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PubMed1999

Functional pharmacology of cloned heterodimeric GABAB receptors expressed in mammalian cells.

1. In this study we report a new assay of heterodimeric gamma-amino-butanoic acid subtype B (GABAB) receptors where either GABABR1a or GABABR1b are co-expressed with GABABR2 and the chimeric G-protein Galphaq-z5 in tsA cells. In this manner we obtained a robust response to GABAB agonists measured as increase in phosphoinositide hydrolysis. 2. We used this assay to characterize a number of commonly used GABAB receptor ligands. Both splice variants displayed the same rank order of agonist potency; 3-aminopropyl(methyl)phosphinic acid (SKF-97541)>GABA>(R)-4-amino-3-(4-chlorophenyl)butanoic acid ((R)-baclofen)>(RS)-4-amino-3-(5-chloro-2-thienyl)butanoic acid (BCTG)>3-aminopropylphosphonic acid (3-APPA) and furthermore, the absolute agonist potency values were very close to each other. 3. 3-APPA was a partial agonist displaying maximal responses of 41 and 61% compared to GABA at GABABR1a and GABABR1b, respectively. The antagonist (RS)-3-amino-2-(4-chlorophenyl)-2-hydroxypropylsulphonic acid (2-OH-saclofen) displayed KB values of 15 and 7.8 microM at GABABR1a and GABABR1b, respectively. 4. The rank order of agonist potency as well as the absolute ligand potencies correspond very well with those previously reported in different tissues, and this study thus provides a functional assay of cloned GABAB receptors which should be a valuable tool for further characterization of GABAB ligands. Finally, we can conclude that the functional pharmacological profiles of the two GABABR1 splice variants are very similar.

Alternative Splicing↗
PubMed2006

Profile of executive and memory function associated with amphetamine and opiate dependence.

Cognitive function was assessed in chronic drug users on neurocognitive measures of executive and memory function. Current amphetamine users were contrasted with current opiate users, and these two groups were compared with former users of these substances (abstinent for at least one year). Four groups of participants were recruited: amphetamine-dependent individuals, opiate-dependent individuals, former users of amphetamines, and/or opiates and healthy non-drug taking controls. Participants were administered the Tower of London (TOL) planning task and the 3D-IDED attentional set-shifting task to assess executive function, and Paired Associates Learning and Delayed Pattern Recognition Memory tasks to assess visual memory function. The three groups of substance users showed significant impairments on TOL planning, Pattern Recognition Memory and Paired Associates Learning. Current amphetamine users displayed a greater degree of impairment than current opiate users. Consistent with previous research showing that healthy men are performing better on visuo-spatial tests than women, our male controls remembered significantly more paired associates than their female counterparts. This relationship was reversed in drug users. While performance of female drug users was normal, male drug users showed significant impairment compared to both their female counterparts and male controls. There was no difference in performance between current and former drug users. Neither years of drug abuse nor years of drug abstinence were associated with performance. Chronic drug users display pronounced neuropsychological impairment in the domains of executive and memory function. Impairment persists after several years of drug abstinence and may reflect neuropathology in frontal and temporal cortices.

Adult↗
PubMed1976

Spouses' understanding of the communication disabilities of aphasic patients.

The language skills of 11 aphasic patients were assessed through the use of the PICA. Spuses's understanding of the aphasic partner's language deficits was measured by rating the aphasic spouse on the Functional Communication Profile. Results indicate that spouses of aphasic patients view the patient's communication to be less impaired than it actually is. Implications for the counseling role of the speech pathologist in language therapy are discussed.

Adult↗
PubMed2003

Pharmacokinetics of cathinone, cathine and norephedrine after the chewing of khat leaves.

