Technical advance: spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein

Plant growth responds rapidly to developmental and environmental signals, but the underlying changes in cell division activity are poorly understood. A labile cyclin-GUS reporter was developed to facilitate the spatio-temporal analysis of cell division patterns. The chimeric reporter protein is turned over every cell cycle and hence its histochemical activity accurately reports individual mitotic cells. Using Arabidopsis plants transformed with cyclin-GUS, we visualized patterns of mitotic activity in wounded leaves which suggest a role for cell division in structural reinforcement.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.     

Influence of the whole-cell patch-clamp configuration on electrophysiological properties of the voltage-dependent sodium current expressed in MDA-MB-231 breast cancer cells

Voltage-gated ionic channels are known to be involved in oncogenesis. However, only a few studies describe the functional characteristics of these channels or the mechanisms by which they are involved in the proliferation and invasive processes. Breast cancer cells proliferate and migrate under the constant activation of growth factors, hormones, extracellular matrix interactions, etc. It would thus not be surprising if the activity of the ionic channels was modulated by intracellular regulation pathways such as kinases or phosphatases, which in turn can affect oncogenic properties. In the present study, we investigated some of the electrophysiological properties of the fast inward sodium current found in the breast cancer cell line MDA-MB-231 with two configurations of the patch-clamp technique. With perforated patch, a configuration which allows to keep the cytoplam intact, the mean current amplitude was lower, the relative conductance–voltage relationship was shifted to more positive potentials and the recovery from inactivation was accelerated when compared to ruptured patch, where the cytoplasm is dialysed by the intrapipette solution. There was no difference in availability–voltage (pseudo-steady-state inactivation) relationship and in time to peak of the current. These results suggest that regulation mechanisms, possibly involving kinases or phosphatases, are switched off when the cytoplasm is diluted. We propose that such a regulation can modulate the functioning of the channels even in the absence of membrane voltage changes, which in turn can affect oncogenic properties. This finding is of importance when evaluating the physiopathological role of ionic channel in cancer development.Abbreviations DMEM Dulbeccos modified Eagles medium - FBS fetal bovine serum - PP perforated patch - RP ruptured patch Presented at the Biophysical Society Meeting on Ion Channels—from structure to disease held in May 2003, Rennes, France     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides derived from 4-isothiocyanato-benzolamide

A series of sulfonamides incorporating 4-thioureido-benzolamide moieties have been prepared from aminobenzolamide and thiophosgene followed by the reaction of the thiocyanato intermediate with aliphatic/aromatic amines or hydrazines. The new derivatives have been investigated as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely of the cytosolic isozymes hCA I and II, as well as the tumor-associated isozyme hCA IX (all of human origin). The new compounds showed excellent inhibitory properties against all three isozymes with inhibition constants in the range of 0.6-62 nM against hCA I, 0.5-1.7 nM against hCA II and 3.2-23 nM against hCA IX, respectively. These derivatives are interesting candidates for the development of novel therapies targeting hypoxic tumors.     

Natural products and combinatorial chemistry: back to the future

The introduction of high-throughput synthesis and combinatorial chemistry has precipitated a global decline in the screening of natural products by the pharmaceutical industry. Some companies terminated their natural products program, despite the unproven success of the new technologies. This was a premature decision, as natural products have a long history of providing important medicinal agents. Furthermore, they occupy a complementary region of chemical space compared with the typical synthetic compound library. For these reasons, the interest in natural products has been rekindled. Various approaches have evolved that combine the power of natural products and organic chemistry, ranging from the combinatorial total synthesis of analogues to the exploration of natural product scaffolds and the design of completely unnatural molecules that resemble natural products in their molecular characteristics.     

Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.     

Involvement of a novel fast inward sodium current in the invasion capacity of a breast cancer cell line

This work reports the finding of a unique fast inward sodium current (I(Na)) in MDA-MB-231 cells which is missing in MDA-MB-468 cells and in MCF-7 cells. This current is high-voltage-activated and displays a window current at the membrane potential of MDA-MB-231 cells. This current is blocked by high concentrations of tetrodotoxin (TTX). In MDA-MB-231 cells, which are the most invasive cells among the three cell lines tested, proliferation and migration were not sensitive to TTX while invasion was reduced by approximately 30%. These experiments suggest that I(Na) is involved in the invasion process, probably through its participation to the regulation of the intracellular sodium homeostasis.     

New N-pyridinyl(methyl)-indole-2- and 3-(alkyl)carboxamides and derivatives acting as systemic and topical inflammation inhibitors

A series of novel N-substituted-(indol-2-yl)carboxamides (12-18) and (indol-3-alkyl)carboxamides (25-31) were synthesized and evaluated as inhibitors of the inflammation process. Pharmacomodulation at the level of the amidic nitrogen by incorporation of the previously described pharmacophoric moieties 6-aminolutidine, beta-picolylamine, 4-aminopyridine and piperazine was investigated; only two compounds (12) and (31) exhibited significant (approximately 40%) inhibitory effect in the carrageenan-induced rat paw edema after oral administration of a dose of 0.1 mM kg(-1). Replacement of the indole core by indazole failed to increase activity. Incorporation of an alkyl chain spacer led to more efficient compounds (46-52) especially in the indolepropanamide sub-series. Determination of the efficiency of the most active compounds on topical inflammation, by measuring reduction of ear thickness in the acute tetradecanoyl phorbol acetate (TPA)-induced mouse ear swelling assay, confirmed the high potency of propanamides (49) and (51) after oral administration: ID50 = 0.041 +/- 0.013 and 0.042 +/- 0.016 mM kg(-1) respectively. The less toxic propanamide (51) exerted a high level of inhibitory activity after topical application of 2 x 100 microg/ear: 78 +/- 2%.