Na+/K+ ATPase α1 and ß3 subunits are localized to the basolateral membrane of trophectoderm cells in human blastocysts.

STUDY QUESTION: Is there a relation between specific Na+/K+ ATPase isoform expression and localization in human blastocysts and the developmental behavior of the embryo? SUMMARY ANSWER: Na+/K+ ATPase α1, ß1 and ß3 are the main isoforms expressed in human blastocysts and no association was found between the expression level of their respective mRNAs and the rate of blastocyst expansion. WHAT IS KNOWN ALREADY: In mouse embryos, Na+/K+ ATPase α1 and ß1 are expressed in the basolateral membrane of trophectoderm (TE) cells and are believed to be involved in blastocoel formation (cavitation). STUDY DESIGN, SIZE, DURATION: A total of 20 surplus embryos from 11 patients who underwent IVF and embryo transfer at a university hospital between 2009 and 2018 were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: After freezing and thawing Day 5 human blastocysts, their developmental behavior was observed for 24 h using time-lapse imaging, and the expression of Na+/K+ ATPase isoforms was examined using quantitative RT-PCR (RT-qPCR). The expressed isoforms were then localized in blastocysts using fluorescent immunostaining. MAIN RESULTS AND THE ROLE OF CHANCE: RT-qPCR results demonstrated the expression of Na+/K+ ATPase α1, ß1 and ß3 isoforms in human blastocysts. Isoforms α1 and ß3 were localized to the basolateral membrane of TE cells, and ß1 was localized between TE cells. A high level of ß3 mRNA expression correlated with easier hatching (P = 0.0261). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The expression of mRNA and the localization of proteins of interest were verified, but we have not been able to perform functional analysis. WIDER IMPLICATIONS OF THE FINDINGS: Of the various Na+/K+ ATPase isoforms, expression levels of the α1, ß1 and ß3 mRNAs were clearly higher than other isoforms in human blastocysts. Since α1 and ß3 were localized to the basolateral membrane via fluorescent immunostaining, we believe that these subunits contribute to the dilation of the blastocoel. The ß1 isoform is localized between TE cells and may be involved in tight junction formation, as previously reported in mouse embryos. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the JSPS KAKENHI (https://www.jsps.go.jp/english/index.html), grant number 17K11215. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflicts of interest.

Pediatric myxopapillary ependymoma treated with subtotal resection and radiation therapy: a case report and review of the literature.

STUDY DESIGN: Case report and review of the literature. OBJECTIVES: Myxopapillary ependymoma (MPE) is a relatively rare glioma that develops from the spinal part of the filum terminale, usually in adulthood. While it is generally benign, MPE can disseminate intraspinally, and this malignant behavior requires a multidisciplinary response with surgery and radiotherapy. We report here a case of MPE occurring in the lumbosacral spine area of an 8-year-old boy. SETTING: Japan, Tokyo. METHODS: We report here a case of MPE, treated with subtotal surgical resection followed by craniospinal irradiation (CSI), in an 8-year-old boy. The patient was referred to our hospital with a 6-month history of severe pain in the lower back and legs, paralysis of the legs and dysuria. Magnetic resonance imaging images showed a large tumor that filled the entire spinal canal below L1. After subtotal resection of the tumor, the pathological findings established a diagnosis of MPE. Since the tumor had perforated its capsule, increasing the risk of intraspinal dissemination, the patient underwent radiotherapy and CSI after surgery. RESULTS: Magnetic resonance images obtained 3 years after the surgery did not show any recurrence of MPE. CONCLUSION: Although tumor resection followed by CSI can be considered an effective strategy for treating a child with MPE, long-term follow-up is necessary to ensure early detection of any local recurrence or dissemination of the tumor, or of post-radiotherapy scoliosis.

Oncolytic activity of Sindbis virus in human oral squamous carcinoma cells.

