Affinity labelling of rat liver acetyl-CoA car☐ylase by a 2′,3′-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA car☐ylase with a 2′,3′-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO₃⁻ accelerated the process. K_i values for oATP in the absence and presence of HCO₃⁻ were 0.35 ± 0.04 and 0.5 ± 0.06 mM , and those of the modification constant (k_mod) were 0.11 and 0.26 min⁻¹ respectively. oATP completely inhibited the [¹⁴C]ADP ⇌ ATP exchange and did not effect the [¹⁴C]acetyl-CoA ⇌ malonyl-CoA exchange. Incorporation of ∼1 equivalent of [³H]oATP per acetyl-CoA car☐ylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or β-mercaptoethanol containing buffers, and treatment with NaB³H₄ has not led to³H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA car☐ylase by oATP. It was shown that the reagent apparently interacted selectively with the ɛ-amino group of lysine in the ATP-binding site to form a morpholine-like structure.     

Inactivation of chicken liver mevalonate 5-diphosphate decar☐ylase by sulfhydryl-directed reagents: evidence of a functional dithiol

Chicken liver mevalonate 5-diphosphate decar☐ylase (ATP:(R)-5-diphosphomevalonate car☐y-lyase (dehydrating), EC 4.1.1.33) is inactivated by methylmethanethiosulfonate and 5,5′-dithiobis(2-nitrobenzoate). The presence of the substrates ATP or mevalonate 5-diphosphate protect very effectively against inactivation. The inactivation is second order with respect to methylmethanethiosulfonate, with an inactivation rate constant of (7.6 ± 0.1) · 10⁻⁵ μM⁻² ·s⁻¹, implying that the modifier may be reacting with more than one thiol in the enzyme. The enzyme is also inactivated by a number of dithiol-specific reagents, suggesting the presence of a functional dithiol. The determined pK_app values for enzyme modification by methyl methanethiosulfonate and phenylarsine oxide are 7.3 ± 0.1 and 7.6 ± 0.3, respectively. From the data presented, it is concluded that the enzyme posseses a functional dithiol that is important for substrate binding.     

Partial purification and characterization of intracellular car☐ypeptidase ofCandida albicans

Intracellular car☐ypeptidase was partially purified from the yeast form ofCandida albicans H-317 by acid dialysis, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Peptidase activity was measured with an enzyme-coupled colorimetric assay. Fractionation by native polyacrylamide gel electrophoresis and Sephacryl S-200 gel filtration indicated the presence of a single peak of car☐ypeptidase, with the isoelectric point at pH 4.6 and a molecular weight of 100,000. The pH optimum and apparentK_m usingN-carbobenzoxy-l-phenylalanine-l-leucine (N-Cbz-l-Phe-l-Leu) as substrate were 6.5 and 2 × 10⁻⁴M, respectively. The best substrates for the enzyme wereN-Cbz-Ala-X peptides, withN-Cbz-Ala-Leu giving the highest rate of hydrolysis. Substrates that gave 7% or less of the control (N-Cbz-Phe-Leu) rate of hydrolysis were Gly-Leu, Gly-Phe, Ile-Phe, Ile-Met, Glu-Phe, Glu-Tyr, Val-Phe, and Pro-Phe. There was no detectable hydrolysis of the followingN-Cbz peptides; Gly-Met, Gly-Val, Gly-Tyr, Gly-Ile, or Ile-Val. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride,p-chloromercuribenzoate, benzyloxy-carbonyl-l-phenylalanine chloromethyl ketone, and tosyl-l-phenylalanine chloromethyl ketone, but was not affected by EDTA, tosyl-l-lysine chloromethyl ketone, pepstatin A, leupeptin, bestatin, or antipain. Although secretory proteinases are thought to play a role in the pathogenesis of this organism, the role of this intracellular car☐ypeptidase has yet to be determined.     

A stable analogue of throm☐ane A2, 9,11-epithio-11,12-methanothrom☐ane A2, stimulates bone resorption in vitro and osteoclast-like cell formation in mouse marrow culture

