonic decreasing amplitude distribution, regardless of whether the underlying events are stereotyped. To test this prediction we developed, implemented, and verified an automated computer algorithm for objective detection and measurement of calcium sparks in raw image data. When the sensitivity and reliability of the algorithm were set appropriately, we observed highly left-skewed or monotonic decreasing amplitude distributions in skeletal muscle cells and cardiomyocytes, confirming the theoretical predictions. The previously reported modal or Gaussian distributions of sparks detected by eye must therefore be the result of subjective detection bias against small amplitude events. In addition, we discuss possible situations when a modal distribution might be observed.

Algorithms↗
PubMed1999

Clonal analysis of mouse intestinal epithelial progenitors.

BACKGROUND & AIMS: Little is known about the cell lineages leading from stem cells to the various terminally differentiated cell types of the intestinal epithelium. In particular, the existence and characterization of intermediate progenitor types remain open issues, which are discussed in this study. METHODS: Chemical mutagenesis was used to genetically mark random intestinal epithelial cells by somatic mutation of the Dlb-1 locus. Intact epithelium was isolated at various times thereafter, and the composition, size, and location of mutant clones were scored. RESULTS & CONCLUSIONS: Analysis of clone dynamics showed short-lived (days) progenitors (C1, M1, and Mix) yielding one or two cell types and long-lived (months) mucous cell progenitors (M0), columnar cell progenitors (C0), and pluripotential stem cells (S) capable of giving rise to all epithelial cell types. Furthermore, study of clonal dispersion, during crypt branching morphogenesis or cell migration, shows that mutant progenitors usually partition into only one of the two daughter crypts and that cells are often widely dispersed in spite of the extensive intercellular junctions in the epithelium.

Animals↗
PubMed1999

The National Diabetic Retinopathy Laser Treatment Audit. III. Clinical outcomes.

PURPOSE: To describe the short-term clinical outcomes for a cohort of patients undergoing first photocoagulation treatment for proliferative retinopathy or maculopathy in the United Kingdom. METHOD: Nine-month follow-up of the Royal College of Ophthalmologists' national audit of laser treatment for diabetic retinopathy. RESULTS: For eyes with maculopathy, 9.2% had had a deterioration in visual acuity equivalent to a doubling of the visual angle and 3.3% of eyes had a visual acuity of less than 6/60 at follow-up. There had been an improvement in the macular oedema or exudate in 64.6% and 77.3% respectively. Prognostic factors for a poorer visual acuity at follow-up were worse visual acuity at baseline, the presence of diffuse (vs focal) oedema and grid (vs focal) treatment. For eyes with proliferative retinopathy, the retinal neovascularisation had regressed fully in 50.8% of cases, whilst there had been no change or a deterioration in 10.3%. A visual acuity of less than 6/60 at follow-up was present in 8.6% of eyes. There was a poor morphological outcome at follow-up (as defined by rubeosis, new tractional detachment or having had a vitrectomy) in 7.2%. Risk factors for poor morphological outcome were the presence of 'high-risk characteristics', female sex and the presence of concurrent maculopathy at baseline. Regression of neovascularisation was associated with greater areas of retinal ablation at the initial treatment session. Although some eyes with proliferative retinopathy appeared to be undertreated initially compared with DRS and ETDRS protocols, some of these eyes did respond to lower amounts of treatment. CONCLUSION: For maculopathy, poorer outcome was related to worse visual acuity at baseline, diffuse (vs focal) maculopathy, and grid treatment. For proliferative retinopathy, poorer outcome was related to 'high-risk characteristics' and coexistence of maculopathy at baseline, and improvement was related to larger areas of ablation. The relationship of poor outcome with worse initial disease argues for earlier detection of retinopathy.

Diabetic Retinopathy↗
PubMed1999

Local control models of cardiac excitation-contraction coupling. A possible role for allosteric interactions between ryanodine receptors.

In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through ryanodine receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation-contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation-contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.

Algorithms↗
PubMed1999

Mismatch repair gene defects contribute to the genetic basis of double primary cancers of the colorectum and endometrium.

