An Anti-β-Amyloid Vaccine for Treating Cognitive Deficits in a Mouse Model of Down Syndrome

In Down syndrome (DS) or trisomy of chromosome 21, the β-amyloid (Aβ) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aβ as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aβ is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer’s disease (AD). At present, no treatment targets Aβ–related pathogenesis in people with DS. Herein we used a vaccine containing the Aβ 1–15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aβ vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aβ IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aβ without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aβ levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aβ as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aβ immunotherapeutic approach may act to target Aβ-related pathology in a mouse model of DS.     

Size changes in single muscle fibers during fixation and embedding.

During fixation of single muscles fibers with glutaraldehyde, the volume of the fiber shrinks 20%, recovers in rinse and osmium tetroxide to near normal volume and shrinks 20% again when staining with uranyl acetate. This suggest that osmotic properties of membranes may not have been completely lost during fixation, post-fixation and en bloc staining. Dehydration in ethanol and propylene oxide produces a further 10% shrinkage in volume. Infiltration and embedding with Epon causes an additional 15% change in volume. This gives a total shrinkage in volume of 45% which is nearly twice that of the apparent shrinkage in the volume of the myosin lattice as determined by electron microscopy.     

Eukaryotic origins: String analysis of 5S ribosomal RNA sequences from some relevant organisms

Summary Using the PHYLOGEN tree-forming programs, we evaluate the published 5S rRNA sequences in certain of the files in the Berlin DataBank in an attempt to identify the connection between archaebacteria and the eukaryotic protists. These programs are based on methods of string analysis developed by Sankoff and others. Their discriminatory power is derived from their continuous realignment of sequences through repeated assessment of insertions and deletions as well as substitutions. The programs demonstrate that even these small molecules (ca. 120 bases) retain substantial records of evolutionary events that occurred over a billion years ago. The eukaryotes seem to have been derived from ancestors near the common origins of the halobacterial and Methanococcales groups. Identifying what might have been a primordial eukaryote is more difficult because several of the species considered as early derivatives from the common root are isolated species with large genetic differences from each other and from all other extant forms that have been sequenced. The ameboid, flagellated, and ciliated protists seem to have emerged nearly simulataneously from an ancient cluster, but the sarcodinid protozoa have preference as the group of most ancient origin. The euglenozoa and the ciliates are of later derivation. Our ability to tease plausible trees from such small molecules suggests that the mode of analysis rather than the size of the molecule is often a major limitation in the reconstruction of acceptable ancient phylogenies. The residual uncertainty with respect to the conclusions of the 5S analysis may indicate a real limit on the informational content of such small molecules; the period of evolutionary time during which the primary eukaryotic radiation occurred may have been very short relative to the rate of fixation of changes in this highly conserved molecule. Much of even this limitation may be resolved, however, when a sufficiently dense sample of the problematic taxa is examined.     

beta-NGF-endopeptidase: structure and activity of a kallikrein encoded by the gene mGK-22

Mouse nerve growth factor (NGF) is cleaved at a histidine-methionine bond to release an NH2-terminal octapeptide (NGF1-8). The enzyme responsible, beta-NGF-endopeptidase, is structurally and functionally similar to gamma-NGF and epidermal growth factor-binding protein (EGF-BP) and cleaves mouse low molecular weight kininogen to produce bradykinin-like activity. These data have suggested that, like gamma-NGF and EGF-BP, beta-NGF-endopeptidase is a mouse glandular kallikrein. Evidence for a physiological role for NGF1-8 encouraged studies to further characterize the structure and function of this enzyme. Purified beta-NGF-endopeptidase migrated as a single band on isoelectric focusing and reducing SDS-polyacrylamide gels. As was expected, it removed NGF1-8 from NGF. Interestingly, enzymatic activity on an artificial substrate, and on NGF, was inhibited by NGF1-8 and by bradykinin. These studies further supported the view that beta-NGF-endopeptidase acts on both NGF and kininogen. The first 30 NH2-terminal amino acids of beta-NGF-endopeptidase were sequenced. This analysis demonstrated that the enzyme is encoded by the gene designated mGK-22 (Evans et al., 1987). The sequence of this gene corresponds to that of EGF-BP type A (Anundi et al., 1982; Drinkwater et al., 1987), and so studies were performed to determine whether or not beta-NGF-endopeptidase participates in EGF complex formation. Chromatographic and kinetic data gave no evidence that beta-NGF-endopeptidase is an EGF-binding protein. Our studies suggest that contamination of high molecular weight (HMW) EGF preparations with beta-NGF-endopeptidase erroneously led to earlier designation of the product of mGK-22 as an EGF-BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His^nuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (His^gab), which resolves the Tdp1-DNA linkage. A His^nuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His^gab to Arg. However, here we report that expression of the yeast His^nucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His^gab mutants, including H432N and the SCAN1-related H432R. Moreover, the His^nucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His^nucPhe mutant was catalytically inactive and suppressed His^gab mutant-induced toxicity. These data suggest that the activity of another nucleophile when His^nuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His^nuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.     

The use of microwave tissue fixation to demonstrate the in vivo phosphorylation of an acidic 80,000 molecular weight protein in the rat neocortex following treatment with soman

Studies were conducted to determine if soman, a cholinesterase inhibitor, could activate the protein kinase C system in the rat neocortex. Using microwave radiation for rapid tissue fixation, it was demonstrated that treatment with soman increased 32P incorporation into an acidic 80,000 molecular weight, heat-stable protein in vivo. Based on relative molecular weight and isoelectric point this protein appears to be identical to a protein identified as a substrate for protein kinase C. Additionally, a protein of the same molecular weight and isoelectric point could be phosphorylated in tissue slices prepared from the neocortex by cholinergic dependent mechanisms. Also, treatment with soman decreased protein kinase C in the soluble fraction of this brain region; however, no corresponding increase was observed in the particulate fraction. These results suggest that soman can activate protein kinase C in vivo, and demonstrate the utility of using microwave tissue fixation to study protein phosphorylation events in vivo.     

Adherence to uroepithelial cells of Providencia stuartii isolated from the catheterized urinary tract

The long-term catheterized urinary tract appears to offer a niche for Providencia stuartii, otherwise an unusual clinical isolate. P. stuartii, the most frequent and persistent isolate from the urine of 51 long-term catheterized patients, was recovered from 761 of 1230 (62%) weekly urine specimens. To test the hypothesis that prevalence of this species may be due to adherence properties of the organism, 20 selected strains from 14 patients at two nursing homes, representing six distinct serotypes and harbouring combinations of nine different plasmid species, were tested for adherence to uroepithelial cells (UEC). Optimal conditions were determined for differentiating strains on the basis of in vitro adherence to UEC. These strains, grown in nutrient broth, were incubated with UEC isolated from the urine of a healthy adult female (10(8) bacteria per 10(5) cells). Washed UEC, retained on 8 micron pore diameter filters, were transferred to slides, fixed and stained; bacteria were counted on each of 40 cells. Fourteen of the 20 strains were defined as adherent to UEC by comparison of mean adherent bacteria and percentage of uroepithelial cells with more than 10 bacteria. Adherence was compared to that of a P-fimbriated strain of Escherichia coli. It was not inhibited by 50 mM-mannose. We conclude that the majority of P. stuartii isolates are adherent to UEC in vitro and suggest that this may play a role in the persistence of this organism in the catheterized urinary tract.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.