Correlated Confocal and Intermediate Voltage Electron Microscopy Imaging of the Same Cells Using Sequential Fluorescence Labeling, Fixation, and Critical Point Dehydration

Confocal laser scanning microscopy (CLSM) and intermediate voltage transmission electron microscopy (IVEM) each has its own particular advantages. CLSM can examine living cells, but is particularly useful when applied to cells that have been lightly fixed, permeabilized, and stained with fluorescent-labeled antibodies for localization of specific molecular species at the resolution of the light microscope while still in the hydrated state. IVEM provides much higher resolution images, but requires more drastic preparation procedures, including dehydration. This paper presents methods for combining these complementary approaches to examine exactly the same cells sequentially by CLSM and IVEM. Cells are grown in culture on sterile formvar films spread over gold index grids on cover glasses, which are mounted on larger cover glasses or microscope slides with spacers to prevent compression of the cells. Light and epifluorescence microscopy, and CLSM are performed concentrating on cells in grid openings. Then the grids are fixed with aldehydes followed by OsO4, dehydrated and critical point dried (CPD) from liquid CO2. Immediately following CPD, the grids are ready for examination in the IVEM. Low magnification (300-600x) survey images allow correlation of the IVEM images with the light microscopic images. In higher power images, structures that are fluorescent labeled can be related to corresponding regions in the IVEM images

Calcium oscillation associated with reduced protein kinase C activities in ras-transformed NIH3T3 cells

AbstractWe show here novel intracellular Ca2+ oscillation in ν-K-ras-transformed NIH3T3 cells induced by mitogenic peptide hormones, bradykinin and bombesin, as well as fetal calf serum. Induction of the Ca2+ oscillation is strongly correlated with the malignant properties and inversely with PKC activities in vitro and in vivo. These results suggest that the mitogen-induced Ca2+ oscillation is negatively regulated by PKC, which modulates Ca2+ influx in ν-K-ras-transformed NIH3T3 cells

Roles for E1-independent replication and E6-mediated p53 degradation during low-risk and high-risk human papillomavirus genome maintenance.

Human papillomaviruses (HPV) have genotype-specific disease associations, with high-risk alpha types causing at least 5% of all human cancers. Despite these conspicuous differences, our data show that high- and low- risk HPV types use similar approaches for genome maintenance and persistence. During the maintenance phase, viral episomes and the host cell genome are replicated synchronously, and for both the high- and low-risk HPV types, the E1 viral helicase is non-essential. During virus genome amplification, replication switches from an E1-independent to an E1-dependent mode, which can uncouple viral DNA replication from that of the host cell. It appears that the viral E2 protein, but not E6 and E7, is required for the synchronous maintenance-replication of both the high and the low-risk HPV types. Interestingly, the ability of the high-risk E6 protein to mediate the proteosomal degradation of p53 and to inhibit keratinocyte differentiation, was also seen with low-risk HPV E6, but in this case was regulated by cell density and the level of viral gene expression. This allows low-risk E6 to support genome amplification, while limiting the extent of E6-mediated cell proliferation during synchronous genome maintenance. Both high and low-risk E7s could facilitate cell cycle re-entry in differentiating cells and support E1-dependent replication. Despite the well-established differences in the viral pathogenesis and cancer risk, it appears that low- and high-risk HPV types use fundamentally similar molecular strategies to maintain their genomes, albeit with important differences in their regulatory control. Our results provide new insights into the regulation of high and low-risk HPV genome replication and persistence in the epithelial basal and parabasal cells layers. Understanding the minimum requirement for viral genome persistence will facilitate the development of therapeutic strategies for clearance.MR

Role of Heat Shock Protein 70 in Induction of Stress Fiber Formation in Rat Arterial Endothelial Cells in Response to Stretch Stress

We investigated the mechanism by which endothelial cells (ECs) resist various forms of physical stress using an experimental system consisting of rat arterial EC sheets. Formation of actin stress fibers (SFs) and expression of endothelial heat-shock stress proteins (HSPs) in response to mechanical stretch stress were assessed by immunofluorescence microscopy. Stretch stimulation increased expression of HSPs 25 and 70, but not that of HSP 90. Treatment with SB203580, a p38 MAP kinase inhibitor that acts upstream of the HSP 25 activation cascade, or with geldanamycin, an inhibitor of HSP 90, had no effect on the SF formation response to mechanical stretch stress. In contrast, treatment with quercetin, an HSP 70 inhibitor, inhibited both upregulation of endothelial HSP 70 and formation of SFs in response to tensile stress. In addition, treatment of stretched ECs with cytochalasin D, which disrupts SF formation, did not adversely affect stretch-induced upregulation of endothelial HSP 70. Our data suggest that endothelial HSP 70 plays an important role in inducing SF formation in response to tensile stress