BACKGROUND: Sindbis virus (SIN) infection causes no or only mild symptoms (fever, rash, and arthralgia) in humans. However, SIN has a strong cytopathic effect (CPE) on various cancer cells. This study focuses on the oncolytic activity of SIN AR399 on oral cancer cells compared with reovirus, a well-known oncolytic virus that targets cancer cells. METHODS: We analysed the cytotoxicity and growth of SIN in 13 oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, H-1, Sa-3, KON, KOSC-2, OK-92, HO-1-N1, SCC-4, SAT, SKN-3) and normal human oral keratinocytes (NHOKs). RESULTS: Sindbis virus infection induced CPE in 12 OSCC cell lines at a low multiplicity of infection (MOI) of 0.01, but not in the OSCC cell line, HSC-4 or NHOKs. Sindbis viral growth was not observed in NHOKs, whereas high SIN growth was observed in all OSCC cell lines, including HCS-4. The cytotoxicity and growth of SIN was the same as reovirus at an MOI of 20 in 12 OSCC cell lines. The CPE was shown, by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assays, to be apoptotic cell death. Furthermore, quantitative RT-PCR of mRNA in HSC-3 and HSC-4 cells after SIN infection showed that activation of caspases, cytochrome c, and IkappaBalpha was associated with SIN-induced apoptosis. CONCLUSION: As a replication-competent oncolytic virus, SIN may be a useful therapeutic modality for oral cancers.

Effects of 6 months of walking training on lower limb muscle and tendon in elderly.

The purpose of this study was to investigate the effects of 6 months of walking training on muscle strength, muscle thickness and tendon stiffness on various parts of the lower limbs in the elderly. Subjects were assigned to training (n=35) and control (n=10) groups. Maximal isometric torque (MVC) and muscle thickness for knee extensors (KE), knee flexors (KF), dorsi flexors (DF) and plantar flexors (PF) were measured. Tendon stiffness for KE and PF was measured using ultrasonography while subjects performed isometric contraction. No significant changes occurred in any measured variables in the control group. In the training group, muscle thickness increased significantly for KF and DF, but not for PF. For KE, significant increases of muscle thickness at the proximal and medial sides were observed, although mean relative increase of the eight measured sites for KE was not significant. MVC increased significantly for KF, DF, and PF, but not for KE. In addition, tendon stiffness for KE and PF did not change after training. These results indicated that walking training brought about increments of muscle thickness and strength in most of the lower limbs in the elderly, but it did not result in any changes in tendon stiffness.

Effects of 20-day bed rest with and without strength training on postural sway during quiet standing.

AIM: To examine the effect of unweighting as a possible contributory factor to a reduced calf muscle volume on postural sway during quiet standing, changes in postural sway following bed rest with or without strength training were investigated. METHODS: Twelve young men participated in a 20-day bed-rest study. Subjects were divided into a non-training group (BR-Con) and a strength training group (BR-Tr). For the BR-Tr group, training was comprised of dynamic calf-raise and leg-press exercises to maintain the muscle volume of the plantar flexors. Before and after bed rest, subjects maintained quiet standing in a barefoot position on a force platform with their eyes open or closed. During the quiet stance, foot centre-of-pressure (CoP) and the mean velocity of CoP was calculated. Muscle volume of the plantar flexors was computed using axial magnetic resonance images of the leg. RESULTS: After the bed-rest period, the muscle volume decreased in the BR-Con group but not in the BR-Tr group. The mean velocity of CoP as an assessment of postural sway, however, increased in both groups. These results indicate that the strength training during bed rest cannot counteract the increase in postural sway. CONCLUSION: We concluded that postural sway increases following 20 days of bed rest despite maintenance of the muscle volume of plantar flexors as the main working muscles for the human postural standing.

Papillomatosis in a European bison.

Five European bison (Bison bonasus) from three European zoos were shipped to the Bukovské Vrchy Hills (Slovakia) in June 2004 and kept together in an acclimatization enclosure. The European bison were released into the wild in December 2004. At that time, papillomas were found at the medial canthus of the left eye of a 12-yr-old female bison. Cutaneous papillomatosis was confirmed histologically. Negative stain transmission electron microscopic examination revealed papillomavirus in the papillomas, and papillomavirus DNA also was detected using the polymerase chain reaction with FAP59 and FAP64 primers. The amplified 413 bp DNA sequence was identical to that of BAPV2 bovine papillomavirus. This paper is the first report of papillomatosis in European bison.

Dynamic changes in AP-1 transcription factor DNA binding activity in rat brain following administration of antidepressant amitriptyline and brain-derived neurotrophic factor.

