DNA- and chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec: H1t is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

DNA-condensing and chromatin-condensing properties of rat testes hla and hit compared to those of rat-liver hlbdec - hlt is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in /the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization.Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.

Ion-regulatory proreins in neuronal development and communication

Brain function is critically dependent on the ionic homeostasis in both the extra- and intracellular compartment. The regulation of brain extracellular ionic composition mainly relies on active transport at blood brain and at blood cerebrospinal fluid interfaces whereas intracellular ion regulation is based on plasmalemmal transporters of neurons and glia. In addition, the latter mechanisms can generate physiologically as well as pathophysiologically significant extracellular ion transients. In this work I have studied molecular mechanisms and development of ion regulation and how these factors alter neuronal excitability and affect synaptic and non-synaptic transmission with a particular emphasis on intracellular pH and chloride (Cl-) regulation. Why is the regulation of acid-base equivalents (H+ and HCO3-) and Cl- of such interest and importance? First of all, GABAA-receptors are permeable to both HCO3- and Cl-. In the adult mammalian central nervous system (CNS) fast postsynaptic inhibition relies on GABAA-receptor mediated transmission. Today, excitatory effects of GABAA-receptors, both in mature neurons and during the early development, have been recognized and the significance of the dual actions of GABA on neuronal communication has become an interesting field of research. The transmembrane gradients of Cl- and HCO3- determine the reversal potential of GABAA-receptor mediated postsynaptic potentials and hence, the function of pH and Cl- regulatory proteins have profound consequences on GABAergic signaling and neuronal excitability. Secondly, perturbations in pH can cause a variety of changes in cellular function, many of them resulting from the interaction of protons with ionizable side chains of proteins. pH-mediated alterations of protein conformation in e.g. ion channels, transporters, and enzymes can powerfully modulate neurotransmission. In the context of pH homeostasis, the enzyme carbonic anhydrase (CA) needs to be taken into account in parallel with ion transporters: for CO2/HCO3- buffering to act in a fast manner, CO2 (de)hydration must be catalyzed by this enzyme. The acid-base equivalents that serve as substrates in the CO2 dehydration-hydration reaction are also engaged in many carrier and channel mediated ion movements. In such processes, CA activity is in key position to modulate transmembrane solute fluxes and their consequences. The bicarbonate transporters (BTs; SLC4) and the electroneutral cation-chloride cotransporters (CCCs; SLC12) belong the to large gene family of solute carriers (SLCs). In my work I have studied the physiological roles of the K+-Cl- cotransporter KCC2 (Slc12a5) and the Na+-driven Cl--HCO3- exchanger NCBE (Slc4a10) and the roles of these two ion transporters in the modualtion of neuronal communication and excitability in the rodent hippocampus. I have also examined the cellular localization and molecular basis of intracellular CA that has been shown to be essential for the generation of prolonged GABAergic excitation in the mature hippocampus. The results in my Thesis provide direct evidence for the view that the postnatal up-regulation of KCC2 accounts for the developmental shift from depolarizing to hyperpolarizing postsynaptic EGABA-A responses in rat hippocampal pyramidal neurons. The results also indicate that after KCC2 expression the developmental onset of excitatory GABAergic transmission upon intense GABAA-receptor stimulation depend on the expression of intrapyramidal CA, identified as the CA isoform VII. Studies on mice with targeted Slc4a10 gene disruption revealed an important role for NCBE in neuronal pH regulation and in pH-dependent modulation of neuronal excitability. Furthermore, this ion transporter is involved in the basolateral Na+ and HCO3- uptake in choroid plexus epithelial cells, and is thus likely to contribute to cerebrospinal fluid production.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

Genotype-Phenotype Relationships in Long QT Syndrome: Role of Mental Stress, Adrenergic Activity and a Common KCNH2 Polymorphism

