In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

Variance Heterogeneity in Saccharomyces cerevisiae Expression Data: Trans-Regulation and Epistasis

Here, we describe the results from the first variance heterogeneity Genome Wide Association Study (VGWAS) on yeast expression data. Using this forward genetics approach, we show that the genetic regulation of gene-expression in the budding yeast, Saccharomyces cerevisiae, includes mechanisms that can lead to variance heterogeneity in the expression between genotypes. Additionally, we performed a mean effect association study (GWAS). Comparing the mean and variance heterogeneity analyses, we find that the mean expression level is under genetic regulation from a larger absolute number of loci but that a higher proportion of the variance controlling loci were trans-regulated. Both mean and variance regulating loci cluster in regulatory hotspots that affect a large number of phenotypes; a single variance-controlling locus, mapping close to DIA2, was found to be involved in more than 10% of the significant associations. It has been suggested in the literature that variance-heterogeneity between the genotypes might be due to genetic interactions. We therefore screened the multi-locus genotype-phenotype maps for several traits where multiple associations were found, for indications of epistasis. Several examples of two and three locus genetic interactions were found to involve variance-controlling loci, with reports from the literature corroborating the functional connections between the loci. By using a new analytical approach to re-analyze a powerful existing dataset, we are thus able to both provide novel insights to the genetic mechanisms involved in the regulation of gene-expression in budding yeast and experimentally validate epistasis as an important mechanism underlying genetic variance-heterogeneity between genotypes.     

Catalytic Turnover Triggers Exchange of Subunits of the Magnesium Chelatase AAA+ Motor Unit

The ATP-dependent insertion of Mg²⁺ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg²⁺ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity.     

Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.     

The Effect on Rat Embryonic Heart Rate of Na+, K+, and Ca2+ Channel Blockers,and the Human Teratogen Phenytoin,Changes with Gestational Age

In this study, we compared the effects of four ion channel blockers on rat embryonic heart function during the organogenic period from gestational day (GD) 10 to 15, to determine the changes in dependence on ion channels during rat cardiac development. Rat embryos in culture were exposed to either the human ether‐á‐go‐go‐related gene potassium channel blocker, dofetilide (400 nM); the sodium channel blocker, lidocaine (250 μM); the L‐type calcium channel blocker, nifedipine (1.8 μM); or the multichannel blocker, phenytoin (200 μM). Lidocaine slowed the heart rate (HR) with the effect becoming more severe with increasing GD. Dofetilide slowed the embryonic HR and caused arrhythmias with the most severe effect on GD 11 to 13. Nifedipine primarily caused a negative inotropic effect except on GD 10 when it stopped the heart in most embryos. Phenytoin stopped the heart of most GD 10 to 12 embryos while on GD 13 to 15 phenytoin slowed the heart. The results demonstrate that as the rat heart develops during the organogenic period its functional dependence on ion channels changes markedly. These changes are important for understanding drug effects on the embryo during pregnancy and the methodology used provides a simple procedure for assessing drug effects on the developing heart.     

SSF of steam-pretreated wheat straw with the addition of saccharified or fermented wheat meal in integrated bioethanol production

Background

Integration of second-generation (2G) bioethanol production with existing first-generation (1G) production may facilitate commercial production of ethanol from cellulosic material. Since 2G hydrolysates have a low sugar concentration and 1G streams often have to be diluted prior to fermentation, mixing of streams is beneficial. Improved ethanol concentrations in the 2G production process lowers energy demand in distillation, improves overall energy efficiency and thus lower production cost. There is also a potential to reach higher ethanol yields, which is required in economically feasible ethanol production. Integrated process scenarios with addition of saccharified wheat meal (SWM) or fermented wheat meal (FWM) were investigated in simultaneous saccharification and (co-)fermentation (SSF or SSCF) of steam-pretreated wheat straw, while the possibility of recovering the valuable protein-rich fibre residue from the wheat was also studied.

Results

The addition of SWM to SSF of steam-pretreated wheat straw, using commercially used dried baker’s yeast, S. cerevisiae, resulted in ethanol concentrations of about 60 g/L, equivalent to ethanol yields of about 90% of the theoretical. The addition of FWM in batch mode SSF was toxic to baker’s yeast, due to the ethanol content of FWM, resulting in a very low yield and high accumulation of glucose. The addition of FWM in fed-batch mode still caused a slight accumulation of glucose, but the ethanol concentration was fairly high, 51.2 g/L, corresponding to an ethanol yield of 90%, based on the amount of glucose added.In batch mode of SSCF using the xylose-fermenting, genetically modified S. cerevisiae strain KE6-12, no improvement was observed in ethanol yield or concentration, compared with baker’s yeast, despite the increased xylose utilization, probably due to the considerable increase in glycerol production. A slight increase in xylose consumption was seen when glucose from SWM was fed at a low feed rate, after 48 hours, compared with batch SSCF. However, the ethanol yield and concentration remained in the same range as in batch mode.