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome caused by germline defects of mismatch repair (MMR) genes. Endometrial cancer is the most common extracolonic neoplasm in HNPCC and is the primary clinical manifestation of the syndrome in some families. The cumulative incidence of endometrial cancer among HNPCC mutation carriers is high, estimated to be from 22 to 43%. We hypothesized that women with double primary cancers of the colorectum and endometrium are likely to be members of HNPCC families. In order to determine how frequently HNPCC manifests in the context of double primary cancers, we examined alterations of two MMR genes, hMSH2 and hMLH1, in 40 unrelated women affected with double primary cancers. These cases were identified using hospital-based and population-based cancer registries in Ontario, Canada. MMR gene mutations were screened by single-strand conformation polymorphism analysis and confirmed by direct sequencing. Eighteen percent (seven of 40) were found to harbor mutations of one of the two MMR genes. Analysis of colorectal and/or endometrial tumors of mutation-negative probands found microsatellite instability in seven of 20 cases. Six of seven mutation-positive probands had strong family histories suggestive of HNPCC. First degree relatives of mutation-positive probands had a very high relative risk (RR) of colorectal cancer (RR = 8.1, CI 3. 5-15.9) and endometrial cancer (RR = 23.8, CI 6.4-61.0). The relative risk of mutation-negative cases was 2.8 (CI 1.7-4.5) for colorectal cancer and 5.4 (CI 2.0-11.7) for endometrial cancer. We recommend that all double primary patients with cancers at these sites should have a genetic evaluation, including molecular analysis for HNPCC where appropriate.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Spontaneous beta(2)-adrenergic signaling fails to modulate L-type Ca(2+) current in mouse ventricular myocytes.

A receptor can be activated either by specific ligand-directed changes in conformation or by intrinsic, spontaneous conformational change. In the beta(2)-adrenergic receptor (AR) overexpression transgenic (TG4) murine heart, spontaneously activated beta(2)AR (beta(2)-R*) in the absence of ligands has been evidenced by elevated basal adenylyl cyclase activity and cardiac function. In the present study, we determined whether the signaling mediated by beta(2)-R* differs from that of a ligand-elicited beta(2)AR activation (beta(2)-LR*). In ventricular myocytes from TG4 mice, the properties of L-type Ca(2+) current (I(Ca)), a major effector of beta(2)-LR* signaling, was unaltered, despite a 2.5-fold increase in the basal cAMP level and a 1.9-fold increase in baseline contraction amplitude as compared with that of wild-type (WT) cells. Although the contractile response to beta(2)-R* in TG4 cells was abolished by a beta(2)AR inverse agonist, ICI118,551 (5 x 10(-7) M), or an inhibitory cAMP analog, Rp-CPT-cAMPS (10(-4) M), no change was detected in the simultaneously recorded I(Ca). These results suggest that the increase in basal cAMP due to beta(2)-R*, while increasing contraction amplitude, does not affect I(Ca) characteristics. In contrast, the beta(2)AR agonist, zinterol elicited a substantial augmentation of I(Ca) in both TG4 and WT cells (pertussis toxin-treated), indicating that L-type Ca(2+) channel in these cells can respond to ligand-directed signaling. Furthermore, forskolin, an adenylyl cyclase activator, elicited similar dose-dependent increase in I(Ca) amplitude in WT and TG4 cells, suggesting that the sensitivity of L-type Ca(2+) channel to cAMP-dependent modulation remains intact in TG4 cells. Thus, we conclude that beta(2)-R* bypasses I(Ca) to modulate contraction, and that beta(2)-LR* and beta(2)-R* exhibit different intracellular signaling and target protein specificity.

Adenylate Cyclase Toxin↗
PubMed1999

Regulation of c-Fes tyrosine kinase and biological activities by N-terminal coiled-coil oligomerization domains.

The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal region did not affect the transforming activity of v-Src in Rat-2 cells, arguing against a nonspecific suppressive effect. Taken together, these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of the first coiled-coil domain may activate Fes by disturbing intramolecular coiled-coil interaction, allowing for oligomerization via the second coiled-coil domain. Deletion of the second coiled-coil domain blocks fibroblast transformation by an activated form of c-Fes, consistent with this model. These results provide the first evidence for regulation of a nonreceptor protein-tyrosine kinase by coiled-coil domains.

Animals↗
PubMed1999

Characterization of receptors mediating AVP- and OT-induced glucagon release from the rat pancreas.

We characterized the receptors that mediate arginine vasopressin (AVP)- and oxytocin (OT)-induced glucagon release by use of a number of antagonists in the perfused rat pancreas and the fluorescence imaging of the receptors. AVP and OT (3 pM-3 nM) increased glucagon release in a concentration-dependent manner. The antagonist with potent V(1b) receptor-blocking activity, CL-4-84 (10 nM), abolished AVP (30 pM)-induced glucagon release but did not alter OT (30 pM)-induced glucagon release. d(CH(2))(5)[Tyr(Me)(2)]AVP (10 nM), a V(1a) receptor antagonist, and L-366,948 (10 nM), a highly specific OT-receptor antagonist, failed to inhibit AVP-induced glucagon release. In contrast, L-366,948 (10 nM) abolished OT (30 pM)-induced glucagon release but did not change the effect of AVP. Fluorescent microscopy of rat pancreatic sections showed that fluorescence-labeled AVP and OT bound to their receptors in the islets of Langerhans and that the bindings were inhibited by 1 microM of Cl-4-84 and L-366,948, respectively. Because AVP and OT at physiological concentrations (3-30 pM) increased glucagon release, we conclude that AVP and OT increase glucagon release under the physiological condition through the activation of V(1b) and OT receptors, respectively.

Animals↗