Calreticulin binding and other biological activities of survival peptide Y-P30 including effects of systemic treatment of rats

Neuron survival-promoting peptide Y-P30, purified from oxidatively stressed neural cell lines, inhibits the appearance of microglia and rescues neurons 1 week after direct application to lesions of the rat cerebral cortex (7). Y-P30 affinity matrices treated with solubilized membranes from a variety of cell lines including human neuroblastoma SY5Y, mouse hippocampal cells HN 33.1, and human promonocytes HL-60, as well as with cerebral cortex tissue from both humans and rats, showed highly specific binding to calreticulin, a ubiquitous calcium binding protein that may be critical for integrin function. Treatment of cultures with 0.1 nM Y-P30 stabilized all these cell types whether differentiated or not, while 1 microM peptide also inhibited the morphological differentiation of the HL-60 cells into macrophages. Western analysis of the medium of SY5Y cell cultures suggested Y-P30-stimulated release of calreticulin, a result consistent with its other biological activities. Likewise, single dose systemic application of Y-P30 in unoperated rats and in rats with cerebral cortex lesions produced significant reductions in cerebral cortex membrane-associated calreticulin. Both direct and intravenous treatment with peptide also reduced cortical neuron atrophy 4 days after these lesions but only direct application consistently inhibited the appearance of ED-1(+) monocyte derivatives. We suggest that in vitro and in vivo mechanisms of Y-P30 effects are similar and involve the targeting of calreticulin. The results also suggest that some of these activities are apparent in the cerebral cortex after systemic application of this peptide.     

Convection-enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach

Using an approach that combines gene therapy with aromatic l-amino acid decarboxylase (AADC) gene and a pro-drug (l-dopa), dopamine, the neurotransmitter involved in Parkinson's disease, can be synthesized and regulated. Striatal neurons infected with the AADC gene by an adeno-associated viral vector can convert peripheral l-dopa to dopamine and may therefore provide a buffer for unmetabolized l-dopa. This approach to treating Parkinson's disease may reduce the need for l-dopa/carbidopa, thus providing a better clinical response with fewer side effects. In addition, the imbalance in dopamine production between the nigrostriatal and mesolimbic dopaminergic systems can be corrected by using AADC gene delivery to the striatum. We have also demonstrated that a fundamental obstacle in the gene therapy approach to the central nervous system, i.e., the ability to deliver viral vectors in sufficient quantities to the whole brain, can be overcome by using convection-enhanced delivery. Finally, this study demonstrates that positron emission tomography and the AADC tracer, 6-[(18)F]fluoro-l-m-tyrosine, can be used to monitor gene therapy in vivo. Our therapeutic approach has the potential to restore dopamine production, even late in the disease process, at levels that can be maintained during continued nigrostriatal degeneration.     

Children of mothers with mental ilness: attachment, emotional and behavioural problems

This study describes the pattern of emotional and behavioural difficulties of children whose mothers have mental illness, and explores the relationship between children's behavioural and emotional difficulties and maternal perceptions of attachment. Thirteen mothers previously admitted to psychiatric hospital for mental illness completed a measure of their own symptoms (Brief Symptom Inventory), their children's emotional and behavioural problems (Strengths and Difficulties Questionnaire (SDQ)), and attachment security (Parent/Child Reunion Inventory) (n = 21). Mean scores for child SDQ profiles were found to be within the 'normal' range, although (on some indices) mothers reported more 'case' scores for their children, than would be expected from standardized norms. It was found that there were significant positive correlations between 'insecurity' scores and all problem scales of the SDQ. Best predictors from the Parent/Child Reunion Inventory factors for each SDQ scale are reported and discussed.     

Differential expression of CYP1A, 2B, and 3A genes in the F344 rat following exposure to a polybrominated diphenyl ether mixture or individual components.

Polybrominated diphenyl ethers (PBDEs), used as flame retardants, have been detected in the environment and in mammalian tissues and fluids. Evidence indicates that PBDE mixtures induce CYPs through aryl hydrocarbon receptor (AhR)-dependent and -independent pathways. The present work has investigated the effects of individual components of a commercial PBDE mixture (DE71) on expression of CYP1A1, a biomarker for activation of the AhR (dioxin-like), and CYP2B and CYP3A, biomarkers for activation of the constitutive androstane and pregnanexreceptors (CAR and PXR), respectively, in the rat. Male F344 rats were dosed orally on three consecutive days with either DE71, PBDE components, 2,2',4,4'-tetraBDE (BDE47), 2,2',4,4',5-pentaBDE (BDE99), 2,2',4,4',5,5'-hexaBDE (BDE153), representative polybrominated dibenzofurans (PBDFs) present in DE71, or reference PCBs. Differential expression of target genes was determined in liver 24 h after the last dose. Quantitative PCR analysis indicated up-regulation of CYP1A1 by DE71; however, the response was weak compared to that for dioxin-like PCB126. Individual PBDE components of DE71 up-regulated CYP1A1 only at the highest administered dose (100 micromol/kg/day). Representative PBDFs efficiently up-regulated CYP1A1; therefore, they, along with other PBDFs and polybrominated dibenzodioxins detected in DE71 and individual PBDE components, may be responsible for most, if not all, dioxin-like properties previously observed for PBDEs. Conversely, PBDEs appear capable of up-regulating CYP2B and CYP3A in rats at doses similar to that for non-dioxin-like PCB153. These results indicate that in vivo PBDE-mediated toxicity would be better categorized by AhR-independent mechanisms, rather than the well-characterized AhR-dependent mechanism associated with exposure to dioxin-like chemicals.     

In vivo therapeutic responses contingent on Fanconi anemia/BRCA2 status of the tumor.

PURPOSE: BRCA2, FANCC, and FANCG gene mutations are present in a subset of pancreatic cancer. Defects in these genes could lead to hypersensitivity to interstrand cross-linkers in vivo and a more optimal treatment of pancreatic cancer patients based on the genetic profile of the tumor. EXPERIMENTAL DESIGN: Two retrovirally complemented pancreatic cancer cell lines having defects in the Fanconi anemia pathway, PL11 (FANCC-mutated) and Hs766T (FANCG-mutated), as well as several parental pancreatic cancer cell lines with or without mutations in the Fanconi anemia/BRCA2 pathway, were assayed for in vitro and in vivo sensitivities to various chemotherapeutic agents. RESULTS: A distinct dichotomy of drug responses was observed. Fanconi anemia-defective cancer cells were hypersensitive to the cross-linking agents mitomycin C (MMC), cisplatin, chlorambucil, and melphalan but not to 5-fluorouracil, gemcitabine, doxorubicin, etoposide, vinblastine, or paclitaxel. Hypersensitivity to cross-linking agents was confirmed in vivo; FANCC-deficient xenografts of PL11 and BRCA2-deficient xenografts of CAPAN1 regressed on treatment with two different regimens of MMC whereas Fanconi anemia-proficient xenografts did not. The MMC response comprised cell cycle arrest, apoptosis, and necrosis. Xenografts of PL11 also regressed after a single dose of cyclophosphamide whereas xenografts of genetically complemented PL11(FANCC) did not. CONCLUSIONS: MMC or other cross-linking agents as a clinical therapy for pancreatic cancer patients with tumors harboring defects in the Fanconi anemia/BRCA2 pathway should be specifically investigated.