Spatial correlations between the vacuolation, prion protein deposition and surviving neurons in variant Creutzfeldt-Jakob Disease (vCJD)

The histological features of cases of variant Creutzfeldt-Jakob disease (vCJD) are often distributed in the brain in clusters. This study investigated the spatial associations between the clusters of the vacuoles, surviving neurons, and prion protein (PrP) deposits in various brain areas in 11 cases of vCJD. Clusters of vacuoles and surviving neurons were positively correlated in the cerebral cortex but negatively correlated in the dentate gyrus. Clusters of the florid and diffuse type of PrP deposit were not positively correlated with those of either the vacuoles or the surviving neurons although a negative correlation was observed between the florid plaques and surviving neurons in some cortical areas. Clusters of the florid and diffuse deposits were either negatively correlated or uncorrelated. These data suggest: 1) that clusters of vacuoles in the cerebral cortex are associated with the presence of surviving neuronal cell bodies, 2) that the clusters of vacuoles are not spatially related to those of the PrP deposits, and 3) different factors are involved in the pathogenesis of the florid and diffuse PrP deposits.

Quantification of vacuolation ('spongiform change'), surviving neurones and prion protein deposition in eleven cases of variant Creutzfeldt-Jakob disease

Vacuolation ('spongiform change') and prion protein (PrP) deposition were quantified in the cerebral cortex, hippocampus, dentate gyrus and molecular layer of the cerebellum in 11 cases of variant Creutzfeldt-Jakob disease (vCJD). The density of vacuoles was greater in the cerebral cortex compared to the hippocampus, dentate gyrus and cerebellum. Within the cortex, vacuole density was significantly greater in the occipital compared to the temporal lobe and the density of surviving neurones was greatest in the occipital lobe. The density of the non-florid PrP plaques was greater in the cerebellum compared to the other brain areas. There were significantly more florid-type PrP plaques in the cerebral cortex compared to the hippocampus and the molecular layer of the cerebellum. No significant correlations were observed between the densities of the vacuoles and the PrP plaques. The densities of vacuoles in the parietal cortex and the non-florid plaques in the frontal cortex were positively correlated with the density of surviving neurones. The densities of the florid and the non-florid plaques were positively correlated in the parietal cortex, occipital cortex, inferior temporal gyrus and dentate gyrus. The data suggest: (i) vacuolation throughout the cerebral cortex, especially in the occipital lobe, but less evident in the hippocampus and molecular layer of the cerebellum; (ii) the non-florid plaques are more common than the florid plaques and predominate in the molecular layer of the cerebellum; and (iii) either the florid plaques develop from the non-florid plaques or both types are morphological variants resulting from the same degenerative process.

Quantification of the vacuolation (spongiform change) and prion protein deposition in 11 patients with sporadic Creutzfeldt-Jakob disease

The vacuolation (spongiform change) and prion protein (PrP) deposition were quantified in the cerebral cortex, hippocampus and cerebellum of 11 patients with sporadic Creutzfeldt-Jakob disease (CJD). The density of the vacuolation, averaged over patients, was greatest in the occipital cortex and cerebellum and least in the dentate gyrus. The degree of PrP deposition was similar in the different cortical areas and in the cerebellum but significantly lower in the hippocampus and absent in the dentate gyrus. There were no significant differences in the extent of the vacuolation and PrP deposition in the upper and lower cortical laminae. Vacuolation and PrP deposition in the upper cortex were both positively correlated with corresponding levels in the lower cortex. In addition, in the parietal cortex and parahippocampal gyrus, the density of the vacuolation was positively correlated with the level of PrP deposition but no such correlations were observed in the remaining areas studied. This quantitative study suggested that: (1) the pathological changes were most severe in the occipital cortex and cerebellum, while the hippocampus was least affected, (2) the pathological changes affect the upper and lower cortical laminae, and (3) the degree of correlation between the density of the vacuolation and PrP deposition may be dependent on brain region.

