Mesenteric lymph reperfusion exacerbates spleen injury caused by superior mesenteric artery occlusion shock

The intestinal lymph pathway plays an important role in the pathogenesis of organ injury following superior mesenteric artery occlusion (SMAO) shock. We hypothesized that mesenteric lymph reperfusion (MLR) is a major cause of spleen injury after SMAO shock. To test this hypothesis, SMAO shock was induced in Wistar rats by clamping the superior mesenteric artery (SMA) for 1 h, followed by reperfusion for 2 h. Similarly, MLR was performed by clamping the mesenteric lymph duct (MLD) for 1 h, followed by reperfusion for 2 h. In the MLR+SMAO group rats, both the SMA and MLD were clamped and then released for reperfusion for 2 h. SMAO shock alone elicited: 1) splenic structure injury, 2) increased levels of malondialdehyde, nitric oxide (NO), intercellular adhesion molecule-1, endotoxin, lipopolysaccharide receptor (CD14), lipopolysaccharide-binding protein, and tumor necrosis factor-α, 3) enhanced activities of NO synthase and myeloperoxidase, and 4) decreased activities of superoxide dismutase and ATPase. MLR following SMAO shock further aggravated these deleterious effects. We conclude that MLR exacerbates spleen injury caused by SMAO shock, which itself is associated with oxidative stress, excessive release of NO, recruitment of polymorphonuclear neutrophils, endotoxin translocation, and enhanced inflammatory responses.

Apoptose, proliferação e histomorfometria do baço de ratas adultas com hipofunção tireoidiana e ovariana / Apoptosis, proliferation and spleen histomorphometry of adult female rats with thyroid and ovarian hypofunction

Apoptose, proliferação e histomorfometria do baço foram investigados em ratas Wistar adultas ovariectomizadas e não-ovariectomizadas, mantidas em hipotireoidismo induzido pela administração diária de propiltiouracil (PTU) por 120 dias. Dois grupos eutireóideos ovariectomizados e não-ovariectomizados serviram como controle. Foi colhido o plasma para dosagem de T4 livre e o baço para análise da histomorfometria, do índice apoptótico e da expressão imunohistoquímica de caspase 3 e CDC47. Valores de T4 livre foram menores nas ratas tratadas com PTU (p < 0,05). Nos grupos hipotireóideos houve redução do peso do baço, do número e do tamanho dos folículos linfóides e aumento do índice apoptótico e da expressão de caspase 3 (p < 0,05). Porém, o baço de ratas hipotireóideas ovariectomizadas apresentou aumento menos acentuado do índice apoptótico e da expressão de caspase 3 do que o baço de ratas hipotireóideas não-ovariectomizadas (p < 0,05). O grupo eutireóideo ovariectomizado apresentou hiperplasia da polpa branca em relação ao grupo eutireóideo não-ovariectomizado. Não houve diferença na expressão de CDC47 entre os grupos. Conclui-se que a hipofunção tireoidiana e gonadal apresentam efeitos distintos no baço e que na associação hipotireoidismo-hipogonadismo há aumento menos acentuado do índice apoptótico e da expressão de caspase-3 esplênica do que no hipotireoidismo isolado.

Apoptosis, proliferation and histomorphometry of spleen were investigated in ovariectomized and non-ovariectomized adult Wistar rats maintained in hypothyroidism induced by daily administration of propylthiouracil (PTU) during 120 days. Two groups ovariectomized euthyroid and non-ovariectomized euthyroid were used as controls. Plasma was collected for free T4 dosage and the spleen for histomorphometry analysis, apoptosis index and the immunohistochemistry expression of caspase 3 and CDC47. Values of free T4 were lower in rats treated with PTU (p<0.05). In the hypothyroid groups there was some decrease in the spleen weight as well as the number and size of lymphoid follicles and there was some increase in the apoptotic index and the caspase 3 expression (p<0.05). However, the increase in the apoptosis index and the expression of caspase 3 in ovariectomized hypothyroid rats spleen was less accentuated than non-ovariectomized hypothyroid ones (p<0.05). The ovariectomized euthyroid group presented white pulp hyperplasia in comparison to the non-ovariectomized euthyroid group. There was no difference in the CDC47 expression between groups. It was concluded that the thyroid and ovarian hypofunction have distinct effects on the spleen and that in the hypothyroidism-hypogonadism association, the increase in the apoptosis index and in the expression of splenic caspase 3 is not as much as in isolated hypothyroidism.

La segunda bomba de sodio: de la proteína al gen / The second sodium pump: from protein to gene

En la membrana laterobasal del epitelio del intestino delgado y del túbulo renal proximal han sido descrito dos mecanismos diferentes de transporte activo primario de Na+: (1) uno dependiente de K+, sensible a la ouabaina e insensible a la furosemida, correspondiente a la bomba de Na+/K+; y (2) otro independiente de K+, insensible a la ouabaina pero inhibida por furosemida, el cual es referido como la segunda bomba de sodio. De igual modo, dos actividades ATPásicas, dependientes de Mg2+, estimuladas por Na+ e inhibidas por vanadato, han sido descritas en esta membrana: (1) una dependiente de K+, sensible a la ouabaina e insensible a la furosemida, correspondiente a la ATPasa de Na+/K+; y (2) otra independiente de K+, insensible a la iuabaina pero inhibida por furosemida, la cual ha sido denominada como la ATPasa de Na+. Dadas las similitudes bioquímicas, se considera que la bomba de Na+/K+ y la segunda bomba de sodio están asociadas con las ATPasas de Na+/K+ y de Na+, respectivamente, como una entidad bioquímica única. No obstante, no había sido posible la separación óptima de ambas enzimas. Recientemente, se logró solubilizar ambas ATPasas utilizando un detergente no-iónico (C12E9), preservando sus actividades, y purificar la ATPasa de Na+ por selección negativa a través de una cromatografía de afinidad con Concanavalina-A-sefarosa. La ATPasa de Na+ esta constituida por dos subunidades: una subunidad alfa de 98 KDa y una subunidad beta de 50 KDa. La subunidad alfa fue parcialmente secuenciada por espectrometría de masa en serie, identificándose tres péptidos que están presentes en la subunidad alfa1 de la ATPasa de Na+/K+. La formación de intermediarios fosforilados durante el ciclo de reacción de la ATPasa de Na+, así como su dependencia de Mg2+ y sensibilidad a vanadato, identifican a esta enzima como integrante de las ATPasas tipo P.

Basolateral plasma membranes of small intestine and proximal renal tubule present two active Na+-transportmechanisms: (1) The Na+/K+-pump, which depends on K+, is inhibited by ouabain but insensitive to furosemide and (2) The Second sodium pump, which is K+-independent, insensitive to ouabain but inhibited by furosemide. Thse two transport mechanisms have been associated with two different Mg2+-dependent Na+-ATPases, inhibited by vanadate: (1) The K+-dependent Na+/K+-ATPase, sensitive to ouabain and insensitive to furosemide, and (2) The K+ independent, Na+-ATPase, which is inhibitable by furosemide and insensitiveto ouabain. There exist multiple biochemical and functional evidences indicating that these two ATPases are different but only recently it has been possible to identify the Na+-ATPase as a unique biochemical entity. Both ATPases can be solubilized in an active form using C12E9 as detergent and separated by exclusion chromatography in sepharose 6-B and affinity chromatography in concanavalinA-sepharose. The Na+-ATPase is constituted by two sub-units: an alpha subunit of 98 KDa and a beta subunit of 50 KDa. The alpha subunit was partially sequenced by Tandem Mass Spectrometry, evidenced three peptides that are also present in the alpha1 subunit of the Na+/K+-ATPase. Na+-ATPase is Mg2+-dependent, inhibited by vanadate and forms phosphorylated intermediate during its reaction cycle ATP, indicating that it si a P-type ATPase. These facts induced us to design degenerated primers against the most preserves motifs present in these ATPases and to intent the cloning of the Na+-ATPase. Thus, we identified a cDNA for a new P-type ATPase probably related with this enzyme.

Alterations in activities of acid phosphatase, alkaline phosphatase, ATPase and ATP content in response to seasonally varying Pi status in okra (Abelmoschus esculentus).

Phosphorus (P) is the second most important macronutrient for plant growth. Plants exhibit numerous physiological and metabolic adaptations in response to seasonal variations in phosphorus content. Activities of acid and alkaline phosphatases, ATPase and ATP content were studied in summer, rainy and winter seasons at two different developmental stages (28 and 58 days after sowing) in Okra. Activities of both acid and alkaline phosphatases increased manifold in winter to cope up with low phosphorus content. ATP content and ATPase activity were high in summer signifying an active metabolic period. Phosphorus deficiency is characterized by low ATP content and ATPase activity (which are in turn partly responsible for a drastic reduction in growth and yield) and enhanced activities of acid and alkaline phosphatases which increase the availability of P in P-deficient seasons.

Regenerative ability of gastrocnemius muscle under diabetic condition with special reference to SDH & m-ATPase.

The present study analyzes the regeneration of skeletal muscle in streptozotocin (STZ) induced diabetic rats. Two weeks later the gastrocnemius muscle from diabetic rats were transplanted into diabetic and normal host to initiate regeneration and the normal gastrocnemius muscle was transplanted in normal and diabetic hosts for comparison. The regenerates were analyzed after 15 and 30 days of transplantation for histochemical (with respect to SDH and m-ATPase) and 7, 14, 21 and 28 days after transplantation for biochemical studies (with respect to SDH and m-ATPase). Least enzymatic activity and the poorest regenerative ability in case of normal muscle in diabetic host (NM-DbH) and comparatively higher enzymatic activity and better regenerative ability in diabetic muscle in normal host (DbM-NH) was observed. The result of this study strongly supports that the normal host environment is crucial for the muscle recovery.

Effect of thyroidectomy on fast and slow muscle fibres of rat gastrocnemius muscle.

A combined histochemical, biochemical and electrophoretic study with respect to the enzymes succnic dehydrogenase(SDH), myofibrillar adenosine triphosphatase (m-ATPase), lactate dehydrogenase (LDH) isozymes and myosin light chains was carried out to investigate the response of rat gastrocnemius muscle (medial head). Twelve weeks after thyroidectomy, the results indicated a shift from fast to slow type pattern of LDH isozymes, fibre type transformation from Type II to Type I and a decrease in SDH and m-ATPase activity. The results suggest, possible thyroidal involvement in determining the phenotypic properties of skeletal muscle.

Isolation and characterization of the basal cell membranes of the human term placenta.

A relatively simple and rapid method is described for the isolation of basal cell membranes (BCM) from the human placenta at term which showed considerable improvement in the yield, purity and membrane characteristics as compared to the earlier described methods. The method is based on thorough washings of the syncytium in balanced salt solution, selective grinding, hypotonic lysis, sonication, incubation with EDTA and then more conventional differential centrifugation and ultracentrifugation. The isolated material showed smooth surfaced vesicular structure of various sizes as revealed by both positive and negative staining and transmission electron microscopic analysis. The membrane was highly enriched in Na+/K+, Ca2+ and Mg2+ dependent ATPase activities while the cross contamination with brush border surfaces was low as revealed by the marker enzyme assays specific for the brush border membrane (BBM) such as the disaccharide hydrolases, aminopeptidase and alkaline phosphatase. The membrane showed a relatively low lipid/protein ratio and the lipid composition represented by a variety of phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol and phosphatidyl serine), neutral lipids (cholesterol, triacyl glycerol, free fatty acids) and glycosphingolipids (ganglioside, cerebroside and sulfatide). It also contained plasmalogens. On SDS-PAGE analysis and Coomassie blue staining reaction, the isolated membrane showed 14 major bands with as many minor ones with a molecular weight ranging between 30-110 kDa.

Effect of diabetes mellitus on epididymal enzymes of adult rats.

Diabetes mellitus caused significant reduction in serum testosterone and accessory sex glands weight. The sperm content of epididymal regions also decreased. Among the epididymal regions, the cauda epididymidal tissue alone showed significant reduction in Na(+)-K+ ATPase activity. However, Mg2+ ATPase activity was lowered in caput epididymidis only. Specific activity of Ca2+ ATPase significantly decreased in caput and cauda epididymides. All three ATPases decreased significantly in caput epididymidal spermatozoa leaving cauda epididymidal spermatozoa unaffected. Specific activity of alkaline phosphatase was suppressed in caput epididymidis and in the spermatozoa collected from caput and cauda epididymides, while the acid phosphatase was unaffected. In general, the results are suggestive of definite influence of diabetes on epididymal phosphatases which is region specific. Diabetes induced decrease in phosphatases may have an impact on secretory and absorptive functions of epididymis and thus on sperm maturation.