Throm☐ane A₂ (TXA₂) is a powerful promoter of platelet aggregation and smooth muscle contraction. However, this compound is highly unstable and is rapidly hydrated to a more stable metabolite, throm☐ane B₂ (TXB₂). TXA₂ has been considered to be involved in bone resorption, in particular bone loss caused by inflammatory diseases and by orthodontic treatment. However precise mechanisms of bone resorption caused by TXA₂ have not yet been proved because of its highly unstable nature.Recently, a chemically stable analogue of TXA₂, 9,11-epithio-11,12-methanothrom☐ane A₂ (STA₂), was successfully synthesized. Using this synthetic compound, we examined its in vitro bone resorbing activity and induction of osteoclast-like cells in a mouse marrow culture system in comparison with related compounds with bone resorbing activity. Like prostaglandin E₂ (PGE₂), a well-known bone resorbing agent. STA₂ time-and dose-dependently stimulated the release of ⁴⁵Ca from prelabelled mouse calvariae. Both STA₂ and PGE₂ induced the accumulation of cAMP in mouse calvariae. The TXA₂, agonist. ONO-3708, inhibited STA₂-induced release of ⁴⁵Ca, TXB₂ induced neither bone resolor cAMP accumulation. When mouse marrow cells were cultured with STA₂ for 8 days, osteoclast-like multinucleated cells appeared in parallel with the increase of the amount of STA₂ added. Again TXB₂ showed no effect on osteoclast-like cell formation. These results indicate a role for TXA₂ in some form of bone resorption.     

Quantitative determination of imidazenil,a novel imidazobenzodiazepine car☐amide derivative,by normal-phase high-performance liquid chromatography

Imidazenil, an imidazobenzodiazepine car☐amide derivative, is a novel anxiolytic and anti-convulsant agent recently characterized as a partial allosteric modulator of GABA_A receptors. Owing to the pharmacological and pharmacokinetic importance of plasma-level determination, a HPLC method has been developed. Imidazenil was extracted from a plasma sample after a partition with diethyl ether, using alprazolam as internal standard. The analysis was performed by a normal-phase HPLC method with UV detection at 255 nm. The limit of quantitation was 6 ng corresponding to 30 ng/ml of plasma concentration. This procedure has been successfully applied to the quantitation of imidazenil plasma levels in primarily pharmacokinetic studies after a single i.v. and an oral administration of the compound to the rat.     

Syntheses and spectroscopic properties of α- and β-phosphatidylcholines and phosphatidylethanolamines

The widely used partial synthesis of phospholipids via deacylation of naturally occurring phospholipids, followed by reacylation with fatty acid anhydrides, is accompanied by phosphoryl migration. The resulting mixture of α- and β-phospholipids was separated by short-column chromatography. Milder acylation procedures in which no phosphoryl migration occurs, were developed. 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine was prepared in 50% yield by acylation of sn-glycero-3-phosphocholine (GPC) with N-linoleoylimidazole. Detailed NMR and infrared spectra of α- and β-phosphatidylcholines (PCs) and -ethanolamines (PEs) are reported and the differences between isomers discussed.     

Phenotypic plasticity and longevity in plants and animals: cause and effect?

Immobile plants and immobile modular animals outlive unitary animals. This paper discusses competing but not necessarily mutually exclusive theories to explain this extreme longevity, especially from the perspective of phenotypic plasticity. Stem cell immortality, vascular autonomy, and epicormic branching are some important features of the phenotypic plasticity of plants that contribute to their longevity. Monocarpy versus polycarpy can also influence the kind of senescent processes experienced by plants. How density-dependent phenomena affecting the establishment of juveniles in these immobile organisms can influence the evolution of senescence, and consequently longevity, is reviewed and discussed. Whether climate change scenarios will favour long-lived or short-lived organisms, with their attendant levels of plasticity, is also presented.     

Dogmatisms and Catechisms — Ethics and Companion Animals

Abstract

The current reconsideration of the place in nature of human beings unfortunately continues to be an acrimonious one. All too often the debate is more akin to a warlike encounter where each side attempts to gain control or the upper hand than a search for points of agreement. Given this context, it is important to entertain views that emanate from different cultural traditions as a way to infuse the debate with new life. Students of Native American culture have consistently pointed out that the essential concepts of life balance and reciprocity represented there may serve as useful points of consideration as we struggle with the appropriate relationships with animals and nature. This article presents a representative Zuni story, told by Governor Robert E. Lewis, that illustrates these notions.     

DCCP and DICP： Construction and Analyses of Databases for Copper- and Iron-Chelating Proteins

Copper and iron play important roles in a variety of biological processes, especially when being chelated with proteins. The proteins involved in the metal binding, transporting and metabolism have aroused much interest. To facilitate the study on this topic, we constructed two databases （DCCP and DICP） containing the known copper- and iron-chelating proteins~ which are freely available from the website http：//sdbi.sdut.edu.cn/en. Users can conveniently search and browse all of the entries in the databases. Based on the two databases, bioinformatic analyses were performed, which provided some novel insights into metalloproteins.     