Protein deposition at polymer surfaces:the bulk and surface structure-property effects of hydrogels

The primary objective of this research has been to investigate the interfacial phenomenon of protein adsorption in relation to the bulk and surface structure-property effect s of hydrogel polymers. In order to achieve this it was first necessary to characterise the bulk and surface properties of the hydrogels, with regard to the structural chemistry of their component monomers. The bulk properties of the hydrogels were established using equilibrium water content measurements, together with water-binding studies by differential scanning calorimetry (D.S.C.). Hamilton and captive air bubble-contact angle techniques were employed to characterise the hydrogel-water interface and from which by a mathematical derivation, the interfacial free energy (ðsw) and the surface free energy components (ð psv, ðdsv, ðsv) were obtained. From the adsorption studies using the radio labelled iodinated (125I) proteins of human serum albumin (H.S.A.) and human fibrinogen (H.Fb.), it was Found that multi-layered adsorption was occurring and that the rate and type of this adsorption was dependent on the physico-chemical behaviour of the adsorbing protein (and its bulk concentration in solution), together with the surface energetics of the adsorbent polymer. A potential method for the invitro evaluation of a material's 'biocompatibility' was also investigated, based on an empirically observed relationship between the adsorption of albumin and fibrinogen and the 'biocompatibility' of polymeric materials. Furthermore, some consideration was also given to the biocompatibility problem of proteinaceous deposit formation on hydrophilic soft' contact lenses and in addition a number of potential continual wear contact lens formulations now undergoing clinical trials,were characterised by the above techniques.

Coexistence of protease sensitive and resistant prion protein in 129VV homozygous sporadic Creutzfeldt-Jakob disease: a case report

Introduction: The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported. Case presentation: A 74-year-old Caucasian woman showed a sporadic Creutzfeldt-Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77. Neuropathological findings showed: spongiform change in the patient"s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous. Conclusion: Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt-Jakob disease. This highlights the importance of molecular analyses of several brain regions in order to correctly diagnose rare and atypical prionopathies

Protein utilisation and turnover in lines of chickens selected for different aspects of body composition

1. Protein utilisation and turnover were measured in male chickens sampled from a line selected for high breast yield and a randombred control line (lines QL and CL, experiment 1) and in male chickens sampled from lines selected for either high or low abdominal fatness (lines FL and LL, experiment 2). In each experiment, 18 birds per line were given iso-energetic (12.9 MJ ME/kg) diets containing either 120 or 220 g CP/kg from 21 to 29 d (experiment 1) and 33 to 43 d (experiment 2). 2. Measurements were made of growth rate, food intake, body composition, excreta production and N-tau-methylhistidine excretion as a measure of myofibrillar protein breakdown, and fractional rates (%/d) of protein deposition, breakdown and synthesis were calculated. 3. In experiment 1, there were no significant differences between the line means for the fractional measures of protein turnover, but there was marked differential response in the two lines in the fractional rates of protein deposition, breakdown and synthesis, to increase in protein intake. The positive slope of the regressions of fractional (%/d) protein deposition and synthesis rates on protein intake (g/d/kg BW) were approximately 1.4- and 2.0-fold higher respectively in the QL than the CL line birds, and the negative slope of the regression of fractional breakdown rate on protein intake was approximately threefold greater in the CL than the QL line birds. 4. In experiment 2, fractional deposition rate was 6.2% lower, but fractional breakdown rate 9.4% higher in the LL than the FL birds, whilst there was essentially no difference in response of the FL and LL birds in the components of protein turnover to increase in protein intake. Line differences in deposition and breakdown rates were thus a reflection of the considerably higher (20%) food and hence protein intake in the FL than the LL birds. 5. The differential line responses in protein turnover in the two experiments suggest that selection for increased breast muscle yield and for reduced body fatness manipulate different physiological pathways in relation to protein turnover, but neither selection strategy results in an improvement in net protein utilisation at typical levels of protein intake by birds on commercial broiler diets, through a reduction in protein breakdown rate.

Níveis de lisina digestível para frangos de corte machos no período de 37 a 49 dias de idade

Foram utilizados 1.015 frangos de corte machos, linhagem Ross, dos 37 aos 49 dias de idade, para avaliar os efeitos de diferentes níveis de lisina digestível nas dietas experimentais. O delineamento experimental foi inteiramente casualizado, com 5 níveis de lisina digestível (0,90; 0,95; 1,00; 1,05; 1,10%), cada um com 7 repetições e 29 aves por unidade experimental. Utilizaram-se dietas isoenergéticas com 3.250 de kcal de EM/kg e isoprotéicas, com 18% de PB, à base de milho e farelo de soja. Foram avaliados o ganho de peso, o consumo de ração, a conversão alimentar, as características de carcaça, o rendimento de cortes, a composição e a deposição de nutrientes corporais. Os níveis de lisina digestível influenciaram, entre as características de desempenho, apenas a conversão alimentar, que melhorou de forma linear de acordo com níveis de lisina da ração. Das características de carcaça e rendimento de cortes, apenas gordura abdominal aumentou de forma quadrática conforme os níveis de lisina. Os níveis de lisina digestível tiveram efeito quadrático no teor da matéria mineral da carcaça, mas não influenciaram a composição química das vísceras e do sangue. Observou-se, contudo, tendência a aumento linear na deposição de proteína da carcaça e do corpo vazio com o aumento no nível de lisina digestível. Os resultados de desempenho indicam que o nível de lisina digestível da ração de frangos de corte machos no período de 37 a 49 dias de idade deve ser igual ou superior a 1,10%.

