Molecular analyses of the agouti allele in the Japanese house mouse identify a novel variant of the agouti gene.

It has been thought that the Japanese house mouse carries the Aw allele at the agouti locus causing light-colored bellies, but they do not always show this coloration. Thus, the presence of the Aw allele seems to be doubtful in them. To ascertain whether the Aw allele is present, a two-pronged approach was used. First, we compared lengths of DNA fragments obtained from three PCRs conducted on them to the known fragment sizes generated from mouse strains exhibiting homozygosities of either a/a, A/A, or Aw/Aw. PCR I, PCR II, and PCR III amplify only in the A and Aw alleles, the a and Aw alleles, and the a allele, respectively, and we detected amplifications in strains with A/A and Aw/Aw by PCR I, in those with a/a and the Japanese house mouse by PCR II, and in those with a/a by PCR III. Second, we sequenced the exon 1A region of the agouti gene and obtained sequences corresponding to the above strains and the Japanese house mouse, but their sequences were similar to those of the a allele. We concluded that their agouti allele is not identical to the Aw allele and seems to be a novel type similar to the a allele.

Colonization and Differentiation Traits of the Japanese House Mouse, Mus musculus (Rodentia, Muridae), Inferred from Mitochondrial Haplotypes and External Body Characteristics.

To evaluate the colonization histories of the Japanese house mice (Mus musculus), phenotypic and genotypic admixtures of the subspecific traits were studied by evaluation of external body characteristics and mitochondrial gene elements. We analyzed mitochondrial Cytb gene and coat colorations and body dimensions as subspecific characteristics in mice from four areas of the Japanese Islands, the Sorachi, Ishikari and Iburi areas of Hokkaido, the Hidaka area of Hokkaido, and northeastern and central Honshu. Three occurrence patterns of the subspecific haplotypes of Cytb-the castaneus type only, the musculus type only, and the castaneus, musculus, and domesticus types together-were observed in the study areas. In central Honshu, the properties of haplotypes were in accord with the external characteristics as reported in previous findings. In contrast, complicated external characteristics were observed in the Hidaka area, where mice showed multiple haplotype properties. In addition, in northeastern Honshu, coat colorations were not in accord with haplotype properties and such discordance was also observed in most mice in the Sorachi, Ishikari and Iburi areas of Hokkaido. These complexities and discordances suggest that the genetic and phenotypic properties have been caused by different processes, not only through founder effects by migrations and subsequent subspecific hybridizations but also through differentiation in each study area.

Anxiolytic effects of Enterococcus faecalis 2001 on a mouse model of colitis.

Ulcerative colitis (UC) is a refractory inflammatory bowel disease, which is known to cause psychiatric disorders such as anxiety and depression at a high rate in addition to peripheral inflammatory symptoms. However, the pathogenesis of these psychiatric disorders remains mostly unknown. While prior research revealed that the Enterococcus faecalis 2001 (EF-2001) suppressed UC-like symptoms and accompanying depressive-like behaviors, observed in a UC model using dextran sulfate sodium (DSS), whether it has an anxiolytic effect remains unclear. Therefore, we examined whether EF-2001 attenuates DSS-induced anxiety-like behaviors. Treatment with 2% DSS for seven days induced UC-like symptoms and anxiety-like behavior through the hole-board test, increased serum lipopolysaccharide (LPS) and corticosterone concentration, and p-glucocorticoid receptor (GR) in the prefrontal cortex (PFC), and decreased N-methyl-D-aspartate receptor subunit (NR) 2A and NR2B expression levels in the PFC. Interestingly, these changes were reversed by EF-2001 administration. Further, EF-2001 administration enhanced CAMKII/CREB/BDNF-Drebrin pathways in the PFC of DSS-treated mice, and labeling of p-GR, p-CAMKII, and p-CREB showed colocalization with neurons. EF-2001 attenuated anxiety-like behavior by reducing serum LPS and corticosterone levels linked to the improvement of UC symptoms and by facilitating the CAMKII/CREB/BDNF-Drebrin pathways in the PFC. Our findings suggest a close relationship between UC and anxiety.

