Quantifiable biomarkers of normal aging in the Japanese medaka fish (Oryzias latipes)

Background

Small laboratory fish share many anatomical and histological characteristics with other vertebrates, yet can be maintained in large numbers at low cost for lifetime studies. Here we characterize biomarkers associated with normal aging in the Japanese medaka (Oryzias latipes), a species that has been widely used in toxicology studies and has potential utility as a model organism for experimental aging research.

Principal Findings

The median lifespan of medaka was approximately 22 months under laboratory conditions. We performed quantitative histological analysis of tissues from age-grouped individuals representing young adults (6 months old), mature adults (16 months old), and adults that had survived beyond the median lifespan (24 months). Livers of 24-month old individuals showed extensive morphologic changes, including spongiosis hepatis, steatosis, ballooning degeneration, inflammation, and nuclear pyknosis. There were also phagolysosomes, vacuoles, and residual bodies in parenchymal cells and congestion of sinusoidal vessels. Livers of aged individuals were characterized by increases in lipofuscin deposits and in the number of TUNEL-positive apoptotic cells. Some of these degenerative characteristics were seen, to a lesser extent, in the livers of 16-month old individuals, but not in 6-month old individuals. The basal layer of the dermis showed an age-dependent decline in the number of dividing cells and an increase in senescence-associated β-galactosidase. The hearts of aged individuals were characterized by fibrosis and lipofuscin deposition. There was also a loss of pigmented cells from the retinal epithelium. By contrast, age-associated changes were not apparent in skeletal muscle, the ocular lens, or the brain.

Significance

The results provide a set of markers that can be used to trace the process of normal tissue aging in medaka and to evaluate the effect of environmental stressors.     

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.     

Proteomic changes in the gills of wild-type and transgenic radiosensitive medaka following exposure to direct irradiation and to X-ray induced bystander signals

The directly irradiated and bystander gill proteome was examined in wild-type and radiosensitive transgenic medaka. Direct irradiation increased the expression of annexin max 3, creatine kinase (CK), and lactate dehydrogenase (LDH) in both strains and reduced annexin A4 in wild-type medaka only. In bystander fish, same strain pairings increased CK and LDH in both strains and increased annexin max 3 and annexin A4 in radiosensitive medaka. Mixed strain pairings revealed that, in bystander fish, annexin max 3 was only increased by a bystander signal originating from a radiosensitive source, annexin A4 was increased in radiosensitive bystanders irrespective of the signal source, and CK and LDH were increased if either the bystander signal origin or the recipient bystander fish was radiosensitive. Warm-temperature acclimation related 65-kDa protein (Wap65) was increased in all bystander medaka, whether they were paired with the same or opposite strain and chromosome 5 SR-like CTD-associated factor (SR=serine-argenine-rich, CTD=C-terminal domain) (SCAF) protein was increased in radiosensitive bystander medaka only. Annexin A4, CK and LDH are associated with apoptosis and mirror the increase in apoptotic bodies previously reported in irradiated and bystander medaka, whereas increased Wap65 and LDH suggest a protective response. Thus the proteomic changes reported here could indicate both immediate protection and longer term adaptation to subsequent radiation exposure. In addition this investigation provides further evidence to show that the bystander signal can override the intrinsic genetically determined response and also that signal production and response can be modulated independently.     

Testicular lumicrine factors regulate ERK, STAT, and NFKB pathways in the initial segment of the rat epididymis to prevent apoptosis

The initial segment of the epididymis is vital for male fertility; therefore, it is important to understand the mechanisms that regulate this important region. Deprival of testicular luminal fluid factors/lumicrine factors from the epididymis results in a wave of apoptosis in the initial segment. In this study, a combination of protein array and microarray analyses was used to examine the early changes in downstream signal transduction pathways following loss of lumicrine factors. We discovered the following cascade of events leading to the loss of protection and eventual apoptosis: in the first 6 h after loss of lumicrine factors, down-regulation of the ERK pathway components was observed at the mRNA expression and protein activity levels. Microarray analysis revealed that mRNA levels of several key components of the ERK pathway, Dusp6, Dusp5, and Etv5, decreased sharply, while the analysis from the protein array revealed a decline in the activities of MAP2K1/2 and MAPK1. Immunostaining of phospho-MAPK3/1 indicated that down-regulation of the ERK pathway was specific to the epithelial cells of the initial segment. Subsequently, after 12 h of loss of lumicrine factors, levels of mRNA expression of STAT and NFKB pathway components increased, mRNA levels of several genes encoding cell cycle inhibitors increased, and levels of protein expression of several proapoptotic phosphatases increased. Finally, after 18 h of loss of protection from lumicrine factors, apoptosis was observed. In conclusion, testicular lumicrine factors protect the cells of the initial segment by activating the ERK pathway, repressing STAT and NFKB pathways, and thereby preventing apoptosis.     

Factors supporting intrathecal humoral responses following viral encephalomyelitis

Central nervous system (CNS) infections and autoimmune inflammatory disorders are often associated with retention of antibody-secreting cells (ASC). Although beneficial or detrimental contributions of ASC to CNS diseases remain to be defined, virus-specific ASC are crucial in controlling persistent CNS infection following coronavirus-induced encephalomyelitis. This report characterizes expression kinetics of factors associated with ASC homing, differentiation, and survival in the spinal cord, the prominent site of coronavirus persistence. Infection induced a vast, gamma interferon (IFN-γ)-dependent, prolonged increase in chemokine (C-X-C motif) ligand 9 (CXCL9), CXCL10, and CXCL11 mRNA, supporting a role for chemokine (C-X-C motif) receptor 3 (CXCR3)-mediated ASC recruitment. Similarly, CD4 T cell-secreted interleukin-21, a critical regulator of both peripheral activated B cells and CD8 T cells, was sustained during viral persistence. The ASC survival factors B cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferating-inducing ligand (APRIL) were also significantly elevated in the infected CNS, albeit delayed relative to the chemokines. Unlike IFN-γ-dependent BAFF upregulation, APRIL induction was IFN-γ independent. Moreover, both APRIL and BAFF were predominantly localized to astrocytes. Last, the expression kinetics of the APRIL and BAFF receptors coincided with CNS accumulation of ASC. Therefore, the factors associated with ASC migration, differentiation, and survival are all induced during acute viral encephalomyelitis, prior to ASC accumulation in the CNS. Importantly, the CNS expression kinetics implicate rapid establishment, and subsequent maintenance, of an environment capable of supporting differentiation and survival of protective antiviral ASC, recruited as plasmablasts from lymphoid organs.     

Juvenile hormone esterase (JHE) from Tenebrio molitor: full-length cDNA sequence,in vitro expression,and characterization of the recombinant protein

Juvenile hormone regulates the development and reproduction in a variety of insects. Juvenile hormone esterase (JHE) is a selective enzyme, which hydrolyzes the methyl ester of JH and alters its activity. In Tenebrio molitor, JHE has been previously purified from pupae and a partial cDNA was amplified by RT-PCR using fat body mRNA. The previous report indicated that several forms of the JHE protein were present in pupal homogenate. In this study, we report the full-length cDNA, which was obtained by RACE methods. The deduced protein sequence corresponds to peptides from two proteins of different molecular weights in the previous study. The coding region of the full-length cDNA was subcloned into the AcMNPV genome and high levels of expression of the JHE enzyme from the viral p10 promoter were demonstrated in cell culture. The majority of JHE is secreted from the cells as a soluble enzyme. The recombinant JHE enzyme was biochemically characterized. The recombinant protein appears by PAGE analysis as a monomer of approximately the same MW (66000) and pI (4.9) as was expected from the deduced amino acid sequence of the cDNA.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Mathematical and computational modeling in biology at multiple scales

A variety of topics are reviewed in the area of mathematical and computational modeling in biology, covering the range of scales from populations of organisms to electrons in atoms. The use of maximum entropy as an inference tool in the fields of biology and drug discovery is discussed. Mathematical and computational methods and models in the areas of epidemiology, cell physiology and cancer are surveyed. The technique of molecular dynamics is covered, with special attention to force fields for protein simulations and methods for the calculation of solvation free energies. The utility of quantum mechanical methods in biophysical and biochemical modeling is explored. The field of computational enzymology is examined.