Possible Mechanisms of Axonal Transport Disturbances in Mouse Spinal Motoneurons Induced by Hypogravity.

The data obtained by transcriptome analysis of lumbar spinal cord segments, sciatic nerve, and the respiratory diaphragm of the mice performed after a space flight on board Bion-M1 biosatellite were processed by bioinformatic methods aimed at elucidation of the regularities in hypogravity-induced transcriptome changes in various compartments of motor neurons. The study revealed abnormalities of axonal transport in spinal motor neurons provoked by weightlessness. These data agree with the results of electron microscopy examination of the spinal cord in experimental animals. In space group mice sacrificed on the landing day, the content of perinuclear ribosomes in lumbar motoneurons surpassed that in control mice or in the recovery group examined 1 week after the flight. The data corroborate our hypothesis on contribution of axonal transport disturbances into pathogenesis of hypogravity motor syndrome. They can be employed as a launching pad for further study of hypogravity-triggered motor disorder mechanisms in order to elaborate the preventive therapy against the development of hypogravity motor syndrome in space flights.

Backscattered electron image of osmium-impregnated/macerated tissues as a novel technique for identifying the cis-face of the Golgi apparatus by high-resolution scanning electron microscopy.

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three-dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis-trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis- or trans-faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis-Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis-elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron-mode SEM. Prolonged osmication by osmium impregnation (2% OsO4 solution at 40°C for 40 h) and osmium maceration (0.1% OsO4 solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.

Development of nanomanipulator using a high-speed atomic force microscope coupled with a haptic device.

The atomic force microscope (AFM) has been widely used for surface fabrication and manipulation. However, nanomanipulation using a conventional AFM is inefficient because of the sequential nature of the scan-manipulation scan cycle, which makes it difficult for the operator to observe the region of interest and perform the manipulation simultaneously. In this paper, a nanomanipulation technique using a high-speed atomic force microscope (HS-AFM) is described. During manipulation using the AFM probe, the operation is periodically interrupted for a fraction of a second for high-speed imaging that allows the topographical image of the manipulated surface to be periodically updated. With the use of high-speed imaging, the interrupting time for imaging can be greatly reduced, and as a result, the operator almost does not notice the blink time of the interruption for imaging during the manipulation. This creates a more intuitive interface with greater feedback and finesse to the operator. Nanofabrication under real-time monitoring was performed to demonstrate the utility of this arrangement for real-time nanomanipulation of sample surfaces under ambient conditions. Furthermore, the HS-AFM is coupled with a haptic device for the human interface, enabling the operator to move the HS-AFM probe to any position on the surface while feeling the response from the surface during the manipulation.

Differences in the pharmacokinetics of Cyp3a substrates in TSOD and streptozotocin-induced diabetic mice.

The pharmacokinetics of drugs can change in diabetes mellitus and even among diabetics. They may differ between type I diabetes (T1DM) and type 2 diabetes (T2DM). As triazolam was administered orally to Tsumura, Suzuki, obese, diabetes (TSOD) mice and streptozotocin (STZ) mice, clearance per body (CL/F) in TSOD mice did not differ compared with Tsumura, Suzuki, non-obesity (TSNO) mice. In STZ mice, CL/F was greater than in control mice. Small intestinal cytochrome P450 (Cyp) 3a expression in TSOD mice was significantly lower than in TSNO mice. No significant difference existed in small intestinal Cyp3a expression between STZ mice and control mice. In insulin-treated mice, small intestinal Cyp3a expression was significantly lower than in control mice. These results suggested that the differences in changes in small intestinal Cyp3a expression between T1DM and T2DM may be due to differences in plasma insulin concentrations. This may be a factor in the difference in the drug pharmacokinetics between T2DM and T1DM patients.

High-speed AFM of human chromosomes in liquid.

Further developments of the previously reported high-speed contact-mode AFM are described. The technique is applied to the imaging of human chromosomes at video rate both in air and in water. These are the largest structures to have been imaged with high-speed AFM and the first imaging in liquid to be reported. A possible mechanism that allows such high-speed contact-mode imaging without significant damage to the sample is discussed in the context of the velocity dependence of the measured lateral force on the AFM tip.

The olfactory route for cerebrospinal fluid drainage into the peripheral lymphatic system.

Drainage of the cerebrospinal fluid through the olfactory nerves into the nasal lymphatics has been suggested repeatedly. To investigate precisely the morphology of this pathway, India ink was injected into the subarachnoidal space of the rat brain, and samples including the olfactory bulbs, olfactory tracts and the nasal mucosa were observed by light and electron microscopy. Under the dissecting microscope, ink particles were found within the subarachnoid space and along the olfactory nerves. At the nasal mucosa, a lymphatic network stained in black was identified near the olfactory nerves, which finally emptied into the superficial and deep cervical lymph nodes. Light microscopically, ink particles were found in the subarachnoid space, partially distributed around the olfactory nerves and within the lymphatic vessels. By electron microscopy, the subarachnoid space often formed a pocket-like space in the entrance of the fila olfactoria. The olfactory nerves were partially surrounded by ink particles within the space between perineurial cells and epineurial fibroblasts. At the nasal mucosa, the lymphatics were frequently located close to the nerves. These results indicate that the cerebrospinal fluid drains from the subarachnoid space along the olfactory nerves to the nasal lymphatics, which in turn, empties into the cervical lymph nodes. This anatomical communication, thus, allows the central nervous system to connect with the lymphatic system. The presence of this route may play an important role in the movement of antigens from the subarachnoidal space to the extracranial lymphatic vessels, resulting in inducement of an immune response of the central nervous system.

Ahl3, a third locus on mouse chromosome 17 affecting age-related hearing loss.

Genetic variation in humans probably plays a role in determining the range of individual susceptibility to age-related hearing loss (AHL), but no contributing loci have been identified because of the difficulties of dissecting complex traits in humans. This paper reports mapping of an AHL locus using a panel of consomic mice between C57BL/6J (B6) and MSM strains, which covered more than a half of chromosome sets. B6 strain exhibited AHL beginning at 10 months of age whereas MSM strain, derived from Japanese wild mice, had normal hearing throughout life. Individuals in the panel were examined with auditory brainstem response (ABR) at various months of age, revealing that one particular strain (B6-Chr17(MSM)) substituting the chromosome 17 with the MSM-derived one showed a prominent resistance, having still good hearing at 18 months of age. Subsequent mapping using 89 individuals in the cross between B6-Chr17(MSM) and B6 was performed, which showed a significant association of ABR thresholds with loci in the vicinity of D17Mit119. These results show a novel AHL-resistant locus, designated as Ahl3, on the chromosome 17.

Imaging of human metaphase chromosomes by atomic force microscopy in liquid.

Human metaphase chromosomes were observed using an intermittent contact mode of atomic force microscopy (AFM) in a phosphate-buffered saline solution to clarify their conformation close to that in the physiological state. In the AFM images in liquid, symmetric alternating ridges and grooves were evident on their surface of the paired sister chromatids. The number of the ridges and grooves were rather specific to the type of the chromosome. The structural changes of chromosomes caused by trypsin treatment were also directly observable using AFM in liquid. These results suggest that the intermittent contact mode AFM is useful not only for analyzing the structure of chromosomes in a liquid condition but also for studying the effect of chemical treatments on chromosomes in relation to their structural changes.

Simultaneous activation of natural killer T cells and autoantibody production in mice injected with denatured syngeneic liver tissue.

Denatured syngeneic liver tissue prepared by mechanical procedures was intraperitoneally injected into adult C57BL/6 mice. In parallel with a decrease in the total number of lymphocytes in the liver, spleen, and thymus from days 1-7 after the injection, the proportion of the CD4+NK1.1+CD3(int) subset of these cells (i.e. natural killer T or NKT cells) increased in the liver. Even the absolute number of these NKT cells increased in the liver on days 14 and 21. In response to the injection of denatured liver tissue, tissue damage was induced in the liver, as shown by elevated levels of serum transaminases and hepatocyte degeneration observed by electron microscopy. Sera obtained on days 7 and 14 contained autoantibodies including anti-DNA antibodies. The proportion of CD1d(high)B cells in the liver was found to decrease on days 1-7. In other words, denatured liver tissue stimulated both NKT cells and certain B cells in the liver. These results suggest that liver lymphocytes might contain not only autoreactive T cells (e.g. CD3(int) or NKT cells) but also some B cells (e.g. B-1 cells) which produce autoantibodies and that the denatured tissue had the potential to stimulate these lymphocytes and to evoke an autoimmune-like state.

A KOH-collagenase digestion method for scanning electron microscopic studies of vascular smooth muscle fibers in the human heart.

The three-dimensional cytoarchitecture of smooth muscle fibers of human cardiac arterial vessels was studied by scanning electron microscopy after removal of extracellular connective tissue matrices with a KOH-collagenase digestion method. Arterioles with an outer diameter of 30-100 microm had a well-developed compact media consisting commonly of circularly oriented smooth muscle fibers. There were also arterioles with oblique or longitudinal muscle fibers in some places. In terminal arterioles with an outer diameter of less than 30 microm, muscle fibers became branched and commonly encircled the endothelial tube. There were some terminal arterioles covered with obliquely arranged smooth muscle fibers. The presence of the regional difference in arrangement of vascular smooth muscle fibers indicates the heterogeneity of blood flows in the level of cardiac microcirculation.

Atomic force microscopy and its related techniques in biomedicine.

The atomic force microscope (AFM), invented in 1986, is a new instrument which provides three-dimensional surface images of samples by scanning a sharp probing tip over the sample surface. Unlike electron microscopes (EM), the AFM has the advantage to obtain high-resolution pictures not only in a vacuous but also in a non-vacuous (i.e., air or liquid) environment. This paper reviews our recent studies on the AFM application to the biomedical fields. AFM is useful for observing biological structures such as DNA, collagen molecules, collagen fibrils and chromosomes. AFM images of living cultured cells in liquid can be used for investigating the movement of cellular processes in relation to subcellular cytoskeletal elements. Recently, numerous AFM-related microscopes, or scanning probe microscopes (SPM), have been invented in parallel with the development of the AFM itself. These microscopes allow the simultaneous collection of topographical and other (e.g., viscoelastic, near-field optical) images of samples in the same portions. Thus, the combination of AFM and the other SPM has great potential for providing valuable new findings on structure and function of cells and tissues.

Cytoarchitecture of the bronchiolar wall in the rat lung: scanning electron microscopic studies.

The cytoarchitecture of rat bronchiolar walls was directly visualized from the adventitial side by scanning electron microscopy after removal of collagen and elastic fibers with a KOH-collagenase digestion method. The bronchioles were surrounded by cord-shaped smooth muscle fibers, which ran circularly to the long axis of the bronchiolar tree. Branches of the bronchial artery were located outside the bronchiolar smooth muscle layer, ran toward the periphery, gave off smaller branches one after another, and finally formed capillary networks both inside and outside the smooth muscle layer. Collecting venules arising from the capillary network ran independently of arterioles, but were gradually accompanied with arterioles as they became thickened. Peripheral nerves covered with a perineurial sheath were also present outside the bronchiolar smooth muscle layer; they gave off branches toward the periphery, and finally entered the bronchiolar smooth muscle layer. Lymphatic vessels were sometimes found at the bifurcation of bronchioles. These findings provide important basic data on the structure and functions of the bronchiolar wall.

The three-dimensional structure of the neonatal mouse kidney as revealed by scanning electron microscopy after KOH treatment.

The shape and arrangement of the developing nephrons were studied three-dimensionally by scanning electron microscopy (SEM) of the neonatal mouse kidney. The specimens were treated with the KOH digestion method in order to remove extracellular connective tissue components, thus enabling the direct observation of the developing nephrons at various stages. At the subcapsular region of the renal cortex, the ureteric ducts were observed as branched tubules with terminal swellings or ampullae. Newly formed blood vessels were often associated with terminals of these ureteric ducts. The cup-shaped renal corpuscles had aggregations of mesangial cells with blood vessels in the groove. At the vascular pole of mature nephrons, extraglomerular mesangial cells were observed as a cellular sheet, which was continuous with the smooth muscle layer of afferent and efferent blood vessels. The present study also demonstrated the shape of the immature podocytes in relation to the endothelial morphology of glomerular capillaries.

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.

Myocardium extending from the left atrium onto the pulmonary veins: a comparison between subjects with and without atrial fibrillation.

Rapid discharges from the myocardium extendingfrom the left atrium onto the pulmonary vein (PV) have been shown to initiate AF, and AF may be eradicated by the catheter ablation within the PV. However, if there is any difference in the distribution patterns of the myocardial sleeve onto the PV between the subjects with and without AF is to be determined. Twenty-one autopsied hearts were examined. Eleven patients previously had AF before death and another 10 patients had normal sinus rhythm as confirmed from the medical records including ECGs before death. After exposing the heart, the distance to the peripheral end of the myocardium was measured from the PV-atrial junction in each PV. Then, the PVs were sectioned and stained and the distal end of myocardium and the distribution pattern were studied. The anteroposterior diameter of the left atrium was also measured. In 74 of 84 PVs, the myocardium extended beyond the PV-atrial junction. The myocardium was localized surrounding the vascular smooth muscle layerforming a myocardial sleeve. The peripheral end of the myocardial sleeve was irregular and the maximal and minimal distances were measured in each PV. The myocardium extended most distally in the superior PVs compared to the inferior ones and the maximal distance to the peripheral end was similar between the AF and non-AF subjects (8.4 +/- 2.8 vs 8.7 +/- 4.4 mm for the left superior and 6.5 +/- 3.5 vs 5.1 +/- 3.9 mm for the right superior PV, respectively). A significant difference was found in the maximal distance in the inferior PVs: 7.3 +/- 4.6 vs 3.3 +/- 2.8 mm for the left (P < 0.05) and 5.7 +/- 2.4 vs 1.7 +/- 1.9 mm for the right inferior PV (P < 0.001) in the subjects with and without AF, respectively. The diameter of left atrium was slightly dilated in AF patients but insignificantly (4.1 +/- 0.1 vs 3.6 +/- 0.1 cm, P > 0.07). The myocytes on the PV were less uniform and surrounded by more fibrosis in patients with AF compared to those without AF. In conclusion, the myocardium extended beyond the atrium-vein junction onto the PVs. The distribution patterns of the myocardium was almost similar between subjects with and without AF, but the histology suggested variable myocytes in size and fibrosis in patients with AF.

Three-dimensional analysis of nephrogenesis in the neonatal rat kidney: light and scanning electron microscopic studies.

In order to clarify the process of renal development more precisely than previously, the present study observed the rat neonatal kidney by scanning electron microscopy (SEM) of KOH digested tissue as well as by light microscopy of plastic sections. In the subcapsular region, aggregation of the mesenchymal cells was closely associated with the upper side of the ureteric duct ampulla. These mesenchymal cells projected a number of fine irregular processes at the basal portion facing the ureteric duct. A spherical cluster transformed from the mesenchymal cell aggregation was found on the lower side of the terminal ampulla, and was differentiated into the renal vesicle. Some cells at the top of the renal vesicle formed a cone-shaped projection and invaded the ureteric duct ampulla, forming a connection with it. In the advanced stage, a shallow transverse cleft appeared on the outer lateral side of the renal vesicle, and a second cleft was formed on the opposite side close to the junction between the renal vesicle and the ampulla. As the two clefts deepened, the vesicle assumed the well-known S-shaped body. In the advanced S-shaped body, the lower limb became cup-shaped, while the segment between the middle and lower limbs of the "S" elongated to form a tubular structure (i.e., the prospective proximal tubule and Henle's loop). The upper limb of the "S" also increased its length to form a distal tubule. The middle limb of the "S", however, was attached firmly to the cup-shaped lower limb (i.e., the prospective renal corpuscle) and was considered to become the macula densa of the mature nephron. In the maturing renal corpuscle, irregularly shaped cells were observed as a sheet-like aggregation at its vascular pole and were continuous with the vascular smooth muscle cells. These findings will help toward a better understanding of the morphological complexities of nephrogenesis.

