Enzymes that hydrolyze adenine nucleotides of patients with hypercholesterolemia and inflammatory processes

The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in platelets from patients with increasing cholesterol levels. A possible association between cholesterol levels and inflammatory markers, such as oxidized low-density lipoprotein, highly sensitive C-reactive protein and oxidized low-density lipoprotein autoantibodies, was also investigated. Lipid peroxidation was estimated by measurement of thiobarbituric acid reactive substances in serum. The following groups were studied: group I, < 150 mg.dL(-1) cholesterol; group II, 151-200 mg.dL(-1) cholesterol; group III, 201-250 mg.dL(-1) cholesterol; and group IV, > 251 mg.dL(-1) cholesterol. The results demonstrated that both ATP hydrolysis and ADP hydrolysis were enhanced as a function of cholesterol level. Low-density lipoprotein levels increased concomitantly with total cholesterol levels. Triglyceride levels were increased in the groups with total cholesterol above 251 mg.dL(-1). Oxidized low-density lipoprotein levels were elevated in groups II, III, and IV. Highly sensitive C-reactive protein was elevated in the group with cholesterol levels higher than 251 mg.dL(-1). Oxidized low-density lipoprotein autoantibodies were elevated in groups III and IV. Thiobarbituric acid reactive substance content was enhanced as a function of cholesterol level. In summary, hypercholesterolemia is associated with enhancement of inflammatory response, oxidative stress, and ATP and ADP hydrolysis. The increased ATP and ADP hydrolysis in group IV was confirmed by an increase in CD39 expression on its surface. The increase in CD39 activity is possibly related to a compensatory response to the inflammatory and pro-oxidative state associated with hypercholesterolemia.     

Causal role of oxidative stress in liver preconditioning by thyroid hormone in rats

Hepatic ischemia-reperfusion (IR) injury, a major clinical drawback during surgery, is abolished by L-3,3',5-triiodothyronine (T(3)) administration. Considering that the triggering mechanisms are unknown, the aim of this study is to assess the role of oxidative stress in T(3) preconditioning using N-acetylcysteine (NAC) before T(3) administration. Male Sprague-Dawley rats given a single dose of 0.1 mg of T(3)/kg were subjected to 1 h ischemia followed by 20 h reperfusion, in groups of animals pretreated with 0.5 g of NAC/kg 0.5 h before T(3) or with the respective control vehicles. At the end of the reperfusion period, blood and liver samples were taken for analysis of serum aspartate aminotransferase (AST) and hepatic histology, glutathione (GSH) and protein carbonyl contents, and nuclear factor-kappaB (NF-kappaB) and activating protein 1 (AP-1) DNA binding. The IR protocol used led to a 4.5-fold increase in serum AST levels and drastic changes in liver histology, with significant GSH depletion and enhancement of protein carbonyl levels and of the protein carbonyl/GSH content ratio, whereas NF-kappaB and AP-1 DNA binding was decreased and enhanced, respectively. In a time window of 48 h, T(3) exerted protection against hepatic IR injury, with 88% reduction in the protein carbonyl/GSH ratio and normalization of NF-kappaB and AP-1 DNA binding, changes that were suppressed by NAC administration before T(3). Data presented suggest that a transient increase in the oxidative stress status of the liver is an important trigger for T(3) preconditioning, evidenced in a warm IR injury model through antioxidant intervention.     

Australia's oldest marsupial fossils and their biogeographical implications

Background

We describe new cranial and post-cranial marsupial fossils from the early Eocene Tingamarra Local Fauna in Australia and refer them to Djarthia murgonensis, which was previously known only from fragmentary dental remains.

Methodology/Principal Findings

The new material indicates that Djarthia is a member of Australidelphia, a pan-Gondwanan clade comprising all extant Australian marsupials together with the South American microbiotheres. Djarthia is therefore the oldest known crown-group marsupial anywhere in the world that is represented by dental, cranial and post-cranial remains, and the oldest known Australian marsupial by 30 million years. It is also the most plesiomorphic known australidelphian, and phylogenetic analyses place it outside all other Australian marsupials.

Conclusions/Significance

As the most plesiomorphic and oldest unequivocal australidelphian, Djarthia may approximate the ancestral morphotype of the Australian marsupial radiation and suggests that the South American microbiotheres may be the result of back-dispersal from eastern Gondwana, which is the reverse of prevailing hypotheses.     