AIM: The stimulating herbal drug khat is habitually used in East Africa and the Arabian peninsula but is also imported into other countries. The aim was to study the pharmacokinetics of its alkaloids cathinone, cathine and norephedrine. METHODS: Four volunteers chewed khat leaves in an amount equivalent to one-quarter of that used in a typical khat session. Blood samples were collected up to 80 h and the alkaloids were assayed using gas chromatography-mass spectrometry. The data were evaluated using computerized pharmacokinetic compartmental analysis. RESULTS: The plasma concentration-time data for the alkaloids could be described using a two-compartment model with two-segment absorption. The mucosa of the oral cavity is considered to be the first absorption segment, where the major proportion of the alkaloids is absorbed (mean +/- SD 59 +/- 21% for cathinone and 84 +/- 6% for cathine). The extraction of the alkaloids from the leaves by chewing was very effective with only 9.1 +/- 4.2% remaining as a residue. Cathinone was eliminated from the central compartment with a mean half-life of 1.5 +/- 0.8 h. The half-life of cathine was 5.2 +/- 3.4 h. The metabolism of cathinone to norephedrine had a substantial influence on its plasma concentration profile. Psychophysical functions were essentially unaffected by the chewing of khat. CONCLUSIONS: The pharmacokinetics of khat alkaloids in humans explain why chewing is the preferred form of khat ingestion. Subjects absorbed a mean dose of 45 mg of cathinone, and did not suffer any severe adverse reactions.

Adult↗
PubMed2000

Ibuprofen inhibits adhesiveness of monocytes to endothelium and reduces cellular oxidative stress in smokers and non-smokers.

BACKGROUND: Cigarette smoking is a major risk factor in atherosclerosis and a useful model from which to study chronic inflammation. We compared monocyte function, lipid profiles and inflammatory markers in smokers and non-smokers, before and after oral ibuprofen intake. The adhesion of freshly isolated monocytes to native and tumour necrosis factor alpha (TNFalpha) stimulated human umbilical vein endothelial cells (HUVEC), as well as superoxide anion (O2-) levels and hydrogen peroxide (H2O2) production in resting and phorbol myristate acetate (PMA) stimulated monocytes were determined. MATERIALS AND METHODS: A group of nine smokers without any other coronary risk factor was compared with an age-matched group of 9 non-smokers. Tests were performed before and after a two-week course of oral ibuprofen (600 mg day-1). RESULTS: In smokers before ibuprofen, monocyte adhesion to native and TNFalpha-stimulated HUVEC was increased (P < 0001 and P < 0.01, respectively), and so were O2- levels in native and PMA-stimulated monocytes (P < 0.01 and P < 0.001, respectively). Ibuprofen reduced the adhesion of monocytes to native and stimulated HUVEC (P < 0.001) and O2- generation by resting and PMA-stimulated cells (P < 0.01) in both groups. H2O2 production by resting and PMA-stimulated monocytes was reduced in smokers and non-smokers (P < 0.01). Interestingly, ibuprofen increased HDL cholesterol levels in smokers (P < 0.01) and non-smokers (P < 0.001), and reduced the level of triglycerides in smokers (P < 0.05). CONCLUSION: Oral administration of ibuprofen reduced the adhesion of monocytes to HUVEC, suppressed oxidative stress and increased HDL cholesterol levels in smokers and non-smokers.

Adult↗
PubMed2001

Combination of subcutaneous levonorgestrel implants and transdermal dihydrotestosterone gel for male hormonal contraception.