Long QT syndrome is a congenital or acquired arrhythmic disorder which manifests as a prolonged QT-interval on the electrocardiogram and as a tendency to develop ventricular arrhythmias which can lead to sudden death. Arrhythmias often occur during intense exercise and/or emotional stress. The two most common subtypes of LQTS are LQT1, caused by mutations in the KCNQ1 gene and LQT2, caused by mutations in the KCNH2 gene. LQT1 and LQT2 patients exhibit arrhythmias in different types of situations: in LQT1 the trigger is usually vigorous exercise whereas in LQT2 arrhythmia results from the patient being startled from rest. It is not clear why trigger factors and clinical outcome differ from each other in the different LQTS subtypes. It is possible that stress hormones such as catecholamines may show different effects depending on the exact nature of the genetic defect, or sensitivity to catecholamines varies from subject to subject. Furthermore, it is possible that subtle genetic variants of putative modifier genes, including those coding for ion channels and hormone receptors, play a role as determinants of individual sensitivity to life-threatening arrhythmias. The present study was designed to identify some of these risk modifiers. It was found that LQT1 and LQT2 patients show an abnormal QT-adaptation to both mental and physical stress. Furthermore, as studied with epinephrine infusion experiments while the heart was paced and action potentials were measured from the right ventricular septum, LQT1 patients showed repolarization abnormalities which were related to their propensity to develop arrhythmia during intense, prolonged sympathetic tone, such as exercise. In LQT2 patients, this repolarization abnormality was noted already at rest corresponding to their arrhythmic episodes as a result of intense, sudden surges in adrenergic tone, such as fright or rage. A common KCNH2 polymorphism was found to affect KCNH2 channel function as demonstrated by in vitro experiments utilizing mammalian cells transfected with the KCNH2 potassium channel as well as QT-dynamics in vivo. Finally, the present study identified a common β-1-adrenergic receptor genotype that is related a shorter QT-interval in LQT1 patients. Also, it was discovered that compound homozygosity for two common β-adrenergic polymorphisms was related to the occurrence of symptoms in the LQT1 type of long QT syndrome. The studies demonstrate important genotype-phenotype differences between different LQTS subtypes and suggest that common modifier gene polymorphisms may affect cardiac repolarization in LQTS. It will be important in the future to prospectively study whether variant gene polymorphisms will assist in clinical risk profiling of LQTS patients.

Who really ate the fruit? A novel approach to camera trapping for quantifying frugivory by ruminants

Tropical forest ruminants disperse several plants; yet, their effectiveness as seed dispersers is not systematically quantified. Information on frequency and extent of frugivory by ruminants is lacking. Techniques such as tree watches or fruit traps adapted from avian frugivore studies are not suitable to study terrestrial frugivores, and conventional camera traps provide little quantitative information. We used a novel time-delay camera-trap technique to assess the effectiveness of ruminants as seed dispersers for Phyllanthus emblica at Mudumalai, southern India. After being triggered by animal movement, cameras were programmed to take pictures every 2 min for the next 6 min, yielding a sequence of four pictures. Actual frugivores were differentiated from mere visitors, who did not consume fruit, by comparing the number of fruit remaining across the time-delay photograph sequence. During a 2-year study using this technique, we found that six terrestrial mammals consumed fallen P. emblica fruit. Additionally, seven mammals and one bird species visited fruiting trees but did not consume fallen fruit. Two ruminants, the Indian chevrotain Moschiola indica and chital Axis axis, were P. emblica's most frequent frugivores and they accounted for over 95% of fruit removal, while murid rodents accounted for less than 1%. Plants like P. emblica that are dispersed mainly by large mammalian frugivores are likely to have limited ability to migrate across fragmented landscapes in response to rapidly changing climates. We hope that more quantitative information on ruminant frugivory will become available with a wider application of our time-delay camera-trap technique.

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis.

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.

Cereal alkylresorcinols as dietary biomarkers-absorption and occurrence in biological membranes

Several studies link the consumption of whole-grain products to a lowered risk of chronic diseases, such as certain types of cancer, type II diabetes, and cardiovascular diseases. However, the final conclusions of the exact protective mechanisms remain unclear, partly due to a lack of a suitable biomarker for the whole-grain cereals intake. Alkylresorcinols (AR) are phenolic lipids abundant in the outer parts of wheat and rye grains usually with homologues of C15:0- C25:0 alkyl chains, and are suggested to function as whole-grain biomarkers. Mammalian lignan enterolactone has also previously been studied as a potential whole-grain biomarker. In the present work a quantified gas chromatography-mass spectrometry method for the analysis of AR in plasma, erythrocytes, and lipoproteins was developed. The method was used to determine human and pig plasma AR concentrations after the intake of whole-grain wheat and rye products compared to low-fibre wheat bread diets to assess the usability of AR as biomarkers of whole-grain intake. AR plasma concentrations were compared to serum ENL concentrations. AR absorption and elimination kinetics were investigated in a pig model. AR occurrence in human erythrocyte membranes and plasma lipoproteins were determined, and the distribution of AR in blood was evaluated. Plasma AR seem to be absorbed via the lymphatic system from the small intestine, like many other lipophilic compounds. Their apparent elimination half-life is relatively short and is similar to that of tocopherols, which have a similar chemical structure. Plasma AR concentrations increased significantly after a one- to eight-week intake of whole-grain wheat and further on with whole-grain rye bread. The concentrations were also higher after habitual Finnish diet compared to diet with low-fibre bread. Inter-individual variation after a one-week intake of the same amount of bread was high, but the mean plasma AR concentrations increased with increasing AR intake. AR are incorporated into erythrocyte membranes and plasma lipoproteins, and VLDL and HDL were the main AR carriers in human plasma. Based on these studies, plasma AR could function as specific biomarkers of dietary whole-grain products. AR are exclusively found in whole-grains and are more suitable as specific biomarkers of whole-grain intake than previously investigated mammalian lignan enterolactone, that is formed from several plants other than cereals in the diet. Plasma AR C17:0/C21:0 -ratio could distinguish whether whole-grain products in the diet are mainly wheat or rye. AR could be used in epidemiological studies to determine whole-grain intake and to better assess the role of whole-grains in disease prevention.

CNS injury, γ1 laminin, and its KDI peptide

Nisäkkäillä keskushermoston uudistuminen on rajallista. Keskushermostovamman jälkeen aktivoituu monien paranemista edistävien tekijöiden lisäksi myös estäviä tekijöitä. Monella molekyylillä, kuten laminiinilla, on keskushermoston paranemista tehostava vaikutus. Laminiinit ovat myös kehon tyvikalvojen oleellisia rakennuskomponentteja. Keskushermoston laminiinit ovat tärkeitä sikiökehityksen aikana, esimerkiksi hermosäikeiden ohjauksessa. Myöhemmin ne osallistuvat veriaivoesteen ylläpitoon sekä vammojen jälkeiseen kudosreaktioon. Väitöskirjatutkimuksessani olen selvittänyt lamiiniinien, erityisesti γ1 laminiinin ja sen KDI peptidin, ekspressiota keskushermoston vammatilanteissa. Kokeellisessa soluviljelmäasetelmassa, joka simuloi vammautunutta keskushermostoympäristöä, osoitimme että KDI peptidi voimistaa sekä hermosolujen selviytymistä että hermosäikeiden kasvua. Kainihappo on glutamaattianalogi, ja glutamaattitoksisuudella uskotaan olevan tärkeä merkitys keskushermoston eri vamma- ja sairaustilanteissa tapahtuvassa hermosolukuolemassa. Toisessa väitöskirjani osatyössä osoitimme eläinmallissa KDI peptidin suojaavan rotan aivojen hippokampuksen hermosoluja kainihapon aiheuttamalta solutuholta. Elektrofysiologisilla mittauksilla osoitimme kolmannessa osatyössäni, että KDI peptidi estää glutamaattireseptorivirtoja ja suojaa siten glutamaattitoksisuudelta. Aivoveritulpan aiheuttama aivovaurio on yleinen syy aivohalvaukseen. Viimeisessä osatyössäni tutkimme eläinmallissa laminiinien ekspressiota iskemian vaurioittamassa aivokudoksessa. Laminiiniekspression todettiin voimistuvan vaurion jälkeen sekä tyvikalvo- että soluväliainerakenteissa. Vaurion ympärillä havaittiin astrosyyttejä, jotka jo melko aikaisessa vaiheessa vamman jälkeen ekspressoivat γ1 laminiinia ja KDI peptidiä. Tästä voidaan päätellä laminiinien osallistuvan aivoiskeemisen vaurion patofysiologiaan. Yleisesti väitöskirjatyöni kartoitti laminiinien ekspressiota sekä terveessä että vammautuneessa keskushermostossa. Väitöskirjatyöni tukee hypoteesia, jonka mukaan KDI peptidi suojaa keskushermostoa vaurioilta.

