C5orf42 is the major gene responsible for OFD syndrome type VI

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the “Joubert syndrome related disorders”. Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.     

Determinants of the Over-Anticoagulation Response during Warfarin Initiation Therapy in Asian Patients Based on Population Pharmacokinetic-Pharmacodynamic Analyses

To clarify pharmacokinetic-pharmacodynamic (PK-PD) factors associated with the over-anticoagulation response in Asians during warfarin induction therapy, population PK-PD analyses were conducted in an attempt to predict the time-courses of the plasma S-warfarin concentration, Cp(S), and coagulation and anti-coagulation (INR) responses. In 99 Chinese patients we analyzed the relationships between dose and Cp(S) to estimate the clearance of S-warfarin, CL(S), and that between Cp(S) and the normal prothrombin concentration (NPT) as a coagulation marker for estimation of IC₅₀. We also analyzed the non-linear relationship between NPT inhibition and the increase in INR to derive the non-linear index λ. Population analyses accurately predicted the time-courses of Cp(S), NPT and INR. Multivariate analysis showed that CYP2C9*3 mutation and body surface area were predictors of CL(S), that VKORC1 and CYP4F2 polymorphisms were predictors of IC₅₀, and that baseline NPT was a predictor of λ. CL(S) and λ were significantly lower in patients with INR≥4 than in those with INR<4 (190 mL/h vs 265 mL/h, P<0.01 and 3.2 vs 3.7, P<0.01, respectively). Finally, logistic regression analysis revealed that CL(S), ALT and hypertension contributed significantly to INR≥4. All these results indicate that factors associated with the reduced metabolic activity of warfarin represented by CL(S), might be critical determinants of the over-anticoagulation response during warfarin initiation in Asians.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02065388","term_id":"NCT02065388"}}NCT02065388     

Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1^WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp³² residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp³² residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1^WT and hSOD1^G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp³² residue in the process. The results showed that Trp³² residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp³² residue (bovine SOD1 and hSOD1^W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp³² residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.     

Identification of a Hydrolyzable Tannin,Oenothein B,as an Aluminum-Detoxifying Ligand in a Highly Aluminum-Resistant Tree,Eucalyptus camaldulensis

Eucalyptus camaldulensis is a tree species in the Myrtaceae that exhibits extremely high resistance to aluminum (Al). To explore a novel mechanism of Al resistance in plants, we examined the Al-binding ligands in roots and their role in Al resistance of E. camaldulensis. We identified a novel type of Al-binding ligand, oenothein B, which is a dimeric hydrolyzable tannin with many adjacent phenolic hydroxyl groups. Oenothein B was isolated from root extracts of E. camaldulensis by reverse-phase high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry analyses. Oenothein B formed water-soluble or -insoluble complexes with Al depending on the ratio of oenothein B to Al and could bind at least four Al ions per molecule. In a bioassay using Arabidopsis (Arabidopsis thaliana), Al-induced inhibition of root elongation was completely alleviated by treatment with exogenous oenothein B, which indicated the capability of oenothein B to detoxify Al. In roots of E. camaldulensis, Al exposure enhanced the accumulation of oenothein B, especially in EDTA-extractable forms, which likely formed complexes with Al. Oenothein B was localized mostly in the root symplast, in which a considerable amount of Al accumulated. In contrast, oenothein B was not detected in three Al-sensitive species, comprising the Myrtaceae tree Melaleuca bracteata, Populus nigra, and Arabidopsis. Oenothein B content in roots of five tree species was correlated with their Al resistance. Taken together, these results suggest that internal detoxification of Al by the formation of complexes with oenothein B in roots likely contributes to the high Al resistance of E. camaldulensis.Aluminum (Al) toxicity is a major factor that limits plant growth in acid soils and affects approximately 30% of the total ice-free land area of the world (von Uexküll and Mutert, 1995). Although Al in soils exist in nonphytotoxic silicate or oxide forms at neutral pH, it is solubilized into a phytotoxic form, mainly as Al³⁺, at a pH of less than 5 (Kinraide, 1991; Kochian, 1995). The accumulation of Al in root tips causes rapid inhibition of root elongation, which is a characteristic symptom of Al toxicity in plants (Delhaize and Ryan, 1995; Ma, 2007). In general, plants exhibit an inhibition of root elongation as early as 30 to 120 min after exposure to excessive Al (Barceló and Poschenrieder, 2002). Inhibition of root elongation leads to decreased water and nutrient uptake and, eventually, to restriction of growth of the whole plant.Plants have evolved different levels of Al resistance mediated by two distinct classes of mechanisms (Kochian et al., 2004; Ma, 2007). One strategy is the exclusion of Al from the root tips (exclusion mechanism), and the other is tolerance to Al that enters the root tips (internal tolerance mechanism). The secretion of organic acid anions from roots in response to exposure to Al is the best-documented mechanism for Al exclusion. Organic acid anions (i.e. malate, citrate, and oxalate) can form a complex with Al in the rhizosphere and thereby prevent Al from entering the root tips. The genes encoding transporters for the Al-induced secretion of malate and citrate have been identified and characterized in several plant species (Ryan et al., 2011; Delhaize et al., 2012). Organic acid anions also play a role in the detoxification of Al that enters the roots by means of internal formation of complexes with Al (Ma et al., 1998). However, findings in recent studies increasingly suggest that the Al resistance of some plant species and cultivars cannot be explained solely by these two functions of organic acid anions (Wenzl et al., 2001, 2002; Piñeros et al., 2005; Zheng et al., 2005; Famoso et al., 2010). In addition to organic acid anions, flavonoid-type phenolics (Kidd et al., 2001), phenolic compounds (Ofei-Manu et al., 2001), cyclic hydroxamates (Poschenrieder et al., 2005), and proanthocyanidins (Osawa et al., 2011) in roots or root exudates are proposed as potential organic ligands for Al. The mechanisms by which these additional ligands confer Al resistance remain poorly understood.Eucalyptus camaldulensis is an evergreen tree belonging to the Myrtaceae family and is cultivated in tropical and subtropical regions of the world on account of its superior growth, broad adaptability, and multipurpose wood properties. E. camaldulensis can grow in acid soils and even in acid sulfate soils, where the pH is often lower than 3.5 and the Al concentration in the soil solution often reaches the millimolar level (van Breemen and Pons, 1978). Indeed, seedlings of this species show no inhibition of root elongation and plant growth when exposed to 1 mm Al for 20 d under hydroponic conditions (Tahara et al., 2005). Such Al resistance is considerably higher than that reported for a variety of herbaceous crops and model plants in studies of Al resistance mechanisms; such plants exhibit an inhibition of root elongation at 1 to 50 μm Al (Wenzl et al., 2001). Although our understanding of Al resistance mechanisms in some crops and model plants has improved recently, that for extremely Al-resistant species such as E. camaldulensis is limited.In E. camaldulensis, citrate secretion from roots and its content in the root tips are increased by exposure to Al, suggesting that citrate may contribute to its Al resistance (Tahara et al., 2008a). However, the amounts of organic acid anions, including citrate, secreted from roots and contained within the root tips are lower than those of more sensitive species (Tahara et al., 2008a). Therefore, the high Al resistance of E. camaldulensis cannot be explained only by the presence of organic acid anions. Roots of E. camaldulensis can accumulate large amounts of Al (11 mg g⁻¹ dry weight) with no symptoms of Al toxicity (Tahara et al., 2005), suggesting the existence of additional mechanisms for internal tolerance. In this study, we investigated the presence of novel Al-binding ligands other than organic acid anions in E. camaldulensis roots and identified a hydrolyzable tannin, oenothein B, as a novel type of Al-binding ligand. We also examined the role of the ligand in the internal Al tolerance of E. camaldulensis.     

