Executive Function Deficits and Social-Behavioral Abnormality in Mice Exposed to a Low Dose of Dioxin In Utero and via Lactation

An increasing prevalence of mental health problems has been partly ascribed to abnormal brain development that is induced upon exposure to environmental chemicals. However, it has been extremely difficult to detect and assess such causality particularly at low exposure levels. To address this question, we here investigated higher brain function in mice exposed to dioxin in utero and via lactation by using our recently developed automated behavioral flexibility test and immunohistochemistry of neuronal activation markers Arc, at the 14 brain areas. Pregnant C57BL/6 mice were given orally a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at a dose of either 0, 0.6 or 3.0 µg/kg on gestation day 12.5. When the pups reached adulthood, they were group-housed in IntelliCage to assess their behavior. As a result, the offspring born to dams exposed to 0.6 µg TCDD/kg were shown to have behavioral inflexibility, compulsive repetitive behavior, and dramatically lowered competitive dominance. In these mice, immunohistochemistry of Arc exhibited the signs of hypoactivation of the medial prefrontal cortex (mPFC) and hyperactivation of the amygdala. Intriguingly, mice exposed to 3.0 µg/kg were hardly affected in both the behavioral and neuronal activation indices, indicating that the robust, non-monotonic dose-response relationship. In conclusion, this study showed for the first time that perinatal exposure to a low dose of TCDD in mice develops executive function deficits and social behavioral abnormality accompanied with the signs of imbalanced mPFC-amygdala activation.     

A novel serum metabolomics-based diagnostic approach for colorectal cancer

Background

To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer.

Methodology/Principal Findings

We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0–2 colorectal cancer (82.8%).

Conclusions/Significance

Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer.     

Reduced retinal function in amyloid precursor protein-over-expressing transgenic mice via attenuating glutamate-N-methyl-d-aspartate receptor signaling

Here, we examined whether amyloid-beta (Abeta) protein participates in cell death and retinal function using three types of transgenic (Tg) mice in vivo [human mutant amyloid precursor protein (APP) Tg (Tg 2576) mice, mutant presenilin-1 (PS-1) knock-in mice, and APP/PS-1 double Tg mice]. ELISA revealed that the insoluble form of Abeta(1-40) was markedly accumulated in the retinas of APP and APP/PS-1, but not PS-1 Tg, mice (vs. wild-type mice). In APP Tg and APP/PS-1 Tg mice, immunostaining revealed accumulations of intracellular Abeta(1-42) in retinal ganglion cells and in the inner and outer nuclear layers. APP Tg and APP/PS-1 Tg, but not PS-1 Tg, mice had less NMDA-induced retinal damage than wild-type mice, and the reduced damage in APP/PS-1 Tg mice was diminished by the pre-treatment of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a gamma-secretase inhibitor. Furthermore, the number of TUNEL-positive cells was significantly less in ganglion cell layer of APP/PS-1 Tg mice than PS-1 Tg mice 24 h after NMDA injection. The phosphorylated form of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha), but not total CaMKIIalpha or total NMDA receptor 1 (NR1) subunit, in total retinal extracts was decreased in non-treated retinas of APP/PS-1 Tg mice (vs. wild-type mice). CaMKIIalpha and NR2B proteins, but not NR1, in retinal membrane fraction were significantly decreased in APP/PS-1 Tg mice as compared with wild-type mice. The NMDA-induced increase in p-CaMKIIalpha in the retina was also lower in APP/PS-1 Tg mice than in wild-type mice. In electroretinogram and visual-evoked potential recordings, the implicit time to each peak from a light stimulus was prolonged in APP/PS-1 mice versus wild-type mice. Hence, Abeta may impair retinal function by reducing activation of NMDA-receptor signaling pathways.     

Involvement of Sema4A in the progression of experimental autoimmune myocarditis

Dilated cardiomyopathy often results from autoimmunity triggered by microbial infections during myocarditis. However, it remains unclear how immunological disorders are implicated in pathogenesis of autoimmune myocarditis. Here, we demonstrated that Sema4A, a class IV semaphorin, plays key roles in experimental autoimmune myocarditis (EAM). Dendritic cells pulsed with myosin heavy chain-α peptides induced severe myocarditis in wild-type mice, but not in Sema4A-deficient mice. In adoptive transfer experiments, CD4⁺ T-cells from wild-type mice induced severe myocarditis, while CD4⁺ T-cells from Sema4A-deficient mice exhibited considerably attenuated myocarditis. Our results indicated that Sema4A is critically involved in EAM by regulating differentiation of T-cells.     

Restoration of spermatogenesis and fertility in azoospermic mutant mice by suppression and reelevation of testosterone followed by intracytoplasmic sperm injection.

