Uronema marinum: identification and biochemical characterization of phosphatidylcholine-hydrolyzing phospholipase C

Phosphatidylcholine (PC)-specific phospholipase D (PC-PLD) and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) activities have been detected in Uronema marinum. Partial purification of PC-PLC revealed that two distinct forms of PC-PLC (named as mPC-PLC and cPC-PLC) were existed in membrane and cytosol fractions. The two PC-PLC enzymes showed the preferential hydrolyzing activity for PC with specific activity of 50.4 for mPC-PLC and 28.3 pmol/min/mg for cPC-PLC, but did not hydrolyze phosphatidylinositol or phosphatidylethanolamine. However, the biochemical characteristics and physiological roles of both enzymes were somewhat different. mPC-PLC had a pH optimum in the acidic region at around, pH 6.0, and required approximately 0.4 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC had a pH optimum in the neutral region at around, pH 7.0, and required 1.6 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC, but not mPC-PLC, showed a dose-dependent inhibitory effect on the luminal-enhanced chemiluminescence (CL) responses and the viability of zymosan-stimulated phagocytes of olive flounder, indicating that cPC-PLC may contribute to the parasite evasion against the host immune response. Our results suggest that U. marinum contains PC-PLD as well as two enzymatically distinct PC-PLC enzymes, and that mPC-PLC may play a role in the intercellular multiplication of U. marinum and cPC-PLC acts as a virulence factor, serving to actively disrupt the host defense mechanisms.     

Amphiphilic beta-sheet cobra cardiotoxin targets mitochondria and disrupts its network

Recent advance in understanding the role of toxin proteins in controlling cell death has revealed that pro-apoptotic viral proteins targeting mitochondria contain amphiphilic alpha-helices with pore-forming properties. Herein, we describe that the pore-forming amphiphilic beta-sheet cardiotoxins (or cytotoxins, CTXs) from Taiwan cobra (Naja atra) also target mitochondrial membrane after internalization and act synergistically with CTX-induced cytosolic calcium increase to disrupt mitochondria network. It is suggested that CTX-induced fragmentation of mitochondria play a role in controlling CTX-induced necrosis of myocytes and cause severe tissue necrosis in the victims.     

The effect of synthetic glucocorticoid, dexamethasone on CYP1A1 inducibility in adult rat and human hepatocytes

Glucocorticoids act synergistically with polycyclic aromatic hydrocarbons in increasing mRNA and protein levels of CYP1A1 in rat liver. The action of dexamethasone to modify CYP1A1 expression has been investigated in adult human hepatocytes. The effect of dexamethasone on the induction of CYP1A1 by 3-methylcholanthrene is different in rat and human liver cells. Dexamethasone potentiates the induction of CYP1A1 about 3- to 4-fold in rat cells. In human hepatocytes, it reduces CYP1A1 induction by 50-60% at enzyme protein level, while it does not have an effect on CYP1A1 mRNA amount.     

Synchronous intra-Golgi transport induces the release of Ca from the Golgi apparatus

The mechanisms of secretory transport through the Golgi apparatus remain an issue of debate. The precise functional importance of calcium ions (Ca²⁺) for intra-Golgi transport has also been poorly studied. Here, using different approaches to measure free Ca²⁺ concentrations in the cell cytosol ([Ca²⁺]_cyt) and inside the lumen of the Golgi apparatus ([Ca²⁺]_GA), we have revealed transient increases in [Ca²⁺]_cyt during the late phase of intra-Golgi transport that are concomitant with a decline in the maximal [Ca²⁺]_GA restoration ability. Thus, this redistribution of Ca²⁺ from the Golgi apparatus into the cytosol during the movement of cargo through the Golgi apparatus appears to have a role in intra-Golgi transport, and mainly in the late Ca²⁺-dependent phase of SNARE-regulated fusion of Golgi compartments.     

Decreased oxidative stress during glycolytic inhibition enables maintenance of ATP production and astrocytic survival

Depressed energy metabolism and oxidative stress are common features in many pathological situations in the brain, including stroke. In order to investigate astrocytic responses to such stress, we induced metabolic depression in cultured rat astrocytes. Iodoacetate (IA), an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used and resulted in a rapid inhibition of GAPDH activity. After 1h of GAPDH inhibition the ATP levels started to decrease and were completely abolished at 4h. In parallel, the activity of reactive oxygen species (ROS) was significantly increased, followed by extensive cell death involving flipping of phosphatidylserine and translocation of apoptosis-inducing factor, but not caspase-3 activation. When IA was combined with azide, a respiratory chain complex IV inhibitor, the ATP levels decreased immediately. Interestingly, with azide present, the ROS activity remained low and the astrocytes remained viable even at very low ATP levels. Addition of exogenous ROS-scavengers prevented the IA-induced ROS activity, the ATP levels were maintained and cell death was prevented. Similar protection could be obtained when astrocytes, prior to addition of IA, were incubated with substances known to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated endogenous antioxidant system. When IA was washed out, after a relatively moderate ATP depression, massive cell death occurred. This was efficiently prevented by addition of azide or ROS scavengers during the IA treatment or by pre-activation of the Nrf2 system. Our results demonstrate that astrocytes in culture can endure and recover from glycolytic inhibition if the ROS activity remained at a low level and suggest that oxidative stress can be an important component for astrocytic cell death following metabolic stress.     

Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages

Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H₂O₂. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments.     

Expression of TEM-induced damage to postmeiotic stages of spermatogenesis of the mouse during early embryogenesis. I. Investigations with in vitro embryo culture.

After treatment of postmeiotic stages of spermatogenesis of the mouse with TEM, dose and stage of spermatogenesis-dependent disturbances of the early embryonic development can be observed both in vivo and after in vitro culture of the embryos. The observations in both systems can be correlated. The embryo-culture system thereby enables analysis of the expression of mutagen-induced damage more accurately than the in vivo dominant lethal test. With the doses used (0.2 and 0.4 mg/kg) TEM-treatment of the fathers did not affect the rate of fertilized and cleaving eggs during the first three weeks post-treatment but severely disturbed the further development of the embryos at all stages up to implantation, exhibiting a maximum effect on morulae.     

Inactivation of glycogen synthase kinase 3β (GSK-3β) enhances mitochondrial biogenesis during myogenesis

Background

Mitochondrial biogenesis is crucial for myogenic differentiation and regeneration of skeletal muscle tissue and is tightly controlled by the peroxisome proliferator-activated receptor-γ co-activator 1 (PGC-1) signaling network. In the present study, we hypothesized that inactivation of glycogen synthase kinase (GSK)-3β, previously suggested to interfere with PGC-1 in non-muscle cells, potentiates PGC-1 signaling and the development of mitochondrial biogenesis during myogenesis, ultimately resulting in an enhanced myotube oxidative capacity.

A study of triflupromazine in dominant-lethal, cytogenetic and host-mediated assays

The phenothiazine psychotherapeutant, triflupromazine (TFP), was studied for mutagenic potential in dominant-lethal, in vivo and in vitro cytogenetic and host-mediated assay procedures. No evidence of gross chromosomal aberrations or point mutations was detected in these assays even at dosage regimens which produced substantial lethality. The effect of the drug on body temperature was measured at the same doses used for mutagenicity testing. A marked and sustained temperature reduction occurs shortly after administration of as little as 10 mg/kg. Due to the pronounced physiological effects at these levels, the validity of mutagenicity studies conducted at the same levels may be seriously questioned.