Proteins from bovine tissues and biological fluids: defining a reference electrophoresis map for liver,kidney, muscle,plasma and red blood cells

A number of high resolution two-dimensional electrophoresis (2-DE) reference maps for bovine tissues and biological fluids have been determined for animals in basal state. Among the 1863 distinct protein features detected in samples of liver, kidney, muscle, plasma and red blood cells, 509 species were identified and associated to 209 different genes. Difficulties in the identification were related to the poorly characterized Bos taurus genome and were solved by a combined matrix-assisted laser desorption/ionisation-mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry approach. The experimental output allowed us to establish a 2-DE database accessible through the World Wide Web network at the URL address (http://www.iabbam.na.cnr.it/Biochem). These reference maps may serve as a tool in future veterinary medical studies aimed at the evaluation of changes in protein repertoire for altered animal physiological conditions and infectious diseases, to the definition of molecular markers for novel diagnostic kits and vaccines, as well as the characterization of protein modifications in bovine materials following technological processes used in the food industry.     

The presence of a chromatin boundary appears to shield a transgene in tobacco from RNA silencing

We present isogenic transgenic tobacco lines that carry at a given chromosomal position a beta-glucuronidase (GUS) reporter gene either with or without the presence of the matrix-associated region known as the chicken lysozyme A element. Plants were generated with the Cre-lox site-specific recombination system using heterospecific lox sites. Analysis of GUS gene expression in plant populations demonstrates that the presence of the A element can shield against RNA silencing of the GUS gene. Protection was observed in two of three independent tobacco transformants. Plants carrying an A element 5' of the GUS gene always had stable GUS activity, but upon removal of this A element, the GUS gene became silenced over time in two lines, notably when homozygous.     

Peripheral benzodiazepine receptor ligand-melphalan conjugates for potential selective drug delivery to brain tumors

To gain insight into the strategy to target PBR ligand-drug conjugates to brain tumors, novel N-imidazopyridinacetyl-melphalan conjugates and the corresponding ethyl esters have been prepared and evaluated for their cytotoxicity in melphalan-sensitive human (SF126, SF188) and rat (RG-2) glioma cell lines. These conjugates exhibited PBR binding affinity with IC(50) values ranging from 57 and 2614 nM. By a computational approach it can be predicted that these conjugates possess significant brain penetration. The stability of the conjugates in 0.05 M phosphate buffer at pH 7.4 and, in some cases, in dilute human serum solution was determined. All the ethyl ester derivatives were stable in 0.05 M phosphate buffer at pH 7.4 and their half-lives exceeded 28 h. Conversely, under the same conditions, the corresponding acids were found to undergo a fast cleavage within a few minutes. HPLC-MS analysis of the mixture from degradation in buffer and physiological medium of the representative cases allowed the identification of their main degradation products. A plausible degradation pathway accounting for the available experimental data is presented.     

Freshwater foraminiferans revealed by analysis of environmental DNA samples

Sediment-dwelling protists are among the most abundant meiobenthic organisms, ubiquitous in all types of aquatic ecosystems. Yet, because their isolation and identification are difficult, their diversity remains largely unknown. In the present work, we applied molecular methods to examine the diversity of freshwater Foraminifera, a group of granuloreticulosan protists largely neglected until now. By using specific PCR primers, we detected the presence of Foraminifera in all sediment samples examined. Phylogenetic analysis of amplified SSU rDNA sequences revealed two distinct groups of freshwater foraminiferans. All obtained sequences branched within monothalamous (single-chambered), marine Foraminifera, suggesting a repeated colonization of freshwater environments. The results of our study challenge the traditional view of Foraminifera as essentially marine organisms, and provide a conceptual framework for charting the molecular diversity of freshwater granuloreticulosan protists.     