The primary aim of the present study was to determine the therapeutic dose of subcutaneous levonorgestrel (LNG) to induce azoospermia or severe oligozoospermia (<3 x 10(6)/mL) in normal men requiring contraception. Transdermal 5 alpha-dihydrotestosterone (DHT) was combined to the treatments to maintain peripheral androgen level. Forty-three 21-45-year-old healthy men were enrolled in this phase II randomised and comparative clinical performance study. The subjects were allocated to five groups to receive: (1) transdermal DHT (Andractim(R), Besins Iscovesco, Paris, France) and one subdermal LNG implant (Jadelle, Leiras, Turku, Finland); (2) transdermal DHT and two subdermal LNG implants; (3) transdermal DHT and four subdermal LNG implants; (4) transdermal DHT and oral LNG (Microluton, Schering, Germany); or (5) transdermal DHT only. A total of 27 men completed the suppression phase. None of them reached azoospermia. One subject with oral LNG and transdermal DHT reached <3 million/mL at 5 months of suppression, but not repeatedly. Together 2/27 (7%) subjects, one with oral LNG and DHT and the other with four subdermal LNG implants and DHT reached <5 million/mL temporarily. Altogether 9/27 (33%) subjects reached <20 million/mL. Serum testosterone concentrations (S-T) decreased significantly during the first 3 months of treatment with one, two and four LNG implants and DHT and during the next 3 months S-T remained significantly lower with two or four implants. Serum oestradiol concentrations (S-E(2)) decreased significantly during the first 3 months only with four implants, but at 6 months S-E(2) was lower also in the group with two implants. Serum luteinizing hormone (LH) decreased significantly only with two LNG implants and DHT gel at 5 and 6 months. Serum FSH did not decrease in any of the groups. None of the subjects filled the criteria to continue to the efficacy phase. A total of 16 men discontinued for various reasons. Of the 27 men completing the suppression phase, all have recovered to sperm levels >20 million/mL. There were no changes in blood count, lipid profile, liver function tests, prostate-specific antigen (PSA), sex hormone binding globulin (SHBG), prolactin or cortisol. The mixed antiglobulin reaction (MAR)-IgG, MAR-IgA or tray agglutination test (TAT) did not change during any of the treatments. The present study shows that the LNG implants themselves are well-tolerated by men and safe, and might be suitable for replacing part of the testosterone used as injections to reduce the androgen dose during male hormonal contraception. The DHT gel was considered as quite or very uncomfortable by 66% of the men because of feeling cold during the time it was on the skin, but noncompliance in using the gel was not reported by the men.

Adult↗
PubMed1997

Comparison of three measures of quality of life outcome in the evaluation of continuous positive airways pressure therapy for sleep apnoea.

Treatment of obstructive sleep apnoea (OSA) with nasal continuous positive airway pressure (NCPAP) has become a standard treatment since its introduction in 1981. Following such treatment the apnoeas disappear, sleep quality improves as apparently do daytime symptoms of sleepiness. Sleepiness is an unusual symptom and its impact on conventional indices of quality of life has rarely been measured. To allow comparison of NCPAP therapy with treatments for other conditions, measurements of quality of life before and after treatment using standard techniques are required. It is not clear which of the standard measures is most suited to measuring the health gain from nasal NCPAP, and indeed whether the disability of excessive sleepiness is included in all such measures. This study looks at three well recognized quality of life measures in OSA, before and after NCPAP therapy; the Short Form 36 (SF-36), Functional Limitations Profile (FLP), and the EuroQol (EQ-5D). The results were compared with data from normal populations. One hundred and eight patients with OSA undergoing a therapeutic assessment of NCPAP completed the three quality of life questionnaires before and 5 weeks after commencing treatment. Over 90 subjects completed all sections of the three measures on both occasions. The SF-36 revealed substantial adverse effects on subjective health of OSA and that NCPAP treatment produced dramatic positive effects. For example, the effect sizes (difference in score, divided by SD of baseline score) in the Energy/Vitality dimension was 0.98 and for the overall Mental and Physical Component Scores, 0.76 and 0.57, respectively: an effect size over 0.5 is considered moderate and over 0.8 as large. The FLP data showed similar pre treatment decrements in quality of life and substantial improvements following NCPAP. The changes with treatment in the majority of the dimensions from both the SF-36 and FLP were statistically significant (P < 0.01). In contrast the EQ-5D did not show significant improvements with therapy, presumably because of its failure to measure the aspects of quality of life related to severe sleep fragmentation and daytime sleepiness. In conclusion, this study has clearly shown considerable decrements in quality of life in patients with OSA, similar to other chronic disabling conditions. Furthermore, NCPAP therapy returns patients to a quality of life similar to the normal population.