The present study was undertaken to examine the effects of the antidepressant, amitriptyline, and brain-derived neurotrophic factor (BDNF) on activator protein-1 (AP-1) DNA binding activity in the rat brain. Acute administration of amitriptyline (5 or 10 mg/kg) initially increased but then decreased AP-1 DNA binding activity in the rat frontal cortex and hippocampus. Chronic administration of amitriptyline (5 or 10 mg/kg, once daily for 3 weeks) initially decreased AP-1 DNA binding activity but ultimately resulted in its persistent elevation in the rat frontal cortex. In contrast, the chronic administration of amitriptyline did not affect the low activity of AP-1 DNA binding in the hippocampus. However, chronic administration of amitriptyline (10 mg/kg, once daily for 3 weeks) significantly increased BDNF protein levels in the hippocampus (by 26.9%) and frontal cortex (by 24.6%). Direct infusion of BDNF (1 microg) into the hippocampal dentate gyrus significantly increased hippocampal AP-1 DNA binding activity. These results suggest that AP-1 transcription factor may be modulated by BDNF and that it may be an important target for the action of antidepressants.

Immortalization of human esophageal keratinocytes by E6 and E7 of human papillomavirus type 16.

Transduction of human papillomavirus type 16 (HPV16) E6/E7 into primary culture of human esophageal keratinocytes using a recombinant adenovirus prolonged the life-span, while untreated cells senesced within 14-16 population doublings (PDLs). Up-regulation of telomerase activity and acquisition of serum-resistant growth were observed in the esophageal keratinocytes with extended life-span between 50 and 100 PDLs, and drastically increased after 100 PDLs. A keratinocyte sample with a polymorphism of Pro/Pro at codon 72 of p53 showed resistance to HPV16 E6/E7-induced life-span-extension and immortalization, in contrast to others with p53 polymorphisms of Arg/Arg or Arg/Pro, which did not. The high efficiency of E6/E7-induction by adenovirus vector also revealed the M1 and M2 stages of keratinocyte immortalization first described in this report.

Detection of human papillomaviruses in cervical neoplasias using multiple sets of generic polymerase chain reaction primers.

OBJECTIVE: The aim of this study was to evaluate precisely the differences in the spectra of human papillomavirus (HPV) types detected by different generic primer pairs commonly used for detection of this extraordinarily heterogeneous virus. METHODS: Three sets of polymerase chain reaction (PCR) primers for the L1 open reading frame (ORF) and two sets for E6/E7 ORFs were used to detect HPVs in DNAs from 107 cervical tissues, including 77 cervical neoplasias. HPV types were determined by analysis of restriction fragment length polymorphisms (RFLPs) and nucleotide sequencing. RESULTS: A high overall detection rate of HPV in cervical neoplasias (76/77, 98.7%) was achieved by polymerase chain reaction (PCR) amplification with multiple sets of generic primers, while the detection rate for each individual primer pair varied from 48/77 (62%) to 70/77 (91%). Only in 34 of 77 cases (44%) were HPV DNAs positive for all sets of primer pairs. Further determination of HPV types by RFLPs and nucleotide sequencing showed inconsistencies between the PCR primer pairs used. CONCLUSION: Our study revealed that the HPV detection rate is critically affected by the choice of PCR primers, and that appropriate use of combinations of generic PCR primer sets followed by RFLP analyses is both necessary and sufficient for typing most HPVs in cervical lesions. More precise methods such as sequencing would be necessary in only a few cases.

Association of tumor necrosis factor alpha gene promoter polymorphism with the presence of chronic obstructive pulmonary disease.

Tumor necrosis factor alpha (TNF-alpha), a potent proinflammatory cytokine, may be involved in the development of chronic obstructive pulmonary disease (COPD). The production of TNF-alpha is elevated in the airways of these patients. A polymorphism at position -308 of the TNF-alpha gene promoter (TNF-alpha-308*1/2) is known to be associated with alteration of TNF-alpha secretion in vitro. In this study we examined the differences in TNF-alpha-308*1/2 allele frequency to investigate the association of this polymorphism with the presence of smoking-related COPD. TNF-alpha-308*1/2 allele frequency in 106 patients (73 men and 33 women) was compared with 110 asymptomatic smoker/ex-smoker control subjects matched for sex and age and population control subjects consisting of 129 blood donors. Genotype was analyzed by the polymerase chain reaction-restriction fragment length polymorphism technique on genomic DNA isolated from peripheral blood lymphocytes. TNF-alpha-308*1/2 allele frequencies were significantly different among the groups: 0.835/0.165 in patients with COPD, 0.918/0.082 in smoker/ex-smoker control subjects, and 0.922/0.078 in population control subjects. These results indicate that TNF-alpha-308*1/2 alleles are significantly associated with the presence of smoking-related COPD.

Alveolar soft part sarcoma of the tongue. Report of a case.

The clinicopathologic, immunohistological, and ultrastructural features of an alveolar soft part sarcoma of the tongue occurring in a 2-year-old girl are described. A primary alveolar soft part sarcoma arising in the dorsum part of the tongue is quite rare. There has been no recurrence or metastasis as of 7 years postoperatively.

[Hepatocellular carcinoma with tumor thrombus in the portal and hepatic veins treated successfully with hepatectomy and chemotherapy--a case report].

A 39-year-old woman was admitted to our hospital because of advanced hepatocellular carcinoma. She had good liver function with clinical Stage I. Abdominal ultrasonographic study and CT scan revealed a huge tumor of 12 cm in diameter in the left lobe of the liver, with tumor thrombi in the portal and hepatic veins. A chest CT scan demonstrated multiple bilateral lung metastases from 5 to 10 mm in size. An extended left hemihepatectomy with extirpation of the portal and hepatic venous tumor thrombi was performed. On postoperative day 7, low-dose cisplatin (10 mg/day-5 days/week) and 5-fluorouracil (250 mg/day-continuous for 7 days/week) were administered intravenously. Four weeks after chemotherapy, CT scan revealed no recurrence in the liver and no change in the lung metastases. The patient is now being treated on an outpatient basis with no change in the metastatic tumors.

Enhanced expression of N-myc messenger RNA in neuroblastomas found by mass screening.

A substantial fraction of neuroblastomas found by mass screening have been suggested to regress spontaneously because of the high incidence of infantile neuroblastomas in the screening population. In this study, 70 neuroblastomas were analyzed for expression of proto-oncogenes related to neuronal differentiation to clarify the biological significance of proto-oncogene expression in the screening-positive and -negative tumors. The tumors consisted of 39 neuroblastomas found by screening (group 1), 16 non-N-myc-amplified neuroblastomas found by clinical symptom(s) (group 2), and 15 N-myc-amplified neuroblastomas found by clinical symptom(s) (group 3). The expression of c-src, trk A, and N-myc in tumor tissues was analyzed by quantitative RNA PCR. Neuronal c-srcN2 expression varied significantly in the following order: group 1 > group 2 > group 3. The level of expression of trk A was markedly reduced in group 3 but did not differ in groups 1 and 2. Most tumors in group 3 overexpressed N-myc. However, N-myc expression in group 1 was significantly higher than that in group 2. Thus, the characteristics of proto-oncogene expression in screening-positive tumors included enhanced expression of c-srcN2 and N-myc mRNA, regardless of nonamplification of N-myc. Our results suggest that the role of N-myc differs in neuroblastomas detected by screening and in N-myc-amplified tumors.

Human papillomavirus-positive well-differentiated villoglandular adenocarcinoma of the uterine cervix: A case report and review of the literature.

OBJECTIVE: A case of well-differentiated villoglandular adenocarcinoma of the uterine cervix, which was positive for human papillomavirus type 18, was reported. METHODS: The patient was a 52-year-old multipara who was referred to our department because of an abnormal Papanicolaou smear. A 4.0-cm exophytic lesion involving the cervix was detected. She was staged as FIGO IIa and radical hysterectomy combined with bilateral pelvic lymphadenectomy was performed. In addition to histopathological examination of the resected tumor, immunohistochemical studies of estrogen and progesterone receptors were performed using monoclonal antibodies. Detection of human papillomavirus DNA was attempted by polymerase chain reaction using consensus primers. RESULTS: The tumor was a typical well-differentiated villoglandular adenocarcinoma involving the vaginal wall. Both estrogen and progesterone receptors were negative. Human papillomavirus type 18 DNA was detected in the resected tumor. CONCLUSION: 'This is the first report of a case of typical well-differentiated villoglandular adenocarcinoma which was positive for human papillomavirus.

Study of the association between the serotonin transporter gene regulatory region polymorphism and personality traits in a Japanese population.

