Ultrastructural immuno-localization of tropoelastin in the chick eye

Summary Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.     

Role of superoxide and angiotensin II suppression in salt-induced changes in endothelial Ca2+ signaling and NO production in rat aorta

Male Sprague-Dawley rats were maintained on a low-salt (LS) diet (0.4% NaCl) or changed to a high-salt (HS) diet (4% NaCl) for 3 days. Increases in intracellular Ca2+ ([Ca2+]i) in response to methacholine (10 microM) and histamine (10 microM) were significantly attenuated in aortic endothelial cells from rats fed a HS diet, whereas thapsigargin (10 microM)-induced increases in [Ca2+]i were unaffected. Methacholine-induced nitric oxide (NO) production was eliminated in endothelial cells of aortas from rats fed a HS diet. Low-dose ANG II infusion (5 ng x kg(-1) x min(-1) iv) for 3 days prevented impaired [Ca2+]i signaling response to methacholine and histamine and restored methacholine-induced NO production in aortas from rats on a HS diet. Adding Tempol (500 microM) to the tissue bath to scavenge superoxide anions increased NO release and caused N(omega)-nitro-L-arginine methyl ester-sensitive vascular relaxation in aortas from rats fed a HS diet but had no effect on methacholine-induced Ca2+ responses. Chronic treatment with Tempol (1 mM) in the drinking water restored NO release, augmented vessel relaxation, and increased methacholine-induced Ca2+ responses significantly in aortas from rats on a HS diet but not in aortas from rats on a LS diet. These findings suggest that 1) agonist-induced Ca2+ responses and NO levels are reduced in aortas of rats on a HS diet; 2) increased vascular superoxide levels contribute to NO destruction, and, eventually, to impaired Ca2+ signaling in the vascular endothelial cells; and 3) reduced circulating ANG II levels during elevated dietary salt lead to elevated superoxide levels, impaired endothelial Ca2+ signaling, and reduced NO production in the endothelium.     

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

Caffeine Impairs Myocardial Blood Flow Response to Physical Exercise in Patients with Coronary Artery Disease as well as in Age-Matched Controls

Background

Caffeine is one of the most widely consumed pharmacologically active substances. Its acute effect on myocardial blood flow is widely unknown. Our aim was to assess the acute effect of caffeine in a dose corresponding to two cups of coffee on myocardial blood flow (MBF) in coronary artery disease (CAD).

Methodology/Principal Findings

MBF was measured with ¹⁵O-labelled H2O and Positron Emission Tomography (PET) at rest and after supine bicycle exercise in controls (n = 15, mean age 58±13 years) and in CAD patients (n = 15, mean age 61±9 years). In the latter, regional MBF was assessed in segments subtended by stenotic and remote coronary arteries. All measurements were repeated fifty minutes after oral caffeine ingestion (200 mg). Myocardial perfusion reserve (MPR) was calculated as ratio of MBF during bicycle stress divided by MBF at rest. Resting MBF was not affected by caffeine in both groups. Exercise-induced MBF response decreased significantly after caffeine in controls (2.26±0.56 vs. 2.02±0.56, P<0.005), remote (2.40±0.70 vs. 1.78±0.46, P<0.001) and in stenotic segments (1.90±0.41 vs. 1.38±0.30, P<0.001). Caffeine decreased MPR significantly by 14% in controls (P<0.05 vs. baseline). In CAD patients MPR decreased by 18% (P<0.05 vs. baseline) in remote and by 25% in stenotic segments (P<0.01 vs. baseline).

Conclusions

We conclude that caffeine impairs exercise-induced hyperaemic MBF response in patients with CAD to a greater degree than age-matched controls.     

The amino acid tryptophan prevents the biosynthesis of dermatan sulfate

An important part of the biosynthesis of proteoglycans is the epimerization of glycosaminoglycan chains. As a consequence of the conversion of chondroitin sulfate (CS) to dermatan sulfate (DS), the glycosaminoglycans become more flexible and enable DS to perform more sophisticated signaling functions. In a recent study, we generated a chimera (S222A) composed of a truncated form of a DS (decorin) and CS (CSF-1) containing proteoglycan and analyzed the influence of the core protein on the extent of epimerization. C-terminal truncation constructs from S222A enabled us to identify an amino acid segment that lies within the CSF-1 part which prevents DS synthesis. Co-localization experiments using S222A-HA and DCN-Flag showed different intracellular localizations for the proteoglycans during biosynthesis. A data base search revealed a sequence motif (TNWVP) within the CSF-1 moiety that is found to be important in other proteoglycans. A single substitution of tryptophan-216 to leucine (W216L) in the chimera S222A increased the amount of l-IdoA to 12-16%. Co-localization with an ER-marker demonstrated that the biosynthesis of recombinant decorin is similar to the chimera S222A and S222A(W216L) in HEK293 cells. Co-staining of S222A-HA and S222A(W216L)-Flag showed different intracellular localizations for the proteoglycans. A more detailed analysis of the glycosaminoglycans reflects a similar total sulfate content for S222A and S222A(W216L). The 4/6 sulfation ratio was similar for decorin and S222A, but altered for S222A(W216L). However, the binding of fibroblasts growth factor-1 to CS/DS was only partially dependent on epimerization. These results are consistent with the model in which the core protein, via the amino acid tryptophan, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase.     

A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.     

The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.     

Trophic role of rotifers in the plankton of lake Kozjak (Plitvice Lakes)

The trophic role of rotifers in the zooplankton community of dimictic, oligotrophic lake Kozjak, the largest lake of the Plitvice Lakes, NW Dinarid Mountains, is analyzed. Their spatial and temporal biomass distribution in relation to that of protozoans, cladocerans and copepods shows that they form a significant part of the non-predatory zooplankton of this karstic standing water.     

Native tree regeneration in native tree plantations: understanding the contribution of Araucaria angustifolia to biodiversity conservation in the threatened Atlantic Forest in Argentina

Deforestation is a global process that has strongly affected the Atlantic Forest in South America, which has been recognised as a threatened biodiversity hotspot. An important proportion of deforested areas were converted to forest plantations. Araucaria angustifolia is a native tree to the Atlantic Forest, which has been largely exploited for wood production and is currently cultivated in commercial plantations. An important question is to what extent such native tree plantations can be managed to reduce biodiversity loss in a highly diverse and vulnerable forest region . We evaluated the effect of stand age, stand basal area, as a measure of stand density, and time since last logging on the density and richness of native tree regeneration in planted araucaria stands that were successively logged over 60 years, as well as the differences between successional groups in the response of plant density to stand variables. We also compared native tree species richness in planted araucaria stands to neighbouring native forest. Species richness was 71 in the planted stands (27 ha sampled) and 82 in native forest (18 ha sampled) which approximate the range of variation in species richness found in the native forests of the study area. The total abundance and species richness of native trees increased with stand age and time since last logging, but ecological groups differed in their response to such variables. Early secondary trees increased in abundance with stand age 3–8 times faster than climax or late secondary trees. Thus, the change in species composition is expected to continue for a long term. The difference in species richness between native forest and planted stands might be mainly explained by the difference in plant density. Therefore, species richness in plantations can contribute to local native tree diversity if practices that increase native tree density are implemented.