Replication of a linear mini-chromosome with terminal inverted repeats from the Kluyveromyces lactis linear DNA plasmid k2 in the cytoplasm of Saccharomyces cerevisiae

The k1 and k2 linear DNA plasmids of Kluveromyces lactis replicate in the cytoplasm under the control of plasmid-encoded genes. These plasmids can also replicate autonomously in the cytoplasm of mitochondrial DNA-deficient strains of Saccharomyces cerevisiae. Essential for replication are plasmid-specific terminal inverted repeats (TIRs) to which a terminal protein (TP) is attached at the 5' ends. A plasmid was constructed with k2 TIRs in opposite orientations and with a selectable marker (URA3) under the control of k1UCS2 (upstream conserved sequence 2, the promoter of k1 open reading frame 2) in between the TIRs. Transformation of k1- and k2-containing S. cerevisiae with a fragment generated by releasing the TIR-flanked fragment from the plasmid by restriction digestion was very efficient, despite the absence of a TP. Transformation was also achieved with a fragment generated by PCR. Southern blotting demonstrated that transformants contained multiple copies of DNA fragments with the same size as the transforming DNA, supporting the hypothesis that these were replicating linear mini-chromosomes. The high frequency of transformation strongly suggests that these mini-chromosomes readily replicate supported by k2. Derivatives with a heterologous gene, firefly luciferase (LUC), expressed luciferase at high levels provided the gene was adjacent to a cytoplasmic plasmid promoter (k2UCS5).     

Protein kinase D is a downstream target of protein kinase Ctheta

Protein kinase D (PKD/PKCmu immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCtheta AE (PKCthetaAE) exhibited a marked increase in basal activity. In contrast, coexpression of constitutively active mutant of PKCzeta does not induce PKD activation in both types of cells. PKCthetaAE does not induce kinase activity in immunocomplexes of PKD kinase-deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCthetaAE signaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Our results show that PKD is a downstream target of the theta isoform of PKC in both COS-7 cells and lymphocytes. The regulation of PKD by PKCtheta reveals a new pathway in the signaling network existing between multiple members of the PKC superfamily and PKD.     

Cardiac fibroblasts and the mechano-electric feedback mechanism in healthy and diseased hearts

Cardiac arrhythmia is a serious clinical condition, which is frequently associated with abnormalities of mechanical loading and changes in wall tension of the heart. Recent novel findings suggest that fibroblasts may function as mechano-electric transducers in healthy and diseased hearts. Cardiac fibroblasts are electrically non-excitable cells that respond to spontaneous contractions of the myocardium with rhythmical changes of their resting membrane potential. This phenomenon is referred to as mechanically induced potential (MIP) and has been implicated in the mechano-electric feedback mechanism of the heart. Mechano-electric feedback is thought to adjust the frequency of spontaneous myocardial contractions to changes in wall tension, which may result from variable filling pressure. Electrophysiological recordings of single atrial fibroblasts indicate that mechanical compression of the cells may activate a non-selective cation conductance leading to depolarisation of the membrane potential. Reduced amplitudes of MIPs due to pharmacological disruption of F-actin and tubulin suggest a role for the cytoskeleton in the mechano-electric signal transduction process. Enhanced sensitivity of the membrane potential of the fibroblasts to mechanical stretch after myocardial infarction correlates with depression of heart rates. It is assumed that altered electrical function of cardiac fibroblasts may contribute to the increased risk of post-infarct arrhythmia.     

Deletion of mtDNA disrupts mitochondrial function and structure, but not biogenesis

The role of nuclear DNA (nDNA)-encoded proteins in the regulation of mitochondrial fission and fusion has been documented, yet the role of mitochondrial DNA (mtDNA) and encoded proteins in mitochondrial biogenesis remains unknown. Long-term treatment of a lymphoblastoid cell line Molt-4 with ethidium bromide generated mtDNA-deficient rho0 mutants. Depletion of mtDNA in rho0 cells produced functional and morphological changes in mitochondria without affecting the nuclear genome and encoded proteins. Indeed, the gene encoding subunit II of mitochondrial cytochrome c oxidase (COX II), a prototypical mitochondrial gene, was reduced in rho0 mutants blunting the activity of mitochondrial cytochrome coxidase. Yet, the amount of the nuclear beta-actin gene and the activity of citrate synthase, a mitochondrial matrix enzyme encoded by nDNA, remained unaffected in rho0 cells. Loss of mtDNA in rho0 cells was associated with significant distortion of mitochondrial structure, decreased electron density of the matrix and disorganized inner and outer membranes, resulting in the appearance of 'ghost-like' mitochondria. However, the number of mitochondria-like structures was not significantly different between mtDNA-deficient and parental cells. Thus, we conclude that cells lacking mtDNA still generate mitochondrial scaffolds, albeit with aberrant function.     

The third P-loop domain in cytoplasmic dynein heavy chain is essential for dynein motor function and ATP-sensitive microtubule binding

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.     

Cellular remodeling in heart failure disrupts K(ATP) channel-dependent stress tolerance

ATP-sensitive potassium (K(ATP)) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on K(ATP) channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor alpha induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, K(ATP) channels responded aberrantly to metabolic signals despite intact intrinsic channel properties, implicating defects proximal to the channel. Indeed, cardiomyocytes from failing hearts exhibited mitochondrial and creatine kinase deficits, and thus a reduced potential for metabolic signal generation and transmission. Consequently, K(ATP) channels failed to properly translate cellular distress under metabolic challenge into a protective membrane response. Failing hearts were excessively vulnerable to metabolic insult, demonstrating cardiomyocyte calcium loading and myofibrillar contraction banding, with tolerance improved by K(ATP) channel openers. Thus, disease-induced K(ATP) channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.     

Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells

The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-₁. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-₁and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-₁ (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-₁ stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-₁ andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-₁, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC.