Band Of Molecular Mass Research Articles

Tumor Necrosis Factor-α Induces Caspase-mediated Cleavage of Peroxisome Proliferator-activated Receptor γ in Adipocytes

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that acts as a primary regulator of adipogenesis and controls adipocyte metabolism and insulin action. Increased expression of tumor necrosis factor (TNFalpha) in adipose tissue of obese subjects potently suppresses the expression of PPARgamma and attenuates adipocyte functions. Here we show that PPARgamma is a substrate of caspase-3 and caspase-6 during TNFalpha receptor signaling in adipocytes, and the consequent PPARgamma cleavage disrupts its nuclear localization. TNFalpha treatment of 3T3-L1 adipocytes decreases full-length PPARgamma while increasing the level of a 45-kDa immunoreactive PPARgamma fragment. Specific inhibitors of caspase-3 and caspase-6 attenuate the cleavage of PPARgamma protein in response to TNFalpha in cultured adipocytes. Incubation of nuclear fractions with recombinant caspase-3 and caspase-6 also generates a 45-kDa PPARgamma cleavage product. Dispersion of nuclear PPARgamma to the cytoplasm in response to TNFalpha treatment occurs in parallel with detection of activated caspase-3. We suggest that activation of the caspase cascade by TNFalpha down-regulates PPARgamma protein and PPARgamma-mediated metabolic processes in adipose cells.

Ion Channel Subunit Expression Changes in Cardiac Purkinje Fibers

Purkinje fibers (PFs) play key roles in cardiac conduction and arrhythmogenesis. Congestive heart failure (CHF) causes well-characterized atrial and ventricular ion channel subunit expression changes, but effects on PF ion channel subunits are unknown. This study assessed changes in PF ion channel subunit expression (real-time PCR, immunoblot, immunohistochemistry), action potential properties, and conduction in dogs with ventricular tachypacing-induced CHF. CHF downregulated mRNA expression of subunits involved in action potential propagation (Nav1.5, by 56%; connexin [Cx]40, 66%; Cx43, 56%) and repolarization (Kv4.3, 43%, Kv3.4, 46%). No significant changes occurred in KChIP2, KvLQT1, ERG, or Kir3.1/3.4 mRNA. At the protein level, downregulation was seen for Nav1.5 (by 38%), Kv4.3 (42%), Kv3.4 (57%), Kir2.1 (26%), Cx40 (53%), and Cx43 (30%). Cx43 dephosphorylation was indicated by decreased larger molecular mass bands (pan-Cx43 antibody) and a 57% decrease in Ser368-phosphorylated Cx43 (phospho-specific antibody). Immunohistochemistry revealed reduced Cx40, Cx43, and phospho-Cx43 expression at intercalated disks. Action potential changes were consistent with observed decreases in ion channel subunits: CHF decreased phase 1 slope (by 56%), overshoot (by 32%), and phase 0 dV/dt(max) (by 35%). Impulse propagation was slowed in PF false tendons: conduction velocity decreased significantly from 2.2+/-0.1 m/s (control) to 1.5+/-0.1 m/s (CHF). His-Purkinje conduction also slowed in vivo, with HV interval increasing from 35.5+/-1.2 (control) to 49.3+/-3.4 ms (CHF). These results indicate important effects of CHF on PF ion channel subunit expression. Alterations in subunits governing conduction properties may be particularly important, because CHF-induced impairments in Purkinje tissue conduction, which this study is the first to describe, could contribute significantly to dyssynchronous ventricular activation, a major determinant of prognosis in CHF-patients.

Changes of soluble proteins in leaf and thylakoid exposed in high saline condition of a mangrove taxa Bruguiera gymnorrhiza

