Main biomarkers associated with age-related plasma zinc decrease and copper/zinc ratio in healthy elderly from ZincAge study.

PURPOSE: Zinc (Zn) plays an essential role in many biological processes including immune response. Impaired Zn status promotes immune dysfunction, and it has been associated with enhanced chronic inflammation during aging. It has been suggested that the measurement of circulating Zn by itself could not reflect the real Zn status of an individual. It is therefore necessary to identify other determinants associated with plasma Zn to better understanding how physiopathological conditions during aging may affect the concentration of this metal. METHODS: We have investigated the association between Zn levels and some biomarkers in 1090 healthy elderly from five European countries to increase the accuracy in the assessment of the Zn status. Stepwise multivariate linear regression models were used to analyze the influence of factors such as age, dietary intake, inflammatory mediators, laboratory parameters and polymorphisms previously associated with Zn homeostasis. RESULTS: Plasma Zn decrement was most strongly predicted by age, while positive correlations were found with albumin, RANTES and Zn intake after adjustment for multiple confounders. HSP70 +1267 AA genotype was an independent factor associated with Zn plasma concentrations. Cu/Zn ratio was positively associated with markers of systemic inflammation and age and negatively associated with albumin serum levels. CONCLUSIONS: Our findings show the most important independent determinants of plasma Zn concentration and Cu/Zn ratio variability in elderly population and suggest that the decline with age of Zn circulating levels is more dependent on physiopathological changes occurring with aging rather than to its nutritional intake.

Modeling of turing structures in the chlorite--iodide--malonic Acid--starch reaction system.

Recent experiments on the chlorite-iodide-malonic acid-starch reaction in a gel reactor give the first evidence of the existence of the symmetry breaking, reaction-diffusion structures predicted by Turing in 1952. A five-variable model that describes the temporal behavior of the system is reduced to a two-variable model, and its spatial behavior is analyzed. Structures have been found with wavelengths that are in good agreement with those observed experimentally. The gel plays a key role by binding key iodine species, thereby creating the necessary difference in the effective diffusion coefficients of the activator and inhibitor species, iodide and chlorite ions, respectively.

Transient turing structures in a gradient-free closed system.

Transient, symmetry-breaking, spatial patterns were obtained in a closed, gradient-free, aqueous medium containing chlorine dioxide, iodine, malonic acid, and starch at 4 degrees to 5 degrees C. The conditions under which these Turing-type structures appear can be accurately predicted from a simple mathematical model of the system. The patterns, which consist of spots, stripes, or both spots and stripes, require about 25 minutes to form and remain stationary for 10 to 30 minutes.

A novel non-opioid binding site for endomorphin-1.

Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the µ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the µ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the µ-opioid receptor. Our recent data indicate that a radiolabelled [3H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no µ-opioid receptors, such as rodent cerebellum, or brain of µ-opioid receptor deficient (MOPr-/-) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.

Characterization of protein kinase and phosphatase systems in chick ciliary ganglion.

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary ganglion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase activities were detected in homogenates of ciliary ganglion dissected from 15-16-day-old embryos. Autophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+/calmodulin or 5 mM ZnSO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose molecular weights corresponded to the regulatory subunits of cyclic AMP-dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following incubation with [gamma-32P]ATP under most conditions had an apparent molecular weight of 65,000 which corresponds to the chicken form of myristoylated alanine-rich C kinase substrate, a known substrate of protein kinase C. Another substrate for protein kinase C was a 45,000 molecular weight protein which was tentatively identified as neuromodulin (B-50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP-dependent protein kinase were detected, protein kinase A strongly labelled a 40,000 molecular weight protein. Using 32P(i)-labelled glycogen phosphorylase, protein phosphatases 1 and 2A were identified in ciliary ganglia homogenates at levels which were indistinguishable from forebrain at the same age. The major endogenous protein substrates in ciliary ganglion homogenates from 15-16-day-old embryos were also labelled to a similar extent in homogenates of ciliary ganglia from newly hatched chickens. Intact ciliary ganglia remained viable for several hours after dissection and, after incubation with 32P(i), responded to phorbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterization of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms responsible for regulating synaptic transmission in this tissue.

Phosphorylation of proteins in chick ciliary ganglion under conditions that induce long-lasting changes in synaptic transmission: phosphoprotein targets for nitric oxide action.