Biosynthesis of geraniol and nerol and their β-d-glucosides in Perlargonium graveolens and Rosa dilecta

1. 3R-[2-(14)C]Mevalonate was incorporated into geranyl and neryl beta-d-glucosides in petals of Rosa dilecta in up to 10.6% yield, and the terpenoid part was specifically and equivalently labelled in the moieties derived from isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate. A similar labelling pattern, with incorporations of 0.06-0.1% was found for geraniol or nerol formed in leaves of Pelargonium graveolens The former results provide the best available evidence for the mevalonoid route to regular monoterpenes in higher plants. 2. Incorporation studies with 3RS-[2-(14)C,(4R)-4-(3)H(1)]-mevalonate and its (4S)-isomer showed that the pro-4R hydrogen atom of the precursor was retained and the pro-4S hydrogen atom was eliminated in both alcohols and both glucosides. These results suggest that the correlation of retention of the pro-4S hydrogen atom of mevalonate with formation of a cis-substituted double bond, such as has been found in certain higher terpenoids, does not apply to the biosynthesis of monoterpenes. It is proposed that either nerol is derived from isomerization of geraniol or the two alcohols are directly formed by different prenyltransferases. Possible mechanisms for these processes are discussed. 3. The experiments with [(14)C,(3)H]mevalonate also show that in these higher plants, as has been previously found in animal tissue and yeast, the pro-4S hydrogen atom of mevalonate was lost in the conversion of isopentenyl pyrophosphate into 3,3-dimethylallyl pyrophosphate.     

Zinc and diabetes — clinical links and molecular mechanisms

Zinc is an essential trace element crucial for the function of more than 300 enzymes and it is important for cellular processes like cell division and apoptosis. Hence, the concentration of zinc in the human body is tightly regulated and disturbances of zinc homeostasis have been associated with several diseases including diabetes mellitus, a disease characterized by high blood glucose concentrations as a consequence of decreased secretion or action of insulin. Zinc supplementation of animals and humans has been shown to ameliorate glycemic control in type 1 and 2 diabetes, the two major forms of diabetes mellitus, but the underlying molecular mechanisms have only slowly been elucidated. Zinc seems to exert insulin-like effects by supporting the signal transduction of insulin and by reducing the production of cytokines, which lead to beta-cell death during the inflammatory process in the pancreas in the course of the disease. Furthermore, zinc might play a role in the development of diabetes, since genetic polymorphisms in the gene of zinc transporter 8 and in metallothionein (MT)-encoding genes could be demonstrated to be associated with type 2 diabetes mellitus. The fact that antibodies against this zinc transporter have been detected in type 1 diabetic patients offers new diagnostic possibilities. This article reviews the influence of zinc on the diabetic state including the molecular mechanisms, the role of the zinc transporter 8 and MT for diabetes development and the resulting diagnostic and therapeutic options.     

siRNA, miRNA and HIV： promises and challenges

Small interfering RNA （siRNA） and microRNA （miRNA） are small RNAs of 18-25 nucleotides （nt） in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex （RISC） and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 （HIV-1） which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi （RNA interference）. On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells＇ antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs （vmiRNAs）.     

Toxoplasmosis and Epilepsy — Systematic Review and Meta Analysis

Background

Toxoplasmosis is an important, widespread, parasitic infection caused by Toxoplasma gondii. The chronic infection in immunocompetent patients, usually considered as asymptomatic, is now suspected to be a risk factor for various neurological disorders, including epilepsy. We aimed to conduct a systematic review and meta-analysis of the available literature to estimate the risk of epilepsy due to toxoplasmosis.

Methods

A systematic literature search was conducted of several databases and journals to identify studies published in English or French, without date restriction, which looked at toxoplasmosis (as exposure) and epilepsy (as disease) and met certain other inclusion criteria. The search was based on keywords and suitable combinations in English and French. Fixed and random effects models were used to determine odds ratios, and statistical significance was set at 5.0%.

Principal findings

Six studies were identified, with an estimated total of 2888 subjects, of whom 1280 had epilepsy (477 positive for toxoplasmosis) and 1608 did not (503 positive for toxoplasmosis). The common odds ratio (calculated) by random effects model was 2.25 (95% CI 1.27–3.9), p = 0.005.

Conclusions

Despite the limited number of studies, and a lack of high-quality data, toxoplasmosis should continue to be regarded as an epilepsy risk factor. More and better studies are needed to determine the real impact of this parasite on the occurrence of epilepsy.     

Rise and Demise of Bioinformatics? Promise and Progress

The field of bioinformatics and computational biology has gone through a number of transformations during the past 15 years, establishing itself as a key component of new biology. This spectacular growth has been challenged by a number of disruptive changes in science and technology. Despite the apparent fatigue of the linguistic use of the term itself, bioinformatics has grown perhaps to a point beyond recognition. We explore both historical aspects and future trends and argue that as the field expands, key questions remain unanswered and acquire new meaning while at the same time the range of applications is widening to cover an ever increasing number of biological disciplines. These trends appear to be pointing to a redefinition of certain objectives, milestones, and possibly the field itself.     

