The plant alkaloid and anti-leukemia drug homoharringtonine sensitizes resistant human colorectal carcinoma cells to TRAIL-induced apoptosis via multiple mechanisms

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic ligand from the TNF-alpha family that is under consideration, along with agonistic anti-TRAIL receptor antibodies, as a potential anti-tumor agent. However, most primary human tumors are resistant to monotherapy with TRAIL apoptogens, and thus the potential applicability of TRAIL in anti-tumor therapy ultimately depends on its rational combination with drugs targeting these resistances. In our high-throughput screening for novel agents/drugs that could sensitize TRAIL-resistant colorectal cancer cells to TRAIL-induced apoptosis, we found homoharringtonine (HHT), a cephalotaxus alkaloid and tested anti-leukemia drug, to be a very effective, low nanomolar enhancer of TRAIL-mediated apoptosis/growth suppression of these resistant cells. Co-treatment of TRAIL-resistant RKO or HT-29 cells with HHT and TRAIL led to the effective induction of apoptosis and the complete elimination of the treated cells. HHT suppressed the expression of the anti-apoptotic proteins Mcl-1 and cFLIP and enhanced the TRAIL-triggered activation of JNK and p38 kinases. The shRNA-mediated down-regulation of cFLIP or Mcl-1 in HT-29 or RKO cells variably enhanced their TRAIL-induced apoptosis but it did not markedly sensitize them to TRAIL-mediated growth suppression. However, with the notable exception of RKO/sh cFLIP cells, the downregulation of cFLIP or Mcl-1 significantly lowered the effective concentration of HHT in HHT + TRAIL co-treatment. Combined HHT + TRAIL therapy also led to the strong suppression of HT-29 tumors implanted into immunodeficient mice. Thus, HHT represents a very efficient enhancer of TRAIL-induced apoptosis with potential application in TRAIL-based, anti-cancer combination therapy.     

Bioluminescent bioreporter Pseudomonas putida TVA8 as a detector of water pollution. Operational conditions and selectivity of free cells sensor

Conditions influencing bioluminescence output from Pseudomonas putida TVA8 harboring chromosomal tod-luxCDABE fusion were followed. In complex media, cell growth was not influenced by the presence of toluene at 53 mg/L. Bioluminescence induction was tested in minimal medium. At 15 °C the highest bioluminescence induced with toluene (1.325 mg/L) was reached after 6 h. At 25 °C the bioluminescence maximum was approximately 20% lower but this was reached after 3.5 h, and at temperatures of 7 °C, 28 °C, 30 °C and 34 °C, bioluminescence peaked at ≤60% of the maximum. Time courses of bioluminescence were dependent on cell concentrations. The heights of bioluminescence maxima were proportional to toluene concentration in the range 0–26 mg/L. Twenty-three organic pollutants (10³× diluted saturated solutions) were tested as bioluminescent inducers. The bioluminescence maximum decreased in the order: ethylbenzene > toluene > phenol > benzene > 4-ethyltoluene > 4-fluorotoluene > cumene > isobutylbenzene > styrene > trichloroethylene > o-, p-xylene > cresol > m-xylene > 2-methylnaphthalene > benzylchloride > naphthalene > salicylic acid > hexachlorobenzene > 2-chloronaphthalene > biphenyl > 2-bromonaphthalene > 1,3,5-triethylbenzene. Bioluminescence was also induced with ethanol and methanol and the presence of these alcohols in concentrations of ≤1% increased bioluminescence of toluene. The induction of bioluminescence from samples of wastewater and groundwater contaminated with BTEX (benzene, toluene, ethylbenzene and xylene) from 0.5 to 120 mg/L was demonstrated.     

Sample preparation in separation of the extracellular chitinolytic enzymes of the human intestinal bacterium Clostridium paraputrificum J4 from the culture fluids

Membrane ultrafiltration (UF) was used in sample preparation of the culture fluids of the human intestinal bacterium Clostridium paraputrificum strain J4 containing seven extracellular chitinolytic isoenzymes (38-90 kDa). The subsequent filtration of the bacteria-free supernatants was carried out through Millipore membranes with cut-off 100 and 30 kDa for separation of undigested components of the culture medium and bacterial metabolites with molecular weight higher and lower than that of the target enzymes. The chitinolytic enzymes, which were the minor components in the culture fluids, were concentrated at UF as well. The aim of the research consisted in evaluation of the effect of component composition of bacteria-free supernatants and the chemical nature of membrane active layer on partial fractionation of the chitinolytic enzymes, their recovery in retentates and purification degree. On the basis of the obtained experimental results, the sample preparation procedure of the culture fluids of C. paraputrificum J4 was established to be used further in chromatographic separations of the chitinolytic enzymes.     

