Interferon-γ inhibits group B Streptococcus survival within human endothelial cells

Endothelial dysfunction is a major component of the pathophysiology of septicaemic group B Streptococcus (GBS) infections. Although cytokines have been shown to activate human umbilical vein endothelial cells (HUVECs), the capacity of interferon (IFN)-γ to enhance the microbicidal activity of HUVECs against GBS has not been studied. We report that the viability of intracellular bacteria was reduced in HUVECs activated by IFN-γ. Enhanced fusion of lysosomes with bacteria-containing vacuoles was observed by acid phosphatase and the colocalisation of Rab-5, Rab-7 and lysosomal-associated membrane protein-1 with GBS in IFN-γ-activated HUVECs. IFN-γ resulted in an enhancement of the phagosome maturation process in HUVECs, improving the capacity to control the intracellular survival of GBS.

Purification and Partial Characterization of a Non-specific Acid Phosphatase Degrading NAD from Aspergillus oryzae NRRL447.

A non specific acid phosphatase from Aspergillus oryzae NRRL447 catalyzes the phosphate hydrolysis from nicotinamide adenine dinucleotide forming nicotinamide riboside, adenosine and Pi as the final products of the reaction. The enzyme was purified to homogeneity by a sequential treatment of acetone fractionation, DEAE-cellulose chromatography and gel filtration chromatography. The enzyme was purified 400-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 52 kDa. The enzyme displayed maximum activity at pH 5.0 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg2+, Ca2+ whereas inhibited strongly by F-, Mo04 -, Cu2+ and Fe2+. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km for NAD+ was 6.25 x 10-4 M.

Regulation of matrix metalloproteinase-9 protein expression by 1alpha,25-(OH)2D3 during osteoclast differentiation

To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.

Ultrastructural changes and oxidative stress markers in wild and cultivar Sesamum orientale L. following Alternaria sesami (Kawamura) Mohanty and Behera inoculation.

Alternaria sesami causes leaf spot disease in Sesamum orientale. Conidium germination, inoculation, penetration and colonization of the pathogen on the plant surfaces were studied using scanning electron microscopy. Electron microscopy analysis revealed multiple germ tubes from conidium that spread in all direction across the leaf surfaces. Penetration in the plant surface occured, directly through the epidermis or via stomata with or without the appressoria formation. Hyphal penetration continued through the substomata cavity and some of hyphal branches grew in the intercellular space of mesophyll tissue. Hyphal toxin, caused cell and cell wall damages. Changes in different biochemical parameters in the diseased sesame plants (both in wild and cultivar) were compared to control. Transmission electron microscopy showed structural changes in the chloroplast of diseased plants. Isozyme pattern and assays of different enzymes, namely catalase, acid phosphatase and peroxidase expressed varied level of activities. Meanwhile, esterase, polyphenol oxidase and superoxide dismutase in diseased plants showed remarkable levels compared to control. Due to the infection, chlorophyll content, carbohydrates and total soluble protein decreased whereas free amino acid, proline, phenols and disease-related proteins increased in the host plants. Differential SDS-PAGE band profiling of total soluble proteins were also observed in plants due to the infection.

A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

Participación de MT1-MMP en la remodelación del ligamento periodontal durante la movilización dentaria / Role of MT1-MMP in the remodeling of the periodontal ligament during tooth movement

La movilización dentaria involucra una serie de cambios en los tejidos de soporte caracterizados por la activa remodelación de estos. La MT1-MMP o MMP-14 es una potente enzima proteolítica capaz de degradar colágeno tipo I, la principal molécula estructural del ligamento periodontal. La migración dentaria requiere de la degradación controlada del colágeno constituyente del ligamento periodontal. Sin embargo, no existen evidencias de la participación de MT1-MMP en la remodelación del tejido periodontal durante este proceso. En el presente estudio hemos analizado la expresión de MT1 -MMP y del marcador de actividad osteoclástica Fosfatasa Acida Tartrato Resistente (TRAP) en un modelo de migración dentaria en ratas. La migración dentaria fue activada mediante la inserción de una banda separadora entre los incisivos superiores. La expresión y distribución de TRAP y MT1-MMP fue evaluada a través de citoquímica e inmunohistoquímica a los días 1, 3, 5 y 7. La producción de TRAP fue identificada principalmente en osteoclastos ubicados en la zona de compresión del ligamento periodontal. La producción de MT1-MMP fue observada en fibroblastos de la zona de compresión del ligamento periodontal y osteoclastos ubicados en esta misma región. Nuestros resultados permiten proponer que tanto MT1 -MMP como TRAP participan en la remodelación de los tejidos de soporte periodontal durante la migración dentaria.

Tooth movement involves a series of changes of the supporting periodontal tissues characterized by the active connective tissue remodeling. MT1-MMP or MMP-14 belongs to the family of matrix metalloproteinases that are able to degrade type I collagen, the main molecule involved in periodontal attachment. Tooth migration requires the controlled degradation of periodontal ligament collagen fibers. However, evidences linking MT1 -MMP expression with periodontal tissue remodeling are lacking. In the present study, we have evaluated the expression of MT1-MMPand of the osteoclast marker Tartrate Resistant Acid Phosphatase (TRAP) in a model of tooth migration in rats. Tooth migration was induced after the insertion of a rubber band between the upper incisors. The distribution of TRAP and MT1 -MMP was evaluated by means of cytochemistry and immunohistochemistry respectively at days 1, 3, 5 and 7. TRAP production was identified in osteoclasts at the area of compression of the periodontal ligament. MT1-MMP distribution was observed in fibroblastsatthe compressed area of the periodontal ligament and also in osteoclasts of the same region. Our findings allow us to propose that MT1-MMP and TRAP take part of the tissue remodeling events observed during tooth movement.

Histoenzymological study on the toxicity of copper sulphate in the digestive glands of Lymnaea luteola.

During 24 and 48 hr of exposure, the digestive glands of Lymnaea treated with a lethal concentration of 0.038 mgl(-1) CuSO4 revealed intense activity of acid phosphatase in perilobular margin. On the other hand, same area of the gland showed moderate activity of ATPase during 24 and 48 hr of exposure. However, alkaline phosphatase showed average activity in perialveolar region and perilobular margin during 24 and 48, and 72 hr of exposure respectively The changes in the activity of these enzymes were nonsignificant in alveolar margin and perialveolar region of the gland. It is interesting to note moderate activity of acid phosphatase in perialveolar region during 24 hr of exposure.

Effect of malathion on reproductive system of male rats.

The pesticides are one of the most potentially harmful chemicals liberated in the environment in an unplanned manner Malathion is widely used as a potent pesticide in many countries and has been shown to produce some adverse health effects. A study was conducted to asses the effects of malathion on the male reproductive system of wistar rats. The pesticide was administered to rats orally at dose levels of 50, 150 and 250 mg/kg/body wt/day for 60 days. In comparison to the control rats, there was a significant reduction in the weight of testes, epididymis, seminal vesicle and ventral prostate. Testicular and epididymal sperm density were decreased in the animals treated with malathion. Pre and post fertility test showed 80% negative results after treatment Biochemical profile of the testis revealed a significant decline in the contents of sialic acid and glycogen. Whereas a significant increase in the protein content of testis and testicular cholesterol was observed. The activity of testicular enzyme acid phosphatase increased significantly while decreased alkaline phosphatase activity was found. Malathion also suppressed the level of testosterone significantly Results of the present study clearly suggest that malathion induce toxic effects on the male reproductive system of rats.

Programmed cell death in salivary glands of Drosophila arizonae and Drosophila mulleri

Programmed cell death (PCD) in insect metamorphosis assumes a great diversity of morphology and controlling processes that are still not well understood. With the objective of obtaining information about the PCD process, salivary glands of Drosophila arizonae and D. mulleri were studied during larval-pupal development. From the results, it can be concluded that the type of the PCD that occurs in these organs is morphologically typical of apoptosis (formation of apoptotic nuclei, followed by fragmentation into apoptotic bodies). Histolysis happens in both species, between 22 and 23 h after pupation. There were no significant differences between the species studied. Apoptosis does not occur simultaneously in all cells. Cytoplasmic acid phosphatase activity gradually increases during development, suggesting the existence of acid phosphatases that are only expressed during the apoptotic stage. Twenty hours after pupation, salivary glands already show biochemical alterations relative to nuclear permeability such as acidification, possibly due to the fusion of lysosomes with the nucleus a few hours before apoptosis. Autophagy seems to act together with apoptosis and has a secondary role in cell death.

Biomolecular changes during in vitro organogenesis of Asteracantha longifolia (L.) Nees--a medicinal herb.

High frequency plant regeneration in A. longifolia (L.) was achieved from leaf explant implanted on MS basal medium supplemented with NAA (0.5 mg/l) + BA (2.0 mg/l) through intervening callus phase. Well-developed shoots (>3cm) were successfully rooted on MS medium supplemented with NAA (0.1 mg/l). Protein and total soluble sugar contents were maximum during organogenesis and multiple shoot induction phase compared with non-organogenic callus and root induction phase. Esterase and catalase activities were maximum during organogenic differentiation, while activities were minimum at non-differentiated callus stages. Peroxidase activities were higher during rhizogenesis. Contradiction to peroxidase activity, acid phosphatase activities were high during organogenesis and declined during rhizogenesis. SDS-PAGE analysis of total soluble proteins revealed expression of non-organogenic callus (97.9 kDa), organogenic callus (77.2, 74.1, 21.9 kDa), multiple shoot induction phase (106.6, 26.9, 11.6 kDa) and root induction phase (15.9 kDa) specific polypeptides. Esterase zymogram revealed one band (Rm 0.204) appeared in both organogenic callus and multiple shoot induction phase. Peroxidase zymogram detected two stage specific bands, one band (Rm 0.42) was specific to root induction phase, while another (Rm 0.761) was specific to multiple shoot induction. Catalase and acid phosphatase zymogram resolved one band (Rm 0.752 and 0.435, respectively) in differentiated stages including both multiple shoot induction phase and root induction phase, but absent in undifferentiated phases.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae).

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine,the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy.Tissue sections were subsequently stained with haematoxylin -eosin,bromophenol blue,silver,or a variant of the critical electrolyte concentration (CEC) method.Ultrathin sections were contrasted with uranyl acetate and lead citrate.Glands were processed for the histochemical and cytochemical localization of acid phosphatase,as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed.The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and,additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen.The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed.The nuclei were pyknotic,with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

Acid phosphatase activity distribution in salivary glands of triatomines (Heteroptera, Reduviidae, Triatominae)

Acid phosphatase activity (Gömori technique) in salivary gland cells was investigated in adult insects (males and females) of four species of triatomines: Triatoma infestans, Panstrongylus megistus, Rhodnius neglectus, and Rhodnius prolixus. Binucleated cells with bulky and polyploidy nuclei were detected, with acid phosphatase activity in the heterochromatin and nucleolus, which showed the most intense response. Thus, the activity of these phosphatases during rRNA molecule transcription, possibly in the nucleolar fibrillar center, is suggested. The difference in reactivity found among salivary glands is associated with the cellular metabolism of these regions and, probably, with the biosynthesis of their different secretions. This must be essential in maintaining the hematophagy of triatomines

In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood

Mesenchymal stem cells (MSCs) have the capabilities for self-renewal and differentiation into cells with the phenotypes of bone, cartilage, neurons and fat cells. These features of MSCs have attracted the attention of investigators for using MSCs for cell-based therapies to treat several human diseases. Because bone marrowderived cells, which are a main source of MSCs, are not always acceptable due to a significant drop in their cell number and proliferative/differentiation capacity with age, human umbilical cord blood (UCB) cells are good substitutes for BMCs due to the immaturity of newborn cells. Although the isolation of hematopoietic stem cells from UCB has been well established, the isolation and characterization of MSCs from UCB still need to be established and evaluated. In this study, we isolated and characterized MSCs. UCB-derived mononuclear cells, which gave rise to adherent cells, exhibited either an osteoclast or a mesenchymal-like phenotype. The attached cells with mesenchymal phenotypes displayed fibroblast-like morphologies, and they expressed mesenchymal-related antigens (SH2 and vimentin) and periodic acid Schiff activity. Also, UCB-derived MSCs were able to transdifferentiate into bone and 2 types of neuronal cells, in vitro. Therefore, it is suggested that the MSCs from UCB might be a good alternative to bone marrow cells for transplantation or cell therapy.

Hydrolytic enzyme activity in rhesus monkey placenta during early gestational malaria: histochemical studies.

BACKGROUND & OBJECTIVES: Early gestational malaria is found to be more fatal than late gestational infection but the pathophysiology of early gestational placenta, the maternofoetal organ responsible for maintenance of pregnancy, remains unexplored. Present study dealing with hydrolytic enzymes in early gestational placenta of rhesus monkeys during Plasmodium cynomolgi infection was anticipated to provide a better insight into the functional impairment of this organ during early gestational maternal malaria. METHODS: Experimental monkeys (Macaca multtta) at 2-2 1/2 months of pregnancy were inoculated with P. cynomolgi bastianelli. After attaining first peak of parasitaemia the animals were anesthetised and placentae were collected for histochemical studies. The snap-frozen, cryostat sections were subjected to histochemical reactions for acid phosphatase and alkaline phosphatase. RESULTS: The placental syncytiotrophoblast showed a loss in alkaline phosphatase activity, while the trophoblast layers and phagocytic cells of the maternal blood showed increased acid phosphatase activity during early gestational malarial infection. Morphological damage to the placental tissue whenever occurred was associated with altered Alk pase activity. INTERPRETATION & CONCLUSION: The altered distribution of Ac pase and Alk pase in malaria infected early gestational placenta has been discussed in the light of placental function. It could be concluded by present studies that these malaria induced changes in hydrolytic enzyme activities in monkey placenta have a direct bearing on functional and morphological integrity of the placental tissue. These changes are apparently responsible for early gestational foetal death and abortions as reported in literature.

Extracellular breakdown of collagen by mice decidual cells. A cytochemical and ultrastructural study

The interaction of antimesometrial decidual cells and collagen fibrils was studied by light microscopy and ultrastructural cytochemistry in fed and acutely fasted mice on days 9-11 of pregnancy. Fibrillar elements in the extracellular space consisted of collagen fibrils and filamentous aggregates (disintegrating collagen fibrils). Intracellular vacuoles exhibited typical collagen immersed in electron-translucent material (clear vacuoles) and faint cross-banded collagen immersed in electron-opaque material (dark vacuoles). Fibrillar elements showed extracellular acid phosphatase activity which was stronger in the region of mature decidua than in predecidual cells region in all animals; it was conspicuous in mature decidua of fasted animals. Intracellular acid phosphatase activity was observed in dark vacuoles and lysosomes, and was absent in clear vacuoles in all cells studied. Since acid phosphatase activity reflects the presence of lysosomal hydrolases in general, the results indicate that breakdown of extracellular collagen occurs by release of lysosomal enzymes by decidual cells and also by internalization of collagen for intracellular degradation in fed and fasted mice. Collagen breakdown may be part of the process of tissue remodeling in mature and predecidual regions, however, in mature decidua, collagen breakdown is enhanced and may therefore contribute to nutrition of the fetus, specially in acutely fasted mice.

Cybil induced hepatobiochemical changes in wistar rats.

Cybil, 25%EC formulation of Cypermethrin, induces biochemical changes in the liver of wistar rats after oral intubation of the same at acute (one day) and subacute (7, 14 and 21 days) levels. The changes were tallied with the controls run simultaneously. LD50 of Cybil was estimated to be 622mg/kg body weight The acute dose is 80mg/kg body weight exposed for one day i.e. 24 hours and subacute dose is 4mg/kg body weight exposed for 7, 14 and 21 days. When compared with the control values, both the doses enhanced the level of glycogen, cholesterol, total lipid and acid phosphatase activities, while decreased activity of alkaline phosphatase. Alterations in glycogen, cholesterol, total lipid, acid phosphatase and alkaline phosphatase resulted in the impairement in liver physiology.

Biochemical and histological studies on H2-receptor antagonist ranitidine-induced hepatotoxicity in rats.

This study was designed to investigate the hepatotoxicity of ranitidine treatment in dose levels of 10, 30, and 50 mg/kg b.wt. for 3 weeks period in male rats. The results showed some adverse changes in rats treated with either 10 or 30 mg/kg. Treatment with dose of 50 mg/kg produced marked increase in the activity of both acid phosphatase in liver and aspartate aminotransferase in serum and liver, with a tendency for increase in serum alanine aminotransferase activity. Also, a significant decrease in the serum activity of both amylase and alkaline phosphatase was noted. Microscopic examination of livers of the same animals revealed absence of some hepatic cells, pyknotic nuclei, dilatation of blood sinusoids, binucleated cells, and infiltration of lymphocytes. These biochemical and histological changes indicate that ranitidine when given chronically in high dose could produce hepatotoxicity in rats.

Metabolic dysregulation and inhibition of spermatogenesis by gibberellic acid in rat testicular cells.

Enzymatic and histological change in the testicular cells of rats treated orally and intradermally for 45 days with gibberellic acid (GBA) in independent studies is reported. Assay of hexokinase (HK), acid phosphatase (AcP) and alkaline phosphatase (AkP) in rat testicular tissue homogenate preparations yielded results that suggested changes in these enzyme activities relative to their respective controls. Histological studies showed loss of germ cells, derangement of the germinal cells, and reduction in the size of the seminiferous tubules and dystrophy of Leydig cells. More importantly decreased sperm count in the lumen was observed. A dysregulatory role is thus established for GBA in rat testicular cell function. This compound may serve as an inhibitor of testicular cell function.

Influence of insecticidal derivative (cartap hydrochloride) from the marine polycheate on certain enzyme systems of the fresh water fish Oreochromis mossambicus.

The activities of phosphatases and transaminases were studied in muscle and liver of the fresh water fish, Oreochromis mossambicus on exposure to different sublethal concentrations (0.25, 0.5, 0.75 and 1 mgl(-1)) of cartap hydrochloride (insecticidal derivative from marine polycheate) for 96 h. There was an overall decrease in phosphatases and transaminases activity in muscle and liver of the fish subjected to cartap hydrochloride.

Modulatory potential of Spirulina fusiformis on testicular phosphatases in Swiss albino mice against mercury intoxication.

Administration of mercuric chloride (HgCl2; 5.0 mg/kg body weight) to male Swiss albino-mice resulted in significantly higher levels of testicular acid phosphatase (ACP) and alkaline phosphatase (ALP) activities as compared to control. In combination group where S. fusiformis (800 mg/kg body weight) was given before and after HgCl2 treatment, the mercury induced toxicity reduced in terms of decreased levels of ACP and ALP activities in the testis. The animal treated with only Spirulina did not show any alteration in ACP and ALP values. It is suggested that oral administration of Spirulina can modulate mercury induced testicular toxicity.