Establishment of a polymerase chain reaction-based method for strain-level management of Enterococcus faecalis EF-2001 using species-specific sequences identified by whole genome sequences.

In the development and manufacture of fermented foods, it is crucial to control and manage the bacterial species used in the products. We previously reported a complete genome sequence analysis of the Enterococcus faecalis EF-2001 strain that was used for supplements. By comparing this sequence to the publicly available complete genome sequence of E. faecalis strains, we were able to identify specific sequences of the EF-2001 strain. We designed primer sets to amplify these specific regions and performed a polymerase chain reaction (PCR). We confirmed that the DNA fragments were specifically amplified in the genome of the EF-2001 strain, but not those of other lactic acid bacteria (LABs) or strains of the same genus. Furthermore, these primers amplified DNA fragments even in genomic DNA extracted from heat-treated bacteria at 121°C and foods containing the EF-2001 strain. These results suggest that this method allows for simple and highly accurate identification of specific fermentation strains, such as LABs at the strain level, which will be useful for controlling the quality of fermented foods.

Evidence of late root formation of molars in Anderson's red-backed vole, Eothenomysandersoni (Thomas, 1905) (Cricetidae, Rodentia), and arguments for its generic allocation.

We evaluated the molars in Anderson's red-backed vole (n = 114) from the Kii Peninsula of Honshu, Japan. Two of the specimens are considered extremely old aged based on their dimensions and on the loss of alveolar capsules of M2, and a third one is also old based on its strongly worn left M3 and M1. Of the former two individuals, one showed an incipient closure of re-entrant angles at its basal end, as estimated from the difference between the occlusal patterns of the occlusal and basal surfaces of the left M2. The latter individual also showed a complete closure of the basal end in the left M3. These patterns differ from incipient roots observed in other vole taxa but were similar to a previous example of incipient roots in Anderson's red-backed vole. Therefore, we suggest that molar roots in this species form at an extremely late age or by strong wear. Root formation in molars is considered an important diagnostic character, as Eothenomys molars lack roots, while Craseomys molars develop roots at a late age. However, this dental character may be particularly difficult to assess in voles under natural conditions. Considering previous phylogenetic findings based on molecular analyses, Craseomys is the most appropriate genus for Anderson's and other Asiatic red-backed voles.

Antidepressant effects of Enterococcus faecalis 2001 through the regulation of prefrontal cortical myelination via the enhancement of CREB/BDNF and NF-κB p65/LIF/STAT3 pathways in olfactory bulbectomized mice.

A therapeutic strategy through the gut-brain axis has been proven to be effective in treatment for depression. In our previous study, we demonstrated that Enterococcus faecalis 2001 (EF-2001) prevents colitis-induced depressive-like behavior through the gut-brain axis in mice. More recently, we found that demyelination in the prefrontal cortex (PFC) was associated with depressive-like behavior in an animal model of major depressive disorder, olfactory bulbectomized (OBX) mice. The present study investigated the effects of EF-2001 on depressive-like behaviors in OBX mice and the underlying molecular mechanisms from the perspective of myelination in the PFC. OBX mice exhibited depressive-like behaviors in the tail-suspension, splash, and sucrose preference tests, and decreased myelin and paranodal proteins along with mature oligodendrocytes in the PFC. These behavioral and biochemical changes were all prevented by treatment with EF-2001. Further, EF-2001 treatment increased brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) in the PFC. Interestingly, an immunohistochemical analysis revealed enhanced phospho (p) -cAMP-responsive element binding protein (CREB) expression in neurons, p-nuclear factor-kappa B (NFκB) p65 (Ser536) expression in astrocytes, and p-signal transducer and activator of transcription 3 (STAT3) (Ty705) expression in mature oligodendrocytes in the PFC of OBX mice. From these results, we suggest that EF-2001 administration prevents depressive-like behaviors by regulating prefrontal cortical myelination via the enhancement of CREB/BDNF and NFκB p65/LIF/STAT3 pathways. Our findings strongly support the idea that a therapeutic strategy involving the gut microbiota may be a promising alternative treatment for alleviating symptoms of depression.

Heat-Killed Enterococcus faecalis Prevents Adipogenesis and High Fat Diet-Induced Obesity by Inhibition of Lipid Accumulation through Inhibiting C/EBP-α and PPAR-γ in the Insulin Signaling Pathway.

Increasing consumption of food with high caloric density and a sedentary lifestyle have influenced the increasing obesity prevalence worldwide. The recent pandemic has contributed to this problem. Obesity refers to a state in which lipid accumulates excessively in adipocytes and adipose tissues. Dried heat-killed Enterococcus faecalis (EF-2001) prevents allergic mechanisms, inflammation, and tumor progression. In the present study, we investigated the effects of EF-2001 on high fat diet (HFD)-induced obese rats. The degree of obesity in experimental rats was reduced after 6 weeks of oral administration of 3 mg/kg or 30 mg/kg dosages of EF-2001, indicating regulating effects in rats with HFD-induced obesity. We found that EF-2001 decreased the amounts of total cholesterol, triglyceride, and non-high density lipoprotein (HDL) in HFD-induced obese rats. The effects of EF-2001 on 3T3-L1 adipocytes stained with Oil red O stain are shown in reductions of lipid accumulation, respectively. In addition, we examined the relationships between EF-2001 treatment and mechanisms for the insulin signaling of adipogenesis in 3T3-L1 cells. EF-2001 induced down-regulation in phosphorylation of Erk, JNK, and Akt through the inhibition of insulin receptor phosphorylation. EF-2001 inhibits the expressions of C/EBP-α and PPAR-γ, a lipid metabolism-related transcription factor through confocal microscope observation and Western blot on 3T3-L1 adipocytes and HFD-induced obese rats. Based on our results, intake of EF-2001 significantly prevented HFD-induced obesity in rats through inhibition of C/EBP-α and PPAR-γ in the insulin signaling pathway on lipid accumulation.

A genetic typing method for albino mutation in the Mongolian gerbil by PCR-RFLP analysis.

1. We established a PCR-RFLP analysis targeting R77H mutation in the Tyr gene as a more effective genotyping to identify carrier (C/c) with the albino allele and the agouti phenotypes. 2. Our breeding system, which targets the R77H site, is a useful cue for detecting C/c carriers with the agouti-phenotype and helps us to obtain albinos by mating agouti-phenotype carriers.

Heat-Killed and Live Enterococcus faecalis Attenuates Enlarged Prostate in an Animal Model of Benign Prostatic Hyperplasia.

In the present study, we investigated the inhibitory effect of heat-killed Enterococcus faecalis (E. faecalis) and live E. faecalis on benign prostatic hyperplasia (BPH). The BPH rat model was established by administering male rats with testosterone propionate (TP, 5 mg/kg, in corn oil) via subcutaneous injections daily for four weeks after castration. The rats were divided into five groups: Con, corn oil-injected (s.c.) + DW administration; BPH, TP (5 mg/kg, s.c.) + DW administration; BPH+K_EF, TP (5 mg/kg, s.c.) + heat-killed E. faecalis (7.5 × 1012 CFU/g, 2.21 mg/kg) administration; BPH+L_EF, TP (5 mg/kg, s.c.) + live E. faecalis (1 × 1011 CFU/g, 166 mg/kg) administration; BPH+Fi, TP (5 mg/kg, s.c.) + finasteride (1 mg/kg) administration. In both of BPH+K_EF and BPH+L_EF groups, the prostate weight decreased and histological changes due to TP treatment recovered to the level of the Con group. Both of these groups also showed regulation of androgen-signaling factors, growth factors, and apoptosis-related factors in prostate tissue. E. faecalis exhibited an inhibitory effect on benign prostatic hyperplasia, and even heat-killed E. faecalis showed similar efficacy on the live cells in the BPH rat model. As the first investigation into the effect of heat-killed and live E. faecalis on BPH, our study suggests that heat-killed E. faecalis might be a food additive candidate for use in various foods, regardless of heat processing.

Activation of cholinergic system partially rescues olfactory dysfunction-induced learning and memory deficit in mice.

Deficits in olfaction are associated with neurodegenerative disorders such as Alzheimer's disease. A recent study reported that intranasal zinc sulfate (ZnSO4)-treated mice show olfaction and memory deficits. However, it remains unknown whether olfaction deficit-induced learning and memory impairment is associated with the cholinergic system in the brain. In this study, we evaluated olfactory function by the buried food find test, and learning and memory function by the Y-maze and passive avoidance tests in ZnSO4-treated mice. The expression of choline acetyltransferase (ChAT) protein in the olfactory bulb (OB), prefrontal cortex, hippocampus, and amygdala was assessed by western blotting. Moreover, we observed the effect of the acetylcholinesterase inhibitor physostigmine on ZnSO4-induced learning and memory deficits. We found that intranasal ZnSO4-treated mice exhibited olfactory dysfunction, while this change was recovered on day 14 after treatment. Both short-term and long-term learning and memory were impaired on days 4 and 7 after treatment with ZnSO4, whereas the former, but not the latter, was recovered on day 14 after treatment. A significant correlation was observed between olfactory function and short-term memory, but not long-term memory. Treatment with ZnSO4 decreased the ChAT level in the OB on day 4, and increased and decreased the ChAT levels in the OB and hippocampus on day 7, respectively. Physostigmine improved the ZnSO4-induced deficit in short-term, but not long-term, memory. Taken together, the present results suggest that short-term memory may be closely associated with olfactory function via the cholinergic system.

Intra- and interspecific nuclear ribosomal gene variation in the two Japanese Eothenomys species, E. andersoni and E. smithii.

We analyzed partial sequences (approx. 630 bp) of the 18S nuclear ribosomal RNA gene (rDNA) in the Japanese Eothenomys voles, E. andersoni and E. smithii, to evaluate their evolutionary complexity. There were two rDNA types of sequences with a few substitutions and an indel, and both species alternatively carried one of the two rDNA types, irrespective of their classifications. One of the rDNA types was mainly distributed in Eastern Japan, and the other in Western Japan. Such rDNA type distributions were not related to the species classifications. The complex genomic elements across both species were confirmed by previous studies of nuclear, mitochondrial, and Y chromosomal genes, and our current data agree with previous findings. Previous studies indicated that the genome constitutions of the Japanese Eothenomys species may have been caused by interspecific genetic exchanges at an early period after the speciation into E. andersoni and E. smithii from their common ancestor, as neither species crosses at present. In addition, the present results also suggest the additional hypothesis that their common ancestor might have two (or more) rDNA gene types, and that one of the rDNA types was fixed in each population of both species during their colonization processes.

Enterococcus faecalis EF-2001 protects DNBS-induced inflammatory bowel disease in mice model.

Recent studies have demonstrated the immunomodulatory effects of heat-killed lactic acid bacteria. The aim of this study was to evaluate the protective effect of heat-killed Enterococcus faecalis EF-2001 (EF-2001) on a model of inflammatory bowel disease (IBD). A total of 28 female NC/Nga mice were divided into 4 treatment groups. Controls were fed a normal commercial diet. In the experimental groups, colitis was induced by rectal administration of dinitrobenzene sulfonic acid. Two groups were orally administered 2 and 17 mg/kg EF-2001, respectively. EF-2001 treatment decreased the expression of several cytokines, including cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, interleukin (IL)-1ß, and IL-6 in inflamed colon compared to the DNBS alone group. In addition, EF-2001 suppressed DNBS-induced colonic tissue destruction. Therefore, this study strongly suggests that EF-2001 could alleviate the inflammation associated with mouse IBD.

[An approach to the safety management of ambulant cancer chemotherapy].

As for ambulant cancer chemotherapy at Tokai University Hachioji Hospital, we have been employing computerized methods with electronic medical charts for a series of operations, such as receiving medication instructions by doctors, preparation of drugs, chemotherapy regimen-checks and quantitative calculations for admixtures of anticancer drugs. Additionally, we have introduced new features that print information such as chemical data needed for regimen-checks and doctors' answers to questions about prescriptions on the prescriptions of injections. The system is enabled by pharmacists accurately and effectively to check prescriptions of injections, to calculate the precise quantity for admixtures of drugs, and also to share information among pharmacists. It has contributed to the efficiency and medical safety of the ambulant cancer chemotherapy.

