Number of lymph nodes dissected in colorectal cancer and probability of positive nodes, angiolymphatic/perineural invasion, and intracellular mucin in a referral service / Número de Linfonodos Dissecados no Câncer Colorretal e Probabilidade de Nodos Positivos, Invasão Angiolinfática, Perineural e Mucina Intracelular em Serviço de Referência

Among the malignancies, colorectal cancer ranks fourth in incidence in Brazil. The main prognostic measure is related to the amount of affected lymph nodes. Thus, many studies try to correlate the number of extracted lymph nodes, with the probability of obtaining positive nodes. Study objectives: Determine whether dissection ≥12 lymph nodes increases probability of finding neoplastic involvement in relation to resection of fewer. Assess the presence of angiolymphatic invasion; perineural and intracelluar mucin and correlate it with tumor differentiation and TNM classification. Correlate the average of positive nodes with angiolymphatic and perineural involvement. Methods: Pathological reports of patients operated for CRC from 1997 to 2013 were analyzed. A probability (p) less than 0.05 was considered to indicate statistical significance. Results: Median of lymph nodes sent to analysis was 12 nodes. Average number of lymph nodes affected was higher when a number ≥12 lymph nodes were dissected (p = 0.001) (Kruskal-Wallis). There was positive association between average of affected lymph nodes and presence of angiolymphatic (p < 0.0001) or perineural invasion (p = 0.024). Angiolymphatic and intracellular mucin are less present in well-differentiated adenocarcinomas. Perineural and angiolymphatic were more present in T4 stages. Conclusions: Dissection ≥ 12 lymph nodes increases chances of finding positive nodes. There is relation between angiolymphatic invasion; perineural and intracellular mucin and type of tumor differentiation, as well as TNM classification. Average number of lymph nodes affected was higher in presence of perineural or angiolymphatic invasion.

Dentre as neoplasias malignas, o câncer colorretal ocupa o quarto lugar em incidência no Brasil. Uma das principais medidas de prognóstico está relacionada à quantidade de linfonodos acometidos. Sendo assim, muitos trabalhos estudam meios de correlacionar o número de linfonodos dissecados, com a probabilidade de se obterem linfonodos positivos. Objetivos do estudo: Determinar se a dissecção ≥ 12 linfonodos aumenta a probabilidade de se encontrar acometimento neoplásico nos mesmos em relação à menor ressecção. Avaliar a presença de invasão angiolinfática; perineural e mucina intracelular e correlacioná-la com diferenciação tumoral e classificação TNM. Correlacionar a média de nodos positivos com acometimento angiolinfático e perineural. Métodos: Foram analisados laudos anatomopatológicos de pacientes operados por câncer colorretal (CCR) de 1997 a 2013. A probabilidade (p) menor que 0,05 foi considerada para indicar significância estatística. Resultados: A média de linfonodos comprometidos foi maior quando um número ≥ 12 linfonodos foram dissecados (p = 0,001) (Kruskal-Wallis). Houve associação positiva entre a média de linfonodos afetados e a presença de invasão angiolinfática (p < 0,0001) ou perineural (p = 0,024). A invasão angiolinfática e a mucina intracelular estavam menos presentes em adenocarcinomas bem diferenciados. Invasão perineural e angiolinfática estiveram mais presentes nos estádios T4. Conclusões: A dissecção ≥ 12 linfonodos aumenta as chances de se encontrar nodo positivo. Existe relação entre invasão angiolinfática; perineural e mucina intracelular e o tipo de diferenciação tumoral, bem como a classificação TNM. A média de linfonodos comprometidos foi maior na presença de invasão perineural ou angiolinfática.

