Therapeutic effects of Semecarpus anacardium Linn. nut milk extract on the changes associated with collagen and glycosaminoglycan metabolism in adjuvant arthritic Wistar rats

The effect of milk extract of Semecarpus anacardium Linn. nut milk extract (SA) was studied to gain some insight into this intriguing disease with reference to collagen metabolism. Arthritis was induced in rats by injecting Freund's complete adjuvant containing 10mg of heat killed mycobacterium tuberculosis in 1 ml paraffin oil (0.1 ml) into the left hind paw of the rat intradermally. After 14 days of induction, SA (150 mg/kg body weight/day) was administered orally by gastric intubations for 14 days. Decreased levels of collagen and glycosaminoglycans (GAGS) components (chondroitin sulphate, heparan sulphate, hyaluronic acid) and increase in the levels of connective tissue degrading lysosomal glycohydrolases such as acid phosphatase, beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin-D observed in arthritic animals were reverted back to near normal levels upon treatment with SA. The drug effectively regulated the uriniray markers of collagen metabolism namely hexosamine, hexuronic acid, hydroxyproline and total GAGS. Electron microscopic studies also revealed the protective effect of SA. Hence, it can be suggested that SA very effectively regulate the collagen metabolism that derange during arthritic condition.     

Effect of Semecarpus anacardium Linn. nut milk extract on rat neutrophil functions in adjuvant arthritis

Neutrophils play an important role in the pathogenesis of rheumatoid arthritis (RA) and various inflammatory conditions, by accumulation and liberation of active proteolytic enzymes. The effect of milk extract of Semecarpus anacardium Linn. nuts (SA) at a dosage of 150 mg kg(-1) body weight day(-1) for 14 days on adjuvant arthritis was studied to gain some insight into this intriguing disease in relation to neutrophil functions. The decreased phagocytic function of neutrophils (phagocytic index and avidity index) found in adjuvant arthritis was significantly increased by the administration of the drug SA. Increased levels of reactive oxygen species (superoxide radical, hydroxyl radical, H2O2 and myeloperoxidase), lysosomal enzymes (acid phosphatase and cathepsin D) and increased accumulation of neutrophils in the joints observed in adjuvant arthritic animals were reverted back to near normal levels by treatment with SA. The results of this study indicate that SA can be considered to be a good therapeutic agent for inflammation and arthritis.     

Restoration of energy metabolism in leukemic mice treated by a siddha drug--Semecarpus anacardium Linn. nut milk extract

Chronic myeloid leukemia (CML) is a clonal disorder characterized by proliferation of hematopoietic cells that possess the BCR-ABL fusion gene resulting in the production of a 210 kDa chimeric tyrosine kinase protein. CML, when left untreated, progresses to a blast phase during which the disease turns aggressive and shows poor response to known treatment regimens. We have studied a Siddha herbal agent, Semecarpus anacardium Linn. nut milk extract (SA) for its antileukemic activity and its effect on the changes in energy metabolism in leukemic mice. Leukemia was induced in BALB/c mice by tail vein injection of BCR-ABL(+) 12B1 murine leukemia cell line. This resulted in an aggressive leukemia, similar to CML in blast crisis, myeloid subtype, confirmed by histopathological study and RT-PCR for the p210 mRNA in the peripheral blood, spleen and liver. Leukemia-bearing mice showed a significant increase in lipid peroxides, glycolytic enzymes, a decrease in gluconeogenic enzymes and significant decrease in the activities of TCA cycle and respiratory chain enzymes as compared to control animals. SA treatment was compared with standard drug imatinib mesylate. SA administration to leukemic animals resulted in clearance of the leukemic cells from the bone marrow and internal organs on histopathological examination and this was confirmed by RT-PCR for the p210 mRNA. Treatment with SA significantly reversed the changes seen in the levels of the lipid peroxides, the glycolytic enzymes, the gluconeogenic enzymes and the mitochondrial enzymes. These effects are probably due to the flavonoids, polyphenols and other compounds present in SA which result in total regression of leukemia and correction of the alterations in energy metabolism. Study of animals treated with SA alone did not reveal any adverse effects. On the basis of the observed results, SA can be considered as a readily accessible, promising and novel antileukemic chemotherapeutic agent.     

