Quantitative analysis of mammalian chromosome by scanning transmission soft X-ray microscopy.

Soft X-ray spectromicroscopy was applied to study the quantitative distribution of DNA and protein in a mammalian chromosome at the spatial resolution of 100 nm. The quantities of DNA and protein were evaluated using 1s-π* transition in the NEXAFS spectra at the nitrogen K absorption edge. DNA was not uniformly distributed in the chromosome and DNA/protein ratio was less than 0.497. The present analysis revealed the clues to identify other molecules that contribute to the absorption spectrum of the sample. The results suggested that accumulation of the absorption spectra of relevant molecules would support the refinement of the analysis.

[Ventricular septal defect, pulmonary hypertension and 3C (cranio-cerebello-cardiac) syndrome: report of a case].

We report a case with the rare association of craniofacial anomalies, cerebellar vermis hypoplasia (Dandy-Walker variant) and congenital heart disease referred to as 3C (cranio-cerebello-cardiac) syndrome. A male infant was born at 40 weeks' gestation. The birth weight was 2,896 g. At birth he had macrocephaly, widely open metopic suture, ocular hypertelorism, cleft palate, apparently low-set ears, cerebellar vermis hypoplasia and ventricular septal defect (VSD) with pulmonary hypertension. Chromosomes were normal and we diagnosed 3C syndrome. At 8 months of age we performed a cardiac catheterization. The pulmonary artery pressure was 70/26 (48) mmHg and pulmonary vascular resistance (RpU) was 8.5 U/m2. But under the oxygenation RpU decreased to 1.5 U/m2. At 10 months of age we performed VSD patch closure. After operation his pulmonary artery pressure was 39/13 (25) mmHg under the oxygen therapy. He was on good course and discharged at 58 post operative day.

Distribution and origin of bovine major histocompatibility complex class II DQA1 genes in Japan.

We sequenced the major histocompatibility complex (MHC) class II DQA1 gene in 352 Japanese cattle (95 Japanese Black, 91 Holstein, 102 Japanese Shorthorn and 64 Jersey cattle) using a new sequence-based typing method. In total, 19 bovine MHC (BoLA)-DQA1 alleles, of which two were novel alleles, were detected. The Holstein, Jersey, Japanese Shorthorn and Japanese Black breeds had 13, 12, 10 and 15 alleles, respectively. The dendrogram that was constructed by the neighbor-joining method on the basis of the DQA1 gene allele frequencies of the four Japanese cattle breeds showed that the Holstein and Japanese Black breeds were closest to each other, with Jersey being farther from these two breeds than Japanese Shorthorn. In addition, Wu-Kabat analysis showed that the DQA1 alleles of the Holstein and Japanese Black were the most and least polymorphic, respectively. Phylogenetic analyses indicated that the DQA1 gene of Bovidae such as cattle, sheep, bison and goat were more similar to pig SLA-DQA genes than to human HLA-DQA1 and dog DLA-DQA genes. The cattle, goat, bison, sheep, human and pig DQA1 molecules had similar rates of amino acid sequence polymorphism, but the distribution of their polymorphic residues differed from that in the dog DQA1 protein. However, the Bovidae DQA1 molecule had more polymorphic residues than the human, pig and dog DQA molecules at two regions, namely positions 52-53 and 65-66. This indicates that the Bovidae DQA1 locus is more polymorphic than the DQA loci of other species.

Establishment of a sequence-based typing system for BoLA-DQA1 exon 2.

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for bovine diseases and immunological traits. Here, we developed a rapid, high-resolution sequence-based typing (SBT) system for BoLA-DQA1. We amplified 355 bp of BoLA-DQA1 by fully nested polymerase chain reaction (PCR) using the newly constructed primers and then performed direct sequencing of each product. Using this method, we investigated the locus in 51 animals whose BoLA haplotypes had been characterized at the Fifth International BoLA Workshop. We identified 15 distinct DQA1 alleles, and there is no conflict between the typing result of PCR-SBT and restriction fragment length polymorphism analysis. Together with the previously developed method for typing BoLA-DRB3, the PCR-SBT for BoLA-DQA1 clearly provides a useful tool for detailed class IIa haplotype analysis.

Estimation of photon dose generated by a short pulse high power laser.

