Search

Tonic current through hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels is influencing neuronal firing properties and channel function is strongly influenced by the brain-specific auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Since Kv1.2 channels and TRIP8b were also suggested to interact, we assessed brain Kv1.2 mRNA and protein expression as well as the reduction of K+ outward currents by Kv1.2-blocking compounds (Psora-4; tityustoxin-Kα, TsTX-Kα) in different brain areas of TRIP8b-deficient (TRIP8b−/−) compared to wildtype (WT) mice. We found that transcription levels of Kv1.2 channels were not different between genotypes. Furthermore, Kv1.2 current amplitude was not affected upon co-expression with TRIP8b in oocytes. However, Kv1.2 immunofluorescence was stronger in dendritic areas of cortical and hippocampal neurons. Furthermore, the peak net outward current was increased and the inactivation of the Psora-4-sensitive current component was less pronounced in cortical neurons in TRIP8b−/− mice. In current clamp recordings, application of TsTX increased the excitability of thalamocortical (TC) neurons with increased number of elicited action potentials upon step depolarization. We conclude that TRIP8b may not preferentially influence the amplitude of current through Kv1.2 channels but seems to affect current inactivation and channel localization. In TRIP8b−/− a compensatory upregulation of other Kv channels was observed. [ABSTRACT FROM AUTHOR]

Tauroursodeoxycholate (TUDC) is well known to protect against glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes. In the present study, we analyzed whether TUDC also exerts protective effects by modulating GCDC-induced gene expression changes. For this, gene array-based transcriptome analysis and quantitative polymerase chain reaction (qPCR) were performed on RNA isolated from rat livers perfused with GCDC, TUDC or a combination of both (each 20 μm for 2 h). GCDC led to a significant increase of lactate dehydrogenase (LDH) into the effluent perfusate, which was prevented by TUDC. GCDC, TUDC and co-perfusion induced distinct gene expression changes. While GCDC upregulated the expression of several pro-inflammatory genes, co-perfusion with TUDC increased the expression of pro-proliferative and anti-apoptotic p53 target genes. In line with this, levels of serine20-phosphorylated p53 and of its target gene p21 were elevated by GCDC in a TUDC-sensitive way. GCDC upregulated the oxidative stress surrogate marker 8OH(d)G and the pro-apoptotic microRNAs miR-15b/16 and these effects were prevented by TUDC. The upregulation of miR-15b and miR-16 in GCDC-perfused livers was accompanied by a downregulation of several potential miR-15b and miR-16 target genes. The present study identified changes in the transcriptome of the rat liver which suggest, that TUDC is hepatoprotective by counteracting GCDC-induced gene expression changes. [ABSTRACT FROM AUTHOR]

Tauroursodeoxycholate (TUDC) is well known to protect against glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes. In the present study, we analyzed whether TUDC also exerts protective effects by modulating GCDC-induced gene expression changes. For this, gene array-based transcriptome analysis and quantitative polymerase chain reaction (qPCR) were performed on RNA isolated from rat livers perfused with GCDC, TUDC or a combination of both (each 20 μm for 2 h). GCDC led to a significant increase of lactate dehydrogenase (LDH) into the effluent perfusate, which was prevented by TUDC. GCDC, TUDC and co-perfusion induced distinct gene expression changes. While GCDC upregulated the expression of several pro-inflammatory genes, co-perfusion with TUDC increased the expression of pro-proliferative and anti-apoptotic p53 target genes. In line with this, levels of serine20-phosphorylated p53 and of its target gene p21 were elevated by GCDC in a TUDC-sensitive way. GCDC upregulated the oxidative stress surrogate marker 8OH(d)G and the pro-apoptotic microRNAs miR-15b/16 and these effects were prevented by TUDC. The upregulation of miR-15b and miR-16 in GCDCperfused livers was accompanied by a downregulation of several potential miR-15b and miR-16 target genes. The present study identified changes in the transcriptome of the rat liver which suggest, that TUDC is hepatoprotective by counteracting GCDC-induced gene expression changes. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is an unsolved health problem which seriously affects human health around the world. miR-34a-5p acting as a tumor-suppressor is associated with left ventricular remodeling. We aimed to explore the functional roles of miR-34a-5p in cardiomyocytes. Hypoxia-induced cell injury in H9c2, HL-1 and human cardiac myocytes was analyzed according to the decrease of cell viability and increase of apoptosis. Expression of miR-34a-5p was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) when the concentration of O2 was decreased. Then, the effects of aberrantly expressed miR-34a-5p on proliferation and apoptosis of cardiomyocytes incubated under hypoxia were assessed. Finally, the downstream protein and signaling pathways of miR-34a-5p were explored. The hypoxic model was successfully constructed after incubation under hypoxia for 48 h. When the concentration of O2 decreased, the miR-34a-5p level was increased significantly. Then, we found miR-34a-5p aggravated hypoxia-induced alterations of proliferation and apoptosis in cardiomyocytes. Zinc finger E-box binding homeobox 1 (ZEB1) was identified as a target of miR-34a-5p, and miR-34a-5p conferred its function via targeting ZEB1. Finally, miR-34a-5p inhibition reversed hypoxia-induced decreases of phosphorylated kinases in the JAK/STAT and PI3K/AKT pathways through up-regulating ZEB1. Our study revealed that miR-34a-5p inhibition protected cardiomyocytes against hypoxia-induced cell injury through activating the JAK/STAT and PI3K/AKT pathways by targeting ZEB1. [ABSTRACT FROM AUTHOR]