Hypoxia and its downstream targets in DMBA induced mammary carcinoma: protective role of Semecarpus anacardium nut extract

Tumors are usually exposed to a hypoxic microenvironment due to their irregular growth and abnormal vascular supply. Under hypoxia, gene regulation (selective activation and inactivation of genes) plays an important role in maintenance of tumor. Multiple hypoxic and angiogenic growth factors are expressed for tumor cell survival. In search of novel anticancer drug, Semecarpus anacardium nut extract (SA) was tried against breast cancer. Mammary carcinoma was induced in vivo by 7,12-dimethyl benz(a) anthracene (DMBA) (25mg/kg b.w., p.o.). Tumor development and vascular structures were accelerated by DMBA. Hypoxia inducible factor-1 alpha (HIF-1) was coexpressed with its downstream genes in mammary tissue. Cancer rats were then treated with S. anacardium nut extract (SA) (250mg/kg b.w., p.o.). Delay in the tumor growth was paralleled with a drastic reduction in vascularization by SA treatment. Activities of glycolytic enzymes were normalized with decreased expression of glucose transporter-1 and carbonic anhydrase IX by drug treatment. Inhibition of HIF-1, vascular endothelial growth factor and inducible nitric oxide synthase by SA may in part explain its antiangiogenic action. SA also inhibits endothelial cell proliferation by blocking the overexpressed survival cytokines. In conclusion, our study demonstrates that at least some part of the antitumor activity of SA is due to the suppression of hypoxic and angiogenic factors. The mechanism of this inhibition seems to be through an action of SA on expression of HIF-1 and its downstream targets.     

The differential effect of Eriobotrya japonica hydrophilic leaf extract on cytokines production and modulation

Stimulating or modulating the release of cytokines by immunomodulators or immunostimulating agents is an attractive mode for treating several diseases such as viral infections. For instance, patients with viral infections may be in need of increasing or inducing T helper 1 (Th1) or proinflammatory cytokines, which ultimately activate T cytotoxic and Natural killer lymphocytes to kill virally infected cells. Of these agents, we found that Eriobotrya japonica hydrophilic leaf extract (EJHE) can induce and modulate cytokines in dose-dependent manner. Twenty-four hour exposure of increasing concentrations of EJHE increased significantly (p < 0.001) the production of IFN-γ and TNF-α, from PHA+LPS-stimulated whole blood. However, the production of IFN-γ and TNF-α plateaued at high EJHE concentrations (10–100 μg/ml). No significant changes in the production of IL-10 were seen. In addition, EJHE at 1 and 10 μg/ml reversed significantly (p < 0.01) the inhibitory effect of hydrocortisone on the IL-12 p70, IFN-γ and TNF-α production from PHAS+LPS stimulated whole blood. Without PHA and LPS, EJHE was found to induce significantly (p < 0.001) IFN-γ, IL-12 p70, TNF-α, and IL-10 from whole blood culture in concentration dependent manner. The maximum induction of IFN-γ, IL-12 p70, and TNF-α by EJHE was at 1 and 10 μg/ml. On the other hand, IL-10 induction kept increasing even at the highest concentration used (100 μg/ml) of EJHE. Furthermore, intra-peritoneal injection of EJHE into mice increased significantly serum cytokines level mainly at 10 and 100 μg/ml. Two-hour post i.p. injection, EJHE increased serum IFN-γ, TNF-α, and IL-10 to 750, 1000, and 250 pg/ml, respectively. However, 24 h post i.p. injection, the levels of TNF-α, and IL-10 were similar to basal levels but IFN-γ levels were 200 pg/ml. These results indicate that EJHE induces proinflammatory and Th1 cytokines in concentration dependent manner and the effect of this induction should be studied further in viral models to check the efficacy of such cytokine induction.     

