Characterization of cytotoxic compound from marine sediment derived actinomycete Streptomyces avidinii strain SU4

Objective

To investigate the cytotoxic activity of actinomycete isolated from marine sediment.

Methods

In the present study the DNA was isolated and the ITS region of 16s rRNA was amplified by polymerase chain reaction, using two universal bacterial primers, 1492R (5′-GGTTACCTTGTTAC GACTT-3′) and Eubac27F (5′-AGAGTTTGATCCTGGCTC AG-3′). The amplified products were purified using TIANgel mini purification kit, ligated to MD18-T simple vector (TaKaRa), and transformed into competent cells of Escherichia coli DH5α. 16S rRNA gene fragment was sequenced using forward primer M13F (-47) and reverse primer M13R (-48). Blast search sequence similarity was found against the existing non-redundant nucleotide sequence database thus, identified as Streptomyces sp SU, Streptomyces rubralavandulae strain SU1, Streptomyces cacaoi strain SU2, Streptomyces cavourensis strain SU3, Streptomyces avidinii strain SU4, Streptomyces globisporus strain SU5, Streptomyces variabilis strain SU6, Streptomyces coelicolor strain SU 7. Among the eight identified isolates, one actinomycete Streptomyces avidinii strain SU4 was selected for further study.

Results

Crude extract of the actinomycete isolate exhibited IC₅₀ in 64.5 µg against Hep-2 cell line, 250 µg in VERO cell line. This value is very close to the criteria of cytotoxicity activity for the crude extracts, as established by the American National Cancer Institute (NCI) is in IC₅₀ < 30 µg/mL. The GC MS analysis showed that the active principle might be 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (12.17%), isooctyl phthalate (15.29%) with the retention time 15.642 and 21.612, respectively.

Conclusions

This study clearly proves that the marine sediment derived actinomycetes with bioactive metabolites can be expected to provide high quality biological material for high throughout biochemical and anticancer screening programs. These results help us to conclude that the potential of using metabolic engineering and post genomic approaches to isolate more bioactive compounds and make their possible commercial application is not far off.     

Long-term regulation of proximal tubule acid-base transporter abundance by angiotensin II

In the proximal tubule, angiotensin II (Ang-II) regulates HCO(-)(3) reabsorption and H+ secretion by binding the type 1 Ang-II (AT1) receptor, stimulating Na(+)/HCO(-)(3) cotransport and Na(+)/H(+) exchange. Studies were carried out to determine if long-term changes in Ang-II receptor occupation alter the abundance of the basolateral Na(+)/HCO(-)(3) cotransporter (NBC1) or the apical membrane type 3 Na(+)/H(+) exchanger (NHE3). In the first set of experiments, rats eating a low-sodium diet were infused with the AT1 blocker, candesartan, or vehicle. In the second, lisinopril-infused rats were infused with either Ang II or vehicle. Transporter abundances were determined in whole kidney homogenates (WKH) and in brush border membrane (BBM) preparations by semiquantitative immunoblotting. Tissue distribution of transporters was assessed by immunocytochemistry. Blockade of the AT1 receptor by candesartan caused decreased abundance of NBC1 in WKH (59 +/- 9% of control; P<0.05) and Ang-II infusion increased abundance (130 +/- 7% of control; P<0.05). Changes in NBC1 in response to candesartan were confirmed immunohistochemically. Neither candesartan nor Ang II infusion affected the abundance of NHE3 in WKH or cortical homogenates. Candesartan decreased type 2 sodium-phosphate cotransporter abundance in both WKH (52 +/- 7% of control; P<0.05) and BBM (32 +/- 7% of control; P<0.05). Serum bicarbonate was decreased by candesartan and increased by Ang-II. Candesartan also decreased urinary ammonium excretion (P<0.05). The long-term effects of Ang-II in the proximal tubule may be mediated in part by regulation of NBC1 abundance, modifying bicarbonate reabsorption.     

The CD94/NKG2 family of receptors

Immune responses must be tightly regulated to avoid hyporesponsiveness on one hand or excessive inflammation and the development of autoimmunity (hyperresponsiveness) on the other hand. This balance is attained through the throttling of activating signals by inhibitory signals that ideally leads to an adequate immune response against an invader without excessive and extended inflammatory signals that promote the development of autoimmunity. The CD94/NKG2 family of receptors is composed of members with activating or inhibitory potential. These receptors are expressed predominantly on NK cells and a subset of CD8+T cells, and they have been shown to play an important role in regulating responses against infected and tumori genic cells. In this review, we discuss the current knowledge about this family of receptors, including ligand and receptor interaction, signaling, membrane dynamics, regulation of gene expression and their roles in disease regulation, infections, and cancer, and bone marrow transplantation.     

The cell biology of the human natural killer cell CD94/NKG2A inhibitory receptor

To avoid destruction of normal bystander cells, natural killer (NK) cells must provide a continuous supply of functional inhibitory receptors to their cell surface. After interaction with its ligand HLA-E, which is expressed on normal cells, the C-type lectin inhibitory receptor CD94/NKG2A suppresses activation signaling processes. CD94/NKG2A receptors continuously recycle from the cell surface through endosomal compartments and back again in a process that requires energy and the cytoskeleton. This steady state process appears to be largely unaffected by exposure to ligand. CD94/NKG2A receptors move freely within the plasma membrane and accumulate at the site of contact with the ligand bearing target cells (or monoclonal antibodies (mAb) coated beads). As expected, ligated CD94/NKG2A receptors are less mobile than the nonligated receptors, and the lipid raft marker cholera toxin B is excluded from the CD94/NKG2A enriched target cell contact sites. Also, methylcyclodextrin does not interfere with CD94/NKG2A accumulation at these contact sites. The constant renewal of CD94/NKG2A receptors at the cell surface and their free mobility within the plasma membrane likely facilitates and insures inhibitory capacity.     

Reduction of soluble complement receptor 2/CD21 in systemic lupus erythomatosus and Sjogren's syndrome but not juvenile arthritis

A soluble form of the complement receptor CD21 (sCD21) is shed from the lymphocyte surface. The amount of sCD21 in serum may modulate immunity as sCD21 levels are correlated with several clinical conditions. We report here the serum levels of sCD21 in juvenile arthritis (JA), systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS). Using enzyme-linked immunosorbent assay, we determined sCD21 levels in SLE, SS and JA patients. Mann-Whitney test for nonparametric two-tail P value was performed to obtain statistical significance. Cytometrical analysis of synovial fluid leucocytes of JA patients was done on a FACSsort. While sCD21 levels in SLE and SS are reduced to levels previously found in rheumatoid arthritis (RA), JA sCD21 levels were normal. sCD21 levels did not correlate with clinical parameters and immunophenotype of synovial cells. CD4 T cells in the synovium were almost all of the CD45RO memory type and 13 of 40 patients displayed synovial expansion of gammadeltaT cells. CD21 shedding in JA differs from RA/SS/SLE. JA sCD21 levels in synovial fluid are always lower compared to blood levels of the same patients. Analysis of JA synovial T cells indicates a T-cell driven response.     

Salivary biomarkers of stress among teachers in an urban setting

The aim of this study was to determine the prevalence of job stress among secondary school teachers using Karasek Job Content Questionnaire (JCQ), the association between salivary cortisol, salivary IgA, and sociodemographic characteristics, and the association between log cortisol, IgA levels, and job strain categories. A cross-sectional study was undertaken using JCQ and salivary cortisol and IgA samples. Cluster sampling was done yielding 302 respondents. The prevalence of stress among all teachers was 20.2%. Being a Malay, teaching experience of 5 to 10 years, and those without a supervisor's support had higher prevalence of high job strain. Teachers in the 31 to 40 years age bracket, educating handicapped children with the absence of supervisor support exhibited higher stress levels with lower log salivary IgA levels. Further studies must be conducted using salivary biomarkers to study the in-depth relationship of stress, extending into other occupational groups.     

Juvenile growth reduces the influence of epithelial sodium channels on myogenic tone in skeletal muscle arterioles

Previous studies have documented that rapid juvenile growth is accompanied by functional changes in the arteriolar endothelium, but much less is known about functional changes in arteriolar smooth muscle over this period. In this study, we investigate the possible contribution of epithelial sodium channels (ENaC) to the myogenic behaviour of arterioles at two stages of juvenile growth. The effects of the ENaC inhibitor benzamil on different levels of myogenic tone were studied in isolated gracilis muscle arterioles from rats aged 21‐28 days (“weanlings”) and 42‐49 days (“juveniles”). ENaC subunit expression in the arteriolar wall was also determined, and the interaction between ENaC and nitric oxide (NO) in regulating vascular tone was explored by combined use of benzamil and N^G‐monomethyl‐l ‐arginine (l ‐NMMA). At physiological pressures, both steady‐state myogenic tone and the dynamic adjustments in this tone triggered by acute pressure changes were less in juvenile arterioles than in weanling arterioles. α, β and γ ENaC protein was present in arterioles at both ages, but benzamil only had an effect on myogenic tone in weanling arterioles. In these vessels, benzamil increased, rather than decreased, myogenic tone, and this effect was prevented by l ‐NMMA or endothelial removal. These findings suggest that although ENaC is present in gracilis muscle arterioles of both weanling and juvenile rats, it is not obligatory for the genesis of myogenic activity in these vessels at either age. However, ENaC activity can significantly modulate the level of myogenic tone through stimulation of endothelial NO release at an early stage of growth.     

A new CD21low B cell population in the peripheral blood of patients with SLE

A hallmark of systemic lupus erythematosus (SLE) is the production of autoantibodies. Recent reports suggest an abnormal peripheral blood B cell homeostasis in SLE patients without being conclusive. We analyzed by four color flow-cytometry peripheral blood B cell subpopulations of SLE patients, healthy donors, and patients with other systemic autoimmune diseases. IgM memory but not switched memory B cells of SLE patients were significantly decreased compared to healthy donors, whereas transitional B cells, characterized by CD19+IgMhiIgD+CD24hiCD38hi, were significantly expanded in SLE patients but also found in other autoimmune disorders. The population of plasmablasts (CD19loCD21loCD27++CD38++) was increased in active disease. Most interestingly, B cells in autoimmune disorders contain a so far uncharacterized subpopulation with an activated phenotype (CD19hiCD21loCD38loCD86int). None of the identified subpopulations was associated with current or previous therapy and therefore may represent different aspects of the disturbed B cell homeostasis in patients with SLE.