The authors obtain a new equation to estimate the forward component of a photon dose generated through the interaction between a target and a short pulse high power laser. As the equation is quite simple, it is useful for calculating the photon dose. The equation shows that the photon dose is proportional to the electron temperature in the range>3 MeV and proportional to the square of the electron temperature in the range<3 MeV. The dose estimated with this method is roughly consistent with the result of Monte Carlo simulation. With some assumptions and corrections, it can reproduce experimental results obtained and the dose result calculated at other laboratories.

Picosecond snapshot of the speckles from ferroelectric BaTiO3 by means of x-ray lasers.

A picosecond x-ray laser speckle has been conducted to study the dynamics of a disordered surface domain structure (BaTiO3 with 90 degrees c/a domains) as a function of temperature for the first time. The transient surface structures induced by ferroelectric domains decrease as temperature increases towards the Curie temperature T(c) and completely disappear above T(c). The dramatic change of the spatial configuration of the c/a domains was observed to occur from a temperature 2 degrees C below T(c), near which the average correlated domain size at equilibrium decreases as (T(c)-T)(0.37+/-0.02).

Antibodies recognizing CD24 LAP epitope on human T cells enhance CD28 and IL-2 T cell proliferation.

Membrane expression of the CD24 molecule on activated T lymphocytes is not elucidated fully. We previously described the intracellular and cell-surface expression of the CD24 sialic acid-dependent epitope(s) on phytohemagglutinin-activated peripheral blood mononuclear cells. However, the CD24 core protein was not detected previously on human T cells. This study reinvestigated the expression and role of CD24 in T cell subsets. We analyzed binding of anti-CD24 monoclonal antibodies (mAbs) to sialic and leucine-alanine-proline (LAP) epitopes in resting and activated, normal T lymphocytes. CD24 LAP and CD24 sialic epitopes were detected on activated CD4- and CD8-positive cells. Although expression of CD24 sialic epitopes remained stably expressed in interleukin (IL)-2-dependent cultures, T cell expression of the LAP epitope was transient. Anti-LAP antibodies strongly enhanced the response of T cells to a combination of anti-CD3/CD28 mAbs and enhanced proliferative response induced by recombinant IL-2. We found similarities in the tissue distribution and function of the human CD24 LAP molecule and the murine, heat-stable antigen, which suggests that CD24 might function as a signaling molecule on human T cells.

Surgical repair of double-orifice of the mitral valve in cases with an atrioventricular canal defects.

OBJECTIVE: A Double-orifice in the mitral valve is an uncommon congenital cardiac lesion which occurs as an isolated anomaly or in association with other cardiac malformation. This report deals with our surgical experience of a double-orifice of the mitral valve in cases with an atrioventricular canal defect. PATIENTS AND METHODS: From 1991 through 1999, ten patients were diagnosed to have a double-orifice of the mitral valve at Shizuoka Children's Hospital. Each patient had associated major cardiac malformations, among which atrioventricular canal defect underwent surgical management, with five of these undergoing complete correction with or without previous pulmonary artery banding. Of these 10, the five cases were enrolled in this study. Two of these had a complete type, and the other three had a partial type. The cleft in the left-sided atrioventricular valve was closed partially in four and left untouched in one. Bridging tissue, when present, was left intact. There was no regurgitation from any accessory orifice and no repair for an accessory orifice was needed. RESULT: There was no late death and no replacement of the valve with prosthesis. During follow-up ranging from 1 to 4 years, none of the patients developed severe stenosis or progressive regurgitation in the left-sided atrioventricular valve. CONCLUSION: Meticulous surgical management of a double-orifice in the mitral valve in association with atrioventricular canal defect an achieve an acceptable midterm result without developing severe dysfunction in the left-sided atrioventricular valve.

Effects of peroxynitrite on rabbit cornea.

BACKGROUND: Nitric oxide (NO) reacts rapidly with the superoxide anion to generate peroxynitrite, which has been found in the aqueous humor in eyes with uveitis. We evaluated the functional and anatomic effects of peroxynitrite on rabbit cornea. METHODS: One eye of each rabbit received an anterior chamber injection of 3-morpholinosydonimine N-ethylcarbamide (SIN-1), which simultaneously generates both NO and the superoxide anion. The corneal thickness was measured using an ultrasonic pachymeter before and after the injection. The eyes were fixed and the corneal specimens were prepared for electron microscopy. RESULTS: Anterior chamber injections of SIN-1 caused a significant increase in the corneal thickness (25.1+/-3.0 microm) 30 min after injection. Transmission electron microscopy showed swollen mitochondria and large vacuoles in the cytoplasm, and scanning electron microscopy revealed obscuring of the mosaic pattern by increased ruffling of endothelial cell surface and borders. CONCLUSION: The results suggest that peroxynitrite generated in the aqueous humor may cause corneal endothelial cell damage, which leads to transient corneal edema.

Intravitreal injection of hyaluronidase cannot induce posterior vitreous detachment in the rabbit.

PURPOSE: To investigate whether intravitreal injection of hyaluronidase can induce posterior vitreous detachment (PVD) in the rabbit. METHODS: One eye each of 12 New Zealand white rabbits received intravitreal injection via the pars plana of 20 IU of hyaluronidase (0.1 mL reconstituted in sterile balanced salt solution [BSS]) into the midvitreous cavity. The fellow eye of each rabbit received a vitreous injection of 0.1 mL of BSS. At 3 and 6 months after intravitreal injection, four and eight rabbits were killed, respectively, and the eyes were enucleated. After fixation, scanning electron microscopy was performed to study the vitreoretinal interface. RESULTS: At 3 and 6 months after injection, scanning electron microscopy showed that the retinal surfaces in eyes that received either hyaluronidase or BSS were covered with vitreous collagen fibers. No eyes, even those that received hyaluronidase over a period of 6 months, had the smooth retinal surface consistent with a bare internal limiting lamina that suggests the development of PVD. CONCLUSION: Hyaluronidase cannot induce PVD in the rabbit over a 6-month period after vitreous injection.

[The repair of congenital coronary artery fistula in the neonatal period].

We operated on 10 cases diagnosed as congenital coronary artery fistula, including 2 critical neonates. Such neonates needed special care for perfusion pressure drop at the beginning of cardiopulmonary bypass and imperfect delivery of cardioplegia. This phenomenon is likely to depend on the shunt size of a fistula, that is much larger in such neonates than in older patients who could be operated on electively. It was helpful in the situation to crossclamp the main PA, compress the fistula and crossclamp the aorta quickly for a standstill. We prefer the closure of a fistula in the recipient chamber especially when an important area is supplied distally to the origin of the fistula. However, if a ventricle is a recipient chamber, the closure through coronary incision should be done as the neonates in our series. There were two hospital deaths due to vascular leakage syndrome and hypoxia, respectively. Aortic regurgitation (AR) got worse in two after the operation. They were found to have more dilatation and distortion in the Valsalva's sinus which appeared to affect the aortic annulus to some extent. It is likely due to long-standing larger shunt. Contrary to them, the patient operated on during neonatal period was followed by no increase of AR, though she had the largest shunt of our series. We have an impression it could be prevented by earlier operation.

Light and electron microscopic immunocytochemical study on the innervation of the pineal gland of the tree shrew (Tupaia glis), with special reference to peptidergic synaptic junctions with pinealocytes.

Conventional and immunocytochemical, light- and electron-microscopic studies on the innervation of the pineal gland of the tree shrew (Tupaia glis) were made. Neuropeptide Y (NPY)-immunoreactive fibers, which were abundantly distributed in the gland, disappeared almost completely after superior cervical ganglionectomy, suggesting that these fibers are mostly postganglionic sympathetic fibers. By contrast, tyrosine hydroxylase (TH)-immunoreactive fibers, which were less numerous than NPY-fibers, remained in considerable numbers in ganglionectomized animals, indicating the innervation of TH-positive fibers from extrasympathetic sources. Bundles of substance P (SP)- or calcitonin gene-related peptide (CGRP)-immunoreactive fibers, entering the gland at its distal end, were left intact after ganglionectomy. SP-fibers were numerous, but CGRP-fibers were scarce in the gland. SP-immunoreactive fibers were myelinated and nonmyelinated, and were regarded as peripheral fibers because of the presence of a Schwann cell sheath. NPY- and SP-immunoreactive fibers and endings were mainly localized in the pineal parenchyma. NPY-immunoreactive endings synapsed frequently, and SP-positive ones did less frequently, with the cell bodies of pinealocytes. The results suggest that NPY and SP directly control the activity of pinealocytes. Sections stained for myelin showed that thick and less thick bundles of myelinated fibers entered the gland by way of the habenular and posterior commissures, respectively. Under the electron microscope, the bundles were found to contain also unmyelinated fibers. A considerable number of nerve endings synapsing with the cell bodies of pinealocytes remained in ganglionectomized animals; these endings were not immunoreactive for TH or SP. Such synaptic endings may be the terminals of commissural fibers.

Posterior vitreous detachment induced by injection of plasmin and sulfur hexafluoride in the rabbit vitreous.

PURPOSE: To investigate whether an injection of plasmin and sulfur hexafluoride (SF6) can induce posterior vitreous detachment (PVD) without vitrectomy. METHODS: One eye each of 15 New Zealand white rabbits was assigned to one of three groups. Eyes in group 1 received a vitreous injection of 1 unit of human plasmin (0.1 mL reconstituted in balanced salt solution) and 0.5 mL of SF6; eyes in group 2 received a vitreous injection of plasmin alone; eyes in group 3 received a vitreous injection of SF6 alone. Seven days after injection, all animals were monitored electroretinographically and killed, and the eyes were enucleated. After fixation, scanning electron microscopy was performed. RESULTS: In group 1 eyes, the retinal surface was smooth except for the vitreous base, which showed complete separation of the vitreous cortex from the retina, indicating PVD. In group 2 and 3 eyes, sparse collagen fibers remained on the retinal surface. CONCLUSION: Vitreous injection of plasmin combined with SF6 can induce PVD without vitrectomy.

Direct ultrastructural imaging of macrophages using a novel x-ray contact microscopy.

A compact, high-resolution, laser-plasma, x-ray contact microscopy method using a table-top Nd:glass laser system has been developed. This x-ray microscopy system was applied for the observation of macrophage ultrastructures. These images were produced using proximity imaging in which a 5-ns pulse of soft x-rays with wavelengths near and inside the water windows (23A-44A) produced by the laser-plasma were absorbed by the specimen and then registered on a photo resist. The x-ray images imprinted on the photo resist were then developed and analyzed with an atomic force microscope (AFM). Mouse thioglycollate-elicited peritoneal macrophages in suspension were examined by this new x-ray microscope. The x-ray images of the macrophages were compared with those observed by conventional transmission electron microscopy (TEM). The x-ray images showed no obvious organelles, including the nucleus and endoplasmic reticulum, as can be seen with TEM, but high- and low-contrast structures caused by mass distribution of carbon were observed. Thus, using the x-ray microscopy we visualized the first x-ray images of macrophage ultrastructures. The successful x-ray imaging of macrophage ultrastructure indicates that proximity x-ray microscopy may be of value in studying physiology linked to the dynamics of a cell.

Variations of posterior vitreous detachment.

AIMS: To identify variations in posterior vitreous detachment (PVD) and establish a clinical classification system for PVD. METHODS: 400 consecutive eyes were examined using biomicroscopy and vitreous photography and classified the PVD variations-complete PVD with collapse, complete PVD without collapse, partial PVD with thickened posterior vitreous cortex (TPVC), or partial PVD without TPVC. RESULTS: In each PVD type, the most frequently seen ocular pathologies were as follows: in complete PVD with collapse (186 eyes), age related changes without vitreoretinal diseases (77 eyes, 41.4%) and high myopia (55 eyes, 29.6%); in complete PVD without collapse (39 eyes), uveitis (23 eyes, 59.0%) and central retinal vein occlusions (8 eyes, 20.5%); in partial PVD with TPVC (64 eyes), proliferative diabetic retinopathy (30 eyes, 46.9%); and inpartial PVD without TPVC (111 eyes), age related changes without vitreoretinal diseases (62 eyes, 55.9%). This PVD categorisation was significantly associated with the prevalence of each vitreoretinal disease (p < 0.0001, chi 2 test on contingency table). CONCLUSIONS: PVD variations can be classified into four types, which is clinically useful because each type corresponds well to specific vitreoretinal changes.

X-ray micrography and imaging of Escherichia coli cell shape using laser plasma pulsed point x-ray sources.