Levels of pro-inflammatory cytokines produced from cord blood in-vitro are pathogen dependent and increased in comparison to adult controls

BACKGROUND: Overproduction of pro-inflammatory cytokines may play a role in increased morbidity and mortality from neonatal sepsis. Objective of this study was to compare secretion of pro-inflammatory cytokines by the cord blood cells of healthy term neonates to the venous blood cells of healthy adults in vitro after stimulation with common neonatal pathogens. METHOD: Blood samples were cultured in the presence of heat-killed group B beta-hemolytic streptococci (GBS), Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epi). Concentrations of secreted cytokines (interleukine-6, IL-6, tumor necrosis factor-alpha, TNF-alpha, interleukine-1 beta, IL-1beta and interleukine-8, IL-8) were measured after 0, 1, 2 and 4 h of incubation using chemiluminescent immunometric automated assay. RESULTS: Blood samples from 22 neonates and 16 adults were compared. After stimulation by GBS and E. coli, cord blood cells secreted significantly higher levels of IL-6 and IL-8 than blood cells of healthy adults. In cord blood, E. coli induced secretion of higher concentration of IL-6, TNF-alpha, IL-1beta and IL-8 than S. epi, and more IL-6 than GBS; GBS induced more IL-1beta than S.epi. CONCLUSIONS: Response of cord blood to microbial activators is different from that of adult controls. Each isolate of heat-killed bacteria induced different amount of pro-inflammatory cytokines in vitro. This may represent a useful in vitro virulence test.     

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-α and its effect on the respiratory burst activity of phagocytes

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-α (rbTNF-α) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-α on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-α was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-α gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-α, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-α was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-α might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.     

Upregulation of tumor necrosis factor-alpha expression by trans10-cis12 conjugated linoleic acid enhances phagocytosis of RAW macrophages via a peroxisome proliferator-activated receptor gamma-dependent pathway

The aim of this study was to examine whether tumor necrosis factor (TNF)-alpha expression in the phagocytic activity of RAW macrophages by trans10-cis12 (10t-12c) conjugated linoleic acid (CLA) is associated with peroxisome proliferator-activated receptor gamma (PPARgamma) activation. 10t-12c CLA induced the TNF-alpha expression in RAW macrophages. Phagocytic activity of naive RAW macrophages was increased either by recombinant mouse (rm) TNF-alpha or by culture supernatant from 10t-12c CLA-treated RAW macrophages. This phagocytic activity was inhibited by addition of anti-rmTNF-alpha polyclonal antibody (pAb). 10t-12c CLA also increased the level of PPARgamma protein and mRNA in RAW macrophages. When naive RAW macrophages were incubated with the culture supernatant from RAW macrophages treated with 10t-12c CLA plus GW 9662, a PPARgamma antagonist, their phagocytic activity was significantly inhibited. In addition, GW 9662 antagonized the effect of 10t-12c CLA in stimulating TNF-alpha expression. These results suggest that 10t-12c CLA modulates the phagocytic activity of RAW macrophages by upregulating TNF-alpha expression via a PPARgamma-dependent pathway.     

Transformation of mouse mammary epithelial cells with the Ha-ras but not with the neu oncogene results in a gene dosage-dependent increase in transforming growth factor-α production

An enhanced expression of transforming growth factor-α (TGFα) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha-ras oncogene. The amount of TGFα production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21^ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGFα secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGFα production or change in EGF receptor expression.     

