Cytochemical and immunocytochemical investigations on epidermal mitochondria-rich cells in Salamandra salamandra salamandra (L.) larvae

In the present study we set out to investigate the expression of E-cadherin, N-cadherin, β₁-integrin, fibronectin and vitronectin in the mitochondria-rich cells (MRC) of the skin of Salamandra salamandra salamandra. Moreover MRC were stained with five lectins (Triticum vulgaris; Dolichos biflorus; Glycine max; Arachis hypogaea and Canavalia ensiformis). Larval MRC expressed both adhesion molecules and extracellular matrix glycoproteins and bound all lectins tested. Juvenile MRC did not react with the antisera utilized, but they stained with the lectins. Both the lectins and the regulatory molecules proved to be good cytochemical markers for distinguishing morphologically differentiated MRC during the larval life of Salamandra salamandra salamandra. The adhesion molecules and matrix glycoproteins are of great utility for maintaining the correct tissue architecture. In Salamandra salamandra salamandra larvae these molecules may be crucial for stability and for the correct localization and fate of all skin elements, including specialized cells such as larval MRC.     

Sylvatic plague reduces genetic variability in black-tailed prairie dogs

Small, isolated populations are vulnerable to loss of genetic diversity through in-breeding and genetic drift. Sylvatic plague due to infection by the bacterium Yersinia pestis caused an epizootic in the early 1990s resullting in declines and extirpations of many black-tailed prairie dog (Cynomys ludovicianus) colonies in north-central Montana, USA. Plague-induced population bottlenecks may contribute to significant reductions in genetic variability. In contrast, gene flow maintains genetic variability within colonies. We investigated the impacts of the plague epizootic and distance to nearest colony on levels of genetic variability in six prairie dog colonies sampled between June 1999 and July 2001 using 24 variable randomly amplified polymorphic DNA (RAPD) markers. Number of effective alleles per locus (n(e)) and gene diversity (h) were significantly decreased in the three colonies affected by plague that were recovering from the resulting bottlenecks compared with the three colonies that did not experience plague. Genetic variability was not significantly affected by geographic distance between colonies. The majority of variance in gene fieqnencies was found within prairie clog colonies. Conservation of genetic variability in black-tailed prairie dogs will require the preservation of both large and small colony complexes and the gene flow amonog them.     

New generation QuIC assays for prion seeding activity

The ability of abnormal TSE-associated forms of PrP to seed the formation of amyloid fibrils from recombinant PrP^Sen has served as the basis for several relatively rapid and highly sensitive tests for prion diseases. These tests include rPrP-PMCA (rPMCA), standard quaking-induced conversion (S-QuIC), amyloid seeding assay (ASA), real-time QuIC (RT-QuIC) and enhanced QuIC (eQuIC). Here, we summarize recent improvements in the RT-QuIC-based assays that enhance the practicality, sensitivity and quantitative attributes of assays QuIC and promote the detection of prion seeding activity in dilute, inhibitor-laden fluids such as blood plasma.     

The role of activity in synaptic degeneration in a protein misfolding disease, prion disease

In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer''s, Parkinson''s and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in naïve mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration.     

Genotoxicity evaluation in chronic renal patients undergoing hemodialysis and peritoneal dialysis, using the micronucleus test

Patients with chronic renal disease have an increased incidence of cancer. It is well known that long periods of hemodialysis treatment are linked to DNA damage due to oxidative stress. This genotoxic effect may cause the loss of chromosome fragments, or even entire chromosomes, which form micronuclei after cell division, and can be detected by the micronucleus test. In the present case-control study, we evaluated the genotoxic effect of hemodialysis treatment in 20 patients undergoing hemodialysis, and 20 subjected to peritoneal dialysis, matched for gender and age with 40 controls. Genetic damage was assessed by examining the frequency of micronuclei in 2000 exfoliated buccal cells per individual. Our results revealed that patients undergoing hemodialysis treatment have a significantly higher frequency of micronucleated cells (MNC; 5.60 +/- 5.31) compared to control subjects (1.50 +/- 2.01, p < 0.01). Interestingly, the same was not observed for the peritoneal dialysis patients who showed no significant differences in MNC (2.85 +/- 2.96) frequency compared to control individuals (3.25 +/- 3.85). In addition, we evaluated the possible association between creatine levels, smoking, alcohol intake, age, duration of treatment, and incomes of the individuals (separately analyzed according to their gender) and the frequency of micronuclei. The results reported here indicate that the duration of treatment is the only factor associated with increased MNC frequency among hemodialysis patients (Spearman coefficient of 0.414, p = 0.01). The number of MNC found in individuals with six years or less of treatment was significantly lower (2.91 +/- 2.74) compared to patients with seven or more years of treatment (8.89 +/- 5.96, p < 0.05). Overall, peritoneal dialysis may be a safer choice of treatment, but further studies need to be performed to investigate the risks and benefits of both treatments.     