Surgery and angiogenesis

Surgery may be regarded as an angiogenesis-inducing condition since it evokes the release of many angiogenic factors. Regarding the mechanistic overlap between tumor-associated neovascularisation and (physiological) angiogenesis in response to injury and hypoxia, surgery may promote the uncontrolled growth of residual dormant tumor cells. With the advent of anti-angiogenic agents, surgeons will be faced with more patients undergoing surgery for primary and secondary tumors under anti-angiogenic treatment. This could present problems with regard to angiogenesis-dependent phenomena such as wound repair, healing of intestinal anastomoses and liver regeneration. In this review we will discuss these matters from a biomedical and clinical point of view.     

Heavy browsing affects the hydraulic capacity of Ceanothus rigidus (Rhamnaceae)

Defoliation by herbivores can reduce carbon assimilation, change plant water relations, and even shift the biotic structure of plant communities. In this study, we took advantage of a long-term deer exclosure experiment to examine the consequences of persistent deer herbivory on plant water relations and the xylem structure–function relationships in Ceanothus rigidus, a maritime chaparral shrub in coastal California. Browsed plants had thicker stems with many intertwined short distal twigs, and significantly higher sapwood-to-leaf area ratios than their non-browsed counterparts. Leaf area-specific hydraulic conductivity was similar in both browsed and non-browsed plants, but xylem area-specific conductivity was significantly lower in the browsed plants. Vessel diameters were equivalent in both plant groups, but the number of vessels on a transverse area basis was nearly 40 % lower in the browsed plants, accounting for their lower transport efficiency. Mid-day in situ water potentials and losses of hydraulic conductivity due to embolism were similar in both groups of plants but stomatal conductance was higher in the browsed shrubs in the early part of the growing season. We discuss our findings in the context of whole-plant ecophysiology, and explore the consequences of herbivory on hormonal signals, wood anatomy, and xylem function.     

Energetics,conformation, and recognition of DNA duplexes containing a major adduct of an anticancer azolato-bridged dinuclear Pt complex

Background

The design of anticancer metallodrugs is currently focused on platinum complexes which form on DNA major adducts that cannot readily be removed by DNA repair systems. Hence, antitumor azolato-bridged dinuclear Pt^II complexes, such as [{cis-Pt(NH₃)₂}₂(μ‐OH)(μ-pyrazolate)]²⁺ (AMPZ), have been designed and synthesized. These complexes exhibit markedly higher toxic effects in tumor cell lines than mononuclear conventional cisplatin.

Methods

Biophysical and biochemical aspects of the alterations induced in short DNA duplexes uniquely and site-specifically modified by the major DNA adduct of AMPZ, namely 1,2-GG intrastrand cross-links, were examined. Attention was also paid to conformational distortions induced in DNA by the adducts of AMPZ and cisplatin, associated alterations in the thermodynamic stability of the duplexes, and recognition of these adducts by high-mobility-group (HMG) domain proteins.

Results

Chemical probing of DNA conformation, DNA bending studies and translesion synthesis by DNA polymerase across the platinum adduct revealed that the distortion induced in DNA by the major adduct of AMPZ was significantly less pronounced than that induced by similar cross-links from cisplatin. Concomitantly, the cross-link from AMPZ reduced the thermodynamic stability of the modified duplex considerably less. In addition, HMGB1 protein recognizes major DNA adducts of AMPZ markedly less than those of cisplatin.

General significance

The experimental evidence demonstrates why the major DNA adducts of the new anticancer azolato-bridged dinuclear Pt^II complexes are poor substrates for DNA repair observed in a previously published report. The relative resistance to DNA repair explains why these platinum complexes show major pharmacological advantages over cisplatin in tumor cells.     

Excretome of the chitinolytic bacterium <Emphasis Type="Italic">Clostridium paraputrificum</Emphasis> J4

A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-d-glucosaminide, N,N′-diacetyl-β-d-chitobiose, or N,N′,N˝-triacetyl-β-d-chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.