The chemical ecology of seed dispersal in monoecious and dioecious figs

In the nursery pollination system of figs (Ficus, Moraceae), flower-bearing receptacles called syconia breed pollinating wasps and are units of both pollination and seed dispersal. Pollinators and mammalian seed dispersers are attracted to syconia by volatile organic compounds (VOCs). In monoecious figs, syconia produce both wasps and seeds, while in (gyno)dioecious figs, male (gall) fig trees produce wasps and female (seed) fig trees produce seeds. VOCs were collected using dynamic headspace adsorption methods on freshly collected figs from different trees using Super Q® collection traps. VOC profiles were determined using gas chromatography–mass spectrometry (GC–MS).The VOC profile of receptive and dispersal phase figs were clearly different only in the dioecious mammal-dispersed Ficus hispida but not in dioecious bird-dispersed F. exasperata and monoecious bird-dispersed F. tsjahela. The VOC profile of dispersal phase female figs was clearly different from that of male figs only in F. hispida but not in F. exasperata, as predicted from the phenology of syconium production which only in F. hispida overlaps between male and female trees. Greater difference in VOC profile in F. hispida might ensure preferential removal of seed figs by dispersal agents when gall figs are simultaneously available.The VOC profile of only mammal-dispersed female figs of F. hispida had high levels of fatty acid derivatives such as amyl-acetates and 2-heptanone, while monoterpenes, sesquiterpenes and shikimic acid derivatives were predominant in the other syconial types. A bird- and mammal-repellent compound methyl anthranilate occurred only in gall figs of both dioecious species, as expected, since gall figs containing wasp pollinators should not be consumed by dispersal agents.

Thiol cofactors for selenoenzymes and their synthetic mimics

The importance of selenium as an essential trace element is now well recognized. In proteins, the redox-active selenium moiety is incorporated as selenocysteine (Sec), the 21st amino acid. In mammals, selenium exerts its redox activities through several selenocysteine-containing enzymes, which include glutathione peroxidase (GPx), iodothyronine deiodinase (ID), and thioredoxin reductase (TrxR). Although these enzymes have Sec in their active sites, they catalyze completely different reactions and their substrate specificity and cofactor or co-substrate systems are significantly different. The antioxidant enzyme GPx uses the tripeptide glutathione (GSH) for the catalytic reduction of hydrogen peroxide and organic peroxides, whereas the larger and more advanced mammalian TrxRs have cysteine moieties in different subunits and prefer to utilize these internal cysteines as thiol cofactors for their catalytic activity. On the other hand, the nature of in vivo cofactor for the deiodinating enzyme ID is not known, although the use of thiols as reducing agents has been well-documented. Recent studies suggest that molecular recognition and effective binding of the thiol cofactors at the active site of the selenoenzymes and their mimics play crucial roles in the catalytic activity. The aim of this perspective is to present an overview of the thiol cofactor systems used by different selenoenzymes and their mimics.

Role of Magmas in protein transport and human mitochondria biogenesis

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of delta pam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

If dung beetles (Scarabaeidae: Scarabaeinae) arose in association with dinosaurs, did they also suffer a mass co-extinction at the K-Pg boundary?

The evolutionary success of beetles and numerous other terrestrial insects is generally attributed to co-radiation with flowering plants but most studies have focused on herbivorous or pollinating insects. Non-herbivores represent a significant proportion of beetle diversity yet potential factors that influence their diversification have been largely unexamined. In the present study, we examine the factors driving diversification within the Scarabaeidae, a speciose beetle family with a range of both herbivorous and non-herbivorous ecologies. In particular, it has been long debated whether the key event in the evolution of dung beetles (Scarabaeidae: Scarabaeinae) was an adaptation to feeding on dinosaur or mammalian dung. Here we present molecular evidence to show that the origin of dung beetles occurred in the middle of the Cretaceous, likely in association with dinosaur dung, but more surprisingly the timing is consistent with the rise of the angiosperms. We hypothesize that the switch in dinosaur diet to incorporate more nutritious and less fibrous angiosperm foliage provided a palatable dung source that ultimately created a new niche for diversification. Given the well-accepted mass extinction of non-avian dinosaurs at the Cretaceous-Paleogene boundary, we examine a potential co-extinction of dung beetles due to the loss of an important evolutionary resource, i.e., dinosaur dung. The biogeography of dung beetles is also examined to explore the previously proposed "out of Africa" hypothesis. Given the inferred age of Scarabaeinae as originating in the Lower Cretaceous, the major radiation of dung feeders prior to the Cenomanian, and the early divergence of both African and Gondwanan lineages, we hypothesise that that faunal exchange between Africa and Gondwanaland occurred during the earliest evolution of the Scarabaeinae. Therefore we propose that both Gondwanan vicariance and dispersal of African lineages is responsible for present day distribution of scarabaeine dung beetles and provide examples.