Functional consequences of ligand-linked dissociation in hemoglobin from the sea cucumber molpadia arenicola

It has been established that Molpadia hemoglobin tends to dissociate into subunits as oxygen is bound. The kinetics and equilibria of carbon monoxide and ehtylisocyanide binding reported here show a dependence on protein concentration that supports the conclusions that the aggregated hemoglobin has a lower ligand affinity than the dissociated subunits. This is true for the isolated D-chain as well as for the unfractionated hemolysate that contains four distinct polypeptide chains (A-D). This indicates that even homopolymers of Molpadia hemoglobin have lower ligand affinity than the dissociated subunits. At high protein concentration hemolysates of Molpadia hemoglobin show slight cooperativity. The time course of ligand binding to the deoxy D-chain also suggests cooperative interactions, The low affinity of the aggregated state may have a different molecular explanation than in human hemoglobin were tetramers of identical subunits (as in Hb H) show high oxygen affinity. The absence of tyrosine and histidine at the C-tremini of the Molpadia D-chains also suggests a different stabilization of the low affinity deoxy state. An additional functional difference between Molpadia hemoglobin and human hemoglobin is that organic phosphate do not alter the ligand affinity of the sea cucumber hemoglobin.     

Spot hemoglobin. Studies on the Root effect hemoglobin of a marine teleost.

The Spot, Leiostomus xanthrus, has a single tetrameric hemoglobin. Structural studies indicate the presence of alpha- and beta-like chains with COOH-terminal sequences of --Arg and --TYR-His, respectively, the same as is found in human hemoglobin. Spot hemoglobin possesses a Root effect: a heterotropic control mechanism like the Bohr effect but with more extreme pH dependence in the equilibria and kinetics of O2 and CO binding. The Root effect seems to be a molecular adaptation, in that pH- and anion-sensitive hemoglobins may help fish achieve neutral buoyancy by facilitating O2 delivery to the swim bladder. Changes in the kinetics of both "on" and "off" processes contribute to the greatly decreased ligand affinity of Spot hemoglobin at low pH. The time course ofligand combination at low pH is biphasic and wavelength dependent, suggesting a differential effect of pH on the alpha- and beta-like chains. The change in the shape of the ligand-binding curve with pH may be interpreted in terms of a proton-dependent transition between low (T) and high (R) affinity conformations. However, this may not be the only mechanism, since differential pH effects on the two types of chains may also contribute to the observed pH dependence.     

UVB radiation prevents skeleton growth and stimulates the expression of stress markers in sea urchin embryos

Ozone depletion results in an increased flux of biologically damaging radiations reaching the earth. Although ultraviolet (UV) penetration is attenuated by the seawater, harmful effects can be still observed at low depths where sea urchin embryos are living. We have used Paracentrotus lividus embryos to study the impacts of UV radiation on their development. Blastula cultures were exposed to different doses of UVB (312 nm) radiations and the resulting endpoint effects were evaluated in terms of embryonic morphological abnormalities, variations in specific gene expression, and changes in the levels of stress proteins. We found that embryos were moderately sensitive to 50 J/m² UVB radiation; an increase in the number of developmentally delayed and malformed embryos was detected when increasing doses, up to 1000 J/m², were used. Major developmental defects, observed 24 and 48 h after exposure, consisted in the failure of skeleton elongation and patterning. Accordingly, we found a reduction in the number of primary mesenchyme cells that expressed Pl-SM30, a gene coding for one of the specific matrix proteins of the skeleton. The morphological effects observed 1, 24, and 48 h after exposure were correlated with a dose-dependent increase in the level and in the activation of two recognized stress markers, namely hsp70 and p38 MAPk, respectively, consistent with their role in mediating cellular response to stress and suggesting a function in embryo survival.     

Overproduction of alpha chains provides a proton-insensitive component to the bluefish hemoglobin system

Expression of alpha and beta chains and their post-translational assembly into alpha(2)beta(2) tetramers is fundamental to the formation and function of most vertebrate hemoglobins. There is a strong evolutionary bias that favors expression of equal amounts of the two types of chains, because cooperativity, pH sensitivity, and anionic control of function occurs only for the alpha(2)beta(2) tetramers. Remarkably, an over-production of alpha chains, as in the pathological condition known as beta thalassemia in humans, is adaptive rather than pathological in the bluefish hemoglobin system. The thalassemia of the bluefish is a novel means of providing for oxygen uptake and delivery when low pH conditions incapacitate the highly pH-sensitive Root effect hemoglobins of the fish. Although fish often have pH-insensitive along with highly pH-sensitive hemoglobins, having pH-insensitive alpha chain monomers in circulation is an unusual structural variation. The role of bluefish alpha chains in oxygen transport is enabled by their remarkably lower oxygen affinity relative to human alpha chains. This is the first reported case of a thalassemic condition that is maintained in a species as an adaptive advantage.     

