Decreased mortality in a rat model of acute postinfarction heart failure

INTRODUCTION: The rat model of postinfarction heart failure (HF) has been very valuable in experimental cardiology. One disadvantage of this model is the very high acute mortality (70-80%). The aim of this study was to evaluate whether measures of intensive cardiac care applied to rats with acute myocardial infarction (MI) would reduce mortality. METHODS: Male Sprague-Dawley rats weighing approximately 300 g were used. The animals were randomized into two groups. The intensive care group (IC) n=20 and conventional care group (CC) n=20. Experimental MI was induced by ligation of the left coronary artery producing large anterolateral MI. Animals in the IC group received isoflurane anesthesia and respiratory support postoperatively. The heart rhythm was monitored continuously and ventricular arrhythmias were treated with amiodarone and cardioversion. RESULTS: Mortality rate within 24 h was 4/20 (20%) in the IC group and 14/20 (70%) in the CC group (p<0.01). This represents a 3.5-fold reduction in acute mortality rate. CONCLUSIONS: The use of amiodarone, respiratory support, isoflurane gas anesthesia, and electrical cardioversion of malignant arrhythmias are simple and effective measures to reduce mortality in rats with acute MI and HF. Improving survival rates increases cost-efficiency and ethical acceptance of this important experimental HF model.     

Elementary steps of proton translocation in the catalytic cycle of cytochrome oxidase

Proton translocation in the catalytic cycle of cytochrome c oxidase (CcO) proceeds sequentially in a four-stroke manner. Every electron donated by cytochrome c drives the enzyme from one of four relatively stable intermediates to another, and each of these transitions is coupled to proton translocation across the membrane, and to uptake of another proton for production of water in the catalytic site. Using cytochrome c oxidase from Paracoccus denitrificans we have studied the kinetics of electron transfer and electric potential generation during several such transitions, two of which are reported here. The extent of electric potential generation during initial electron equilibration between CuA and heme a confirms that this reaction is not kinetically linked to vectorial proton transfer, whereas oxidation of heme a is kinetically coupled to the main proton translocation events during functioning of the proton pump. We find that the rates and amplitudes in multiphase heme a oxidation are different in the OH-->EH and PM-->F steps of the catalytic cycle, and that this is reflected in the kinetics of electric potential generation. We discuss this difference in terms of different driving forces and relate our results, and data from the literature, to proposed mechanisms of proton pumping in cytochrome c oxidase.     

Microsomal preparations from plant and yeast acylate free fatty acids without prior activation to acyl-thioesters

Acylation of fatty acids to hydroxy groups in cells generally require activation to a thioester (ACP or CoA) or transacylation from another oxygen ester. We now show that microsomal membranes from Arabidopsis leaves efficiently acylate free fatty acids to long chain alcohols with no activation of the fatty acids to thioesters prior to acylation. Studies of the fatty alcohol and fatty acids specificities of the reaction in membranes from Arabidopsis leaves revealed that long chain (C18-C24) unsaturated fatty alcohols and C18-C22 unsaturated fatty acids were preferred. Microsomal preparations from Arabidopsis roots and leaves and from yeast efficiently synthesized ethyl esters from ethanol and free fatty acids. This reaction also occurred without prior activation of the fatty acid to a thioester. The results presented strongly suggest that wax ester and ethyl ester formation are carried out by separate enzymes. The physiological significance of the reactions in plants is discussed in connection to suberin and cutin synthesis. The results also have implication regarding the interpretation of lipid metabolic experiments done with microsomal fraction.     

A hydrophobic patch in the competence-stimulating Peptide, a pneumococcal competence pheromone, is essential for specificity and biological activity

Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic alpha-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the alpha-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified alpha-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.     

Perfused hearts from Type 2 diabetic (db/db) mice show metabolic responsiveness to insulin

Diabetic (db/db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult.     

IdeE, an IgG-endopeptidase of Streptococcus equi ssp. equi

Streptococcus equi ssp. equi is the causative agent of strangles, a highly contagious and serious disease in the upper respiratory tract of horses. The present study describes the characterization of IdeE, a homolog of the secreted IgG-specific protease IdeS/Mac of Streptococcus pyogenes. The activity of IdeE is compared with the activity of IdeZ, the corresponding enzyme of the closely related S. equi ssp. zooepidemicus. A study of the proteolytic activity of recombinant IdeE and IdeZ on IgG from a selection of mammals shows that only antibodies containing the substrate site of IdeS/Mac are cleaved, indicating that the specificities of these enzymes are similar. Interestingly, IgG from horse is less effectively cleaved than IgG from e.g. dog or humans, as the dominating IgG isotype in horse sera (IgG4) lacks a distinct substrate site for IdeE/IdeZ. IgG-degradation is observed when S. equi ssp. equi is grown in the presence of horse serum, but not when grown with purified IgG. As the fraction of degraded IgG contains IgG4, the observed activity might be due to the expression of an unknown enzyme rather than IdeE. In a similar assay, no proteolysis of IgG was detected in the growth media of S. equi ssp. zooepidemicus.     