Human dyskerin is an evolutively conserved protein that participates in diverse nuclear complexes: the H/ACA snoRNPs, that control ribosome biogenesis, RNA pseudouridylation, and stability of H/ACA snoRNAs; the scaRNPs, that control pseudouridylation of snRNAs; and the telomerase active holoenzyme, which safeguards telomere integrity. The biological importance of dyskerin is further outlined by the fact that its deficiency causes the X-linked dyskeratosis congenita disease, while its over-expression characterizes several types of cancers and has been proposed as prognostic marker. The role of dyskerin in telomere maintenance has widely been discussed, while its functions as H/ACA sno/scaRNP component has been so far mostly overlooked and represent the main goal of this review. Here we summarize how increasing evidence indicates that the snoRNA/microRNA pathways can be interlaced, and that dyskerin-dependent RNA pseudouridylation represents a flexible mechanism able to modulate RNA function in different ways, including modulation of splicing, change of mRNA coding properties, and selective regulation of IRESdependent translation. We also propose a speculative model that suggests that the dynamics of pre-assembly and nuclear import of H/ACA RNPs are crucial regulatory steps that can be finely controlled in the cytoplasm in response to developmental, differentiative and stress stimuli. [ABSTRACT FROM AUTHOR]

A role for the kinin B1 receptor in energy-homeostatic processes was implicated in previous studies; notably, the studies where kinin B1 receptor knockout mice (B1-/-) were shown to have impaired adiposity, impaired leptin and insulin production, lower feed efficiency, protection from liver steatosis and diet-induced obesity when fed a high fat diet (HFD). In particular, in a model where the B1 receptor is expressed exclusively in the adipose tissue, it rescues the plasma insulin concentration and the weight gain seen in wild type mice. Taking into consideration that leptin participates in the formation of hypothalamic nuclei, which modulate energy expenditure, and feeding behavior, we hypothesized that these brain regions could also be altered in B1-/- mice. We observed for the first time a difference in the gene expression pattern of cocaine and amphetamine related transcript (CART) in the (lateral hypothalamic area (LHA) resulting from the deletion of the kinin B1 receptor gene. The correlation between CART expression in the LHA and the thwarting of diet-induced obesity corroborates independent correlations between CART and obesity. Furthermore, it seems to indicate that the mechanism underlying the 'lean' phenotype of B1-/- mice does not stem solely from changes in peripheral tissues but may also receive contributions from changes in the hypothalamic machinery involved in energy homeostasis processes. [ABSTRACT FROM AUTHOR]

To look for oral proteasome inhibitors, daily injested food is the best source for cancer chemoprevention. A combination of active components from vegetables, coffee, tea, and fruit could be more efficient to inhibit 26S proteasome activities for preventing cancer diseases. Tannic acid and quercetin have been shown to strongly inhibit 26S proteasome activity, but the molecular target involved remains unknown. Overlay assay, peptide assay, Western blot, and 2-D gels were used to assess the combination of quercetin and tannic acid as a potential inhibitor. Here, we demonstrated that the combination of quercetin and tannic acid (1) synergistically suppresses chymotrypsin-, caspase-, and trypsin-like proteolytic activities, (2) are tightly binding substrates, (3) do not perturb the proteasome structure, (4) inhibit the 26S proteasome affected by ubiquitin, ATP, or β-casein, and (5) inhibit β-casein degradation by the 26S proteasome in vitro. Finally, the inhibition of the proteasome by a combination of quercetin plus tannic acid in Hep-2 cells resulted in the induction of S5a at low dose, accumulation of ubiquitin, and the cleavage of pro-caspase-3, followed by the induction of apoptotic cell death. Evaluating the combination of quercetin and tannic acid as an oral drug to prevent cancer may provide a pharmacological rationale to pursue preclinical trials of this combination. [ABSTRACT FROM AUTHOR]

Chloroplast biogenesis often requires a tight orchestration between gene expression (both plastidial and nuclear) and translocation of ∼3000 nuclear-encoded proteins into the organelle. Protein translocation is achieved via two multimeric import machineries at the outer (TOC) and inner (TIC) envelope of chloroplast, respectively. Three components constitute the core element of the TOC complex: a β-barrel protein translocation channel Toc75 and two receptor constituents, Toc159 and Toc34. A diverse set of distinct TOC complexes have recently been characterized and these diversified TOC complexes have evolved to coordinate the translocation of differentially expressed proteins. This review aims to describe the recent discoveries relating to the typical characteristics of these distinct TOC complexes, particularly the receptor constituents, which are the main contributors for TOC complex diversification. [ABSTRACT FROM AUTHOR]

SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH. [ABSTRACT FROM AUTHOR]

The cAMP response element (enhCRE) in the distal enhancer regulatory region of renin gene is believed to play a major role in the control of renin transcription. enhCRE binds the CRE-binding protein (CREB), which is the main transcription factor target of cAMP signaling. Using the mouse renin-producing cell line As4.1 we found that activating transcription factor-2 (ATF2) also binds to enhCRE. N-terminal phosphorylation of ATF2, which controls its transactivation, is associated with downregulation of renin gene expression by the cytokine tumor necrosis factor-α (TNFα). The ubiquitin proteasome inhibitor MG132 also phosphorylates ATF2 and inhibits renin expression. Knockdown of ATF2 attenuated the suppression of renin gene expression by MG132, thus demonstrating that ATF2 mediates the inhibitory effect of MG132. In addition, MG132 increased the DNA-binding of ATF2 as well as the ratio of bound ATF2 to CREB. Using ATF2- and CREB-Gal4 fusion protein constructs coupled with luciferase reporter system we showed that ATF2 has a weaker transactivating capacity than CREB. These data suggest that ATF2 represses renin expression by drifting the transcriptional control of renin gene away from CREB. Accordingly, TNFα completely abrogated the cAMP-dependent stimulation of renin gene expression. [ABSTRACT FROM AUTHOR]

Trypanothione synthetase, a validated drug target, synthesizes trypanothione from glutathione and spermidine. Here we report the gene cloning, expression, characterization and inhibition studies of trypanothione synthetase from Leishmania donovani (LdTryS). The purified recombinant LdTryS enzyme obeyed Michaelis-Menten kinetics. High substrate inhibition was observed with glutathione ( Km=33.24 μ m, kcat=1.3 s-1, Ki=866 μ m). The enzyme shows simple hyperbolic kinetics with fixed glutathione concentration and with other substrates limiting Km values for Mg. ATP and spermidine of 14.2 μ m and 139.6 μ m, respectively. LdTryS was also screened for inhibitors. Tomatine, conessine, uvaol and betulin were identified as inhibitors of the enzyme and were tested for leishmanicidal activity. Finally, the effect of LdTryS inhibitors on redox homeostasis of the parasite gives a broader picture of their action against leishmaniasis. [ABSTRACT FROM AUTHOR]