Phylogenetic relationships of rodent pinworms (genus Syphacia) in Japan inferred from mitochondrial CO1 gene sequences.

Species of the genus Syphacia are considered to have generally co-evolved with their rodent hosts. This study determined partial sequences of the CO1 gene from several species in the genus Syphacia and discuss the relationships between pinworms and their hosts. Syphacia montana, which parasitizes Microtinae, was closely related to S. frederici and S. obvelata, which parasitize Murinae. Although both S. obvelata and S. ohtaorum parasitize rodents in the genus Mus, these two species were not found to be closely related to each other. Syphacia frederici, S. emileromani and S. agraria are all pinworms of the Apodemus species, but genetic affiliation between these three species was not indicated. These facts suggest that the co-evolutionary relationship between species of the genus Syphacia and their host rodents may not so strict and host switching has probably occurred during the course of evolution.

[An approach to ambulant cancer chemotherapy by the pharmaceutical department system].

In recent years, cancer chemotherapy has been switching from hospital treatment to ambulant treatment. In such a situation, pharmacists are required to carry out their work unfailingly and smoothly in checking chemotherapy regimens, admixtures of drugs and providing information, for example. At Tokai University Hachioji Hospital, we have been trying to computerize a regimen-check, quantitative calculations for admixtures of anticancer drugs and the comment-input function of the pharmaceutical department so that our work will be more efficient and precise. The regimen-check system receives automatically, the patients' own data and the information of injection prescriptions and of the clinical laboratory from the Hospital Information System. Then pharmacists put the information to the regimen master file for recording in order to manage dosages, the accumulated dose and dose schedule. Also by registering the amount of dissolution of each anticancer drug beforehand, the quantitative calculation system makes it possible to automatically convert dosages on electronic medical charts to a liquid measure needed for the admixtures of drugs and to print the information on the prescription of injections. Additionally, the comment-input function makes it possible to print information on injections such as chemical information needed for regimen-checks and doctors' answers to questions about prescriptions. The pharmaceutical department system enables pharmacists to check regimens accurately and quickly to calculate the precise quantity for admixtures of drugs and to share information among pharmacists. The system has also contributed to the efficiency and medical safety of the ambulant cancer chemotherapy.

Pharmaceutical Production of Anti-tumor and Immune-potentiating Enterococcus faecalis-2001 ß-glucans: Enhanced Activity of Macrophage and Lymphocytes in Tumor-implanted Mice.

BACKGROUND: Enterococcus faecalis 2001 is a probiotic lactic acid bacterium and has been used as a biological response modifier (BRM). From physiological limitation of bacterial preservation in storage and safety, the live E. faecalis 2001 has been heat-treated and the BRM components containing high level of ß-glucan, named EF-2001, were prepared. METHOD: The heat-treated EF-2001 has been examined for the antioxidative potential for radical scavenging and anti-tumor activities as well as immune-enhancing response in mice. Lymphocyte versus polymorphonuclear leukocyte ratio was increased in mice upon treatment with EF-2001. The number of lymphocytes was increased in the EF-2001-treated group. In the mice bearing two different Ehrlich solid and Sarcoma-180 carcinomas, the treatment with EF-2001 resulted in anti-tumor action. Tumor-suppressive capacity upon treatment with EF-2001 was significantly increased compared to normal controls. RESULTS: During the time interval administration of 5 weeks between the priming and secondary administration of EF-2001, the expression and production levels of TNF-α were also observed in the EF- 2001-administered mice. Additionally, anti-tumor activity examined with the intravenous administration of EF 2001 with a 34 times interval was also observed, as the growth of Sarcoma180 cells was clearly inhibited by the EF-2001. CONCLUSION: From the results, it was suggested that the immune response is enhanced due to antioxidative activity caused by the EF-2001 and anti-tumor activity by NK cells and TNF-α.

