Spontaneous combined hyperlipidemia, coronary heart disease and decreased survival in Dahl salt-sensitive hypertensive rats transgenic for human cholesteryl ester transfer protein

The acceleration of atherosclerosis by polygenic (essential) hypertension is well-characterized in humans; however, the lack of an animal model that simulates human disease hinders the elucidation of pathogenic mechanisms. We report here a transgenic atherosclerosis-polygenic hypertension model in Dahl salt-sensitive hypertensive rats that overexpress the human cholesteryl ester transfer protein (Tg[hCETP]DS). Male Tg[hCETP]DS rats fed regular rat chow showed age-dependent severe combined hyperlipidemia, atherosclerotic lesions, myocardial infarctions and decreased survival. These findings differ from various mouse atherosclerosis models, demonstrating the necessity of complex disease modeling in different species. The data demonstrate that cholesteryl ester transfer protein can be proatherogenic. The interaction of polygenic hypertension and hyperlipidemia in the pathogenesis of atherosclerosis in Tg[hCETP]DS rats substantiates epidemiological observations in humans.     

A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions.     

Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-alpha, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (Kd) of 2 and 0.7 pM, and a receptor density of about 10, 000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. The Kd values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with 125I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.     

Non-functional conserved residues in globins and their possible role as a folding nucleus.

Structure-based sequence alignment of 728 sequences of different globin subfamilies shows that in each subfamily there are two clusters of consensually conserved residues. The first is the well-known "functional" cluster which includes six heme-binding conserved residues (Phe CD1, His F8; aliphatic E11, FG5; hydrophobic F4, G5) and seven other conserved residues (Pro C2; aliphatic H19; hydrophobic B10, B13, B14, CD4, E4) that do not bind the heme but belong to its immediate neighborhood. The second cluster revealed here (aliphatic A8, G16, G12; aromatic A12; hydrophobic H8 and possibly H12) is distant from the heme. It is entirely non-polar and includes one turn (i, i+4 positions) from each of helices A, G, and H. It is known that A, G, and H helices formed at the earliest stage of apomyoglobin folding remain relatively stable in the equilibrium molten globule state, and are likely to be tightly packed with each other in this state. We have shown the existence of two similar conserved clusters in c -type cytochromes, heme-binding and distal from the heme. The second cluster in c -cytochromes includes one turn from each of the N and C-terminal alpha-helices. These N and C-terminal helices in cytochrome c are formed at the earliest stage of protein folding, remain relatively stable in the molten globule state, and are tightly packed with each other in this state, similar to the observed behavior of the globins. At least these two large protein families (c -type cytochromes and globins) have a close similarity in the existence and mutual positions of non-functional conserved residues. We assume that non-functional conserved residues are requisite for the fast and correct folding of both of these protein families into their stable 3D structures.     

Neuroprotection by pramipexole against dopamine- and levodopa-induced cytotoxicity

Pramipexole, a novel non-ergoline dopamine (DA) agonist, has been applied successfully for treatment of Parkinson's disease (PD). We report here that pramipexole can protect dopaminergic cell line Mes23.5 against dopamine- and levodopa-induced cytotoxicity possibly through a mechanism related to antioxidant activity. In the MES 23.5 cultures, DA and L-DOPA induce a dose- and time-dependent cytotoxicity, as determined by tetrazolium salt and trypan blue assays. Furthermore, an in situ terminal deoxynucleotidyl transferase assay demonstrates that DA-induced cell death is apoptotic. Pretreatment with pramipexole in a concentration range (4-100 microM) significantly attenuates DA- or L-DOPA-induced cytotoxicity and apoptosis, an action which is not blocked by D3 antagonist U-99194 A or D2 antagonist raclopride. Pramipexole also protects MES 23.5 cells from hydrogen peroxide-induced cytotoxicity in a dose-dependent manner. In cell-free system, pramipexole can effectively inhibit the formation of melanin, an end product resulting from DA or L-DOPA oxidation. These results indicate that pramipexole exerts its neuroprotective effect possibly through a mechanism, which is independent of DA receptors but related to antioxidation or scavenging of free radicals (e.g. hydrogen peroxide). As a direct DA agonist and potentially neuroprotective agent, pramipexole remains attractive in the treatment of PD.     