Adult↗
PubMed1999

N-glycan structures of matrix metalloproteinase-1 derived from human fibroblasts and from HT-1080 fibrosarcoma cells.

Matrix metalloproteinase-1 (MMP-1) is a collagenolytic metalloproteinase capable of cleaving native triple-helical forms of several collagen subtypes, as well as a number of non-collagenous substrates. The role of MMP-1 in various diseases affecting the connective tissue is well characterized. MMP-1 is secreted as both glycosylated and unglycosylated species, and the two forms have been shown to be identical with respect to substrate specificity, specific activity and inhibitory profile. No function for the glycan moiety of the enzyme has been ascribed to date. In the present study, we report on the detailed characterization of MMP-1-derived oligosaccharides. Using strategies based on sequential exoglycosidase digestion combined with matrix-assisted laser desorption ionization-time of flight MS and electrospray tandem MS, we have characterized the N-glycan structures of MMP-1, derived from human dermal fibroblasts and from the HT-1080 fibrosarcoma cell line. MMP-1 derived from fibroblasts was found to carry mainly alpha 2,3-sialylated complex-type diantennary glycans. On the other hand, HT-1080 cells produce MMP-1 that has a heterogeneous glycosylation pattern, comprising diantennary glycans carrying Lewis X, LacdiNAc, sialylated LacdiNAc and GalNAc beta 1,4 (Fuc alpha 1,3)GlcNAc (LacdiNAc analogue of Lewis X) as terminal elements. We also show that, of the two potential glycosylation sites in the MMP-1 sequence, only Asn120 is used.

Carbohydrate Sequence↗
PubMed1998

Randomized long-term evaluation of bicarbonate-buffered CAPD solution.

BACKGROUND: Over the past 15 years, lactate has been used successfully as a buffer in peritoneal dialysis solutions, although its effectiveness in the correction of uremic acidosis and its biocompatibility on peritoneal resident cells have been questioned. In addition, some investigators have suggested other potential adverse metabolic effects resulting from the unphysiologically high lactate flux into the body during CAPD. These potential problems associated with lactate-containing CAPD solution prompted the search for alternative buffer-containing solutions. Bicarbonate, the physiological buffer, was considered when the problem of calcium and magnesium carbonate solubility was solved by the use of a two-compartment bag system, allowing the mixing of bicarbonate and divalent cations immediately before infusion. The long-term tolerance, safety, efficacy and therapeutic value of a bicarbonate-buffered peritoneal dialysis solution were evaluated in this study. METHODS: This open, randomized, controlled, multicenter study comparing a 34 mmol/liter bicarbonate- with a 35 mmol/liter lactate-buffered peritoneal dialysis solution was performed in two consecutive 12-week-treatment phases. Fourteen Centers participated in this trial. RESULTS: A total of 69 out of initially 123 randomized patients completed the six-month study period (36 patients in the bicarbonate group and 33 in the lactate group). While the arterial acid base status of the total study population did not change during the study period and no significant difference was observed between the two treatment groups, the acid-base status of patients in the bicarbonate group entering the study with a metabolic acidosis significantly improved (mean +/- SD; blood pH: baseline = 7.361 +/- 0.05, week 12 = 7.380 +/- 0.04, P < 0.05; week 24 = 7.388 +/- 0.03 P < 0.05; plasma bicarbonate: baseline = 19.49 +/- 3.01 mmol/liter, week 12 = 21.16 +/- 2.63 mmol/liter, P < 0.01; week 24 = 21.51 +/- 2.42 mmol/liter, P < 0.01). No significant changes were recorded in acidotic patients treated with the conventional lactate-buffered solution. The changes in plasma bicarbonate from baseline during the study was significantly different between the groups (week 12: lactate = +0.11 +/- 2.21 mmol/liter, bicarbonate = +1.69 +/- 2.55 mmol/liter, P < 0.05, 95% confidence interval for the difference 0.21 to 2.94 mmol/liter; week 24: lactate = +0.03 +/- 2.48 mmol/liter, bicarbonate = +1.82 +/- 2. 96 mmol/liter, P < 0.05, 95% confidence interval for the difference 0.16 to 3.42 mmol/liter). The normalized protein catabolic rate (nPCR) slightly but significantly decreased in the lactate group (baseline -0.90 +/- 0.23 g/kg/day, week 24 -0.83 +/- 0.21 g/kg/day, P < 0.01) and increased in the bicarbonate group (baseline +0.89 +/- 0.28 g/kg/day, week 24 +0.92 +/- 0.26 g/kg/day, P < 0.05). Changes from baseline between groups were significant (week 24, lactate = -0. 099 +/- 0.15 g/kg/day, bicarbonate = 0.049 +/- 0.12 g/kg/day, P < 0. 01, 95% confidence interval for the difference 0.068 to 0.229 g/kg/day). Other evaluated parameters (biochemical profile, peritoneal function parameters, dialysate protein loss) did not differ significantly between the two groups. No adverse effects related to the study solution were recorded. CONCLUSIONS: These results support the efficacy and safety of bicarbonate-buffered peritoneal solutions in a controlled randomized comparison for up to six months. Peritoneal dialysis solutions containing the physiological buffer bicarbonate might effectively replace conventional lactate-buffered CAPD solutions.

