Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.     

BDNF-overexpression regulates the reactivity of small pulmonary arteries to neurokinin A

Brain-derived neurotrophic factor (BDNF) is a key modulator during the development of jugular and nodose ganglia neurons, which represent the origin of a large proportion of the sensory innervation of the lung. It belongs to the family of neurotrophins, which have been shown to induce the expression of tachykinins. To assess the interactions of BDNF and the tachykinin neurokinin A (NKA) in small pulmonary vessels, BDNF-transgenic mice were examined for tachykinin contents in the airways, heart, trigeminal ganglion and jugular and nodose ganglion complex (JNC) using reverse phase HPLC (rpHPLC) and radioimmunoassay. BDNF-overexpression led to increased NKA levels in the heart and the JNC, whereas only slightly enhanced levels in the trigeminal ganglion were detected. Lower NKA levels were found in the lung. To assess vasoreactivity in small arteries, precision cut lung slices were subjected to videomorphometry and the response to NKA was examined, which showed significantly stronger effects in the BDNF-transgenic mice, while NK-2 receptor mRNA expression, assayed by real-time RT-PCR, was reduced. In conclusion, BDNF-overexpression results in decreased levels of NKA in the lung with subsequently increased NKA-sensitivity of small arteries. These findings point to a modulatory role of neurotrophins in small respiratory vessel tone regulation.     

Molecular basis of bacterial resistance to chloramphenicol and florfenicol

Chloramphenicol (Cm) and its fluorinated derivative florfenicol (Ff) represent highly potent inhibitors of bacterial protein biosynthesis. As a consequence of the use of Cm in human and veterinary medicine, bacterial pathogens of various species and genera have developed and/or acquired Cm resistance. Ff is solely used in veterinary medicine and has been introduced into clinical use in the mid-1990s. Of the Cm resistance genes known to date, only a small number also mediates resistance to Ff. In this review, we present an overview of the different mechanisms responsible for resistance to Cm and Ff with particular focus on the two different types of chloramphenicol acetyltransferases (CATs), specific exporters and multidrug transporters. Phylogenetic trees of the different CAT proteins and exporter proteins were constructed on the basis of a multisequence alignment. Moreover, information is provided on the mobile genetic elements carrying Cm or Cm/Ff resistance genes to provide a basis for the understanding of the distribution and the spread of Cm resistance--even in the absence of a selective pressure imposed by the use of Cm or Ff.     

Quantify this! Report on a round table discussion on quantitative mass spectrometry in proteomics

Following the success of the first round table in 2001, the Swiss Proteomic Society has organized two additional specific events during its last two meetings: a proteomic application exercise in 2002 and a round table in 2003. Such events have as their main objective to bring together, around a challenging topic in mass spectrometry, two groups of specialists, those who develop and commercialize mass spectrometry equipment and software, and expert MS users for peptidomics and proteomics studies. The first round table (Geneva, 2001) entitled "Challenges in Mass Spectrometry" was supported by brief oral presentations that stressed critical questions in the field of MS development or applications (St?cklin and Binz, Proteomics 2002, 2, 825-827). Topics such as (i) direct analysis of complex biological samples, (ii) status and perspectives for MS investigations of noncovalent peptide-ligant interactions; (iii) is it more appropriate to have complementary instruments rather than a universal equipment, (iv) standardization and improvement of the MS signals for protein identification, (v) what would be the new generation of equipment and finally (vi) how to keep hardware and software adapted to MS up-to-date and accessible to all. For the SPS'02 meeting (Lausanne, 2002), a full session alternative event "Proteomic Application Exercise" was proposed. Two different samples were prepared and sent to the different participants: 100 micro g of snake venom (a complex mixture of peptides and proteins) and 10-20 micro g of almost pure recombinant polypeptide derived from the shrimp Penaeus vannamei carrying an heterogeneous post-translational modification (PTM). Among the 15 participants that received the samples blind, eight returned results and most of them were asked to present their results emphasizing the strategy, the manpower and the instrumentation used during the congress (Binz et. al., Proteomics 2003, 3, 1562-1566). It appeared that for the snake venom extract, the quality of the results was not particularly dependant on the strategy used, as all approaches allowed Lication of identification of a certain number of protein families. The genus of the snake was identified in most cases, but the species was ambiguous. Surprisingly, the precise identification of the recombinant almost pure polypeptides appeared to be much more complicated than expected as only one group reported the full sequence. Finally the SPS'03 meeting reported here included a round table on the difficult and challenging task of "Quantification by Mass Spectrometry", a discussion sustained by four selected oral presentations on the use of stable isotopes, electrospray ionization versus matrix-assisted laser desorption/ionization approaches to quantify peptides and proteins in biological fluids, the handling of differential two-dimensional liquid chromatography tandem mass spectrometry data resulting from high throughput experiments, and the quantitative analysis of PTMs. During these three events at the SPS meetings, the impressive quality and quantity of exchanges between the developers and providers of mass spectrometry equipment and software, expert users and the audience, were a key element for the success of these fruitful events and will have definitively paved the way for future round tables and challenging exercises at SPS meetings.     

