60 resultados para Structure-function
Resumo:
Cytochrome P450 proteins are involved in metabolism of drugs and xenobiotics. In the endoplasmic reticulum a single nicotinamide adenine dinucleotide phosphate (NADPH) P450 oxidoreductase (POR) supplies electrons to all microsomal P450s for catalytic activity. POR is a flavoprotein that contains both flavin mononucleotide and flavin adenine dinucleotide as cofactors and uses NADPH as the source of electrons. We have recently reported a number of POR mutations in the patients with disordered steroidogenesis. In the first report we had described missense mutations (A287P, R457H, V492E, C569Y, and V608F) identified in four patients with defects in steroid production. Each POR variant was produced as recombinant N-27 form of the enzyme in bacteria and as full-length form in yeast. Membranes from bacteria or yeast expressing normal or variant POR were purified and their activities were characterized in cytochrome c and CYP17A1 assays. Later we have published a larger study that described a whole range of POR mutations and characterized the mutants/polymorphisms A115V, T142A, M263V, Y459H, A503V, G539R, L565P, R616X, V631I, and F646del from the sequencing of patient DNA. We also studied POR variants Y181D, P228L, R316W, G413S, and G504R that were available in public databases or published literature. Three-dimensional structure of rat POR is known and we have used this structure to deduce the structure-function correlation of POR mutations in human. The missense mutations found in patients with disordered steroidogenesis are generally in the co-factor binding and functionally important domains of POR and the apparent polymorphisms are found in regions with lesser structural importance. A variation in POR can alter the activity of all microsomal P450s, and therefore, can affect the metabolism of drugs and xenobiotics even when the P450s involved are otherwise normal. It is important to study the genetic and biochemical basis of POR variants in human population to gain information about possible differences in P450 mediated reactions among the individuals carrying a variant or polymorphic form of POR that could impact their metabolism.
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The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.
Resumo:
The alveolated structure of the pulmonary acinus plays a vital role in gas exchange function. Three-dimensional (3D) analysis of the parenchymal region is fundamental to understanding this structure-function relationship, but only a limited number of attempts have been conducted in the past because of technical limitations. In this study, we developed a new image processing methodology based on finite element (FE) analysis for accurate 3D structural reconstruction of the gas exchange regions of the lung. Stereologically well characterized rat lung samples (Pediatr Res 53: 72-80, 2003) were imaged using high-resolution synchrotron radiation-based X-ray tomographic microscopy. A stack of 1,024 images (each slice: 1024 x 1024 pixels) with resolution of 1.4 mum(3) per voxel were generated. For the development of FE algorithm, regions of interest (ROI), containing approximately 7.5 million voxels, were further extracted as a working subunit. 3D FEs were created overlaying the voxel map using a grid-based hexahedral algorithm. A proper threshold value for appropriate segmentation was iteratively determined to match the calculated volume density of tissue to the stereologically determined value (Pediatr Res 53: 72-80, 2003). The resulting 3D FEs are ready to be used for 3D structural analysis as well as for subsequent FE computational analyses like fluid dynamics and skeletonization.
Resumo:
RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.
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Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.
