Search PubMed⌕ Search

Biomedical subjects

J Ma

Publications and source records attributed to J Ma.

At least 307 records · Page 17Linked to original sources
PubMed1998

Tryptophan-136 in subunit II of cytochrome bo3 from Escherichia coli may participate in the binding of ubiquinol.

In the cytochrome c oxidases, the role of subunit II is to provide the electron entry site into the enzyme. This subunit contains both the binding site for the substrate, cytochrome c, and the CuA redox center, which is initially reduced by cytochrome c. Cytochrome bo3 and other quinol oxidases that are members of the heme-copper oxidase superfamily have a homologous subunit II, but the CuA site is absent, as is the docking site for cytochrome c. Speculation that subunit II in the quinol oxidases may also be important as an electron entry site is supported by the demonstration several years ago that a photoreactive substrate analogue, azido-Q, covalently labeled subunit II in cytochrome bo3. In the current work, a sequence alignment of subunit II of heme-copper quinol oxidases is used as a guide to select conserved residues that might be important for the binding of ubiquinol to cytochrome bo3. Results are presented for point mutants in 24 different residue positions in subunit II. The membrane-bound enzymes were examined by optical spectroscopy and by determining the activity of ubiquinol-1 oxidase. In each case, the Km for ubiquinol-1 was determined as a measure of possible perturbation to a quinol binding site. The only mutant that had a noticeably altered Km for ubiquinol-1 was W136A, in which the Km was about sixfold increased. Thus, W136 may be at or close to a substrate (ubiquinol)-binding site in cytochrome bo3. In the cytochrome c oxidases, the equivalent tryptophan (W121 in Paracoccus denitrificans) has been identified as the "electron entry site".

Amino Acid Sequence↗
PubMed1998

The allosteric mechanism of the chaperonin GroEL: a dynamic analysis.

Normal mode calculations on individual subunits and a multisubunit construct are used to analyze the structural transitions that occur during the GroEL cycle. The normal modes demonstrate that the specific displacements of the domains (hinge bending, twisting) observed in the structural studies arise from the intrinsic flexibility of the subunits. The allosteric mechanism (positive cooperativity within a ring, negative cooperativity between rings) is shown to be based on coupled tertiary structural changes, rather than the quaternary transition found in classic allosteric proteins. The results unify static structural data from x-ray crystallography and cryoelectron microscopy with functional measurements of binding and cooperativity.

Allosteric Regulation↗
PubMed1998

Molecular cloning of the salamander red and blue cone visual pigments.

PURPOSE: Salamander retinas are known to contain at least three cone pigments and two rod pigments. The purpose of this study was to clone and characterize the visual pigments from salamander cones. METHODS: cDNA fragments of cone pigments were amplified from a salamander retina cDNA library by PCR using a pair of primers with consensus for visual pigments. These fragments were cloned and used as probes for library-screening. The full-length cDNAs were isolated from the retinal library using the cloned PCR products as probes. DNA sequences were determined by the dideoxynucleotide chain termination method. RESULTS: Two pigment cDNAs were cloned and sequenced from the salamander library. The global GenBank search showed that they do not match any existing sequences but have significant sequence similarity to visual pigments. One of the pigment cDNAs showed a high sequence homology with red cone pigments from other species and thus, was designated as a red cone opsin. The other pigment was designated as a blue cone opsin as it is most homologous to the chicken and goldfish blue cone pigments. Both cDNAs contain a full-length coding region encoding 365 amino acids in the red and 363 amino acids in the blue cone pigment. Hydropathy analysis predicted that both pigments could form seven hydrophobic transmembrane helices. Both pigments retain the key amino acid residues critical for maintaining the structure and function of opsins and have similar G-protein interaction sequences which differ from that of rod opsin. Phylogenetic analysis indicates that the red opsin belongs to the L group and the blue opsin belongs to the M1 group of visual pigments. CONCLUSIONS: The salamander red and blue cone pigments share high sequence homology with the cone pigments of other species.

Amino Acid Sequence↗
PubMed1998

pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation.

