CLPB Variants Associated with Autosomal-Recessive Mitochondrial Disorder with Cataract,Neutropenia, Epilepsy,and Methylglutaconic Aciduria

3-methylglutaconic aciduria (3-MGA-uria) is a nonspecific finding associated with mitochondrial dysfunction, including defects of oxidative phosphorylation. 3-MGA-uria is classified into five groups, of which one, type IV, is genetically heterogeneous. Here we report five children with a form of type IV 3-MGA-uria characterized by cataracts, severe psychomotor regression during febrile episodes, epilepsy, neutropenia with frequent infections, and death in early childhood. Four of the individuals were of Greenlandic descent, and one was North American, of Northern European and Asian descent. Through a combination of homozygosity mapping in the Greenlandic individuals and exome sequencing in the North American, we identified biallelic variants in the caseinolytic peptidase B homolog (CLPB). The causative variants included one missense variant, c.803C>T (p.Thr268Met), and two nonsense variants, c.961A>T (p.Lys321^∗) and c.1249C>T (p.Arg417^∗). The level of CLPB protein was markedly decreased in fibroblasts and liver of affected individuals. CLPB is proposed to function as a mitochondrial chaperone involved in disaggregation of misfolded proteins, resulting from stress such as heat denaturation.     

Closed testing with Globaltest,with application in metabolomics

The Globaltest is a powerful test for the global null hypothesis that there is no association between a group of features and a response of interest, which is popular in pathway testing in metabolomics. Evaluating multiple feature sets, however, requires multiple testing correction. In this paper, we propose a multiple testing method, based on closed testing, specifically designed for the Globaltest. The proposed method controls the familywise error rate simultaneously over all possible feature sets, and therefore allows post hoc inference, that is, the researcher may choose feature sets of interest after seeing the data without jeopardizing error control. To circumvent the exponential computation time of closed testing, we derive a novel shortcut that allows exact closed testing to be performed on the scale of metabolomics data. An R package ctgt is available on comprehensive R archive network for the implementation of the shortcut procedure, with applications on several real metabolomics data examples.     

Working with,not against,coral-reef fisheries

The fisheries policies of some Pacific island nations are more appropriate to the biology of their resources than are some of the fisheries policies of more industrialized countries. Exclusive local ownership of natural resources in Palau encourages adjustive management on biologically relevant scales of time and space and promotes responsibility by reducing the tragedy of the commons. The presence of large individuals in fish populations and adequate size of spawning aggregations are more efficient and meaningful cues for timely management than are surveys of abundance or biomass. Taking fish from populations more than halfway to their carrying capacity is working favorably with the fishery because removing fish potentially increases resource stability by negative feedback between stock size and population production. Taking the same amount of fish from a population below half its carrying capacity is working against the fishery, making the population unstable, because reducing the reproductive stock potentially accelerates reduction of the population production by positive feedback. Reef fish are consumed locally, while Palauan laws ban the export of reef resources. This is consistent with the high gross primary production with little excess net production from undisturbed coral-reef ecosystems. The relatively rapid growth rates, short life spans, reliable recruitment and wide-ranging movements of open-ocean fishes such as scombrids make them much more productive than coral-reef fishes. The greater fisheries yield per square kilometer in the open ocean multiplied by well over a thousand times the area of the exclusive economic zone than that of Palau’s coral reefs should encourage Palauans to keep reef fishes for subsistence and to feed tourists open-ocean fishes. Fisheries having only artisanal means should be encouraged to increase the yield and sustainability by moving away from coral reefs to bulk harvesting of nearshore pelagics.     

Melittin: structure, properties, interaction with a membrane

The paper is concerned with the analysis of the present data as to the structure and properties of melittin, a biologically active peptide of bee venom, able to be inserted spontaneously into the natural and synthetic membranes and to destruct cells in micromolar concentrations. This property is a result of the peptide structure peculiarities. Attention is focused on the mechanism of melittin insertion into the phospholipid bilayer as well as on the possibility of its use to study the nature of protein-lipid interactions and formation of ionic channels.     

