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The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonic anhydrase II (HCA II) has been examined by kinetic, X-ray, and simulation approaches. The X-ray crystallography studies, which were previously reported, and molecular dynamics (MD) simulations indicate that the proton shuttling residue, His64, predominantly resides in the outward orientation with a significant disruption of the ordered water in the active site for the dehydration pathway. While disorder is seen in the active-site water, water cluster analysis indicates that the N67L mutant may form water clusters similar to those seen in the wild-type (WT). For the hydration pathway of the enzyme, the active site water cluster analysis reveals an inability of the N67L mutant to stabilize water clusters when His64 is in the inward orientation, thereby favoring PT when His64 is in the outward orientation. The preference of the N67L mutant to carry out the PT when His64 is in the outward orientation for both the hydration and dehydration pathway is reasoned to be the main cause of the observed reduction in the overall rate. To probe the mechanism of PT, solvent H/D kinetic isotope effects (KIEs) were experimentally studied with catalysis measured by the exchange of (18)O between CO(2) and water. The values obtained from the KIEs were determined as a function of the deuterium content of solvent, using the proton inventory method. No differences were detected in the overarching mechanism of PT between WT and N67L HCA II, despite changes in the active-site water structure and/or the orientation of His64.

The GIGYF2 (Grb10-Interacting GYF Protein-2) gene has recently been proposed to be the responsible gene for the PARK11 locus. Ten different putative pathogenic variants were identified in cohorts of Parkinson's disease (PD) patients from Italy and France. Among these variants Asn56Ser and Asn457Thr were found repeatedly. In the present study we screened 669 PD patients (predominantly of central European origin) and 1051 control individuals for the presence of these two variants. Asn56Ser was found in one patient with a positive family history of the disease and in one control individual. The affected sister of the patient did not carry this variant. Asn457Thr was found in one patient, who was exceptional for his Egyptian origin and in three control individuals. This variant was not found in 50 control individuals from Egypt. We conclude that neither of these two variants plays a major role in the pathogenesis of PD in our study population.

The rates of deamidation of alpha-synuclein and single Asn residues in 13 Asn-sequence mutants have been measured for 5 x 10(-5)M protein in both the absence and presence of 10(-2)M sodium dodecyl sulfate (SDS). In the course of these experiments, 370 quantitative protein deamidation measurements were performed and 37 deamidation rates were determined by ion cyclotron resonance Fourier transform mass spectrometry, using an improved whole protein isotopic envelope method and a mass defect method with both enzymatic and collision-induced fragmentation. The measured deamidation index of alpha-synuclein was found to be 0.23 for an overall deamidation half-time of 23 days, without or with SDS micelles, owing primarily to the deamidation of Asn(103) and Asn(122). Deamidation rates of 15 Asn residues in the wild-type and mutant proteins were found to be primary sequence controlled without SDS. However, the presence of SDS micelles slowed the deamidation rates of nine N-terminal region Asn residues, caused by the known three-dimensional structures induced through protein binding to SDS micelles.

We analyzed the envelope proteins in pathogenic flaviviruses to determine whether there are sequence signatures associated with the tendency of viruses to produce hemorrhagic disease (H-viruses) or encephalitis (E-viruses). We found that, at the position corresponding to the glycosylated Asn-67 in dengue virus, asparagine (Asn) occurs in all seven viral species that cause hemorrhagic disease in humans. Furthermore, Asn was extremely rare at position 67 in six flaviviruses that cause encephalitis, being replaced by Asp in four of them. Of the 3,246 sequences from H- and E-viruses, we found that 2,916 sequences (90%) contained Asn in position 67 for H-viruses or Asp in position 67 for E-viruses. The change from Asn-67 that is prevalent in H-viruses to Asp-67 (common in E-viruses) contributes to a stronger electrostatically negative surface in the E-viruses as compared to the H-viruses. These findings should help predicting the disease potential of emerging and re-emerging flaviviruses and understanding the relationship between protein structure and disease outcome.

Objective: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) causes multiple endocrine deficiencies, oral candidiasis and different forms of ectodermal dystrophy including enamel hypoplasia, documented in permanent teeth. Our purpose was to examine dental aberrations associated with APECED, including possible manifestations in primary teeth., Design: We studied clinically, radiographically, and by scanning electron microscopy (SEM) teeth of children belonging to two APECED families with different mutations in the AIRE gene., Results: In addition to enamel defects in the permanent teeth we observed hypoplastic pits and hypomaturated patches in the deciduous teeth with underlying changes in the prismatic ultrastructure. The enamel of the permanent molars exhibited a layered arrangement with included whirl-like formations., Conclusions: Our findings confirm that APECED causes enamel defects that are individually but chronologically distributed, and can alter enamel development early enough to affect deciduous teeth.

Mutations in LGI1 have been reported in several families with autosomal dominant lateral temporal epilepsy. In a family in which three patients also experienced migraine-like episodes we found a novel three base-pair deletion (c.377_379delACA), resulting in the deletion of an asparagine residue in the second leucine-rich repeat. Functional studies showed that the mutated protein was not secreted when transfected in COS cells, consistent with a causative role in the disease.

Many lines of evidences have shown that Panax ginseng exhibits beneficial effects on cardiovascular systems. We previously demonstrated that ginsenoside Rg(3) (Rg(3)), one of active ingredients of Panax ginseng, inhibits Ca(2+) channel currents in a stereospecific manner and affects the steady-state activation but not inactivation. This points a possibility that Rg(3) regulates Ca(2+) channels through specific interaction site(s) for Ca(2+) influx inhibition through Ca(2+) channels. However, it was not known how Rg(3) interacts with Ca(2+) channel proteins. In the current study, we sought to identify these site(s) in Xenopus oocytes expressing cardiac wild-type and mutant L(alpha(1C))-type Ca(2+) channels using the two-microelectrode voltage-clamp technique. To this end, we assessed how various point mutations of the L-type Ca(2+) channel affected the Rg(3) action. Mutations of L427R, N428R and L431K in transmembrane domain-I-segment 6 (IS6) of the channel significantly attenuated the Rg(3) action and caused rightward shifts in dose-response curves. Rg(3) treatment produced a negative shift in the inactivation voltage but did not alter the steady-state activation voltage, and none of the mutant channels affected the Rg(3)-induced negative shift of inactivation voltage. Rg(3) had no effects on inactivation time constant in wild-type and mutant channels. These results indicate that Rg(3) inhibition of L-type Ca(2+) channel currents is attenuated by mutations of Leu427, Asn428 and Leu431 in transmembrane IS6 residues. Leu427, Asn428 and Leu431 residues of the L-type Ca(2+) channel play important roles in the Rg(3) effect on channel properties.

Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of L-aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of L-aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which L-aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of L-aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for L-aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the lyases in the aspartase/fumarase superfamily.

Biotin synthase catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This reaction is initiated by the reductive cleavage of the sulfonium center of S-adenosyl-L-methionine (AdoMet), generating methionine and a transient 5'-deoxyadenosyl radical that functions as an oxidant by abstracting hydrogen atoms from DTB. Biotin synthase contains a highly conserved sequence motif, YNHNLD, in which Asn153 and Asp155 form hydrogen bonds with the ribose hydroxyl groups of AdoMet. In the present work, we constructed four individual site-directed mutations to change each of these two residues in order to probe their role in the active site. We used molecular weight filtration assays to show that for most of the mutant enzymes binding of the substrates was only slightly affected. In vitro assays demonstrate that several of the mutant enzymes were able to reductively cleave AdoMet, but none were able to produce a significant amount of biotin. Several of the mutants, especially Asn153Ser, were able to produce high levels of the stable intermediate 9-mercaptodethiobiotin. Some of the mutants, such as Asp155Asn and Asn153Ala, produced instead an alternate product tentatively identified by mass spectrometry as 5'-mercapto-5'-deoxyadenosine, generated by direct attack of the 5'-deoxyadenosyl radical on the [4Fe-4S](2+) cluster. Collectively, these results suggest that the protein residues that form hydrogen bonds to AdoMet and DTB are important for retaining intermediates during the catalytic cycle and for targeting the reactivity of the 5'-deoxyadenosyl radical.

The single nucleotide polymorphism 118A>G of the human micro-opioid receptor gene OPRM1, which leads to an exchange of the amino acid asparagine (N) to aspartic acid (D) at position 40 of the extracellular receptor region, alters the in vivo effects of opioids to different degrees in pain-processing brain regions. The most pronounced N40D effects were found in brain regions involved in the sensory processing of pain intensity. Using the mu-opioid receptor-specific agonist DAMGO, we analyzed the micro-opioid receptor signaling, expression, and binding affinity in human brain tissue sampled postmortem from the secondary somatosensory area (SII) and from the ventral posterior part of the lateral thalamus, two regions involved in the sensory processing and transmission of nociceptive information. We show that the main effect of the N40D micro-opioid receptor variant is a reduction of the agonist-induced receptor signaling efficacy. In the SII region of homo- and heterozygous carriers of the variant 118G allele (n=18), DAMGO was only 62% as efficient (p=0.002) as in homozygous carriers of the wild-type 118A allele (n=15). In contrast, the number of [3H]DAMGO binding sites was unaffected. Hence, the micro-opioid receptor G-protein coupling efficacy in SII of carriers of the 118G variant was only 58% as efficient as in homozygous carriers of the 118A allele (p<0.001). The thalamus was unaffected by the OPRM1 118A>G SNP. In conclusion, we provide a molecular basis for the reduced clinical effects of opioid analgesics in carriers of mu-opioid receptor variant N40D.

Mouse models of human disease are helpful for understanding the pathogenesis of the disorder and ultimately for testing potential therapeutic agents. Here, we describe the engineering and characterization of a mouse carrying the I268N mutation in Egr2, observed in patients with recessively inherited Charcot-Marie-Tooth (CMT) disease type 4E, which is predicted to alter the ability of Egr2 to interact with the Nab transcriptional coregulatory proteins. Mice homozygous for Egr2(I268N) develop a congenital hypomyelinating neuropathy similar to their human counterparts. Egr2(I268N) is expressed at normal levels in developing nerve but is unable to interact with Nab proteins or to properly activate transcription of target genes critical for proper peripheral myelin development. Interestingly, Egr2(I268N/I268N) mutant mice maintain normal weight and have only mild tremor until 2 weeks after birth, at which point they rapidly develop worsening weakness and uniformly die within several days. Nerve electrophysiology revealed conduction block, and neuromuscular junctions showed marked terminal sprouting similar to that seen in animals with pharmacologically induced blockade of action potentials or neuromuscular transmission. These studies describe a unique animal model of CMT, whereby weakness is due to conduction block or neuromuscular junction failure rather than secondary axon loss and demonstrate that the Egr2-Nab complex is critical for proper peripheral nerve myelination.

This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of alpha-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO43- allosteric site where PO43- functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

Parkinson's disease (PD) is a common movement disorder which may arise from mutations in the parkin gene. To date, more than 100 different parkin mutations have been reported. The aim of the present study was to determine the frequency of point mutations and homozygous exon deletions in the parkin gene in a group of 91 South African patients diagnosed with PD. Mutation screening of the 12 exons of parkin was performed using single strand conformation polymorphism analysis and the high-resolution melt technique. Six different mutations were identified: four putative disease-causing missense heterozygous changes (H200Q, D280N, E310D and R402C) and two homozygous exon deletions (exons 3 and 4, and exon 4). The D280N and R402C variants have both previously been described but their pathogenic status has been equivocal. In the present study, the D280N variant was observed in three early onset PD-affected siblings and was not present in a 63-year-old unaffected sibling. This data provide further support for the pathogenicity of this variant which is situated within the first RING finger of the RING-box. None of the four missense variants were detected in over 100 ethnic-matched control chromosomes. We conclude that point mutations and homozygous exon deletions in the parkin gene are not a major cause of PD in the South African population. Further studies on this group of patients are needed to determine the contribution of heterozygous exon deletions and insertions in parkin. The present study is the first report on the molecular etiology of PD in South African patients.

Purpose: To present the light and electron microscopic findings of a unique corneal dystrophy never before described in a German family carrying the Gly623Asp Mutation of the TGFBI gene with late clinical onset., Design: Experimental study., Participants: Four affected and 6 nonaffected family members., Methods: Slit-lamp examination, photographic documentation, and isolation of genomic DNA from peripheral blood leucocytes obtained from each family member examined. Exons 3, 4, 5, and 11 to 14 of the TGFBI gene were amplified and sequenced in these family members. Five corneal buttons of 3 affected siblings were excised at the time of penetrating keratoplasty. Light and electron microscopic examination were performed including immunohistochemistry with antibodies against keratoepithelin (KE) 2 and 15., Main Outcome Measures: Clinical and histologic characteristics of corneal opacification in affected patients and presence of coding region changes in the TGFBI gene., Results: The specimens showed destructive changes in Bowman's layer and the adjacent stroma. Patchy Congo red-positive amyloid deposits were found within the epithelium in 1 cornea, in Bowman's layer and in the anterior stroma of all specimens also showing KE2, but not KE15, immunostaining. Electron microscopy revealed deposits mainly located in the anterior stroma and Bowman's layer and in small amounts in the basal area of some epithelial cells. The destroyed areas were strongly Alcian blue-positive, the Masson Trichrome stain proved mainly negative for the deposits. All affected but none of the unaffected family members had a heterozygous missense mutation in exon 14 of the TGFBI gene (G-->A transition at nucleotide 1915) replacing glycin by aspartic acid amino acid (Gly623Asp) at position 623 of the KE protein., Conclusions: In contrast with the patient carrying the Gly623Asp mutation of the TGFBI gene described by Afshari et al, our cases presented with Salzmann's nodular degeneration-like clinical features and their specimens contained KE2-positive amyloid. The reason for this now "meeting the expectation histologic phenotype" is unclear. The histologic findings emphasize that this is a unique corneal dystrophy, which shares no clinical characteristics with Reis-Bücklers' dystrophy and should be treated as a distinct entity., Financial Disclosure(s): The authors have no proprietary or commercial interest in any materials discussed in this article.

Background: Rs619203 (Cys2229Ser) and rs529038 (Asp2213Asn) polymorphisms in the ROS1 gene have been studied in relation to myocardial infarction (MI) yielding inconsistent results. We investigated the role of ROS1 rs529038 polymorphism in coronary artery disease (CAD) in Greeks using a case-control study., Methods: Genotyping for rs529038 polymorphism was performed using a multiplex PCR technique in patients with CAD (n=294) and controls (n=311). Logistic regression analysis was used to calculate crude and adjusted odds ratios (ORs)., Results: Logistic regression analysis did not show any statistically significant effect of ROS1 polymorphism in the occurrence of CAD (AG vs. AA, OR: 1.08, p=0.635; GG vs. AA, OR: 0.62, p=0.220). Adjustment for confounding factors gave similar results, irrespective of the type of disease (i.e., stable coronary artery disease vs. acute coronary syndrome)., Conclusions: Our findings do not support the hypothesis that ROS1 rs529038 polymorphism is an important contributing factor in the etiology of CAD in the Greek population.

Lowe syndrome is a multisystem disorder characterized by anomalies of the eye, the nervous system, and the kidney. It is an uncommon, X-linked disease. Bilateral cataract and severe hypotonia are present at birth. Psychomotor retardation is evident in childhood, while renal complications arise in adolescence. The mutation of the gene OCRL1 localized at Xq26.1 is responsible for the disease. The authors report on a 12-year-old male with mental retardation, facial dysmorphism as prominent forehead, long and slender-shaped face, prominent eyebrows, epicanthus, microphthalmia, low-posterior set ears with prominent helix and antihelix, long philtrum, and mild prognathia. He also had history of neonatal hypotonia and congenital cataracts. His cranial magnetic resonance imaging showed increased signal intensity in white matter on T2-weighted images, and magnetic resonance spectroscopy revealed elevation of the myoinositol peak at 3.56 ppm. Molecular analysis of OCRL1 gene revealed novel N574K mutation on 17th exon.

Two AmpC variants harboring the S287N substitution were obtained by mutagenesis from cephalosporinases representative of the phylogenetic groups A and B2 of Escherichia coli. Their biochemical characterization revealed that the S287N replacement led to an important increase in the catalytic efficiency toward extended-spectrum cephalosporins in the AmpC beta-lactamase of group A only.

Loss-of-function mutations in the human ERG1 potassium channel (hERG1) frequently underlie the long QT2 (LQT2) syndrome. The role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous, loss-of-function LQT2 syndrome mutation. The hERG N629D mutation was introduced into the orthologous mouse gene, mERG, by homologous recombination in mouse embryonic stem cells. Intact homozygous embryos showed abrupt cessation of the heart beat. N629D/N629D embryos die in utero by embryonic day 11.5. Their developmental defects include altered looping architecture, poorly developed bulbus cordis, and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of I(Kr) function, depolarized resting potential, prolonged action potential duration (LQT), failure to repolarize, and propensity to oscillatory arrhythmias. N629D/N629D myocytes manifest calcium oscillations and increased sarcoplasmic reticulum Ca(+2) content. Although the N629D/N629D protein is synthesized, it is mainly located intracellularly, whereas +/+ mERG protein is mainly in plasmalemma. N629D/N629D embryos show robust apoptosis in craniofacial regions, particularly in the first branchial arch and, to a lesser extent, in the cardiac outflow tract. Because deletion of Hand2 produces apoptosis, in similar regions and with a similar final developmental phenotype, Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. In conclusion, loss of I(Kr) function in N629D/N629D cardiovascular system leads to defects in cardiac ontogeny in the first branchial arch, outflow tract, and the right ventricle.

Role of the conserved Asn345 and Asn435 residues of Bacillus kaustophilus leucine aminopeptidase (BkLAP) was investigated by performing computer modeling and site-directed mutagenesis. Replacement of BkLAP Asn345 with Gln or Leu resulted in a dramatic reduction in enzymatic activity. A complete loss of the LAP activity was observed in Asn435 variants. Circular dichroism spectra were nearly identical for wild-type and all mutant enzymes, while measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in Asn345 and Asn435 replacements. Except for N435R and N435L, wild-type and other mutant enzymes showed a similar sensitivity towards temperature-induced denaturation. Computer modeling of the active-site structures of wild-type and mutant enzymes exhibits a partial or complete loss of the hydrogen bonding in the variants.

The formation of signal-promoting dimeric or oligomeric receptor complexes at the cell surface is modulated by self-interaction of their transmembrane (TM) domains. To address the importance of TM domain packing density for receptor functionality, we examined a set of asparagine mutants in the TM domain of the erythropoietin receptor (EpoR). We identified EpoR-T242N as a receptor variant that is present at the cell surface similar to wild-type EpoR but lacks visible localization in vesicle-like structures and is impaired in efficient activation of specific signaling cascades. Analysis by a molecular modeling approach indicated an increased interhelical distance for the EpoR-T242N TM dimer. By employing the model, we designed additional mutants with increased or decreased packing volume and confirmed a correlation between packing volume and biological responsiveness. These results propose that the packing density of the TM domain provides a novel layer for fine-tuned regulation of signal transduction and cellular decisions.

The widely abused psychostimulant cocaine is thought to elicit its reinforcing effects primarily via inhibition of the neuronal dopamine transporter (DAT). However, not all DAT inhibitors share cocaine's behavioral profile, despite similar or greater affinity for the DAT. This may be due to differential molecular interactions with the DAT. Our previous work using transporter mutants with altered conformational equilibrium (W84L and D313N) indicated that benztropine and GBR12909 interact with the DAT in a different manner than cocaine. Here, we expand upon these previous findings, studying a number of structurally different DAT inhibitors for their ability to inhibit [(3)H]CFT binding to wild-type, W84L and D313N transporters. We systematically tested structural intermediates between cocaine and benztropine, structural hybrids of benztropine and GBR12909 and a number of other structurally heterologous inhibitors. Derivatives of the stimulant desoxypipradrol (2-benzhydrylpiperidine) exhibited a cocaine-like binding profile with respect to mutation, whereas compounds possessing the diphenylmethoxy moiety of benztropine and GBR12909 were dissimilar to cocaine-like compounds. In tests with specific isomers of cocaine and tropane analogues, compounds with 3alpha stereochemistry tended to exhibit benztropine-like binding, whereas those with 3beta stereochemistry were more cocaine-like. Our results point to the importance of specific molecular features--most notably the presence of a diphenylmethoxy moiety--in determining a compound's binding profile. This study furthers the concept of using DAT mutants to differentiate cocaine-like inhibitors from atypical inhibitors in vitro. Further studies of the molecular features that define inhibitor-transporter interaction could lead to the development of DAT inhibitors with differential clinical utility.

A FAD-dependent glucose dehydrogenase (FADGDH) mutant with narrow substrate specificity was constructed by site-directed mutagenesis. Several characteristics of FADGDH, such as high catalytic activity and high electron transfer ability, make this enzyme suitable for application to glucose sensors. However, for further applications, improvement of the broad substrate specificity is needed. In this paper, we mutated two residues, Asn475 and Ala472, which are located near the putative active site of the catalytic subunit of FADGDH and have been predicted from the alignment with the active site of glucose oxidase. Of the 38 mutants constructed, Ala472Phe and Asn475Asp were purified and their activities were analyzed. Both mutants showed a higher specificity toward glucose compared to the wild type enzyme.

Purpose: We tested the hypothesis that genetic variants in vasoactive and angiogenic factors regulating the retina vasculature contribute to the development of diabetic retinopathy (DR)., Methods: A case-control study was performed to study the genetic association between DR and polymorphic variants of EDN1 (Lys198Asn), LTA (IVS1-80C>A, IVS1-206G>C, IVS1-252A>G), eNOS (Glu298Asp), and ITGA2 (BgI II) in a Chinese population with type 2 diabetes mellitus. A well defined population with type 2 diabetes, consisting of 127 controls and 216 DR patients, was recruited., Results: A higher frequency of the Asn/Asn genotype of EDN1 was found in individuals with at least 10 years of diabetes and no retinopathy (controls) compared with DR patients with any duration of diabetes (DR: 2.3%; control: 11.0%; p=0.0002). The Asn allele was also more frequent in controls than DR patients (DR: 16.4%; control: 29.5%; p=0.007). Multiple logistic regression analysis showed that the Asn/Asn genotype was the factor most significantly associated with reduced risk of DR (odds ratio=0.19; 95% CI: 0.07-0.53; p=0.002) and with late onset of diabetes (Asn/Asn: 59 years; Lys/Lys + Lys/Asn: 53 years; p=0.02). Moreover, the Lys/Lys genotype was more common among patients with nonproliferative (75.7%) than proliferative DR (56.9%; p=0.008). The distributions of Lys198Asn alleles in hypertension did not differ from normotensive subjects. No associations between DR and polymorphisms of LTA, eNOS, or ITGA2 were detected, and there were no detectable gene-gene or gene-environmental interactions among the polymorphisms., Conclusions: The Asn/Asn genotype of EDN1 was associated with a reduced risk of DR and with delayed onset of type 2 diabetes.

The deprotonation of nitroethane catalyzed by Neurospora crassa 2-nitropropane dioxygenase was investigated by measuring the formation and release of ethylnitronate formed in turnover as a function of pH and through mutagenesis studies. Progress curves for the enzymatic reaction obtained by following the increase in absorbance at 228 nm over time were visibly nonlinear, requiring a logarithmic approximation of the initial reaction rates for the determination of the kinetic parameters of the enzyme. The pH dependence of the second-order rate constant k cat/ K m with nitroethane as substrate implicates the presence of a group with a p K a of 8.1 +/- 0.1 that must be unprotonated for nitronate formation. Mutagenesis studies suggest that this group is histidine 196 as evident from the inability of a H196N variant form of the enzyme to catalyze the formation of ethylnitronate from nitroethane. Replacement of histidine 196 with asparagine resulted in an approximately 15-fold increase in the k cat/ K m with ethylnitronate as compared to the wild-type, which results from the inability of the mutant enzyme to undergo nonoxidative turnover. The results presented herein are consistent with a branched catalytic mechanism for the enzyme in which the ethylnitronate intermediate formed from the H196-catalyzed deprotonation of nitroethane partitions between release from the active site and oxidative denitrification to yield acetaldehyde and nitrite.

Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique bisubunit DNA-topoisomerase IB (LdTopIB). The catalytically active enzyme is a heterodimer constituted by a large subunit (LdTopIL) containing a non-conserved N-terminal end and the phylogenetically conserved core domain, whereas the small subunit (LdTopIS) harbors the C-terminal domain with the characteristic tyrosine residue in the active site. Site-directed mutagenesis was used to substitute the basic amino acid (Arg-314, Lys-352, Arg-410 and His-453) of the LdTopIL subunit by the neutral amino acid alanine. The expression of these mutants in a topoisomerase-free yeast strain produced inactive proteins. Similarly, when the Tyr-222 from small subunit, involved in DNA cleavage, was substituted by Phe no topoisomerase activity was detected in yeast overexpressing extracts. In addition two substitutions involved in camptothecin inhibition were also analyzed. Asp-353 located in the core domain of the large subunit and Asn-221 which heads Tyr-222 in the small subunit, were replaced by Ala and Ser, respectively. These mutants were insensitive to the inhibitor; despite they displayed significant relaxation activity.

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.

The halorhodopsin (hR)-overproducing mutant strain KM-1 was isolated from the extremely haloalkaliphilic archaeon Natronomonas pharaonis type strain DSM2160(T). hR-enriched membranes were easily obtained by washing the cells with distilled water. The membranes were claret colored owing to two pigments: hR and bacterioruberin. The hR component in the absorption spectra changed from blue to purple upon the addition of Cl(-) and had a K(m) value of 1.7 mM. Overexpression of hR in strain KM-1 might be caused by the point mutation Asp324-->Asn in the bacteriorhodopsin activator homologues of N. pharaonis. The mutation changed the hR-expression pattern from inducible to constitutive in the late exponential phase.

Human carbamoyl-phosphate synthetase (hCPS) has evolved three features that allow it to remove excess, potentially neurotoxic ammonia via the urea cycle: inability to use glutamine as an alternative nitrogen donor; a K(m) for ammonia 100-fold lower than for CPSs that also use glutamine; and required allosteric activation by N-acetylglutamate (AGA), a sensor of excess amino acids. To determine the structural features of hCPS that allow its unique functioning, we have developed the first recombinant expression system for hCPS, utilizing Schizosaccharomyces pombe. Of several common single-nucleotide polymorphisms identified in the gene encoding hCPS, only the one resulting in substitution of threonine at position 1406 with asparagine has been linked to phenotypic effects. We have expressed and characterized both variants of hCPS. The asparagine polymorph, hCPS&#95;N, consistently displayed inferior catalytic properties, but the K(m) and k(cat) values for overall and partial reactions varied only by a factor of 1.7 or less. We have designed and characterized an hCPS construction from which the N-terminal domain A is deleted. hCPS&#95;DeltaA was competent to bind AGA, demonstrating that domain A does not contain the AGA binding site. Thus, the site at the C/D boundary previously identified by AGA analogue labelling appears to be the functionally significant initial binding site for AGA. However, hCPS&#95;DeltaA was not able to fully assume the catalytically competent conformation, with specific activity of CP formation decreased 700-fold.

In eukaryotes, ORC (origin recognition complex), a six-protein complex, is the most likely initiator of chromosomal DNA replication. ORC belongs to the AAA(+) (ATPases associated with a variety of cellular activities) family of proteins and has intrinsic ATPase activity derived from Orc1p, one of its subunits. To reveal the role of this ATPase activity in Saccharomyces cerevisiae (baker's yeast) ORC, we mutated the Orc1p sensor 1 and sensor 2 regions, which are important for ATPase activity in AAA(+) proteins. Plasmid-shuffling analysis revealed that Asn(600), Arg(694) and Arg(704) are essential for the function of Orc1p. In yeast cells, overexpression of Orc1R694Ep inhibited growth, caused inefficient loading of MCM (mini-chromosome maintenance complex of proteins) and slowed the progression of S phase. In vitro, purified ORC-1R [ORC with Orc1R694Ep (Orc1p Arg(694)-->Glu mutant)] has decreased ATPase activity in the presence or absence of origin DNA. However, other activities (ATP binding and origin DNA binding) were indistinguishable from those of wild-type ORC. The present study showed that Arg(694) of the Orc1p subunit is important for the ATPase activity of ORC and suggests that this ATPase activity is required for efficient MCM loading on to origin DNA and for progression of S phase.

