Decolorization of an industrial effluent by free and immobilized cells of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> AAP56<Emphasis Type="Italic">.</Emphasis> Implementation of efficient down flow column reactor

A bacterial strain AAP56, isolated from a polluted soil (from Kelibia city) and identified as Stentrophomonas maltophilia, was particularly interesting for its ability to decolorize recalcitrant dyes of an industrial effluent: SITEX Black. The final percentage decolorization 60% was shown by bacterial culture after incubation in LB medium at 30°C under shaking conditions. The decolorization was closely correlated with the metabolic bacterial growth. The replacement of yeast extract in LB medium composition by soya flour was clearly efficient to enhance the percentage decolorization by 20% and also to reduce the growth medium cost 60-fold. The bacteria were able to reduce 23% from the initial COD and 28% from the initial BOD₅ of the effluent. The immobilization of bacterial cells in calcium alginate beads improved by 25% the effectiveness of the biotransformation within 24 h in batch conditions. The potential of a downflow fixed column reactor (DFCR) to decolorize SITEX Black was evaluated under dilution rate. The best decolorization percentage (82%) was recorded at 0.3 h⁻¹. This bioprocess seems to be a potentially useful method to remediate the colored textile wastewater.     

Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo

In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem. 274, 20826-20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1 or TfR2-alpha cDNA. TfR2-alpha-expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression of TfR2 mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2 mRNA was regulated in the cell cycle with the highest expression in late G(1) phase and no expression in G(0)/G(1). DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-alpha-expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-alpha developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth both in vitro and in vivo.     

Total calcium-sensing receptor expression in circulating monocytes is increased in rheumatoid arthritis patients with severe coronary artery calcification

Introduction

Human circulating monocytes express the calcium-sensing receptor (CaSR) and are involved in atherosclerosis. This study investigated the potential association between vascular calcification in rheumatoid arthritis (RA) and CaSR expression in circulating monocytes.

Methods

In this cross-sectional study, 50 RA patients were compared to 25 control subjects matched for age and gender. Isolation of peripheral blood mononuclear cells and flow cytometry analysis were performed to study the surface and total CaSR expression in circulating monocytes. Coronary artery calcium (CAC) and abdominal aortic calcification (AAC) scores were evaluated by computed tomography and an association between these scores and the surface and/or total CaSR expression in circulating monocytes in RA patients was investigated.

Results

The two groups were similar in terms of age (RA: 60.9 ± 8.3 years, versus controls: 59.6 ± 5.3 years) and gender (RA: 74.0% females versus 72.0% females). We did not find a higher prevalence and greater burden of CAC or AAC in RA patients versus age- and gender-matched controls. When compared with control subjects, RA patients did not exhibit greater total CaSR (101.6% ± 28.8 vs. 99.9% ± 22.0) or surface CaSR (104.6% ± 20.4 vs. 99.9% ± 13.7) expression, but total CaSR expression in circulating monocytes was significantly higher in RA patients with severe CAC (Agatston score ≥200, n = 11) than in patients with mild-to-moderate CAC (1 to 199, n = 21) (P = 0.01).

Conclusions

This study demonstrates for the first time that total CaSR expression in human circulating monocytes is increased in RA patients with severe coronary artery calcification.     

Involvement of histidine residues and sulfhydryl groups in the function of the biotin transport carrier of rabbit intestinal brush-border membrane.

Possible involvement of histidine residues and sulfhydryl groups in the function of the intestinal brush-border membrane (BBM) transporter of biotin was investigated. This was done by examining the effects of pretreatment of BBM vesicle (BBMV) isolated from rabbit intestine with the histidine-specific reagent diethyl pyrocarbonate (DEPC) and the sulfhydryl group-specific reagents p-chloromercuribenzenesulfonic acid (p-CMBS) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) on carrier-mediated biotin transport. Pretreatment of BBMV with DEPC caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with DEPC provided significant protection to biotin transport. Treatment of DEPC-pretreated vesicles with the reducing agents dithiothreitol and 2,3-dimercaptopropanol failed to reverse the inhibitory effect of DEPC on biotin transport. The inhibitory effect of DEPC was found to be mediated through a marked decrease in the number of the functional biotin transport carriers with no change in their affinity, as indicated by the severe inhibition in the Vmax but not the apparent Km of the biotin transport process, respectively. Pretreatment of BBMV with p-CMBS and NBD-Cl also caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with p-CMBS (or NBD-Cl) failed to protect biotin transport from inhibition. On the other hand, treatment of vesicles pretreated with p-CMBS (or NBD-Cl) with the reducing agents dithiothreitol and mercaptoethanol caused significant reversal in the inhibition of biotin transport. The inhibitory effects of p-CMBS (and NBD-Cl) on biotin transport was also found to be mediated through inhibition in the Vmax, but not the apparent Km, of biotin transport process. These results indicate the involvement of histidine residues and sulfhydryl groups in the normal function of the biotin transport system of rabbit intestinal BBM. Furthermore, the results also suggest that the histidine residues are probably located at (or near) the substrate-binding site while the sulfhydryl groups are located at a site other than the substrate binding region.     