Fluorescent labeling of nitrocellulose-bound proteins at the nanogram level without changes in immunoreactivity

Proteins blotted on nitrocellulose were stained with either 5-dimethylamino-1-naphthalene-sulfonylchloride (dansyl chloride) or fluorescein isothiocyanate. In both cases the staining procedure can be completed in less than 30 min. The sensitivity for detecting fluorescent-labeled proteins on nitrocellulose was 0.5 ng using a dot test. This was accomplished by transparentizing the nitrocellulose with either immersion oil or toluene. Dansylated proteins were successfully utilized for optimizing the electroblotting procedure. In the presence of 0.2% sodium dodecyl sulfate and 20% methanol the distribution of proteins on the nitrocellulose was an exact replica of the protein pattern seen in the polyacrylamide gel. The fluorescent labeling did not affect the antigenic properties of proteins allowing the subsequent probing with antisera. For this procedure, only one set of samples is needed to obtain accurate photographic records of the gel, the nitrocellulose blot, and the probed blot.     

Phasenverschiebungen der endogenen Tagesrhythmik durch Reduktion der Atmung

Zusammenfassung der Ergebnisse Untersucht wurden die Phasenverschiebungen durch atmungsreduzierende Einflüsse bei den endogen-tagesperiodischen Primärblattbewegungen vonPhaseolus multiflorus und den Öffnungs- und Schließungsbewegungen vonKalanchoe blossfeldiana.Selbst eine 4stündige Einwirkung einer Temperatur von +2°, die schon Turgorverlust hervorruft, bedingt beiPhaseolus in einigen Phasen des Cyclus nur eine Verzögerung um etwa 2 Std, in anderen Phasen Verzögerungen, die die Dauer der Kälteeinwirkung übersteigen.Eine 4stündige Sauerstoffverdrängung durch Stickstoff bedingt bei beiden Versuchsobjekten nach der Einwirkung in einigen Phasen keine signifikante Verschiebung, nach der Einwirkung in anderen Phasen Verzögerungen, die 4 Std oder etwas mehr erreichen können.Das unterschiedliche Ansprechen der einzelnen Phasen des Cyclus auf Sauerstoffverdrängung durch Stickstoff geht dem unterschiedlichen Ansprechen auf niedrige Temperatur nicht parallel.DNP-Vergiftung wirkt beiPhaseolus ähnlich wie Sauerstoffverdrängung. Jedoch treten hierbei, noch extremer bei KCN-Vergiftung, nicht nur Phasenverzögerungen, sondern auch Vorverlegungen auf.

---

The induction of phase shifts in endogenous diurnal rhythms through the reduction of respiration

Summary Experiments were carried out in order to determine the effect of reducing respiration on the phases of endogenous diurnal movements inPhaseolus multiflorus (primary leaves) andKalanchoe blossfeldiana (opening and closing of the flowers). Such an effect was examined by measuring the delay occurring in peak formation, 2 or more days after treatment.In the case ofPhaseolus, treatment for 4 hours at +20°C caused a phase shift of only about 2 hours when given at certain times in the 24 hours cycle. The same treatment at other points in the cycle resulted in a phase shift which greatly exceeded the duration of the treatment (Fig. 1).Periods of anaerobic conditions were provided at different times during the 24 hour cycle. It was found that, in both species, there were points in the 24 hour cycle at which the treatment did not result in phase shifts in the following peaks, and also points at which the treatment resulted in marked phase shifts (Figs. 2, 3 and 4).These points in the 24 hour cycle with no significant response to the anaerobic conditions are not identical with those at which little or no response is obtained after low temperature treatment.Poisons, such as DNP and KCN, can also induce phase shifts. However, both delays and advances in the clock can result from such treatments (Fig. 5). This is interpreted as being due to a more extreme interruption of the energy supply, which not only delays, but also partially upsets the processes involved in the working of the clock.

---

---

Mit 5 Textabbildungen     

Phylogeography and population genetics of the endangered Amazonian manatee, Trichechus inunguis Natterer, 1883 (Mammalia, Sirenia)

We used mitochondrial DNA control region sequences to examine phylogeography and population differentiation of the endangered Amazonian manatee Trichechus inunguis. We observe lack of molecular differentiation among localities and we find weak association between geographical and genetic distances. However, nested clade analysis supports restricted gene flow and/or dispersal with some long-distance dispersal. Although this species has a history of extensive hunting, genetic diversity and effective population sizes are relatively high when compared to the West Indian manatee Trichechus manatus. Patterns of mtDNA haplotype diversity in T. inunguis suggest a genetic disequilibrium most likely explained by demographic expansion resulting from secession of hunting and enforcement of conservation and protective measures. Phylogenetic analysis of T. manatus and T. inunguis haplotypes suggests that T. inunguis is nested within T. manatus, effectively making T. manatus a paraphyletic entity. Paraphyly of T. manatus and recent divergence times of T. inunguis and the three main T. manatus lineages suggest a possible need for a taxonomic re-evaluation of the western Atlantic Trichechus.     

The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.