The mechanisms by which agonists and other ligands bind ligand-gated ion channels are important determinants of function in neurotransmitter receptors. The partial agonist, kainic acid (KA) activates a less desensitized, and more robust AMPA receptor (AMPAR) current than full agonists, glutamate or AMPA. Cyclothiazide (CTZ), the allosteric modulator of AMPARs, potentiates receptor currents by inhibiting receptor desensitization resulting from agonist activation. We have constructed an AMPAR GluR1 subunit deletion mutant GluR1L3T(Delta739-784) by deleting the splice-variable "flip/flop" region of the L3 domain in the wild-type receptor and compared its function to that of the wild-type GluR1 receptor and an AMPAR substitution mutant GluR1A782N. When compared to GluR1, the potency of glutamate activation of GluR1L3T was increased, in contrast to a decrease in potency of activation and reduced sensitivity to optimal concentrations of KA. Furthermore, GluR1L3T was totally insensitive to CTZ potentiation of KA and glutamate-activated currents in Xenopus laevis oocytes. The potency of glutamate and KA activation of GluR1A782N was not significantly different from that of the wild-type GluR1 receptor although the mutant receptor currents were more sensitive to CTZ potentiation than the wild-type receptor current. This result is an indication that glutamate and KA binding to the agonist (S1/S2) domain on AMPAR can be modulated by an expendable splice-variable region of the receptor. Moreover, the effect of the allosteric modulator, CTZ on agonist activation of AMPAR can also be modified by a non-conserved amino acid residue substitution within the splice-variable "flip/flop" region.

Four missense mutations, that target highly conserved amino acid residues in the small GTPase Rab7, have been associated with the Charcot-Marie-Tooth (CMT) type 2B phenotype. CMT2B peripheral axonal neuropathies are characterized by severe sensory loss, often complicated by infections, arthropathy, and amputations. Here, we have investigated the biochemical and functional properties of the Rab7 K157N mutated protein. Interestingly, Rab7 K157N showed altered nucleotide exchange rate and GTP hydrolysis compared to the wild type protein. Consistently, the majority of the expressed protein in HeLa cells was bound to GTP. In addition, Rab7 K157N was able to restore EGF degradation, previously inhibited by Rab7 silencing. Altogether these data indicate that Rab7 K157N, similarly to the other three mutated proteins causative of CMT2B, is predominantly in the GTP-bound form and behaves as an active mutant. Therefore, activated forms of Rab7 protein cause the CMT2B disease.

Thrombospondins (THBSs) are multimodular, secreted proteins characterized by a signature domain comprising a unique set of 13 calcium-binding repeats flanked by epidermal growth factor (EGF)-like and lectin-like modules. A polymorphism that changes a conserved Asn to Ser at residue 700 in the most N-terminal calcium-binding repeat of THBS-1 (repeat 1C) is found in 8-10% of European populations and has been linked to increased risk of premature coronary artery disease. The Ser substitution leads to altered stability in the EGF-like and wire modules of the THBS-1 signature domain as assessed by differential scanning calorimetry carried out in 2 mm or 200 mum calcium. Studies of the melting profiles of the THBS-2 signature domain proteins with Asn or Ser at position 702 (homologous to 700 in THBS-1) revealed that the impact of the Ser allele is similar in both THBS-1 and THBS-2. Structure determination of the Ser(702) THBS-2 variant in 2 mm calcium showed that repeat 1C contains two bound calcium ions as in the crystal of the Asn(702) protein, including the ion that is coordinated by Asn(702), and is associated with changes in conformation of repeat 1C and the adjacent EGF-like modules. The Ser substitution leads to the decreased ability of soluble THBS-2 signature domain protein to bind 4B6.13, a conformation-sensitive monoclonal antibody that recognizes an epitope in repeat 1C. These results indicate that although THBS harboring the Ser allele binds a full complement of calcium ions, repeat 1C is altered, leading to destabilization of surrounding structures.

Met467, the axial ligand to type I Cu in a multicopper oxidase, Myrothecium verrucaria bilirubin oxidase was substituted with a non-coordinating Phe and Leu to transform the spectral and magnetic properties and oxidase activities of the enzyme into those of fungal laccases, but the mutated type I Cu center showed properties characteristic of phytocyanins, blue copper proteins with an axial coordination of Gln, due to compensatory binding of the distal Asn459 as evidenced by a double mutation.

In Escherichia coli, interactions between the nascent TnaC-tRNA(Pro) peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA(Pro) cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of tna operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 or Pro24 of TnaC of E. coli with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA(Pro) cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNA(Pro) peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome.

Mitochondrial DNA depletion syndromes are a heterogeneous group of childhood neurological disorders characterised by a quantitative abnormality of mitochondrial DNA. We describe two siblings who presented at 8 months and 14 months with myopathy, which rapidly progressed and resulted in death by respiratory failure at age 14 and 18 months, respectively. Muscle biopsy revealed marked respiratory chain defects, with real-time PCR confirming a dramatic depletion of mitochondrial DNA. Sequencing of the thymidine kinase 2 (TK2) gene revealed two, novel heterozygous mutations (p.Q87X and p.N100S) with parental DNA analysis confirming the transmission of mutated alleles.

The binding sites of Cys-loop receptors are formed from at least six loops (A-F). Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of two residues in loop A of the 5-HT3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC50 (from approximately 10-fold more potent to approximately 10-fold less potent than that of the wild type), increased the maximal peak current for mCPBG compared to 5-HT (R max) 2-19-fold, and decreased n H, indicating this residue is involved in receptor gating; we propose Asn128 faces away from the binding pocket and plays a role in facilitating transitions between conformational states. Substitutions of Glu129 resulted in functional receptors only when the residue could accept a hydrogen bond, but with both these and other substitutions, no [(3)H]granisetron binding could be detected, indicating a role in ligand binding. We propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Thus, the data support a modified model of the 5-HT3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor.

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder resulting from CAG repeat expansion in the gene that encodes for the protein huntingtin. To identify neuroprotective compound (s) that can slow down disease progression and can be administered long term with few side effects in Huntington's disease, we investigated the effect of sertraline, a selective serotonin reuptake inhibitor (SSRI) which has been shown to upregulate BDNF levels in rodent brains. We report here that in HD mice sertraline increased BDNF levels, preserved chaperone protein HSP70 and Bcl-2 levels in brains, attenuated the progression of brain atrophy and behavioral abnormalities and thereby increased survival. Sertraline also enhanced neurogenesis, which appeared to be responsible for mediating the beneficial effects of sertraline in HD mice. Additionally, the effective levels of sertraline are comparable to the safe levels achievable in humans. The findings suggest that sertraline is a potential candidate for treatment of HD patients.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by chorea, incoordination, and shortened life-span, and by huntingtin inclusions and neurodegeneration. We previously screened the 1040 FDA-approved compounds from the NINDS compound library and found that a compound, nipecotic acid, significantly reduced mutant huntingtin aggregations and blocked cell toxicity in an inducible cell model of HD. Because nipecotic acid does not cross the blood-brain barrier (BBB), we studied its analogue, tiagabine, which is able to cross the BBB, in both N171-82Q and R6/2 transgenic mouse models of HD. Tiagabine was administered intraperitoneally at 2 and 5 mg/kg daily in HD mice. We found that tiagabine extended survival, improved motor performance, and attenuated brain atrophy and neurodegeneration in N171-82Q HD mice. These beneficial effects were further confirmed in R6/2 HD mice. The levels of tiagabine at effective doses in mouse serum are comparable to the levels in human patients treated with tiagabine. These results suggest that tiagabine may have beneficial effects in the treatment of HD. Because tiagabine is an FDA-approved drug, it may be a promising candidate for future clinical trials for the treatment of HD.

Prion protein (PrP) contains two N-linked glycosylation sites. It is unknown which amino acid substitution contributes most efficiently to the abolishment of N-linked glycosylations. To define the influence of amino acid substitution at the N-linked glycosylation sites on the conversion efficiency of mouse PrP, we tested each of all 19 amino acid substitutions at either one of the N-linked glycosylation sites (codon 180, 182, 196 or 198). The conversion efficiency of the mutagenized PrP was highly dependent on the newly introduced amino acid itself regardless of the absence of N-linked glycosylation in scrapie-infected mouse neuroblastoma cells. The majority of mutant PrP with substitutions at the Asn residues of the N-linked glycosylation sites were conversion-competent, whereas most mutant PrP with substitutions at the Thr residues were conversion-incompetent. These findings emphasize that the Asn residues of the N-linked glycosylation sites are replaceable to abolish N-linked glycosylations without directly affecting the protein function.

The neutral amino acid transporter 2 (SNAT2), which belongs to the SLC38 family of solute transporters, couples the transport of amino acid to the cotransport of one Na(+) ion into the cell. Several polar amino acids are highly conserved within the SLC38 family. Here, we mutated three of these conserved amino acids, Asn(82) in the predicted transmembrane domain 1 (TMD1), Tyr(337) in TMD7, and Arg(374) in TMD8; and we studied the functional consequences of these modifications. The mutation of N82A virtually eliminated the alanine-induced transport current, as well as amino acid uptake by SNAT2. In contrast, the mutations Y337A and R374Q did not abolish amino acid transport. The K(m) of SNAT2 for its interaction with Na(+), K(Na(+)), was dramatically reduced by the N82A mutation, whereas the more conservative mutation N82S resulted in a K(Na(+)) that was in between SNAT2(N82A) and SNAT2(WT). These results were interpreted as a reduction of Na(+) affinity caused by the Asn(82) mutations, suggesting that these mutations interfere with the interaction of SNAT2 with the sodium ion. As a consequence of this dramatic reduction in Na(+) affinity, the apparent K(m) of SNAT2(N82A) for alanine was increased 27-fold compared with that of SNAT2(WT). Our results demonstrate a direct or indirect involvement of Asn(82) in Na(+) coordination by SNAT2. Therefore, we predict that TMD1 is crucial for the function of SLC38 transporters and that of related families.

Epithelial cell adhesion molecule EpCAM is strongly over-expressed in a variety of carcinomas where it is involved in signalling events resulting in increased expression of target genes such as c-Myc, cyclins and others, eventually conferring cells an oncogenic phenotype. However, EpCAM is also expressed in a series of healthy epithelia, albeit generally to a far lesser extend. We have uncovered differential glycosylation of EpCAM as a means to discriminate normal from malignant tissues. EpCAM was hyperglycosylated in carcinoma tissue as compared with autologous normal epithelia. All three N-glycosylation consensus sequences within EpCAM's extracellular domain were used in human and murine cells. We show that glycosylation at asparagine198 is crucial for protein stability. Mutants of EpCAM that substitute asparagine198 for alanine showed a decreased overall expression and half-life of the molecule at the plasma membrane. This is of considerable importance with respect to EpCAM variants expressed in normal tissue, where it might reveal to be less stable and thus may have repercussions on functionality.

The major-histocompatibility-complex protein UAP56 (BAT1) is a DEAD-box helicase that is deposited on mRNA during splicing. UAP56 is retained on spliced mRNA in an exon junction complex (EJC) or, alternatively, with the TREX complex at the 5' end, where it might facilitate the export of the spliced mRNA to the cytoplasm. Using confocal microscopy, UAP56 was found to be concentrated in RNA-splicing speckled domains of nuclei but was also enriched in adjacent nuclear regions, sites at which most mRNA transcription and splicing occur. At speckled domains, UAP56 was in complexes with the RNA-splicing and -export protein SRm160, and, as measured by FRAP, was in a dynamic binding equilibrium. The application of an in vitro FRAP assay, in which fluorescent nuclear proteins are photobleached in digitonin-extracted cells, revealed that the equilibrium binding of UAP56 in complexes at speckled domains was directly regulated by ATP binding. This was confirmed using a point mutant of UAP56 that did not bind ATP. Point mutation of UAP56 to eliminate ATP binding did not affect RNA splicing, but strongly inhibited the export of mRNA to the cytoplasm.

