Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells

The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.     

The small heat-shock chaperone protein, alpha-crystallin, does not recognize stable molten globule states of cytosolic proteins

The small heat-shock protein (sHsp), alpha-crystallin, acts as a molecular chaperone by interacting with destabilized 'substrate' proteins to prevent their precipitation from solution under conditions of stress. alpha-Crystallin and all sHsps are intracellular proteins. Similarly to other chaperones, the 'substrate' protein is in an intermediately folded, partly structured molten globule state when it interacts and complexes with alpha-crystallin. In this study, stable molten globule states of the cytosolic proteins, gamma-crystallin and myoglobin, have been prepared. Within the lens, gamma-crystallin naturally interacts with alpha-crystallin and myoglobin and alpha-crystallin are present together in muscle tissue. The molten globule states of gamma-crystallin and myoglobin were prepared by reacting gamma-crystallin with glucose 6-phosphate and by removing the haem group of myoglobin. Following spectroscopic characterisation of these modified proteins, their interaction with alpha-crystallin was examined by a variety of spectroscopic and protein chemical techniques. In both cases, there was no interaction with alpha-crystallin that led to complexation. It is concluded that alpha-crystallin does not recognise stable molten globule states of cytosolic 'substrate' proteins and only interacts with molten globule states of proteins that are on the irreversible pathway towards an aggregated and precipitated form.     

The luteinising hormone-releasing hormone analogue triptorelin with or without the aromatase inhibitor formestane in premenopausal breast cancer: effects on bone metabolism markers

BACKGROUND: the combination of a luteinising hormone-releasing hormone (LH-RH) analogue and an aromatase inhibitor (AI) induces greater oestrogen suppression than the analogue alone in premenopausal breast cancer. However, very few data on the biological effects of such a combination are currently available. AIM OF THE STUDY: the short-term effects of treatment with the LH-RH analogue triptorelin alone or in association with the AI formestane on bone metabolism were investigated in premenopausal breast cancer. Circulating levels of the bone formation markers carboxy-terminal and amino-terminal propeptides of type I procollagen (PICP and PINP) and the bone resorption marker cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) were assessed. In addition, serum levels of insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3 and interleukin 6 (IL-6) were evaluated. PATIENTS AND METHODS: twenty-one patients with advanced breast cancer were randomly given triptorelin monthly alone (n=10, arm A) or in combination with formestane fortnightly (n=11, arm B). Blood samples were collected over a 3-month period. RESULTS: serum PICP and PINP levels increased significantly over time (P=0.0065 and 0.0197 in arm A and B, respectively); no change in ICTP levels was observed. A rise in IGF-I and IGFBP-3 levels was seen in each treatment group, but only the increase in IGF-I was significant (P=0.0138, always). The on-treatment levels of the bone turnover markers and IGF-system components were inversely correlated with serum oestrogens. Neither treatment modalities significantly affected serum IL-6 levels over time. No difference in the behaviour of any of the assessed biomarkers was observed between patients with or without skeletal metastases. CONCLUSION: it is worth noting that complete oestrogen depletion, at least in our case series, seems to increase only osteoblastic activity markers. The observed modifications appear to be related to oestrogen depletion per se rather than the degree of oestrogen suppression or the different therapeutic regimen administered.     

Ligand-induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small-angle scattering data

Small-angle neutron and x-ray scattering experiments have been performed on type 2 tissular transglutaminase to characterize the conformational changes that bring about Ca(2+) activation and guanosine triphosphate (GTP) inhibition. The native and a proteolyzed form of the enzyme, in the presence and in the absence of the two effectors, were considered. To describe the shape of transglutaminase in the different conformations, a Monte Carlo method for calculating small-angle neutron scattering profiles was developed by taking into account the computer-designed structure of the native transglutaminase, the results of the Guinier analysis, and the essential role played by the solvent-exposed peptide loop for the conformational changes of the protein after activation. Although the range of the neutron scattering data is rather limited, by using the Monte Carlo analysis, and because the structure of the native protein is available, the distribution of the protein conformations after ligand interaction was obtained. Calcium activation promotes a rotation of the C-terminal with respect to the N-terminal domain around the solvent-exposed peptide loop that connects the two regions. The psi angle between the longest axes of the two pairs of domains is found to be above 50 degrees, larger than the psi value of 35 degrees calculated for the native transglutaminase. On the other hand, the addition of GTP makes possible conformations characterized by psi angles lower than 34 degrees. These results are in good agreement with the proposed enzyme activity regulation: in the presence of GTP, the catalytic site is shielded by the more compact protein structure, while the conformational changes induced by Ca(2+) make the active site accessible to the substrate.     

Constitutive expression of heat shock proteins hsp25 and hsp70 in the rat oviduct during neonatal development, the oestrous cycle and early pregnancy

