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PubMed2004

Chondrogenic potential of multipotential cells from human adipose tissue.

The use of stem cells for cell-based tissue-engineering strategies represents a promising alternative for the repair of cartilaginous defects. The multilineage potential of a population of putative mesodermal stem cells obtained from human lipoaspirates, termed processed lipoaspirate cells, was previously characterized. The chondrogenic potential of those cells was confirmed with a combination of histological and molecular approaches. Processed lipoaspirate cells under high-density micromass culture conditions, supplemented with transforming growth factor-beta1, insulin, transferrin, and ascorbic acid, formed well-defined nodules within 48 hours of induction and expressed the cartilaginous markers collagen type II, chondroitin-4-sulfate, and keratan sulfate. Reverse transcription polymerase chain reaction analysis confirmed the expression of collagen type II and the cartilage-specific proteoglycan aggrecan. In summary, human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis in vitro.

Adipose Tissue↗
PubMedDate not supplied

Effect of cardiopulmonary bypass on the pharmacokinetics of drugs.

The cardiopulmonary bypass apparatus must temporarily substitute for the cardiac and pulmonary function of the patient undergoing heart surgery. In order to meet the metabolic needs of the patient and the technical demands of the surgeon, within the limits of engineering technology, a number of major alterations are made in normal physiology. The patient is typically cooled to 27 degrees C and perfused with a non-pulsatile flow of blood which has been diluted with saline to a haematocrit in the mid-20s. Blood flow and pressure are often considerably less than normal. Blood coagulation is prevented by administration of a massive dose of heparin. Central redistribution of blood flow, elaboration of stress-reactant hormones, and fluid and electrolyte shifts occur in response to these changes. In the postoperative period, these alterations are reversed, and normal physiology is restored. Effects upon the pharmacokinetics of drugs are anticipated. The clearance of many drugs may be reduced. Protein binding is diminished by haemodilution, but may rise above normal in the postoperative period for basic drugs which bind to alpha 1-acid glycoprotein. Changes in volume of distribution depend upon the opposing influences of protein binding and reduced peripheral perfusion. Previous studies on the pharmacokinetics of drugs during and after cardiopulmonary bypass illustrate many of these effects. The clearance of digoxin, fentanyl, and the cephalosporins is reduced after cardiopulmonary bypass, and the volume of distribution of cefazolin is increased during cardiopulmonary bypass. Studies of digitoxin and propranolol are also reviewed. Many of the investigations in this area of study have been limited by logistical and methodological factors. Thus, the effects of cardiopulmonary bypass on the pharmacokinetics of drugs are incompletely understood, and the subject merits further attention.

Animals↗
PubMed2005

Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database.

HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.

Algorithms↗
PubMed2001

Fast hydride transfer in proton-translocating transhydrogenase revealed in a rapid mixing continuous flow device.

Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Coupling is achieved through changes in protein conformation. Upon mixing, the isolated nucleotide-binding components of transhydrogenase (dI, which binds NAD(H), and dIII, which binds NADP(H)) form a catalytic dI(2).dIII(1) complex, the structure of which was recently solved by x-ray crystallography. The fluorescence from an engineered Trp in dIII changes when bound NADP(+) is reduced. Using a continuous flow device, we have measured the Trp fluorescence change when dI(2).dIII(1) complexes catalyze reduction of NADP(+) by NADH on a sub-millisecond scale. At elevated NADH concentrations, the first-order rate constant of the reaction approaches 21,200 s(-1), which is larger than that measured for redox reactions of nicotinamide nucleotides in other, soluble enzymes. Rather high concentrations of NADH are required to saturate the reaction. The deuterium isotope effect is small. Comparison with the rate of the reverse reaction (oxidation of NADPH by NAD(+)) reveals that the equilibrium constant for the redox reaction on the complex is >36. This high value might be important in ensuring high turnover rates in the intact enzyme.

Crystallography, X-Ray↗
PubMed1999

Inhibition of human immunodeficiency virus type 1 by Tat/Rev-regulated expression of cytosine deaminase, interferon alpha2, or diphtheria toxin compared with inhibition by transdominant Rev.

