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PubMed2004

Understanding and improving transgene stability and expression in insects for SIT and conditional lethal release programs.

Genetically transformed insect pests provide significant opportunities to create strains for improved sterile insect technique and new strategies based on conditional lethality. A major concern for programs that rely on the release of transgenic insects is the stability of the transgene, and maintenance of consistent expression of genes of interest within the transgene. Transgene instability would influence the integrity of the transformant strain upon which the effectiveness of the biological control program depends. Loss or intra-genomic transgene movement would result in strain attributes important to the program being lost or diminished, and the mass-release of such insects could significantly exacerbate the insect pest problem. Instability resulting in intra-genomic movement may also be a prelude to inter-genomic transgene movement between species resulting in ecological risks. This is less of a concern for short-term releases, where transgenic insects are not expected to survive in the environment beyond two or three generations. Transgene movement may occur, however, into infectious agents during mass-rearing, and the potential for movement after release is a possibility for programs using many millions of insects. The primary methods of addressing potential transgene instability relate to an understanding of the vector system used for gene transfer, the potential for its mobilization by the same or a related vector system, and methods required to identify transformants and determine if unexpected transgene movement has occurred. Methods also exist for preventing transposon-mediated mobilization, by deleting or rearranging vector sequences required for transposition using recombination systems. Stability of transgene expression is also a critical concern, especially in terms of potential epigenetic interactions with host genomes resulting in gene silencing that have been observed in plants and fungi, and it must be determined if this or related phenomena can occur in insects.

Animals↗

PubMed1995

The measurement of ionic conductivities and mobilities of certain less common organic ions needed for junction potential corrections in electrophysiology.

Junction potential corrections are often required in electrophysiological measurements, particularly when using the patch-clamp technique. The ionic conductivities and mobilities of certain less common organic ions were measured for use in junction potential corrections in electrophysiological measurements. The mobilities of these ions relative to K+ were: aspartate (0.30), choline (0.51), gluconate (0.33), glutamate (0.26), HEPES (0.30), isethionate (0.52), MES (0.37), MOPS (0.35), N-methyl-D-glucamine (0.33), and Tris (0.40). In addition, junction potentials were directly measured for a number of typical simplified patch-clamp solutions containing these ions. The measured junction potentials generally agreed well with the values calculated using the Generalized Henderson Equation with the appropriate ionic mobility. The accuracy of the measurements is discussed.

Electric Conductivity↗

PubMed2025

Whole-Genome Sequencing Uncovers Chromosomal and Plasmid-Borne Multidrug Resistance and Virulence Genes in Poultry-Associated Escherichia coli from Nigeria.

