A Bacterial Phosphatase-Like Enzyme of the Malaria Parasite Plasmodium falciparum Possesses Tyrosine Phosphatase Activity and Is Implicated in the Regulation of Band 3 Dynamics during Parasite Invasion

Eukaryotic parasites of the genus Plasmodium cause malaria by invading and developing within host erythrocytes. Here, we demonstrate that PfShelph2, a gene product of Plasmodium falciparum that belongs to the Shewanella-like phosphatase (Shelph) subfamily, selectively hydrolyzes phosphotyrosine, as shown for other previously studied Shelph family members. In the extracellular merozoite stage, PfShelph2 localizes to vesicles that appear to be distinct from those of rhoptry, dense granule, or microneme organelles. During invasion, PfShelph2 is released from these vesicles and exported to the host erythrocyte. In vitro, PfShelph2 shows tyrosine phosphatase activity against the host erythrocyte protein Band 3, which is the most abundant tyrosine-phosphorylated species of the erythrocyte. During P. falciparum invasion, Band 3 undergoes dynamic and rapid clearance from the invasion junction within 1 to 2 s of parasite attachment to the erythrocyte. Release of Pfshelph2 occurs after clearance of Band 3 from the parasite-host cell interface and when the parasite is nearly or completely enclosed in the nascent vacuole. We propose a model in which the phosphatase modifies Band 3 in time to restore its interaction with the cytoskeleton and thus reestablishes the erythrocyte cytoskeletal network at the end of the invasion process.     

Predictive Factors of Late Venous Aortocoronary Graft Failure: Ultrastructural Studies

Background

Venous aortocoronary graft arterialization may precede a preterm occlusion in some coronary artery bypass grafting (CABG) patients. The aim of the present study was to identify ultrastructural variations in the saphenous vein wall that may have an impact on the development of venous graft disease in CABG patients.

Methods

The study involved 365 consecutive patients with a mean age of 62.9±9.4 years who underwent isolated CABG. The thickness and area of the whole venous wall, the tunica intima, the tunica media and the adventitia and the number and shape (length, thickness and length/thickness ratio) of the nuclei in the medial smooth muscle cells nuclei in the distal saphenous vein segments were evaluated by ultrastructural studies. Patients were followed up for 41 to 50 months (mean 45.1±5.1). Saphenous vein graft patency was assessed by follow-up coronary angiography. Logistic regression models were used to identify independent risk factors for late graft failure.

Results

In 71 patients significant lesions in the saphenous vein grafts were observed. The whole venous wall thickness (437.5 µm vs. 405.5 µm), tunica media thickness (257.2 µm vs. 211.5 µm), whole venous wall area (2.23 mm² vs. 2.02 mm²) and tunica media area (1.09 mm² vs. 0.93 mm²) were significantly larger for this group of patients than for those without graft disease. In the latter group more elongated smooth muscle cell nuclei (higher length/thickness ratio) were found in the tunica media of the saphenous vein segments. Thickening of the saphenous vein tunica media and chunky smooth muscle cell nuclei were identified as independent risk factors for graft disease development.

Conclusions

Saphenous vein tunica media hypertrophy (resulting in wall thickening) and chunky smooth muscle cell nuclei might predict the development of venous graft disease.     

Cyclooxygenase-2 contributes to elevated renin in the early postnatal period in rats

We asked whether cyclooxygenase (COX) activity controls the renin-angiotensin system in the postnatal period. During kidney development, renin peaked at postnatal days 0-1 at the mRNA, tissue protein [renal renin concentration (RRC)], and plasma renin concentration (PRC) levels and was widely expressed along preglomerular vessels. PRC and renin mRNA expression was elevated until weaning in the 4th postnatal week compared with adult rats. Renocortical COX-2 was restricted to Tamm-Horsfall protein-positive cells in the thick ascending limb of Henle's loop, and cortical COX-2 mRNA and protein expression were elevated along with PRC in the 2nd and 3rd postnatal weeks. In contrast, cortical COX-1 expression was constant, but medullary COX-1 expression increased eightfold from the 1st to 4th postnatal week. A COX-2-selective blocker, parecoxib, and a nonselective blocker, indomethacin, given in a period with COX-2 induction from postnatal day 6 to day 12, markedly decreased PRC, but not renin mRNA or RRC. Inhibition of angiotensin AT(1) receptors by candesartan from postnatal day 1 to day 5 increased COX-2 mRNA (2.5-fold), protein, and distribution, renin mRNA (7-fold) and PRC (20- to 70-fold), but had no influence on COX-1 mRNA. Thus, due to very low levels of expression, COX-2 is unlikely to be responsible for the birth peak of renin, but COX-2 activity supports renin secretion later in the suckling period. ANG II negatively feeds back on renocortical COX-2 expression in the 1st postnatal days with high activity of the renin system. We suggest that suckling in the rat is correlated to an enhanced, COX-2-mediated, secretory activity of renin-producing juxtaglomerular cells.     

