Altered dose response to beta-agonists in SERCA1a-expressing hearts ex vivo and in vivo

In this study we evaluated the contractile characteristics of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)1a-expressing hearts ex vivo and in vivo and in particular their response to beta-adrenergic stimulation. Analysis of isolated, work-performing hearts revealed that transgenic (TG) hearts develop much higher maximal rates of contraction and relaxation than wild-type (WT) hearts. Addition of isoproterenol only moderately increased the maximal rate of relaxation (+20%) but did not increase contractility or decrease relaxation time in TG hearts. Perfusion with varied buffer Ca(2+) concentrations indicated an altered dose response to Ca(2+). In vivo TG hearts displayed fairly higher maximal rates of contraction (+ 25%) but unchanged relaxation parameters and a blunted but significant response to dobutamine. Our study also shows that the phospholamban (PLB) level was decreased (-40%) and its phosphorylation status modified in TG hearts. This study clearly demonstrates that increases in SERCA protein level alter the beta-adrenergic response and affect the phosphorylation of PLB. Interestingly, the overall cardiac function in the live animal is only slightly enhanced, suggesting that (neuro)hormonal regulations may play an important role in controlling in vivo heart function.     

Effects of glucose on contractile function, [Ca2+]i, and glycogen in isolated mouse skeletal muscle

Extensor digitorum longus muscles were stimulated to contract to fatigue and allowed to recover for 2 h in the absence or presence of 5.5 or 11 mM extracellular glucose. This was followed by a second fatigue run, which ended when the absolute force was the same as at the end of the first run. During the first fatigue run, the fluorescence ratio for indo 1 increased [reflecting an increase in myoplasmic free Ca2+ concentration ([Ca2+]i)] during the initial tetani, peaking at approximately 115% of the first tetanic value, followed by a continuous decrease to approximately 90% at fatigue. During the first fatigue run, myofibrillar Ca2+ sensitivity was significantly decreased. During the second run, the number of tetani was 57 +/- 6% of initial force in muscles that recovered in the absence of glucose and 110 +/- 6 and 119 +/- 2% of initial force in muscles that recovered in 5.5 and 11 mM glucose, respectively. Fluorescence ratios during the first, peak, and last tetani did not differ significantly between the first and second fatigue runs during any of the three conditions. Glycogen decreased by almost 50% during the first fatigue run and did not change further after recovery in the absence of glucose. After recovery in the presence of 5.5 and 11 mM glucose, glycogen increased 32 and 42% above the nonstimulated control value (P < 0.01). These data demonstrate that extracellular glucose delays the decrease of tetanic force and [Ca2+]i during fatiguing stimulation and that glycogen supercompensation following contraction can occur in the absence of insulin.     

Utilization of Energy Nutrients by Cerebellar Slices

We performed an ontogenetic study about the utilization of glycine, glutamine, -hydroxybutyrate and glycerol as energy nutrients by rat cerebellum slices. Production of CO₂ from glycerol and glutamine increased with the animals' age and glutamine was the most used nutrient for CO₂ production. In adult age, glutamine oxidation to CO₂ was 15 to 35 times higher than all other nutrients studied. CO₂ production from glycine decreased markedly with age and 10 day-old rats showed an oxidation 7.5 times higher than that of adult rats. At fetal age and at 10 postnatal days, glycine oxidation to CO₂ was only 2 times lower than glutamine oxidation to CO₂. Lipid synthesis from -hydroxybutyrate was highest in adult rats. We did not observe any difference in the utilization of -hydroxybutyrate between slices of cerebral cortex and cerebellum at the ages of 10 days and adult. The main nutrients used for lipid synthesis were glycerol and -hydroxybutyrate.     

A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast

Yeast SUMO (Smt3) and its mammalian ortholog SUMO-1 are ubiquitin-like proteins that can reversibly be conjugated to other proteins. Among the substrates for SUMO modification in vertebrates are RanGAP1 and RanBP2/Nup358, two proteins previously implicated in nucleocytoplasmic transport. Sumoylated RanGAP1 binds to the nuclear pore complex via RanBP2/Nup358, a giant nucleoporin, which was recently reported to act as a SUMO E3 ligase on some nuclear substrates. However, no direct evidence for a role of the SUMO system in nuclear transport has been obtained so far. By the use of conditional yeast mutants, we examined nuclear protein import in vivo. We show here that cNLS-dependent protein import is impaired in mutants with defective Ulp1 and Uba2, two enzymes involved in the SUMO conjugation reaction. In contrast, other transport pathways such as rgNLS-mediated protein import and mRNA export are not affected. Furthermore, we find that the yeast importin-alpha subunit Srp1 accumulates in the nucleus of ulp1 and uba2 strains but not the importin-beta subunit Kap95, indicating that a lack of Srp1 export might impair cNLS import. In summary, our results provide evidence that SUMO modification in yeast, as has been suspected for vertebrates, plays an important role in nucleocytoplasmic trafficking.     