A retroviral vector was designed to express toxic proteins only in the presence of the HIV-1 Rev and/or Tat protein(s). The design of this vector incorporates an HIV-specific expression cassette that consists of three elements: the U3R region of the HIV-1 IIIB LTR provides the promoter and Tat-responsive element, a modified intron derived from the human c-src gene facilitates the splicing of inserted genes, and the HIV-1 RRE region enhances the transport of unspliced mRNAs. To further limit potential readthrough transcription, the expression cassette was inserted in the reverse transcriptional orientation relative to the retroviral vector LTR. Three different genes, interferon alpha2, diphtheria toxin (DT-A), and cytosine deaminase, were inserted into this vector. Tat and Rev inducibility was demonstrated directly by a >300-fold induction of interferon production and functionally by a decrease in colony-forming units when a Tat and Rev expression vector was titered on HeLa cells harboring the inducible DT-A cassette. The Tat-inducible cytosine deaminase gene was tested in the Sup-T1 T cell line and shown to inhibit HIV-1 production only when engineered cells were grown in the presence of 5-fluorocytosine. To test the ability of this system to inhibit HIV-1 infection in bulk PBL cultures, a series of transduction and challenge experiments was initiated with both the interferon and DT-A vectors. Protection against infection was documented against three HIV strains in PBLs. Last, the interferon and DT-A vectors were compared with a vector encoding a transdominant Rev protein and were shown to mediate equal or greater inhibition of HIV-1.

Blotting, Northern↗
PubMed2006

Gefitinib modulates the function of multiple ATP-binding cassette transporters in vivo.

The 4-anilinoquinazoline (4-AQ) derivative gefitinib (Iressa) is an oral epidermal growth factor receptor tyrosine kinase inhibitor. Oral administration of 4-AQ molecules, such as gefitinib, inhibits ATP-binding cassette (ABC) transporter-mediated drug efflux and strongly increases the apparent bioavailability of coadministered drug molecules that are transporter substrates. Based on in vitro studies investigating 4-AQ interactions with several transporters, these effects have primarily been attributed to the inhibition of breast cancer resistance protein (BCRP; ABCG2). Although 4-AQ shows in vitro inhibition of P-glycoprotein [multidrug resistance protein (MDR1); ABCB1], the in vivo effect on this and other transporters is not known. In our studies, pretreatment of Abcg2(-/-) and Mdr1(a/b)(-/-) mice with gefitinib increased oral absorption and decreased systemic clearance of topotecan, a model substrate, indicating that additional transporters were inhibited. These results were extended to human orthologues using engineered cell lines to show that gefitinib inhibited the efflux of BCRP and MDR1 substrates and restored vincristine sensitivity in MDR1-expressing cells. Although gefitinib inhibited BCRP more potently than MDR1 (10-fold), the inhibition of both transporters occurred at clinically relevant concentrations (e.g., 1-5 micromol/L). These studies illustrate the broad implications for the therapeutic combination of gefitinib or other 4-AQ molecules with agents that are BCRP and MDR1 substrates. 4-AQ molecules may offer a means to increase the low and variable oral drug absorption of transporter substrates while decreasing interpatient variability and reversing tumor drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Biological approaches to bone regeneration by gene therapy.

Safe, effective approaches for bone regeneration are needed to reverse bone loss caused by trauma, disease, and tumor resection. Unfortunately, the science of bone regeneration is still in its infancy, with all current or emerging therapies having serious limitations. Unlike current regenerative therapies that use single regenerative factors, the natural processes of bone formation and repair require the coordinated expression of many molecules, including growth factors, bone morphogenetic proteins, and specific transcription factors. As will be developed in this article, future advances in bone regeneration will likely incorporate therapies that mimic critical aspects of these natural biological processes, using the tools of gene therapy and tissue engineering. This review will summarize current knowledge related to normal bone development and fracture repair, and will describe how gene therapy, in combination with tissue engineering, may mimic critical aspects of these natural processes. Current gene therapy approaches for bone regeneration will then be summarized, including recent work where combinatorial gene therapy was used to express groups of molecules that synergistically interacted to stimulate bone regeneration. Last, proposed future directions for this field will be discussed, where regulated gene expression systems will be combined with cells seeded in precise three-dimensional configurations on synthetic scaffolds to control both temporal and spatial distribution of regenerative factors. It is the premise of this article that such approaches will eventually allow us to achieve the ultimate goal of bone tissue engineering: to reconstruct entire bones with associated joints, ligaments, or sutures. Abbreviations used: BMP, bone morphogenetic protein; FGF, fibroblast growth factor; AER, apical ectodermal ridge; ZPA, zone of polarizing activity; PZ, progress zone; SHH, sonic hedgehog; OSX, osterix transcription factor; FGFR, fibroblast growth factor receptor; PMN, polymorphonuclear neutrophil; PDGF, platelet-derived growth factor; IGF, insulin-like growth factor; TGF-beta, tumor-derived growth factor beta; CAR, coxsackievirus and adenovirus receptor; MLV, murine leukemia virus; HIV, human immunodeficiency virus; AAV, adeno-associated virus; CAT, computer-aided tomography; CMV, cytomegalovirus; GAM, gene-activated matrix; MSC, marrow stromal cell; MDSC, muscle-derived stem cell; VEGF, vascular endothelial growth factor.

Biomimetics↗
PubMed2006

Cell-gene delivery of brain-derived neurotrophic factor to the mouse inner ear.

Sensorineural hearing loss is a common disability, but treatment options are currently limited to cochlear implants and hearing aids. Studies are therefore being conducted to provide alternative means of biological therapy, including gene therapy. Safe and effective methods of gene delivery to the cochlea need to be developed to facilitate the clinical application of these therapeutic treatments for hearing loss. In this study, we examined the potential of cell-gene therapy with nonviral vectors for delivery of therapeutic molecules into the cochlea. NIH3T3 cells were transfected with the brain-derived neurotrophic factor (Bdnf) gene using lipofection and then transplanted into the mouse inner ear. Immunohistochemistry and Western blotting demonstrated the survival of grafted cells in the cochlea for up to 4 weeks after transplantation. No significant hearing loss was induced by the transplantation procedure. A Bdnf-specific enzyme-linked immunosorbent assay revealed a significant increase in Bdnf production in the inner ear following transplantation of engineered cells. These findings indicate that cell-gene delivery with nonviral vectors may be applicable for the local, sustained delivery of therapeutic molecules into the cochlea.

Animals↗
PubMed2003

Dynamic simulation and metabolic re-design of a branched pathway using linlog kinetics.

This paper presents a new mathematical framework for modeling of in vivo dynamics and for metabolic re-design: the linlog approach. This approach is an extension of metabolic control analysis (MCA), valid for large changes of enzyme and metabolite levels. Furthermore, the presented framework combines MCA with kinetic modeling, thereby also combining the merits of both approaches. The linlog framework includes general expressions giving the steady-state fluxes and metabolite concentrations as a function of enzyme levels and extracellular concentrations, and a metabolic design equation that allows direct calculation of required enzyme levels for a desired steady state when control and response coefficients are available. Expressions giving control coefficients as a function of the enzyme levels are also derived. The validity of the linlog approximation in metabolic modeling is demonstrated by application of linlog kinetics to a branched pathway with moiety conservation, reversible reactions and allosteric interactions. Results show that the linlog approximation is able to describe the non-linear dynamics of this pathway very well for concentration changes up to a factor 20. Also the metabolic design equation was tested successfully.

Algorithms↗
PubMed1991

Reengineering the catalytic lysine of aspartate aminotransferase by chemical elaboration of a genetically introduced cysteine.