The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo

The Caenorhabditis elegans early embryo is widely used to study the regulation of microtubule-related processes. In a screen for mutants affecting the first cell division, we isolated a temperature-sensitive mutation affecting pronuclear migration and spindle positioning, phenotypes typically linked to microtubule or centrosome defects. In the mutant, microtubules are shorter and chromosome segregation is impaired, while centrosome organization appears normal. The mutation corresponds to a strong loss of function in mbk-2, a conserved serine/threonine kinase. The microtubule-related defects are due to the postmeiotic persistence of MEI-1, a homologue of the microtubule-severing protein katanin. In addition, P-granule distribution is abnormal in mbk-2 mutants, consistent with genetic evidence that mbk-2 has other functions and with the requirement of mbk-2 activity at the one-cell stage. We propose that mbk-2 potentiates the degradation of MEI-1 and other proteins, possibly via direct phosphorylation.     

Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

Background

Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, themerAgene coding for the mercuric reductase. We report on the development of a profiling method formerAand its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation.

Results

Based on an alignment of 30merAsequences from Gram negative bacteria, conserved primers were designed for amplification ofmerAfragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with differentmerAsequences. ThemerAprofiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of themerAcommunity profile was also detected in a biocatalyzer effluent isolate, which was identified asPseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing.

Conclusions

ThemerAprofiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducingPs. aeruginosastrain was identified by its unique mercuric reductase gene.     

A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 3. Monitoring oxidative processes

The fluorescence-based CCOA method for determination of carbonyl group profiles in cellulosic substrates was employed to study the mechanisms of various oxidative and degradation processes involving celluloses in greater detail. The approach comprises labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide (CCOA), followed by gel permeation chromatography in DMAc/LiCl with fluorescence, multiangle laser light scattering, and refractive index detection. At first, the CCOA method was applied to study solutions of pulp in N-methylmorpholine-N-oxide monohydrate (NMMO), as occurring in the production of Lyocell-type fibers. NMMO is a rather strong oxidant that on one hand converts reducing end groups to carboxyl structures, thus lowering the overall carbonyl content, but generates new keto structures along the chain by nonselective oxidation on the other hand. The CCOA method allowed for the first time to distinguish the carbonyl course in different molecular weight ranges. Second, alkalization and aging of pulp, which are used in the industrial preparation of cellulose derivatives, e.g., as an element of the preripening process in viscose rayon production, were investigated. The CCOA method shows a clear reduction of the molecular weight, accompanied by a fast loss of carbonyls in the first phase, which is due to removal of low-molecular weight material by dissolution, and a slow decrease in the second phase, which is caused by further oxidation of carbonyl groups. Also here, differences in the carbonyl course in different molecular weight regions were monitored. Third, electron beaming, proposed as a means of pulp activation, was shown to decrease and narrow the molecular weight distribution, under generation of comparatively low amounts of carbonyls, which, however, are also introduced into high molecular weight, crystalline domains, as shown by a comparison of homogeneous and heterogeneous CCOA labeling approach. Finally, as the fourth application, thermal treatment of cellulose at temperatures between 105 and 165 degrees C was shown to bring about a small reduction of the molecular weight, which only at higher drying temperatures is accompanied by an introduction of carbonyls over the whole molecular weight range.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane, cancer-associated isozyme IX with lipophilic sulfonamides

A series of new compounds was obtained by reaction of aromatic/heterocyclic sulfonamides incorporating amino groups with N,N-diphenylcarbamoyl chloride and diphenylacetyl chloride. These sulfonamides were assayed for the inhibition of three carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic CA I and CA II, and the transmembrane, cancer-associated isozyme CA IX. Good inhibitors against all these isoforms were detected, and the inhibition profile of the newly investigated isozyme IX was observed to be different from that of the cytosolic isozymes, I and II. This may lead to the development of novel anticancer therapies based on the selective inhibition of CA IX.     

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating succinyl hydroxamate and iminodiacetic acid hydroxamate moieties

A series of succinyl hydroxamates/bishydroxamates as well as a new structural type of matrix metalloproteinase (MMP)/bacterial protease (BP) inhibitors, incorporating iminodiacetic (IDA) hydroxamate/bishydroxamate moieties, has been synthesized and tested for interaction with four vertebrate proteases, MMP-1, MMP-2, MMP-8 and MMP-9, and a BP, the collagenase isolated from Clostridium histolyticum (ChC). The new derivatives generally showed inhibition constants in the range of 8-62 nM against the five proteases mentioned above.