Transient kinetic analysis of N-phenylmaleimide-reacted myosin subfragment-1

Alkylation of myosin's Cys-707 (SH1) and Cys-697 (SH2) has profound consequences for myosin's ability to interact with actin and hydrolyze MgATP. Pre-steady-state measurements of myosin-S1 alkylated at SH1 and SH2 by N-phenylmaleimide (NPM) in the presence of ATP were taken to identify the steps of the reaction that are altered. It was found that the rate constant most affected by this modification is the apparent rate of the ATP hydrolysis step. This rate constant is reduced 20000-fold, an effect comparable in magnitude to the effect of the same modification on the binding of MgATP to S1 or acto-S1 [Xie, L., and Schoenberg, M. (1998) Biochemistry 37, 8048]. In contrast, the rate constants of phosphate release and dissociation of acto-S1 by ATP were reduced <20-fold. For unmodified S1, the enhancement of fluorescence seen after addition of ATP had the same rate constant as the ATP hydrolysis step (S1.ATP if S1.ADP.Pi) measured by single-turnover experiments in a quench-flow experiment. This is consistent with results previously observed [Johnson, K. A., and Taylor, E. W. (1978) Biochemistry 17, 3432]. However, NPM-modified S1 exhibited virtually no fluorescence enhancement upon ATP binding. This provides further evidence that M.ATP is the predominant intermediate of NPM-S1-catalyzed ATP hydrolysis.     

Mitochondrial Dysfunction in the Pathogenesis of Necrotic and Apoptotic Cell Death

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca²⁺ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca²⁺, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP.     

Mycotoxin Production by Fusarium proliferatum isolates from rice with Fusarium sheath rot disease

Twenty samples of unpolished (rough) rice collected in Arkansas and Texas during the 1995 harvesting season from fields exhibiting Fusarium sheath rot disease or panicle blight were previously shown to include 8 samples positive for fumonisin B₁(FB₁) in the range 2.2–5.2 ppm, and moniliformin (MON), but no beauvericin (BEA), deoxynivalenol, its derivatives or zearalenone were detected. Fifteen cultures of F. proliferatum were established from the 20 rough rice samples. Single spore isolates of each culture were grown on rice and tested for the production of fumonisins (FB₁, FB₂, FB₃, etc.), MON and BEA. All 15 isolates produced FB₁, FB₂, MON and BEA in culture on rice. No deoxynivalenol, its derivatives orzearalenone were detected. Seven cultures produced FB₁ at >50ppm (range 80–230 ppm), with therest producing FB₁ in the range 14–43 ppm.FB₂ was produced in the range 5–47 ppm, and those cultures which produced the most FB₁ also produced the most FB₂. Of the 15 cultures producing MON, 11 produced it at >100 ppm in the range 188–6018 ppm, with the rest producing in the range 7–64 ppm. BEA was produced in the range 109–1350 ppm. Other derivatives of fumonisins, including FA₁, FA₂ and partially hydrolyzed FB₁, as well asseveral unknown metabolites including a compound with MW 414, were identified in culture extracts by continuous flow fast atom bombardment with ion spraymass spectrometry (CF/FAB/MS). Further study is needed to identify the factors that control production of FB₁, MON and BEA by F.proliferatu in culture and in field samples. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nucleocytoplasmic shuttling of JAZ, a new cargo protein for exportin-5

Exportin-5 is a nuclear export receptor for certain classes of double-stranded RNA (dsRNA), including pre-micro-RNAs, viral hairpin RNAs, and some tRNAs. It can also export the RNA binding proteins ILF3 and elongation factor EF1A. However, the rules that determine which RNA binding proteins are exportin-5 cargoes remain unclear. JAZ possesses an unusual dsRNA binding domain consisting of multiple C2H2 zinc fingers. We found that JAZ binds to exportin-5 in a Ran-GTP- and dsRNA-dependent manner. Exportin-5 stimulates JAZ shuttling, and gene silencing of exportin-5 reduces shuttling. Recombinant exportin-5 also stimulates nuclear export of JAZ in permeabilized cells. JAZ also binds to ILF3, and surprisingly, this interaction is RNA independent, even though it requires the dsRNA binding domains of ILF3. Exportin-5, JAZ, and ILF3 can form a heteromeric complex with Ran-GTP and dsRNA, and JAZ increases ILF3 binding to exportin-5. JAZ does not contain a classical nuclear localization signal, and in digitonin-permeabilized cells, nuclear accumulation of JAZ does not require energy or cytosol. Nonetheless, low temperatures prevent JAZ import, suggesting that nuclear entry does not occur via simple diffusion. Together, these data suggest that JAZ is exported by exportin-5 but translocates back into nuclei by a facilitated diffusion mechanism.