Biomedical subjects

L Yu

Publications and source records attributed to L Yu.

At least 343 records · Page 19Linked to original sources

PubMed1998

Gp91(phox) is the heme binding subunit of the superoxide-generating NADPH oxidase.

The phagocyte NADPH oxidase flavocytochrome b558 is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91(phox), and a nonglycosylated subunit, p22(phox). It contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O2), resulting in the generation of superoxide ion (O2-). However, the location of the hemes within the flavocytochrome heterodimer remains controversial. In this study, we have used transgenic COS7 cell lines expressing gp91(phox), p22(phox), or both polypeptides to examine the relative role of each flavocytochrome b558 subunit in heme binding and O2- formation. A similar membrane localization was observed when gp91(phox) and p22(phox) were either expressed individually or coexpressed, as analyzed by confocal microscopy and immunoblotting of subcellular fractions. Spectral analysis of membranes prepared from COS7 cell lines expressing either gp91(phox) or both gp91(phox) and p22(phox) showed a b-type cytochrome with spectral characteristics identical to those of human neutrophil flavocytochrome b558. In contrast, no heme spectrum was detected in wild-type COS7 membranes or those containing only p22(phox). Furthermore, redox titration studies suggested that two heme groups were contained in gp91(phox) expressed in COS7 membranes, with midpoint potentials of -264 and -233 mV that were very similar to those obtained for neutrophil flavocytochrome b558. These results provide strong support for the hypothesis that gp91(phox) is the sole heme binding subunit of flavocytochrome b558. However, coexpression of gp91(phox) and p22(phox) in COS7 membranes was required to support O2- production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome b558.

Animals↗

PubMed1998

Inhibitor binding changes domain mobility in the iron-sulfur protein of the mitochondrial bc1 complex from bovine heart.

We have analyzed crystal structures of cytochrome bc1 complexes with electron transfer inhibitors bound to the ubiquinone binding pockets Qi and/or Qo in the cytochrome b subunit. The presence or absence of the Qi inhibitor antimycin A did not affect the binding of the Qo inhibitors. Different subtypes of Qo inhibitors had dramatically different effects on the mobility of the extramembrane domain of the iron-sulfur protein (ISP): Binding of 5-undecyl-6-hydroxy-4, 7-dioxobenzothiazol and stigmatellin (subtype Qo-II and Qo-III, respectively) led to a fixation of the ISP domain on the surface of cytochrome b, whereas binding of myxothiazol and methoxyacrylate-stilbene (subtype Qo-I) favored release of this domain. The native structure has an empty Qo pocket and is intermediate between these extremes. On the basis of these observations we propose a model of quinone oxidation in the bc1 complex, which incorporates fixed and loose states of the ISP as features important for electron transfer and, possibly, also proton transport.

Animals↗

PubMed1998

Polysomnographic characteristics of sleep disorders in chronic pelvic pain.

Objective: We performed a pilot study to determine if patients with chronic pelvic pain have a consistent or unique sleep disorder by a polysomnographic sleep study.Methods: Overnight polysomnography was performed prospectively in 11 women with chronic pelvic pain (ages 33-44 years, mean 35). None of the women were taking sedating or psychoactive medications in the month prior to testing. Sleep indices were derived using polysomnography scoring guidelines currently recommended by the American Sleep Disorder Association.Results: Statistical comparisons with age-matched normative data (Williams and Karacan, 1974) revealed increased stage 1 sleep (P < 10(-7)), reduced REM sleep (P < 10(-6)), increased slow wave sleep (P <.001), and REM sleep onset latencies (P <.01) in women with chronic pelvic pain. One woman had periodic leg movements, but none had obstructive sleep apnea.Conclusion: Previously, sleep disorders in patients with chronic pelvic pain (CPP) have been characterized by history alone. We have characterized sleep disturbances in individuals with CPP by polysomnographic sleep studies. Patients with CPP have different sleep patterns than patients with depression. Our preliminary findings and review of the literature conclude that patients with CPP have a unique sleep disorder. We are currently studying the effects of low doses of amitriptyline in altering sleep patterns to determine if there is a clinical response.

Journal Article↗

PubMed1998

Correlation of color Doppler flow in the prostate with tissue microvascularity.

BACKGROUND: The pathophysiology of increased color Doppler (CD) flow has not previously been addressed in histologic evaluations of microvascular parameters. In this study, the authors attempted to define the differences between benign and malignant biopsy cores found in regions of the prostate with normal and high CD flow. METHODS: Forty patients were retrospectively chosen for CD histologic comparison, each of whom had a core from a sextant biopsy with the following characteristics: malignant tissue with distinct increased CD flow (n=11), malignant tissue with normal CD flow (n=10), benign tissue with distinctly increased CD flow (n=9), or benign tissue with normal CD flow (n=10). All biopsy cores were stained with factor VIII-related antigen to identify microvasculature and to determine the number of microvessels per square millimeter (mm2) in an average cross-sectional area of microvessels, the percentage of tissue occupied by microvasculature, and the Gleason score. RESULTS: In biopsies of benign tissue, high CD flow was associated with greater numbers (P < 0.025) of vessels of similar size than in normal flow benign biopsies. Biopsies of malignant tissue contained significantly greater numbers (P < 0.01) of much smaller vessels (P < 0.0005) than biopsies of benign tissue. In biopsies of malignant tissue, no significant differences in microvasculature parameters were noted between high and normal CD flow, yet biopsies with high CD flow had average Gleason score of 6.7 compared with only 5.9 for biopsies with normal CD flow (P < 0.025). CONCLUSIONS: Increased CD flow in biopsies of benign tissue was correlated with a greater number of vessels/mm2, yet all biopsies of malignant tissue had more vessels/mm2 than those of benign tissue. Increased CD flow in biopsies of malignant tissue cannot be explained by standard microvasculature analysis but significantly guides biopsies to regions with a greater Gleason score.

Humans↗

PubMed1998

Structural basis of functions of the mitochondrial cytochrome bc1 complex.

The crystal structure of the cytochrome bc1 complex (ubiquinol-cytochrome c reductase) from bovine heart submitochondria was determined at 2.9 A resolution. The bc1 complex in crystal exists as a closely interacting dimer, suggesting that the dimer is a functional unit. Over half of the mass of the complex, including subunits core 1 and core 2, are on the matrix side of the membrane, while most of the cytochrome b subunit is located within the membrane. There are 13 transmembrane helices in each monomer, eight of them belonging to cytochrome b. Two large cavities are made of the transmembrane helices D, C, F and H in one monomer and helices D' and E' from the other monomer of cytochrome b, and the transmembrane helices of c1, iron-sulfur protein (ISP), and subunits 10 and 11. These cavities provide entrances for ubiquinone or inhibitor and connect the Qi pocket of one monomer and the Qo pocket of the other monomer. Ubiquinol made at the Qi site of one monomer can proceed to the nearby Qo site of the other monomer without having to leave the bc1 complex. The soluble parts of cytochrome c1 and ISP, including their redox prosthetic groups, are located on the cytoplasmic side of the membrane. The distances between the four redox centers in the complex have been determined, and the binding sites for several electron transfer inhibitors have been located. Structural analysis of the protein/inhibitor complexes revealed that the extramembrane domain of the Rieske iron-sulfur protein may undergo substantial movement during the catalytic cycle of the complex. The Rieske protein movement and the larger than expected distance between FeS and cytochrome c1 heme suggest that electron transfer reaction between FeS and cytochrome c1 may involve movements or conformational changes in the soluble domain of iron-sulfur protein. The inhibitory function of E-beta-methoxyacrylate-stilbene and myxothiazol may result from the increase of mobility in ISP, whereas the function of stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole may result from the immobilization of ISP.

Animals↗

PubMed1998

[Chromosome assignment of a novel microsatellite polymorphic marker (D14S1435) by fluorescence in situ hybridization].

OBJECTIVE: Chromosome assignment of a novel microsatellite marker (D14S1435) containing CA repeats and showing 11 alleles and 0.85 of PIC value in Chinese population) isolated from microdissected human chromosome 14q24.3 DNA library by fluorescence in situ hybridization(FISH). METHODS: The polymorphic marker was used as probe to screen a human Lambda/DASH genome library, a positive clone was isolated and the recombinant phage DNA was extracted as primary material. The insert was recovered through BamH I digestion to complete and low melting point agarose gel electro- phoresis, after processed with Sau3A I the probe was prepared by linker- catch PCR method and the following fluorescence in situ hybridization(FISH) was conducted to metaphase chromosomes. RESULTS: this novel STR was precisely mapped to human chromosome 14q24.3, and this demonstrated the validity of isolating chromosome band-specific genetic markers from probe pool made by chromosome microdissection. CONCLUSION: Confirmed by chromosome fluorescence in situ hybridization, the novel STR obtained by chromosome microdissection, PCR and subcloning method was really from the micro- dissected region. This not only increases the numbers of genetic markers used for linkage analysis in the region,but also provides a novel valuable genetic marker for the gene diagnosis and gene cloning of the possible disease-causing genes located in the region of 14q24.3.

Chromosome Mapping↗

PubMed1998

Site-directed mutations in the vnd/NK-2 homeodomain. Basis of variations in structure and sequence-specific DNA binding.

Secondary structures, DNA binding properties, and thermal denaturation behavior of six site-directed mutant homeodomains encoded by the vnd/NK-2 gene from Drosophila melanogaster are described. Three single site H52R, Y54M, and T56W mutations, two double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutation were investigated. These positions were chosen based on their variability across homeodomains displaying differences in secondary structure and DNA binding specificity. Multidimensional NMR, electrophoretic mobility shift assays, and circular dichroism spectropolarimetry studies were carried out on recombinant 80-amino acid residue proteins containing the homeodomain. Position 56, but more importantly position 56 in combination with position 52, plays an important role in determining the length of the recognition helix. The H52R mutation alone does not affect the length of this helix but does increase the thermal stability. Introduction of site mutations at positions 52 and 56 in vnd/NK-2 does not modify their high affinity binding to the 18-base pair DNA fragment containing the vnd/NK-2 consensus binding sequence, CAAGTG. Site mutations involving position 54 (Y54M, Y54M/T56W, and H52R/Y54M/T56W) all show a decrease of 1 order of magnitude in their binding affinity. The roles in structure and sequence specificity of individual atom-atom interactions are described.

Amino Acid Sequence↗

PubMed1998

A primitive pathway of porphyrin biosynthesis and enzymology in Desulfovibrio vulgaris.

Culture of Desulfovibrio vulgaris in a medium supplemented with 5-aminolevulinic acid and L-methionine-methyl-d3 resulted in the formation of porphyrins (sirohydrochlorin, coproporphyrin III, and protoporphyrin IX) in which the methyl groups at the C-2 and C-7 positions were deuterated. A previously unknown hexacarboxylic acid was also isolated, and its structure was determined to be 12, 18-didecarboxysirohydrochlorin by mass spectrometry and 1H NMR. These results indicate a primitive pathway of heme biosynthesis in D. vulgaris consisting of the following enzymatic steps: (i) methylation of the C-2 and C-7 positions of uroporphyrinogen III to form precorrin-2 (dihydrosirohydrochlorin); (ii) decarboxylation of acetate groups at the C-12 and C-18 positions of precorrin-2 to form 12,18-didecarboxyprecorrin-2; (iii) elimination of acetate groups of the C-2 and C-7 positions of 12,18-didecarboxyprecorrin-2 to form coproporphyrinogen III; and (iv) conversion of coproporphyrinogen III to protoporphyrin IX via protoporphyrinogen IX. We isolated the following three enzymatic activities involved in steps i-iii from the soluble fraction of the cells by anion-exchange chromatography: S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase, precorrin-2 12,18-acetate decarboxylase, and 12, 18-didecarboxyprecorrin-2 2,7-decarboxymethylase; all enzymic products were converted into autooxidized methyl esters and analyzed by thin-layer chromatography, UV-visible (UV-VIS) absorption, and mass spectrometry. The enzymatic reactions in D. vulgaris shed new light on porphyrin biosynthesis at an early stage in the evolution of prokaryotes.

Chromatography, Thin Layer↗

PubMed1998

Probing the role of the carboxyl terminus of the gp91phox subunit of neutrophil flavocytochrome b558 using site-directed mutagenesis.

Site-directed mutagenesis was used to generate a series of substitutions and deletions in the carboxyl-terminal 11 residues of gp91phox, the 91-kDa subunit of the phagocyte NADPH oxidase flavocytochrome b558. This region encompasses 559RGVHFIF565, implicated as a contact point for the cytosolic oxidase subunit p47phox during oxidase activation, and a carboxyl-terminal phenylalanine (Phe570), which corresponds in position to a highly conserved aromatic residue that interacts with the flavin group in the ferredoxin-NADP+ reductase flavoenzyme family, of which gp91phox is a member. Mutant proteins were expressed in human myeloid leukemia cells which lack expression of endogenous gp91phox due to targeted disruption of the X-linked gp91phox gene. Although specific residues within 559RGVHFIF565 had previously been identified by alanine scanning as essential for peptide inhibition of oxidase activity in a cell-free assay, comparable substitutions in the gp91phox polypeptide had either no or only a modest effect on oxidase activity in whole cells. Replacement of nonpolar with polar or charged residues had greater effects on oxidase activity, but were also associated with decreased gp91phox expression, suggesting that overall protein structure was perturbed. No stable gp91phox protein was detected upon deletion of the terminal 11 amino acids. Alanine substitution or deletion of the carboxyl-terminal Phe570 in gp91phox resulted in a 2-fold reduction in superoxide production. This contrasts with a approximately 300-800-fold reduction reported for comparable mutations in pea ferredoxin-NADP+ reductase, which suggests that structural or functional differences exist between the carboxyl terminus of gp91phox and other ferredoxin-NADP+ reductases.

Amino Acid Sequence↗

PubMed1998

An important role for the retinoblastoma protein in staurosporine-induced G1 arrest in murine embryonic fibroblasts.

In this study, we investigated the molecular basis of the ability of staurosporine to induce G1 arrest in murine embryonic fibroblasts (MEFs). We used MEFs from transgenic mice lacking several negative regulators of the G1/S phase transition including cells from mice lacking p53, p21, retinoblastoma (Rb), or p16 genes. We found that p53 function was not essential for staurosporine-induced G1 arrest. In contrast, MEFs from mice lacking Rb genes showed approximately a 70% reduced capacity to arrest in the G1 phase following staurosporine treatment. In support of a role for Rb in staurosporine-induced G1 arrest, rat embryonic fibroblasts stably overexpressing cyclin D1/Cdk4(R24C) exhibited approximately a 50% reduced G1 arrest response to staurosporine. The role of Rb in determining the degree of staurosporine-induced G1 arrest did not depend on the function of the cyclin-dependent kinase inhibitors p16 or p21 because MEFs lacking either of these genes were still capable of undergoing G1 arrest following staurosporine exposure. Our studies provide evidence of an important role for the Rb protein in determining the degree of staurosporine-induced G1 arrest in the first cell cycle.

Animals↗

PubMed1998

[Rapid selection of the gradually shortened DNA clones by using PCR amplification].

OBJECTIVE: To establish a simple technique with which the gradually shortened DNA clones can be selected rapidly and accurately so that the sequencing and identification of the full length cDNA or longer DNA fragments can be performed as quick as possible. METHODS: Primers were designed according to the sequence flanking the multiple cloning site of pGEM vector and were used to amplify the inserted fragments in a series of deletion sub-clones originated from a clone. with longer DNA insert fragment. These sub-clones which contain the gradually shortened fragments can be selected directly. This method was compared with the routine method in which the restriction endonuclease was used to digest DNA samples. RESULTS: A series of deletion sub-clones were identified accurately with this new method and the cycle sequencing could be performed on these PCR products as well. This method was characteristic of more accurate, more simple and less time-consuming, compared with the routine method. CONCLUSION: The method presented is very effective for rapid selection of the gradually shortened inserted fragments constructed in pGEM vector. It can greatly save time and materials, and the strategy in the report could also fit for selecting the clones containing gradually shortened fragments in other plasmid vectors.