Immunomodulatory activity of Sulforaphane, a naturally occurring isothiocyanate from broccoli (Brassica oleracea)

The effect of Sulforaphane on the immune system was studied using BALB/c mice. Intraperitoneal administration of five doses of Sulforaphane (500 microg/dose/animal/day) was found to enhance the total WBC count (12,950 cells/mm3) on 9th day. Bone marrow cellularity (23 x 10(6) cells/femur) and number of alpha-esterase positive cells (1346.66/4000 cells) were also increased by the administration of Sulforaphane. Treatment with Sulforaphane along with the antigen, sheep red blood cells (SRBC), produced an enhancement in the circulating antibody titre and the number of plaque forming cells (PFC) in the spleen. Maximum number of PFC (315.83 PFC/10(6) spleen cells) was obtained on the 6th day. Administration of Sulforaphane also showed an enhancement in the phagocytic activity of peritoneal macrophages. Moreover administration of Sulforaphane significantly reduced the elevated level of TNF-alpha production by LPS stimulated macrophages. These results indicate the immunomodulatory activity of Sulforaphane.     

Free radical scavenging activity of Cleome gynandra L. leaves on adjuvant induced arthritis in rats

The generation of free radicals has been implicated in the causation of several diseases of known and unknown etiologies such as, rheumatoid arthritis, diabetes, cancer, etc., and compounds that can scavenge free radicals have great potential in ameliorating these disease processes. The present study was aimed to investigate the possible anti-oxidant potential of Cleome gynandra leaf extract at a dose of 150 mg/kg body weight for 30 days on adjuvant induced arthritis in experimental rats. Oral administration of C. gynandra leaf extract significantly increased the levels of lipid peroxides and activities of catalase, glutathione peroxidase and decreased the levels of reduced glutathione and superoxide dismutase activity in arthritis induced rats. The free radical scavenging activity of the plant was further evidenced by histological observations made on the limb tissue. The presence of biologically active ingredients and vital trace elements in the leaves readily account for free radical scavenging property of C. gynandra. (Mol Cell Biochem 276: 71–80, 2005)     

Expression of Stra13 during mouse endochondral bone development

We have examined the expression of the basic helix-loop-helix factor Stra13 (DEC1/Sharp2) during endochondral bone development in the mouse. Stra13 expression was examined by in situ hybridization in the tibia from E14.5-E18.5, and at post-natal day 24. At E14.5, expression of Stra13 mRNA was very low, with expression limited to scattered hypertrophic chondrocytes. At E15.5 Stra13 mRNA was present in post-mitotic hypertrophic chondrocytes, co-localizing with collagen X expression. At E16.5-E18.5, Stra13 was expressed in both the proliferating chondrocytes and in the late hypertrophic chondrocytes. At E15.5-E18.5, Stra13 expression was also observed in the primary spongiosa. Stra13 expression was also maintained in the 24-day post-natal tibia, with expression detectable only in the late hypertrophic chondrocytes. Because Stra13 has been shown to be induced by hypoxia, and the growth plate is hypoxic during embryonic development, we compared the expression pattern of Stra13 and the HIF1-alpha target gene VEGF. VEGF is expressed predominantly in the late hypertrophic chondrocytes, with lower expression in the proliferating chondrocytes. Thus, there was a large degree of overlap in the expression patterns of Stra13 and VEGF in chondrocytes during embryonic development.     

Molecular variation of Spiranthes sinensis (Orchidaceae) in Japan, with special reference to systematic treatment of seasonally differentiated groups and a dwarf form, f. gracilis, from Yakushima Island

Molecular variations of Spiranthes sinensis Ames var. australis (R.Br.) H. Hara et Kitam. ex Kitam. in Japan were examined to evaluate the validity of the seasonally differentiated groups and a dwarf form of the species, which is endemic to Yakushima Island, Japan. Sequence differences in the plastid trnL-F locus clearly distinguished Japanese S. sinensis var. australis from S. sinensis var. sinensis collected from Ryukyu. In contrast, the trnL-F sequence of S. sinensis var. australis from Sabah, Malaysia, clearly differed from that of Japanese S. sinensis var. australis, suggesting genetic heterogeneity of Spiranthes sinensis var. australis in Asia. Moreover, a molecular analysis based on the sequences of nuclear ITS1 regions indicated that there are two major groups of S. sinensis var. australis in Japan, with a geographic distribution boundary on Kyushu Island. However, the trnL-F and ITS1 sequences did not support the genetic differentiation of the seasonally differentiated groups or the dwarf form from the other Japanese individuals. Based on these molecular data, the systematic treatment of physiological and morphological variations in the Japanese population of S. sinensis. var. australis is discussed.     

Molecular characterization of the alpha-glucosidase activity in Enterobacter sakazakii reveals the presence of a putative gene cluster for palatinose metabolism

Enterobacter sakazakii is considered an opportunistic pathogen for premature infants and neonates. Although E. sakazakii has been isolated from various types of food, recontaminated dried infant formula has been epidemiologically identified as the major source of infection. Amongst others, alpha-glucosidase activity is one of the most important biochemical features, which differentiates E. sakazakii from other species in the family Enterobacteriaceae and has therefore been used as a selective marker in the development of differential media. However, it has been shown, that methods based on this biochemical feature are prone to producing false-positive results for presumptive E. sakazakii colonies due to the presence of this enzymatic activity in other species of the Enterobacteriaceae. Therefore, elucidation of the molecular basis responsible for the biochemical feature in E. sakazakii would provide novel targets suitable for the development of more specific and direct identification systems for this organism. By applying the bacterial artificial chromosome (BAC) approach, along with heterologous gene expression in Escherichia coli, the molecular basis of the alpha-glucosidase activity in E. sakazakii was characterized. Here we report the identification of two different alpha-glucosidase encoding genes. Homology searches of the deduced amino acid sequences revealed that the proteins belong to a cluster of gene products putatively responsible for the metabolism of isomaltulose (palatinose; 6-O-alpha-d-glucopyranosyl-d-fructose). The glycosyl-hydrolyzing activity of each protein was demonstrated by subcloning the respective open reading frames and screening of E. coli transformants for their ability to hydrolyze 4-methyl-umbelliferyl-alpha-d-glucoside. Analysis at the protein level revealed that both enzymes belong to the intracellular fraction of cell proteins. The presence of the postulated palatinose metabolism was proven by growth experiments using this sugar as a sole carbon source.     

Production of IL-6, in contrast to other cytokines and chemokines, in macrophage innate immune responses: effect of serum and fungal (Blastomyces) challenge

Murine peritoneal macrophages, after adherence and establishment in culture in vitro in the presence of medium containing fetal bovine serum (FBS) for 20 h, then cultured for 20 h, produced several cytokines. If, in the second 20 h period, a fungus (heat-killed Blastomyces, HK-Bd) was introduced, a more complex pattern of cytokine (particularly TNF) and chemokine production ensued. The cytokine production, assayed by antibody array and also quantitation in supernatants, was depressed (particularly TNF) by the addition of mouse serum to these cultures, with the exception of IL-6. Macrophages could be cultured in the presence or absence of serum during the initial 20 h adherence and establishment period, enabling study of the effect of serum factors. In the absence of serum, with or without fungal stimulation, cytokine and chemokine production was more restricted, largely to TNF and IL-6. The addition of mouse serum [corrected] resulted in marked depression of TNF and enhancement of IL-6. The combination of HK-Bd and mouse serum resulted in more IL-6 production than either component alone. The enhancement of IL-6 by mouse serum was concentration-dependent and maximal at 8 h. The effects of fungus or serum on macrophage production of cytokines were similar in an outbred and an inbred mouse strain. The larger repertoire of cytokine production in the macrophages that had been cultured longer (20 h+20 h) in serum may be related to maturation of cell receptors. IL-6 production in vivo in response to fungal-serum complexes could affect pathogenesis by opposing the host defense modulation by proinflammatory cytokines or by modulating the destructive effects of inflammation on host tissues.