Production of nitric oxide and the activation of protein kinases are required for long-term potentiation of synaptic transmission at the giant synapses in chicken ciliary ganglion. In the present study, we investigated the ability of nitric oxide to regulate the phosphorylation of endogenous proteins under conditions that induced long-term potentiation in intact ciliary ganglion and the protein kinases responsible for the phosphorylation of these proteins in lysed ciliary ganglion. Using Calcium Green-1 we showed that the nitric oxide donor sodium nitroprusside did not change the intraterminal Ca2+ dynamics in ciliary ganglion. Two dimensional phosphopeptide analysis of 32Pi-labelled intact ciliary ganglion showed that the sodium nitroprusside (300 microM) increased the phosphorylation of several phosphopeptides (P50a, P50b and P41) derived from proteins at 50,000 and 41,000 mol. wts which we have called nitric oxide-responsive phosphoproteins. A similar stimulation of phosphorylation was achieved by 8-bromo-cyclic AMP (100 microM), which also induced long-term potentiation, but not by phorbol dibutyrate (2 microM) that does not induce long-term potentiation in ciliary ganglion. When subcellular fractions from lysed ciliary ganglion were labelled in vitro by [gamma-32P]ATP in the presence of purified cGMP-dependent, cAMP-dependent or Ca2+-phospholipid-dependent protein kinases, we identified cyclic GMP-dependent protein kinase substrates that gave rise to phosphopeptides co-migrating with P50a, P50b and P41 from 32Pi-labelled intact ciliary ganglion. P50a and P41 were derived from soluble proteins while P50b was derived from a membrane-associated protein. The proteins giving rise to P50a, P50b and P41 were also substrates for cyclic AMP-dependent protein kinase, but not for calcium and phospholipid-dependent protein kinase in vitro, suggesting that nitric oxide-responsive phosphoproteins are convergence points in information processing in vivo and their phosphorylation might represent an important mechanism in nitric oxide-mediated synaptic plasticity in ciliary ganglion.

Regulation of transendothelial transport in the cerebral microvessels: the role of second messengers-generating systems.

Different elements of the intracellular signaling messenger systems have been detected in the course of our studies in the cerebral endothelial cells. It has been shown that the synthesizing enzymes of and substrate proteins for the second messenger molecules are present in the cerebral endothelial cells, and their activity and/or amount can change in pathological circumstances, i.e., during the formation of brain oedema. Pharmacological treatments interfering with the second messenger systems proved to be effective in the prevention of brain oedema formation.

Early X-irradiation of rats-4. Decrease in phosphorylation of low molecular weight proteins in the olfactory bulb accompanies the loss of GABAergic microneurons.

X-irradiation of neonatal rats by low, fragmented and repeated doses reduces or eliminates several phosphoproteins (16, 18 and 19 Da) in an extract of the olfactory bulb. This coincides with a dramatic reduction of the number of GABAergic granule cells. The phosphorylation of these proteins was specifically stimulated in control animals by Ca(2+)/calmodulin, but not by cyclic adenosine triphosphate. The reduced inhibitory function, observed previously in other "granuloprived" bulbs, might be the consequence of loss of those cells expressing these low molecular weight phosphoproteins. To assess the cellular changes caused by X-irradiation, low molecular weight phosphoproteins can serve as specific markers in the olfactory bulb.

Alpha and beta subunits of CaM-kinase II are localized in different neurons in chick ciliary ganglion.

The ciliary ganglion of the chicken contains only two types of neurons. Using monoclonal antibodies against the alpha and the beta subunits of Ca2+/calmodulin-stimulated protein kinase II (CaMPK-II) we found that the alpha-subunit was localized to the choroid neurons while beta subunit was associated with the ciliary neurons. As both neurons receive their inputs from the oculomotor nerve, while their postganglionic axons leave via different nerves, the ciliary ganglion of the chicken is a neuronal system in which the functional differences between alpha and beta CaMPK-II homopolymers in the regulation of synaptic transmission can be investigated.

Simultaneous measurement of tyrosine hydroxylase activity and phosphorylation in bovine adrenal chromaffin cells.