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O₂ ^•-) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O₂ ^•- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O₂ ^•- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O₂ ^•- than those from controls. Nifedipine treatment considerably decreased O₂ ^•- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O₂ ^•- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O₂ ^•- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.     

Cre recombinase-mediated gene targeting of mesenchymal cells

Loss-of-function approaches by the Cre/loxP technology have provided powerful tools for functional analyses of genes of interest expressed preferentially in a particular tissue. Here we describe the generation of transgenic mouse lines expressing Cre recombinase under the control of the promoter/enhancer unit of the gene for the alpha2 chain of collagen type I (Col1alpha2). As an expression vector, we used a P1-derived artificial chromosome (PAC), which harbors approximately 100 kb carrying the col1alpha2 gene. The improved coding sequence of the Cre recombinase was introduced to replace the first exon of col1alpha2. Cre expression was determined by immunohistochemistry and Cre-mediated onset of beta-galactosidase expression in ROSA26R-Cre reporter mice. In four analyzed transgenic lines, Cre recombinase was efficiently expressed during embryogenesis and in adult animals in cells of mesenchymal origin, such as dermal fibroblasts, mesenchymal cells of blood vessel walls, and cells in fibrous connective tissues surrounding internal organs.     

The ability of protein tyrosine phosphatase SHP-1 to suppress NFkappaB can be inhibited by dominant negative mutant of SIRPalpha

In contrast with hematopoietic cells and fibroblasts, which express mainly one form of protein tyrosine phosphatase (PTP) SHP-1 or SHP-2, epithelial cells like A431, HeLa, and 293 express both forms of PTP. These two PTP regulate NFkappaB activity differently; SHP-1 inhibits and SHP-2 stimulates NFkappaB activation. In epithelial cells the process of NFkappaB activation depends on the combination of two PTP activities. The activity of PTP SHP-1 dominates in this tandem according to our data. The signal regulatory protein (SIRPalpha) is the adapter and the substrate of PTP SHP-1 and SHP-2. We investigated the role of SIRPalpha and its dominant negative mutant in PTP activities in 293 cells. The overexpression of wild-type SIRPalpha suppresses the activities of both PTP, but has a stronger effect on PTP SHP-2, especially when this protein is overexpressed in 293 cells. In contrast with wild-type SIRPalpha, its dominant negative mutant acts predominantly against PTP SHP-1, and can be detected in the complex with PTP SHP-1. The expression of dominant negative mutant of SIRPalpha has an effect similar to the expression of dominant negative PTP SHP-1 in the process of NFkappaB activation.     

Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX

We developed an integrated method to identify aptamers with only 10 fixed nucleotides through ligation and removal of primer binding sites within the systematic evolution of ligands by exponential enrichment (SELEX) process. This Tailored-SELEX approach was validated by identifying a Spiegelmer (‘mirror-image aptamer’) that inhibits the action of the migraine-associated target calcitonin gene-related peptide 1 (α-CGRP) with an IC₅₀ of 3 nM at 37°C in cell culture. Aptamers are oligonucleotide ligands that can be generated to bind to targets with high affinity and specificity. Stabilized aptamers and Spiegelmers have shown activity in vivo and may be used as therapeutics. Aptamers are isolated by in vitro selection from combinatorial nucleic acid libraries that are composed of a central randomized region and additional fixed primer binding sites with ~30–40 nt. The identified sequences are usually not short enough for efficient chemical Spiegelmer synthesis, post-SELEX stabilization of aptamers and economical production. If the terminal primer binding sites are part of the target recognizing domain, truncation of aptamers has proven difficult and laborious. Tailored-SELEX results in short sequences that can be tested more rapidly in biological systems. Currently, our identified CGRP binding Spiegelmer serves as a lead compound for in vivo studies.     

Histone modifications: an assembly line for active chromatin?

Histone modifications play a major role in the control of genes in their natural chromatin context. A detailed, time-resolved analysis of histone modifications during gene activation has now revealed the order of appearance of particular histone modifications on a promoter during regulatory processes.