Resumo:
BACKGROUND Delayed enhancement (DE) MRI can assess the fibrotic substrate of scar-related VT. MDCT has the advantage of inframillimetric spatial resolution and better 3D reconstructions. We sought to evaluate the feasibility and usefulness of integrating merged MDCT/MRI data in 3D-mapping systems for structure-function assessment and multimodal guidance of VT mapping and ablation. METHODS Nine patients, including 3 ischemic cardiomyopathy (ICM), 3 nonischemic cardiomyopathy (NICM), 2 myocarditis, and 1 redo procedure for idiopathic VT, underwent MRI and MDCT before VT ablation. Merged MRI/MDCT data were integrated in 3D-mapping systems and registered to high-density endocardial and epicardial maps. Low-voltage areas (<1.5 mV) and local abnormal ventricular activities (LAVA) during sinus rhythm were correlated to DE at MRI, and wall-thinning (WT) at MDCT. RESULTS Endocardium and epicardium were mapped with 391 ± 388 and 1098 ± 734 points per map, respectively. Registration of MDCT allowed visualization of coronary arteries during epicardial mapping/ablation. In the idiopathic patient, integration of MRI data identified previously ablated regions. In ICM patients, both DE at MRI and WT at MDCT matched areas of low voltage (overlap 94 ± 6% and 79 ± 5%, respectively). In NICM patients, wall-thinning areas matched areas of low voltage (overlap 63 ± 21%). In patients with myocarditis, subepicardial DE matched areas of epicardial low voltage (overlap 92 ± 12%). A total number of 266 LAVA sites were found in 7/9 patients. All LAVA sites were associated to structural substrate at imaging (90% inside, 100% within 18 mm). CONCLUSION The integration of merged MDCT and DEMRI data is feasible and allows combining substrate assessment with high-spatial resolution to better define structure-function relationship in scar-related VT.
Resumo:
BACKGROUND Functional characterization of mutations involving the SCN5A-encoded cardiac sodium channel has established the pathogenic mechanisms for type 3 long QT syndrome and type 1 Brugada syndrome and has provided key insights into the physiological importance of essential structure-function domains. OBJECTIVE This study sought to present the clinical and biophysical phenotypes discerned from compound heterozygosity mutations in SCN5A on different alleles in a toddler diagnosed with QT prolongation and fever-induced ventricular arrhythmias. METHODS A 22-month-old boy presented emergently with fever and refractory ventricular tachycardia. Despite restoration of sinus rhythm, the infant sustained profound neurological injury and died. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, comprehensive open-reading frame/splice mutational analysis of the 12 known long QT syndrome susceptibility genes was performed. RESULTS The infant had 2 SCN5A mutations: a maternally inherited N-terminal frame shift/deletion (R34fs/60) and a paternally inherited missense mutation, R1195H. The mutations were engineered by site-directed mutagenesis and heterologously expressed transiently in HEK293 cells. As expected, the frame-shifted and prematurely truncated peptide, SCN5A-R34fs/60, showed no current. SCN5A-R1195H had normal peak and late current but abnormal voltage-dependent gating parameters. Surprisingly, co-expression of SCN5A-R34fs/60 with SCN5A-R1195H elicited a significant increase in late sodium current, whereas co-expression of SCN5A-WT with SCN5A-R34fs/60 did not. CONCLUSIONS A severe clinical phenotype characterized by fever-induced monomorphic ventricular tachycardia and QT interval prolongation emerged in a toddler with compound heterozygosity involving SCN5A: R34fs/60, and R1195H. Unexpectedly, the 94-amino-acid fusion peptide derived from the R34fs/60 mutation accentuated the late sodium current of R1195H-containing Na(V)1.5 channels in vitro.
Resumo:
Mammalian members of the proton-coupled oligopeptide transporter family (SLC15) are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs. The driving force for uphill electrogenic symport is the chemical gradient and membrane potential which favors proton uptake into the cell along with the peptide/mimetic substrate. The peptide transporters are responsible for the absorption and conservation of dietary protein digestion products in the intestine and kidney, respectively, and in maintaining homeostasis of neuropeptides in the brain. They are also responsible for the absorption and disposition of a number of pharmacologically important compounds including some aminocephalosporins, angiotensin-converting enzyme inhibitors, antiviral prodrugs, and others. In this review, we provide updated information on the structure-function of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4) and PhT2 (SLC15A3), and their expression and localization in key tissues. Moreover, mammalian peptide transporters are discussed in regard to pharmacogenomic and regulatory implications on host pharmacology and disease, and as potential targets for drug delivery. Significant emphasis is placed on the evolving role of these peptide transporters as elucidated by studies using genetically modified animals. Whenever possible, the relevance of drug-drug interactions and regulatory mechanisms are evaluated using in vivo studies.