The GABAergic network in the suprachiasmatic nucleus as a key regulator of the biological clock: does it change during senescence?

GABA is the main neurotransmitter of the hypothalamic suprachiasmatic nucleus (SCN) and plays a key role in the function of this master circadian pacemaker. Despite the evidence that disturbances of biological rhythms are common during aging, little is known about the GABAergic network in the SCN of the aging brain. We here provide a brief overview of the GABAergic structures and the role of GABA in the SCN. We also review some age-related changes of the GABAergic system occurring in the brain outside the SCN. Finally, we present preliminary data on the GABAergic system within the SCN comparing young and aging mice. In particular, our study on age-related changes in the SCN focused on the daily expression of the alpha3 subunit of the GABA(A) receptor and on the density of GABAergic axon terminals. Interestingly, our preliminary findings point to alterations of the GABAergic network in the biological clock during senescence.     

Determination of eumelanin in human urine

Normal and malignant melanocytes produce melanins and melanin-related metabolites, most of which are retained in the cells but some are secreted into the blood and then excreted in the urine. In this study, we developed a method to measure levels of eumelanin in urine samples and evaluated its clinical significance in comparison with the melanin-related metabolites 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) and 5-S-cysteinyldopa (5-S-CD), and with pheomelanin, measured after degradation as 4-amino-3-hydroxyphenylalanine (4-AHP). The method is based on the production of pyrrole-2,3,5-tricarboxylic acid (PTCA) on permanganate oxidation of eumelanin, followed by quantification by liquid chromatography. For 118 urine samples from 10 control subjects, mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD and 4-AHP were 19, 67, 37 and 59 micromol/mol creatinine respectively. In melanoma patients (n = 45), the mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD, and 4-AHP were 91, 926, 4070 and 3530 micromol/mol creatinine respectively. Median level of PTCA in melanoma patients was elevated 2.1-fold compared with control subjects. The degrees of elevation for 6H5MI2C, 5-S-CD, and 4-AHP were 1.8-, 22- and 6.2-fold respectively. Thus, although urinary PTCA is of little clinical value in following the progression of melanoma, urinary 4-AHP appears to be of considerable value in this respect.     

Photosynthesis and UV-B Tolerance of the Marine Alga Fucus vesiculosus at Different Sea Water Salinities

The marine algal species in the Baltic Sea are few due to the low sea water salinity. One of the few species that can be found is Fucus vesiculosus. Even this species is affected by the low salinity and becomes smaller in size in the Baltic. In present work the photosynthesis of F. vesiculosus in the northern Baltic (Bothnian Sea) was compared to the photosynthesis of F. vesiculosus in the Atlantic. Oxygen evolution was measured before and after exposure to 2.3 W of UV-B (280–320 nm) radiation for 5 h, as well as after 48 h recovery in low light. The plants were kept in their own sea water salinity as well as in a changed salinity, this to examine possible correlations between salinity and photosynthesis. The results show a significant higher initial maximal photosynthesis (P _max) for Atlantic plants (10.3 nmol O₂ g⁻¹ FW s⁻¹) compared to Baltic plants (4.0 nmol O₂ g⁻¹ FW s⁻¹). The Baltic plants were found more sensitive to UV-B with a 40–50% decrease of P _max as well as a lower degree of recovery (60–70% compared to 75–95% for the Atlantic plants). The higher salinity (35 psu) had a positive effect on the Baltic F. vesiculosus with increased P _max as well as increased tolerance to UV-B. The lower salinity (5 psu) had a negative effect on the Atlantic plants with a decreased P _max as well as a lower tolerance to UV-B. Pigment content was found higher in Atlantic F. vesiculosus. The pigment content decreased then the Atlantic plants were transferred to 5 psu. The concentration of Chl a as well as the total content of violaxanthin, diadinoxanthin and zeaxanthin in Baltic plants increased when transferred to 35 psu. The Atlantic F. vesiculosus can not survive the low salinity in the northern Baltic (died within 8 weeks). It is likely that a long time acclimation or adaptation to low salinity has taken place for F. vesiculosus in northern Baltic. If this is an ecotypic or genotypic development it is too early to say.