The role of aquaporins in cellular and whole plant water balance

Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. More than 150 MIPs have been identified in organisms ranging from bacteria to animals and plants. In plants, aquaporins are present in the plasma membrane and in the vacuolar membrane where they are abundant constituents. Functional studies of aquaporins have hitherto mainly been performed by heterologous expression in Xenopus oocytes. A main issue is now to understand their role in the plant, where they are likely to be important both at the cellular and at the whole plant level. Plants contain a large number of aquaporin isoforms with distinct cell type- and tissue-specific expression patterns. Some of these are constitutively expressed, whereas the expression of others is regulated in response to environmental factors, such as drought and salinity. At the protein level, regulation of water transport activity by phosphorylation has been reported for some aquaporins.     

Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis

Development of the gastrointestinal (GI) tract depends on reciprocal epithelial-mesenchymal cell signaling. Here, we demonstrate a role for platelet-derived growth factor-A (PDGF-A) and its receptor, PDGFR-(alpha), in this process. Mice lacking PDGF-A or PDGFR-(alpha) were found to develop an abnormal GI mucosal lining, including fewer and misshapen villi and loss of pericryptal mesenchyme. Onset of villus morphogenesis correlated with the formation of clusters of PDGFR-(alpha) positive cells, 'villus clusters', which remained located at the tip of the mesenchymal core of the growing villus. Lack of PDGF-A or PDGFR-(alpha) resulted in progressive depletion of PDGFR-(alpha) positive mesenchymal cells, the formation of fewer villus clusters, and premature expression of smooth muscle actin (SMA) in the villus mesenchyme. We found that the villus clusters were postmitotic, expressed BMP-2 and BMP-4, and that their formation correlated with downregulated DNA synthesis in adjacent intestinal epithelium. We propose a model in which villus morphogenesis is initiated as a result of aggregation of PDGFR-(&agr;) positive cells into cell clusters that subsequently function as mesenchymal centers of signaling to the epithelium. The role of PDGF-A seems to be to secure renewal of PDGFR-(alpha) positive cells when they are consumed in the initial rounds of cluster formation.     

The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants

Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes. MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants. In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes. Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns. Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons. Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies. The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species. Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed. The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species.     

Natural phenological variation in aspen (Populus tremula): the SwAsp collection

The genus Populus is currently the main model system for genetic, genomic, and physiological research in trees. Phenotypic variation in aspen (Populus tremula) populations growing in different environments across Sweden is expected to reflect genetic variation that is important for local adaptation. To analyze such natural phenotypic and genetic variation, the Swedish Aspen (SwAsp) Collection was established. Trees were taken from 12 different populations across Sweden, from 56° to 66° latitude north and planted in two common gardens in Ekebo (55.9°N) and Sävar (63.4°N). Data related to phenological and growth traits were collected during the second year of growth. Some traits like the date of bud set and leaf area duration showed strong clinal variation patterns with latitude in both field trials, but the date of bud flush did not change along a latitudinal cline. The phenological traits showed moderate within-populations heritabilities, although growth traits showed weaker clinal patterns and lower heritabilities than the phenological traits. This research forms the starting point for the development of the SwAsp collection, a resource facilitating analysis of the natural genetic variation in aspen, the elucidation of the structure and dynamics of aspen populations, and the future identification of the genes controlling adaptive traits using association mapping of selected candidate genes.     

Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: adsorption, sugar production pattern, and synergism of the enzymes

Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40 degrees C) and enzyme loading (0.16 micromol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 micromol/g at 4 degrees C and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60-90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40 degrees C when the enzymes were added together they produced 27-45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4 degrees C, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11-15%, 82-89%, and <6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain.     

Gene expression in autumn leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.     

Criterion validation of surface EMG variables as fatigue indicators using peak torque: a study of repetitive maximum isokinetic knee extensions.

A number of studies have been published that have used variables of the electromyogram (EMG) power spectrum during dynamic exercise. Despite these studies there is a shortage of studies of the validity of surface EMG registrations during repetitive dynamic contractions with respect to fatigue. The aim of this study was to investigate if the surface EMG variables mean frequency (MNF [Hz]) and the signal amplitude (RMS [microV]) are valid indicators of muscular fatigue (defined as "any exercise-induced reduction in the capacity to generate force or power output") during maximum repeated isokinetic knee extensions (i.e. criterion validity using peak torque). Twenty-one healthy volunteers performed 100 isokinetic knee extensions at 90 degrees s(-1). EMG signals were recorded from the vastus lateralis, the rectus femoris and the vastus medialis of the right thigh by surface electrodes. MNF and RMS of the EMG together with peak torque (PT [Nm]) were determined for each contraction. MNF showed consequently higher correlation coefficients with PT than RMS did. Positive correlations generally existed between MNF and PT. The majority of the subjects had positive correlations between RMS and PT (i.e. decreases both in PT and in RMS).In conclusion, at the individual level MNF generally - in contrast to RMS - showed good criterion validity with respect to biomechanical fatigue during dynamic maximum contractions.     

Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors

There are five subtypes of muscarinic acetylcholine receptors (M(1) to M(5)) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M(1), M(2) and M(4) receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M(1), M(3) and M(4) receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M(1) and M(4) selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M(4) receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M(4) receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M(4) receptors for memory is that injection of the M(4) selective antagonist muscarinic toxin 3 (M(4)-toxin 1) into rat hippocampus produced amnesia.     

Separation of native and latent forms of human antithrombin by hydrophobic interaction high-performance liquid chromatography

Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains albumin as the main stabilizer. The AT was reconstituted and heat treated in order to generate L-AT. This latent form of AT has been shown to exhibit a strong antiangiogenic activity and also to suppress tumor growth. The HPLC system included a TSK Phenyl 5PW column and a segmented gradient, 4.5-0 mol/L sodium chloride. Antithrombin was eluted at about 13 min, and L-AT, at 30 min, corresponding to about 4.2 and 1.6 mol/L sodium chloride, respectively. A reference sample gave 42% L-AT when analyzed by the HIC method and 41% L-AT when analyzed by the heparin affinity chromatography method. The resolution between AT and L-AT was higher with the HIC method than with the heparin affinity method. Incubation of Atenativ at 45 degrees C for 15 h gave about 18% L-AT and was shown by native polyacrylamide gel electrophoresis to contain only monomeric AT. A good resolution between AT and L-AT, but not between albumin and L-AT, was also achieved by a linear gradient of 2-0 mol/L ammonium sulfate, in 25 mmol/L Tris/HCl, pH 8.0.     

Discovery and SAR studies of a novel series of noncovalent cathepsin S inhibitors

A novel series of competitive, reversible cathepsin S (CatS) inhibitors was discovered and optimized. The 4-(2-keto-1-benzimidazolinyl)-piperidin-1-yl moiety was found to be an effective replacement for the 4-arylpiperazin-1-yl group found in our earlier series of CatS inhibitors. This replacement imparted improved PK properties as well as decreased off-target activity. Optimization of the ketobenzimidazole moiety led to the discovery of the lead compound JNJ 10329670, which represents a novel class of selective, noncovalent, reversible, and orally bioavailable inhibitors of cathepsin S.