Biomedical subjects

J Xing

Publications and source records attributed to J Xing.

At least 19 recordsLinked to original sources

PubMed2000

Kinesin has three nucleotide-dependent conformations. Implications for strain-dependent release.

Although crystallographic information is available on several nucleotide-induced states in myosin, little is known about the corresponding structural changes in kinesin, since a crystallographic model is only available for the kinesin:ADP complex. This makes it difficult to characterize at a molecular level the structural changes that occur in this motor through the course of its ATPase cycle. In this study, we report on the production of a series of single tryptophan mutants of a monomeric human kinesin motor domain, which demonstrate nucleotide-dependent changes in microtubule affinity that are similar to wild type. We have used these mutations to measure intramolecular distances in both strong and weak binding states, using fluorescence resonance energy transfer. This work provides direct evidence that movement of the switch II loop and helix are essential to mediate communication between the catalytic and microtubule binding sites, evidence that is supported as well by molecular modeling. Kinetic studies of fluorescent nucleotide binding to these mutants are consistent with these distance changes, and demonstrate as well that binding of ADP produces two structural transitions, neither of which are identical to that produced by the binding of ATP. This study provides a basis for understanding current structural models of the kinesin mechanochemical cycle.

Adenosine Diphosphate↗

PubMed2000

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle.

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.

Actins↗

PubMed2000

Investigations of the active site of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase using fluorescent labeled dolichyl-phosphate derivatives.

Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity.

Amino Acid Sequence↗

PubMed2000

Synthesis of dolichyl phosphate derivatives with fluorescent label at the omega-end of the chain, new tools to study protein glycosylation.

Derivatives of dolichyl phosphate (Dol-P) with 2-aminopyridine or 1-aminonaphtalene fluorophore groups at the omega-end of the chain were synthesized. These products serve as substrates for recombinant yeast Dol-P-mannose synthase. Fluorescence resonance energy transfer between a Trp residue of the enzyme and the 1-aminonaphtalene group of the Dol-P analogue was demonstrated.

1-Naphthylamine↗

PubMed2000

Allograft tolerance induced by cyclophosphamide without prior inoculation of donor cells--immune suppression and redirection.

OBJECTIVES: To determine the possibility and cellular mechanism of inducing allograft tolerance by multiple injection of a lower dose of cyclophosphamide without prior infusion of donor cells. METHODS AND RESULTS: Heterotopic heart grafts were performed in MHC mismatched strain combinations (C57/B6 vs. BALB/c). Cyclophosphamide (40 mg/kg) was given intravenously on days 0, 2, 4 and 7 without prior infusion of donor cells. Long-term (> 100 days) allograft survival with normal histology was achieved. The long-term survivors accepted the donor skin grafts, but rejected the third-party skin grafts. Cyclophosphamide treatment initially led to profound lymphocytopenia, inhibition of spontaneous blastogenesis and low levels of lymphocyte proliferation response to both donor and third-party antigens. Ultimately, donor-specific tolerance occurred demonstrated by normal levels of peripheral lymphocytes, spontaneous blastogenesis and lymphocyte proliferation response to third-party antigens, and low levels of lymphocyte proliferation response to donor antigen. A switch of cytokines from IFNgamma dominant to IL-4 dominant, a low level of IgM and a high level of IgG1 were found in tolerant mice. CONCLUSIONS: Allograft tolerance can be induced by a short course of cyclophosphamide without prior donor cell inoculation. Tolerance induced is characterized initially by non-specific immunosuppression, which progresses to donor-specific hyporesponsiveness associated with the development of a Th2 dominant cytokine response.

Animals↗

PubMed2000

Effect of urea on volatile generation from Maillard reaction of cysteine and ribose.

Urea occurs naturally in many food products, and its presence affects food quality. However, little is known about its impact on flavor generation in food production. In this study, the urea contents in beef, pork, and chicken were determined. The effects of urea and pH on thermal flavor generation were investigated using the model system of cysteine with ribose, which was heated to the roasting temperature of 180 degrees C for 2 h at pH 5 and pH 8.5. The results revealed relatively large amounts of urea in these meats and demonstrated that pH affects aroma generation. Volatiles identified from the reaction system of ribose and cysteine showed that sulfur-containing compounds such as thiophenes, thiazoles, and thiophenethiols were the most abundant compounds. The addition of urea into the reaction mixture caused the disappearance or reduction in content of some sulfur-containing compounds but resulted in the generation of several important nitrogen-containing volatiles, like pyrazine, methylpyrazine, 2,5- (and 2,6-)dimethylpyrazine and other alkylpyrazines, which are known to elicit roasty, nutty flavor notes. A plausible explanation for this phenomenon is that ammonia can be released from urea upon heating and the formed ammonia competes with hydrogen sulfide to react with Maillard reaction precursors to produce nitrogen-containing compounds such as alkylpyrazines.

