NO enhances presynaptic currents during cerebellar mossy fiber-granule cell LTP

Nitric oxide (NO) is a candidate retrograde messenger in long-term potentiation (LTP). The NO metabolic pathway is expressed in the cerebellar granule cell layer but its physiological role remained unknown. In this paper we have investigated the role of NO in cerebellar mossy fiber-granule cell LTP, which has postsynaptic N-methyl-d-aspartate (NMDA) receptor-dependent induction. Pre- and postsynaptic current changes were simultaneously measured by using extracellular focal recordings, and NO release was monitored with an electrochemical probe in P21 rat cerebellar slices. High-frequency mossy fiber stimulation induced LTP and caused a significant NO release (6.2 +/- 2.8 nM; n = 5) in the granular layer that was dependent on NMDA receptor as well as on nitric oxide synthase (NOS) activation. Preventing NO production by perfusing the NOS inhibitor 100 microM NG-nitro-l-arginine (L-NNA), blocking extracellular NO diffusion by 10 microM MbO2, or inhibiting the NO target guanylyl cyclase (sGC) with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-dione (ODQ) prevented LTP. Moreover, the NO donor 10 microM 2-(N,N-diethylamino)-diazenolate-2-oxide.Na (DEA-NO) induced LTP, which was mutually occlusive with LTP generated by high-frequency stimulation, prevented by ODQ, and insensitive to NMDA channel blockade (50 microM APV + 25 microM 7-Cl-kyn) or interruption of mossy fiber stimulation. Thus NO is critical for LTP induction at the cerebellar mossy fiber-granule cell relay. Interestingly, LTP manipulations were accompanied by consensual changes in the presynaptic current, suggesting that NO acts as a retrograde signal-enhancing presynaptic terminal excitability.     

Primary osteoblasts response to shock wave therapy using different parameters

Over the past decade extracorporeal shock-wave therapy (ESWT) has been increasingly applied to orthopaedic and musculoskeletal pathologies, the aim of this study was to assess how the energy density of the shock waves and the number of impulses affect viability, differentiation and synthetic activity of osteoblasts. Primary sheep osteoblasts cultures were treated with ESWT with an electro-hydraulic shock wave generator by selecting three different energy levels (14-21-28 kV corresponding at 0.15-0.31-0.40 mJ/mm2) and two different total numbers of impulses (500, 1000) for each level. At the end of treatment, cell counts and viability were recorded. Cells were then cultivated for 48 hours starting from a concentration of 1 x 10(4) cells/ml. The biological activity and viability were evaluated at 24 and 48 hours after treatment. No cytodestructive effects were observed in Group A, while a cytodestructive effect of ESWT was seen in cultures receiving the highest energy treatments. The different shock wave treatment induced differences in MTT assays after 24 and 48 hours, in particular the highest level showed a detrimental effect on cell respiration at both experimental times as compared to the Control Group and the protein metabolism was generally depressed by ESWT with impulses at the highest energy level. After 24 hours such effect further increased with the growing number of impulses. The lowest energy level appeared to significantly improve the metabolic parameter in primary cell cultures as compared to controls when 500 impulses were selected. The current study has demonstrated that one of the most important aspects to be considered is not the total number of impulses used but the energy level of the shock waves, thus confirming that ESWT has a dose-dependent effect on cells.     

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.     

Neural precursor proliferation and newborn cell survival in the adult rat dentate gyrus are affected by vitamin E deficiency

The adult hippocampal neurogenesis is affected by vitamin E deficiency. In the present investigation we examined if neural precursor proliferation, newborn cell survival or both are altered by vitamin E deficiency. 5-Bromo-2'-deoxyuridine (BrdU) was employed as a marker of proliferating cells. BrdU-labelled cells were revealed 1 and 30 days after BrdU administration in order to evaluate proliferation and newborn cell survival, respectively. Cell proliferation decreased in controls from juvenile to adult age, and the decrease was lesser in vitamin E deficiency. Thus we found a higher number of proliferating cells in vitamin E-deficient rats than in age-matched controls at 5 months of age. Comparing the number of BrdU-positive cells between 1 and 30 days after the last BrdU injection revealed a remarkable decrease in all groups; this is the greatest in vitamin E-deficient rats and the lowest in control rats. Consistently cell death in the dentate gyrus, assessed by TUNEL technique, was found to decrease from 1 to 5 months of age, but at 5 months it was significantly higher in vitamin E-deficient rats than in age-matched controls. These data show that vitamin E deficiency enhances neural precursor proliferation and cell death during adult neurogenesis.     

XX sex reversal, palmoplantar keratoderma, and predisposition to squamous cell carcinoma: genetic analysis in one family

We describe a large inbred Sicilian family that includes four 46, XX (SRY-) brothers. Palmoplantar hyperkeratosis (PPK) and an associated predisposition to squamous cell carcinoma (SCC) of the skin, segregates as a recessive trait within the family. Interestingly, all the PPK-affected members of the family are phenotypic males (46,XY or 46,XX) while seven XX sibs are healthy phenotypic females with no signs of PPK. We propose that homozygosity for a single mutational event, possibly including contiguous genes, may cause PPK/SCC in both XY or XX individuals and sex reversal in XX individuals. The family is informative for linkage analysis for the PPK trait and allows linkage exclusion for the sex reversal trait. Here we show that 15 loci involved in PPK etiology, skin differentiation, function or malignancy, and nine loci involved in sex determination/differentiation are not implicated in the phenotype of this family.     