The active-site essential catalytic residue of aspartate aminotransferase, Lys 258, has been converted to Cys (K258C) by site-directed mutagenesis. This mutant retains less than 10(-6) of the wild-type activity with L-aspartate. The deleted general base was functionally replaced by selective (with respect to the other five cysteines in wild type) aminoethylation of the introduced Cys 258 with (2-bromoethyl)amine following reversible protection of the nontarget sulfhydryl groups at different stages of unfolding. The chemically elaborated mutant (K258C-EA) is 10(5) times more reactive than is K258C and has a kcat value of approximately 7% of that of wild type (WT). Km and KI values are similar to those for WT. The acidic pKa controlling V/KAsp is shifted from 7.3 (WT) to 6.0 (mutant). V/K values for amino acids are approximately 3% of those found for WT, whereas they are approximately 20% for keto acids. The value of DV increases from 1.6 for WT to 3.4 for the mutant, indicating that C alpha proton abstraction constitutes a more significant kinetic barrier for the latter enzyme. A smaller, but still significant, increase in D(V/KAsp) from 1.9 in WT to 3.0 in the mutant shows that the forward and reverse commitment factors are inverted by the mutation. The acidic limb of the V/KAsp versus pH profile, is lowered by 1.3 pH units, probably reflecting the similar difference in the basicity of the epsilon-NH2 group in gamma-thialysine versus that in lysine.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed2004

Differences in transductional tropism of adenoviral and lentiviral vectors in the rat brainstem.

Adenoviral vectors (AVVs) and lentiviral vectors (LVVs) are highly useful research tools which can be used to investigate the function of specific cell phenotypes in the brain. The transductional tropism of viral vectors has a critical impact upon the transgene expression in different brain areas. This largely depends on the properties of the viral particles, which for AVVs are most commonly analogous to the serotype 5 adenovirus and, in the case of LVVs, are determined by the envelope used for pseudotyping, for example the vesicular stomatitis virus coat (VSVG). We have created a matching set of shuttle plasmids that allow a one-step transfer of an entire expression cassette between the backbones of AVVs and LVVs. This has permitted a fair assessment of the impact of the vector type on tropism for both AVVs and LVVs. Thus, the aims of this study were twofold: (i) to develop and demonstrate the validity of a transgene 'swap' system between AVVs and LVVs; and (ii) using this system, to assess the tropism of AVVs and LVVs for neuronal versus glial cell types. We have constructed AVVs and VSVG-coated LVVs to express monomeric red fluorescent protein (mRFP) driven by the human cytomegalovirus promoter (hCMV). Transgene expression in neurones and glia in the hypoglossal and dorsal vagal motor nuclei of the rat brainstem was compared by determining the colocalization with immunostaining for the neuronal marker NeuN (neuronal nuclear antigen) and the glial marker GFAP (glial fibrillatory acidic protein). We found that 55% of mRFP-expressing cells transduced with AVVs were immunopositive for GFAP, while only 38% were NeuN-immunoreactive. In contrast, when the same expression cassette was delivered by VSVG-coated LVVs, the neurone/glia ratio of mRFP expression was reversed with 56% of mRFP-positive cells identified as neurones and 26% as glia. Thus, the present study provides compelling evidence that VSVG-coated LVVs significantly shift transgene expression towards neurones while transduction with AVVs favours glia.

Adenoviridae↗
PubMed2006

The cumate gene-switch: a system for regulated expression in mammalian cells.

BACKGROUND: A number of expression systems have been developed where transgene expression can be regulated. They all have specific characteristics making them more suitable for certain applications than for others. Since some applications require the regulation of several genes, there is a need for a variety of independent yet compatible systems. RESULTS: We have used the regulatory mechanisms of bacterial operons (cmt and cym) to regulate gene expression in mammalian cells using three different strategies. In the repressor configuration, regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a strong constitutive promoter. Addition of cumate, a small molecule, relieves the repression. In the transactivator configuration, a chimaeric transactivator (cTA) protein, formed by the fusion of CymR with the activation domain of VP16, is able to activate transcription when bound to multiple copies of CuO, placed upstream of the CMV minimal promoter. Cumate addition abrogates DNA binding and therefore transactivation by cTA. Finally, an adenoviral library of cTA mutants was screened to identify a reverse cumate activator (rcTA), which activates transcription in the presence rather than the absence of cumate. CONCLUSION: We report the generation of a new versatile inducible expression system.