Cysteine↗

PubMed2000

An interdomain distance in cardiac troponin C determined by fluorescence spectroscopy.

The distance between Ca2+-binding site III in the C-terminal domain and Cys35 in the N-terminal domain in cardiac muscle troponin C (cTnC) was determined with a single-tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12-residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48-49 A regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half-width >9 A) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 A for cTnC, but without an effect on the half-width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.

Amino Acid Substitution↗

PubMed2000

Memory activity of LIP neurons for sequential eye movements simulated with neural networks.

Many neurons in macaque lateral intraparietal cortex (LIP) maintain elevated activity induced by visual or auditory targets during tasks in which monkeys are required to withhold one or more planned eye movements. We studied the mechanisms for such memory activity with neural network modeling. Recurrent connections among simulated LIP neurons were used to model memory responses of LIP neurons. The connection weights were computed using an optimization procedure to produce desired outputs in memory-saccade tasks. One constraint for the training process is the "single-purpose" rule, which mimics the fact that once LIP neurons hold the memory activity of a saccade, they are insensitive to further stimuli until the motor action is completed. After training, excitatory connections were developed between units with similar preferred saccade directions, while inhibitory connections were formed between units with dissimilar directions. This "push-pull" mechanism enables the network to encode the next intended eye movement and is essential for programming sequential saccades. In simulating double saccades, the push-pull connections locked the on-going activity in the network for the first saccade until the saccade was made, then a new population of units became active to prepare for the second saccade. The simulated LIP neurons exhibited sensory responses and memory activities similar to those recorded in LIP neurons. We propose that push-pull recurrent connections might be the basic structure mediating the memory activity of area LIP in planning sequential eye movements.

Animals↗

PubMed2000

Models of the posterior parietal cortex which perform multimodal integration and represent space in several coordinate frames.

Many neurons in the posterior-parietal cortex (PPC) have saccadic responses to visual and auditory targets. The responses are modulated by eye position and head position. These findings suggest that PPC integrates multisensory inputs and may provide information about saccadic targets represented in different coordinate frames. In addition to an eyecentered output representation, PPC may also project to brain areas which contain head-centered and body-centered representations of the space. In this report, possible coordinate transformations in PPC were examined by comparing several sets of models of PPC, each having different representations in the output layer: (i) an eye-centered map only; (ii) a head-centered map only; (iii) an eye-centered map and a head-centered map; and (iv) an eye-centered map, a head-centered map, and a body-centered map. These output maps correctly encoded saccades to visual and auditory targets through training. The units in the hidden layers of the models exhibited the following properties: (1) The units had gain fields (GFs) for eye position, and also for head position if the model had a body-centered output representation; (2) As the result of the GF and the nonlinear activation function of the units, the hidden layers often employed "intermediate" coding, e.g., the hidden units coded targets partially in eye-centered coordinates and, partially, in head-centered coordinates; (3) Different types of coordinate transformations in these models were carried out by different relationships between the receptive fields (RFs) and the GFs of the hidden units; and (4) The properties of PPC neurons are in better accordance with the hidden units of the models that had multiple-output representations than the models that had only one single-output representation. In conclusion, the results show that the GF is an effective mechanism for performing coordinate transformations. The models also suggest that neurons with intermediate coding are to be expected in the process of coordinate transformations.

Algorithms↗

PubMed1999

Complement inhibition with an anti-C5 monoclonal antibody prevents hyperacute rejection in a xenograft heart transplantation model.

BACKGROUND: The present study was undertaken to determine whether anti-complement 5 (C5) monoclonal antibodies (mAb) prevent hyperacute rejection (HAR) in a rat-to-presensitized mouse heart transplantation model and whether these mAb, combined with cyclosporine (CsA) and cyclophosphamide (CyP), can achieve long-term graft survival. METHODS: BALB/c mice were presensitized with 2x10(7) splenocytes from Lewis rats 14 days before grafting. Heart grafts from Lewis rats were heterotopically transplanted into BALB/c mice. Presensitized mice were treated with either anti-C5 mAb or a combination of anti-C5 mAb, CsA, and CyP. Controls included: presensitized mice with no treatment, presensitized mice treated with either CsA + CyP or IgG, and nonpresensitized mice with either no treatment or with CsA + CyP treatment. RESULTS: Although typical features of HAR were evident in the presensitized grafts, the mAb completely inhibited complement activation and successfully prevented HAR. Despite complement inactivation, the graft was rejected on postoperative day 6 with acute vascular rejection (AVR) also known as delayed xenograft rejection (DXR). Notably, this type of rejection cannot be effectively overcome by CsA and CyP. CONCLUSIONS: We conclude that (1) anti-C5 mAb prevents HAR, (2) AVR/DXR still occurs when HAR is prevented by complement inactivation, and (3) AVR/DXR cannot be overcome by conventional immunosuppression. These data suggest that anti-C5 mAb may be valuable for preventing HAR in future clinical xenotransplantation and that additional interventions may be required to address AVR/DXR.

Acute Disease↗

PubMed1999

Conformation of the regulatory domain of cardiac muscle troponin C in its complex with cardiac troponin I.

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca(2+)-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca(2+). However, in the presence of bound cardiac TnI (cTnI), activator Ca(2+) induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC.cTnI complex has properties favorable for the Ca(2+)-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca(2+)-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship.

Animals↗

PubMed1999

How stable is stable? Function versus community composition.

The microbial community dynamics of a functionally stable, well-mixed, methanogenic reactor fed with glucose were analyzed over a 605-day period. The reactor maintained constant pH and chemical oxygen demand removal during this period. Thirty-six rrn clones from each of seven sampling events were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) for the Bacteria and Archaea domains and by sequence analysis of dominant members of the community. Operational taxonomic units (OTUs), distinguished as unique ARDRA patterns, showed reproducible distribution for three sample replicates. The highest diversity was observed in the Bacteria domain. The 16S ribosomal DNA Bacteria clone library contained 75 OTUs, with the dominant OTU accounting for 13% of the total clones, but just 21 Archaea OTUs were found, and the most prominent OTU represented 50% of the clones from the respective library. Succession in methanogenic populations was observed, and two periods were distinguished: in the first, Methanobacterium formicicum was dominant, and in the second, Methanosarcina mazei and a Methanobacterium bryantii-related organism were dominant. Higher variability in Bacteria populations was detected, and the temporal OTU distribution suggested a chaotic pattern. Although dominant OTUs were constantly replaced from one sampling point to the next, phylogenetic analysis indicated that inferred physiologic changes in the community were not as dramatic as were genetic changes. Seven of eight dominant OTUs during the first period clustered with the spirochete group, although a cyclic pattern of substitution occurred among members within this order. A more flexible community structure characterized the second period, since a sequential replacement of a Eubacterium-related organism by an unrelated deep-branched organism and finally by a Propionibacterium-like species was observed. Metabolic differences among the dominant fermenters detected suggest that changes in carbon and electron flow occurred during the stable performance and indicate that an extremely dynamic community can maintain a stable ecosystem function.

Bacteria↗

PubMed1998

Regulation of the Escherichia coli sheA gene and characterization of its encoded hemolytic activity.

Escherichia coli K-12 carries the cryptic hemolysin gene sheA which is under the control of positive and negative transcriptional regulators. The objectives of the present study were to further analyze the regulation of the sheA gene in E. coli, to compare the sheA genes from E. coli K-12 and a pathogenic E. coli strain, and to characterize the SheA hemolytic activity. Northern blot analysis demonstrated that the transcriptional regulator SlyA activates the E. coli K-12 sheA gene. The main transcriptional start site of the sheA gene was 56 nucleotides upstream from the start codon as determined by primer extension analysis. The sheA genes from E. coli K-12 and a pathogenic E. coli strain were identical. SheA hemolytic activity was cell associated and Ca2+ independent.

Animals↗

PubMed1998

Structural and kinetic studies of phosphorylation-dependent regulation in smooth muscle myosin.

In this study, we have examined the mechanism of phosphorylation-dependent regulation in smooth muscle myosin through the use of structural and kinetic methodologies applied to several myosin fragments. Fluorescence anisotropy decay measurements demonstrate that regulatory light chain phosphorylation significantly reduces the rotational correlation time of regulatable myosin preparations, whereas minimally regulated ones show little effect in this assay. Sedimentation equilibrium studies show that the regulatory domain can dimerize with a dissociation constant that is unaffected by regulatory light chain phosphorylation. Finally, kinetic studies on the interactions of myosin-ADP constructs with actin are also consistent with a model in which interactions occur between the two heads, which are lost with regulatory light chain phosphorylation. We propose that in the absence of regulatory light chain phosphorylation, the two heads of myosin interact with each other, due to a weak intrinsic dimerization of the regulatory domains that is significantly stabilized by the proximal rod. Regulatory light chain phosphorylation abolishes the stabilizing effect of the proximal rod, leading to a loss of this interaction.

