The E46K mutation modulates α-synuclein prion replication in transgenic mice

In multiple system atrophy (MSA), the α-synuclein protein misfolds into a self-templating prion conformation that spreads throughout the brain, leading to progressive neurodegeneration. While the E46K mutation in α-synuclein causes familial Parkinson’s disease (PD), we previously discovered that this mutation blocks in vitro propagation of MSA prions. Recent studies by others indicate that α-synuclein adopts a misfolded conformation in MSA in which a Greek key motif is stabilized by an intramolecular salt bridge between residues E46 and K80. Hypothesizing that the E46K mutation impedes salt bridge formation and, therefore, exerts a selective pressure that can modulate α-synuclein strain propagation, we asked whether three distinct α-synuclein prion strains could propagate in TgM47^+/- mice, which express human α-synuclein with the E46K mutation. Following intracranial injection of these strains, TgM47^+/- mice were resistant to MSA prion transmission, whereas recombinant E46K preformed fibrils (PFFs) transmitted neurological disease to mice and induced the formation of phosphorylated α-synuclein neuropathology. In contrast, heterotypic seeding following wild-type (WT) PFF–inoculation resulted in preclinical α-synuclein prion propagation. Moreover, when we inoculated TgM20^+/- mice, which express WT human α-synuclein, with E46K PFFs, we observed delayed transmission kinetics with an incomplete attack rate. These findings suggest that the E46K mutation constrains the number of α-synuclein prion conformations that can propagate in TgM47^+/- mice, expanding our understanding of the selective pressures that impact α-synuclein prion replication.     

Studies ofH-2K b mutant mice

Three newH-2 ^b mutant strains, B6.C-H-2 ^bm9 , B6.C-H-2 ^bm10 and B6.C-H–2 ^bm11 , are described. The three mutant strains are of the gain and loss type as they reject skin grafts reciprocally with the parental C57BL/6Kh. The mutations, which arose independently, are all allelic at the same locus as 11 other mutant strains already described. By complementation and other studies the mutated gene has been shown to beH-2K ^b . The strains were typed directly and by absorption with antisera specific for H-2K^b and H-2D^b private and public specificities and for Ia^b specificities. Each strain typed differently with these sera. The strain B6.C-H-2 ^bm9 was found to be serologically identical with C57BL/6. The strains B6.C-H-2 ^bm10 and B6.C-H-2 ^bm11 were found to have alterations in the private H-2K^b specificity, H-2.33, and in the public specificity, H-2.5, but to a different extent. B6.C-H- 2^bm10 had a marked decrease in the amount of H-2.33 expressed on the splenic cell surface as compared to C57BL/6 and also has a marked decrease in the expression of H-2.5 on both spleen and red blood cells. In comparison, B6.C-H-2 ^bm11 has a decrease in the expression of H-2.33 but an increase in the expression of H-2.5 on both splenic and red blood cells. The other H-2^b specificities appeared to be unaltered as compared with C57BL/6.     

Pak regulates calpain-dependent degradation of E3b1

E3b1, a binding partner of Eps8, plays a critical role in receptor tyrosine kinase (RTK)-mediated Rac activation by facilitating the interaction of Eps8 with Sos-1 and the consequent activation of the Rac-specific guanine nucleotide exchange factor activity of Sos-1. Here we present evidence that E3b1 levels are regulated by the Ca(2+)-activated protease calpain, and also by Pak, a downstream target of Rac signaling. Serum starvation of Rat2 or COS7 cells resulted in rapid loss of E3b1 that was reversed by calpain inhibitors. Loss was also prevented by expressing the constitutively active Pak1 mutant, Pak1(H83,86L). Activation of endogenous Pak by platelet-derived growth factor or the constitutively active Rac1 mutant, Rac1(G12V), also inhibited degradation. In contrast, inhibition of endogenous Pak activity by expressing the Pak auto-inhibitory domain caused degradation of over-expressed E3b1 even in the presence of serum. Taken together, these findings indicate that E3b1 is down-regulated by calpain activation and stabilized by Pak activation. They also suggest that RTK-mediated Rac activation can be modulated by changes in the level of E3b1 in response to signals that affect the activity of calpain or Pak.     

Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses

Background

Helper‐dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high‐titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo.

Methods

Replication‐defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7‐cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre‐terminal protein, and with a cre‐recombinase plasmid.

Results

We show that C7‐cre cells allow efficient production of gutted Ad using ΔE1 + ΔE2b + ΔE3 helper viruses whose growth can be limited by cre‐loxP‐mediated excision of the packaging signal. Gutted Ad vectors carrying ～28 kb cassettes expressing full‐length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%.

Conclusions

These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b‐deleted vectors display a profound reduction in viral gene expression. Copyright © 2002 John Wiley & Sons, Ltd.

     

Blocking the apolipoprotein E/Amyloid β interaction in triple transgenic mice ameliorates Alzheimer's disease related amyloid β and tau pathology

Inheritance of the apolipoprotein E4 (apoE4) genotype has been identified as the major genetic risk factor for late‐onset Alzheimer's disease (AD). Studies have shown that the binding between apoE and amyloid‐β (Aβ) peptides occurs at residues 244–272 of apoE and residues 12–28 of Aβ. ApoE4 has been implicated in promoting Aβ deposition and impairing clearance of Aβ. We hypothesized that blocking the apoE/Aβ interaction would serve as an effective new approach to AD therapy. We have previously shown that treatment with Aβ12‐28P can reduce amyloid plaques in APP/PS1 transgenic (Tg) mice and vascular amyloid in TgSwDI mice with congophilic amyloid angiopathy. In the present study, we investigated whether the Aβ12‐28P elicits a therapeutic effect on tau‐related pathology in addition to amyloid pathology using old triple transgenic AD mice (3xTg, with PS1_M146V, APP_Swe and tau_P30IL transgenes) with established pathology from the ages of 21 to 26 months. We show that treatment with Aβ12‐28P substantially reduces tau pathology both immunohistochemically and biochemically, as well as reducing the amyloid burden and suppressing the activation of astrocytes and microglia. These affects correlate with a behavioral amelioration in the treated Tg mice.

     

Production and Characterization of Improved Adenovirus Vectors with the E1, E2b, and E3 Genes Deleted

Adenovirus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems. Most Ad vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted E1 gene functions in trans. Unfortunately, the use of high titers of E1-deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors. We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions. The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA. We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome. The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.     

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.     

Multiple calvarial defects in lmx1b mutant mice

The vertebrate cranial vault, or calvaria, forms during embryonic development from cranial mesenchyme of multiple embryonic origins. Inductive interactions are thought to specify the number and location of the calvarial bones, including interactions between the neuroepithelium and cranial mesenchyme. An important feature of calvarial development is the local inhibition of osteogenic potential which occurs between specific bones and results in the formation of the cranial sutures. These sutures allow for postnatal growth of the skull to accommodate postnatal increase in brain size. The molecular genetic mechanisms responsible for the patterning of individual calvarial bones and for the specification of the number and location of sutures are poorly understood at the molecular genetic level. Here we report on the function and expression pattern of the LIM-homeodomain gene, lmx1b, during calvarial development. Lmx1b is expressed in the neuroepithelium underlying portions of the developing skull and in cranial mesenchym which contributes to portions of the cranial vault. Lmx1b is essential for proper patterning and morphogenesis of the calvaria since the supraoccipital and interparietal bones of lmx1b mutant mice are either missing or severely reduced. Moreover, lmx1b mutant mice have severely abnormal sutures between the frontal, parietal, and interparietal bones. Our results indicate that lmx1b is required for multiple events in calvarial development and suggest possible genetic interaction with other genes known to regulate skull development and suture formation. Dev. Genet. 22:314–320, 1998. © 1998 Wiley-Liss, Inc.     

The relationship between region E1a and E1b of human adenoviruses in cell transformation

Baby rat kidney (BRK) cells were transfected either with intact region E1 DNA of adenovirus type 5 (Ad5) or with mixtures of DNA fragments containing the separated E1a and E1b regions. The results showed that mixtures of regions E1a and E1b transform with a similar efficiency as intact region E1. DNA fragments containing region E1b alone have no detectable transforming activity in primary BRK cells nor in established rat cell lines. When region E1a and Ad5 was combined with region E1b and Ad12 complete transformation was also obtained. Characterization of the cell lines transformed by separated E1a and E1b regions have led to the following conclusions: (1) Expression of region E1b is not dependent on specific linkage to region E1a as it occurs in the intact E1 region. (2) Region E1b is normally expressed into the corresponding major adenovirus T antigens (65,000 and 19,000 Mr with region E1b of Ad5; 60,000 and 19,000 Mr with E1b or AD12). (3) Region E1b of Ad12 can be activated by region E1a of Ad5 indicating that the Ela regions of both serotypes are functionally similar in transformation. (4) Cell lines containing region E1b of Ad5 are weakly oncogenic in nude mice whereas cells containing E1b of Ad12 are highly oncogenic in nude mice, indicating that the degree of oncogenicity is determined by region E1b.     

Wnt7b regulates placental development in mice.

Secreted Wnt proteins regulate many developmental processes in multicellular organisms. We have generated a targeted mutation in the mouse Wnt7b gene. Homozygous Wnt7b mutant mice die at midgestation stages as a result of placental abnormalities. Wnt7b expression in the chorion is required for fusion of the chorion and allantois during placental development. The alpha4 integrin protein, required for chorioallantoic fusion, is not expressed by cells in the mutant chorion. Wnt7b also is required for normal organization of cells in the chorionic plate. Thus, Wnt7b signaling is central to the early stages of placental development in mammals.     

Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice

Gel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The “nonmammary” mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr ^−/− mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr ^−/− mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen’s kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis.     

Rapporto Tra le Clorofille a E b in Cuscuta Australis

Abstract

Chlorophylls a/b ratio in seedlings of CUSCUTA AUSTRALIS. — The ratio between chlorophylls a/b in seedlings of Cuscuta australis increases after a 12 hr. period of darkness preceding the extraction, owing to for a decrease of chlorophyll b greater than chlorophyll a.     

Spontaneously hyperlipidemic (SHL) mice: Japanese wild mice with apolipoprotein E deficiency

During inbreeding of Japanese wild mice (Mus musculus molossinus), we established a strain of mice with severe cutaneous xanthomatous lesions. Since those mice showed high plasma cholesterol values, we named them spontaneously hyperlipidemic (SHL) mice; total cholesterol values of these mice (even when fed on conventional low-fat diet) are unusually high throughout the life span. The xanthomatous lesions appear in palms and distal extremities of forelimbs as early as 4 weeks after birth, and continue to expand to chest, abdomen, and face until the mice die before 14 months of age. Histological examination of these lesions revealed cholesterol crystal deposits, an infiltration of foam cells or macrophages, while that of the vascular system revealed atherosclerosis in the aortic sinus. Immunoblot and Northern blot analyses failed to detect apolipoprotein E (APOE) expression in these animals. Consistent with these findings, Southern blot analysis found disruption of the Apoe gene in SHL mice. Phenotypes of SHL mice, however, were distinct from those of Apoe ^tm1Unc (hereafter Apoe ^−/− ) mice, whose Apoe gene was disrupted by homologous recombination; hypercholesterolemia and xanthoma were more severe in SHL mice than in Apoe ^−/− mice, while atherosclerosis was milder in SHL mice. These distinctions suggest that there are modifier genes for the phenotypes. Alternatively, other gene(s), besides the Apoe gene, may be mutated in SHL mice. In either case, comparative genetic and molecular dissection of SHL mice will provide a good opportunity to understand the genetic basis for hyperlipidemia and atherosclerosis. Received: 2 October 1998 / Accepted: 2 December 1998     