The expression of cathepsin L, a lysosomal protease, is known to be elevated in cancer and other pathologies. Multiple splice variants of human cathepsin L with variable 5′UTRs exist, which encode for the same protein. Previously we have observed that variant hCATL A (bearing the longest 5′UTR) was translated in vitro with significantly lower efficiency than variant hCATL AIII (bearing the shortest 5′UTR). Contrary to these findings, results of the present study reveal that in cancer cells, hCATL A mRNA exhibits higher translatability in spite of having lower stability than AIII. This is the first report demonstrating a highly contrasting trend in translation efficiencies of hCATL variants in rabbit reticulocytes and live cells. Expression from chimeric mRNAs containing 5′UTRs of A or AIII upstream to luciferase reporter cDNA established the A UTR to be the sole determinant for this effect. Transient transfections of bicistronic plasmids and mRNAs confirmed the presence of a functional Internal Ribosome Entry Site in this UTR. Our data suggest that differential stability and translation initiation modes mediated by the 5′UTRs of human cathepsin L variants are involved in regulating its expression. [ABSTRACT FROM AUTHOR]

Dipeptidyl peptidase IV (DPPIV)/CD26 is by far the most extensively studied member of the prolyl oligopeptidase family of serine proteases. The discovery of the related enzymes DPP8 and DPP9 necessitates a (re-)evaluation of the DPPIV-like enzymatic activity in cells and organs. In this study, we aimed (1) to investigate the expression of the individual dipeptidyl peptidases in different types of endothelial cells (ECs) and (2) to reconsider published data in relation to our findings. Examination of DPP expression in rat primary ECs of aortic, endocardial and cardiac microvascular origin revealed the presence of DPPIV-like activity in all cell lysates. More than half of this activity could be attributed to DPP8/9. Western blot analysis revealed an abundance of the DPP8 protein as compared to DPP9. The expression of DPPIV and DPP8 was significantly higher in the cardiac microvascular endothelium than in the other ECs, suggesting a more pronounced role of these DPPs in the microvasculature. In situ, DPP activity in ventricular microvasculature was completely inhibited by sitagliptin, indicating that DPPIV is the predominant DPPIV-like enzyme in this organ. By contrast, immunohistochemical studies indicated DPP9 as the predominant DPP in human carotid artery ECs. In conclusion, our results support a highly regulated expression of individual DPPs in ECs, with a spatial heterogeneity in the cardiovascular tree. [ABSTRACT FROM AUTHOR]

Two families of proteins, the bestrophins (Best) and the recently cloned TMEM16 proteins (anoctamin, Ano), recapitulate properties of Ca2+-activated Cl- currents. Best1 is strongly expressed in the retinal pigment epithelium and could have a function as a Ca2+-activated Cl- channel as well as a regulator of Ca2+ signaling. It is also present at much lower levels in other cell types including epithelial cells, where it regulates plasma membrane localized Cl- channels by controlling intracellular Ca2+ levels. Best1 interacts with important Ca2+-signaling proteins such as STIM1 and can interact directly with other Ca2+-activated Cl- channels such as TMEM16A. Best1 is detected in the endoplasmic reticulum (ER) where it shapes the dynamic ER structure and regulates cell proliferation, which could be important for renal cystogenesis. Ca2+-activated Cl- channels of the anoctamin family (TMEM16A) show biophysical and pharmacological properties that are typical for endogenous Ca2+-dependent Cl- channels. TMEM16 proteins are abundantly expressed and many reports demonstrate their physiological importance in epithelial as well as non-epithelial cells. These channels are also activated by cell swelling and can therefore control cell volume, proliferation and apoptosis. To fully understand the function and regulation of Ca2+-activated Cl- currents, it is necessary to appreciate that Best1 and TMEM16A are embedded in a protein network and that they probably operate in functional microdomains. [ABSTRACT FROM AUTHOR]

In the past year and a half, five studies have independently established a direct connection between the hypoxic response transcription factor, HIF-1, and aging in Caenorhabditis elegans. These studies demonstrated that HIF-1 can both promote and limit longevity via pathways that are mechanistically distinct. Here, we review the current state of knowledge regarding modulation of aging by HIF-1 and speculate on potential aspects of HIF-1 function that could be relevant for mammalian longevity and healthspan. [ABSTRACT FROM AUTHOR]

Recently, epithelial to mesenchymal transition (EMT) has been shown to represent a feature of dedifferentiating hepatocytes in vitro. Three-dimensional soft collagen gels can antagonize but not completely abolish this effect. Hormonal additives to culture media are known to maintain differentiated hepatocyte functions. Therefore, we studied whether insulin and dexamethasone antagonize EMT in cultured hepatocytes. Both hormones antagonized but not completely abolished certain morphological features of EMT. Dexamethasone antagonized acquisition of fibroblastoid shape, whereas insulin favored bile canaliculi formation. In a subsequent step, we analyzed expression of a battery of EMT-related genes. Of all markers tested, vimentin and snail-1 correlated best with morphological features of EMT. Interestingly, dexamethasone reduced expression levels of both vimentin and snail-1, whereas the influence of insulin was less pronounced. An important result of this study is that 12 out of 17 analyzed EMT markers were transcriptionally influenced by dexamethasone (vimentin, snail-1, snail-2, HNF4α, Twist-1, ZEB2, fibronectin, occludin, MMP14, claudin-1, cytokeratin-8, and cytokeratin-18), whereas the remaining factors seemed to be less dependent on dexamethasone. In conclusion, EMT markers in hepatocytes can be classified as dexamethasone-dependent versus -independent. [ABSTRACT FROM AUTHOR]

In genome-wide screens we studied CA/C1 peptidases of malaria-causing plasmodia and their hosts (man and mouse). For Plasmodium falciparum and P. berghei, several new CA/C1 peptidase genes encoding proteases of the L- and B-family with specific promoter modules were identified. In addition, two new human CA/C1 peptidase loci and one new mouse gene locus were found; otherwise, the sets of CA/C1 peptidase genes in man and mouse seem to be complete now. In each species studied there is a multitude of CA/C1 peptidases with lysosomal localization signals and partial functional overlap according to similar but subfamily-specific structures. Individual target structures in plasmodia include residues specifically different in CA/C1 peptidase subsite 2. This is of medical interest considering CA/C1 peptidase inhibition for chemotherapy in malaria, malignancies and other diseases. Promoter structures and mRNA regulation differ widely among CA/C1 peptidase subfamilies and between mammals and plasmodia. We characterized promoter modules conserved in mouse and man for the CA/C1 peptidase families B and L (with the L-like subfamily, F-like subfamily and mouse-specific J-like subfamily). RNA motif searches revealed conserved regulatory elements such as GAIT elements; plasmodial CA/C1 peptidase mRNA elements include ARE elements and mammalian mRNAs contain 15-lox DICE elements. [ABSTRACT FROM AUTHOR]

Fanconi anemia is a fatal, hereditary chromosome instability syndrome of early childhood with progressive pancytopenia and cancer-proneness. Hypersensitivity to alkylating agents points to DNA repair inefficiency. Excess reactive oxygen intermediates and hypersensitivity to oxygen, all features of Fanconi anemia cells, give evidence for a disturbed oxidative metabolism. Here, we report that expression of the inducible heme oxygenase-1, an essential antioxidative defense protein, is impaired in Fanconi anemia cells and can be reinstated with the transfection of Fanconi A wild-type cDNA. A causative interaction of Fanconi anemia proteins with transcription of selected proteins is indicated. The results enlighten the oxygen sensitivity in Fanconi anemia. [ABSTRACT FROM AUTHOR]

The klotho gene functions as an aging-suppressor gene that extends life span when overexpressed and accelerates aging-like phenotypes when disrupted in mice. The klotho gene encodes a single-pass transmembrane protein that binds to multiple fibroblast growth factor (FGF) receptors and functions as a co-receptor for FGF23, a bone-derived hormone that suppresses phosphate reabsorption and vitamin D biosynthesis in the kidney. In addition, the extracellular domain of Klotho protein is shed and secreted, potentially functioning as a humoral factor. The secreted Klotho protein can regulate multiple growth factor signaling pathways, including insulin/IGF-1 and Wnt, and the activity of multiple ion channels. Klotho protein also protects cells and tissues from oxidative stress, yet the precise mechanism underlying these activities remains to be determined. Thus, understanding of Klotho protein function is expected to provide new insights into the molecular basis for aging, phosphate/vitamin D metabolism, cancer and stem cell biology. [ABSTRACT FROM AUTHOR]

Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns that encode reverse transcriptase-like genes, and group I introns and archaeal introns that encode homing endonuclease genes (HEGs). Although rDNA-embedded protein genes are widespread in nuclei, organelles and bacteria, there is surprisingly little information available on how these genes are expressed. Exceptions include a handful of HEGs from group I introns. Recent studies have revealed unusual and essential roles of group I and group I-like ribozymes in the endogenous expression of HEGs. Here we discuss general aspects of rDNA-embedded protein genes and focus on HEG expression from group I introns in the nucleolus. [ABSTRACT FROM AUTHOR]

The ligand-activated aryl hydrocarbon receptor (AHR) is known to modulate many genes in a highly cell-specific manner, either directly or indirectly via secondary effects. In contrast, little is known about the effects of AHR deficiency on gene expression balance. We compared the transcriptome of CD4 T cells from AHR-/- mice and wild-type mice; 390 genes, many of them immunotypic, were deregulated in AHR-deficient CD4 cells. TCDD-induced transcriptome changes correlated with the AHR expression level in immune cells. However, there was little overlap in AHR-dependent transcripts found in T lineage cells or dendritic cells. Our results demonstrate flexible gene accessibility for the AHR in immune cells. The idea of a universal battery of AHR-responsive genes is not tenable. [ABSTRACT FROM AUTHOR]

Five new genes belonging to the pathogenesis-related (PR) 4 family have been cloned and characterised in Triticum aestivum. Two full-length genes, named wPR4e and wPR4f-b, were isolated by library screening, demonstrating the presence of a small intron only in wPR4f-b. Two other PR4 genes ( wPR4f-a and wPR4f-c) were isolated by PCR, showing very high sequence identity with wPR4f-b and constituting a new sub-family. Transcription start analysis was performed by RLM-RACE, leading to the isolation of a fifth gene, named wPR4g, that is highly homologous to wPR4e; both encode putative vacuolar PR4 proteins (Wheatwin7 and Wheatwin5, respectively). wPR4e and wPR4f sub-family genes are induced by F. culmorum infection, by chemicals that lead to systemic acquired resistance and by wounding, showing different spatial and temporal induction pathways. In silico analysis of the 5′ untranslated regions of wPR4e and wPR4f-b revealed the presence of several abiotic and biotic stress-responsive elements. wPR4e and wPR4f-b putative promoters were fused to the β-glucuronidase ( GUS) reporter gene, and transient and stable expression assays demonstrated that both are able to drive expression of GUS. Characterisation of these new PR4 genes and particularly of their 5′ untranslated regions, as well as the determination of their expression patterns, will contribute to our understanding of the responsiveness of this gene family to various stress conditions and of its role in plant defence. [ABSTRACT FROM AUTHOR]

Our in vitro studies support a functional link between the induction of cathepsin B gene expression and the catabolic restructuring associated with myotube formation during myogenesis in vivo. We have tested two predictions that are basic to this hypothesis: (1) that active cathepsin B is localized to plasma membrane caveolae of fusing myoblasts; and (2) that active cathepsin B is secreted from fusing myoblasts at physiological pH. During differentiation, L6 rat myoblasts demonstrated a fusion-related increase in activity associated with the 25/26-kDa, fully processed, active form of cathepsin B. Immunocytochemical studies demonstrated a redistribution of lysosomal cathepsin B protein toward the membrane of fusing myoblasts, and a colocalization of cathepsin B with caveolin-3, the muscle-specific structural protein of membrane caveolae. Sucrose density fractionation and Western blot analysis demonstrated that an active form of cathepsin B localizes to caveolar fractions along with caveolin-3, annexin-VII, β-dystroglycan and dystrophin. Finally, ‘real-time’ activity assays and Western blot analysis demonstrated that active cathepsin B is secreted from fusing myoblasts at physiological pH. Collectively, these studies support an association of active cathepsin B with plasma membrane caveolae and the secretion of active cathepsin B from differentiating myoblasts during myoblast fusion. [ABSTRACT FROM AUTHOR]