High-resolution x-ray microscopy is a relatively new technique and is performed mostly at a few large synchrotron x-ray sources that use exposure times of seconds. We utilized a bench-top source of single-shot laser (ns) plasma to generate x-rays similar to synchrotron facilities. A 5 microlitres suspension of Escherichia coli ATCC 25922 in 0.9% phosphate buffered saline was placed on polymethylmethyacrylate coated photoresist, covered with a thin (100 nm) SiN window and positioned in a vacuum chamber close to the x-ray source. The emission spectrum was tuned for optimal absorption by carbon-rich material. Atomic force microscope scans provided a surface and topographical image of differential x-ray absorption corresponding to specimen properties. By using this technique we observed a distinct layer around whole cells, possibly representing the Gram-negative envelope, darker stained areas inside the cell corresponding to chromosomal DNA as seen by thin section electron microscopy, and dent(s) midway through one cell, and 1/3- and 2/3-lengths in another cell, possibly representing one or more division septa. This quick and high resolution with depth-of-field microscopy technique is unmatched to image live hydrated ultrastructure, and has much potential for application in the study of fragile biological specimens.

Copper-ion-catalyzed vitreous liquefaction in vivo.

To investigate copper-ion (Cu2+)-catalyzed vitreous liquefaction in vivo, Cu2+ solution (10 mumol) was injected into the vitreous cavity of rabbits. At 24 h after the injection, the gel and liquid vitreous were weighed, and the percent of vitreous liquefaction was calculated. Cu2+ injection resulted in liquefaction of 58% of the vitreous, although control eyes had 12% liquefaction (p < 0.1). The vitreous liquefaction was more pronounced in the presence of exogenous ascorbic acid. However, the addition of mannitol, a hydroxyl-radical-specific scavenger, significantly suppressed the Cu(2+)-catalyzed vitreous liquefaction. The free radicals generated by the Cu(2+)-catalyzed oxidation system may cause vitreous liquefaction in vivo.

Transient corneal edema induced by nitric oxide synthase inhibition.

The aim of the study is to identify nitric oxide synthase (NOS) in the rabbit cornea and further investigate the physiological role of nitric oxide in the rabbit cornea. For histological identification, an immunohistochemical technique using anti-NOS monoclonal antibodies was employed. For the physiological study, we measured the corneal thickness in vivo as an indicator of corneal edema by ultrasonic pachymetry. The measurements were repeated before and after ipsilateral injections of N(G)-nitro-L-arginine methyl ester (L-NAME) or N(G)-nitro-D-arginine methyl ester (D-NAME) or 6-anilino-5,8-quinolinedione (LY-83583) with contralateral injection of vehicle (balanced salt solution) into the anterior chamber of the rabbit. We also monitored intraocular pressure (IOP) by pneumatonometry. Endothelial NOS (eNOS) immunoreactivity was demonstrated both in the corneal epithelium and the endothelium. The corneal thickness significantly increased after L-NAME or LY-83583 without significant rise of IOP, whereas no change was detected after vehicle or D-NAME. These results suggest that NO is spontaneously produced in the corneal endothelium and the NO/cyclic GMP pathway is involved in maintainance of corneal thickness.

X-ray microscopy and imaging of Escherichia coli, LPS and DNA.

Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X-ray microscopy can take instant images of specimens but is mostly restricted to a few synchrotron X-ray sources. We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material. To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 microns wavelength, 5 x 10(12) W cm-2 intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist). This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10-ns burst of laser energy (at 20J) radiates strong emission lines in the region of 300 eV. The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E.coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 A level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.

[Fluorophotometry of the animal eye].

We assessed the usefulness of the Fluorotron Master fitted with a small animal adapter. We also discuss the measurement conditions for the tupai, which is a promising experimental mammal. A good concentration for measurements with this instrument ranged from 0.5 x 10(-6) to 1.0 x 10(-6) g/ml. A suitable time for fluorophotometry of the tupai was 30 min. after injection of fluorescein-Na, and a suitable dose of the fluorescein-Na was 2 mg/kg. We could do ultrafiltration for measurement of the protein unbound fluorescein concentration from a minimal sample of the blood using a hematocrit tube, and thus could reduce the deleterious effects of blood sampling on the animal. This instrument is useful for the estimation of the blood-ocular barrier permeability of animal models.