One-year-old seedlings of Bruguiera gymnorrhiza (L) Savingay were exposed to 500 mM NaCl for 6d under hydroponic culture condition to characterize the changes in leaf and thylakoid protein profiles in response to short-term salt exposures. Significant changes in leaf dry mass, chlorophylls and soluble leaf proteins were observed in short term of salt exposures, as it happens under tidal situations in nature. Chlorophyll a/b ratio showed decrease of light harvesting efficiency in salt treatment. Total soluble proteins in leaves were extracted from control and NaCl-treated plants at 2d intervals and were analyzed by SDS-PAGE. Intensity of several protein bands of different molecular mass of leaf protein profile ranging from 10 to 86 kDa (10, 16, 23, 33, 37, 42, 44, 50 and 86 kDa) were decreased due to high salt treatment. Out of these, 16, 23 and 33 kDa protein bands decreased dramatically from 1-3 fold but recovered in 7d growth, except the 33 kDa band. SDSPAGE profile of thylakoid protein revealed that both number and the intensity of several protein bands got altered by salt concentration. However, 33 kDa protein band of thylakoid reappeared in recovery that might not be of the same characteristics with same molecular mass as shown in total leaf protein profile. The numbers of major bands found in SDS-PAGE were reduced when analyzed in urea-SDS-PAGE to minimize protein aggregations by high salt. It was noted that 47 kDa disappeared while some proteins of apparent molecular mass like 23 kDa, 33 kDa, 37 kDa and 50 kDa degraded to minor bands. Partial restoration of protein bands occurred when the salt-treated plants were brought back to initial growth condition. These results clearly demonstrate that short term high salt concentration could cause major alterations to photosynthetic apparatus of a true non salt-secreting tree mangrove Bruguiera gymnorrhiza and adapted against fluctuation of salinity by altering leaf protein pool relatively more than the thylakoid proteins.

Sequence Differences in the IQ Motifs of CaV1.1 and CaV1.2 Strongly Impact Calmodulin Binding and Calcium-dependent Inactivation

The proximal C terminus of the cardiac L-type calcium channel (Ca(V)1.2) contains structural elements important for the binding of calmodulin (CaM) and calcium-dependent inactivation, and exhibits extensive sequence conservation with the corresponding region of the skeletal L-type channel (Ca(V)1.1). However, there are several Ca(V)1.1 residues that are both identical in six species and are non-conservatively changed from the corresponding Ca(V)1.2 residues, including three of the "IQ motif." To investigate the functional significance of these residue differences, we used native gel electrophoresis and expression in intact myotubes to compare the binding of CaM to extended regions (up to 300 residues) of the C termini of Ca(V)1.1 and Ca(V)1.2. We found that in the presence of Ca(2+) (either millimolar or that in resting myotubes), CaM bound strongly to C termini of Ca(V)1.2 but not of Ca(V)1.1. Furthermore, replacement of two residues (Tyr(1657) and Lys(1662)) within the IQ motif of a C-terminal Ca(V)1.2 construct with the divergent residues of Ca(V)1.1 (His(1532) and Met(1537)) led to a weakening of CaM binding (native gels), whereas the reciprocal substitution in Ca(V)1.1 caused a gain of CaM binding. In full-length Ca(V)1.2, substitution of these same two divergent residues with those of Ca(V)1.1 (Y1657H, K1662M) eliminated calcium-dependent inactivation of the heterologously expressed channel. Thus, our results reveal that a conserved difference between the IQ motifs of Ca(V)1.2 and Ca(V)1.1 has a profound effect on both CaM binding and calcium-dependent inactivation.

Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.

Demonstration and partial characterization of ecto-ATPase inBalamuthia mandrillarisand its possible role in the host-cell interactions

To investigate the presence and partial characterization of ecto-ATPase in Balamuthia mandrillaris. In vitro assays were used to demonstrate that live B. mandrillaris hydrolyses extracellular AtP. Using nondenaturing polyacrylamide gel electrophoresis, B. mandrillaris exhibited a single ecto-ATPase band of molecular mass of more than 545 kDa. This ecto-ATPase was insensitive to ouabain, levamisole, sodium azide and sodium orthovanadate but stimulated by MgCl2. The ecto-ATPase was heat stable, but labile to detergent, sodium dodecyl sulphate. Suramin, an antagonist of P2 purinoreceptors and an inhibitor of some ecto-ATPases, inhibited B. mandrillaris binding to and cytotoxicity of HBMEC (human brain microvascular endothelial cello), in vitro. For the first time, we describe that live B. mandrillaris hydrolyses extracellular ATP and exhibits a > 545kDa ecto-ATPase. This surface enzyme may play a role in the salvage of purines from the extracellular medium and may be important for the pathogenesis of B. mandrillaris.