Luminal surface microgeometry affects platelet adhesion in small-diameter synthetic grafts

One of the major problems when using small-diameter vascular grafts in arterial reconstruction is the development of platelet-rich thrombi as a consequence of blood contact with artificial surfaces. The degree of occlusion is certainly affected by the thrombogenicity of the internal surface that seems to play a key role in patency and long-term wound healing of grafts. In this study, the blood compatibility of Cardiothane (CA) vascular grafts was investigated. The CA material, a blend of polyurethane and polydimethylsiloxane that has shown relatively good physical and biocompatibility properties, was manufactured into vascular grafts by the instrument named "spray-machine". Grafts with different luminal surface porosity were produced using increasing CA concentrations by the "spray-machine" and the blood compatibility was evaluated in vitro by a circulation system in which the human blood was allowed to interact with the material in a well-controlled setting. The samples of circulating blood were collected at different times of circulation and platelet adhesion and activation were studied. Grafts with a highly porous luminal surface induced a lower adhesion and activation of platelets in vitro than the low-porosity ones. These results underlined the importance of the microgeometry of the graft luminal surface in the interaction with blood.     

Novel MC1R variants in Ligurian melanoma patients and controls

Several variant forms of the melanocortin-1 receptor gene (MC1R) have been associated with red hair, fair skin and an increased risk for melanoma. Their involvement in melanoma susceptibility is apparently linked both to skin sensitivity and to non-pigmentary pathways. We investigated the frequency of the MC1R variants in the Italian region of Liguria, where the occurrence and penetrance of melanoma are low and primary susceptibility is characterized by prevalence of the CDKN2A c.301G>T [p.G101W] founder mutation. Additionally, we attempted to establish the frequency of the red hair/fair skin phenotype in our region. As predicted by anecdotal evidence, the frequency of red hair/phototype I was very low (0.7%). Screening of 17 red-haired individuals and their red-haired relatives, 207 controls and 214 melanoma patients unselected for hair color but all of Ligurian descent, led to the detection of 8 novel substitutions (c.133T>C [p.F45L], c.248C>T [p.S83L], c.332C>T [p. A111V], c.479G>A [p.R160Q], c.637C>T [p.R213W], c.793G>A [p. V265I], c.923C>T [p. T308M], c.943T>C [p.C315R]), 1 novel deletion (c.520_523delGTC [p.V174del]) and 3 novel synonymous variants (c.366G>C [p. V122V], c.684G>A [p. Q228Q], c.726C>T [p.T241T]). Preliminary genotype/phenotype correlation seems to indicate that other genes involved in the regulation of human pigmentation may mask the recessive action of high-penetrance MC1R alleles, thus determining the low frequency of at-risk phototypes and of incidence and/or penetrance of melanoma in Liguria.     

The detection by cellular electrophoresis of surface antibodies in human lymphocytes

The electrophoretic mobility of peripheral blood lymphocytes from human subjects with positive Mantoux tests was measured before and after treatment with purified protein derivative. Lymphocytes from subjects with negative Mantoux reactions were used as controls. A large number of the first group lymphocytes showed a reduction of electrophoretic mobility and hence of the surface electrical charge. The mobility of the control cells submitted to the same treatment was unchanged. The possible presence of cytophilic antibodies or of cellular specific receptors on the lymphocyte surface is discussed.     

A bone substitute composed of polymethylmethacrylate and alpha-tricalcium phosphate: results in terms of osteoblast function and bone tissue formation

The biological properties of a composite polymeric matrix (PMMA + alpha-TCP) made of polymethylmethacrylate (PMMA) and alfa-tricalciumphosphate (alpha-TCP) was tested by means of in vitro and in vivo investigations. PMMA was used as a comparative material. Osteoblast cultures (MG 63) demonstrated that PMMA + alpha-TCP significantly and positively affected osteoblast viability, synthetic activity and interleukin-6 level as compared to PMMA. At 12 weeks, the PMMA + alpha-TCP implants in rabbit bone successfully osteointegrated in trabecular and cortical tissue (affinity index: 57.14+/-8.84% and 68.31+/-6.18%, respectively). The newly formed bone after tetracycline labelling was histologically observed inside PMMA + alpha-TCP porosity. The microhardness test at the bone-PMMA + alpha-TCP interface showed a significantly higher rate of newly formed bone mineralization compared with PMMA (+83.5% and +58.5%, respectively), but differences still existed between newly formed and pre-existing normal bone. It is herein hypothesized that the present positive results may be ascribed to the porous macroarchitecture of PMMA + alpha-TCP and the presence of the bioactive ceramic material that could have a synergic effect and be responsible for the improvement of (a) the material colonization by bone cells, (b) osteoblast activity, (c) osteoinduction and osteoconduction processes, (d) bone remodelling.