MSG lesions decrease body mass of suckling-age rats by attenuating circadian decreases of energy expenditure

Suckling-age rats display endogenous circadian rhythmicity of metabolic rate (MR) with energy-saving, torpor-like decreases, which are sympathetically controlled and suppressed by leptin treatment. We investigated whether neonatal monosodium glutamate (MSG) treatment, known to cause arcuate nucleus damage and adult-age obesity, alters energy balance in the first two postnatal weeks. Continuously recorded MR and core temperatures (T(c)) show that MSG treatment disinhibits the periodic, sympathetically controlled, energy-saving drops of T(c) and MR. Increased energy expenditure thus explains reduced body fat at normal lean body mass found in MSG-treated pups artificially nourished identically to controls. In MSG-treated mother-reared pups, lean body mass is additionally reduced, suggesting that MSG also reduces suckling. Plasma leptin levels are similar in controls and MSG-treated pups but higher per unit of fat mass in the latter. We conclude that the postweaning development of MSG obesity and depressed thermogenesis are preceded by an early phase of increased energy expenditure with decreased fat deposition during suckling age and hypothesize cell damage in the arcuate nucleus to be involved in both.     

Gravity and cyclic GMP levels in melanocytic cells

Guanosine 3',5'-cyclic monophosphate (cyclic GMP) is a major second messenger molecule, that is believed to play a role in various physiological and pathophysiological processes. Here we report that hypergravity induces differential effects on cyclic GMP turnover in melanocytic cells. Nonmetastatic melanoma cells responded to long-time exposure (24 h) of hypergravity (up to 5 x g) with decrease in intracellular cyclic GMP accumulation in the presence of an universal inhibitor of phosphodiesterases (IBMX), whereas the extracellular cyclic GMP increase. In contrast, there were no changes in cyclic GMP turnover in metastatic melanocytes. The expression of the guanylyl cyclases appeared to be not affected. These results suggest that cyclic GMP signaling may be involved in adaptation of human melanocytes to altered gravity conditions.     

HPLC preparation of fish waste hydrolysate fractions. Effect on guinea pig ileum and ACE activity

The effect of RP-HPLC-purified fractions of fish waste hydrolysates issued from three fish industries was tested on guinea pig ileum in order to examine the presence of opioid molecules. The evaluation of anti-hypertensive activities of whole hydrolysates and fractions were also tested, monitoring the ability of the fraction to inhibit the activity of angiotensin I-converting enzyme involved in hypertension regulation. Sardine autolysate and cod head hydrolysate powder (50 microg) were able to inhibit near 30% of ACE activity, whereas 50 microg of shrimp hydrolysate allows the inhibition of 57% of ACE activity. HPLC fractionation of cod head hydrolysate and sardine autolysate was necessary to evidence biological activity, whereas HPLC separation of shrimp hydrolysate exhibited low biological activity fractions. Further studies are necessary to characterise bioactive molecules from cod head alcalase hydrolysate and from sardine autolysate.     

Selenoprotein P in subjects exposed to mercury and other stress situations such as physical load or metal chelation treatment

In plasma, Se is found in plasma glutathione peroxidase (pGSH-Px), selenoprotein P (Sel-P), and albumins. The aim of the present study was to determine the influence of lifelong exposure to various levels of mercury vapor (Hg⁰) on plasma Se content and the fraction bound to Sel-P. Second, a pilot study was performed on the influence of short-term excessive physical stress and metal chelation (DIMAVAL) treatment. Samples of human plasma/serum obtained from a control group, Idrija residents living in a Hg-polluted environment because of the vicinity of the Idrija mercury mine (closed 1994), a few Idrija residents exposed to excessive physical stress, and two retired miners treated with the drug DIMAVAL were investigated. Selenoprotein P was isolated by affinity chromatography (heparin-Sepharose), and the concentrations of selenium were determined by radiochemical neutron activation analysis and hydride generation-atomic fluorescence spectrometry. Regardless of the group investigated, plasma Se average values were very similar (about 70–90 ngSe/g). A significant change of Sel P (7–24% decrease) was noted only in the group exposed to physical stress as compared to the same subjects before the test, to the control group, and to the Hg exposed group. The decrease of Se bound on Sel-P was accompanied by its increase in fraction of pGSH-Px with albumin.     

Copper-containing monooxygenases: enzymatic and biomimetic studies of the O-atom transfer catalysis

This review reports our recent studies or the mechanism of O-atom transfer to a benzylic C-H bond promoted by Dopamine beta-Hydroxylase (DBH) and its biomimetic models. We demonstrate that it is possible to carry out parallel and comparative studies on this enzyme (DBH) and its biomimetic models with the same substrate: 2-aminoindane (3). It was chosen because its two stereogenic centers, both in benzylic positions, make it very powerful for studying the stereochemistry of an O-atom transfer reaction. DBH-catalyzed hydroxylation of 3 produced exclusively 14% of trans-(1S,2S)-2-amino-1-indanol (4) (93% ee). Studies with stereospecifically deuterium-labeled 2-aminoindanes (1R,2S)-3b and (1S,2S)-3a showed that the formation of 4 was the rcsult of an overall process with retention of configuration where an O-atom is stereospecifically inserted in the trans pro-S position of the substrate. With copper(I) and (II) complexes of IndPY2 ligands we studied the reaction with dioxygen and observed an O-atom transfer to a benzylic C-H bond which was performed in the same manner as that of DBH. With the deuterium-labeled cis-2-d-IndPY2 ligand, we demonstrated that the reaction occurs by a stereospecific process with retention of configuration. In both cases (enzymatic vs. biomimetic) the O-atom transfers occur in a two-step process involving radical intermediates.