Epidemiological analyses on animal parasitoses: recent activity of the I.M.I.P.P.V

The paper presents a selected review of the epidemiological analyses performed in recent years (1984-1988) in the Institute of Malattie Infettive, Profilassi e Polizia Veterinaria of the University of Bologna on animal parasitoses, with special reference to the evaluation of some risk factors of bovine, swine and canine helminthoses, and to the validity of the coprological test for some parasites of the red fox.     

Studies on the antigenic properties of serum albumins

Bovine serum albumin (BSA) is one ofthe most widely studied proteins; its structure iswell known and its antigenic properties have beendescribed in animal models. The aimof our work was to evaluate the role of conformationon antigenicity of serum albumins. This study was performed using electrophoresisassociated with the immunoblotting technique, wheresera from children allergic to BSA were used.Data obtained in this research indicatethat serum albumin antigenicity is only partiallycorrelated to its native three-dimensional structure.Heat treatment and chemical denaturation(SDS treatment) are not able to significantly decrease its capability to bind circulating IgEs. Only thereducing treatment is able to modify the antigenicityof this protein. Moreover, a direct correlationbetween the cross-reactivity observed inimmunoblotting between different serum albumins andthe percentage of their sequence identity(phylogenetic similarity of the species) was shown.     

Xanthophore migration from the dermis to the epidermis and dermal remodeling during Salamandra salamandra salamandra (L.) larval development

During larval development of Salamandra salamandra salamandra chromatophores organize to form the definitive pigment pattern constituted by a black background with yellow patches that are characterized by epidermal xanthophores and dermal iridophores. Simultaneously the dermis undergoes remodeling from the larval stage to that typical of the adult. In the present study we ultrastucturally and immunocytochemically examined skin fragments of S. s. salamandra larvae and juveniles in order to investigate the modalities of xanthophore migration and differentiation in the context of dermal remodeling from the larval to adult stage. Semithin and thin sections showed that the dermis in newly born larvae consists of a compact connective tissue (basement lamella), to which fibroblasts and xanthophores adhere, and of a loose deep collagen layer. As larval development proceeds, fibroblasts and xanthophores invade the basement lamella, skin glands develop and the adult dermis forms. At metamorphosis, xanthophores reach the epidermis crossing through the basal lamina. We examined immunocytochemically the expression of signal molecules, such as fibronectin, vitronectin, beta1-integrin, chondroitin sulfate, E-cadherin, N-cadherin and plasminogen activator, which are known to be involved in regulating morphogenetic events. Their role in dermal remodeling and in pigment pattern formation is discussed.     

Cycloheximide blocks the fall of plasma and tissue tryptophan levels after tryptophan-free amino acid mixtures

The hypothesis that incorporation of tryptophan (TRY) into proteins is the mechanism underlying the decrease in plasma and tissue TRY levels after a TRY-free amino acid mixture was investigated. Rats fasted 15 hours were pretreated with saline or with the protein synthesis inhibitor cycloheximide (CHEX) and treated with saline or a TRY-free amino acid mixture. In a first experiment, in saline pretreated rats the TRY-free mixture caused a decrease of 49% in total plasma TRY, of 64% in free plasma TRY, of 66% in brain TRY and of 42% in liver TRY. After 5 mg/kg of CHEX the same TRY-free diet caused a decrease of 5% in total plasma TRY, 14% in free plasma TRY, 18% in brain TRY and 9% in liver TRY. In a second experiment, the TRY-free diet caused a 43% decrease of total plasma TRY in saline pretreated animals and a decrease of 15%, 6% and 2% respectively after the pretreatment with 0.3, 1.0 and 5.0 mg/kg of CHEX. In brain TRY, the TRY-free diet caused a 62% decrease in saline pretreated rats and a decrease of 38%, 20% and 19% respectively after the pretreatment with 0.3, 1.0 and 5.0 mg/kg of CHEX. Since 5.0 mg/kg of CHEX almost completely block protein synthesis and since doses of CHEX from 0.3 to 5.0 mg/kg cause a dose-dependent inhibition of protein synthesis, our data support the hypothesis that protein synthesis is the mechanism through which TRY-free mixtures decrease TRY levels.