Chloroperoxidase,a peroxidase with potential

Summary Chloroperoxidase is an extracellular heme glycoprotein produced by the imperfect fungusCaldariomyces fumago. The enzyme can catalyse chlorination reactions as well as act as a catalase or a peroxidase. As a peroxidase, it has a wide substrate specificity and we are interested in some applied aspects of this activity, requiring the production and purification of moderate quantities of the enzyme. High levels of chloroperoxidase are produced in a fructose synthetic medium, and highest enzyme production occurs in a low-shear environment. fungal pellets produce enzyme continuously at low medium replacement rates and at up to 0.6 g enzyme per 1: chloroperoxidase is essentially the only extracellular enzyme produced. Enzyme purification is uncomplicated and gives good yields of high purity. Pure enzyme is stable for weeks at room temperature and under pH control. Chloroperoxidase can be ionically bound to aminopropyl glass, then covalently immobilized by glutaraldehyde crosslinking. Immobilized preparations have been washed and re-used five times, and are most stable at pH 5.5-6. Like many peroxidases, chloroperoxidase will oxidize phenols and phenolics, often causing a precipitate, and can totally remove phenols at low aqueous concentrations. Chloroperoxidase incubation with the petroporphyrin component of crude oil asphaltene (fraction 5) causes a reduction or removal of the Soret band (410 nm) and the -peak (573 nm). This petroporphyrin fraction is enriched with vanadium which poisons the chemical catalyst used in cracking crude oil.     

MICAL, a novel CasL interacting molecule, associates with vimentin

CasL/HEF1 belongs to the p130(Cas) family. It is tyrosine-phosphorylated following beta(1) integrin and/or T cell receptor stimulation and is thus considered to be important for immunological reactions. CasL has several structural motifs such as an SH3 domain and a substrate domain and interacts with many molecules through these motifs. To obtain more insights on the CasL-mediated signal transduction, we sought proteins that interact with the CasL SH3 domain by far Western screening, and we identified a novel human molecule, MICAL (a Molecule Interacting with CasL). MICAL is a protein of 118 kDa and is expressed in the thymus, lung, spleen, kidney, testis, and hematopoietic cells. MICAL has a calponin homology domain, a LIM domain, a putative leucine zipper motif, and a proline-rich PPKPP sequence. MICAL associates with CasL through this PPKPP sequence. MICAL is a cytoplasmic protein and colocalizes with CasL at the perinuclear area. Through the COOH-terminal region, MICAL also associates with vimentin that is a major component of intermediate filaments. Immunostaining revealed that MICAL localizes along with vimentin intermediate filaments. These results suggest that MICAL may be a cytoskeletal regulator that connects CasL to intermediate filaments.     

Titin-cap associates with,and regulates secretion of,Myostatin

Myostatin, a secreted growth factor, is a key negative regulator of skeletal muscle growth. To identify modifiers of Myostatin function, we screened for Myostatin interacting proteins. Using a yeast two-hybrid screen, we identified Titin-cap (T-cap) protein as interacting with Myostatin. T-cap is a sarcomeric protein that binds to the N-terminal domain of Titin and is a substrate of the titin kinase. Mammalian two-hybrid studies, in vitro binding assays and protein truncations in the yeast two-hybrid system verified the specific interaction between processed mature Myostatin and full-length T-cap. Analysis of protein-protein interaction using surface plasmon resonance (Biacore, Uppsala, Sweden) kinetics revealed a high affinity between Myostatin and T-cap with a KD of 40 nM. When T-cap was stably overexpressed in C(2)C(12) myoblasts, the rate of cell proliferation was significantly increased. Western analyses showed that production and processing of Myostatin were not altered in cells overexpressing T-cap, but an increase in the retention of mature Myostatin indicated that T-cap may block Myostatin secretion. Bioassay for Myostatin confirmed that conditioned media from myoblasts overexpressing T-cap contained lower levels of Myostatin. Given that Myostatin negatively regulates myoblast proliferation, the increase in proliferation observed in myoblasts overexpressing T-cap could thus be due to reduced Myostatin secretion. These results suggest that T-cap, by interacting with Myostatin, controls Myostatin secretion in myogenic precursor cells without affecting the processing step of precursor Myostatin.     

Research with animals: requirement, responsibility, welfare

Recognition of unacceptable cruelty to animals in pasttimes such as bull-baiting, dates in Britain from the early 19th century. The Society for the Prevention of Cruelty to Animals was founded in 1824. Several bills to curb cruelty were discussed in Parliament, and the Cruel and Improper Treatment of Cattle Act was passed in 1822. Other Acts have followed over the years. Cruelty in the form of painful scientific experiments, including dissection of living, conscious animals, vivisection, was proscribed by the Cruelty to Animals Act 1876. That Act required anyone wishing to experiment with animals to obtain a licence from the Secretary of State. Conditions for issue of licences were strict and remain so to this day. The Act is still valid, and is enforced by the Home Office, with its medical and veterinary Inspectors. The Cruelty to Animals Act 1876 allows experiments on animals under strictly controlled conditions. Experiments must have the clear objective of improving the welfare of man and/or animals. Benefits from experiments carried out under the Act have been enormous, covering every aspect of diagnosis, treatment, and prophylaxis in human and veterinary medicine. Coincidentally, the welfare of laboratory animals has also been greatly improved. There has always been some opposition to the use of animals in biomedical research. The subject is emotive but, by and large, discussion has been rational and within the law. In recent years, however, the morality of using experimental animals has been examined more closely. The possibility of replacing them by alternative methods has been investigated. Where these alternatives are applicable, they are used and further research on them continues. The questioning of animal experiments has emphasized the need to look constantly at animal welfare to ensure humane treatment of all animals, especially those restricted in a laboratory or on a farm. Attention has been drawn in this work to our existing laws protecting animals, but new legislation is being demanded, not only by some lay welfare groups but also by scientists. Hence, it has become very important to discuss various ways of ensuring animal welfare, including by legislation, especially with those knowledgeable in laboratory animal science and animal experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor (K(I) of 914 mM) but it interacts with hCA II (K(I) of 27.5mM). Isozyme IV is well inhibited by sulfate (K(I) of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate (K(I) of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate (K(I) of 680 mM) and carbonate (K(I) of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here (K(I) of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition (K(I) of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate (K(I) in the range of 8.6-9.6 microM). This constitutes another proof that this isozyme best works at acidic pH values present in tumors, being inhibited substantially at higher pH values when more carbonate may be present. Bromate and chlorate are quite weak CA IX inhibitors (K(I) s of 147-274 mM).     

LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes

The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogen es EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes .     

VE-cadherin-derived cell-penetrating peptide, pVEC, with carrier functions

Cell-penetrating peptides, CPPs, have been shown to translocate into living cells by a receptor-independent mechanism and to carry macromolecules over the plasma membrane. This article reports studies of the internalization of pVEC, an 18-amino acid-long peptide derived from the murine sequence of the cell adhesion molecule vascular endothelial cadherin, amino acids 615-632. Fluorophore-labeled pVEC entered four different cell lines tested: human aortic endothelial cells, brain capillary endothelial cells, Bowes melanoma cells, and murine brain endothelial cells. In order to evaluate the translocation efficiency of pVEC, we performed a side-by-side comparison with penetratin, a well-characterized CPP. The cellular uptake of pVEC was highest for murine brain endothelial cells. All cell lines tested contained equal or slightly higher concentrations of pVEC than penetratin. pVEC mainly accumulated in nuclear structures but was also found throughout the cells. Furthermore, pVEC functioned as a transporter of both a hexameric peptide nucleic acid molecule of 1.7 kDa and a 67-kDa protein, streptavidin-FITC, and cellular uptake of fluorophore-labeled pVEC took place at 4 degrees C, suggesting a nonendocytotic mechanism of translocation. In conclusion, our results indicate that pVEC is efficiently and rapidly taken up into cells and functions as a potent carrier peptide.     

Studies with a plant fungicide,imazalil, with vapor-phase activity,in the therapy of human alternariosis

The use of a plant fungicide, the imidazole imazalil, in the therapy of human alternariosis is described. The patient's disease involved the palate, nose and sinuses, and had been unresponsive to conventional therapy. Imazalil has been used to treat Alternaria infections of plant materials. The patient's isolate was susceptible in vitro. We also showed that the drug was inhibitory to fungi in vitro as a vapor, not requiring contact by diffusion through agar or broth. The drug was used topically, by installation and irrigation, in this case and administered orally in doses of up to 1 200 mg daily, for 6 months. Peak serum concentrations exceeded 4 meg/ml. The infection was arrested but not cured. The drug was tolerated without evident toxicity, though an unpleasant taste and nausea at high doses were limiting.     

Local vasodilatation with metacholine, but not with nitroprusside, increases forearm glucose uptake

Insulin is known to increase blood flow in parallel to glucose uptake in skeletal muscle. However, it is not known if an increase in blood flow by itself is associated with an increase in glucose uptake in the absence of hyperinsulinemia. To investigate further this matter, the effect of increased blood flow on forearm glucose uptake was studied in the fasting state during intra-arterial infusions of two different vasodilators, metacholine and nitroprusside, in 19 hypertensive subjects. Both metacholine (4 microg/min) and nitroprusside (10 microg/min) increased resting forearm blood flow, measured by venous occlusion plethysmography, to a similar degree (180 % and 170 %, respectively, p<0.0001 for both). However, metacholine infusion increased the forearm glucose uptake from 2.0+/-0.9 (S.D.) during rest to 5.5+/-3.0 umol/min/100 ml tissue (p<0.0001), while no significant change in glucose uptake was seen during nitroprusside infusion (2.3+/-1.4 micromol/min/100 ml tissue). In conclusion, vasodilatation induced by metacholine, but not by nitroprusside, increased glucose uptake in the forearm of hypertensive patients. Thus, an increase in forearm blood flow does not necessarily improve glucose uptake in the forearm during the fasting state.     

Association of endothelial microparticle with NO,eNOS, ET-1, and fractional flow reserve in patients with coronary intermediate lesions

Abstract

Endothelial microparticle (EMP) is a biomarker for endothelial dysfunction. The aim of this study is to investigate the utility of EMP in evaluating coronary intermediate lesions. Participants included 49 patients with coronary intermediate lesions and 24 subjects with normal coronary arteries. Among these subjects, 28 patients accepted fractional flow reserve (FFR). Results showed that level of EMP was significantly higher in the intermediate lesion group. No correlation was found between EMP and FFR value, suggesting that circulating EMP is a systemic marker rather than a focal one.     

Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate, and nitrate

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 A. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.     

Savanna chimpanzees, Pan troglodytes verus, hunt with tools

Although tool use is known to occur in species ranging from naked mole rats [1] to owls [2], chimpanzees are the most accomplished tool users [3-5]. The modification and use of tools during hunting, however, is still considered to be a uniquely human trait among primates. Here, we report the first account of habitual tool use during vertebrate hunting by nonhumans. At the Fongoli site in Senegal, we observed ten different chimpanzees use tools to hunt prosimian prey in 22 bouts. This includes immature chimpanzees and females, members of age-sex classes not normally characterized by extensive hunting behavior. Chimpanzees made 26 different tools, and we were able to recover and analyze 12 of these. Tool construction entailed up to five steps, including trimming the tool tip to a point. Tools were used in the manner of a spear, rather than a probe or rousing tool. This new information on chimpanzee tool use has important implications for the evolution of tool use and construction for hunting in the earliest hominids, especially given our observations that females and immature chimpanzees exhibited this behavior more frequently than adult males.     

Aph2, a protein with a zf-DHHC motif,interacts with c-Abl and has pro-apoptotic activity

c-Abl is a non-receptor tyrosine kinase implicated in DNA damage-induced cell death and in growth factor receptor signaling. To further understand the function and regulation of c-Abl, a yeast two-hybrid screen was performed to identify c-Abl-interacting proteins. Here we report the identification of Abl-philin 2 (Aph2), encoding a novel protein with a unique cysteine-rich motif (zf-DHHC) and a 53-amino acid stretch sharing homology with the creatine kinase family. The zf-DHHC domain is highly conserved from yeast to human. Two proteins containing this motif, Akr1p and Erf2p, have been characterized in Saccharomyces cerevisiae, both implicated in signaling pathways. Deletion analysis by two-hybrid assays revealed that the N-terminal portion of Aph2 interacts with the C terminus of c-Abl. Aph2 was demonstrated to interact with c-Abl by co-immunoprecipitation assays. Aph2 is expressed in most tissues tested and is localized in the cytoplasm, mainly in the endoplasmic reticulum (ER). The sequences required for ER location reside in the N terminus and the zf-DHHC motif of Aph2. It has been reported that a portion of c-Abl is localized in the ER. We demonstrate here that Aph2 and c-Abl are co-localized in the ER region. Overexpression of Aph2 leads to apoptosis as justified by TUNEL assays, and the induction of apoptosis requires the N terminus. Co-expression of c-Abl and Aph2 had a synergistic effect on apoptosis induction and led to a decreased expression of both proteins, suggesting either that these two proteins are mutually down-regulated or that cells expressing both c-Abl and Aph2 rapidly disappeared from the culture. These results suggest that Aph2 may be involved in ER stress-induced apoptosis in which c-Abl plays an important role.     

Therapy with Zoledronic Acid, 5 Mg,for a Patient with Paget Disease of Bone

ObjectiveTo describe the effects of an infusion of zoledronic acid in a patient with Paget disease of bone (PD) who had been treated unsuccessfully with several other bisphosphonates.MethodsThe patient’s treatment history is described, and his response to various bisphosphonates, including zoledronic acid, is discussed.ResultsThe patient was a 61-year-old man when a diagnosis of PD was made on the basis of an elevated serum alkaline phosphatase (ALP) level of 391 U/L (reference range, 45 to 135). Pagetic bone changes were noted on bone scan and x-ray examinations. Treatment with etidronate had no effect on ALP levels. This intervention was followed by 2 courses of intravenous therapy with pamidronate, which decreased ALP levels by 57% and 55% without inducing a remission. Subsequent oral treatment with alendronate and then risedronate yielded unsustained biochemical remissions of 6 months each. Most recently, therapy with an infusion of 5 mg of zoledronic acid induced a remission that is thus far 20 months in duration. With this therapy, the patient has experienced no side effects, and he has noted a decrease in bone pain.ConclusionThis case study shows that a patient with PD who received successive treatments with several bisphosphonates with inadequate responses or only brief remissions was more effectively treated with a single 5-mg infusion of zoledronic acid. This patient’s therapeutic responses illustrate the relative efficacy of available bisphosphonates and the potential for longer-lasting remissions with zoledronic acid in patients with PD. (Endocr Pract. 2008;14:607-610)