Recently a relationship between serotonin transporter transcriptional control region (5-HTTLPR) polymorphism and anxiety related personality traits in Caucasians was reported. We performed PCR of DNAs from the blood for determining the 5-HTTLPR genotypes of 191 Japanese subjects, which were medical staff and students, and obtained Revised NEO Personality Inventory (NEO-PI-R) and the Temperament and Character Inventory (TCI) in 144 subjects. The association was analyzed by one-way ANOVA. The present study demonstrated that allelic frequency of 5-HTTLPR (s allele frequency was 0.785) in our subjects was considerably different from that in Caucasians. No significant differences were found in the anxiety-related personality traits among genotypes, while cooperativeness in TCI showed a significant difference among genotypes. The property of 5-HTTLPR may not be reflected directory on the personality inventories.

Ultrastructural study of intranuclear inclusion bodies of pulmonary adenocarcinoma.

Intranuclear inclusion bodies are sometimes observed in pulmonary adenocarcinoma by light microscopy. Electron microscopic characteristics of lung cancer cells with intranuclear inclusion bodies were studied. In addition, polymerase chain reaction (PCR) was performed using primers coding for human papillomavirus (HPV) types 16, 18, and 33. Eosinophilic intranuclear inclusion bodies were observed in 22 out of 285 cases by light microscopy. Immunohistochemically, cancer cell nuclei stained with PE-10. Three types of intranuclear inclusion bodies were classified electron microscopically. Type A showed aggregation of electron dense particles (30-40 nm) with an electron-dense core and was most frequently observed. Type B consisted of a mass of branching and whirling tubular structures. Type B intranuclear inclusions had a relationship with inner nuclear membrane. In type C, several spherical inclusions were observed in one nucleus. HPV DNA was detected using PCR and type-specific probes in a case with type A inclusion bodies. This study suggests that intranuclear inclusion bodies in pulmonary adenocarcinoma are formed by several different mechanisms.

P16INK4a expression adenovirus vector to suppress pancreas cancer cell proliferation.

The prognoses of pancreatic cancer patients have been miserable even after radical surgery, and adjuvant therapy is necessary to improve the surgical results. p16(INK4a) (p16) is tight-binding and inhibitory protein for cyclin-dependent kinase 4 to induce G1 arrest of the cell cycle. p16 gene deletion is frequently identified in human pancreas cancer. The impaired gene function of p16 might be a major factor of the uncontrolled proliferation and malignancy of pancreas cancer cells. In this study, we investigated the effect of adenovirus p16 expression vector for pancreas cancer cell proliferation to clarify whether the vector might be a promising mode to assist the surgical therapy for pancreas cancer. We constructed the adenovirus p16 expression vector AdexCACSp16 by inserting p16 cDNA to a cassette cosmid containing a nearly full-length adenovirus type 5 genome with E1 and E3 deletions. Thereafter, we assessed the activity of AdexCACSp16 to induce p16 gene mRNA expression in pancreas cancer cell line MIAPaCa-2 and to control cell proliferation. AdexCACSp16 induced a high level of p16 gene mRNA expression in MIAPaCa-2 cells with 1 h contact to the cells. The cell proliferation was significantly suppressed by AdexCACSp16 compared with the control adenovirus group. These data indicate that AdexCACSp16 has the potential to induce p16 gene expression and control pancreas cancer cell proliferation and that the adenovirus p16 expression vector AdexCACSp16 might be a possible method of gene therapy to improve the surgical therapeutic results for pancreas cancer.

Trans-activating activity of the E6 proteins of the human papillomavirus (HPV) type-11 and -16 on the PE1E4 promoter of HPV-11 in C33A cells.

We investigated trans-activating effects of the full-length E6 protein of HPV-16 (16E6) and the E6 protein of HPV-11 (11E6) on the PE1E4 promoter of HPV-11 in C33A cells which lack normal function of p53. 16E6 showed no significant activation of the reporter plasmid containing PE1E4 and the upstream sequence, including the long control region (LCR). In contrast, 11E6 activated the promoter in a dose dependent manner, while relatively high doses of 11E6 were required to activate the promoter. When a reporter plasmid, which lacked LCR was used, however, both 16E6 and 11E6 activated the promoter, though high doses of 16E6 suppressed activity. Using deletion plasmids we further showed that 11E6 activated transcriptions from any mutant reporter plasmids as far as the constructs have promoter activities. Finally, we showed that 11E6 enhanced the expression levels of c-fos protein by infection of C33A cells with 11E6-expressing recombinant adenovirus. These findings suggested that E6 proteins of both HPVs would induce similar protein(s) which is required for an efficient transcription of minimum promoter of viral and cellular genes, and that the 16E6 induce additional protein(s) which suppress PE1E4 in the presence or absence of LCR.