Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.     

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.     

Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe11), KE3 (probe7), KE2 (probe5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the 2(XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb 3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.     

Oxidative stress and aging in Caenorhabditis elegans

Much attention has been focused on the hypothesis that oxidative damage plays in cellular and organismal aging. A mev-1 (kn1) mutant of Caenorhabditis elegans, isolated on the basis of its methyl viologen (paraquat) hypersensitivity, is also hypersensitive to elevated oxygen levels. Unlike the wild type, its life span decreases dramatically as oxygen concentrations are increased from 1% to 60%. Strains, which bear this mutation, accumulate fluorescent materials and protein carbonyl groups, markers of aging, at faster rates than the wild type. We have cloned mev-1 gene by transformation rescue and found that it is, in fact, the previously sequenced gene (cyt-1) that encodes succinate dehydrogenase cytochrome b. A missense mutation abolishes complex II activity in the mitochondrial membrane but not succinate dehydrogenase enzyme activity per se. These data suggest that CYT-1 directly participates in electron transport from FADH₂ to coenzyme Q. Moreover, mutational inactivation of this process renders animals susceptible to oxidative stress and, as a result, leads to premature aging.     

Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis

A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011 bp with predicted 336 amino acids and molecular mass of 38 kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the K_m value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494 ± 1.536 μM. Risedronate inhibited the activity of BbFPPS yielding IC₅₀ value of 8.4 ± 1.2 nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC₅₀ = 4.02 ± 0.91 μM). No regrowth of B. bovis was observed at 10 μM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis.     

A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

Mitochondrial oxidative stress is considered as a key accelerator of fibrosis in various organs including the liver. However, the production of oxidative stress and progression of liver fibrosis may merely represent the independent consequences of hepatocellular injury caused by the primary disease. Because of a lack of appropriate experimental models to evaluate the sole effects of oxidative stress, it is virtually unknown whether this stress is causatively linked to the progression of liver fibrosis. Here, we examined the direct effects of mitochondrial reactive oxygen species (ROS) on the progression of high fat/calorie diet-induced steatohepatitis using Tet-mev-1 mice, in which a mutated succinate dehydrogenase transgene impairs the mitochondrial electron transport and generates an excess amount of ROS in response to doxycycline administration. Wild type and Tet-mev-1 mice that had been continuously given doxycycline-containing water were subsequently fed either normal chow or a cholesterol-free high-fat/high-sucrose diet for 4 months at approximately 1 or 2 years of age. Histopathological examinations indicated that neither the mitochondrial ROS induced in Tet-mev-1 mice nor the feeding of wild type animals with high-fat/high-sucrose diet alone caused significant liver fibrosis. Only when the Tet-mev-1 mice were fed a high-fat/high-sucrose diet, it induced lipid peroxidation in hepatocytes and enhanced hepatic CC chemokine expression. These events were accompanied by increased infiltration of CCR5-positive cells and activation of myofibroblasts, resulting in extensive liver fibrosis. Interestingly, this combinatorial effect of mitochondrial ROS and excess fat/calorie intake on liver fibrosis was observed only in 2-year-old Tet-mev-1 mice, not in the 1-year-old animals. Collectively, these results indicate that mitochondrial ROS in combination with excess fat/calorie intake accelerates liver fibrosis by enhancing CC chemokine production in aged animals. We have provided a good experimental model to explore how high fat/calorie intake increases the susceptibility to nonalcoholic steatohepatitis in aged individuals who have impaired mitochondrial adaptation.     

Babesia bovis: effects of cysteine protease inhibitors on in vitro growth

In the present study, we examined the effects of four kinds of cysteine protease inhibitors (E64, E64d, leupeptin, and ALLN) on the in vitro asexual growth of Babesia bovis. Of these, only the lipophilic inhibitors, E64d and ALLN, were found to effectively inhibit the growth of B. bovis. In further experiments, E64d, but not ALLN, significantly suppressed the parasite’s invasion of host erythrocytes, while both chemicals, especially ALLN, inhibited the parasite’s replication within the infected erythrocytes. These data suggested the presence of cysteine protease(s) derived from B. bovis, in which the protease(s) would play important roles in the erythrocyte invasion and/or replication processes of the parasite.     

Isolation and cDNA cloning of ovarian cortical rod protein in kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae)

Two cortical rod proteins having molecular weights of 28.6 kDa and 30.5 kDa were isolated from the mature ovary of Marsupenaeus japonicus using gel filtration and reversed-phase HPLC. Analysis of the N-terminal amino acid sequence of the 28.6 kDa molecule revealed that amino acid residues 1-21 corresponded to residues 9-29 of the 30.5 kDa molecule. Examination of homology using BLAST showed that 21 amino acids out of 29 residues of the 28.6 kDa molecule, and 14 out of 29 residues of the 30.5 kDa molecule were identical to that of the ovarian cortical rod proteins of Penaeus semisulcatus. Positive immunohistochemical reaction to antiserum raised against the 28.6 kDa protein was observed on cortical rods forming around the periphery of oocytes at the maturation stages. Western blotting analysis revealed that both the 28.6 kDa and 30.5 kDa molecules stained with the anti-28.6 kDa antiserum. Furthermore, the 28.6 kDa and 30.5 kDa proteins were both glycosylated, as evidenced by positive carbohydrate staining using Concanavalin A and production of positive PAS reaction. These results indicate that the cortical rods are comprised of the 28.6 kDa and 30.5 kDa molecules. We subsequently cloned two full-length cDNAs based on the N-terminal sequences of the 28.6 kDa and 30.5 kDa molecules. The open reading frame of 28.6 kDa and 30.5 kDa encoded 276 amino acid residues. Comparison analysis of the two cDNAs revealed that the location of the processing site and sequence of signal peptides differed, indicating that the two cDNAs are products of two separate genes and encode the 28.6 kDa molecule and 30.5 kDa molecule, respectively. Both proteins possessed one potential N-linked glycosylation site. It is considered that both molecules are components of the cortical rods, forming a jelly layer after fertilization.