Actins↗

PubMed1998

Calcium binding to the regulatory domain of skeletal muscle troponin C induces a highly constrained open conformation.

We have used fluorescence resonance energy transfer to investigate the conformation of the apo and calcium-loaded states of the regulatory N-terminal domain of full-length troponin C mutants from skeletal muscle. The mutants studied each contained a single tryptophan residue (position 22 or 90) and a single cysteine residue (position 52 or 101). The intrinsic fluorophore in each mutant served as an energy donor and the cysteine was conjugated to the acceptor probe 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid. The distributions of two intersite distances (between residues 22 and 52, and residues 90 and 52) were broad in the apo state, indicative of considerable structural dynamics. These distributions were shifted to longer distances and considerably sharpened in the calcium-loaded state. The shifts to longer distances by 8 to 11 A indicate a calcium-induced opening of the N-terminal domain conformation. The transition of the troponin C structure from a closed conformation to an open conformation is accompanied by a substantial reduction of structural fluctuations that dominate in the apo structure as evidenced from the large decrease of the widths of the distributions. This highly constrained open conformation is required as part of the structural basis to facilitate productive interaction between troponin C and troponin I to trigger contraction in skeletal muscle.

Animals↗

PubMed1998

Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket.

A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin has been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myosin filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To test this hypothesis, loops derived from different myosin II isoforms that display a range of actin filament sliding velocities were inserted into a smooth muscle myosin backbone. Chimeric myosins were produced by baculovirus/Sf9 cell expression. Although the nature of this loop affected the rate of ADP release (up to 9-fold), in vitro motility (2.7-fold), and the Vmax of actin-activated ATPase activity (up to 2-fold), the properties of each chimera did not correlate with the relative speed of the myosin from which the loop was derived. Rather, the rate of ADP release was a function of loop size/flexibility with the larger loops giving faster rates of ADP release. The rate of actin filament translocation was altered by the rate of ADP release, but was not solely determined by it. Through a combination of solute quenching and transient fluorescence measurements, it is concluded that, as the loop gets smaller, access to the nucleotide pocket is more restricted, ATP binding becomes less favored, and ADP binding becomes more favored. In addition, the rate of ATP hydrolysis is slowed.

Actins↗

PubMed1998

Stimulatory effects of hepatocyte growth factor on hemopoiesis of SCF/c-kit system-deficient mice.

In this study, we report that W/W mutant mice, which have severe macrocytic anemia caused by a deficit of extracellular domain in c-kit molecules and therefore die perinatally, have hemopoietic stem cells (HSCs) and mature hematolymphoid cells in the bone marrow (BM), thymus, and spleen, although there are significant decreases in cell counts. Moreover, the mitogen-induced proliferative response, mixed lymphocyte reaction, and anti-SRBC plaque formation of spleen cells in W/W mice are similar to those in age-matched +/? littermates and normal mice, suggesting that the SCF/c-kit system is necessary for cell proliferation but not essential for HSCs to differentiate. We next examine the stimulatory effects of hepatocyte growth factor (HGF) on hemopoiesis in W/W mice. HGF has a stimulatory effect on the colony formation (CFU-C) of W/W BM cells when cultured using either a methylcellulose assay (containing cytokines) or a long-term culture (LTC) assay. A similar stimulatory effect of HGF is observed in the other W or SI locus-mutant mice (W/Wv and SI/SId mice), which show less severe anemia than W/W. The numbers of nonadherent cells and cobblestone colonies significantly increase in the LTCs using their BM cells. In addition, in vivo administration of HGF shows a transient increase in the CFU-C counts in BM cells and peripheral blood cells. RBC, WBC, and platelet counts also increased. These results suggest that the SCF/c-kit system is not essential to hemopoiesis but that a compensatory system such as the HGF/c-met system functions in the SCF/c-kit system-deficient mice.

Anemia, Macrocytic↗

PubMed1998

Pathogenicity of an enterotoxigenic Escherichia coli hemolysin (hlyA) mutant in gnotobiotic piglets.

Pigs infected with hemolytic F4(+) strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4(+) enterotoxigenic E. coli to cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene (hlyA) did not decrease the incidence of septicemia in the gnotobiotic piglet model.

Animals↗