A method for simultaneous measurement of tyrosine hydroxylase (TH) activation and phosphorylation in permeabilised and intact bovine adrenal chromaffin cells (BACCs) was established. Permeabilised cells were stimulated with cyclic AMP (1--10 microM) in the presence of [32P]ATP and L-[carboxyl-(14)C]tyrosine. Intact BACCs were preincubated with 32P(i) for 3 h and stimulated with forskolin (1--5 microM) in the presence of L-[carboxyl-(14)C]tyrosine. On stimulation each well was covered with a sealed 'chimney' fitted with a small plastic cup containing 300 microl of 1.0 M NaOH that trapped the 14CO(2) released. TH activity was determined by measuring 14C radioactivity. TH phosphorylation was measured in the same cells by separating the solubilized proteins on SDS PAGE followed by autoradiography and/or HPLC analysis. It was found that H89, a protein kinase A inhibitor, significantly blocked both TH phosphorylation and activation in response to cyclic AMP in permeabilised cells. However, in intact cells, H89 was effective only in respect to forskolin-stimulated TH activity and did not block the forskolin-stimulated TH phosphorylation of Ser-40. The reason(s) for this lack of correlation between TH activation and phosphorylation is presently not understood.

Receptor constants for endomorphin-1 and endomorphin-1-ol indicate differences in efficacy and receptor occupancy.

The opioid properties of endomorphin derivatives containing a C-terminal alcoholic(-ol) function were compared to the parent amidated compounds in isolated organs (longitudinal muscle strip of guinea-pig ileum and mouse vas deferens). Similar data were also generated for the mu-opioid receptor selective agonist synthetic peptide (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAMGO) and its Gly5-NH2 congener (DAMGA). Endomorphin-1-ol (Tyr-Pro-Trp-Phe-ol) had an IC50 of 80.6 nM in mouse vas deferens and 61.2 nM in guinea-pig ileum; the corresponding values for endomorphin-2-ol (Tyr-Pro-Phe-Phe-ol) were 49.6 and 48.2 nM, for DAMGO 59.8 and 29.2 nM, respectively. As it was indicated by the antagonism by naltrexone, the agonist actions were exerted exclusively at mu-opioid receptors in both organs. The -ol derivatives were slightly (2.3-4.3 times) less potent than the parent amides in the bioassays: all peptides had, apparently, full agonist properties in intact preparations. With the aim of revealing potential partial agonist properties among the investigated peptides, we partially inactivated the mu-opioid receptor pool in mouse vas deferens by 5x10(-7) M beta-funaltrexamine. The calculated receptor constants indicated a "high-affinity, low intrinsic efficacy" profile (i.e. a potential partial agonist property) for endomorphin-1, an intermediate character for endomorpin-1-ol and full agonism for DAMGA and DAMGO. Apparently, a higher receptor fraction remained accessible for endomorphin-1 (42.8%) than for the -ol congener (14.0%), DAMGO (20.2%) and DAMGA (14.1%) after partial inactivation.

[Hyperuricacidemic nephrogenic diabetes insipidus]. / Hyperuricaemiás nephrogen diabetes insipidus.

Five weeks following the removal of the urate calculus the 58-year-old patient suffered from anadipsia and drank 6-8 litre of water daily. Uric acid level was high in the serum and the carbamide-N and creatinine showed also increased values. Upon the administration of adiuretin the polyuria and polydipsia ceased, yet the specific weight of the urine stayed under 1010.

Agreement between image grading of conventional (45°) and ultra wide-angle (200°) digital images in the macula in the Reykjavik eye study.

PURPOSE: To establish the agreement between image grading of conventional (45°) and ultra wide-angle (200°) digital images in the macula. METHODS: In 2008, the 12-year follow-up was conducted on 573 participants of the Reykjavik Eye Study. This study included the use of the Optos P200C AF ultra wide-angle laser scanning ophthalmoscope alongside Zeiss FF 450 conventional digital fundus camera on 121 eyes with or without age-related macular degeneration using the International Classification System. Of these eyes, detailed grading was carried out on five cases each with hard drusen, geographic atrophy and chorioretinal neovascularisation, and six cases of soft drusen. Exact agreement and κ-statistics were calculated. RESULTS: Comparison of the conventional and ultra wide-angle images in the macula showed an overall 96.43% agreement (κ=0.93) with no disagreement at end-stage disease; although in one eye chorioretinal neovascularisation was graded as drusenoid pigment epithelial detachment. Of patients with drusen only, the exact agreement was 96.1%. The detailed grading showed no clinically significant disagreement between the conventional 45° and 200° images. CONCLUSIONS: On the basis of our results, there is a good agreement between grading conventional and ultra wide-angle images in the macula.