The intrinsic apoptosis apparatus plays a significant role in generating and amplifying cell death signals. In this study we examined whether there are differences in the expression of its components and in its functioning in non-small cell lung carcinoma (NSCLC) and the lung. We show that NSCLC cell lines express Apaf-1 and pro-caspase-9 and -3 proteins and that the expression of Apaf-1 and procaspase-3, but not of procaspase-9 and -7, is frequently up-regulated in NSCLC tissues as compared to the lung. NSCLC tissues and lungs and some NSCLC cell lines expressed also caspase-9S(b) and displayed a high caspase-9S(b)/procaspase-9 expression ratio. Procaspase-3 from NSCLCs and lungs was readily processed to caspase-3 by granzyme B or caspase-8, and the granzyme B-generated caspase-3-like activity was significantly higher in tumor tissues and cells than in lungs. By contrast, cytochrome c plus dATP could induce a significant increase of caspase-3-like activity in cytosol only in some NSCLC cell lines and in subsets of studied NSCLC tissues and lungs, while procaspase-3 and -7 were detectably processed only in NSCLC tissues which showed a high (cytochrome c q dATP)-induced caspase-3-like activity. Taken together, the present study provides evidence that the expression of Apaf-1 and procaspase-3 is up-regulated in NSCLCs and indicates that the tumors have a capability to suppress the apoptosome-driven caspase activation in their cytosol. [ABSTRACT FROM AUTHOR]

Calcineurin (CN), a Ca&sup2+; /calmodulin-dependent protein phosphatase, plays a critical role in T-cell activation by regulating the activity of NF-AT. CN is a heterodimer consisting of a catalytic subunit (CNA) and a Ca&sup2+; -binding regulatory subunit (CNB). CNB is composed of two global domains: the C-terminal domain (DC) and the N-terminal domain (DN), each containing two Ca&sup2+; binding sites. In this study, using purified DN and DC derived from constructed expression systems, we revealed that intact CNB and DC can stimulate the phosphatase activity of CNA, about 2.2 and 1.6 times the phosphatase activity of CNA alone, respectively, DN itself has little effect on the phosphatase activity of CNA. Fluorescence spectroscopy of an ANS-hydrophobic fluorescence probe shows that binding of Ca&sup2+; to CNB, DC or DN leads to expo-sure of the hydrophobic surface of the proteins and that the hydrophobicity of CNB is the greatest, that of DC is less, and that of DN is the least. The hydrophobic surface of CNB may be an important structural basis for stimulating CN phosphatase activity. [ABSTRACT FROM AUTHOR]

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) is present in at least three different isoforms in testis: as a cytosolic, as a mitochondrial, and as a nuclear protein. We have recently shown that a sperm nucleus-specific glutathione peroxidase (snGPx) is identical to the mitochondrial and cytosolic forms of PHGPx apart from its N-terminus. This arginine-rich N-terminus of snGPx, reminiscent of protamines, is encoded by an alternative exon located in the first intron of the PHGPx gene and is responsible for nuclear localisation and chromatin binding of snGPx [Pfeifer et al., FASEB J. 15 (2001), pp. 1236-1238]. By using a combination of techniques including selective cloning of mRNA 5'-ends, RT-PCR, and S1 analyses, we provide evidence that the transcript encoding the nuclear form is generated by transcription initiation at an alternative promoter and not by alternative splicing. We show that the major transcription start region is located at -12 to -14 upstream of the AUG translation initiation site of the sperm nucleus-specific exon and lacks a TATA box. Two minor TATA-less transcription initiation sites are located at around -30 and -45. We have shown by in situ hybridisation that snGPx expression in testis, like protamine expression, is restricted to late stages of spermatogenesis whereas PHGPx expression is only found in spermatocytes and early spermatids. These findings have to be taken into account when studying either the differential regulation of PHGPx and snGPx expression in testis or the impact of putative mutations in snGPx on male fertility in man. [ABSTRACT FROM AUTHOR]

A multivariate analysis of the National Cancer Institute gene expression database is reported here. The soft independent modelling of a class analogy approach achieved cell line classification according to histological origin. With the PCA method, based on the expression of 9605 genes and ESTs, classification of colon, leukaemia, renal, melanoma and CNS cells could be performed, but not of lung, breast and ovarian cells. Another multivariate procedure, called partial least squares discriminant analysis (PLS-DA), provides bioinformatic clues for the selection of a limited number of gene transcripts most effective in discriminating different tumoral histotypes. Among them it is possible to identify candidates in the development of new diagnostic tests for cancer detection and unknown genes deserving high priority in further studies. In particular, melan-A, acid phosphatase 5, dopachrome tautomerase, S100-β and acid ceramidase were found to be among the most important genes for melanoma. The potential of the present bioinformatic approach is exemplified by its ability to identify differentiation and diagnostic markers already in use in clinical settings, such as protein S-100, a prognostic parameter in patients with metastatic melanoma and a screening marker for melanoma metastasis. [ABSTRACT FROM AUTHOR]