The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and nuclear fluorescence was observed. Cytosolic fluorescence constituted over 80% of the total. Excitation scanning of transfected cells revealed two GFPmut1-specific regions that were pH-sensitive over the physiologic range, and each region exhibited a unique pH "bias" in fluorescence emission. Excitation at or near the expected maximum of 488 nm (region II) uniformly resulted in fluorescence that was preferentially altered at acidic pH. In contrast, a novel "wild-type" excitation peak at 400 nm (region I) resulted in alkaline-biased fluorescence similar to that described for the wild-type chromophore in vitro, suggesting that wild-type spectral features disrupted in vitro by mutagenesis may be recovered in intact cells. Calibration of intracellular pH (pHi) with in situ fluorescence following excitation in either region revealed a semilogarithmic relationship between fluorescence intensity and pH within the physiologic range. We therefore measured pHi changes attributable to altered Na/HCO3 cotransport (NBC) activity both in GFPmut1-expressing cells and in paired untransfected cells loaded with BCECF. Basal NBC activity was the same in each group, as was the stimulation of activity by 10% CO2, thus validating the utility of GFPmut1 as a fluorescent probe for pHi and establishing a novel, useful, and practical application for GFPmut1 in monitoring pHi in real time.

Animals↗
PubMed1998

Regulation of Bcl-xl channel activity by calcium.

Recent studies have demonstrated that the anti-apoptotic proteins, Bcl-2 and Bcl-xl, with the carboxyl-terminal hydrophobic domain removed, form cation-selective channels in the lipid bilayer reconstitution system. However, the regulatory properties of these channels are unknown. In this study, we investigated the ion-conducting properties of full-length Bcl-xl in the lipid bilayer reconstitution system. Our findings indicate that Bcl-xl forms a cation-selective channel that conducts sodium but not calcium and that Bcl-xl channel activity is reversibly inhibited by luminal calcium with a half-dissociation constant of approximately 60 microM. This calcium-dependent regulation of the Bcl-xl channel provides new insights into the roles of calcium and Bcl-2-related proteins in the programmed cell death pathway.

Apoptosis↗
PubMed1998

A role of subicular and hippocampal afterdischarges in initiation of locomotor activity in rats.

The possible role of a hippocampal afterdischarge (AD) episode in eliciting locomotor movements was evaluated in freely moving rats. Electrical stimulation of either the ventral subiculum (VSB) or the hippocampal CA1 region evoked an AD of 6-50 s in duration, which was followed by an increase in locomotor activity. Similar results were also observed after unilateral injection of N-methyl-d-aspartic acid (NMDA, 0.25 microg or 1 microg), a glutamate receptor agonist, into the VSB. Locomotor activity was not observed when either electrical or chemical stimulation of the VSB, or electrical stimulation of the CA1 region did not elicit an AD. In addition, the duration of the AD was positively correlated with the number of locomotor movements induced by stimulation of VSB or CA1 region. It is suggested that the hippocampal/subicular AD may be a necessary condition to induce locomotor activity by either chemical or electrical stimulation of the hippocampus in rats.

Animals↗
PubMed1998

Overexpression, purification, and stereochemical studies of the recombinant (S)-adenosyl-L-methionine: delta 24(25)- to delta 24(28)-sterol methyl transferase enzyme from Saccharomyces cerevisiae.

The ERG6 gene that encodes (S)-adenosyl-L-methionine: delta 24(25)-to delta 24(28)-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae was introduced into plasmid pET23a(+) and the resulting native protein was overexpressed in BL21 (DE3) host cells under control of a T7 promoter. This enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography. N-Terminal sequence analysis of the first 10 amino acids of the purified SMT protein confirmed the identity of the start triplet and expected primary structure. The enzyme exhibited a turnover number of 0.01/s and an isoelectric point of 5.95. A combination of Superose 6 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified SMT enzyme possessed a native molecular weight of 172,000 and was tetrameric. The purified SMT enzyme generated kinetics in which velocity versus substrate curves relative to zymosterol (preferred sterol acceptor molecule) and AdoMet were sigmoidal rather than hyperbolic, indicating enzyme cooperativity among the subunits. Studies on product formation using [27-13C]zymosterol and [2H3-methyl]AdoMet incubated with the pure SMT enzyme confirmed the reaction mechanism of sterol methylation to involve a 1,2-hydride shift of H-24 to C-25 from the Re-face of the original 24,25- double bond. Deduced amino acid sequence comparisons of the SMT polypeptide from S. cerevisiae with related sterol methyl transferase enzymes of plant and fungal origin indicate that there is a significant degree of similarity between these enzymes. Specifically, there is a conserved sequence (in yeast from amino acids ca. 79 to 92 which contains an YEXGWG motif; referred to as Region I) that is not present in other AdoMet-dependent methyl transferase enzymes.

Amino Acid Sequence↗
PubMed1998

Mapping of the CYP2J cytochrome P450 genes to human chromosome 1 and mouse chromosome 4.

CYP2J subfamily cytochromes P450 catalyze the NADPH-dependent oxidation of arachidonic acid to several unique eicosanoids that possess numerous biological activities including modulation of ion transport, control of bronchial and vascular smooth muscle tone, and stimulation of peptide hormone secretion. We have identified sequence variants in the 3' untranslated regions of two mouse Cyp2j genes (Cyp2j5 and Cyp2j6) and used a PCR-based oligonucleotide hybridization assay to map both genes to the central region of chromosome 4 distal to the Jun oncogene. The corresponding human CYP2J gene (CYP2J2) has been assigned to human chromosome 1 on a panel of somatic hybrid cell lines and to 1p31.3-p31.2 by fluorescence in situ hybridization analysis. The proximity of the Cyp2j cluster to the Cyp4a cluster suggests that these genes may be part of a cassette of P450 genes involved in the oxidation of fatty acids.

Animals↗
PubMed1998

Direct activation of cystic fibrosis transmembrane conductance regulator channels by 8-cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX).

8-Cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX) are xanthine adenosine antagonists which activate chloride efflux from cells expressing either wild-type or mutant (DeltaF508) cystic fibrosis transmembrane conductance regulator (CFTR). These drugs are active in extremely low concentrations, suggesting their possible therapeutic uses in treating cystic fibrosis. However, knowledge of the mechanism of action of these compounds is lacking. We report here that the same low concentrations of both CPX and DAX which activate chloride currents from cells also generate a profound activation of CFTR channels incorporated into planar lipid bilayers. The process of activation involves a pronounced increase in the total conductive time of the incorporated CFTR channels. The mechanism involves an increase in the frequency and duration of channel opening events. Thus, activation by these drugs of chloride efflux in cells very likely involves direct interaction of the drugs with the CFTR protein. We anticipate that this new information will contribute fundamentally to the rational development of these and related compounds for cystic fibrosis therapy.

Cell Line↗
PubMed1998

Post-ischemic changes in the expression of Alzheimer's APP isoforms in rat cerebral cortex.

A significant porportion (25%) of patients with Alzheimer's disease (AD) also shows vascular pathology. Recent ultrastructural studies demonstrated characteristic and extensive angio-architectural distortions of cerebral capillaries in AD brains. We examined the expression of APP mRNA isoforms of cerebral cortex after transient ischemia by middle cerebral artery occlusion, using RT-PCR. Neuronal damage and glial fibrillary acidic protein immunohistochemistry were also examined histologically. After transient ischemia, the Kunitz protease inhibitor-bearing isoforms (KPI-APP) were increased whereas APP 695, which lacks KPI domain, was decreased. Neuronal damage and GFAP-immunoreactive astrocytes were also observed. These results show that focal, transient ischemia alters KPI-APP/APP 695 ratio in cerebral cortex and this shift in APP isoforms could be related to neurodegeneration and/or activation of astrocytes during the ischemic process.

Alzheimer Disease↗
PubMed1998

Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study.

Insulin-like growth factor-I (IGF-I) is a mitogen for prostate epithelial cells. To investigate associations between plasma IGF levels and prostate cancer risk, a nested case-control study within the Physicians' Health Study was conducted on prospectively collected plasma from 152 cases and 152 controls. A strong positive association was observed between IGF-I levels and prostate cancer risk. Men in the highest quartile of IGF-I levels had a relative risk of 4.3 (95 percent confidence interval 1.8 to 10.6) compared with men in the lowest quartile. This association was independent of baseline prostate-specific antigen levels. Identification of plasma IGF-I as a predictor of prostate cancer risk may have implications for risk reduction and treatment.

Adult↗
PubMed1998

Investigation of the Microenvironment in Nonionic Reverse Micelles Using Methyl Orange and Methylene Blue as Absorption Probes

The microenvironment in nonionic reverse micelles of Triton X-100, n-hexanol, and water in cyclohexane is investigated by using methyl orange (MO) and methylene blue (MB) as absorption probes. Information about the states of water in the polar core of the reverse micelles is obtained through the solvatochromic behavior of MO and the sensitivity of the complex formation between MB and Triton X-100 in reverse micelles to the state of water in the polar core. The results obtained from the spectra of MO and MB in the TX-100 reverse micelles with different amounts of water are compatible with the proposal that there are three types of water present in the polar core: the primary bound water, which interacts directly with the ethylene oxide (EO) groups of the surfactant to form a primary hydration shell, the second bound water, which is next to the primary hydration shell of the EO groups and bound less tightly, and the free water, which is present in the water pool and resembles the bulk water. It is found that there is an equilibrium between two states of MB in the reverse micelles: the bound MB and the free MB, and the presence of secondary bound water and free water will lead to the equilibrium shift from bound MB to free MB. In the Triton X-100 reverse micellar system studied, the secondary bound water is present at about W = 1.8 and water pools begin to form when the water content reaches W = 5.3. The strong interaction between MB and nonionic surfactants, which results in the complex formation, and its sensitivity to the state of water in the polar core are found to be also present in the reverse micelles of either Triton X-114 or C12E4 in cyclohexane. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗
PubMed1998

Measurement of R'2 in the presence of multiple spectral components using reference spectrum deconvolution.

A method is described for measuring R'2, the RF reversible contribution to the effective transverse relaxation rate in yellow trabecular marrow, as a means to evaluate trabecular bone structure and density. The method exploits the similarity in spectral composition of the marrow and fat in subcutaneous tissue. Under these conditions the gradient echo envelope of the marrow signal can be regarded as a convolution of a function describing the bone marrow intravoxel line broadening (R'2) with a function expressing chemical shift modulation, which is obtained from the echo envelope of the subcutaneous fat signal in a reference region. Simple division of each of a series of echoes by the reference signal is shown to afford a smooth decay which can be fitted to a model to extract R'2. The method has been evaluated in the upper femur of test subjects and a strong correlation of the thus derived R'2 values with those obtained by the GESFIDE technique is demonstrated. The close correspondence in spectral composition of proximal femur marrow and subcutaneous fat is further illustrated by means of localized spectroscopy. The major potential error source is global inhomogeneity in the reference region which can lead to an underestimation of the demodulation-derived R'2.

Absorptiometry, Photon↗
PubMed1998

Treatment of hepatocellular carcinoma with the cellular tumor vaccines generated by in vitro modification of tumor cells with non gene transfer approaches.

Anti-tumor immune responses are mediated primarily by T cells. Down regulation of major histocompatibility complex (MHC) and the molecules that costimulate the immune responses is associated with defective signaling of tumor cells for T cell activation. In vitro fusion of autologous tumor cells with antigen presenting cells (APCs) or treatment of tumor cells with a combination of cytokines significantly increased the expression of MHC class I and adhesion molecules on tumor cell surfaces that costimulate host immune responses. The hybrid cells generated by fusion of tumor cells with APCs and the tumor cells treated in vitro with a combination of cytokines and pre-incubated with a bispecific monoclonal antibody (bi-Mab) cross-linking antigen on tumor cells to CD28 on T cells, become immunogenic and able to stimulate naive T cells with generation of tumor specific cytotoxic T cells both in vitro and in vivo. Immunization with the modified tumor cells elicits an immune response mediated by both CD4+ and CD8+ T cells. This response protected against a parental tumor cell challenge and cured established tumors. The approach was effective in both low immunogenic and non-immunogenic tumor systems. Modification of tumor cells with tumor:APC fusion or the two-step procedure may provide a strategy for development of tumor vaccines that is effective for cancer immunotherapy.

Animals↗
PubMed1998

Induction of anti-idiotypic humoral and cellular immune responses by a murine monoclonal antibody recognizing the ovarian carcinoma antigen CA125 encapsulated in biodegradable microspheres.

The use of biodegradable poly(DL-lactic-co-glycolic acid) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses was investigated using a murine monoclonal antibody B43.13 that recognizes the human ovarian cancer antigen CA125. Immunization of mice with mAb B43.13 encapsulated in poly(DL-lactic-co-glycolic acid) microspheres resulted in enhanced humoral and cellular immune responses compared with mAb B43.13 alone or mAb B43.13 mixed with microspheres. The antibody responses could be further enhanced by the co-encapsulation of mAb B43.13 with monophosphoryl lipid A, a non-toxic adjuvant, in microspheres. Anti-idiotypic humoral responses were shown to result in Ab2 antibodies mimicking the nominal antigen CA125 and Ab3 antibodies recognizing CA125. Further, microsphere delivery of mAb B43.13 also resulted in induction of T cell responses involving T2 cells reactive with mAb B43.13 epitopes and T3 cells recognizing CA125. These results indicate that microsphere delivery of Abl can induce both humoral and cellular anti-idiotypic responses relevant to cancer antigens. This raises the possibility of the use of such formulations for anti-idiotypic induction immunotherapy for cancer.

Animals↗
PubMed1998

Abrogated energy-dependent uptake of cisplatin in a cisplatin-resistant subline of the human ovarian cancer cell line IGROV-1.

The parental IGROV-1 human ovarian adenocarcinoma cell line was intermittently exposed to increasing concentrations of cisplatin to obtain resistant sublines. A stable resistant subline with a resistance factor of 8.4 had been developed after 9 months and 28 passages, which was denoted IGROV(CDDP). A high correlation coefficient of 0.97 was found between the log cell survival and the DNA-adduct peak level during the process of resistance development. IGROV(CDDP) was strongly cross-resistant to carboplatin and doxorubicin and moderately cross-resistant to etoposide, docetaxel, and topotecan. Only minor resistance against 5-fluorouracil was observed, whereas IGROV(CDDP) was not cross-resistant to methotrexate. Intracellular accumulation of cisplatin was 65% lower in IGROV(CDDP) as compared with parental IGROV-1 at 37 degrees C under normal conditions. Coincubation of cisplatin with the Na+/K+-ATPase inhibitor ouabain resulted in a more pronounced decrease in platinum accumulation in IGROV-1 (44% decrease) than in IGROV(CDDP) (26% decrease). Under energy-depleting conditions the accumulation of cisplatin in the parental cell line was approximately 60% lower than that observed under normal (energy [i.e., ATP] rich) culture conditions. In contrast, the accumulation in IGROV(CDDP) was not affected by ATP-depletion. There appeared to be no significant difference between the intracellular accumulation of platinum in the resistant and sensitive cells under conditions of energy deprivation or when the uptake was studied at 0 degrees C. In conclusion, abrogation of energy-dependent accumulation in IGROV(CDDP) seems to be a major mechanism of resistance to cisplatin in this cell line.

Adenocarcinoma↗
PubMed1998

Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro.

The cytotoxicity of cisplatin alone and in combination with topotecan (TPT) or SN-38, two novel topoisomerase I (topo I) inhibitors, was determined in a panel of eight well-characterized human solid-tumor cell lines. Interactions between cisplatin and these topo I inhibitors were investigated using three different administration schedules: (1) simultaneous incubation (C + T and C + S), (2) cisplatin followed by TPT or SN-38 (C --> T and C --> S), and (3) TPT or SN-38 followed by cisplatin (T --> C and S --> C). Median-effect analysis revealed synergistic cytotoxicity in seven of the eight cell lines used. In addition, a significant schedule-dependent synergistic cytotoxicity was found in three of the cell lines used, with C --> T (or C --> S) being the most active schedule. The formation and repair of total cisplatin-DNA adducts in the IGROV-1 ovarian cancer cell line and its cisplatin-resistant subline IGROV(CDDP) was not significantly affected by TPT on simultaneous incubation. In contrast, the number of cisplatin-DNA interstrand cross-links detected in the IGROV-1 and IGROV(CDDP) lines at certain time points was significantly lower after coincubation of the cells with TPT. Assessment of the cell-cycle distribution revealed an accumulation of cells in the G2/M phase after exposure to cisplatin. After exposure to TPT a different pattern was observed that was cell-type-specific and dependent upon the TPT concentration. Although up to 4-fold differences in topo I activity were observed in this panel of cell lines, these differences did not appear to be related to the synergy observed between cisplatin and TPT or SN-38. The observed synergy may at least partly be explained by the increased retention of cisplatin-DNA interstrand cross-links in the presence of topo I inhibitors.

Antineoplastic Combined Chemotherapy Protocols↗