An allosteric activator of glucokinase impairs the interaction of glucokinase and glucokinase regulatory protein and regulates glucose metabolism

Glucokinase (GK) plays a key role in the control of blood glucose homeostasis. We identified a small molecule GK activator, compound A, that increased the glucose affinity and maximal velocity (V(max)) of GK. Compound A augmented insulin secretion from isolated rat islets and enhanced glucose utilization in primary cultured rat hepatocytes. In rat oral glucose tolerance tests, orally administrated compound A lowered plasma glucose elevation with a concomitant increase in plasma insulin and hepatic glycogen. In liver, GK activity is acutely controlled by its association to the glucokinase regulatory protein (GKRP). In order to decipher the molecular aspects of how GK activator affects the shuttling of GK between nucleus and cytoplasm, the effect of compound A on GK-GKRP interaction was further investigated. Compound A increased the level of cytoplasmic GK in both isolated rat primary hepatocytes and the liver tissues from rats. Experiments in a cell-free system revealed that compound A interacted with glucose-bound free GK, thereby impairing the association of GK and GKRP. On the other hand, compound A did not bind to glucose-unbound GK or GKRP-associated GK. Furthermore, we found that glucose-dependent GK-GKRP interaction also required ATP. Given the combined prominent role of GK on insulin secretion and hepatic glucose metabolism where the GK-GKRP mechanism is involved, activation of GK has a new therapeutic potential in the treatment of type 2 diabetes.     

Antibodies for proteomic research: comparison of traditional immunization with recombinant antibody technology

Antibodies play a pivotal role in studying the expression and function of proteins. Proteomics studies require the generation of specific and high‐affinity antibodies against large numbers of proteins. While traditional animal‐based antibody generation is laborious, difficult to automate, and therefore less suited to keep up with the requirements of proteomics research, the use of recombinant in vitro antibody technology might offer a solution to this problem. However, it has not been demonstrated yet that such antibodies are at least as useful as conventional antibodies for typical proteomics applications. Here we generated novel recombinant Fab antibody fragments from the naïve HuCAL® GOLD library against a number of targets derived from a mouse cDNA library. We compared these antibodies with polyclonal antisera produced against the same targets and show that these recombinant antibodies are useful reagents for typical applications like Western blotting or immunohistochemistry.     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.