BACKGROUND: Broad and unregulated antibiotic use in livestock production, particularly poultry farming, has increased the development and persistence of multidrug-resistant (MDR) bacterial strains in animals. These resistant pathogens and their antibiotic resistance genes (ARGs) can spread to humans through environmental exposure and the food chain, posing serious public health risks. Whole-genome sequencing (WGS), alongside phenotypic antimicrobial susceptibility testing (AST), enables a comprehensive understanding of resistance mechanisms and informs antimicrobial stewardship strategies, particularly in resource-limited settings. AIM: This study aimed to characterize the phenotypic and genotypic antimicrobial resistance profiles, plasmid content, and virulence factors of an MDR E. coli strain (S3) isolated from a poultry farm in Enugu State, Nigeria, to elucidate potential risks to public health and the role of poultry as a reservoir for resistance determinants. METHODS: E. coli strain S3 was isolated from chicken droppings using standard microbiological methods and confirmed by MALDI-TOF mass spectrometry. AST was assessed using disc diffusion and broth microdilution to determine minimum inhibitory concentrations (MICs) for ten antibiotics across multiple classes. WGS was performed with a hybrid approach combining Illumina and Nanopore platforms, followed by genome assembly and annotation. ARGs, plasmid replicons, and virulence factors were identified in silico using AMRFinderPlus, starAMR, RGI/CARD, PlasmidFinder, MOB-suite, and the Virulence Factor Database (VFDB). RESULTS: Phenotypic testing revealed extensive resistance, with complete resistance to six of seven tested antibiotics (cefotaxime, ampicillin, erythromycin, gentamicin, ciprofloxacin, and doxycycline). MICs exceeded clinical breakpoints for multiple classes, confirming an MDR phenotype. Genome analysis indicated a 5.33 Mb genome distributed across five contigs, including one chromosome and four plasmid-associated contigs. The strain harboured numerous ARGs, including bla CTX-M-15, bla OXA-1, bla TEM-1, aac(6')-Ib-cr, aadA5, aph(3")-Ib, sul1/sul2, tet(A), dfrA17, and mph(A), co-localized on plasmids indicative of horizontal gene transfer (HGT) potential. Plasmid types included Col156, IncF, and two rep clusters. Virulence profiling revealed genes associated with adhesion (pap cluster, ECP), iron acquisition (enterobactin, yersiniabactin, aerobactin, heme uptake), and toxins (sat, senB), highlighting the isolate's potential for urinary tract and intestinal infections. CONCLUSION: This study highlights the significant role of poultry-associated bacteria as reservoirs of AMR genes, particularly those harboured on mobile plasmids with potential for HGT. E. coli strain S3 exhibits extensive multidrug resistance and carries a complex plasmid repertoire facilitating horizontal transfer of ARGs. Coupled with a rich virulence gene profile, this strain underscores the public health risk posed by poultry-associated E. coli in Nigeria. These findings demonstrate the urgent need for stringent antimicrobial stewardship, regulatory oversight, and genomic surveillance in poultry production milieus to mitigate the dissemination of MDR pathogens.

Escherichia coli↗

PubMed2004

In vivo-mobilized kidney dendritic cells are functionally immature, subvert alloreactive T-cell responses, and prolong organ allograft survival.

BACKGROUND: Migratory antigen-presenting cells resident in kidneys may have tolerogenic potential. Difficulties inherent in their isolation have limited their characterization. The authors examined the phenotype and function of murine kidney dendritic cells (DC) mobilized in vivo by systemic administration of fms-like tyrosine 3 kinase ligand (Flt3L). METHODS: Monoclonal antibody staining was used to characterize DC subsets in situ, immediately after their isolation, and after lipopolysaccharide stimulation. Cytokine and CC chemokine receptor (CCR) gene expression was analyzed by RNase protection assay. Mixed leukocyte reactions were performed to assess DC allostimulatory ability and also the function of putative T-regulatory cells. In vivo DC trafficking was monitored by fluorescence imaging of dye-labeled cells and the influence of renal DC on vascularized heart allograft survival was determined. RESULTS: Flt3L induced a marked increase both in CD11cCD8alpha and in CD11cCD8alpha DC within the renal cortex and medulla. Rarer, CD11cB220 (precursor plasmacytoid) DC were also detected. Bulk freshly isolated DC exhibited no interleukin (IL)-12p35 mRNA, low surface co-stimulatory molecule expression, and CCR transcripts, consistent with immaturity. They elicited only weak allogeneic T-cell proliferative responses, and repeated stimulation induced CD4CD25 IL-10 T cells. In vivo, the freshly isolated DC failed to prime T cells of naive allogeneic hosts for anti-donor cytotoxic T-cell responses. When infused systemically, 1 week before organ transplantation, they prolonged graft survival without immunosuppressive therapy. CONCLUSIONS: Hematopoietin-mobilized renal DC are functionally immature and exhibit tolerogenic potential. Mobilization of DC within kidneys is likely to affect their antigen-handling capacity, immunogenicity, and tolerogenic ability.

Animals↗

PubMed2003

Mathematical modelling of adsorption and transport processes in capillary electrochromatography: open-tubular geometry.

