De novo and salvage pathways of DNA synthesis in primary cultured neurall stem cells

We studied the de novo and salvage pathways of DNA synthesis in sphere-forming neural stem cells obtained from mouse embryos by a neurosphere method. The former pathway needs folic acid (FA) for nucleotide biosynthesis, while the latter requires deoxyribonucleosides (dNS). We examined the proliferative activity of sphere-forming cells in E14.5 embryos by counting the number of spheres formed in media that lacked FA and/or dNS. Proliferation failure and apoptosis occurred in a deficient medium lacking of both FA and dNS. Spheres formed in the deficient medium supplemented with dNS, without FA, did not produce neuron, but rather only seem to generate astrocytes and oligodendrocytes when plated under differentiation condition in culture. On the other hand, a subpopulation of cultured cells formed spheres in the deficient medium supplemented with FA alone in an appropriate concentration, and did possess the self-renewing and multipotential characteristics of neural stem cells. Spheres formed in the media containing low dose Azathioprine and methotrexate, inhibitors of de novo DNA synthesis, were selectively prevented from producing neurons even in the presence of FA. These results suggested that activating de novo DNA synthesis was needed for neural stem cells to proliferate with multipotentiality.     

Presence of Bacterial 16S Ribosomal RNA Gene Segments in Human Intestinal Lymph Follicles

Background: There is currently no information regarding microbial agents inside the intestinal lymph follicles. Methods: Biopsy or resected specimens, mostly from macroscopically normal areas, were sectioned with a cryostat. DNA was extracted from microdissected samples, exclusively from the lymph follicle. Amplification of DNA was performed using universal primers designed from conserved regions of bacterial 16S ribosomal RNA (rRNA). Several clones with inserts of around 400 base pairs were subjected to DNA sequence analysis followed by a database homology search. Results: Bacterial 16S rRNA gene segments were detected in the lymph follicle in 2 of 14 (14%) non-inflammatory bowel disease (IBD) cases, 4 of 14 (28%) Crohn disease cases, and in 2 of 5 (40%) ulcerative colitis cases. Nineteen 16S rRNA gene segments were recognized in the eight positive cases. Five segments showed 100% identity to known bacterial 16S rRNAs, namely staphylococcus species, Streptococcus sanguis, and Paracoccus marcusii. However, the other 14 segments showed below 100% identity, indicating either the presence of unknown bacteria or of bacteria without known DNA data. No single identified or unidentified bacterium, characteristic of IBD, including Mycobacterium paratuberculosis and Listeria monocytogenes, was detected. Conclusions: The present study confirms the presence of bacterial 16S rRNA gene segments in human intestinal lymph follicles and paves the way for new investigations into the microbiology of the lymph follicle. Whether or not bacteria inside the lymph follicle is a primary stimulus in IBD has yet to be clarified.     

Preclinical study of recombinant human bone morphogenetic protein-2: application of hyperbaric oxygenation during bone formation under unfavourable condition

The objective of this study was to evaluate the effect of hyperbaric oxygenation on bone formation by recombinant human bone morphogenetic protein-2 (rhBMP-2) under unfavourable conditions. The calf muscles of 10 rats with low-blood supply were prepared by ligating and cutting the right femoral artery, and 10 micro of rhBMP-2 was implanted in the calf muscle. Five rats each were randomly assigned to the hyperbaric oxygenation group and the control group (untreated). The rats in the hyperbaric oxygenation group were treated with hyperbaric oxygenation at 2.0 atmospheres absolute for 3 weeks. In the histologic evaluation, the number of osteoblasts in the hyperbaric oxygenation group was greater than that in control group. The area of the trabecular bone induced in the hyperbaric oxygenation group was significantly larger than that in the control group. The values of alkaline phosphatase activity and calcium contents in the hyperbaric oxygenation group were significantly higher than those in the control group. The results of the present study suggest that hyperbaric oxygenation increases the partial oxygen pressure in low blood supply tissue and accelerates the activity and rate of osteoinduction by rhBMP-2. Hyperbaric oxygenation therapy may increase the clinical application of rhBMP-2 to unfavourable condition.     

Effects of an hERG Activator,ICA-105574, on Electrophysiological Properties of Canine Hearts

In short QT syndrome, inherited gain-of-function mutations in the human ether a-go-go-related gene (hERG) K⁺ channel have been associated with development of fatal arrhythmias. This implies that drugs that activate hERG as a side effect may likewise pose significant arrhythmia risk. hERG activators have been found to have diverse mechanisms of activation, which may reflect their distinct binding sites. Recently, the new hERG activator ICA-105574 was introduced, which disables inactivation of the hERG channel with very high potency. We explored characteristics of this new drug in several experimental models. Patch clamp experiments were used to verify activation of hERG channels by ICA-105574 in human embryonic kidney cells stably-expressing hERG channels. ICA-105574 significantly shortened QT and QTc intervals and monophasic action potential duration (MAP₉₀) in Langendorff-perfused guinea-pig hearts. We also administered ICA-105574 to anesthetized dogs while recording ECG and drug plasma concentrations. ICA-105574 (10 mg/kg) significantly shortened QT and QTc intervals, with a free plasma concentration of approximately 1.7 μM at the point of maximal effect. Our data showed that unbound ICA-105574 caused QT shortening in dogs at concentrations comparable to the half maximal effective concentration (EC₅₀, 0.42 μM) of hERG activation in the patch clamp studies.     

Molecular genetics of ependymomas and pediatric diffuse gliomas: a short review

Here, we review the recent literature on molecular discoveries in ependymomas and pediatric diffuse gliomas. Ependymomas can now be categorized into three location-related subgroups according to their biological profile: posterior fossa ependymomas, group A (PFA) and B (PFB), and supratentorial ependymomas. Although no recurrently mutated genes were found throughout these groups of ependymomas, PFA exhibited a CpG island methylator phenotype, PFB was associated with extensive chromosomal aberrations, and the C11orf95-RELA fusion gene was frequently observed in supratentorial ependymomas. Meanwhile, it has now become apparent that pediatric diffuse gliomas have a distinct genetic status from their adult counterparts, even though they share an indistinguishable histology. In pediatric low-grade diffuse gliomas, an intragenic duplication of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB/MYBL1 were found recurrently and mutually exclusively. As for non-brainstem high-grade tumors, in addition to H3F3A, TP53, and ATRX mutations, which were frequently observed in older children, recurrent fusions involving NTRK1, NTRK2, and NTRK3 were reported in infants younger than 3 years of age. Moreover, in diffuse intrinsic pontine gliomas (DIPG), recurrent somatic mutations of ACVR1 were found in association with HIST1H3B mutations.     

Intradural extramedullary spinal nerve sheath myxoma: a report of two cases

Nerve sheath myxoma, a myxoid variant of schwannoma, is a dermal tumor that usually occurs in the upper extremities, head and neck region, or trunk; occasionally, however, it has also been reported to develop in the spinal canal. Here, we describe two cases of intraspinal nerve sheath myxoma. Case 1 was a 74-year-old man with left hypochondrial pain. Gadolinium-enhanced magnetic resonance imaging (MRI) of his spine revealed a well-demarcated intradural extramedullary tumor with peripheral enhancement at the Th8 level. Case 2 was a 58-year-old man with lower back and left buttock pain. Gadolinium-enhanced MRI revealed a well-demarcated intradural extramedullary tumor with peripheral enhancement at the Th12-L1 level. Both cases were clinically diagnosed as schwannoma. Histological studies revealed characteristic myxoid lobules which were separated by fibrous septa or bands of more compact cellular area. The tumor cells were diffusely positive for S-100 and focally positive for Schwann/2E, which reacts with Schwann cells and myelin in the peripheral nervous system. The positive reaction to Schwann/2E confirmed the occurrence of peripheral nerve sheath differentiation. Nerve sheath myxoma should be included in differential diagnosis of spinal canal tumors.     

Primary CNS lymphoma arising in the region of the optic nerve presenting as loss of vision: 2 case reports,including a patient with a massive intracerebral hemorrhage

We report 2 cases of primary central nervous system (CNS) lymphoma arising in the region of the optic nerve. For both patients, diagnosis of lymphoma was impossible without histological examination because of the rarity of the lymphoma location. The first case involved an 84-year-old woman who developed loss of vision and hypopituitarism. Intraoperative finding was optic glioma; histological diagnosis was diffuse large B cell lymphoma, however. The second case involved a 67-year-old man who developed loss of vision. The pre-surgical diagnosis was optic nerve neuritis; this was then revised to granuloma. The tumor arose in the optic nerve. Methotrexate and rituximab were administered and the patient remained in complete remission for 3 years. However, a sudden intratumoral hemorrhage occurred. Although most of the lymphoma cells obtained from the initial surgery were negative for vascular endothelial growth factor (VEGF) immunoreactivity, high levels of VEGF immunoreactivity in lymphoma cells was detected in the specimen obtained after intratumoral bleeding at recurrence, and correlation between VEGF reactivity and tumor recurrence was suggested. To date, primary CNS lymphomas with intracerebral hemorrhage have been reported in 3 cases only, and a correlation between intratumoral hemorrhage and the degree of VEGF expression has been suggested. VEGF also might have predictive significance for recurrence.     

Functional analysis of a novel glioma antigen,EFTUD1

Background

A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.

Methods

EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.

Results

EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.

Conclusion

These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.     

Acoustic Radiation Force Impulse Imaging for Reactive and Malignant/Metastatic Cervical Lymph Nodes

The aim of this study was to compare lymph node stiffness using acoustic radiation force impulse (ARFI) imaging in patients with cervical lymph node swelling. Forty-two cervical lymph nodes (reactive, n = 22; metastatic, n = 20) from 19 patients (13 men, 6 women; mean age, 63.68 ± 14.9 y; range, 23–85 y) were examined between September 2011 and March 2012. The shear wave velocity (SWV, m/s) of each lymph node was evaluated by ARFI imaging. SWV of reactive lymph nodes was 1.52 ± 0.48 m/s, and that of metastatic/malignant lymph nodes was 2.46 ± 0.75 m/s. A SWV > 1.9 m/s was very useful metastatic lymph node classification, with 95.0% specificity, 81.8% sensitivity and 88.0% overall accuracy. The area under the receiver operating characteristic curve was 0.923 (95% confidence interval, 0.842–1.000). ARFI imaging can be useful in the differentiation of reactive and malignant/metastatic cervical lymph nodes.