Morphological characteristics of Schwann cells in the islets of Langerhans of the murine pancreas.

The present study demonstrated the three-dimensional architecture of peri-insular nerve plexuses in the murine pancreas by the combined use of light microscopy of S-100 immunostained sections, transmission electron microscopy (TEM) of thin sections, and scanning electron microscopy (SEM) of KOH digested tissues. By light microscopy of thin sections immunostained with anti-S-100 antibody, Schwann cells were often found on the margin of the islets as if delimiting the islet and exocrine parenchyma. In thick sections, Schwann cells of the islet connected their thin and slender processes with each other to form a delicate network on the surface of the islet. By TEM, Schwann cells were observed as an attenuated sheet that invested the surface of the islet. Axon terminals were usually found on the outer surface of these membranous Schwann cells. SEM of KOH digested tissues revealed that nerves reaching the islet spread on the insular surface. Schwann cells in this portion extended their thin membranous processes, which directly covered the basal part of several endocrine cells as a whole. Numerous axons with varicosities were usually found on the surface of these membranous Schwann cells, but sometimes crept beneath them. These findings indicate that "the interstitial cells" described by light microscopists are peculiar-shaped Schwann cells present in the islets. The functional significance of the rich innervation of the islets is also briefly discussed in the present study.

A point mutation in a cadherin gene, Cdh23, causes deafness in a novel mutant, Waltzer mouse niigata.

A novel mouse model for human nonsyndromic hearing loss, Waltzer niigata (v(ngt)), is found and subjected to positional cloning analysis. Genome-wide scan of 1648 backcross mice maps v(ngt) to the D10Mit258 locus near Waltzer (v). Recombination breakpoints are positioned on a physical map consisting of 13 BACs relative to the flanking markers in the vicinity of v(ngt). Allelism test done in parallel shows that v(ngt) and v are allelic. Sequence analysis reveals one-base deletion in the cDNA encoding a cadherin-related protein, Cdh23, mutation of which is recently reported in v mutants. The frame-shift change, producing a truncated protein of 51 amino acids, is ascribed to a base-substitution of G to A in the acceptor site of splicing junction which is predicted to cause one-base shift of the splicing position.

Three-dimensional structure of G-banded human metaphase chromosomes observed by atomic force microscopy.

The structure of G-bands in human metaphase chromosomes was analyzed by comparison between light microscopic and atomic force microscopic (AFM) images of the same chromosomes. G-bands of the chromosomes were made by trypsin treatment followed by staining with a Giemsa solution. The banded chromosomes examined by light microscopy were dried either in air or in a critical point-drier, and observed by non-contact mode AFM. Air-dried chromosomes after G-band staining showed alternating ridges and grooves on their surface, which corresponded to light-microscopically determined G-positive and G-negative bands, respectively. At high magnification, the G-positive ridges were composed of densely packed chromatin fibers, while the fibers were loose in the G-negative grooves. Fibers bridging the gap between sister chromatids of a mitotic pair were often found, especially in the G-positive portions. These findings suggest that the G-banding pattern reflects the high-order structure of human metaphase chromosomes.

Atomic force microscopy of intact and digested collagen molecules.

The present study was performed to analyse the structure of non-digested and digested collagen type I molecules by atomic force microscopy (AFM). Collagen type I molecules from the bovine skin were diluted with 0.05 N acetic acid, spread on a mica plate, air-dried and observed by non-contact mode AFM in air. Collagen molecules digested with Clostridium histolyticum collagenase were also examined by AFM. Intact collagen type I molecules were observed as twisted threads ranging mainly between 280 and 310 nm in length. The surface of the molecules was uneven and both ends usually slightly bulged like a globule. Depressions on the molecules were found throughout the length, and were most prominent approximately 70 nm from one end of the molecules. The collagenase-treated collagen molecules were degraded into fragments with various lengths, which corresponded to the data from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The end of these fragments often appeared like a tuft, suggesting that the triple-helix unraveled at these regions.