Intraspecific differentiation in the lesser Japanese mole in eastern Honshu, Japan, indicated by nuclear and mitochondrial gene analyses.

The lesser Japanese mole, Mogera imaizumii, recognized by Motokawa and Abe (1996), occurs in eastern Honshu, western Honshu and Shikoku. Mitochondrial and nuclear DNA were analyzed for mole samples from eastern Honshu to elucidate intraspecific differentiation. Analyses of sequences of the mitochondrial cytochrome b gene (Cytb) and of a restriction fragment length polymorphism of the nuclear 28S ribosomal RNA gene spacer (rDNA-RFLP) revealed two genetic types, partially corresponding to Hutterer's (1993) taxa, M. wogura (= M. imaizumii) and M. minor. Most samples showed either of two combinations of mitochondrial / nuclear gene types. However, two specimens showed a different combination. This incongruent combination of mitochondrial and nuclear genes might have derived, in part, from an introgression event between genetically differentiated populations after secondary contact during the evolutionary history of the lesser Japanese mole in eastern Honshu.

[The cooperation between pharmacists and nurses in outpatient cancer chemotherapy].

The cooperation between each section and exchange of information are important in outpatient cancer chemotherapy. At Tokai University Hachioji hospital, we adopted a sheet that supports aseptic preparation of anticancer agents and a sheet for exchanging information. The purpose of the sheet is to grasp the information that we get in each section. The main information that we exchanged was its contents about an order input, drip infusion rate and a dosage order of antiemetic or anticancer drugs. By having added the sheet, we could unify a mixture preparation and show a clear presentation to nursing of a preparation method that can be enabled. In addition, acquisition of information obtained in each of the section became easy. In this way, by cooperating between sections and sharing information that we obtain, it is thought that it leads to improving the safety of outpatient cancer chemotherapy.

Effect of Enterococcus faecalis EF-2001 on experimentally induced atopic eczema in mice.

Here, the effects of heat-killed Enterococcus faecalis EF-2001 (EF-2001) on atopic eczema (AE) were assessed. An AE model was established in vivo by repetitious topical exposure to 1-chloro-2,4-dinitrobenzene (CDNB) and dermatophagoidesfarinae extract (DFE) via application on each ear. Mice were administered EF-2001 orally for 4 weeks, dermal and epidermal ear thickness, mast cell infiltration of the ear tissue, and serum IgE and IgG2a levels were evaluated. Moreover, pathogenic cytokines levels of the ears, splenocytes, and cervical lymph nodes were determined. EF-2001 reduced AE symptoms grounded in the ear thickness, histopathological analysis, and serum IgE levels. Furthermore, EF-2001 attenuated mast cell infiltration in the ears and CDNB/DFE-induced various pathogenic cytokines levels of the ears, splenocytes and cervical lymph nodes. Thus, our data suggested that EF-2001 may have potential medicinal applications in the treatment of AE through its immunomodulatory properties.

Heat-Killed Enterococcus faecalis EF-2001 Ameliorates Atopic Dermatitis in a Murine Model.

Recent reports have shown the immunomodulatory effect of heat-killed lactic acid bacteria. Atopic dermatitis (AD) is an allergic skin disease, caused by immune dysregulation among other factors. The aim of this study was to assess the effect of heat-killed Enterococcus faecalis EF-2001 (EF-2001) on AD. We established an in vivo AD model by repeated local exposure of Dermatophagoides farinae extract (DFE; house dust mite extract) and 2,4-dinitrochlorobenzene (DNCB) to the ears of mice. After oral administration of EF-2001 for four weeks, the epidermal and dermal ear thickness, mast cell infiltration, and serum immunoglobulin levels were measured. In addition, the gene expression levels of pathogenic cytokines in the ears, lymph nodes, and splenocytes were assayed. EF-2001 attenuated AD symptoms based on the ear thickness, histopathological analysis, and serum immunoglobulin levels. Moreover, EF-2001 decreased the DFE/DNCB-induced expression of various pathogenic cytokines in the ears, lymph nodes, and splenocytes. These results suggest that EF-2001 has therapeutic potential in the treatment of AD owing to its immunomodulatory effects.