A mathematical modelling approach for open-tubular capillary electrochromatography is presented. The spatially one-dimensional model takes into account (i) a coupling of (non)linear adsorption of positively or negatively charged analyte molecules (at a negatively charged capillary inner surface) with the equilibrium electrokinetics at this solid-liquid interface, (ii) mobile phase transport by electroosmosis and pressure-driven flow, as well as (iii) transport of species by electrophoresis and molecular diffusion. Under these conditions the local zeta-potential and electroosmotic mobility become a function of the concentration of the charged analyte. The resulting inhomogeneity of electroosmotic flow through the capillary produces a compensating pore pressure as requirement for incompressible mobile phase flow (i.e., for constant volumetric flow along the capillary). The results of the simulations are discussed in view of the surface-to-volume ratio of the capillary lumen, the analyte concentration (in combination with a Langmuir isotherm for the adsorption process), and buffer effects.

Adsorption↗

PubMed2006

Distribution and mobility of metals in agricultural soils near a copper smelter in South China.

The distribution and mobility of heavy metals in the paddy soils surrounding a copper smelting plant in south China was investigated. We assessed the degree of metal contamination using an index of geoaccumulation. The metals were divided into two groups: (1) Cu, Zn, Pb and Cd, whose concentrations were heavily affected by anthropogenic inputs, and (2) Ni, Co and Cr, which were mainly of geochemical origin. Concentrations of Cu, Cd, Zn, and Pb in the polluted soils were higher than the Chinese soil quality criteria. The chemical partitioning patterns of Pb, Zn and Cu indicated that Pb was largely associated with the residual and NH(2)OH HCl extractable fractions. In contrast, Cd was predominantly associated with the MgCl(2) extractable fraction. A large proportion of Cu was bound to the acidic H(2)O(2) extractable fractions, while Zn was predominantly found in the residual phase. The fraction of mobile species, which potentially is the most harmful to the environment, was found to be elevated compared to unpolluted soils in which heavy metals are more strongly bound to the matrix. The mobility of the metals was studied by water extraction using a modification of Tessier's procedure, and the order of mobility was Zn > Cd > Cu > Pb.

Cadmium↗

PubMed1989

Bacteriophage introns: parasites within parasites?

Several bacteriophage introns carry the information necessary to perform a complex series of RNA as well as DNA rearrangements. The RNA splicing reactions allow expression of the intron-containing genes, whereas recombination at the DNA level results in the mobility and invasive potential of the introns. The RNA and DNA transactions are temporally regulated to permit timely splicing early in infection, and expression of intron-encoded DNA endonucleases involved in mobility at late times. This is achieved through efficient use of the viral regulatory machinery, ensuring the perpetuation of the 'parasitic' introns.

Animals↗

PubMed2001

Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities.

Hydrophilic interaction chromatography (HILIC) is described as a useful alternative to reversed-phase chromatography for applications involving polar compounds. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal-phase retention behavior. Silica and amino columns with aqueous-acetonitrile mobile phases offer potential for use in the HILIC mode. An examination of the retention and separation of several pyrimidines, purines, and amides on silica and amino columns from three manufacturers revealed that mobile phases should contain a buffer or acid for pH control to achieve similar and reproducible results among columns from different sources. Amino columns may also be used in an anion-exchange mode, which provides an advantage for some applications. In some cases, silica can provide different selectivity and better separation than an amino column. Example applications include: low-molecular-mass organic acids and amides as impurities in non-polar drug substances, 5-fluorouracil in 5-fluorocytosine, guanine in acyclovir, and different selectivity for polar basic compounds compared to an ion-pairing system.

Acetamides↗

PubMed1992

Potentiation of inositol trisphosphate-induced Ca2+ mobilization in Xenopus oocytes by cytosolic Ca2+.

1. The ability of cytosolic Ca2+ ions to modulate inositol 1,4,5-trisphosphate (Insp3)-induced Ca2+ liberation from intracellular stores was studied in Xenopus oocytes using light flash photolysis of caged InsP3. Changes in cytosolic free Ca2+ level were effected by inducing Ca2+ entry through ionophore and voltage-gated plasma membrane channels and by injection of Ca2+ through a micropipette. Their effects on Ca2+ liberation were monitored by video imaging of Fluo-3 fluorescence and by voltage clamp recording of Ca(2+)-activated membrane Cl- currents. 2. Treatment of oocytes with the Ca2+ ionophores A23187 and ionomycin caused a transient elevation of cytosolic Ca2+ level when cells were bathed in Ca(2+)-free solution, which probably arose because of release of Ca2+ from intracellular stores. 3. Membrane current and Fluo-3 Ca2+ signals evoked by photoreleased InsP3 in ionophore-treated oocytes were potentiated when the intracellular Ca2+ level was elevated by raising the Ca2+ level in the bathing solution. 4. Responses to photoreleased InsP3 were similarly potentiated following activation of Ca2+ entry through voltage-gated Ca2+ channels expressed in the plasma membrane. 5. Ca(2+)-activated membrane currents evoked by depolarization developed a delayed 'hump' component during sustained photorelease of InsP3, probably because Ca2+ ions entering through the membrane channels triggered liberation of Ca2+ from intracellular stores. 6. Ba2+ and Sr2+ ions were able to substitute for Ca2+ in potentiating InsP3-mediated Ca2+ liberation. 7. Gradual photorelease of InsP3 by weak photolysis light evoked Ca2+ liberation that began at particular foci and then propagated throughout, but not beyond that area of the oocyte exposed to the light. Local elevations of intracellular Ca2+ produced by microinjection of Ca2+ acted as new foci for the initiation of Ca2+ liberation by InsP3. 8. In resting oocytes, intracellular injections of Ca2+ resulted only in localized elevation of intracellular Ca2+, and did not evoke propagating waves. 9. The results show that cytosolic Ca2+ ions potentiate the ability of InsP3 to liberate Ca2+ from intracellular stores. This process may be important for the positive feedback mechanism underlying the generation of Ca2+ spikes and waves, and for interactions between the InsP3 pathway and Ca2+ ions entering cells through voltage- and ligand-gated channels.

Animals↗

PubMed1984

Effect of oestradiol on catecholamine-stimulated lipolysis.

The authors studied the effect of a single in vivo dose of oestradiol (OE) on adrenergic lipolysis in the epididymal adipose tissue of adult and juvenile male rats, and the effect of OE on plasma free fatty acids (FFA), cholesterol and beta-lipoprotein levels at various intervals after its administration. It was found that OE injected 24 h beforehand in vivo (s.c.), in doses of 100 and 200 micrograms X kg-1 body weight, significantly potentiated the lipid-mobilizing action of the catecholamines noradrenaline (NOR) and isoprenaline (ISO) in adult rats (the action of ISO was potentiated more intensively); in addition, the adipose tissue became more sensitive to the action of NOR, but not of ISO. Raising the dose of OE to 400 micrograms X kg-1 did not enhance the potentiation of the lipolytic action of the catecholamines any further; on the contrary, the lipid mobilizing effect of the catecholamines was potentiated less than after half this dose. Following the s.c. injection of an oily OE solution, the lipolytic effect was potentiated after more than 7 h; the potentiation was strongest after 12 h, but only as far as the maximum attainable degree of lipolysis was concerned. Potentiation of adrenergic lipolysis was found only in adult male rats. In male rats weighing 130-150 g the lipolytic effect of catecholamines (in mumol/g adipose tissue) was significantly greater than in adult animals and the pre-administration of OE did not potentiate adrenergic lipolysis any further. Determination of plasma FFA, cholesterol and beta-lipoprotein levels 1, 2, 4 and 6 hours after the s.c. injection of OE showed only nonsignificant changes (an increase in FFA and a decrease in cholesterol). The authors consider it important to distinguish between the effect of OE on catecholamine-stimulated lipolysis in depot adipose tissue and its effect on lipid metabolism. In their opinion, the dose-dependent effect of OE on muscular and metabolic adrenergic reactions could be one of the factors co-reversible for certain side reactions to steroid contraceptives.