Integrated education of gross anatomy and CT radiology for current advances in medicine

It is essential to learn human anatomy in 3D for advanced medicine. We designed such an education system by integrating anatomy dissection with diagnostic CT radiology. Cadavers were scanned by CT, and students consulted the postmortem CT images while dissecting the cadaver to gain a better understanding of 3D human anatomy and diagnostic radiology. Students used handheld DICOM viewers at the bench-side (OsiriX on iPod touch). Students had lectures and workshops on diagnostic radiology, and study assignments where they discussed findings in anatomy labs in comparison with CT radiology. This teaching method for gross anatomy was used from 2009, and yielded positive students’ perspectives, and significant improvements in radiology skills at clinical courses.This is the pre-peer reviewed version of the following article: Tohru Murakami, Yuki Tajika, Hitoshi Ueno, Sachiko Awata, Satoshi Hirasawa, Maki Sugimoto, Yoshihiko Kominato, Yoshito Tsushima, Keigo Endo, and Hiroshi Yorifuji. An integrated teaching method of gross anatomy and computed tomography radiology. Anat Sci Educ, 2014, which has been published in final form at http://onlinelibrary.wiley.com/ doi/10.1002/ase.1430/abstract

Regulatory expression of uncoupling protein 1 and its related genes by endogenous activity of the transforming growth factor‐β family in bovine myogenic cells

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis, with restricted expression in brown/beige adipocytes in humans and rodents. We have previously shown an unexpected expression of UCP1 in bovine skeletal muscles. This study evaluated factors affecting Ucp1 gene expression in cultured bovine myogenic cells. Myosatellite cells, which were isolated from the bovine musculus longissimus cervicis, were induced to differentiate into myotubes in the presence of 2% horse serum. Previous studies using murine brown/beige adipocytes revealed that Ucp1 expression levels are directly increased by forskolin and all-trans retinoic acid (RA). The transforming growth factor-β (TGF-β)/activin pathway negatively regulated Ucp1 expression, whereas activation of the bone morphogenetic protein (BMP) pathway indirectly increases Ucp1 expression through the stimulation of brown/beige adipogenesis. Neither forskolin nor RA significantly affected Ucp1 mRNA levels in bovine myogenic cells. A-83-01, an inhibitor of the TGF-β/activin pathway, stimulated myogenesis in these cells. A-83-01 significantly increased the expression of some brown fat signature genes such as Pgc-1α, Cox7a1, and Dio2, with a quantitative but not significant increase in the expression of Ucp1. Treatment with LDN-193189, an inhibitor of the BMP pathway, did not affect the differentiation of bovine myosatellite cells. Rather, LDN-193189 increased Ucp1 mRNA levels without modulating the levels of other brown/beige adipocyte-related genes. The current results indicate that the regulation of Ucp1 expression in bovine myogenic cells is distinct from that in murine brown/beige adipocytes, which has been more intensely characterized

Long-term observation of fibrillation cycle length in patients under angiotensin II receptor blocker therapy for chronic atrial fibrillation

AbstractIntroductionThe long-term effect of angiotensin II receptor blockers (ARBs) on atrial fibrillation (AF) is unclear. In this study, we evaluated the change in the fibrillation cycle length (FCL) in patients under long-term ARB therapy for chronic AF.Methods and resultsThe study population consisted of 25 chronic AF patients who were prescribed the same medication for more than 6 years and in whom specific ECG recording for FCL evaluation could be performed before and after the 6-year observation period. The patients were divided into 2 groups: those with and without ARB (ARB group and non-ARB group and n=15 and 10, respectively). FCL was calculated by the spectral analysis of the fibrillation waves in the surface ECG. There was no significant difference in the clinical characteristics between the 2 groups. In the ARB group, the mean FCL was prolonged from 154±20ms to 187±37ms (p=0.005), whereas it remained unchanged in the non-ARB group (150±12ms vs. 149±10ms). In the comparison between patients with and those without FCL prolongation (>30ms; n=6 and 19, respectively), a significant difference was observed only in those prescribed ARBs.ConclusionIn cases of chronic AF, FCL might be prolonged under long-term ARB treatment