Certain heat shock proteins are regulated by steroid hormones and are associated with oestrogen receptor function in reproductive tissues, indicating that these proteins have a role during implantation, decidualization and placentation. In the present study, the expression of hsp25, hsp70 and oestrogen receptor alpha were examined by immunohistochemistry in oviducts from rats during neonatal development, the oestrous cycle and during early pregnancy. Oestrogen receptor alpha was the first protein observed in the neonatal oviduct, and its expression preceded that of hsp70 and hsp25. Although these heat shock proteins have been associated with the oestrogen receptor, this study showed that during early development of the oviduct, the receptor protein was not associated with the concomitant expression of hsp25 and hsp70. However, these heat shock proteins were expressed when oviductal cells became differentiated. In the adult oviduct, hsp70 was more abundant than hsp25, moreover, there were no significant modifications in expression of hsp25 during the oestrous cycle. In contrast, the expression of hsp70 was significantly higher in epithelial cells during dioestrus, when the maximum amount of oestrogen receptor alpha was also observed. Therefore, the present study shows that hsp70, but not hsp25, is an oviductal protein modulated by the oestrous cycle and that it is a protein marker for specific phases of the oestrous cycle. In addition, hsp70 was more responsive to the hormonal changes in the infundibulum and ampullar regions of the oviduct. During early pregnancy, hsp25 expression was downregulated (unlike in the endometrium), whereas hsp70 was relatively abundant in the oviduct. hsp70 was observed in all functional segments of the oviduct during pregnancy, indicating that in the oviduct, this protein is modulated by oestrogens and progesterone and possibly by other pregnancy-related hormones.     

Ginkgo biloba Expresses Calreticulin,the Major Calcium-Binding Reticuloplasmin in Eukaryotic Cells

Calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was characterized in Ginkgo biloba L. pollen and seeds. Electrophoretic analysis of the partly purified extracts showed the presence of two protein bands of 57 and 50kDa apparent molecular masses, which strongly cross-reacted with antibodies against plant calreticulins. Amino acid sequence comparison with other plant and animal calreticulins revealed a much higher similarity of the N-terminus of Ginkgo calreticulins with the homologue from angiosperms rather than with that from mammals. The finding of calreticulin in Ginkgo is indicative of the conservation also in gymnosperms of Ca²⁺ homeostatic mechanisms, which seem to rely on the same molecular components as all eukaryotic cells.     

Health and Economic Outcomes of Introducing the New MenB Vaccine (Bexsero) into the Italian Routine Infant Immunisation Programme

Introduction

In January 2013 a novel type of multicomponent protein-based vaccine against group B meningococcal disease was licensed by the European Medicines Agency. With the widespread use of the meningococcal serogroup C conjugate vaccines, serogroup B remains now the major cause of bacterial meningitis and septicaemia in young children in Europe. The aim of this study is to investigate the health and the economic outcomes of MenB vaccine introduction into the Italian routine mass vaccination programme.

Methods

The present work is structured in two main parts. Firstly, we assess the epidemiological burden of group B meningococcal disease using official hospitalisation and notification data from two of the most populated Italian regions (Lombardia and Piemonte) during a 6-year study period (2007-2012). Secondly, we evaluate the cost-effectiveness of the immunisation programme in Italy from the public health payer perspective under base case parameters assumptions and performing a comprehensive sensitivity analysis to assess the robustness and the uncertainty of our model results.

Results

MenB serotype is responsible for 59% of the 341 cases of Invasive Meningococcal Disease in Lombardia and Piemonte. Incidence rate for MenB infection is estimated to be 0.21/100,000/y resulting at the highest level in children ≤4 years of age. Although the new MenB vaccine can potentially prevent about one third of the disease cases in the Italian population, model results show this strategy is unlikely to be cost-effective (ICER value over €350,000/QALY) with a vaccine that prevents disease only. These results are robust under most of the sensitivity scenarios except when allowing for lower discount rates.

Discussion

The introduction of the novel vaccine into the routine immunisation schedule needs to be carefully evaluated. The new MenB vaccine has the potential to reduce the disease burden at the population level. However, from the Italian Health Service perspective, the immunisation programme is unlikely to be cost-effective at the current incidence levels and vaccine price.     

TNFA Haplotype Genetic Testing Improves HLA in Estimating the Risk of Celiac Disease in Children

Background

TNF-α and IFN-γ play a role in the development of mucosal damage in celiac disease (CD). Polymorphisms of TNFA and IFNG genes, as well as of the TNFRSF1A gene, encoding the TNF-α receptor 1, might underlie different inter-individual disease susceptibility over a common HLA risk background. The aims of this study were to ascertain whether five SNPs in the TNFA promoter (-1031T>C,-857C>T,-376G>A,-308G>A,-238G>A), sequence variants of the TNFRSF1A gene and IFNG +874A>T polymorphism are associated with CD in a HLA independent manner.

Methods

511 children (244 CD, 267 controls) were genotyped for HLA, TNFA and INFG (Real Time PCR). TNFRSF1A variants were studied (DHPLC and sequence).

Results

Only the rare TNFA-1031C (OR=0.65, 95% CI:0.44-0.95), -857T (OR=0.42, 95% CI:0.27-0.65), -376A (OR=2.25, 95% CI:1.12-4.51) and -308A (OR=4.76, 95% CI:3.12-7.26) alleles were significantly associated with CD. One TNFRSF1A variant was identified (c.625+10A>G, rs1800693), but not associated with CD. The CD-correlated TNFA SNPs resulted in six haplotypes. Two haplotypes were control-associated (CCGG and TTGG) and three were CD-associated (CCAG, TCGA and CCGA). The seventeen inferred haplotype combinations were grouped (A to E) based on their frequencies among CD. Binary logistic regression analysis documented a strong association between CD and HLA (OR for intermediate risk haplotypes=178; 95% CI:24-1317; OR for high risk haplotypes=2752; 95% CI:287-26387), but also an HLA-independent correlation between CD and TNFA haplotype combination groups. The CD risk for patients carrying an intermediate risk HLA haplotype could be sub-stratified by TNFA haplotype combinations.

Conclusion

TNFA promoter haplotypes associate with CD independently from HLA. We suggest that their evaluation might enhance the accuracy in estimating the CD genetic risk.