DNA, Complementary↗

PubMed1998

Reconstitution of cytochrome b-560 (QPs1) of bovine heart mitochondrial succinate-ubiquinone reductase.

The QPs1 subunit of bovine heart mitochondrial succinate-ubiquinone reductase was overexpressed in Escherichia coli DH5 alpha cells as a glutathione S-transferase fusion protein (GST-QPs1) using the expression vector, pGEX/QPs1. The yield of soluble active recombinant GST-QPs1 fusion protein depends on the IPTG concentration, induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth with 0.5 mM IPTG at 27 degrees C in an enriched medium containing betaine and sorbitol. QPs1 is released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPs1 shows one protein band in SDS-polyacrylamide gel electrophoresis corresponding to subunit III of mitochondrial succinate-ubiquinone reductase. However, partial N-terminal amino acid sequence analysis of recombinant QPs1 shows two extra amino acid residues, glycine and serine, at the N-terminus of mature QPs1, resulting from the recombinant manipulation. When isolated recombinant QPs1 is dispersed in 0.01% dodecyl maltoside, it is in a highly aggregated form with an apparent molecular mass of over 1 million. Recombinant GST-QPs1 contains little cytochrome b-560 heme. However, addition of hemin chloride restores the spectral characteristics of cytochrome b-560. Cytochrome b-560 restoration varies with the amount of hemin used. Maximum reconstitution is obtained when the molar ratio of heme to fusion protein used in the system is 0.6. Reconstituted cytochrome b-560 shows a EPR signal at g = 2.91 which corresponds to one of the EPR signals of cytochrome b-560 in a QPs preparation. When GST-QPs1 with reconstituted cytochrome b-560 is treated with thrombin to cleave GST from QPs1, no change in the absorption and EPR characteristics of cytochrome b-560 is observed, indicating that the bis-histidine ligands of reconstituted cytochrome b-560 are provided by QPs1.

Amino Acid Sequence↗

PubMed1998

Supercoiling affects the accessibility of glutathione to DNA-bound molecules: positive supercoiling inhibits calicheamicin-induced DNA damage.

DNA superhelical tension, an important feature of genomic organization, is known to affect the interactions of intercalating molecules with DNA. However, the effect of torsional tension on nonintercalative DNA-binding chemicals has received less attention. We demonstrate here that the enediyne calicheamicin gamma1I, a strand-breaking agent specific to the minor groove, causes approximately 50% more damage in negatively supercoiled plasmid DNA than in DNA with positive superhelicity. Furthermore, we show that the decrease in damage in positively supercoiled DNA is controlled at the level of thiol activation of the drug. Our results suggest that supercoiling may affect both the activity of nonintercalating genotoxins in vivo and the accessibility of glutathione and other small physiologic molecules to DNA-bound chemicals or reactions occurring in the grooves of DNA.

Aminoglycosides↗

PubMed1998

A homology identification method that combines protein sequence and structure information.

A new method is presented for identifying distantly related homologous proteins that are unrecognizable by conventional sequence comparison methods. The method combines information about functionally conserved sequence patterns with information about structure context. This information is encoded in stochastic discrete state-space models (DSMs) that comprise a new family of hidden Markov models. The new models are called sequence-pattern-embedded DSMs (pDSMs). This method can identify distantly related protein family members with a high sensitivity and specificity. The method is illustrated with trypsin-like serine proteases and globins. The strategy for building pDSMs is presented. The method has been validated using carefully constructed positive and negative control sets. In addition to the ability to recognize remote homologs, pDSM sequence analysis predicts secondary structures with higher sensitivity, specificity, and Q3 accuracy than DSM analysis, which omits information about conserved sequence patterns. The identification of trypsin-like serine proteases in new genomes is discussed.

Amino Acid Sequence↗

PubMed1998

The mechanical effectiveness of erect and "bent-hip, bent-knee" bipedal walking in Australopithecus afarensis.

It is universally accepted that the postcranial skeleton of the early hominid Australopithecus afarensis shows adaptations, or at least exaptations, towards bipedalism. However, there continues to be a debate concerning the likely form of terrestrial bipedality: whether gait was erect, like our own, or "bent-hip, bent-knee" like the bipedalism of living chimpanzees. In this study we use predictive dynamic modelling to assess the mechanical effectiveness of AL-288-1 under both hypotheses, on the basis of data on segment proportions from the literature. AL-288-1's proportions are incompatible with the kinematics of chimpanzee bipedalism, but compatible with the kinematics of either erect or "bent-hip, bent-knee" human gait. In the latter case, neither the ankle nor the knee joint would have contributed substantial mechanical work to propulsion of the body, and net energy absorption is predicted for these joints, which would have resulted in increased heat load. Such an ineffective gait is unlikely to have lead to selection for "bipedal" features in the postcranial skeleton.

Animals↗

PubMed1998

Langerhans cell histiocytosis: unusual skeletal manifestations observed in thirty-four cases.

OBJECTIVE: Unusual manifestations are occasionally encountered in Langerhans cell histiocytosis and may be a source of confusion. The objective of this study was to determine the frequency of occurrence of the unusual manifestations in our case material. DESIGN AND PATIENTS: Thirty-four children, average age 4.4 years (range 3 months to 16 years) with 262 skeletal lesions of biopsy-proven Langerhans cell histiocytosis (LCH) were retrospectively reviewed to determine the frequency of occurrence of unusual manifestations defined either as an atypical location of a skeletal lesion or an atypical radiographic appearance of the lesion. RESULTS: Twenty-four unusual lesions were found in this retrospective review. Among these were epiphyseal lesions, transphyseal lesions, extracranial 'button' sequestra, posterior vertebral arch lesions, dural extension of vertebral lesions, and fluid-fluid levels. The finding of fluid-fluid levels has not previously been described in the radiologic literature. Involvement of unusual sites included clavicles and small bones of the hands and feet. CONCLUSIONS: Radiographic, computed tomographic, and magnetic resonance imaging of LCH yield a variety of unusual manifestations. Recognition of these varied appearances of LCH may prevent confusion of such appearances with other pathologic processes. When the unusual manifestation occurs as the initial finding of the disease, LCH should be included in the differential diagnosis.

Adolescent↗

PubMed1998

Epitope mapping of monoclonal antibodies by mass spectrometry: identification of protein antigens in complex biological systems.

We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an extract of the lysate of cells overexpressing the Ty1 Gag protein. MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope-containing peptide fragment. Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells. Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS. To our knowledge, this constitutes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system.

Antibodies, Monoclonal↗

PubMed1998

Determination of the glass properties of D-mannitol using sorbitol as an impurity.

Due to its strong tendency to crystallize, the glass properties of mannitol cannot be measured directly. However, because mannitol can exist in a fully or partially amorphous state in drug formulations, it is important to determine the glass properties of mannitol. We obtained the glass properties of mannitol by introducing a small amount of sorbitol, an isomer of mannitol, to delay the onset of crystallization. Extrapolation to zero sorbitol concentration yielded the following properties for the mannitol glass: Tg onset = 10.7(o)C, Tg midpoint= 12.6( o)C, Tg end = 18.4 degreesC and DeltaCp = 1.27 J/g/K. In addition, we estimated the following parameters of the mannitol glass from the width of glass transition using the results of Moynihan (J. Am. Ceram. Soc. 1993, 76, 1081) and Angell (J. Phys. Chem. 1994, 98, 13780): DeltaH (at Tg onset) = 103 kcal/mol, D = 11, and T0 = 222 K. The value of T0 is consistent with the Kauzmann temperature TK (236 K) obtained calorimetrically. The properties of the mannitol glass may be useful for predicting the behavior of amorphous mixtures containing mannitol.

Calorimetry, Differential Scanning↗