Acid-Base Equilibrium↗
PubMed2000

Exercise training for claudicants: changes in blood flow, cardiorespiratory status, metabolic functions, blood rheology and lipid profile.

OBJECTIVE: Exercise training improves the walking distance of claudicants. The aim of this study was to investigate factors associated with the improvement in the maximum walking distance (MWD) in respect to cardiovascular, respiratory and metabolic adaptations. METHODS: Forty claudicants were studied. Common femoral artery blood flow (BF), heart rate (HR), oxygen consumption (VO(2)), respiratory exchange ratio (RER), lactate levels, blood rheology and lipid profiles were measured. Tests were repeated after 3 months of exercise training. RESULTS: Fifteen patients did not complete the exercise program. For patients who did complete the program, MWD improved by 82%. A significant reduction in HR and VO(2)during exercise was demonstrated. No significant changes occurred in BF or RER. Although MWD increased significantly, there was no increase in recovery VO(2)(oxygen debt). A significant reduction in post-exercise lactate levels occurred. Blood rheology was unchanged, but an improvement in HDL levels was noted. CONCLUSIONS: Many claudicants could not complete an exercise program, mainly due to osteoarthritis. Exercise training improved exercise tolerance significantly without any increase in BF. The HR and oxygen cost of similar exercise was reduced. An improved MWD did not correlate with a higher oxygen debt or lactate load. Favourable changes in lipid profiles occurred.

Aged↗
PubMed2001

Prospective study comparing human albumin vs. reinfusion of ultrafiltrate-ascitic fluid after total paracentesis in cirrhotic patients with tense ascites.

Although, total paracentesis associated with human albumin substitution has shown to be a rapid, effective and safe treatment of diuretic refractory ascites in advanced liver cirrhosis, it implies high costs and has a limited availability. Therefore an alternative procedure the reinfusion of concentrated ascites has gained popularity in recent years (Smart et al. 1990; Grazioto et al. 1997). It was the aim of the study to compare human albumin substitution vs. reinfusion of ascitic-ultrafiltrate after total paracentesis. 35 patients with cirrhosis and tense ascites received total paracentesis associated with either human albumin (5-8 g/l ascites) (= group A) or reinfusion of an ascitic-ultrafiltrate fluid by means of hemofiltration technique (= group B). The mean volume of ascites removed was 9.41 (2.1-20.0) in group A and 11.41 (6.5-21.0) in group B. No significant differences in serum electrolytes, liver and renal function, coagulation profiles and hormones of the renin-angiotensin-aldosterone system were observed during hospitalization. In both groups sodium excretion increased significantly. 43% of the patients in group B developed pyrexia and chill after reinfusion of the ascitic-ultrafiltrate fluid. In one patient an anaphylactic bronchospasm occurred requiring IUC-treatment. The treatment cost in case of human albumin were 326.-DM vs. 290.-DM for each patient treated with ascitic-ultrafiltrate fluid reinfusion. The probabilities of hospital readmission and survival were similar in both groups during follow-up. The results indicate that i.v. infusion of ascitic-ultrafiltrate fluid is as effective as total paracentesis and albumin infusion in case of diuretic refractory ascites. However, according to the costs of instruments and staff and due to the significant allergic reactions caused by ascitic fluid it cannot be considered as a real alternative to albumin substitution.