Adenoviridae↗
PubMed2000

Generation of intersubtype human immunodeficiency virus type 1 recombinants in env gene in vitro: influences in the biological behavior and in the establishment of productive infections.

The occurrence of human immunodeficiency virus type 1 (HIV-1) recombinant genomes belonging to different subtypes is a common event in regions where more than two subtypes cocirculate. Although there are accumulating data toward an increase in the number of intersubtype recombinants, little has been addressed about the biological behavior of such mosaic genomes. This work reports the biological characterization of engineered in vitro HIV-1 intersubtype recombinants in the gp120 region. The recombinants possess the entire gp120 of B or F Brazilian isolates in the Z6 (subtype D) backbone. Here we show that this type of recombinant structure results in profound impairment to the establishment of productive infections in CD4-positive cells. The characterization of biological properties of those recombinant viruses demonstrated viral production occurring only during a transient peak early on infection and that they are not able to down-regulate the expression of CD4 receptor on the cell surface. We also report the phenotype reversion of one recombinant virus studied here, after 62 days in culture. Two amino acid substitutions in highly constant gp120 regions (C1 and C4) were identified in the revertant virus. The mutation occurring in the C4 region is localized near two amino acid residues critical for gp120/CD4 interaction. Based on these data, we suggest that failure in CD4 down-modulation by recombinant viruses can be due to a structural dysfunction of gp160 protein unable to block CD4 at the endoplasmic reticule. The possibilities that the establishment of latent infections can be directly related to the continuous expression of CD4 on the infected cell surface and that the occurrence of mutations in amino acid nearby residues critical for gp120/CD4 interaction can restore the fully productive infectious process are discussed.

Amino Acid Sequence↗
PubMed2005

Comparison of rapid liquid chromatography-electrospray ionization-tandem mass spectrometry methods for determination of glycoalkaloids in transgenic field-grown potatoes.

Two rapid methods for highly selective detection and quantification of the two major glycoalkaloids in potatoes, alpha-chaconine and alpha-solanine, were compared for robustness in high-throughput operations for over 1000 analytical runs using potato tuber samples from field trials. Glycoalkaloids were analyzed using liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode. An electrospray interface was used in the detection of glycoalkaloids in positive ion mode. Classical reversed phase (RP) and hydrophilic interaction (HILIC) columns were investigated for chromatographic separation, ruggedness, recovery, precision, and accuracy. During the validation procedure both methods proved to be precise and accurate enough in relation to the high degree of endogenous biological variability found for field-grown potato tubers. However, the RP method was found to be more precise, more accurate, and, more importantly, more rugged than the HILIC method for maintaining the analytes' peak shape symmetry in high-throughput operation. When applied to the comparison of six classically bred potato cultivars to six genetically modified (GM) lines engineered to synthesize health beneficial inulins, the glycoalkaloid content in potato peels of all GM lines was found within the range of the six cultivars. We suggest complementing current unbiased metabolomic strategies by validating quantitative analytical methods for important target analytes such as the toxic glycoalkaloids in potato plants.

Agriculture↗
PubMed2002

Lipid-triggered conformational switch of apolipophorin III helix bundle to an extended helix organization.

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic alpha-helices (H1-H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C-apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C-apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene "excimer" fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene-A8C-apoLp-III/DMPC or pyrene-A138C-apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene-A8C/A138C-apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.

Animals↗
PubMed2001

Hepatic insulin expression improves glycemic control in type 1 diabetic rats.