Functional coupling of oxygen binding and vasoactivity in S-nitrosohemoglobin

S-Nitrosohemoglobin (SNO-Hb) is a vasodilator whose activity is allosterically modulated by oxygen ("thermodyamic linkage"). Blood vessel contractions are favored in the oxygenated structure, and vasorelaxant activity is "linked" to deoxygenation, as illustrated herein. We further show that transnitrosation reactions between SNO-Hb and ambient thiols transduce the NO-related bioactivity, whereas NO itself is inactive. One remaining problem is that the amounts of SNO-Hb present in vivo are so large as to be incompatible with life were all the S-nitrosothiols transformed into bioactive equivalents during each arterial-venous cycle. Experiments were therefore undertaken to address how SNO-Hb conserves its NO-related activity. Our studies show that 1) increased O(2) affinity of SNO-Hb (which otherwise retains allosteric responsivity) restricts the hypoxia-induced allosteric transition that exchanges NO groups with ambient thiols for vasorelaxation; 2) some NO groups released from Cys(beta93) upon transition to T structure are autocaptured by the hemes, even in the presence of glutathione; and 3) an O(2)-dependent equilibrium between SNO-Hb and iron nitrosylhemoglobin acts to conserve NO. Thus, by sequestering a significant fraction of NO liberated upon transition to T structure, Hb can conserve NO groups that would otherwise be released in an untimely or deleterious manner.     

Identification of the two histidine residues responsible for the inhibition by malonyl-CoA in peroxisomal carnitine octanoyltransferase from rat liver

Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 microM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.     

Absence of Helicobacter pylori in tonsillar swabs from dyspeptic patients

Aim of this study was to evaluate whether tonsils might be a potential reservoir for Helicobacter pylori (H. pylori) infection. A total of 72 consecutive dyspeptic patients undergoing endoscopy for the first time were studied. For each patient, a bilateral tonsillar swab was performed, before endoscopy, for microbiological culture and immunochemical analysis. Antral biopsies were also collected at endoscopy for microbiological culture, rapid urease test, and histological examination. Helicobacter pylori infection was detected in 42 of 72 (58.3%) patients. All tonsillar specimens were negative for H. pylori on both microbiological culture and immunochemical analysis, suggesting that the tonsils are not an extragastric reservoir for H. pylori infection.     

Postconflict Behavior Among Male Japanese Macaques

Reconciliation was first described more than 20 years ago. Since then, it has been observed in many mammals (mainly primates) but data on postconflict behavior among males are still scarce because they usually aggressively compete for mating partners, rarely maintain amicable relationships with one another. Accordingly, reconciliation is expected to occur at low rates. Although this is true for Japanese macaque males, the subspecies on Yakushima Island (Macaca fuscata yakui) seems to represent an exception as grooming among males occurs often. We analyzed postconflict behavior among them and discuss the possible factors that may favor the occurrence of grooming and reconciliation. Selective attraction between former opponents—reconciliation—occurred soon after conflicts. Consolation—affiliative interactions between a focal animal and group members other than the former opponents occurring earlier in PCs than in MCs—was absent among males. Conciliatory tendency is higher for Yakushima macaque males (0.31) versus that in studies on the other subspecies Macaca fuscata. We discuss differences in the behavioral ecology of the 2 subspecies, the ecological and social factors that may favor the occurrence of reconciliation, and the possible benefits that males gain from grooming exchange and reconciliation.     

Salmonella typhimurium-endocarditis secondary to an acquired environmental infection: a case report

A diabetic, cardiopathic and anemic 44-year-old farmer presented with a seven-day history of remittent fever with evening peaks. Two months before he had undergone amputation of the V-finger of the left hand secondary to a phlegmon caused by an agricultural injury. Prior to amputation, anaerobic culture analysis of phlegmon-pus and selective procedures used to isolate Gram-positive cocci and/or Pseudomonas spp. resulted negative. The diagnosis of endocarditis was supported by isolation of S. typhimurium from blood and by echocardiography showing endocarditic lesions. The source of infection was identified by PCR ribotyping as the same Salmonella typhimurium strain that was present, but not sought, both in the anatomic explanted tissues and from blood samples of the patient. The infection was successfully treated with a combination of gentamicin and ampicillin with consequent improvement in the general clinical picture of the patient. We believe this is the first reported case of S. typhimurium-endocarditis secondary to a phlegmon resulting from an environmental source of infection.