Clofibrate causes an upregulation of PPAR-{alpha} target genes but does not alter expression of SREBP target genes in liver and adipose tissue of pigs

This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-alpha and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-alpha target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-alpha in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-alpha activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs.     

Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (～1/100 of the exciting wavelength).     

Desferoxamine and ethyl-3,4-dihydroxybenzoate protect myocardium by activating NOS and generating mitochondrial ROS

Protection from a prolyl hydroxylase domain-containing enzyme (PHD) inhibitor, desferoxamine (DFO), was recently reported to be dependent on production of reactive oxygen species (ROS). Ischemic preconditioning triggers the protected state by stimulating nitric oxide (NO) production to open mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels, generating ROS required for protection. We tested whether DFO and a second PHD inhibitor, ethyl-3,4-dihydroxybenzoate (EDHB), might have similar mechanisms. EDHB and DFO increased ROS generation by 50-75% (P < 0.001) in isolated rabbit cardiomyocytes. This increase after EDHB exposure was blocked by N(omega)-nitro-L-arginine methyl ester (L-NAME), an NO synthase (NOS) inhibitor; ODQ, a guanylyl cyclase antagonist; and Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate Rp isomer, a PKG blocker, thus implicating the NO pathway in EDHB's signaling. Glibenclamide, a nonselective K(ATP) channel blocker, or 5-hydroxydecanoate, a selective mitoK(ATP) channel antagonist, also prevented EDHB's ROS production, as did blockade of mitochondrial electron transport with myxothiazol. NOS is activated by Akt. However, neither wortmannin, an inhibitor of phosphatidylinositol-3-kinase, nor Akt inhibitor blocked EDHB-induced ROS generation, indicating that EDHB initiates signaling downstream of Akt. DFO also increased ROS production, and this effect was blocked by ODQ, 5-hydroxydecanoate, and N-(2-mercaptopropionyl)glycine, an ROS scavenger. DFO increased cardiomyocyte production of nitrite, a metabolite of NO, and this effect was blocked by an inhibitor of NOS. DFO also spared ischemic myocardium in intact hearts. This infarct-sparing effect was blocked by ODQ, L-NAME, and N-(2-mercaptopropionyl)glycine. Hence, DFO and EDHB stimulate NO-dependent activation of PKG to open mitoK(ATP) channels and produce ROS, which act as second messengers to trigger entrance into the preconditioned state.     

Species-Specific Viromes in the Ancestral Holobiont Hydra

Recent evidence showing host specificity of colonizing bacteria supports the view that multicellular organisms are holobionts comprised of the macroscopic host in synergistic interdependence with a heterogeneous and host-specific microbial community. Whereas host-bacteria interactions have been extensively investigated, comparatively little is known about host-virus interactions and viral contribution to the holobiont. We sought to determine the viral communities associating with different Hydra species, whether these viral communities were altered with environmental stress, and whether these viruses affect the Hydra-associated holobiont. Here we show that each species of Hydra harbors a diverse host-associated virome. Primary viral families associated with Hydra are Myoviridae, Siphoviridae, Inoviridae, and Herpesviridae. Most Hydra-associated viruses are bacteriophages, a reflection of their involvement in the holobiont. Changes in environmental conditions alter the associated virome, increase viral diversity, and affect the metabolism of the holobiont. The specificity and dynamics of the virome point to potential viral involvement in regulating microbial associations in the Hydra holobiont. While viruses are generally regarded as pathogenic agents, our study suggests an evolutionary conserved ability of viruses to function as holobiont regulators and, therefore, constitutes an emerging paradigm shift in host-microbe interactions.