Glucagon-like peptide-1 regulates lipometabolism by down-regulating adipose triglyceride lipase in 3T3-L1 adipocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of glucagon-like peptide-1 (GLP-1) on glycolipid metabolism in 3T3-L1 adipocytes and explore the mechanism.</p><p><b>METHODS</b>3T3-L1 adipocytes were treated with GLP-1, insulin, or both for 24 h, and Western blotting was used to analyze the expression levels of adipose triglyceride lipase (ATGL), glucose transporter type 4 (GLUT4), Akt1, Akt2 and phosphorylated Akt in the cells. Immunofluorescence was used to observe lipid content in 3T3-L1 adipocytes.</p><p><b>RESULTS</b>Akt1 and Akt2 were not activated by insulin stimulation in 3T3-L1 adipocytes. Akt was phosphorylated by GLP-1 stimulation, which inhibited the expression of ATGL and increased the translocation of GLUT4 from the intracellular membranes to plasma membranes. These changes were more obvious under the synergistic effect of insulin in 3T3-L1 adipocytes.</p><p><b>CONCLUSION</b>GLP-1 decreases lipolysis by inhibiting the expression of ATGL and improves insulin resistance by increasing the translocation of GLUT4 in 3T3-L1 adipocytes.</p>

Efeitos da restrição calórica nas vias de sinalização por insulina e óxido nítrico: implicações para biogênese, morfologia e função mitocondriais / Calorie restriction restriction effects on insulin and nitric oxide signaling: implications to mitochondrial biogenesis, morphology and function

A restrição calórica (RC) estende a expectativa de vida de muitos organismos por mecanismos ainda em estudo. Entre os vários efeitos fisiológicos da RC encontra-se o aumento na biogênese mitocondrial, dependente de óxido nítrico (NO•), sintetizado pela enzima óxido nítrico sintase endotelial (eNOS). Um dos indutores fisiológicos mais potentes da eNOS é a insulina, cujos níveis plasmáticos são consideravelmente reduzidos nos organismos em RC. O objetivo deste trabalho foi investigar os mecanismos associados ao aumento da sinalização por NO• durante a RC in vivo e in vitro, e as conseqüências celulares do aumento de massa mitocondrial no que diz respeito à longevidade e capacidade respiratória celulares. Submetemos camundongos Swiss fêmeas à RC de 40% e observamos um considerável aumento tecido-específico na fosforilação basal de Akt e eNOS em músculo esquelético, tecido adiposo visceral e cérebro, os quais também apresentaram maior massa mitocondrial. A associação entre a sinalização por insulina, NO• e biogênese mitocondrial foi adicionalmente confirmada em um grupo de camundongos tratados com o desacoplador mitocondrial dinitrofenol (DNP), que também reduz a insulinemia e aumenta a longevidade em camundongos. Para o estudo mecanístico deste fenômeno, usamos soros de ratos Sprague-Dawley submetidos à RC de 40% ou alimentados ad libitum (AL) em cultura celular de células vasculares da musculatura lisa (VSMC), reproduzindo um protocolo descrito para RC in vitro. O uso do soro RC aumentou a fosforilação do receptor de insulina e Akt, a expressão de eNOS e nNOS (forma neural da NOS) e a fosforilação de eNOS, o que se refletiu em maior liberação de nitrito (NO2) no meio de cultura. Inibindo-se a Akt, todos os efeitos promovidos pela RC na sinalização por NO• foram revertidos. Ao se imunoprecipitar do soro a adiponectina, citocina conhecida por aumentar a sensibilidade à insulina, aumentada durante a RC, os efeitos do soro RC na via de sinalização de insulina foram abolidos e, conseqüentemente, os efeitos na sinalização por •NO foram prevenidos. Neurônios de células granulosas de cerebelo, que não expressam eNOS, apenas nNOS, foram cultivados com os soros AL ou RC, e também apresentaram considerável aumento na sinalização por •NO. Estas alterações induziram a biogênese mitocondrial e capacidade respiratória, e foram associadas à maior longevidade celular. Os mesmos efeitos mitocondriais foram observados em células secretoras de insulina, INS1, entretanto a secreção de insulina em resposta à glicose tornou-se inibida, por um mecanismo desconhecido, porém associado a reduzidos níveis intracelulares de espécies oxidantes, moléculas-chave para a secreção de insulina; e à alteração da morfologia mitocondrial, provavelmente devido à maior expressão de mitofusina-2 (Mfn-2). Ao se nocautear a Mfn-2, houve um aumento na geração de EROs e as células em RC passaram a secretar insulina a níveis comparáveis aos das células controle. Concluímos que durante a RC a maior sensibilidade à insulina aumenta a atividade de eNOS, via Akt, associada à maior biogênese mitocondrial. A adiponectina é uma molécula-central nestes eventos. A expressão de nNOS também é afetada, por mecanismos desconhecidos. O aumento de biogênese mitocondrial eleva a capacidade respiratória celular e impacta positivamente a longevidade in vitro. A alteração da morfologia mitocondrial associa-se a alterações na produção de oxidantes intracelulares e mudanças na secreção de insulina

Calorie restriction (RC) is known to extend the lifespan in many organisms, and its mechanisms of action are still under investigation. Enhanced mitochondrial biogenesis driven by nitric oxide (•NO), synthesized by the endothelial nitric oxide synthase (eNOS), is proposed to be a CR central effect. Insulin is one of the most potent physiological activators of eNOS. However, plasmatic insulin levels are dramatically reduced in organisms under CR. The goal of this work was uncover the mechanisms associated with enhanced •NO signaling during CR, in vivo and in vitro, as well as the cellular consequences of increased mitochondrial mass, regarding lifespan and reserve respiratory capability. Female Swiss mice were submitted to 40% of CR. A tissue-specific (skeletal muscle, abdominal adipose tissue and brain) increment in basal Akt and eNOS phosphorylation, which was related to enhanced mitochondrial biogenesis, was observed. Indeed, this association was also verified in tissues from mice treated with low doses of a mitochondrial uncoupler, dinitrophenol (DNP). To unveil the mechanism behind the insulin signaling effects on •NO levels, serum from Sprague-Dawley rats submmited to 40% of CR was used to culture in VSMC cells, an in vitro CR protocol. CR sera enhanced insulin receptor (IR) and Akt phosphorylation, as well as nitrite (NO2-) accumulation in the culture media, the expression of eNOS and nNOS (neural NOS isoform) and eNOS phosphorylation. The effects of CR sera were reversed by Akt inhibition. The immunoprecipitation of serum adiponectin, a cytokine known to improve peripheral insulin sensitivity, also reversed the CR serum effects on insulin and •NO signaling. Cerebellar neurons, which do not express eNOS, just nNOS, were also cultured with CR or AL serum and also presented striking increments in •NO signaling, associated with mitochondrial biogenesis, increased reserve respiratory capability and lifespan extension. The mitochondrial effects promoted by CR were also observed in insulin secreting cells (INS1). However, under the CR condition, insulin secretion stimulated by glucose was impaired. The likely explanations are reduced mitochondrial reactive oxygen species (ROS) generation, or the alteration in mitochondrial morphology, associated, in our model, with enhanced mitofusin-2 expression (Mfn-2). In cells which the Mfn-2 was knocked down, insulin secretion in CR and AL groups was responsive to glucose at the same level, and the intracellular oxidants levels were much higher. Overall, CR improves •NO signaling due to enhanced insulin sensitivity, through Akt, and results in mitochondrial biogenesis. Adiponectin is a key molecule in this phenomenon. Increments in mitochondrial mass enhance the cellular reserve respiratory capability and lifespan. Mitochondrial morphology alterations are associated with possible decreases in ROS generation and impaired insulin release, maintained the low levels of plasmatic insulin

Glycosphingolipid Modification: Structural Diversity, Functional and Mechanistic Integration of Diabetes

Glycosphingolipids (GSLs) are present in all mammalian cell plasma membranes and intracellular membrane structures. They are especially concentrated in plasma membrane lipid domains that are specialized for cell signaling. Plasma membranes have typical structures called rafts and caveola domain structures, with large amounts of sphingolipids, cholesterol, and sphingomyelin. GSLs are usually observed in many organs ubiquitously. However, GSLs, including over 400 derivatives, participate in diverse cellular functions. Several studies indicate that GSLs might have an effect on signal transduction related to insulin receptors and epidermal growth factor receptors. GSLs may modulate immune responses by transmitting signals from the exterior to the interior of the cell. Guillain-Barre syndrome is one of the autoimmune disorders characterized by symmetrical weakness in the muscles of the legs. The targets of the immune response are thought to be gangliosides, which are one group of GSLs. Other GSLs may serve as second messengers in several signaling pathways that are important to cell survival or programmed cell death. In the search for clear evidence that GSLs may play critical roles in various biological functions, many researchers have made genetically engineered mice. Before the era of gene manipulation, spontaneous animal models or chemical-induced disease models were used.