Defining and measuring autophagosome flux—concept and reality

The autophagic system is involved in both bulk degradation of primarily long-lived cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and a number of methods are currently utilized to assess autophagic flux. However, despite major advances in measuring various molecular aspects of the autophagic machinery, we remain less able to express autophagic flux in a highly sensitive, robust, and well-quantifiable manner. Here, we describe a conceptual framework for defining and measuring autophagosome flux at the single-cell level. The concept discussed here is based on the theoretical framework of metabolic control analysis, which distinguishes between the pathway along which there is a flow of material and the quantitative measure of this flow. By treating the autophagic system as a multistep pathway with each step characterized by a particular rate, we are able to provide a single-cell fluorescence live-cell imaging-based approach that describes the accurate assessment of the complete autophagosome pool size, the autophagosome flux, and the transition time required to turn over the intracellular autophagosome pool. In doing so, this perspective provides clarity on whether the system is at steady state or in a transient state moving towards a new steady state. It is hoped that this theoretical account of quantitatively measuring autophagosome flux may contribute towards a new direction in the field of autophagy, a standardized approach that allows the establishment of systematic flux databases of clinically relevant cell and tissue types that serve as important model systems for human pathologies.Abbreviations: CMA, chaperone-mediated autophagy; GFP, green fluorescent protein; J, flux; LC3, microtubule-associated protein 1 light chain 3; n_A, number of autophagosomes; τ, transition time; TEM, transmission electron microscopyThe autophagic system is involved in both bulk degradation of primarily long-lived cytosolic proteins as well as in the selective degradation of cytoplasmic organelles. In the past few years the assessment and evaluation of this complete system including its dynamics has received growing attention, as our understanding of autophagosome turnover and kinetic behavior has progressed. Autophagic flux is often defined as a measure of autophagic degradation activity, and a number of methods are currently suggested for assessing autophagic flux, many of which infer whether or not autophagic flux is occuring.¹ However, although we have advanced in the methodological approach to assess whether or not a change in autophagic flux is occurring, and whether autophagic flux goes up or down, we remain less able to express this change in a sensitive, robust, and well-quantifiable manner. Moreover, although the development of novel reporter assays enabled the identification of pharmacological regulators of autophagy, highly sensitive assays that characterize the extent and dynamics of this regulation quantitatively remain a challenge in the in vitro, and even more so the in vivo, environment. Based on the well-established metabolic control analysis approach,^2,3 where the use of the term flux has been reserved for the rate of flow along a metabolic pathway, we describe a methodological concept that allows the definition and measurement of autophagosome flux at the single cell level in a sensitive and quantifiable manner. Here, we treat the autophagic system as a multistep pathway with each step characterized by a particular rate. We distinguish between the vesicular machinery of the autophagic system, and the cargo that is being degraded within this system. Autophagosome flux, the subject of this paper, is the rate of flow along the vesicular pathway, whereas substrate clearance flux is the rate of cargo degradation within the vesicular system. This distinction makes it possible not only to describe whether or not the vesicular part of the autophagic system is at steady state, but also to quantitatively assess autophagosome flux and to calculate the transition time of the system required to turn over its autophagosome pool. The concept described here makes it possible to quantify and to compare treatment interventions or different cellular systems with one another in terms of change in autophagosome pool size, autophagosome flux, and pool size turnover as well as in the responsiveness and sensitivity to the treatment intervention.We will start with our quantitative definition of autophagosome flux and describe a methodological concept for measuring this flux. We will then briefly describe 3 of the major current and predominantly used approaches to measuring autophagic degradation activity, and, by juxtaposing them against our definition of autophagosome flux, attempt to indicate the major inherent challenges to these techniques. We will then describe a step-by-step methodology that describes the accurate assessment of i) the complete autophagosome pool size, ii) the steady state, iii) autophagosome flux, and iv) the transition time. Since our definition of autophagosome flux requires a dynamic assessment over time, this conceptual approach shows how to acquire data that describe both the existence of a steady state and the variables that characterize the steady state, such as the steady-state number of autophagosomes and the associated flux in terms of the change in this number per time per cell. We hope that this approach will provide a robust tool for generating numerical data that are sensitive enough to allow us to change flux incrementally, say by 5%, that allows a comparison of fluxes and steady states of different cellular systems, with the benefit of using data that directly reflect the intracellular autophagosome pool. It is moreover envisaged that the approach described here may contribute towards a standardized means for the establishment of flux databases of clinically relevant cell and tissue types that serve as important model systems for, for example, neurodegenerative disorders, cancer, or heart disease.^4-6 Finally, this concept may lay the foundation for future control analyses, to unravel the degree of control as opposed to regulation that the different steps in the autophagic pathway exert over the autophagosome flux.