ABO blood typing of human skeletal remains in Hungary.

The author reports about the theoretical effects of his paleoserologic investigations on some historical population genetics problems. First he refers to the essence of the two modifications by the help of which the fluorescent antibody method can be made suitable for blood typing or archeological skeletal remains and determines his working units (sample, series, "population") used in the paleoserologic researches. The benefits of the projection of the ABO blood typing results on the map of the cemetaries are demonstrated. The distribution of the several phenotypes are collated to the character or richness of the grave goods and to the taxonomic features of the late individuals. The thorough examination of the serogenetic distances among the several samples of a given historical period may cast more light on the ethnic interrelations of the earlier populations living in the same geographic area. Following up the serogenetic changes of a population during subsequent historical periods, new ideas can be gained about the importance of the environmental, economic, and demographic factors shaping the serogenetic profile of the population.

Histomorphological and pathobiochemical changes of varicose veins. A possible explanation of the development of varicosis.

The authors should like to contribute to the unsettled problem of the development of varicose venous disease with their own experience. They evaluate parallel the histomorphological observations and certain pathobiochemical changes which can be recognized in exstirpated varicose veins. Fourty five stripped saphenas have been studied. They were divided into two groups: a macroscopically normal and a varicose one. The varicose patients were divided again into two subsamples according to the occurrence or lack of thrombophlebitis in their medical case history. The authors were looking for the appropriate pathobiochemical changes of the vessel walls running parallel to the usual histopathological changes. It seems quite possible that the effect which triggers the development of the disease could be anything which causes hypoxia, alters the energy metabolism of the otherwise bradytrophic vascular tissues. Accumulation of proteoglycans, as well as collagen and elastic fibers in place of the smooth muscle cells may cause a decrease in the elasticity of the veins and may produce favourable conditions to thrombus formation and local inflammation.

A chemical approach to designing Turing patterns in reaction-diffusion systems.

A systematic approach is suggested to design chemical systems capable of displaying stationary, symmetry-breaking reaction diffusion patterns (Turing structures). The technique utilizes the fact that reversible complexation of an activator species to form an unreactive, immobile complex reduces the effective diffusion constant of the activator, thereby facilitating the development of Turing patterns. The chlorine dioxide/iodine/malonic acid reaction is examined as an example, and it is suggested that a similar phenomenon may occur in some biological pattern formation processes.

Inosine increases anoxic tolerance and postischaemic restitution of the heart: experimental studies on possible mechanism(s) of action.

Various experimental models were utilized to determine the in vivo cardiac actions underlying the presumptive therapeutic benefit of the naturally occurring adenine nucleoside, inosine. 1. In pentobarbital-anaesthetized dogs coronary vasodilator responses to arterial hypoxia were augmented and postanoxic depression of cardiac contractile force was prevented by i.v. infusion of inosine (5 mg X kg-1 X min-1). 2. Thermographic observations demonstrated in the in situ dog heart a significant inosine-induced epicardial warming (coronary flow increase) which was resistant to P1-purinergic blockade (aminophylline). 3. Following total cardiopulmonary bypass and cardioplegic cardiac arrest of 90 min duration in the dog, inosine administration (5 mg X kg-1 X min-1) evoked a lasting increase in myocardial contractile force and cardiac output. 4. In Langendorff perfused guinea pig hearts subjected to normothermic ischaemia of 1 hour duration and treated with inosine (0.05 mg X min-1), the release of cytoplasmatic enzymes (CK-MB, alpha-HBDH) and myoglobin was significantly less than in untreated ischaemic controls. In the two latter series of experiments (3 and 4) the inosine action was contrasted by the deleterious effect of catecholamines (noradrenaline and isoproterenol) administered in moderate or small doses, respectively. It was concluded that inosine has cardioprotective actions partly related to and partly independent of its influence on coronary blood supply, suggesting that administration of the nucleoside may be useful in various surgical attempts directed to myocardial salvage.