Expression and Translocation of Aquaporin (AQP)-1 after Pelvic Nerve Stimulation in Rat Vagina / 대한남성과학회지

PURPOSE: Aquaporins (AQPs) are membrane proteins that facilitate water movement across biological membranes. The purposes of this study were to investigate the localization and functional roles of AQP-1 water channels in rat vagina. MATERIALS AND METHODS: Female Sprague-Dawley rats (230~240 g, n=40) were anesthetized. To investigate the expression and localization of AQPs in the vagina, the vaginal branch of the pelvic nerve was stimulated for 60 seconds (10 V, 16 Hz, 0.8 msec), and then the animals were sacrificed immediately or 5 minutes later. The expression and cellular localization of AQP-1 in the vaginal tissue was measured by Western blot and immunohistochemistry. The cytosolic (or intracellular membrane) and plasma membrane fractions of AQP-1 in vaginal tissue were studied by immunoblot analysis. RESULTS: Immunolabeling showed that AQP-1 was mainly expressed in the capillaries and venules of the vagina. The translocation of AQP-1 isoforms from the cytosolic compartment to the plasma membrane compartment was observed right after nerve stimulation and had declined at 5 minutes after nerve stimulation. However, when the nerve stimulation was repeated 3 times, the translocation of AQP-1 from the intracellular membrane compartment to the plasma membrane compartment was still observed at 5 minutes. CONCLUSIONS: This study suggests that sexual arousal induced by pelvic nerve stimulation modulates AQP-1 activity in the rat vagina. This result implies that AQP-1 may play an important role in vaginal lubrication.

Papel de la vacuola parasitófora de macrófagos de ratón infectados por Leishmania amazonensis en la adquisición de de moléculas / Role of the parasitophorous vacuole of murine macrophages infected with Leishmania amazonensis in molecule acquisition

Introducción. Leishmania son parásitos intracelulares de macrófagos, confinados en compartimentos denominados vacuolas parasitóforas. La permeabilidad de este compartimento depende de su interacción con el tráfico vesicular y transportadores presentes en su membrana. Objetivo. En este trabajo se estudió la permeabilidad de la membrana de la vacuola parasitófora en la línea celular J774.A1 infectada con Leishmania amazonensis, in situ y en compartimentos aislados. Materiales y métodos. El aislamiento de vacuolas parasitóforas se hizo por gradiente de densidad. La permeabilidad de la membrana de estas se valoró por distribución de sondas fluorescentes y electrofisiología. Para establecer indirectamente el transporte de protones se usó naranja de acridina. La presencia de transportadores ABC sensibles a probenecid se estableció con amarillo lucifer y calceína. Por primera vez con la técnica de patch-clamp se registraron corrientes en la membrana de este compartimento aislado. Resultados. La vacuola parasitófora colorea de rojo con naranja de acridina indicando un pH ácido. Concentra amarillo lucifer a través de un transportador sensible a probenecid, pero excluye la sonda calceína. Vacuolas aisladas se marcan de rojo con naranja de acridina y concentran amarillo lucifer a través de un transportador sensible a probenecid. Estas vacuolas excluyeron calceína y presentaron en su membrana una corriente iónica que se activa a diferencias de potencial cercanas a 60 mV, con una conductancia de 46 ± 3 pS. Conclusiones. Se pueden aislar vacuolas parasitóforas con propiedades de permeabilidad que preservan mecanismos de transporte similares a los encontrados in situ. Se registra por primera vez la presencia de una corriente iónica poco selectiva en la membrana de este compartimiento.