Advances in assisted reproduction techniques such as in vitro fertilization and intracytoplasmic sperm injection have made paternity possible for many patients with male infertility. However, at least some sperm or spermatids are required for these techniques to be successful, and patients incapable of producing spermatids cannot be helped. Male mice homozygous for the mutant juvenile spermatogonial depletion (jsd) gene show spermatogonial arrest and an elevated intratesticular testosterone level like many other experimental infertility models such as those with iradiation- or chemotherapy-induced testicular damage. In this category of infertile males, suppression of the testosterone level induces spermatogonial differentiation to the stage of spermatocytes but no further. In the present study with jsd mutant mice, we induced spermatogenesis first to spermatocytes and then to elongated spermatids by suppression of testosterone levels with a GnRH antagonist, Nal-Glu, at a dose of 2500 microg kg(-1) day(-1) for 4 wk and then withdrawal of Nal-Glu. Spermatids were seen in the cross-sections of seminiferous tubules in all mice treated by administration and subsequent withdrawal of Nal-Glu. Four weeks after withdrawal of Nal-Glu, some of the germ cells differentiated into elongated spermatids. Supplementation with testosterone and Nal-Glu after 4 wk of treatment with Nal-Glu alone also induced spermatogenesis similar to the induction by withdrawal of Nal-Glu. Thus, we ascribe the restoration of the differentiation of spermatocytes to spermatids to reelevation of the testosterone level. Furthermore, we successfully rescued male sterility in jsd mice by subsequent intracytoplasmic sperm injection using the elongated spermatids induced by the programmed hormone therapy.     

Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.     

Failure to prove arenavirus infection among the small mammals from an endemic area of Korean hemorrhagic nephrosonephritis.

In the light of recent knowledge on a complex of diseases caused by a new group of viruses, arenaviruses, virological studies largely directed toward small field mammals were undertaken during 1973-1974 aiming at etiological clarification of Korean hemorrhagic nephrosonephritis (KHNN). Specimens were collected in an endemic area of KHNN located north to northeast of Seoul. Virus isolation tests with 299 urine specimens and 131 mite pools recovered from small mammals and 14 acute stage sera from typical cases yielded negative results. Complement-fixation (CF) tests failed to detect antibodies against the antigens of Congo, lymphocytic choriomeningitis (LCM), Tacaribe, and Pichinde viruses among 366 small mammal sera. In addition, CF tests of 59 of the above sera against Apoi and Lassa virus antigens were negative. The results do not support the likelihood of an arenavirus being transmitted among Korean small field mammals, the overwhelming majority of which were Apodemus agrarius. A hypothesis that KHNN is caused by a virus of small field mammal origin was not proved within the technical limit of relatively unsophisticated methods employed herein.     

Purification and Characterization of Kynurenine Aminotransferase I from Human Brain

Abstract: Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l -kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000-fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2-oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute K _m of 2.0 m M and 10.0 m M for l -kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l -glutamine, l -phenylalanine, and l -tryptophan, using either pyruvate (1 m M ) or 2-oxoisocaproate (1 m M ) as a cosubstrate. l -Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate ( K _i = 480 µ M ) and competitively with regard to l -kynurenine ( K _i = 200 µ M ). Anti-KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine-pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.     

Proceedings: Preservation of bacteria by freezing at moderately low temperatures

In order to get some basic information for the development of a long-term preservation method by freezing at moderately low temperatures, the viability of 259 strains belonging to 32 genera and 135 species was measured. Cells were suspended in 10% glycerol and stored at ?53 °C for 16 months. About 93%, 88%, and 74% of aerobic bacteria gave viable cell counts higher than 10⁵/ml, 10⁶/ml, and 10⁷/ml, respectively. About 10% of gram-positives and 3% of gram-negatives gave viable cell counts lower than 10⁵/ml. There seemed to be some species—and genus—specificity with respect to viability after frozen storage and liquid paraffin-seal storage. Strains of coryneform bacteria, genera of the family Enterobacteriaceae, and the genus Pseudomonas were generally resistant. Pseudomonas putrefaciens proved to be specifically sensitive. Lactic acid bacteria were subject to sublethal injury, requiring special recovery media. Psychrophilic bacteria were very susceptible to frozen storage. All the tested strains of acetic acid bacteria survived frozen storage well both in 10% glycerol and in 10% honey at ?28 °C for 4.5 years. Honey proved to be a better adjuvant for frozen storage than glycerol. It was suggested from the results that for many kinds of bacteria, long-term preservation by freezing at moderately low temperatures might be possible when appropriate procedures are applied.