Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia

High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L‐Ser and/or L‐Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1‐mRNA and AST2‐mRNA by RT‐PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L‐Asp. AST1‐mRNA expression was independent on oxygen and cell line, but AST2‐mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L‐Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells.     

Reactions of *NO2 with chromium(III) complexes with histamine and pyridoxamine ligands studied by the stopped-flow technique

This study demonstrated the direct formation of the nitrogen dioxide (*NO2) radical during the decomposition of 3-morpholinosydnonimine (SIN-1) in biological buffer 4-morpholinoethanosulfone acid solution. Consequently, at approximately pH 4, SIN-1 can be used successfully as a source of *NO2. This conclusion is drawn from a comparison of the reactions of cis-[Cr(C2O4)(L- L)(OH2)2]+, where L-L denotes pyridoxamine (Hpm) or histamine (hm), with the gaseous *NO2 radical obtained by two methods: from SIN-1 and from a simple redox reaction. These reactions were investigated using the stopped-flow technique. The measurements were carried out at temperatures ranging from 5 to 25 degrees C over a pH range from 6.52 to 9.11 for cis-[Cr(C2O4)(Hpm) (OH2)2]+ and from 6.03 to 8.15 for cis-[Cr(C2O4)(hm)(OH2)2] +. We also determined the thermodynamic activation parameter (E(a)) and the uptake mechanism for each of the coordination compounds studied.     

Synthesis of 3'-azido-3'-deoxythymidine (AZT)--Cinchona alkaloid conjugates via click chemistry: Toward novel fluorescent markers and cytostatic agents

Novel nucleoside-Cinchona alkaloid conjugates were synthesized using ‘click’ chemistry approach based on the copper(I) catalyzed Huisgen azide-alkyne cycloaddition. Two series of conjugates were prepared employing 3′-azido-3′-deoxythymidine (AZT) as the azide component and the four 10,11-didehydro Cinchona alkaloids as well as their 9-O-propargyl ethers as the alkyne components. All obtained conjugates showed strong fluorescence emission and some of them exhibited marked cytotoxic activity in vitro.     

Identification of Molecular Markers of Delayed Graft Function Based on the Regulation of Biological Ageing

IntroductionDelayed graft function is a prevalent clinical problem in renal transplantation for which there is no objective system to predict occurrence in advance. It can result in a significant increase in the necessity for hospitalisation post-transplant and is a significant risk factor for other post-transplant complications.MethodologyThe importance of microRNAs (miRNAs), a specific subclass of small RNA, have been clearly demonstrated to influence many pathways in health and disease. To investigate the influence of miRNAs on renal allograft performance post-transplant, the expression of a panel of miRNAs in pre-transplant renal biopsies was measured using qPCR. Expression was then related to clinical parameters and outcomes in two independent renal transplant cohorts.ResultsHere we demonstrate, in two independent cohorts of pre-implantation human renal allograft biopsies, that a novel pre-transplant renal performance scoring system (GRPSS), can determine the occurrence of DGF with a high sensitivity (>90%) and specificity (>60%) for donor allografts pre-transplant, using just three senescence associated microRNAs combined with donor age and type of organ donation.ConclusionThese results demonstrate a relationship between pre-transplant microRNA expression levels, cellular biological ageing pathways and clinical outcomes for renal transplantation. They provide for a simple, rapid quantitative molecular pre-transplant assay to determine post-transplant allograft function and scope for future intervention. Furthermore, these results demonstrate the involvement of senescence pathways in ischaemic injury during the organ transplantation process and an indication of accelerated bio-ageing as a consequence of both warm and cold ischaemia.     

One‐Pot Synthesis of New (1,3‐Thiazolo[5,4‐b]pyridin‐2‐yl)benzenediols and Their Antiproliferative Activities against Human Cancer Cell Lines

A one‐pot synthesis of new 4‐(1,3‐thiazolo[5,4‐b]pyridin‐2‐yl)benzene‐1,3‐diols has been described. The compounds were prepared by the reaction of sulfinylbis[(2,4‐dihydroxyphenyl)methanethione] derivatives, with various substituents in the aryl rings, with 2‐chloropyridin‐3‐amines. Their structures were deduced from IR and, ¹H‐ and ¹³C‐NMR spectroscopic, mass spectrometric, and elemental analyses. The antiproliferative properties of some of the products against human cancer cell lines were comparable to those of cisplatin. Structure? activity analysis showed that the presence of hydrophobic substituents in both heterocyclic fused and phenyl rings of the compounds improves their biological effects. Further, an additional OH group in the resorcinol moiety reduced the antiproliferative activity.     

Characteristic defects in neural crest cell-specific Galphaq/Galpha11- and Galpha12/Galpha13-deficient mice

The endothelin/endothelin receptor system plays a critical role in the differentiation and terminal migration of particular neural crest cell subpopulations. Targeted deletion of the G-protein-coupled endothelin receptors ET(A) and ET(B) was shown to result in characteristic developmental defects of derivatives of cephalic and cardiac neural crest and of neural crest-derived melanocytes and enteric neurons, respectively. Since both endothelin receptors are coupled to G-proteins of the G(q)/G(11)- and G(12)/G(13)-families, we generated mouse lines lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13) in neural crest cells to study their roles in neural crest development. Mice lacking Galpha(q)/Galpha(11) in a neural crest cell-specific manner had craniofacial defects similar to those observed in mice lacking the ET(A) receptor or endothelin-1 (ET-1). However, in contrast to ET-1/ET(A) mutant animals, cardiac outflow tract morphology was intact. Surprisingly, neither Galpha(q)/Galpha(11)- nor Galpha(12)/Galpha(13)-deficient mice showed developmental defects seen in animals lacking either the ET(B) receptor or its ligand endothelin-3 (ET-3). Interestingly, Galpha(12)/Galpha(13) deficiency in neural crest cell-derived cardiac cells resulted in characteristic cardiac malformations. Our data show that G(q)/G(11)- but not G(12)/G(13)-mediated signaling processes mediate ET-1/ET(A)-dependent development of the cephalic neural crest. In contrast, ET-3/ET(B)-mediated development of neural crest-derived melanocytes and enteric neurons appears to involve G-proteins different from G(q)/G(11)/G(12)/G(13).     

Lumican affects actin cytoskeletal organization in human melanoma A375 cells

AIMS: Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line. MAIN METHODS: Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic beta- and gamma-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms. KEY FINDINGS: A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican. SIGNIFICANCE: Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.     

Replisome localization in vegetative and aerial hyphae of Streptomyces coelicolor

Using a functional fusion of DnaN to enhanced green fluorescent protein, we examined the subcellular localization of the replisome machinery in the vegetative mycelium and aerial mycelium of the multinucleoid organism Streptomyces coelicolor. Chromosome replication took place in many compartments of both types of hypha, with the apical compartments of the aerial mycelium exhibiting the highest replication activity. Within a single compartment, the number of "current" ongoing DNA replications was lower than the expected chromosome number, and the appearance of fluorescent foci was often heterogeneous, indicating that this process is asynchronous within compartments and that only selected chromosomes undergo replication.     

Dynamic interplay of ParA with the polarity protein,Scy, coordinates the growth with chromosome segregation in Streptomyces coelicolor

Prior to bacterial cell division, the ATP-dependent polymerization of the cytoskeletal protein, ParA, positions the newly replicated origin-proximal region of the chromosome by interacting with ParB complexes assembled on parS sites located close to the origin. During the formation of unigenomic spores from multi-genomic aerial hyphae compartments of Streptomyces coelicolor, ParA is developmentally triggered to form filaments along the hyphae; this promotes the accurate and synchronized segregation of tens of chromosomes into prespore compartments. Here, we show that in addition to being a segregation protein, ParA also interacts with the polarity protein, Scy, which is a component of the tip-organizing centre that controls tip growth. Scy recruits ParA to the hyphal tips and regulates ParA polymerization. These results are supported by the phenotype of a strain with a mutant form of ParA that uncouples ParA polymerization from Scy. We suggest that the ParA–Scy interaction coordinates the transition from hyphal elongation to sporulation.