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Stuart Smith

Publications and source records attributed to Stuart Smith.

31 records · Page 2Linked to original sources
PubMed2003

Cloning, expression, characterization, and interaction of two components of a human mitochondrial fatty acid synthase. Malonyltransferase and acyl carrier protein.

The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.

Acyl Carrier Protein↗
PubMed2003

Cloning, expression, and characterization of a human 4'-phosphopantetheinyl transferase with broad substrate specificity.

A single candidate 4'-phosphopantetheine transferase, identified by BLAST searches of the human genome sequence data base, has been cloned, expressed, and characterized. The human enzyme, which is expressed mainly in the cytosolic compartment in a wide range of tissues, is a 329-residue, monomeric protein. The enzyme is capable of transferring the 4'-phosphopantetheine moiety of coenzyme A to a conserved serine residue in both the acyl carrier protein domain of the human cytosolic multifunctional fatty acid synthase and the acyl carrier protein associated independently with human mitochondria. The human 4'-phosphopantetheine transferase is also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes. The same human protein also has recently been implicated in phosphopantetheinylation of the alpha-aminoadipate semialdehyde dehydrogenase involved in lysine catabolism (Praphanphoj, V., Sacksteder, K. A., Gould, S. J., Thomas, G. H., and Geraghty, M. T. (2001) Mol. Genet. Metab. 72, 336-342). Thus, in contrast to yeast, which utilizes separate 4'-phosphopantetheine transferases to service each of three different carrier protein substrates, humans appear to utilize a single, broad specificity enzyme for all posttranslational 4'-phosphopantetheinylation reactions.

Amino Acid Sequence↗
PubMed2003

Day-case laparoscopic cholecystectomy: a prospective evaluation of a 6-year experience.

BACKGROUND/PURPOSE: Day-case laparoscopic cholecystectomy (LC) offers convenience to patients and cost saving to the healthcare institutes. This article reviews our prospectively recorded experience with day-case LC to determine its applicability and safety, as well as patient satisfaction. METHODS: Of 744 consecutive patients who underwent LC over a 6-year period, 140 (19%) were scheduled for day-case surgery. Selection criteria included American Society of Anesthesiologists (ASA) score of 1 or 2, absence of morbid obesity, low risk for common bile duct stones, domicile within 50 km of the hospital, age greater than 18 and less than 75, and the ability to admit the patient on the day of surgery for operation during the morning. Patient satisfaction with day-case surgery was assessed by questionnaire at 4-6 weeks after operation. RESULTS: Some 117 of the 140 patients (84%) were discharged home on the same day of the operation. Two patients were re-admitted with abdominal pain, 1 of whom underwent a negative re-laparoscopy. There were no major complications. The reasons for overnight hospital stay were anesthetic in 12 (52%), surgical in 7 (30%), and social or logistic in 4 (18%) patients. There were no conversions. The proportion of patients scheduled for day-case remained static (median, 18.5%; range, 16%-22%). Some 99 of 105 patients (94%) who completed the questionnaire were satisfied with day-case surgery. CONCLUSIONS: In carefully selected patients, day-case LC is achievable and safe, and provides good patient satisfaction.

Adult↗
PubMed2003

Structural and functional organization of the animal fatty acid synthase.

The entire pathway of palmitate synthesis from malonyl-CoA in mammals is catalyzed by a single, homodimeric, multifunctional protein, the fatty acid synthase. Each subunit contains three N-terminal domains, the beta-ketoacyl synthase, malonyl/acetyl transferase and dehydrase separated by a structural core from four C-terminal domains, the enoyl reductase, beta-ketoacyl reductase, acyl carrier protein and thiosterase. The kinetics and specificities of the substrate loading reaction catalyzed by the malonyl/acetyl transferase, the condensation reaction catalyzed by beta-ketoacyl synthase and chain-terminating reaction catalyzed by the thioesterase ensure that intermediates do not leak off the enzyme, saturated chains exclusively are elongated and palmitate is released as the major product. Only in the fatty acid synthase dimer do the subunits adopt conformations that facilitate productive coupling of the individual reactions for fatty acid synthesis at the two acyl carrier protein centers. Introduction of a double tagging and dual affinity chromatographic procedure has permitted the engineering and isolation of heterodimeric fatty acid synthases carrying different mutations on each subunit. Characterization of these heterodimers, by activity assays and chemical cross-linking, has been exploited to map the functional topology of the protein. The results reveal that the two acyl carrier protein domains engage in substrate loading and condensation reactions catalyzed by the malonyl/acetyl transferase and beta-ketoacyl synthase domains of either subunit. In contrast, the reactions involved in processing of the beta-carbon atom, following each chain elongation step, together with the release of palmitate, are catalyzed by the cooperation of the acyl carrier protein with catalytic domains of the same subunit. These findings suggest a revised model for the fatty acid synthase in which the two polypeptides are oriented such that head-to-tail contacts are formed both between and within subunits.

Acyl Carrier Protein↗
PubMed2003

Engineering of an active animal fatty acid synthase dimer with only one competent subunit.

Animal fatty acid synthases are large polypeptides containing seven functional domains that are active only in the dimeric form. Inactivity of the monomeric form has long been attributed to the obligatory participation of domains from both subunits in catalysis of substrate loading and condensation reactions. However, we have engineered a fatty acid synthase containing one wild-type subunit and one subunit compromised by mutations in all seven functional domains that is active in fatty acid synthesis. This finding indicates that a single subunit, in the context of a dimer, is able to catalyze the entire biosynthetic pathway and suggests that, in the natural complex, each of the two subunits forms a scaffold that optimizes the conformation of the companion subunit.

Acetone↗
PubMed2003

Protective effects of intracerebral adenoviral-mediated GDNF gene transfer in a rat model of Parkinson's disease.

This study focuses on the potential protective effects of intracerebral adeno-viral mediated glial cell line derived neurotrophic factor (GDNF) gene transfer in a rat model of Parkinson's disease (PD). Thirty-five SD rats were divided into three groups to receive perinigral injections of recombinant adenovirus encoding GDNF (Ad-GDNF), LacZ (Ad-LacZ) or PBS, respectively. One week later, an intrastriatal injection of 6-hydroxydopamine (6-OHDA) was administered to induce the progressive degeneration of dopaminergic neurons. Immunohistochemistry showed that GDNF treatment prior to neuronal damage could promote survival and morphological recovery of tyrosine hydroxylase (TH)-positive neurons in the midbrain. Approximately 70% of nigral TH-positive cells survived in the Ad-GDNF group, compared to approximately 30% for the Ad-LacZ or PBS control group. Histochemical analysis of monoamine levels in the striatum demonstrated that the dopamine content was higher for the Ad-GDNF group than the control groups. Similarly, Ad-GDNF treated animals showed improved apomorphine-induced rotational behavior. The exogenous GDNF gene was efficiently expressed in the brain as detected by ELISA. This work demonstrates that intracerebral adeno-viral mediated GDNF gene transfer can protect dopaminergic neurons in vivo from 6-OHDA-induced injuries. The approach used in this study could potentially be used therapeutically in patients with PD and further work is required to explore this idea in depth.

Adenoviridae↗
PubMed2003

Visual orientation illusions: global mechanisms involved in hierarchical effects and frames of reference.

Five experiments were conducted in order to determine which of two hypotheses, initially proposed by Rock (1990), accounts for interactions between oriented elements in a visual scene. We also explored the suggestion that two hypothetical processes--namely, frame of reference and hierarchical organization--describe phenomena arising from distinct mechanisms (Spinelli, Antonucci, Daini, Martelli, & Zoccolotti, 1999). Double inducing stimulus versions of one-dimensional and two-dimensional tilt illusions, the rod-and-frame illusion, and combinations of these were used. Our data suggest that both hypotheses can predict orientation interactions in conditions in which only one mechanism--namely, the global visual mechanism of symmetry axes extraction (Wenderoth & Beh, 1977)--is activated. Which hypothesis is appropriate to predict the perceived orientation depends on some physical features of the objects.

Discrimination, Psychological↗
PubMed2003

Thiamine deficiency in congestive heart failure patients receiving long term furosemide therapy.

OBJECTIVE: To assess the presence of thiamine deficiency in congestive heart failure patients receiving furosemide therapy. DESIGN: Prospective, biochemical analysis of thiamine status was performed in outpatients and inpatients of the University of Ottawa Heart Institute. SUBJECTS: Thirty-two patients with congestive heart failure who received at least 40 mg/day of furosemide were included. Patients were then separated into two groups depending on whether the dose of furosemide was greater than or equal to 80 mg/day. METHODS: The primary measure was actual thiamine status as assessed by the erythrocyte transketolase enzyme activity and the degree of thiamine pyrophosphate effect. RESULTS: Biochemical evidence of severe thiamine deficiency was found in 98% (24 of 25) patients receiving at least 80 mg/day of furosemide and in 57% (four of seven) of patients taking 40 mg furosemide daily, odds ratio (OR) 19.0 (1.13<OR<601.29). Thiamine status was not associated with any other clinical variables. CONCLUSIONS: These findings suggest that thiamine deficiency occurs in a substantial proportion of congestive heart failure patients being treated with furosemide.