Introduction. Leishmania are intracellular parasites of macrophages, confined into compartments known as parasitophorous vacuoles. The permeability of this compartment depends on its interaction with the endocytic pathway and transport proteins present on its membrane. Objective. The membrane permeability of the parasitophorous vacuole was studied in J774.A1- macrophage like cells infected with Leishmania amazonensis, in situ and on isolated compartments. Materials and methods. The parasitophorous vacuoles were isolated by density gradients. Fluorescent probe distribution and electrophysiological recordings were used to determine parasitophorous vacuole membrane permeability. Proton transport was evaluated indirectly by acridine orange staining. Probenecid sensitive ABC transporters were detected using the fluorescent probes lucifer yellow and calcein. For the first time ion currents were recorded on the membrane of isolated parasitophorous vacuoles using the patch clamp technique. Results. The parasitophorous vacuole stains red with acridine orange indicating an acidic compartment. It concentrates lucifer yellow by means of a probenecid sensitive transporter but excludes calcein. Isolated vacuoles stained red with acridine orange and concentrated lucifer yellow by means of a probenecid sensitive transporter. These vacuoles excluded calcein and showed an ion current in their membrane which is activated at potentials close to 60 mV with a mean conductance of 46 ± 3 pS. Conclusions. Isolated parasitophorous vacuoles with permeability properties preserving transport mechanisms similar to those found in situ can be purified. A poorly selective ion current on the parasitophorous vacuole membrane is reported for the first time.

Characteristics and physiological role of surfactant-like particles secreted by entrocytes.

Intestinal epithelium secretes novel unilamellar membranes having characteristics similar to lung surfactants and thus has been named Surfactant-like particles (SLP). The chemical analysis of the membranes revealed cholesterol/phospholipid molar ratio of 0.68-0.78, which is much distinct from that of the underlying microvillus membranes (1.34-1.49). The membrane contains 4-6 proteins with a molar weight of 30-120 kDa and is enriched with alkaline phosphatase, contains low amounts of disaccharidases but no Na+, K(+)-ATPase activity. The secretion of SLP is stimulated by fat feeding. Chronic ethanol ingestion also induces the formation of SLP in rat intestine. A number of physiological functions have been attributed to SLP, which include: (i) as a protective lubricant in intestinal lumen, (ii) a role in triacylglycerol transport, (iii) as a vehicle for the transport of luminal proteins into blood, (iv) as a stratum for the adhesion of microorganisms in intestinal lumen, and (v) a role in trans-signalling mechanism across the basolateral surface of enterocytes.

Understanding the mechanisms of lung mechanical stress

Physical forces affect both the function and phenotype of cells in the lung. Bronchial, alveolar, and other parenchymal cells, as well as fibroblasts and macrophages, are normally subjected to a variety of passive and active mechanical forces associated with lung inflation and vascular perfusion as a result of the dynamic nature of lung function. These forces include changes in stress (force per unit area) or strain (any forced change in length in relation to the initial length) and shear stress (the stress component parallel to a given surface). The responses of cells to mechanical forces are the result of the cell's ability to sense and transduce these stimuli into intracellular signaling pathways able to communicate the information to its interior. This review will focus on the modulation of intracellular pathways by lung mechanical forces and the intercellular signaling. A better understanding of the mechanisms by which lung cells transduce physical forces into biochemical and biological signals is of key importance for identifying targets for the treatment and prevention of physical force-related disorders.

Fast kinetics of calcium dissociation from calsequestrin

We measured the kinetics of calcium dissociation from calsequestrin in solution or forming part of isolated junctional sarcoplasmic reticulum membranes by mixing calsequestrin equilibrated with calcium with calcium-free solutions in a stopped-flow system. In parallel, we measured the kinetics of the intrinsic fluorescence changes that take place following calcium dissociation from calsequestrin. We found that at 25°C calcium dissociation was 10-fold faster for calsequestrin attached to junctional membranes (k = 109 s-1) than in solution. These results imply that calcium dissociation from calsequestrin in vivo is not rate limiting during excitation-contraction coupling. In addition, we found that the intrinsic fluorescence decrease for calsequestrin in solution or forming part of junctional membranes was significantly slower than the rates of calcium dissociation. The kinetics of intrinsic fluorescence changes had two components for calsequestrin associated to junctional membranes and only one for calsequestrin in solution; the faster component was 8-fold faster (k = 54.1 s-1) than the slower component (k = 6.9 s-1), which had the same k value as for calsequestrin in solution. These combined results suggest that the presence of calsequestrin at high concentrations in a restricted space, such as when bound to the junctional membrane, accelerates calcium dissociation and the resulting structural changes, presumably as a result of cooperative molecular interactions.

Interactions between secreted GRA proteins and host cell proteins across the paratitophorous vacuolar membrane in the parasitism of Toxoplasma gondii

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the X-alpha-gal positive and PCR, 157 colonies of the X-beta-gal assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.

An overview of the effects of annexin 1 on cells involved in the inflammatory process

The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.

Lysosomal stability in Oreochromis mossambicus (Peters) on exposure to surfactants.

The three commonly used surfactants viz. anionic sodium dodecyl sulfate (SDS), cationic cetyl tri methyl ammonium bromide (CTAB) and non-ionic triton X-100 were toxic even at sub lethal levels (1 ppm for 30 days) to 0. mossambicus. Lysosomal stability index (LSI) was lowest in triton-exposed animals in vitro. In vivo, CTAB was the most toxic. SDS, the anionic surfactant was the least toxic. The possible role of surfactant structure, critical micellar concentration (CMC) and metabolism in influencing the toxicity is discussed and mechanism of action via membrane lipid peroxidation is suggested.

Effects of total flavone of Abelmoschl Manihot L. Medic on the function of platelets and its mechanism / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effects of total flavone of Abelmoschl Manihot L. Medic (TFA) on the function of platelets and to explore its mechanism.</p><p><b>METHODS</b>Rat models of artery-veins bypassing thrombus formation were used. The platelets of rabbits were collected. Platelet aggregation was induced by collagen and intracellular calcium ion concentration ([Ca(2+)]i) was assayed by Fura-2 method.</p><p><b>RESULTS</b>TFA (25, 50, 100 mg/kg) significantly and dose-dependently reduced the weight of thrombus. TFA (0.025, 0.05, 0.1 mg/ml) possessed dose-dependant inhibitory effects on rabbits' platelet aggregation induced by collagen. TFA significantly reduced the resting and CaCl(2)-induced increase of free intracellular calcium concentration ([Ca(2+)]i) in rabbit platelet in vitro.</p><p><b>CONCLUSION</b>TFA has an antiplatelet effect via the inhibition on the influx of Ca(2+).</p>

Contribution to the study on process of lipid peroxydation in cellular membran and modifications of some anti-oxydant enzymes during the storage process of erythrocytes

Study on the peroxidation of membrane lipid and the change of some anti-oxidant enzymes during the storage of erythrocytes by additive system. The invitro study was conducted with two additive systems: one is the AS-T which is the first preparation in VN and the other is the preparation of Terumo, a leading company of the world on the blood preparations. The results demonstrate that the two systems are equally in the anti-peroxidation activation of membrance lipid. The ability to maintain enzyme activation of Superoxyd dismutase (SOD) and Gluathion peroxidase (GCH-Px) of erythrocytes during of storage process (from ) to 35 days), the erythrocyte mass which is preserved by AST solution abilize activity of SOD and GSH-Px better than by Terumo solution, with this result os signficant slower increasing of MDA concentration at the end of storage

Immunomagnetic removal of cryo-damaged human spermatozoa / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To estimate the dissipation of mitochondrial transmembrane potential (mTMP, DeltaPsim) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding.</p><p><b>METHODS</b>The mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB).</p><p><b>RESULTS</b>The cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1 % +/- 7.2 % to 53.5 % +/- 13.1 % and increased the spermatozoa with activated pan-caspases (aCP) from 21.8 % +/- 2.6 % to 47.7 % +/- 5.8 % (n=10; mean +/- SEM; P <0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n=22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 +/- 3.9 (P <0.01) and also those with aCP to 9.3 % +/- 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P <0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions.</p><p><b>CONCLUSION</b>The cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.</p>