Conclusion

Ethanol concentrations of about 6% (w/v) were obtained, which will result in a significant reduction in the cost of downstream processing, compared with SSF of the lignocellulosic substrate alone. As an additional benefit, it is also possible to recover the protein-rich residue from the SWM in the process configurations presented, providing a valuable co-product.

     

B-Cell Epitopes in NTS-DBL1α of PfEMP1 Recognized by Human Antibodies in Rosetting Plasmodium falciparum

Plasmodium falciparum is the most lethal of the human malaria parasites. The virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1α-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1α of IT4_var60) expressed by parasite FCR3S1.2. The plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1α domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. The other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. In conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1α of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. The data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.     

Multi-seasonal barnacle (Balanus improvisus) protection achieved by trace amounts of a macrocyclic lactone (ivermectin) included in rosin-based coatings

Rosin-based coatings loaded with 0.1% (w/v) ivermectin were found to be effective in preventing colonization by barnacles (Balanus improvisus) both on test panels as well as on yachts for at least two fouling seasons. The leaching rate of ivermectin was determined by mass-spectroscopy (LC/MS-MS) to be 0.7?ng cm(-2) day(-1). This low leaching rate, as deduced from the Higuchi model, is a result of the low loading, low water solubility, high affinity to the matrix and high molar volume of the model biocide. Comparison of ivermectin and control areas of panels immersed in the field showed undisturbed colonisation of barnacles after immersion for 35 days. After 73 days the mean barnacle base plate area on the controls was 13?mm(2), while on the ivermectin coating it was 3?mm(2). After 388 days, no barnacles were observed on the ivermectin coating while the barnacles on the control coating had reached a mean of 60?mm(2). In another series of coated panels, ivermectin was dissolved in a cosolvent mixture of propylene glycol and glycerol formal prior to the addition to the paint base. This method further improved the anti-barnacle performance of the coatings. An increased release rate (3?ng cm(-2) day(-1)) and dispersion of ivermectin, determined by fluorescence microscopy, and decreased hardness of the coatings were the consequences of the cosolvent mixture in the paint. The antifouling mechanism of macrocyclic lactones, such as avermectins, needs to be clarified in further studies. Beside chronic intoxication as ivermectin is slowly released from the paint film even contact intoxication occurring inside the coatings, triggered by penetration of the coating by barnacles, is a possible explanation for the mode of action and this is under investigation.     

The matrilins: modulators of extracellular matrix assembly

The matrilins form a family of oligomeric extracellular adaptor proteins that are most strongly expressed in cartilage but also present in many other extracellular matrices. Matrilins bind to different types of collagen fibrils, to other noncollagenous proteins and to aggrecan. They thereby support matrix assembly by connecting fibrillar components and mediating interactions between these and the aggrecan gel. The binding avidity of a matrilin can be varied by alternative splicing, proteolytic processing and formation of homo- and heterooligomers. Such changes in matrilin structure may lead to a modulation of extracellular matrix assembly. Some matrilins bind weakly to α1β1 integrin and cell surface proteoglycans, but even though matrilins play a role in mechanotransduction and matrilin-3 activates the expression of osteoarthritis-associated genes the physiological relevance of matrilin-cell interactions is unclear. Matrilin knockout mice do not display pronounced phenotypes, which points to a redundancy within the protein family or with functionally related proteins. In man, dominant mutations in the von Willebrand factor A like domain of matrilin-3 lead to a protein retention in the endoplasmic reticulum that causes multiple epiphyseal dysplasia by initiating a cell stress response. In contrast, a mutation in an EGF domain of matrilin-3 that is associated with hand osteoarthritis and disc degeneration does not interfere with secretion but instead with extracellular assembly of matrix structures. In this review we summarize such information on matrilin structure and function that we believe is important for the understanding of extracellular matrix assembly and for deciphering pathophysiological mechanisms in diseases causing skeletal malformations or cartilage degeneration.     

Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.