Adipose Tissue↗

PubMed2006

Microtubules as mechanical force sensors.

Microtubules are polymers of tubulin subunits (dimers) arranged on a hexagonal lattice. Each tubulin dimer comprises two monomers, the alpha-tubulin and beta-tubulin, and can be found in two states. In the first state a mobile negative charge is located into the alpha-tubulin monomer and in the second into the beta-tubulin monomer. Each tubulin dimer is modeled as an electrical dipole coupled to its neighbors by electrostatic forces. The location of the mobile charge in each dimer depends on the location of the charges in the dimer's neighborhood. Mechanical forces that act on the microtubule affect the distances between the dimers and alter the electrostatic potential. Changes in this potential affect the mobile negative charge location in each dimer and the charge distribution in the microtubule. The net effect is that mechanical forces affect the charge distribution in microtubules. We propose to exploit this effect and use microtubules as mechanical force sensors. We model each dimer as a two-state quantum system and, following the quantum computation paradigm, we use discrete quantum random walk on the hexagonal microtubule lattice to determine the charge distribution. Different forces applied on the microtubule are modeled as different coin biases leading to different probability distributions of the quantum walker location, which are directly connected to different charge distributions. Simulation results show that there is a strong indication that microtubules can be used as mechanical force sensors and that they can also detect the force directions and magnitudes.

Biomechanical Phenomena↗

PubMed2001

Nitrogen deposition and leaching from two forested catchments in Southwest China--preliminary data and research needs.

Increased nitrogen deposition has resulted in increased nitrogen pools and nitrogen leaching in European and North American forest soils. The development in Asia in general, and China in particular, suggests increased deposition of reduced nitrogen from changes in agricultural practices and of oxidized nitrogen from rapid growth of the transportation sector. Decreased nitrogen retention in forested areas in the future may cause increased NO3- leaching and, thus, acidification and eutrophication in surface waters. The differences in climate, ecosystems, land use, and deposition history make direct application of knowledge from studies in Europe and North America difficult. In Southwest China the potential for nitrogen mobilization from forest soils may be high because of the warm and humid climate, resulting in high decomposition rates of soil organic matter. However, there are very few data available for quantifying the suspected potential for increased nitrogen leaching in forest ecosystems. Here we present data from two forested catchments, dominated by Masson pine (Pinus massoniana), near Guiyang and Chongqing, respectively, in Southwest China. The present nitrogen deposition is moderate, estimated in the range from 10 to 40 kg N ha(-1) year(-1). The C/N ratios of the soils are generally below 15. Nitrate concentrations in soil water are rather variable in space, with highest values of several hundred microequivalents per liter. The turnover rate of nitrogen in the forest ecosystem is quite high compared to the atmospheric deposition rate. At present, nitrate runoff from the catchments is low and intermediate in Guiyang and Chongqing, respectively. More research is needed to improve our ability to predict future nitrogen leaching from subtropical Asian coniferous forests.

Carbon↗

PubMedDate not supplied

[Effect of an electrical field on Alcaligenes eutropha cells].

The electrophoretic mobility and electrokinetic potential of the hydrogen-oxidizing bacterium Alcaligenes eutropha were measured using microelectrophoresis. The cells were shown to bear a negative charge of ca. 2 x 10(-8) coulomb/cm2. The electrokinetic potential causing electrophoresis of the bacterium changes from negative values in an alkaline or neutral medium to positive values in an acid medium, and the isoelectric point is at pH approximately 6.0. The electrophoretic mobility of the bacterium rises with an increase of the ionic strength of solutions. The electric field of a low voltage (up to 5 V/cm) does not induce a noticeable change in the behaviour and growth of the bacterium under the autotrophic conditions.

Alcaligenes↗

PubMed1997

Flt-3 ligand synergizes with granulocyte colony-stimulating factor to increase neutrophil numbers and to mobilize peripheral blood stem cells with long-term repopulating potential.

Flt-3 ligand (FL) shares many features with stem cell factor (SCF), a widely documented cofactor for peripheral blood progenitor cell (PBPC) mobilization. We investigated the mobilization of PBPCs by FL in combination with granulocyte colony-stimulating factor (G-CSF). As a single agent, FL was a relatively modest mobilizer of PBPCs, resulting in 360 granulocyte/macrophage colony-forming cells (GM-CFCs)/mL blood (control, 155 GM-CFCs/mL blood) and no advantage in leukocyte recovery when these PBPCs were transplanted to irradiated recipient mice. G-CSF, on the other hand, mobilized over 20,000 GM-CFCs/mL blood, and the combination of G-CSF + FL resulted in over 100,000 GM-CFCs/mL blood. The combination of G-CSF + FL stimulated increased levels of monocytes and basophils in the peripheral blood. The performance of the mobilized PBPC product in irradiated hosts correlated with progenitor numbers resulting in long-term engraftment in association with accelerated short-term recovery of both leukocytes and platelets. These data demonstrate the potential of FL to synergize with G-CSF to mobilize PBPCs with both short- and long-term engraftment potential. The effect is similar to the synergistic interaction of G-CSF and SCF on PBPC mobilization. The use of FL as opposed to SCF may elicit a different spectrum of toxicities including lymphoid proliferation effects, in contrast to the mast cell degranulation effects of SCF. Clinical studies of FL are needed to evaluate its usefulness in man.

Animals↗

PubMed1999

Mobile echoes on prosthetic valves are not reproducible. Results and clinical implications of a multicentre study.

AIMS: To test the hypothesis that inter-observer variability accounts for the wide variation in reported prevalences of fibrin strands on prosthetic heart valves and to develop criteria for their identification and reporting. METHODS AND RESULTS: A videotape with 30 sequences of prosthetic heart valves imaged by transoesophageal echocardiography and showing abnormalities such as strands, microbubbles, and spontaneous echocardiographic contrast, was assessed in 13 European and three American centres. There were three duplicated examples, unbeknown to the observers. Definitions and reported prevalence rates of the abnormalities were analysed, and inter- and intra-observer agreement estimated with the kappa statistic. Mobile echoes were identified in 40 to 80% of the sequences on the tape. The reported prevalence of mobile echoes correlated with the time spent reporting the tape. There was moderate inter-observer agreement for the identification of any mobile echoes (kappa = 0.38), but no agreement for their labelling (kappa = 0.22), in spite of similar definitions. Intra-observer reproducibility was good (agreement in 76% of the reduplicated sequences). CONCLUSIONS: The true prevalence and potential significance of mobile echoes on prosthetic heart valves cannot be assessed unless inter-observer consensus on echocardiographic criteria for identifying such echoes is reached.

Adult↗

PubMed2006

Extended-duration thromboprophylaxis in acutely ill medical patients with recent reduced mobility: methodology for the EXCLAIM study.

BACKGROUND: Venous thromboembolism (VTE) is a significant cause of mortality and morbidity in medical patients. Although thromboprophylaxis with enoxaparin reduces the risk of VTE in these patients, the optimal duration of therapy is not currently known. The EXCLAIM (EXtended CLinical prophylaxis in Acutely Ill Medical patients) study is designed to compare the efficacy and safety of extended-duration thromboprophylaxis using enoxaparin with the standard regimen of enoxaparin in acutely ill medical patients with recent reduced mobility. METHODS: All enrolled acutely ill medical patients receive enoxaparin 40 mg subcutaneously once daily for 10 +/- 4 days. Eligible patients are then randomized in a blinded manner to receive either extended-duration thromboprophylaxis with enoxaparin 40 mg subcutaneously once daily or placebo subcutaneously once daily for an additional 28 +/- 4 days. The primary efficacy endpoint is the incidence of VTE during the 28 +/- 4 days after randomization. This study utilizes a standardized bilateral compression ultrasonography examination protocol that consists of an intensive interrogation of the deep veins of the lower extremities for proximal deep-vein thrombosis. The secondary efficacy endpoints are the rate of symptomatic VTE during the 3 months after randomization and mortality at 3 and 6 months after enrollment. The primary safety endpoint is the incidence of major hemorrhagic complications during the 28 +/- 4 days after randomization. RESULTS: To date, 3,983 patients, with a broad range of medical conditions, have been included in the study. Almost one third of the enrolled patients with reduced mobility are acutely ill due to respiratory insufficiency and one third have infectious diseases. CONCLUSIONS: The EXCLAIM study is designed to show the efficacy and safety of extended-duration thromboprophylaxis using enoxaparin in acutely ill medical patients with recent reduced mobility, which may potentially lead to a reduction in the incidence of late VTE events in these patients. The EXCLAIM (EXtended CLinical prophylaxis in Acutely Ill Medical patients) study, involving 4,726 acutely ill medical patients with recent reduced mobility, is designed to compare the efficacy and safety of extended-duration thromboprophylaxis using 40 mg once daily enoxaparin (38 +/- 4 days) with the standard regimen for enoxaparin (40 mg once daily for 10 +/- 4 days). The objective of this study is to demonstrate that the extended-duration enoxaparin regimen is an effective and safe thromboprophylaxis regimen out of hospital.

Adult↗

PubMed2006

Solution structure and backbone dynamics of an N-terminal ubiquitin-like domain in the GLUT4-regulating protein, TUG.

The GLUT4-regulating protein, TUG, functions to retain GLUT4-containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10-83 of full-length TUG, and confirms a beta-grasp or ubiquitin-like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG-UBL1 domain has not yet been described. We undertook a limited literature review of similar N-terminal UBL domains and noted that a majority participate in protein-protein interactions, generally functioning as adaptor modules to physically associate the over all activity of the protein with a specific cellular process, such as the ubiquitin-proteasome pathway. In consistent fashion, TUG-UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C-terminal diglycine ("GG") motif required for conjugation to an acceptor lysine, and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG-UBL1 molecular surface reveals a lack of conservation of the "Ile-44 hydrophobic face" typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein-protein interaction site.

Amino Acid Sequence↗

PubMed2002

Defects in calcium control.

METHODS: Multicellular preparations from nonfailing and failing human hearts or animals with cardiac hypertrophy were used to study intracellular calcium mobilization. Left ventricular muscle strips were loaded with the intracellular calcium indicator aequorin. Muscle strips were attached to a force transducer and stretched until there was no further increase in active force and stimulated to contract at varying frequencies. Muscles were placed in an oxygenated bath and studied at 30 degrees C. Pharmacological agents were used to increase intracellular sodium or intracellular calcium directly. Agents with known sites of action were then applied to define the original of resulting changes in the amplitude and shape of the caclium transient. Cellular homogenates were also used to study SR Ca(2+) ATPase activity based in a pyruvate/NADH-coupled reaction. Action potentials were also recorded from isolated muscle strips. Findings from isolated myocytes loaded with an intracellular calcium indicator are also reported. CONCLUSIONS: In failing human cardiomyocytes, decreased SERCA2a activity contributes to abnormal calcium handling, elevated diastolic calcium concentrations, and decreased contractility at higher rates of stimulation. Enhanced sodium calcium exchanger activity when working in the reverse mode (ie, transporting calcium into the cell) can potentially worsen calcium mobilization, induce arrhythmias, and negatively impact muscle contraction. Elevated intracellular sodium concentrations can prolong the action potential duration, as well as the time course of muscle contraction, resulting in increased arrhythmogenesis.

Action Potentials↗