Calmodulin and calcium-release channels

Calmodulin (CaM) is a ubiquitous cytosolic protein that plays a critical role in regulating cellular functions by altering the activity of a large number of ion channels. There are many examples for CaM directly mediating the feedback effects of Ca2+ on Ca2+ channels. Recently the molecular mechanisms by which CaM interacts with voltage-gated Ca2+ channels, Ca2+-activated K+ channels and ryanodine receptors have been clarified. CaM plays an important role in regulating these ion channels through lobe-specific Ca2+ detection. CaM seems to behave as a channel subunit. It binds at low [Ca2+] and undergoes conformational changes upon binding of Ca2+, leading to an interaction with another part of the channel to regulate its gating. Here we focus on the mechanism by which CaM regulates the inositol 1,4,5-trisphosphate receptor (IP3R). Although the IP3R is inhibited by CaM and by other CaM-like proteins in the presence of Ca2+, we conclude that CaM does not act as the Ca2+ sensor for IP3R function. Furthermore we discuss a novel Ca2+-induced Ca2+-release mechanism found in A7r5 (embryonic rat aorta) and 16HBE14o- (human bronchial mucosa) cells for which CaM acts as a Ca2+ sensor.

Signal transduction and gene expression regulated by calcium release from internal stores in excitable cells

Calcium regulation of several transcription factors involves different calcium-dependent signaling cascades and engages cytoplasmic as well as nuclear calcium signals. The study of the specific sources of calcium signals involved in regulation of gene expression in skeletal muscle has been addressed only recently. In this tissue, most cytoplasmic and nuclear calcium signals originate from calcium release from internal stores, mediated either by ryanodine receptor (RyR) or IP3 receptor (IP3R) channels. The latter are located both in the sarcoplasmic reticulum (SR) and in the nuclear membrane, and their activation results in long-lasting nuclear calcium increase. The calcium signals mediated by RyR and IP3R are very different in kinetics, amplitude and subcellular localization; an open question is whether these differences are differentially sensed by transcription factors. In neurons, it is well established that calcium entry through L-type calcium channels and NMDA receptors plays a role in the regulation of gene expression. Increasing evidence, however, points to a role for calcium release from intracellular stores in this process. In this article, we discuss how RyR-mediated calcium release contributes to the activation of the calcium-dependent transcription factor CREB and the subsequent LTP generation. We present novel results from our laboratory showing ERK-mediated CREB activation by hydrogen peroxide. This activation takes place in the absence of extracellular calcium and is blocked by inhibitory ryanodine concentrations, suggesting it is caused by redox activation of RyR-mediated calcium release.

Effects of nitric oxide on mitochondrial permeability transition and cytochrome C of human hepatocellular carcinoma cell lines / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effects of nitric oxide on mitochondrial permeability and cytochrome C (cyt C) of human hepatocellular carcinoma cell lines.</p><p><b>METHODS</b>NO-mediated apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 was investigated by flow cytometry. The growth and proliferation of human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 were evaluted by MTT assay. Mitochondrial transmembrane potential was analyzed by flow cytometry with double staining of Rh123 and PI, and cytoplasmid cyt C was measured by Western blot. The cells were preincubate with cyclosporin A or GSH synthesis blocker BSO to explore their effect on the results of the above experiments.</p><p><b>RESULTS</b>NO donor sodium nitroprusside (SNP) induced apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 and resulted in the decrease of the mitochondrial transmembrane potential and the increase of the amount of cytoplasmid cyt C in time-dependent manner. Cyclosporin A (CsA) specific inhibitor of the mitochondrial permeability transition pore could partially prevent the decrease of delta psi m and the release of cyt C. In contrast, GSH synthesis blocker BSO promoted the decrease of delta psi m and the release of cyt C.</p><p><b>CONCLUSIONS</b>NO may induce apoptosis in human hepatocellular carcinoma cell lines SMMC-7721 and HepG2 by decreasing delta psi m, opening the mitochondrial permeability transition pore, and releasing the cyt C.</p>