Lisina digestível para frangos de corte machos entre 12 e 22 dias de idade

Avaliou-se nível de lisina digestível para 1050 frangos de corte dos 12 aos 22 dias de idade. O delineamento experimental foi inteiramente ao acaso, com cinco tratamentos, sete repetições e 30 aves por unidade experimental. Os tratamentos foram: 1,05; 1,10; 1,15; 1,20 e 1,25% de lisina digestível. Avaliaram-se ganho de peso, consumo de ração, conversão alimentar, composição e deposição de nutrientes corporais. Foram constatados efeitos quadráticos de lisina digestível no consumo de ração e resposta linear ascendente no peso da carcaça. Na composição química da carcaça, houve resposta quadrática do nível de lisina na concentração de proteína. As taxas de deposição proteica, deposição de água, da carcaça e do corpo total tiveram aumento linear em resposta ao acréscimo de lisina na dieta. O aumento da concentração de lisina, todavia, coincidiu com a redução da matéria mineral nas vísceras e sangue e no corpo total. Considerado o desempenho, o nível 1,1% de lisina digestível atendeu às necessidades do frango de corte entre o 12º e o 22º dia de idade. Consideradas a composição química e as taxas de deposição dos nutrientes corporais, a demanda pelo aminoácido digestível torna-se igual ou maior que 1,25%

Lysine and zinc chelate in diets for brown laying hens: effects on egg production and composition

It was evaluated the effects of levels of digestible lysine and chelate zinc combined in the diet for laying on the egg quality. It was used 720 birds, from 48 to 60 weeks of age, distributed in a completely randomized design in a 3 × 5 factorial scheme with three levels of zinc and five levels of lysine, applied into six replicates in the experimental units of eight birds per plot. The levels were: 137, 309 and 655 ppm zinc and 0.482, 0.527, 0.582, 0.644 and 0.732% digestible lysine. It was not observed any interaction among digestible lysine and zinc for the primary variables of fractions and egg composition. Levels of zinc reduced egg weight, suggesting the lowest efficiency in nutrient intake. At the highest dietary concentration of zinc, the addition of digestible lysine coincided with a linear increase in shell weight. However, zinc addition, regardless of lysine level in the diet, resulted in the reduction of egg weight and of the percentage of mineral matter in the yolk, limiting the efficiency of mineral deposition in this fraction of the egg. Concentration of zinc that produced the best results was 137 ppm inasmuch as higher quantities limit the use of digestible lysine, with effects harming composition and egg quality. The study indicates the following requirement for digestible lysine: 0.639% from the 48th to the 52nd week, 0.679% from the 52nd to 56th week, and 0.635% from the 56th to the 60th week. Considering the total period from 48th to the 60th week, the level 0.638% of lysine or the daily intake of 707 mg of the amino acid met the requirement for egg quality of semi-heavy layers used in this study.

Lisina digestível e zinco orgânico para frangos de corte machos na fase de 22 a 42 dias de idade

Os efeitos dietéticos de lisina digestível e zinco quelato para frangos de corte machos entre 22 e 42 dias de idade foram avaliados em dois ensaios experimentais. Adotou-se o delineamento inteiramente casualizado em arranjo fatorial 5 × 2, com cinco níveis de lisina (0,841; 0,876; 0,997; 1,022 e 1,030%) e dois de zinco (43 e 243 ppm). No primeiro ensaio, utilizaram-se 900 aves com peso médio inicial de 957,4 g distribuídas em parcelas experimentais de 30 aves e três repetições e, no segundo ensaio, 180 frangos com peso médio inicial de 866,22 g, divididos em parcelas de três aves e seis repetições. As características avaliadas foram desempenho, rendimento de cortes e composição corporal (1º ensaio), balanço de nitrogênio e digestibilidade aparente das dietas (2º ensaio). Houve interação entre os níveis de lisina e zinco para o ganho de peso, a conversão alimentar, a matéria seca ingerida e o balanço energético. Os rendimentos de peito, coxa e sobrecoxa tiveram aumentos lineares em resposta ao acréscimo no nível de lisina digestível na dieta. O melhor desempenho foi obtido com o nível de 0,997% de lisina digestível (ou 1,134% lisina total) e de 43 ppm de zinco. Para maior rendimento dos cortes, o nível de lisina digestível recomendável deve ser no mínimo 1,002% (ou 1,139% de lisina total), independentemente dos níveis de zinco quelato. A maior inclusão de zinco em dietas para frangos de corte dos 22 aos 42 dias de idade não melhora a utilização da lisina na dieta e aumenta a deposição de gordura corporal.

Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone

Background: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. Methods: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. Results: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. Conclusions: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.

Effect of body composition at selection on reproductive development in large white gilts

Fifty-four Large White gilts were used to determine the effect of body composition at selection (145 d of age) on the onset of puberty and subsequent reproductive development until 202 d of age. Gilts were assigned to one of three groups based on their backfat depth at selection: 10 to 12 mm (L), 13 to 15 mm (M), and 16 to 18 mm (F). All of the F gilts, 92% of the M gilts, and 67% of the L gilts reached puberty by slaughter at 202 d of age. Data from a subgroup (first 67% to reach puberty in each group; L = Lp, M = Mp, and F = Fp) was also used. The M (Mp) and F (Fp) gilts reached puberty at 172 d (166 d) and 170 d (166 d) of age, respectively, but the L (Lp) gilts at 184.5 d were 12 d (18 d) older than M(P < .05), Mp(P < .001), and F(P < .01), Fp (P < .001) gilts. The Lp (97.68 kg) and Mp (98.33 kg) gilts were lighter (P < .01) than Fp (108.72 kg) gilts at puberty. There were no differences (P < .05) among the L, M, and F gilts in terms of backfat depth or weight at puberty. The L (Lp) gilts had a mean of 1.16 (1.75) estrous cycles, which was lower (P < .01) than for M (Mp) and (P < .01) F (Fp) gilts, with 1.96 (2.29) and 2.25 (2.33) cycles, respectively. L (Lp) gilts had fewer (P < .05) follicles, 13.14 (12.63), than either M (Mp), 19.08 (18.71), or F (Fp), 18.25 (17.42) gilts. The number of corpora lutea was not influenced (P > .05) by grouping at selection, but Fp gilts had fewer (P < .05) corpora lutea than Mp or Fp gilts. Live weight at slaughter was not influenced (P > .10) by grouping at selection or subgrouping at puberty. The L gilts with a mean of 18.05 mm of backfat at slaughter were leaner (P < .05) than the F (21.66 mm) but not (P > .10) the M gilts (19.41 mm). Subgrouping had no effect. Fat deposition and protein deposition were higher (P < .05) in those animals that attained puberty. We conclude that the rate of fat and protein deposition seems to be one of the determinants of puberty attainment.

Effect of restricted feed intake on early reproductive development in Large White gilts

Forty-five Large White gilts were used to study the effect of energy intake from 28 to 176 d of age on body composition and reproductive development. From 28 to 60 d, the gilts were fed ad libitum a 16.6 MJ DE/kg, 24% crude protein and 1.3% total lysine diet. From 61 d of age three dietary treatments were used; 1) ad libitum access to feed (15.6 MJ DE/kg, 21% crude protein and 1.07% total lysine) (H), 2) feed offered at 75% (M) of the previous days intake of H, and 3) feed offered at 60% (L) of the previous days intake of H. ADG from 61 to 176 d of age was (p <0.05) affected by treatment. Although live weight at 176 d of age did not differ (p >0.1) the H gilts had higher (p <0.08) carcass weights than the M or L gilts. Back fat depths were similar (p >0.1) for all treatments at 115 d of age, however by 176 d of age M and H gilts were fatter (p <0.1) than L gilts. The mean lipid deposition (LD) from 115 to 176 d of age for L gilts (78.9 g/d) was less (p <0.05) than for M gilts (143.6 g/d) and H gilts (135.6 g/d). There were no differences between treatments for protein deposition (PD) over the same period. More (p <0.05) H gilts (n=8) attained puberty (first observed estrus) than either M gilts or L gilts (n=4 for both). Follicle numbers were similar (p >0.1) across treatments. For gilts that attained puberty, H gilts had fewer (p <0.05) follicles (13.5) than M gilts (19.7) and L gilts (21.3). For gilts with follicular development, H gilts had the heaviest (458.7 g) reproductive tract weight (RTW). However, for those that attained puberty, L gilts had the heaviest RTW. RTW were lowest for those with no follicular development. Energy restriction had a negative impact on puberty attainment, i.e. it took longer to reach puberty. However, for gilts that attained puberty, the number of follicles was greater for those on lower feed intakes. It would appear that rate of fat deposition, but not necessarily the total amount of fat, plays an important role in puberty attainment.

Modulation of α-synuclein aggregation and toxicity

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina