A. Hernanz

Effect of aging on the modulation of macrophage functions by neuropeptides.

The existence of a functional connection between the nervous and the immune system is supported by increasing recent evidence. In previous work we have shown that peptides from the nervous system, such as gastrin-releasing peptide (GRP), neuropeptide Y (NPY) and sulfated cholecystokinin octapeptide (CCK-8s), have modulatory effects on the immune functions in adult animals. Since the immunodepression found in aging organisms may be related to changes in the neuroimmune network, the aim of the present work was to study the changes with aging in the effect of CCK-8s, GRP and NPY on peritoneal macrophage functions (adherence to tissues, mobility, ingestion of foreign particles and superoxide anion production) from BALB/c mice of three different ages: adult (24+/-2 weeks old), mature (50+/-2 weeks old) and old (72+/-2 weeks old). The results show that the increase in adherence capacity produced by neuropeptides in cells from adult and mature animals disappears in old mice. The stimulatory effect of GRP and NPY on mobility, ingestion and superoxide production in macrophages from adult mice disappears (GRP) or changes to inhibition (NPY) in cells from old animals. The decrease of these functions caused by CCK-8s in adult or mature animals continues in old mice. These data suggest that the modulation by neuropeptides of the macrophage function changes with the age of animals.

Age-related changes in the neuropeptide Y effects on murine lymphoproliferation and interleukin-2 production.

Neuropeptide Y (NPY) modulates several aspects of the immune response but it is not known whether NPY responsiveness is altered with aging. In this work, the in vitro effect of NPY at concentrations ranging from 10(-)(14) M to 10(-)(7) M on lymphoproliferation has been studied in spleen, axillary node and thymus leukocytes from young, adult, mature and old BALB/c mice. The spontaneous proliferation of spleen lymphocytes from young mice was significantly stimulated by NPY. In response to the mitogen Con A, lymphoproliferation and IL-2 release by lymphocytes were inhibited significantly by NPY, these effects disappearing with aging. The results show that NPY is a modulator of lymphoproliferation and that this effect disappears progressively with age. Moreover, this regulatory role of NPY may be carried out through a decrease in IL-2 production.

Modulation of murine lymphocyte functions by sulfated cholecystokinin octapeptide.

The effects in vitro of the sulfated octapeptide form of cholecystokinin (CCK-8) at concentrations ranging from 10(-13) M to 10(-6) M on several functions of murine lymphocytes were studied, i.e. adherence to substrate, mobility (spontaneous and directed by chemical gradient or chemotaxis) and spontaneous and phytohemagglutinin (PHA)-mediated proliferation. Lymphocytes were obtained from peritoneal suspension as well as from axillary nodes, spleen and thymus of BALB/c mice. CCK-8, at concentrations from 10(-10) M to 10(-8) M, significantly inhibited the mobility capacity and the PHA-induced proliferation and increased the adherence and the spontaneous proliferation of lymphocytes. A dose-response relationship was observed, with a maximum effect on lymphocyte functions at 10(-10) M. In addition, CCK-8 induced a significant decrease in membrane and cytosol protein kinase C (PKC) activity in murine lymphocytes, as well as an increase of intracellular cyclic AMP levels. These results suggest that CCK-8 is a negative modulator of two important lymphocyte functions in the immune response, i.e. mobility and mitogen-induced proliferation, and that the PKC activity inhibition and cAMP increase could be the mechanisms through which CCK inhibits these lymphocyte activities.

Vibrational analysis and spectra of orotic acid

The IR and Raman spectra of polycrystalline anhydrous orotic acid and its N1, N3, and O12 trideuterated isotopomer are recorded in the 4000-40 cm(-1) spectral interval as part of a series of vibrational analyses of nucleosides, nucleotides, and related compounds carried out in our laboratory. The frequencies of the fundamental transitions and the potential energy distributions of the 39 normal modes of orotic acid and its isotopomer are calculated by an ab initio density functional theory Becke3P86/6-311G** treatment. Assignments of the vibrational modes are proposed that consider the results of these calculations and the observed spectra. The results of the ab initio treatment are related to crystallographic and spectral data, and they are compared with previous assignments for similar molecules.

Normal coordinate analysis and vibrational spectra of adenosine

The Fourier transform infrared and Fourier transform Raman spectra of adenosine in the polycrystalline state were recorded in the 4000- to 30-cm−1 spectral region as part of a series of normal coordinate analyses of nucleic acid components and their analogues carried out in our laboratory. The harmonic frequencies and potential energy distributions (PED) of the vibrational modes of adenosine are calculated by two different methods: a classical molecular mechanics method and the semiempirical molecular orbital (MO) method, PM3. The results of both computational methods, based on Wilsons matrix method, are compared with observed spectra, and an assignment of the vibrational modes of adenosine is proposed on the basis of the PED and the results of calculations for the 1,3-15N2, 2-13C, 8-2H, and 1′-2H isotopomers. It is found that the wavenumbers can be calculated with remarkable accuracy (≈1% deviation in most cases), with the classical mechanics method, by transferring a sufficiently large set of available harmonic force constants, thus permitting a reliable assignment. The semiempirical MO method, PM3, is found to be useful for the assignment of experimental frequencies, although it is less accurate (≈10% deviation). Infrared intensities calculated by this method did not coincide with the experimental values. Certain out-of-plane vibrations in the base, not reported in previous studies, have been observed. The performance of both methods was related to the crystallographic and ab initio data available. Previous normal coordinate calculations for the adenine base and the nucleoside 5′-dGMP are compared with the present results and discussed.

In situ characterization by Raman and X-ray fluorescence spectroscopy of post-Paleolithic blackish pictographs exposed to the open air in Los Chaparros shelter (Albalate del Arzobispo, Teruel, Spain)

An in situ study of post-Palaeolithic blackish pictographs found in an open air rock-shelter, Los Chaparros site (Albalate del Arzobispo, Teruel province, Spain), was carried out to identify the black pigments used. The composition of the pigments was analyzed by means of non-invasive instrumentation, such as a portable Raman spectrometer (RS) and a hand-held energy dispersive X-ray fluorescence (EDXRF) analyzer. In addition, some black natural deposits with a dendritic pattern, typical of manganese compounds, were also in situ analysed with the aforementioned techniques to explore the possibility that post-Paleolithic people used minerals from the surroundings of the Los Chaparros rock-shelter to elaborate the paintings. The results obtained by the EDXRF analyses of black pigments showed differences in composition between a black Levantine deer, in which manganese was present as the main element, and a deep red Schematic pictograph that included manganese as the secondary element. The results of Principal Component Analysis (PCA) of collected EDXRF spectra showed similarities in the elemental composition between the manganese dendrite formations present in the rock-shelter and the black deer. In order to confirm this, the in situ analytical campaign was completed with some analysis in the laboratory by using micro-RS (μ-RS) and X-ray diffraction (XRD) on mineral samples having black crystallisations. Two specimens were taken, one from the black dendrite present in the same rock-shelter and the other from the Los Mases de Crivillen mining area (which is near to Los Chaparros). These analyses revealed that the characteristic bands of Mn–O and Mn–OH bending and stretching vibrations obtained in situ on the black pictograph were the same as those observed in the Raman spectra of the dendrite mineralization of Los Chaparros obtained in the laboratory by μ-RS.

Changes with age in the modulation of natural killer activity of murine leukocytes by gastrin-releasing peptide, neuropeptide Y and sulfated cholecystokinin octapeptide