In the present study we show that the interaction of the CaM (calmodulin)-binding domain (Lys(3614)-Asn(3643)) with the Cys(4114)-Asn(4142) region (a region included in the CaM-like domain) serves as an intrinsic regulator of the RyR1 (type-1 ryanodine receptor). We tested the effects of antibodies raised against the two putative key regions of RyR1 [anti-(Lys(3614)-Asn(3643)) and anti-(Cys(4114)-Asn(4142)) antibodies]. Both antibodies produced significant inhibition of [3H]ryanodine-binding activity of RyR1. This suggests that the inter-domain interaction between the two domains, Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142), activates the channel, and that the binding of antibody to either side of the interacting domain pair interfered with the formation of a 'channel-activation link' between the two regions. In order to spectroscopically monitor the mode of interaction of these domains, the site of inter-domain interaction was fluorescently labelled with MCA [(7-methoxycoumarin-4-yl)acetyl] in a site-directed manner. The accessibility of the bound MCA to a large molecular mass fluorescence quencher, BSA-QSY (namely, the size of a gap between the interacting domains) decreased with an increase of [Ca2+] in a range of 0.03-2.0 microM, as determined by Stern-Volmer fluorescence quenching analysis. The Ca2+-dependent decrease in the quencher accessibility was more pronounced in the presence of 150 microM 4-CmC (4-chlorometacresol), and was reversed by 1 mM Mg2+ (a well-known inhibitor of Ca2+/agonist-induced channel activation). These results suggest that the Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142) regions of RyR1 interact with each other in a Ca2+- and agonist-dependent manner, and this serves as a mechanism of Ca2+- and agonist-dependent activation of the RyR1 Ca2+ channel.

A mechanistic study of the essential allosteric activation of human pancreatic alpha-amylase by chloride ion has been conducted by exploring a wide range of anion substitutions through kinetic and structural experiments. Surprisingly, kinetic studies indicate that the majority of these alternative anions can induce some level of enzymatic activity despite very different atomic geometries, sizes, and polyatomic natures. These data and subsequent structural studies attest to the remarkable plasticity of the chloride binding site, even though earlier structural studies of wild-type human pancreatic alpha-amylase suggested this site would likely be restricted to chloride binding. Notably, no apparent relationship is observed between anion binding affinity and relative activity, emphasizing the complexity of the relationship between chloride binding parameters and the activation mechanism that facilitates catalysis. Of the anions studied, particularly intriguing in terms of observed trends in substrate kinetics and their novel atomic compositions were the nitrite, nitrate, and azide anions, the latter of which was found to enhance the relative activity of human pancreatic alpha-amylase by nearly 5-fold. Structural studies have provided considerable insight into the nature of the interactions formed in the chloride binding site by the nitrite and nitrate anions. To probe the role such interactions play in allosteric activation, further structural analyses were conducted in the presence of acarbose, which served as a sensitive reporter molecule of the catalytic ability of these modified enzymes to carry out its expected rearrangement by human pancreatic alpha-amylase. These studies show that the largest anion of this group, nitrate, can comfortably fit in the chloride binding pocket, making all the necessary hydrogen bonds. Further, this anion has nearly the same ability to activate human pancreatic alpha-amylase and leads to the production of the same acarbose product. In contrast, while nitrite considerably boosts the relative activity of human pancreatic alpha-amylase, its presence leads to changes in the electrostatic environment and active site conformations that substantially modify catalytic parameters and produce a novel acarbose rearrangement product. In particular, nitrite-substituted human pancreatic alpha-amylase demonstrates the unique ability to cleave acarbose into its acarviosine and maltose parts and carry out a previously unseen product elongation. In a completely unexpected turn of events, structural studies show that in azide-bound human pancreatic alpha-amylase, the normally resident chloride ion is retained in its binding site and an azide anion is found bound in an embedded side pocket in the substrate binding cleft. These results clearly indicate that azide enzymatic activation occurs via a mechanism distinct from that of the nitrite and nitrate anions.

The etiologies and clinical spectra of HPE are extremely heterogeneous. Here, we report a Brazilian boy with lobar holoprosencephaly who was ascertained in a sample of 60 patients with HPE and HPE-like phenotypes and screened for molecular analysis of the major HPE causative genes: SHH, PTCH, SIX3, GLI2, and TGIF. This boy presented a p.K44N (c.132G>T) mutation in exon 2 of the TGIF gene which was inherited from his phenotypically normal mother. This mutation leads to lysine to arginine amino acid change and is predicted to be a damaging mutation. Clinical aspects involving variable phenotypical manifestations in different mutations of TGIF are discussed.

Some polymorphisms in the FCRL3 gene, a member of the Fc-receptor like family, have been associated with several autoimmune diseases and recently with multiple sclerosis (MS). We performed a case-control study of three SNPs in FCRL3 gene in 645 MS patients and 786 controls, all Caucasians from the South of Spain. Genotype and allele frequencies of two SNPs (rs7528684/FCRL3&#95;3 and rs7522061/N28D), which were in high linkage disequilibrium (r(2) = 0.87), differed between MS cases and controls. The C allele of FCRL3&#95;3 was found to be protective for MS (per allele OR = 0.81, 95% C.I. = 0.70-0.94; P-value = 0.007) as was the G variant of N28D, but no association was found for rs11264799/FCRL3&#95;4. Haplotype analysis confirmed these associations with highly consistent effect sizes for haplotypes carrying the C allele of FCRL3&#95;3.

Background: Mutations in the glucocerebrosidase (GBA) gene have been reported to modify risk for Parkinson disease (PD) and dementia with Lewy bodies (DLB). However, these findings have not been consistently replicated, and most studies have had substantial methodological shortcomings., Objective: To better assess the role of GBA variants in altering risk for Lewy body disorders., Design: Case-control study., Setting: Four movement disorder clinics in the Seattle, Washington, area., Participants: Seven hundred twenty-one patients with PD, 554 healthy control subjects, and 57 patients with DLB., Main Outcome Measures: Disease status and presence or absence of the 2 most common GBA mutations (N370S and L444P)., Results: We observed a significantly higher heterozygote frequency for the 2 mutations in patients with PD (2.9%; P <.001) and those with DLB (3.5%; P = .045) compared with control subjects (0.4%)., Conclusion: Our findings suggest that GBA mutations exert a large effect on susceptibility for Lewy body disorders at the individual level but are associated with a modest (approximately 3%) population-attributable risk in individuals of European ancestry.

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.