Aged↗
PubMed2002

Mechanism of the beta-ketoacyl synthase reaction catalyzed by the animal fatty acid synthase.

The catalytic mechanism of the beta-ketoacyl synthase domain of the multifunctional fatty acid synthase has been investigated by a combination of mutagenesis, active-site titration, product analysis, and product inhibition. Neither the reactivity of the active-site Cys161 residue toward iodoacetamide nor the rate of unidirectional transfer of acyl moieties to Cys161 was significantly decreased by replacement of any of the conserved residues, His293, His331, or Lys326, with Ala. Decarboxylation of malonyl moieties in the fully-active Cys161Gln background generated equimolar amounts of acetyl-CoA and bicarbonate, rather than carbon dioxide, and was seriously compromised by replacement of any of the conserved basic residues. The ability of bicarbonate to inhibit decarboxylation of malonyl moieties in the Cys161Gln background was significantly reduced by replacement of His293 but less so by replacement of His331. The data are consistent with a reaction mechanism, in which the initial primer transfer reaction is promoted largely through a lowering of the pKa of the Cys161 thiol by a helix dipole effect and activation of the substrate thioester carbon atom by binding of the keto group in an oxyanion hole. The data also indicate that an activated water molecule is present at the active site that is required either for the rapid hydration of carbon dioxide, prior its release as bicarbonate or, alternatively, for an initial attack on the malonyl C3. In the alternative mechanism, a negatively-charged tetrahedral transition state could be generated, stabilized in part by interaction of His293 with the negatively charged oxygen at C3 and interaction of His331 with the negatively charged thioester carbonyl oxygen, that breaks down to generate bicarbonate directly. Finally, the carbanion at C2, attacks the electrophilic C1 of the primer, generating a second tetrahedral transition state, also stabilized through contacts with the oxyanion hole and His331, that breaks down to form the beta-ketoacyl-S-acyl carrier protein product.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2002

Giant cell myocarditis: clinical presentation, bridge to transplantation with mechanical circulatory support, and long-term outcome.

BACKGROUND: The multicenter Giant Cell Myocarditis Registry recorded 64 cases from 36 centers before 1996. The median transplant-free survival of 30 patients without immunosuppression was 3 months. Of 34 patients who received heart transplantations, 9 experienced recurrence of giant cell myocarditis in their transplanted hearts and 1 patient died. METHODS: We reviewed our experience in 340 heart transplantations since 1984. Unexpected giant cell myocarditis was found in the explanted hearts of 7 patients (6 men and 1 female, aged 18-65 years). RESULTS: The duration from the onset of symptoms to assist-device implant or transplantation ranged from 11 days to 9 years, whereas the time interval from referral or deterioration ranged from 2 days to 4 months. Four patients required mechanical circulatory support before surgery (total artificial hearts in 2 and left ventricular assist devices in 2), and 3 patients required inotropic drugs. Six patients are alive with no sign of recurrent giant cell myocarditis at 12 to 113 months after surgery. One patient died suddenly 75 months after surgery, and autopsy showed severe graft vascular disease with no recurrence of giant cell myocarditis. Surveillance, right ventricular endomyocardial biopsy specimens showed recurrent asymptomatic giant cell myocarditis in 3 patients at 5 to 13 months after surgery, and found recurrence in 1 patient 30 months after surgery. This patient received augmented immunosuppression. CONCLUSIONS: Giant cell myocarditis often is not diagnosed before transplantation. It can present as dilated cardiomyopathy with late deterioration, or it can present with rapid hemodynamic deterioration. In our experience, these patients can be bridged successfully to transplant with mechanical circulatory assist. Giant cell myocarditis may recur after transplantation but may respond to augmented immunosuppression.

Adolescent↗
PubMed2002

Modular NRPSs are monomeric.

NRPSs, PKSs, and hybrid NRPS/PKSs are modular proteins with similar assembly-line organizations. Although PKSs function as dimers, new data demonstrate that functional NRPSs are monomeric. This discovery has significant implications for engineering artificial assemblies for the production of novel biotherapeutics.

Bacteria↗