Several investigations have suggested that the interactions between the nervous and immune systems are modified with age. The aim of the present work was to study the effect of three neuropeptides: gastrin-releasing peptide (GRP), neuropeptide Y (NPY) and sulfated cholecystokinin octapeptide (CCK-8 s) on natural killer (NK) activity of spleen, thymus and axillary node leukocytes from BALB/c male, young (8+/-1 weeks), adult (24+/-2 weeks) and old (72+/-2 weeks) mice. We used cells from murine lymphoma YAC-1 as targets for the cytotoxic assay and three physiological concentrations of the neuropeptides (10(-8), 10(-10) and 10(-12) M). In control samples, in the absence of neuropeptide, we observed a decreased NK activity in young and old mice with respect to the adults in the three organs studied. Regarding the effect of the neuropeptides, GRP stimulates the cytotoxic activity of leukocytes from all locations, in adult animals. At the same age, NPY also stimulates the NK activity of leukocytes from axillary nodes and thymus, whereas it decreases the NK activity of spleen leukocytes from young mice. CCK-8 s has an inhibitory effect on the axillary node leukocytes from young mice and spleen leukocytes from old animals. However, CCK-8 s increased the NK activity of thymus leukocytes from young and adult mice. The results indicate that the highest values of NK activity are found in adult mice, and that the stimulating effect of the three neuropeptides studied on NK activity of leukocytes from adult mice are reduced or disappeared, in general, in old as well as in young animals. Furthermore, the changes observed with ageing in the modulation of NK activity by the neuropeptides studied suggest an altered integration of the nervous and immune systems.

Changes with aging and physical exercise in ascorbic acid content and proliferative response of murine lymphocytes

Ascorbic acid content and lymphoproliferative response to phytohemagglutinin were measured in lymphocytes from axillary nodes, spleen and thymus of young (15 +/- 2 weeks) and old (60 +/- 5 weeks) BALB/c mice. Ascorbic acid content in lymphocytes from spleen and thymus was found to be significantly higher and the lymphoproliferative response in the three immunocompetent organs significantly lower in old mice as compared to young mice. Moreover, young and old BALB/c mice were required to maintain a swimming activity until exhaustion (exhaustive exercise) or 90 min of swimming each day for a total of 20 days (continuous exercise). In both young and old mice the stress produced by exhaustive exercise and confirmed by the existence in serum of significantly increased levels of corticosterone compared to controls, caused a significant decrease in ascorbic acid content as well as in lymphoproliferative response. Continuous exercise, characterized by the presence in serum of significantly decreased levels of corticosterone compared to controls, produced the most significant decrease in ascorbic acid content from young and old murine lymphocytes. Moreover, this exercise resulted in a significant increase in lymphoproliferative response. Our results suggest that aging results in an increase in the ascorbic acid content of lymphocytes accompanied by a decline in the lymphoproliferative response in old BALB/c mice.

Micro-Raman spectroscopic investigation of external wall paintings from St. Dumitru’s Church, Suceava, Romania

The external sixteenth century wall paintings of St. Dumitru’s Church in Suceava (Romania) are suffering visually from deterioration. Fragments of these paintings spallated from the external wall have been studied by micro-Raman microscopy in order to elucidate possible causes of this process. Calcite and α-quartz are the components of the substratum indicating that the artists used the Roman fresco technique comprising a limewash putty. No organic binders have been detected in the substrate or pigment application. Amorphous carbon and goethite, α-FeOOH, have been identified in areas containing residues of grey and yellow pigments, respectively. Small amounts of gypsum have been detected in the grey areas which we attribute to special attention being given to surface preparation and pigment application in these areas. An abundance of sodium nitrate, nitratine, microcrystals have been observed on the surfaces of many fragments which suggests that a biodeterioration process originating from guano deposits could have been operating in these frescoes.

Vibrational analysis and spectra of cytidine 3′-monophosphate (3′-CMP)

Abstract The FTIR and FT-Raman spectra of cytidylic acid b (cytidine 3′-monophosphate, 3′-CMP) and its hexadeuterated derivative (deuteration in the amino and hydroxyl groups) in the solid phase and aqueous solutions have been assigned on the basis of normal coordinate analysis. This was carried out using a valence force field based on force fields for simpler molecules and modified for 3′-CMP, as well as applying the semiempirical PM3 method. Assignments are discussed in the light of observed and calculated wavenumbers and isotopic shifts. The wavenumbers calculated with a large set of force constants result in lower deviation than the wavenumbers calculated by the PM3 method. The results obtained indicate vibrational coupling between the cytosine and ribose moieties of the nucleotide for some frequencies below 1400 cm −1 .