An assay for cancer stem cell-induced angiogenesis on chick chorioallantoic membrane

Angiogenesis is generally involved in tumor growth and metastasis. Cancer stem cells (CSCs) are considered to facilitate the angiogenesis. Therefore, CSCs could be the effective targets to stop angiogenesis. Recently, our group successfully generated CSC models from induced pluripotent stem cells (iPSCs) in the presence of conditioned medium derived from cancer derived cells. These novel model CSCs has been characterized by highly tumorigenic, angiogenic and metastatic potentials in vivo. The angiogenic potential of CSCs has been explained by the expression of both angiogenic factors and their receptors implying the angiogenesis in autocrine manner. In this protocol we optimized the method to evaluate tumor angiogenesis with the CSC model, which was described effective to assess sorafenib as an antiangiogenic drug, on chick chorioallantoic membrane (CAM) assay. Our results demonstrate that CSCs developed from iPSCs and CAM assay are a robust and cost-effective tool to evaluate tumor angiogenesis with CSCs. Collectively, CSCs in CAM assay could serve as a very useful model for the screening of potential therapeutic agents targeting tumor angiogenesis.     

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.     

Semisynthesis and in vitro anticancer activities of andrographolide analogues

The plant Andrographis paniculata found throughout Southeast Asia contains Andrographolide 1, a diterpenoid lactone, which has antitumour activities against in vitro and in vivo breast cancer models. In the present study, we report on the synthesis of andrographolide derivatives, 3,19-isopropylideneandrographolide (2), 14-acetyl-3,19-isopropylideneandrographolide (3) and 14-acetylandrographolide (4), and their in vitro antitumour activities against a 2-cell line panel consisting of MCF-7 (breast cancer cell line) and HCT-116 (colon cancer cell line). Compounds 2 and 4 were also screened at the US National Cancer Institute (NCI) for their activities against a panel of 60 human cancer cell lines derived from nine cancer types. Compound 2 was found to be selective towards leukaemia and colon cancer cells, and compound 4 was selective towards leukaemia, ovarian and renal cancer cells at all the dose-response parameters. Compounds 2 and 4 showed non-specific phase of the cell cycle arrest in MCF-7 cells treated at different intervals with different concentrations. NCI's COMPARE and SOM mechanistic analyses indicated that the anticancer activities of these new class of compounds were not similar to that of standard anticancer agents, suggesting novel mechanism(s) of action.     

Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception

Transgenic tomato [Lycopersicon esculentum (=Solanum lycopersicum)] lines overexpressing tomato PHYA, PHYB1, or PHYB2, under control of the constitutive double-35S promoter from cauliflower mosaic virus (CaMV) have been generated to test the level of saturation in individual phytochrome-signalling pathways in tomato. Western blot analysis confirmed the elevated phytochrome protein levels in dark-grown seedlings of the respective PHY overexpressing (PHYOE) lines. Exposure to 4 h of red light resulted in a decrease in phytochrome A protein level in the PHYAOE lines, indicating that the chromophore availability is not limiting for assembly into holoprotein and that the excess of phytochrome A protein is also targeted for light-regulated destruction. The elongation and anthocyanin accumulation responses of plants grown under white light, red light, far-red light, and end-of-day far-red light were used for characterization of selected PHYOE lines. In addition, the anthocyanin accumulation response to different fluence rates of red light of 4-d-old dark-grown seedlings was studied. The elevated levels of phyA in the PHYAOE lines had little effect on seedling and adult plant phenotype. Both PHYAOE in the phyA mutant background and PHYB2OE in the double-mutant background rescued the mutant phenotype, proving that expression of the transgene results in biologically active phytochrome. The PHYB1OE lines showed mild effects on the inhibition of stem elongation and anthocyanin accumulation and little or no effect on the red light high irradiance response. By contrast, the PHYB2OE lines showed a strong inhibition of elongation, enhancement of anthocyanin accumulation, and a strong amplification of the red light high irradiance response.     

Plasma protein advanced glycation end products, carboxymethyl cysteine, and carboxyethyl cysteine, are elevated and related to nephropathy in patients with diabetes

In Diabetes Mellitus (DM), glucose and the aldehydes glyoxal and methylglyoxal modify free amino groups of lysine and arginine of proteins forming advanced glycation end products (AGEs). Elevated levels of these AGEs are implicated in diabetic complications including nephropathy. Our objective was to measure carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC), AGEs formed by modification of free cysteine sulfhydryl groups of proteins by these aldehydes, in plasma proteins of patients with diabetes, and investigate their association with the albumin creatinine ratio (ACR, urine albumin (mg)/creatinine (mmol)), an indicator of nephropathy. Blood was collected from forty-two patients with type 1 and 2 diabetes (18–36 years) and eighteen individuals without diabetes (17–35 years). A liquid chromatography-mass spectrophotometric method was developed to measure plasma protein CMC and CEC levels. Values for ACR and hemoglobin A1C (HbA1C) were obtained. Mean plasma CMC (μg/l) and CEC (μg/l) were significantly higher in DM (55.73 ± 29.43, 521.47 ± 239.13, respectively) compared to controls (24.25 ± 10.26, 262.85 ± 132.02, respectively). In patients with diabetes CMC and CEC were positively correlated with ACR, as was HbA1C. Further, CMC or CEC in combination with HbA1C were better predictors of nephropathy than any one of these variables alone. These results suggest that glucose, glyoxal, and methylglyoxal may all be involved in the etiology of diabetic nephropathy.