Low levels of hepatic insulin production have been shown to prevent lethal ketoacidosis associated with type 1 diabetes. To assess the beneficial effects of sustained hepatic production of insulin on glycemic control in type 1 diabetes, we have employed the adenovirus-mediated gene delivery system to transfer an engineered rat preproinsulin gene to the livers of streptozotocin-induced diabetic nude rats. Hepatic insulin production resulted in the reduction of blood glucose in treated diabetic rats, the degree of blood glucose reduction correlated with both the vector dose and the level of hepatic insulin expression. At moderate vector doses, 0.3-0.7 ng/ml of plasma insulin was produced in treated diabetic animals, resulting in significant reduction of nonfasting hyperglycemia and improvement in glucose tolerance. Furthermore, these animals maintained euglycemia after 12-h fast. At higher vector doses, greater than 1 ng/ml of plasma insulin was produced, completely reversing nonfasting hyperglycemia in treated rats. However, all of the treated animals developed severe hypoglycemia upon fasting. This study has defined the maximal tolerable level of hepatic insulin production that is sufficient to reduce the degree and ameliorate the adverse effects of nonfasting hyperglycemia without risk of fasting hypoglycemia in type 1 diabetic rats.

Amino Acid Sequence↗
PubMed2000

Acute liver failure: targeted artificial and hepatocyte-based support of liver regeneration and reversal of multiorgan failure.

Acute liver failure (ALF) still represents a major therapeutic challenge for hepatologists due to its high mortality rate as a result of multiorgan failure. Although emergency orthotopic liver transplantation represents a major advance in the management of selected patients, it is not applicable to all candidates due to limited organ availability. Therefore, new therapeutic options should be developed to bridge selected patients to transplantation or to treat patients not candidates for liver transplantation. Although new techniques for cell culture and perfusion have resulted in a number of promising devices for the provision of temporary liver support in acute liver failure, their clinical efficacy is as yet uncertain. Controlled trials on a multi-centre basis in well-defined patient groups and with standardised outcome measures, including the extent to which treatment influences cell damage and regeneration and prevents or reverses multiorgan failure, will be essential to properly evaluate the clinical value of current and evolving artificial and bioartificial devices. The same considerations must also apply to the assessment of therapeutic efficacy of hepatocyte transplantation. A better understanding of mechanisms responsible for the development of liver cell death, along with cellular and molecular mechanisms allowing surviving cells to proliferate in a hostile environment, will be required if a more targeted therapeutic approach to decreasing hepatocellular injury and enhancing liver regeneration is to be achieved. Whether extracorporeal devices or the transplantation of primary hepatocytes, stem cells or cells genetically engineered to over-express key metabolic functions, a proliferative phenotype and/or cytoprotective pathways will be best suited to meeting these demanding challenges remains to be determined.

Acute Disease↗
PubMed2002

Identification of human vesicle monoamine transporter (VMAT2) lumenal cysteines that form an intramolecular disulfide bond.

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. To obtain structural information regarding this large membrane protein by analysis of disulfide bonds and other intramolecular cross-links, we engineered a strategic thrombin cleavage site into deglycosylated, HA-tagged human VMAT2. Insertion of this protease site did not disrupt ligand binding or serotonin transport. Thrombin cleavage at an engineered site in the predicted cytoplasmic loop between transmembrane (TM) domains 6 and 7 (loop 6/7) was rapid and quantitative in the absence of any detergent. The loop 6/7 thrombin site allowed assessment of an intramolecular disulfide bond between the N- and C-terminal halves of the transporter. Consistent with this hypothesis, after quantitative loop 6/7 thrombin cleavage, in the absence of reducing agent, VMAT2 migrated on SDS-polyacrylamide gels as a full-length transporter. Addition of dithiothreitol resulted in complete conversion from full-length to thrombin-cleaved size, demonstrating a DTT-reversible covalent bond. The identity of the disulfide-bound cysteine pair was suggested by the observation that replacement of Cys 126 or Cys 333 with serine both reduced [(3)H]serotonin transport. Replacement of either Cys 126 or Cys 333 was found to eliminate the DTT-reversible intramolecular covalent bond. We conclude that human VMAT2 Cys 126 in loop 1/2 and Cys 333 in loop 7/8 form a disulfide bond which contributes to efficient monoamine transport.

Amino Acid Substitution↗