Anaphylaxis↗
PubMed2005

Adsorption of comb copolymers on weakly attractive solid surfaces.

In this work continuum and lattice Monte Carlo simulation methods are used to study the adsorption of linear and comb polymers on flat surfaces. Selected polymer segments, located at the tips of the side chains in comb polymers or equally spaced along the linear polymers, are attracted to each other and to the surface via square-well potentials. The rest of the polymer segments are modeled as tangent hard spheres in the continuum model and as self-avoiding random walks in the lattice model. Results are presented in terms of segment-density profiles, distribution functions, and radii of gyration of the adsorbed polymers. At infinite dilution the presence of short side chains promotes the adsorption of polymers favoring both a decrease in the depletion-layer thickness and a spreading of the polymer molecule on the surface. The presence of long side chains favors the adsorption of polymers on the surface, but does not permit the spreading of the polymers. At finite concentration linear polymers and comb polymers with long side chains readily adsorb on the solid surface, while comb polymers with short side chains are unlikely to adsorb. The simple models of comb copolymers with short side chains used here show properties similar to those of associating polymers and of globular proteins in aqueous solutions, and can be used as a first approximation to investigate the mechanism of adsorption of proteins onto hydrophobic surfaces.

Journal Article↗
PubMed2006

Chemical shift anisotropic mapping of a coherent optical absorber using magnetic field induced quantum beats.

A new method for mapping the spatial structure of optical coherent materials which relies on imposing a set of linear orthogonal gradient magnetic fields for a controlled hyperfine splitting of energy levels to create characteristic quantum beats when illuminated with a laser pulse with sufficient bandwidth to excite these levels is proposed. In this approach, a spectroscopic fingerprint of the dopant sites due to concentration and field susceptibilities in the sample is achieved through a Fourier decomposition of the radiative relaxation decay in an approach analogous to nuclear magnetic resonance spectroscopy due to the imposition of a controlled spatial-spectral encoding scheme. A three pulse sequence necessary to interrogate a gradient resolved voxel is also discussed. This three pulse approach can be combined with the conventional confocal imaging technique to provide information about the underlying chemistry of dopant distribution along each imaging plane which is useful in guiding the design and manufacturing process of optical crystals. In combination with gradient induced quantum beats, the entire inhomogeneous bandwidth can be interrogated. The proposed approach would scan this entire bandwidth at much faster rate enabling characterization of a large number of crystals than is currently possible through mechanical scanning with a confocal microscopy based spectroscopic technique as well as providing functional dopant profiling which is not currently possible with conventional approaches.

Journal Article↗
PubMed2006

Influence of atomistic physics on electro-osmotic flow: an analysis based on density functional theory.

Molecular density profiles and charge distributions determined by density functional theory (DFT) are used in conjunction with the continuum Navier-Stokes equations to compute electro-osmotic flows in nanoscale channels. The ion species of the electrolyte are represented as centrally charged hard spheres, and the solvent is treated as a dense fluid of neutral hard spheres having a uniform dielectric constant. The model explicitly accounts for Lennard-Jones interactions among fluid and wall molecules, hard sphere repulsions, and short range electrical interactions, as well as long range Coulombic interactions. Only the last of these interactions is included in classical Poisson-Boltzmann (PB) modeling of the electric field. Although the proposed DFT approach is quite general, the sample calculations presented here are limited to symmetric monovalent electrolytes. For a prescribed surface charge, this DFT model predicts larger counterion concentrations near charged channel walls, relative to classical PB modeling, and hence smaller concentrations in the channel center. This shifting of counterions toward the walls reduces the effective thickness of the Debye layer and reduces electro-osmotic velocities as compared to classical PB modeling. Zeta potentials and fluid speeds computed by the DFT model are as much as two or three times smaller than corresponding PB results. This disparity generally increases with increasing electrolyte concentration, increasing surface charge density and decreasing channel width. The DFT results are found to be comparable to those obtained by molecular dynamics simulation, but require considerably less computing time.

Journal Article↗
PubMed2004

A vascular cell-restricted RhoGAP, p73RhoGAP, is a key regulator of angiogenesis.

Angiogenesis is a major therapeutic target. Ideal drug targets are genes expressed only in endothelial cells (ECs) or only during the angiogenic process. Here, we describe a gene, p73RhoGAP (p73), that has both of these properties. By using a PCR-based subtraction-hybridization approach to clone cDNAs from ECs undergoing capillary-tube formation, we identified a RhoGAP member, p73. p73 displays GTPase activity to Rho but not to Rac or Cdc42. Knockdown of p73 protein, achieved by adenovirus delivery of p73 antisense and by small interfering RNA into ECs, demonstrated the importance of this protein in EC function. Under such conditions, EC migration, proliferation, and capillary-tube formation were inhibited. Furthermore, angiogenesis in vivo was also inhibited by antisense p73. A mutant R82A alteration achieved a similar phenotype in vitro to the antisense, demonstrating the importance of the GTPase-activating protein activity to p73 function. Expression profiling of p73 shows that it is vascular cell-selective, being highly expressed in ECs and smooth-muscle cells but not in other cell types. Finally, we show that the mRNA of p73 is up-regulated in an angiogenic milieu with little or no regulation seen under nonangiogenic conditions. p73, a vascular cell-specific GTPase-activating protein, is an important modulator of angiogenesis and displays many of features that make it worthy of being a drug target.

Base Sequence↗
PubMed2003

Identifying residue-residue clashes in protein hybrids by using a second-order mean-field approach.

In this article, a second-order mean-field-based approach is introduced for characterizing the complete set of residue-residue couplings consistent with a given protein structure. This information is subsequently used to classify protein hybrids with respect to their potential to be functional based on the presenceabsence and severity of clashing residue-residue interactions. First, atomistic representations of both the native and denatured states are used to calculate rotamer-backbone, rotamer-intrinsic, and rotamer-rotamer conformational energies. Next, this complete conformational energy table is coupled with a second-order mean-field description to elucidate the probabilities of all possible rotamer-rotamer combinations in a minimum Helmholtz free-energy ensemble. Computational results for the dihydrofolate reductase family reveal correlation in substitution patterns between not only contacting but also distal second-order structural elements. Residue-residue clashes in hybrid proteins are quantified by contrasting the ensemble probabilities of protein hybrids against the ones of the original parental sequences. Good agreement with experimental data is demonstrated by superimposing these clashes against the functional crossover profiles of bidirectional incremental truncation libraries for Escherichia coli and human glycinamide ribonucleotide transformylases.

Protein Conformation↗
PubMed2002

Dicer is required for chromosome segregation and gene silencing in fission yeast cells.

RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1+) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into approximately 23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1+ was required for silencing of two genes containing a conserved motif.

Chromosome Segregation↗
PubMed2001

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway.

The differential properties of anion-mediated Fe(3+) release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe(3+) release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe(3+) with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe(3+) release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe(3+). The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 A resolution demonstrated the existence of a single bound SO(4)(2-) in the interdomain cleft, which interacts directly with Thr(461)-OG1, Tyr(431)-OH, and His(592)-NE2 and indirectly with Tyr(524)-OH. The latter three groups are Fe(3+)-coordinating ligands, strongly suggesting the facilitated Fe(3+) release upon the anion occupation at this site. The SO(4)(2-) binding structure supported the single-pathway kinetic model.

Anions↗