Inhibition of immunoglobulin E production in allergic model mice by supplementation with vitamin E and beta-carotene

A diet containing different amounts of vitamin E (alpha-tocopherol; 0.5 mg, 5 mg, 10 mg or 50 mg per 100 g diet) was supplemented to BALB/c mice for 6 weeks. These mice were subcutaneously immunized twice with ovalbumin (OVA). A passive cutaneous anaphylaxis (PCA) analysis demonstrated that the mice fed on the diet containing 5 mg of vitamin E produced the highest level of the OVA-specific immunoglobulin E (IgE) antibody. A lower level of serum IgE was found in the mice supplemented with 0.5 mg, 10 mg and 50 mg of vitamin E. A sandwich ELISA analysis showed that the pattern of the total IgE antibody level among these four groups was the same as that of the allergen-specific IgE. In a separate experiment, 5 mg of vitamin E and/or 50 mg of beta-carotene was supplemented to the basal diet containing vitamin E as alpha-tocopherol acetate (5 mg) in order to evaluate the effect of their combination on OVA-specific and total IgE production in the mice. The supplementation with beta-carotene alone had no effect on OVA-specific or total IgE production. In contrast, supplementation with vitamin E plus beta-carotene effectively suppressed both the antigen-specific and total IgE antibodies. The serum vitamin E and beta-carotene levels were increased by supplementation with the respective compounds. These results strongly suggest that the combination of dietary vitamin E and beta-carotene suppressed IgE production and would therefore help to prevent the type-I allergic reaction.     

Social dominance in male vasopressin 1b receptor knockout mice

We have previously reported that mice with a targeted disruption of their vasopressin 1b receptor gene, Avpr1b, have mild impairments in social recognition and reduced aggression. The reductions in aggression are limited to social forms of aggression, i.e., maternal and inter-male aggression, while predatory aggression remains unaffected. To further clarify the role of the Avpr1b in the regulation of social behavior we first examined anxiety-like and depression-like behaviors in Avpr1b knockout (Avpr1b −/−) mice. We then went on to test the ability of Avpr1b −/− mice to form dominance hierarchies. No major differences were found between Avpr1b −/− and wildtype mice in anxiety-like behaviors, as measured using an elevated plus maze and an open field test, or depression-like behaviors, as measured using a forced swim test. In the social dominance study we found that Avpr1b −/− mice are able to form dominance hierarchies, though in early hierarchy formation dominant Avpr1b −/− mice display significantly more mounting behavior on Day 1 of testing compared to wildtype controls. Further, non-socially dominant Avpr1b −/− mice spend less time engaged in attack behavior than wildtype controls. These findings suggest that while Avpr1b −/− mice may be able to form dominance hierarchies they appear to employ alternate strategies.     

Human keratin 14 driven HPV 16 E6/E7 transgenic mice exhibit hyperkeratinosis

Human papillomavirus type 16 (HPV16) has been known as a major causative factor for the development of uterine cervical carcinomas. To investigate the in vivo activity of HPV16 expressed in squamous epithelia, transgenic mice harboring HPV16 E6/E7 with human keratin 14 (hK14) promoter were generated. Grossly, hK14 driven HPV16 E6/E7 transgenic mice exhibited multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair in neonates, thickened ears, and loss of hair in adults. Transgenic mice with phenotype exhibiting severe wrinkled skin and a lack of hair growth died at the age of 3-4 weeks. Histological analysis revealed that in transgenic mice survived beyond the initial 3-4 weeks, HPV16 E6/E7 causes epidermal hyperplasia in multiple transgenic lineages with high incidence of transgene penetration. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and keratinocytes, and was associated with hyperkeratosis. Such activities were significantly higher in the skin of transgenic mice than that of the normal mice. Thus, these transgenic mice appeared to be useful for the expression of HPV16 E6/E7 gene and subsequent analysis on hyperkeratosis.     

Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.