Effect of lipid-bound apoA-I cysteine mutants on lipopolysaccharide-induced endotoxemia in mice

HDL has been shown to be able to neutralize the toxicity of lipopolysaccharide (LPS). Our previous study (J. Lipid Res. 2005. 46: 1303-1311) characterized the properties of secondary structure and in vitro functions of different cysteine mutants of apolipoprotein A-I. Here, we reconstituted recombinant HDLs (named rHDLwt, rHDL52, rHDL74, rHDL107, rHDL129, rHDL173, rHDL195, and rHDL228) by mixing wild type or those mutants with dipalmitoyl phosphatidylcholine and examined their in vivo effects on LPS-induced endotoxemia in mice. Our results showed that 24 h after injection, mice receiving rHDL74 or rHDL52 had a significant decrease of plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), compared with control mice receiving either saline or rHDLwt (P < 0.05). Administration of rHDL74 to mice injected with LPS also led to a decrease of plasma IL-6, protection of lung against acute injury, and attenuation of endotoxin-induced clinical symptoms in mice, compared with controls injected with LPS only. However, injection of rHDL228 significantly increased plasma concentration of TNF-alpha and exacerbated LPS-induced lung injury. In summary, compared with rHDLwt, rHDL74 and rHDL52 exhibit higher anti-inflammation capabilities, whereas rHDL228 shows hyper-proinflammation by exacerbating LPS-induced endotoxemia in mice.

DKK1 Antagonizes Wnt Signaling without Promotion of LRP6 Internalization and Degradation

DKK1 is a secreted protein that antagonizes Wnt signaling and plays essential roles in vertebrate embryogenesis including head induction, skeletal development, and limb patterning. DKK1 is also implicated in osteoporosis, arthritis, and cancer and represents a potential therapeutic target for the treatment of these diseases. DKK1 is a high affinity antagonistic ligand for LRP6, which is a Wnt coreceptor that acts together with the Frizzled serpentine receptor to initiate Wnt signal transduction. Two different models have been proposed to account for the mechanism by which DKK1 antagonizes LRP6 function. One model suggests that DKK1 binding to LRP6 disrupts Wnt-induced Frizzled-LRP6 complex formation, whereas the other model proposes that DKK1 interaction with LRP6 promotes LRP6 internalization and degradation, thereby reducing the cell surface LRP6 level. To clarify the molecular basis of DKK1 action, we examined how DKK1 affects the endogenous LRP6 in several mammalian cell lines including mouse embryonic fibroblasts. Here we show that DKK1 inhibits Wnt signaling but induces neither LRP6 down-regulation from the cell surface nor reduction of total LRP6 protein level and that DKK1 has no effect on the rate of continuous internalization of LRP6 and the half-life (about 4.7 h) of LRP6. We conclude that DKK1 inhibition of LRP6 is independent of LRP6 internalization and degradation.

Novel extracellular proteolytic activity inPediococcus acidilacticiATCC 8042

Proteolytic systems are common in lactic acid bacteria, but there are few reports about proteases or peptidases in the genus Pediococcus. To evaluate the presence of these types of enzymes, Pediococcus acidilactici ATCC 8042 was cultured in MRS broth. Supernatants collected during the log phase showed proteolytic activity towards an elastin dispersion when assayed using a spectrophotometer. Zn2+ showed a stimulatory effect, and the proteolytic activity reached its maximum when 200 mmol/L NaCl was included in the reaction buffer. On the other hand, activity was reduced when 5 mmol/L EDTA, 10 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L 1,10-phenanthroline were used or when the sample was heat treated. Zymograms showed two different proteolytic bands when gelatin was used as a substrate (>200 and 107 kDa), but only the higher molecular mass band was detected when casein or elastin was used. The gelatinolytic activity was not detected with zymograms of the 107 kDa band, which was the one inactivated by heat treatment. The use of a renaturing SDS-PAGE gel with embedded Micrococcus lysodeikticus cells allowed for the detection of a band with peptidoglycan hydrolase activity migrating at about 110 kDa. This activity was lost when 10 mmol/L EDTA was added to the renaturing buffer. Therefore, Pediococcus showed at least three different extracellular enzymes that were produced during the logarithmic growth phase and acted on peptide substrates. Each showed different substrate specificity, ion requirements, and thermostability.

Localized Regulation of Axonal RanGTPase Controls Retrograde Injury Signaling in Peripheral Nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

Cysteine-674 oxidation and degradation of sarcoplasmic reticulum Ca 2+ ATPase in diabetic pig aorta

The sarcoplasmic reticulum Ca 2+ ATPase (SERCA) is redox-regulated by posttranslational thiol modifications of cysteine-674 to regulate smooth muscle relaxation and migration. To detect oxidation of cysteine-674 that irreversibly prevents redox regulation, a polyclonal, sequence-specific antibody was developed toward a peptide containing cysteine-674 sulfonic acid. The antibody stained intact 110-kDa SERCA in pig cardiac SR that was oxidized in vitro by peroxynitrite in a sequence-specific manner, and histochemically stained atherosclerotic pig and rabbit aorta. Surprisingly, immunoblots of the pig aorta failed to stain intact 110-kDa SERCA protein, but rather, higher molecular mass aggregates and lower molecular mass bands. Of the latter bands at 70 and 60 kDa, the largest were observed in diabetic, hyperlipidemic pigs, and coincided with the most positive histochemical staining. The 70- and 60-kDa molecular mass bands also coincided with the majority of the protein detected by a monoclonal total anti-SERCA antibody, which detected the intact 110-kDa protein in normal pigs. Mass spectrometry identified SERCA in all the major bands detected by the sulfonic acid antibody as well as the oxidation of cysteine-674 in the 70-kDa band. These studies demonstrate a sequence-specific antibody that detects partial degradation products of SERCA, which represent the majority of the protein in some diabetic hypercholesterolemic pig aortae. In addition, the results suggest an association between irreversible oxidation of SERCA and its degradation, and that an important portion of the oxidized protein in tissue samples may be partially degraded.

The iron–sulphur protein Ind1 is required for effective complex I assembly

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.

Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish

The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4β-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4α-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.

The Folding of Human Active and Inactive Extracellular Superoxide Dismutases Is an Intracellular Event

Human extracellular superoxide dismutase (EC-SOD) is a tetrameric glycoprotein responsible for the removal of superoxide generated in the extracellular space. Two different folding variants of EC-SOD exist based on the disulfide bridge connectivity, resulting in enzymatically active (aEC-SOD) and inactive (iEC-SOD) subunits. As a consequence of this, the assembly of the EC-SOD tetramers produces molecules with variable activity and may represent a way to regulate the antioxidant level in the extracellular space. To determine whether the formation of these two folding variants is an intra- or extracellular event, we analyzed the biosynthesis in human embryonic kidney 293 cells expressing wild-type EC-SOD. These analyses revealed that both folding variants were present in the intra- and extracellular spaces, suggesting that the formation is an intracellular event. To further analyze the biosynthesis, we constructed mutants with the capacity to generate only aEC-SOD (C195S) or iEC-SOD (C45S). The expression of these suggested that the cellular biosynthetic machinery supported the secretion of aEC-SOD but not iEC-SOD. The coexpression of these two mutants did not affect the expression pattern. This study shows that generation of the EC-SOD folding variants is an intracellular event that depends on a free cysteine residue not involved in disulfide bonding.

Repetitive Firing Triggers Clustering of Kv2.1 Potassium Channels in Aplysia Neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.

Differential subcellular distribution and colocalization of microsomal and soluble epoxide hydrolases in rat cortical astrocytes.

The purpose of this study was to determine the expression, localization and subcellular distribution of microsomal (mEH) and soluble (sEH) epoxide hydrolases in cultured rat cortical astrocytes. Cultures of astrocytes were probed for mEH and sEH by immunocytochemistry, immunoblotting and enzymatic assays. Our results showed immunofluorescence for mEH and sEH, which colocalized with the astroglial cytoskeletal marker, glial fibrillary acidic protein (GFAP). Western blotting analyses for mEH revealed a protein band of molecular mass ∼50 kDa, the intensity of which was increased about 1.5‐fold, especially in the microsomal fraction. Whereas the polyclonal sEH rabbit serum recognized a protein band of molecular mass ∼62 kDa, which increased 2.0 fold in the 105,000xg supernatant over the cell lysate. The sEH rabbit serum also recognized similar protein bands in mitochondrial and nuclear fractions. The enzymatic analyses, using mEH‐ and sEH‐selective substrates further corroborated these findings. Our results suggest that astroglial cells, by expressing both mEH and sEH, may play a role in the cerebrovascular functions affected by epoxides (Funding Support: G12RR03045‐17 (ACS) and R37ES02710 (BDH)).

