Effects of ovariectomy on the secretory apparatus in the right atrial cardiomyocytes of middle-aged mice

OBJECTIVE : The aim of the present study was to evaluate the effects of ovariectomy on the secretory apparatus of natriuretic peptides in right atrial cardiomyocytes. METHODS: Nine-month-old mice underwent bilateral ovariectomy or sham surgery. The blood exam of the ovariectomized mice showed results consistent with castrated females. Systolic blood pressure was measured after ovariectomy (9 mo of age) and at the moment of sacrifice (12 mo of age). Fragments of the right atrium were collected and prepared for electron microscopy examination. The following variables were quantified: the quantitative density and area of the natriuretic peptide granules, the relative volume of euchromatin in the nucleus, the number of pores per 10 μm of the nuclear membrane and the relative volumes of the mitochondria and Golgi complex. RESULTS: The cardiomyocytes obtained from ovariectomized mice indicated that the quantitative density and the area of secretory granules of natriuretic peptides were significantly lower compared with the sham-operated mice. Furthermore, there was a decrease in the relative volume of euchromatin, a lower density of nuclear pores, and lower relative volumes of the mitochondria and Golgi complex in the ovariectomized mice compared with the sham-operated mice. These findings suggest a pool with a low turnover rate, i.e., low synthesis and elimination of natriuretic peptides. CONCLUSION: A lack of estrogen caused hypotrophy of the secretory apparatus in right atrial cardiomyocytes that could explain the weak synthesis of natriuretic peptides in mice. Furthermore, one of the mechanisms of blood pressure control was lost, which may explain, in part, the elevated blood pressure in ovariectomized mice. .

Variantes de la heterocromatina y la eucromatina en el diagnóstico prenatal citogenético / Variants of heterochromatin and euchromatin in cytogenetic prenatal diagnosis

Algunos cambios en la morfología de los cromosomas, detectados durante el análisis citogenético, no están asociados con defectos clínicos, representan un dilema para el asesor genético principalmente durante la realización de un estudio prenatal; por esta razón es que una apropiada discriminación entre una variante inocua y una verdadera anomalía resulta crucial para llevar a cabo un asesoramiento genético preciso. Los polimorfismos de la heterocromatina son identificados usualmente por técnicas de bandeo específicas y consideradas como variaciones mendelianas sin una significación clínica. De igual modo, en la literatura se expone la presencia de variantes en regiones eucromáticas que después de un análisis detallado resultan ser de naturaleza benigna. Debido a la importancia de este tema en la actualidad se hace necesario proponer un protocolo a seguir en los laboratorios cada vez que una variante cromosómica sea detectada en el diagnóstico prenatal. El objetivo de este trabajo es presentar una revisión de la literatura acerca de los pasos que se siguen ante la aparición de una variante cromosómica y las sugerencias que se brindan para un manejo más adecuado.

Some changes in chromosome morphology, which are detected in cytogenetic diagnostics, are not associated with clinical defects presenting a dilemma for the genetic counsellor, especially during prenatal diagnosis; this is the reason why a proper discrimination between innocuous variants and true anomalies is crucial to allow precise counselling. Polymorphisms of heterochromatin are identified usually by specific banding techniques and considered as Mendelian variations without a clinical significance. Likewise, it has been exposed in the literature the presence of variants in euchromatic regions that after a detailed analysis turns out to be of benign nature. Due to the current importance of this issue it is necessary to propose a protocol to follow in our laboratories every time a chromosome variant is detected while performing a prenatal analysis and supported by experienced specialist in our field. The goal of this work is to present a review of the literature about how a finding of a chromosome variant is handled and the suggestions given for a more proper management.

[Chromosome abnormalities with clinical sign]

Some changes in chromosome morphology, detected during cytogenetic analysis, are not associated with clinical defects. Therefore a proper discrimination of harmless variants from true abnormalities, especially during prenatal diagnosis, is crucial to allow precise counselling. In this review we described chromosome variants and examples of chromosome anomalies that are considered to be unrelated to phenotypic consequences. The correlation between the presence of marker chromosomes and a risk of clinical signs is also discussed. Recently so-called molecular karyotyping, especially by the use of high-resolution array-CGH technique, contributed to revealing a high number of previously unknown small genomic variations, which seem to be asymptomatic, as they are present in phenotypically normal individuals

Las células mamarias en proceso de diferenciación disminuyen su volumen citoplasmático y nuclear / Mammalian cell differentiation process decreasing their cytoplasmatic and nuclear volume

Las modificaciones experimentadas en el proceso biológico de diferenciación celular determinan cambios complejos y fundamentales en la ultraestructura, la bioquímica y la fisiología celular que pueden ser apreciadas claramente utilizando técnicas morfométricas, las cuales traducidas en información cuantitativa permiten evidenciar los cambios que conlleva este mecanismo. Células normales de epitelio mamario de rata, mantenidas en cultivo, estimuladas a proliferar con el factor de crecimiento epidérmico, originan el grupo celular HC11 GM. Estas células normales y proliferantes son inducidas a diferenciarse mediante inducciones de hormonas lactogénicas: dexametasona, prolactina e insulina, determinándose la generación de un tipo celular diferenciado: HC11 IM. Estudiamos a nivel de microscopía electrónica estos tipos celulares, procurando datos morfométricos discriminantes a lo largo de la diferenciación, diagnosticando las fracciones volumétricas de componentes celulares, determinando así mismo la variación en el área de las células involucradas, precisando de este modo, nuevos marcadores en el padrón de modificación que caracteriza este proceso.

Microdeletions in the Y chromosome of patients with idiopathic azoospermia / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To evaluate the occurrence and prevalence of microdeletions in the gamma chromosome of patients with azoospermia.</p><p><b>METHODS</b>DNA from 29 men with idiopathic azoospermia was screened by polymerase chain reaction (PCR) analysis with a set of gamma chromosome specific sequence-tagged sites (STSs) to determine microdeletions in the gamma chromosome.</p><p><b>RESULTS</b>Deletions in the DAZ (deleted in azoospermia) loci sgamma254 and sgamma255 were found in three patients with idiopathic azoospermia, resulting in an estimated frequency of deletions of 10.7% in idiopathic azoospermia men.</p><p><b>CONCLUSION</b>We conclude that PCR analysis is useful for the diagnosis of microdeletions in the Y chromosome, which is important when deciding the suitability of a patient for assisted reproductive technology such as testicular sperm extracion-intracytoplasmic sperm injection (TESE-ICSI).</p>

mRNA is Synthesized Mainly at the Phase between the Euchromatin and Heterochromatin: Proposal of a Phase Theory

BACKGROUND: Malignant cell nuclei, in general, have increased amounts of heterochromatin and decreased electron densities of euchromatin, making the chromatin pattern coarser than that of benign cell nuclei. The chromatin pattern in benign and malignant cells, however, is barely explained in terms of molecular structure. In this study, the chromatin pattern of metaplastic and carcinomatous squamous cells of the uterine cervix was correlated with transcriptional activity by ultrastructural autoradiography. METHODS: Punch-biopsied tissues were cultured with 3H-uridine for 5 minutes and processed for electron microscopy. Thin sections of the tissues on nickel grids were covered with photosensitive emulsion and kept cold in a dark room for 10 to 16 weeks. After development and staining, the tissues were observed by electron microscopy. RESULTS: The nuclei of the metaplastic squamous cells consisted mostly of euchromatin. A few silver grains were observed, mainly at the periphery of the nuclei. The nuclei of the carcinomatous cells had increased amounts of heterochromatin along the nuclear membrane, and also in the euchromatin area. Silver grains were observed mainly at the boundary between the heterochromatin and euchromatin. CONCLUSION: These findings suggest that an increased amount of heterochromatin in carcinomatous cells results in an increase of the boundary area between the heterochromatin and euchromatin, an area which may be a transcriptionally active site.

Cyclosporine Effect on the Expression Pattern of the Myosin Heavy Chain Gene and the Morphologic Changes of Myocardium in Overloaded Left Ventricle of Rats / 대한법의학회지

BACKGROUND: In response to numerous pathologic stimuli, the myocardium undergoes a hypertrophic response characterized by increased myocardial cell size and activation of fetal cardiac genes. Recently, the calcineurin inhibitor, cyclosporine has been reported to prevent the development of cardiac hypertrophy, however, others reported data which are disagreed to the cyclosporine effect on the prevention of cardiac hypertrophy. METHOD: To clarify whether the calcineurin signaling pathway is a critical for overloaded hypertrophy in vivo and to characterize the cyclosporine effect on the develpment of cardiac hypertrophy, I examined the effects of cyclosporine on the left ventricular overload in the experimental model of clipping of abdominal aorta between the diaphragm and renal artery for three weeks in rats. RESULTS: Left ventricular mass was larger in the group of clipping of abdominal aorta than in the group of cyclosporine injection after clipping of abdominal aorta, however, which had larger ventricular mass rather than control group. It means that cyclosporine suppress hypertrophic growth. Both treated and untreated animals showed increased nuclear polymorphism and euchromatin pattern, and also, ultrastructurally, showed degenerative changes in the cardiac myocytes such as swelling of subsarcolemmal cytoplasm with indistinct sarcoplasmic reticulum and "T" tubules, loosening of myofibril bundles with decreased electron density, and electron dense mitochondria with decreased number. Characteristically, the group of cyclosporine injection after clipping of abdominal aorta showed polymorphic electron dense unswollen giant mitochondria which was not characteristic in other groups. alpha-MyHC mRNA including non-spliced mRNA of the group of abdominal aortic clipping was downregulated in the both groups of clipping of abdominal aorta. beta-MyHC mRNA was upregulated in the group of clipping of abdominal aorta and downregulated in the group of cyclosporine injection after clipping of abdominal aorta. From the above results, initial response to overload is a degenerative changes of cardiac myocytes and cyclosporine may suppress hypertrophic response and the fetal gene reactivation such as beta-MyHC mRNA in this experiment.

Electron Microscopic Analysis of Apoptosis of HGPRT- Mouse Myeloma Cell Induced by Aminopterin, a de novo Pathway Blocking Agent / 대한면역학회지

Programrned cell death (PCD), or apoptosis, is a process by which cells die in response to specific physiological and toxicological signals. This genetically programmed form of cellular suicide is intirnately involved in many biological processes including growth, metamorphosis, embryogenesis, and oncogenesis. Cells undergoing PCD in normal and neoplasmic tissues display the following biochemical and morphological features: internucleosomal DNA fragmentation, reduced cell volume, condensed chromatin in nucleus, zeiosis and generation of apoptotic bodies containing intact organelles and plasma rnembrane. Hybridoma cell production, resulting from the fusion of myeloma cells with antibody producing spleen cells, is widely used in various fields of life science. This technique requires hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient mutant myeloma cell line as a fusion partner. When these mutants cell is treated with aminopterin plus hypoxanthine-thymidine (HAT) after the cell fusion they are selectively and efficiently eliminated remaining fused hybridoma celis. But there hasn't been any report regarding the selective elimination mechanism of this HGPRT mutant myeloma cell. By using HGPRT myeloma P3-X 63-Ag8.653 (V653) as a model system, this study demonstrated that PCD was induced by aminopterin treatment of this V653 cell line. And the sequential ultrastructural changes during this death process were observed by using electron microscope. When V653 cells were incubated with 0.4 uM aminopterin, DNA fragmentation was detectable after 3 hours and peaked between 12 and 18 hours of aminopterin treatment and the cell viability decreased in a time dependant manner. V653 cells incubated with amiopterin showed following ultrastructural changes during the death process. Dilatation of rough endoplasmic reticulum (RER) and detachment of ribosomes were the earliest ultrastructural changes and first seen after 30 minute incubation. Dilatation of perinuclear cisternae began to appear after 1 hour and deformation of nucleoplasm such as decreased electron density of perinuclear heterochromatin and increased electron density of euchromatin were seen after 3 hours. Increased electron density of cytoplasm, decreased cell volume, condensation of chromatin and apoptotic bodies were observed in many cells after 9 hours but vacuolation by severe dilatation of RER was seen in some cells. 24 hours after incubation with aminopterin, many cells showed typical form of apoptosis characterized by cell shrinkage, increased electron density of cytoplasm and apoptotic bodies. On the contrary, some cells showed different type of cell death characterized by increased cell volume, decreased electron density of cytoplasm, severely dilated RER and apoptotic bodies. In both types of cells, mitochondrial cristae and limiting membrane of mitochondria are comparatively well preserved. In other cells, nuclei did not show significant changes but there were deformations of mitochondria such as markedly increased electron density and formation of lamella bodies. The death process of V653 cell was not synchronized among cells. The results of this study proved that aminopterin-induced selective elimination of fusion partner V653 myeloma cell is due to PCD. The earliest ultrastructural changes observed in this process were dilatation of RER and detachment of ribosomes. And there were two distinct morphological types in the PCD.

A study of Digital Image Analysis of Chromatin Texture for Discrimination of Thyroid Neoplastic Cells / 대한세포병리학회지

Chromatin texture, which partly reflects nuclear organization, is evolving as an important parameter indicating cell activation or transformation. In this study, chromatin pattern was evaluated by image analysis of the electron micrographs of follicular and papillary carcinoma cells of the thyroid gland and tested for discrimination of the two neoplasms. Digital grey images were converted from the electron micrographs; nuclear images, excluding nucleolus and intranuclear cytoplasmic inclusions, were obtained by segmentation; grey levels were standardized; and grey level histograms were generated. The histograms in follicular carcinoma showed Gaussian or near-Gaussian distribution and had a single peak, whereas those in papillary carcinoma had two peaks(bimodal), one at the black zone and the other at the white zone. In papillary carcinoma. the peak in the black zone represented an increased amount of heterochromatin particles and that at the white zone represented decreased electron density of euchromatin or nuclear matrix. These results indicate that the nuclei of follicular and papillary carcinoma cells differ intheir chromatin pattern and the difference may be due to decondensed chromatin and/or matrix substances.

Fine structure and detoxification kinetics in kupffer cells after injection of endotoxin in rats / 영남의대학술지

The aim of this study was to clarify the role of Kupffer cells in the mechanism of endotoxin-induced liver injury. The study on fine structure of Kupffer cells was performed after the injection of endotoxin. The endotoxin(Escherichia soli lipopolysaccharide 026: B6, 1.5mg/100 g of body weight) was intraperitoneally injected in Sprague-Dewley rats. Animals were sacrificed at 1/4, 1/2, 1, 2, 4, 8, 16, 24, 72 and 120 hours after the injection of endotoxin. Livers were extirpated and processed to be examined by light and electron microscopy. The results obtained were summerized as follows: Early changes observed in liver after endotoxin injection included the increased number and hypertrophy of Kupffer cells, infiltration of neutrophils and presence of fibrin thrombi within the sinusoids. The coritinuous increase of the Kupffer cells in number with hypertrophy, congestion and infiltration of inflammatory cells within the sinusoids were observed. Hepatocytes showed* fatty change and occasional necrosis. At 72 hours the congestion decreased. At 120 hours the number of Kupffer cells was increased, but the morphology of Kupffer cells became similar to that of the control group. The numbers and sizes of primary and secondary lysosomes and amount of euchromatin of Kupffer cells increased. Swellings and increase in number of mitochondria, Golgi complex, smooth endoplasmic reticulum, rough endoplasmic reticulum were evident. Microthrombi were present within the sinusoids. The swelling of rough endoplasmic reticulum and mitochondria, decrease of glycogen particles, fatty change, hypoxic vacuoles, pyknotic nuclei and occasional necrosis were observed in hepatocytes. At 72 hours the number of secondary lysosomes in Kupffer cells decreased. At 120 hours the morphology of Kupffer cells became similar to that of the control group. According to these results, it was postulated that the endotoxin was initially taken up by pinocytosis into Kupffer cells and degraded in secondary lysosomes of activated Kupffer cells. Kupffer cells may play an important role in the defense mechanism of liver during endotoxemia. The dysfunction of Kupffer cells and ischemia by sinusoidal microthrombi may cause liver injury.