Calpain Inhibitors as Potential Therapeutic Modulators in Neurodegenerative Diseases

It is considered a significant challenge to understand the neuronal cell death mechanisms with a suitable cure for neurodegenerative disorders in the coming years. Calpains are one of the best-considered “cysteine proteases activated” in brain disorders. Calpain is an important marker and mediator in the pathophysiology of neurodegeneration. Calpain activation being the essential neurodegenerative factor causing apoptotic machinery activation, it is crucial to develop reliable and effective approaches to prevent calpain-mediated apoptosis in degenerating neurons. It has been recently seen that the “inhibition of calpain activation” has appeared as a possible therapeutic target for managing neurodegenerative diseases. A systematic literature review of PubMed, Medline, Bentham, Scopus, and EMBASE (Elsevier) databases was conducted. The present article reviews the basic pathobiology and role of selective calpain inhibitors used in various neurodegenerative diseases as a therapeutic target.

Graphical Abstract

     

Bile Acids as Modulators of Enzyme Activity and Stability

Bile acids deactivate certain enzymes, such as prolyl endopeptidases (PEPs), which are investigated as candidates for protease-based therapy for celiac sprue. Deactivation by bile acids presents a problem for therapeutic enzymes targetted to function in the upper intestine. However, enzyme deactivation by bile acids is not a general phenomenon. Trypsin and chymotrypsin are not deactivated by bile acids. In fact, these pancreatic enzymes are more efficient at cleaving large dietary substrates in the presence of bile acids. We targeted the origin of the apparently different effect of bile acids on prolyl endopeptidases and pancreatic enzymes by examining the effect of bile acids on the kinetics of cleavage of small substrates, and by determining the effect of bile acids on the thermodynamic stabilities of these enzymes. Physiological amounts (5 mM) of cholic acid decrease the thermodynamic stability of Flavobacterium meningosepticum PEP from 18.5 ± 2 kcal/mol to 10.5 ± 1 kcal/mol, while thermostability of trypsin and chymotrypsin is unchanged. Trypsin and chymotrypsin activation by bile and PEP deactivation can both be explained in terms of a common mechanism: bile acid-mediated protein destabilization. Bile acids, usually considered non-denaturing surfactants, in this case act as a destabilizing agent on PEP thus deactivating the enzyme. However, this level of global thermodynamic destabilization does not account for a more than 50% decrease in enzyme activity, suggesting that bile acids most likely modulate enzyme activity through specific local interactions.     

Gene Regulation by Thyroid Hormone During Amphibian Metamorphosis: Implications on the Role of Cell-Cell and Cell-Extracellular Matrix Interactions

SYNOPSIS. Amphibian metamorphosis is the developmental processinitiated by thyroid hormone which transforms a tadpole intoa frog. This transformation requires extensive remodeling ofalmost every tissue in the animal. One of the more well-studiedtadpole tissues that undergoes remodeling is the small intestine.This tissue requires a shortening in length as well as internalanatomical restructuring to function in the adult frog. Briefly,the tadpole epithelial cells undergo programmed cell death (orapoptosis) and are replaced by a layer of newly formed adultepithelium. About 20 thyroid hormone-regulated genes participatingin this intestinal remodeling have been identified. These genescan be divided into several groups based on the proposed functionsof their products. One of these groups contains several secretedand/or signaling molecules. Most prominent among these are theXenopus homologs of the hedgehog and stromelysin-3 genes. Basedon the expression profiles and cellular localization, hedgehogappears to be involved in adult epithelial morphogenesis. Stromelysin-3may participate in basal lamina modification which is potentiallyinvolved in the apoptosis of the larval epithelium and developmentof the adult epithelium. Here we will review in detail the potentialroles for these secreted factors as well as the proposed molecularmechanisms responsible for their physiological functions. Furthermore,we will examine the effect of these proteins on the extracellularenvironment and how this impacts upon cellular processes involvedin intestinal remodeling.     

Proteinaceous Inhibitor Versus Fructose as Modulators of Pteris deflexa Invertase Activity

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer M r 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had β -fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with K m of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.     

中国林蛙蝌蚪变态过程中甲状腺的发育

两栖动物幼体变态过程是受甲状腺激素所精密调控的。对中国林蛙Rana chensinensis变态前期、临近变态期、变态高峰期和变态完成期的蝌蚪进行了外部形态指标(全长、体长、尾长和后肢长等)的测定,采用解剖和组织学方法对其甲状腺进行了组织学观察,并对蝌蚪外部形态指标与甲状腺形态机能之间的相关性进行了统计分析。结果显示,中国林蛙蝌蚪甲状腺分泌甲状腺激素机能活性的峰值出现于变态高峰期的前肢伸出期。统计分析表明,蝌蚪外部形态指标全长和后肢长的发育信息也可以反映其甲状腺分泌甲状腺激素的机能活性。     

Biological Activity and Antidiabetic Potential of C-Terminal Octapeptide Fragments of the Gut-Derived Hormone Xenin

Xenin is a peptide that is co-secreted with the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), from intestinal K-cells in response to feeding. Studies demonstrate that xenin has appetite suppressive effects and modulates glucose-induced insulin secretion. The present study was undertaken to determine the bioactivity and antidiabetic properties of two C-terminal fragment xenin peptides, namely xenin 18–25 and xenin 18–25 Gln. In BRIN-BD11 cells, both xenin fragment peptides concentration-dependently stimulated insulin secretion, with similar efficacy as the parent peptide. Neither fragment peptide had any effect on acute feeding behaviour at elevated doses of 500 nmol/kg bw. When administered together with glucose to normal mice at 25 nmol/kg bw, the overall insulin secretory effect was significantly enhanced in both xenin 18–25 and xenin 18–25 Gln treated mice, with better moderation of blood glucose levels. Twice daily administration of xenin 18–25 or xenin 18–25 Gln for 21 days in high fat fed mice did not affect energy intake, body weight, circulating blood glucose or body fat stores. However, circulating plasma insulin concentrations had a tendency to be elevated, particularly in xenin 18–25 Gln mice. Both treatment regimens significantly improved insulin sensitivity by the end of the treatment period. In addition, sustained treatment with xenin 18–25 Gln significantly reduced the overall glycaemic excursion and augmented the insulinotropic response to an exogenous glucose challenge on day 21. In harmony with this, GIP-mediated glucose-lowering and insulin-releasing effects were substantially improved by twice daily xenin 18–25 Gln treatment. Overall, these data provide evidence that C-terminal octapeptide fragments of xenin, such as xenin 18–25 Gln, have potential therapeutic utility for type 2 diabetes.     

Thyroid Hormone Receptor Genes of Neotenic Amphibians

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to α and β genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3′,5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRα in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRβ expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRα and TRβ may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis. Received: 23 April 1996 / Accepted: 20 January 1997     

Thyroid Hormone Receptors in the Developing Rat Brain

It is widely believed that the adult mammalian brain is insensitiveto thyroid hormones unlike the neonatal brain which is criticallydependent on these hormones for the development of normal structureand function. Recent studies have demonstrated the presenceof limited capacity, high affinity, triiodothyronine (T3) bindingnuclear sites in tissues that are considered responsive to thyroidhormones. Furthermore, there is evidence from studies on peripheraltissues that these T3 binding sites act as true receptors ininitiating thyroid hormone action. This report examines whetherthe higher sensitivity of neonatal brain to thyroid hormonesand the purported decline in sensitivity in adulthood are relatedto changes in the concentration and affinity characteristicsof thyroid hormone receptors in rat cerebral nuclei. Analysisof Scatchard plots of in vitro T3 binding data indicate thatcerebral nuclei from adult rats contain T3 specific nuclearbinding sites at a concentration comparable to that presentduring the period when the brain is critically dependent onthe presence of thyroid hormones and exceed that in the liver,a tissue generally considered thyroid sensitive. Neonatal thyroidectomysignificantly increased the number of binding sites. The resultsshow that the apparent unresponsiveness of the cerebral cortexof adult rats to thyroid hormones is not due to the absenceor a low density of T3 nuclear binding sites. The significanceof these results is discussed.     

Insect Repellents: Modulators of Mosquito Odorant Receptor Activity

Background

DEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs).

Methodology/Principal Findings

Here we characterize the action of these repellents on two Aedes aegypti ORs, AaOR2 and AaOR8, individually co-expressed with the common co-receptor AaOR7 in Xenopus oocytes; these ORs are respectively activated by the odors indole (AaOR2) and (R)-(−)-1-octen3-ol (AaOR8), odorants used to locate oviposition sites and host animals. In the absence of odorants, DEET activates AaOR2 but not AaOR8, while 2-U activates AaOR8 but not AaOR2; IR3535 and Picaridin do not activate these ORs. In the presence of odors, DEET strongly inhibits AaOR8 but not AaOR2, while 2-U strongly inhibits AaOR2 but not AaOR8; IR3535 and Picaridin strongly inhibit both ORs.

Conclusions/Significance

These data demonstrate that repellents can act as olfactory agonists or antagonists, thus modulating OR activity, bringing concordance to conflicting models.     

Circulating B-Lymphocytes as Potential Biomarkers of Tuberculosis Infection Activity

Accurate biomarkers of Mycobacterium tuberculosis infection activity would significantly improve early diagnosis, treatment and management of M. tuberculosis infection. We hypothesised that circulating B-lymphocytes may be useful biomarkers of tuberculosis (TB) infection status in highly TB-endemic settings. Ex-vivo and in-vitro mycobacteria-specific B-cell ELISPOT assays were used to examine the plasmablast (PB) and memory B-cell (MBC) responses in the peripheral blood of adult, healthy, community controls (n = 151) and of active TB patients (n = 48) living in Uganda. Frequencies of mycobacteria-specific PBs were markedly higher in active TB patients compared to healthy controls, and, conversely, MBCs were markedly higher in the healthy controls compared to active TB patients. In addition, the community controls with evidence of latent TB infection had higher peripheral blood PB and MBC responses than those without evidence of TB infection. These data demonstrate that peripheral blood B-cell responses are differentially modulated during latent and active M. tuberculosis infection, and suggest that the PB to MBC ratio may be a useful biomarker of TB infection activity.     

Modulation of Thyroid Hormone Receptors by Non-Thyroidal Stimuli

Abstract

The ability of non-thyroidal stimuli to affect the binding affinity and capacity of solubilized nuclear receptors for thyroid hormones was studied in a normal homeostatic system (erythropoiesis) and a pathobiologic one (lung-ozone interaction). No significant effects on affinity were found, as Kd control values for receptors derived from rat bone marrow averaged 57 (±28) pM while experimental (hypoxic) values averaged 89 (±55) pM. Kd control values in rat lung were found to average 142 (±22) pM while average values derived from experimental protocols with ozone and methimazole were 267 (±44) pM and 161 (±35) pM respectively. Finally, Kd control values for receptors derived from cultured MEL cells averaged 19 (±2.6) pM while experimental values during exposure to DMSO or IGFl were 23 (±3.6) pM and 26 (±11) pM respectively. In contrast, binding capacity (expressed as fmoles of hormone bound per unit protein of solubilized receptor) was markedly perturbed in several tisses by various agents: ozone effects on lung were shown by an average control value of 3.3 (±0.4) as opposed to an experimental average of 28 (±1.9); and hypoxia effects on erythroid tissue were displayed by an     

Adaptive Activation of Thyroid Hormone and Energy Expenditure

The mechanisms by which thyroid hormone accelerates energy expenditure are poorly understood. In the brown adipose tissue (BAT), activation of thyroid hormone by type 2 iodothyronine deiodinase (D2) has been known to play a role in adaptive energy expenditure during cold exposure in human newborns and other small mammals. Although BAT is not present in significant amounts in normal adult humans, recent studies have found substantial amounts of D2 in skeletal muscle, a metabolically relevant tissue in humans. This article reviews current biological knowledge about D2 and adaptive T3 production and their roles in energy expenditure.     

Gene Specific Actions of Thyroid Hormone Receptor Subtypes

There are two homologous thyroid hormone (TH) receptors (TRs α and β), which are members of the nuclear hormone receptor (NR) family. While TRs regulate different processes in vivo and other highly related NRs regulate distinct gene sets, initial studies of TR action revealed near complete overlaps in their actions at the level of individual genes. Here, we assessed the extent that TRα and TRβ differ in target gene regulation by comparing effects of equal levels of stably expressed exogenous TRs +/− T₃ in two cell backgrounds (HepG2 and HeLa). We find that hundreds of genes respond to T₃ or to unliganded TRs in both cell types, but were not able to detect verifiable examples of completely TR subtype-specific gene regulation. TR actions are, however, far from identical and we detect TR subtype-specific effects on global T₃ response kinetics in HepG2 cells and many examples of TR subtype specificity at the level of individual genes, including effects on magnitude of response to TR +/− T₃, TR regulation patterns and T₃ dose response. Cycloheximide (CHX) treatment confirms that at least some differential effects involve verifiable direct TR target genes. TR subtype/gene-specific effects emerge in the context of widespread variation in target gene response and we suggest that gene-selective effects on mechanism of TR action highlight differences in TR subtype function that emerge in the environment of specific genes. We propose that differential TR actions could influence physiologic and pharmacologic responses to THs and selective TR modulators (STRMs).