Regulation of Myoblast Motility and Fusion by the CXCR4-associated Sialomucin, CD164

Myoblast fusion is fundamental to the development and regeneration of skeletal muscle. To fuse, myoblasts undergo cell-cell recognition and adhesion and merger of membranes between apposing cells. Cell migration must occur in advance of these events to bring myoblasts into proximity, but the factors that regulate myoblast motility are not fully understood. CD164 is a cell surface sialomucin that is targeted to endosomes and lysosomes via its intracellular region. In hematopoietic progenitor cells, CD164 forms complexes with the motility-stimulating chemokine receptor, CXCR4, in response to the CXCR4 ligand, CXCL12/SDF-1 (Forde, S., Tye, B. J., Newey, S. E., Roubelakis, M., Smythe, J., McGuckin, C. P., Pettengell, R., and Watt, S. M. (2007) Blood 109, 1825-1833). We have previously shown that CD164 stimulates myotube formation in vitro. We report here that CD164 is associated with CXCR4 in C2C12 myoblasts. Cells in which CD164 levels are increased or decreased via overexpression or RNA interference-mediated knockdown, respectively, show enhanced or reduced myotube formation and cell migration, the latter both basally and in response to CXCL12/SDF-1. Furthermore, expression of CD164 cytoplasmic tail mutants that alter the endosome/lysosome targeting sequence and, consequently, the subcellular localization in myoblasts, reveals a similar correlation between cell motility and myotube formation. Finally, Cd164 mRNA is expressed in the dorsal somite (the early myogenic compartment of the mouse embryo) and in premuscle masses. Taken together, these results suggest that CD164 is a regulator of myoblast motility and that this property contributes to its ability to promote myoblast fusion into myotubes.

Nuclear Tumor Necrosis Factor Receptor-associated Factor 6 in Lymphoid Cells Negatively Regulates c-Myb-mediated Transactivation through Small Ubiquitin-related Modifier-1 Modification

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-kappaB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5'-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.

Levels of Estrogen Receptors &amp;#945; and &amp;#946; in Frontal Cortex of Patients with Alzheimers Disease: Relationship to Mini-Mental State Examination Scores

Estrogen exerts beneficial effects on the brain throughout life. Studies demonstrate that estrogen is neuroprotective and that reduced brain estrogen activity may influence the clinical course of Alzheimer's disease (AD). Changes in levels of estrogen receptors have been detected in postmortem brain tissue of AD patients. Very little is known about the relationship between clinical stage and levels of estrogen receptors in postmortem brain. We hypothesized that estrogen receptor levels would be related to severity of cognitive impairment assessed proximate to death. Western blotting was used to quantify ER-alpha and ER-beta in nuclear, cytosolic, and crude membrane fractions of superior frontal cortex from 25 AD patients. Multiple linear regression analyses adjusted for age, sex, and education showed a significant linear relationship between Mini-Mental State Examination score (MMSE) and wild-type nuclear ER-alpha (â = 5.463, p = 0.03), but none between MMSE and wild-type nuclear ER-beta (â = 2.29, p = 0.36). We incidentally observed additional higher and lower molecular mass bands for ER-alpha in study subjects. Additional experiments performed on frontal cortex nuclear fractions prepared from subjects enrolled in a different study confirmed that these same bands are present in female and males with and without AD. Together our data show a relationship between wild-type ER-alpha and level of cognitive impairment in AD, and also suggest the possibility that variant isoforms of ER-alpha may be present in frontal cortex of patients with and without AD.

Kidney abnormalities in low density lipoprotein receptor associated protein knockout mice

Mice lacking the LDL receptor associated protein (RAP) have a severe defect of thyroglobulin secretion into the colloid, associated with moderately increased serum TSH levels and histological features of early goiter. RAP is expressed also in renal proximal tubule cells, where it functions as a molecular chaperone for the endocytic receptor megalin, which is responsible for reabsorption of low molecular weight proteins from the glomerular filtrate. Here we investigated whether the thyroid phenotype in RAP knockout (KO) mice is associated with kidney alterations. By immunohistochemistry, we found that in RAP KO mice megalin expression on the apical membrane of renal proximal tubule cells was markedly reduced, with intracellular retention of the receptor. The reduced expression of megalin was associated with its impaired function. Thus, urinary protein concentrations and urinary protein excretion in 24 h were higher in RAP KO than in wild-type mice. Coomassie staining of urine samples revealed an increased intensity of low molecular mass bands in the urine of RAP KO mice, indicating that they had low molecular weight proteinuria. Therefore, we concluded that disruption of the RAP gene determines not only thyroid abnormalities, but also a severe defect of megalin expression and function in the kidney.