Expression and subcellular localization of GSE protein in germ cells and preimplantation embryos

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.     

Surgical and radiotherapy treatment of a spinal cord ependymoma in a dog

A 4-year-old Beagle dog was presented for investigation of a left pelvic limb gait abnormality. Neurolocalisation indicated a lumbar (L2 to L5) spinal cord lesion. On magnetic resonance imaging (MRI), an intramedullary mass was demonstrated at L3. The mass was partially removed under general anaesthesia and a diagnosis of ependymoma was made on histological examination. The dog was treated with postoperative orthovoltage x-ray radiation (total dose; 44 Gy given in 11 fractions over a 4 week period) combined with low dose carboplatin (25 mg/m2). The dog was alive 16 months after surgery without further neurological deficits. No further tumour growth was detected on subsequent MRI evaluations.     

Calreticulin expression in neoplastic versus normal dog mammary glands: a cDNA subtraction-based study

This is the first report describing the expression of canine calreticulin (cCRT) in canine mammary gland tumour (MGT). Using cDNA subtraction method, it is found that mRNAs of CRT, cathepsin A, ovostatin, and lactotransferrin were differentially expressed in mammary adenocarcinoma as compared to hyperplasia, both of which were obtained from the dog. Furthermore, the mRNA expression levels of CRT and cathepsin A were significantly higher in canine MGT samples than in nontumour samples. In contrast, immunohistochemical studies have indicated that the expression of cCRT protein found to be detected in most of mammary gland tissues and was not correlated to the types of canine MGTs. Furthermore, cCRT was molecularly cloned, and the amino acid sequence of cCRT was found to be very similar to those of other species. Further studies are required to elucidate additional roles of cCRT in canine MGT.     

Population size and specific nitrification potential of soil ammonia-oxidizing bacteria under long-term fertilizer management

Population size of soil ammonia-oxidizing bacteria (AOB) was quantified by real-time PCR in a long-term (16 years) field experiment under different fertilizer managements. AOB population sizes in mineral nitrogen-fertilized soils and organic manure-fertilized soil were 10.3 and 3.1 times, respectively, that of the control, while phosphorus and potassium fertilization had no significant effect. On the other hand, the AOB specific nitrification potential (soil nitrification potential per AOB cell) was significantly higher (P < 0.05) in organic manure-fertilized soil than in mineral-fertilized soils and the control, indicating that AOB was likely more metabolically active in organic manure-fertilized soils than in mineral nitrogen-fertilized soils after long-term application.     

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC₅₀ of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC₅₀s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.     

Orally administered rutin inhibits the gene expression of Th2 cytokines in the gut and lung in aged mice

Rutin is one of the flavonoids derived from plants such as buckwheat and is well known as a powerful antioxidant. To determine whether dietary rutin could modulate mucosal immunity, we examined the gene expression of Th1/Th2 cytokines and the receptors in the gut and lung. Aged (18 months old, 18 M) C3H/HeN female mice were orally administered rutin for 10 days. The small intestine and lung were taken and analyzed by real-time PCR for gene expression. Interleukin (IL)-13 and IL-13Rα2 gene expression was significantly low (P<0.05 respectively) in the small intestine of aged rutin-fed mice. Meanwhile, there was no change in interferon γ gene expression between control and rutin-fed mice. IL-13 gene expression was also downregulated in the lung. To examine the mechanism of the inhibitory effect of rutin on Th2 cytokines in aged mice, intestinal nitric oxide synthase (NOS) expression was evaluated. Rutin inhibited inducible NOS (NOS2) gene expression, but not neuronal NOS and endothelial NOS. Gene analysis of cells collected from the small intestine by laser capture dissection revealed that NOS2 expression was significantly inhibited in crypt regions. Thus, rutin might be effective against a Th2-dominant profile through NOS2 inhibition in aged mice.     

Intestinal smooth muscle cells locally enhance stem cell factor (SCF) production against gastrointestinal nematode infections

Smooth muscle cells can produce stem cell factor (SCF) in the normal state for the preservation of mast cells, but it is still unknown whether smooth muscle cells can enhance SCF production in response to the pathological stimuli. The present study showed that smooth muscle cells in mast cell-increased regions around worm cysts of intestinal nematodes significantly enhanced SCF gene expression compared with mast cell non-increased regions in same sample. SCF gene expression in mast cell non-increased regions in nematode-infected mice showed almost the same level as in non-infected control groups. These results indicate that smooth muscle cells can locally enhance SCF gene expression, and may have a role in local immunological reactions as growth factor-producing cells.     

Enhanced protection against Heligmosomoides polygyrus in IL-2 receptor β-chain overexpressed transgenic mice with intestinal mastocytosis

IL-2 receptor β-chain overexpressed transgenic (Tg2Rβ) mice lack NK cells, but the development of other lymphocyte subsets and macrophages remained apparently intact. These mice also exhibit intestinal mastocytosis. Helminth infection induces various immune responses, such as mast cells, goblet cells, eosinophils and IgE, mediated by Th2 cytokines. IL-4 is also important in the regulation of resistance and susceptibility to Heligmosomoides polygyrus infection. However, there are contradictory results about the relation between resistance to H. polygyrus and intestinal mastocytosis. The present study showed that Tg2Rβ mice suppressed worm fecundity with mastocytosis without an increase of the levels of goblet cells, eosinophils and IgE compared with control mice. These results clearly indicated that mast cells have the ability for to protect against H. polygyrus infection. However, additional studies are required to evaluate protective effector mechanisms against H. polygyrus.     

Residual setup error in the canine intracranial region after megavoltage,kilovoltage, or cone‐beam computed tomographic image guidance for radiation therapy

Sources of residual setup error after image guidance include image localization accuracy, errors associated with image registration, and inability of some treatment couches to correct submillimeter translational errors and/or pitch and roll errors. The purpose of this experimental study was to measure setup error after image‐guided correction of the canine intracranial region, using a four degrees‐of‐freedom couch capable of 1 mm translational moves. Six cadaver dogs were positioned 45 times as for clinical treatment using a vacuum deformable body cushion, a customizable head cushion, a thermoplastic mask and an indexed maxillary plate with a dental mould. The location of five fiducial markers in the skull bones was compared between the reference position and after megavoltage (MV), kilovoltage (kV) and cone‐beam computed tomography (CBCT)‐guided correction using orthogonal kV images. The mean three‐dimensional distance vectors (3DDV) after MV, kV and CBCT‐guided correction were 1.7, 1.5 and 2.2 mm, respectively. All values were significantly different (P < .01). The 95th percentiles of the 3DDV after online MV, kV and CBCT‐guided correction were 2.8, 2.6 and 3.6 mm, respectively. Residual setup error in the clinical scenario examined was on the order of millimetres and should be considered when choosing PTV margins for image‐guided radiation therapy of the canine intracranial region.