Resumo:
Abnormal yawning is an underappreciated phenomenon in patients with ischemic stroke. We aimed at identifying frequently affected core regions in the supratentorial brain of stroke patients with abnormal yawning and contributing to the anatomical network concept of yawning control. Ten patients with acute anterior circulation stroke and ≥3 yawns/15 min without obvious cause were analyzed. The NIH stroke scale (NIHSS), Glasgow Coma Scale (GCS), symptom onset, period with abnormal yawning, blood oxygen saturation, glucose, body temperature, blood pressure, heart rate, and modified Rankin scale (mRS) were assessed for all patients. MRI lesion maps were segmented on diffusion-weighted images, spatially normalized, and the extent of overlap between the different stroke patterns was determined. Correlations between the period with abnormal yawning and the apparent diffusion coefficient (ADC) in the overlapping regions, total stroke volume, NIHSS and mRS were performed. Periods in which patients presented with episodes of abnormal yawning lasted on average for 58 h. Average GCS, NIHSS, and mRS scores were 12.6, 11.6, and 3.5, respectively. Clinical parameters were within normal limits. Ischemic brain lesions overlapped in nine out of ten patients: in seven patients in the insula and in seven in the caudate nucleus. The decrease of the ADC within the lesions correlated with the period with abnormal yawing (r = -0.76, Bonferroni-corrected p = 0.02). The stroke lesion intensity of the common overlapping regions in the insula and the caudate nucleus correlates with the period with abnormal yawning. The insula might be the long sought-after brain region for serotonin-mediated yawning.
Resumo:
Preclinical studies using animal models have shown that grey matter plasticity in both perilesional and distant neural networks contributes to behavioural recovery of sensorimotor functions after ischaemic cortical stroke. Whether such morphological changes can be detected after human cortical stroke is not yet known, but this would be essential to better understand post-stroke brain architecture and its impact on recovery. Using serial behavioural and high-resolution magnetic resonance imaging (MRI) measurements, we tracked recovery of dexterous hand function in 28 patients with ischaemic stroke involving the primary sensorimotor cortices. We were able to classify three recovery subgroups (fast, slow, and poor) using response feature analysis of individual recovery curves. To detect areas with significant longitudinal grey matter volume (GMV) change, we performed tensor-based morphometry of MRI data acquired in the subacute phase, i.e. after the stage compromised by acute oedema and inflammation. We found significant GMV expansion in the perilesional premotor cortex, ipsilesional mediodorsal thalamus, and caudate nucleus, and GMV contraction in the contralesional cerebellum. According to an interaction model, patients with fast recovery had more perilesional than subcortical expansion, whereas the contrary was true for patients with impaired recovery. Also, there were significant voxel-wise correlations between motor performance and ipsilesional GMV contraction in the posterior parietal lobes and expansion in dorsolateral prefrontal cortex. In sum, perilesional GMV expansion is associated with successful recovery after cortical stroke, possibly reflecting the restructuring of local cortical networks. Distant changes within the prefrontal-striato-thalamic network are related to impaired recovery, probably indicating higher demands on cognitive control of motor behaviour.
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The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.
Resumo:
CONTEXT The autosomal dominant form of GH deficiency (IGHD II) is characterized by markedly reduced GH secretion combined with low concentrations of IGF-1 leading to short stature. OBJECTIVE Structure-function analysis of a missense mutation in the GH-1 gene converting codon 76 from leucine (L) to proline (P) yielding a mutant GH-L76P peptide. DESIGN, SETTINGS, AND PATIENTS Heterozygosity for GH-L76P/wt-GH was identified in a nonconsanguineous Spanish family. The index patients, two siblings, a boy and a girl, were referred for assessment of their short stature (-3.2 and -3.8 SD). Their grandmother, father, and aunt were also carrying the same mutation and showed severe short stature; therefore, IGHD II was diagnosed. INTERVENTIONS AND RESULTS AtT-20 cells coexpressing both wt-GH and GH-L76P showed a reduced GH secretion (P < .001) after forskolin stimulation when compared with the cells expressing only wt-GH. In silico mutagenesis and molecular dynamics simulations presented alterations of correct folding and mutant stability compared with wt-GH. Therefore, further structural analysis of the GH-L76P mutant was performed using expressed and purified proteins in Escherichia coli by thermofluor assay and fast degradation proteolysis assay. Both assays revealed that the GH-L76P mutant is unstable and misfolded compared to wt-GH confirming the bioinformatic model prediction. CONCLUSIONS This is the first report of a family suffering from short stature caused by IGHD II, which severely affects intracellular GH folding and stability as well as secretion, highlighting the necessity of functional analysis of any GH variant for defining new mechanisms as a cause for IGHD II.
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Recent evidence suggests that individual differences in physical activity (PA) may be associated with individual differences in white matter microstructure and with grey matter volume of the hippocampus. Therefore, this study investigated the association between PA and white matter microstructure of pathways connecting to the hippocampus. A total of 33 young, healthy adults underwent magnetic resonance imaging (MRI). High angular resolution diffusion-weighted imaging and multi-component relaxometry MRI scans (multi-component driven equilibrium pulse observation of T1 and T2) were acquired for each participant. Activity levels (AL) of participants were calculated from 72-h actigraphy recordings. Tractography using the damped Richardson Lucy algorithm was used to reconstruct the fornix and bilateral parahippocampal cinguli (PHC). The mean fractional anisotropy (FA) and the myelin water fraction (MWF), a putative marker of myelination, were determined for each pathway. A positive correlation between both AL and FA and between AL and MWF were hypothesized for the three pathways. There was a selective positive correlation between AL and MWF in the right PHC (r = 0.482, p = 0.007). Thus, our results provide initial in vivo evidence for an association between myelination of the right PHC and PA in young healthy adults. Our results suggest that MWF may not only be more specific, but also more sensitive than FA to detect white matter microstructural alterations. If PA was to induce structural plasticity of the right PHC this may contribute to reverse structural alterations of the right PHC in neuropsychiatric disorder with hippocampal pathologies.
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Severe hereditary coagulation factor XIII deficiency is a rare homozygous bleeding disorder affecting one person in every two million individuals. In contrast, heterozygous factor XIII deficiency is more common, but usually not associated with severe hemorrhage such as intracranial bleeding or hemarthrosis. In most cases, the disease is caused by F13A gene mutations. Causative mutations associated with the F13B gene are rarer.
Resumo:
Recent findings are reported about certain aspects of the structure and function of the mammalian and avian lungs that include (a) the architecture of the air capillaries (ACs) and the blood capillaries (BCs); (b) the pulmonary blood capillary circulatory dynamics; (c) the adaptive molecular, cellular, biochemical, compositional, and developmental characteristics of the surfactant system; (d) the mechanisms of the translocation of fine and ultrafine particles across the airway epithelial barrier; and (e) the particle-cell interactions in the pulmonary airways. In the lung of the Muscovy duck Cairina moschata, at least, the ACs are rotund structures that are interconnected by narrow cylindrical sections, while the BCs comprise segments that are almost as long as they are wide. In contrast to the mammalian pulmonary BCs, which are highly compliant, those of birds practically behave like rigid tubes. Diving pressure has been a very powerful directional selection force that has influenced phenotypic changes in surfactant composition and function in lungs of marine mammals. After nanosized particulates are deposited on the respiratory tract of healthy human subjects, some reach organs such as the brain with potentially serious health implications. Finally, in the mammalian lung, dendritic cells of the pulmonary airways are powerful agents in engulfing deposited particles, and in birds, macrophages and erythrocytes are ardent phagocytizing cellular agents. The morphology of the lung that allows it to perform different functions-including gas exchange, ventilation of the lung by being compliant, defense, and secretion of important pharmacological factors-is reflected in its "compromise design."