Comprehensive Analysis of HAMP Domains: Implications for Transmembrane Signal Transduction

Homodimeric receptors with one or two transmembrane (TM) segments per monomer are universal to life and represent the largest and most diverse group of cellular TM receptors. They frequently share domain types across phyla and, in some cases, have been recombined experimentally into functional chimeras (e.g., the bacterial aspartate chemoreceptor with the human insulin receptor), suggesting that they have a common mechanism. The nature of this mechanism, however, is still being debated. We have proposed a new model for transduction mechanism by axial helix rotation, based on the structure of a widespread domain, HAMP, that frequently occurs in direct continuation of the last TM segment, primarily in histidine kinases and chemoreceptors. Here we show by statistical analysis that HAMP domain sequences have biophysical properties compatible with the two conformations proposed by the model. The analysis also identifies three networks of coevolving residues, which allow the mechanism to subdivide into individual steps. The most extended of these networks is specific for membrane-bound HAMP domains and most likely accepts the signal from the TM helices. In a classification based on sequence clustering, these HAMPs form a central supercluster, surrounded by smaller clusters of divergent HAMPs, which typically combine into arrays of up to 31 consecutive copies and accept conformational input from other HAMP domains. Unexpectedly, the classification shows a division between domains of histidine kinases and those of chemoreceptors; thus, except for a few versatile lineages, HAMP domains are largely specific for one particular output domain. Within proteins using a given output domain, HAMP domains also show extensive coevolution with histidine kinases, but not with chemoreceptors. We attribute the greater capability for recombination among chemoreceptors to their acquisition of a reversible modification system, which acts as a capacitor for the initially deleterious effects of combining domains optimized in different contexts.     

Increase of the IL-1 beta and IL-6 levels in CSF in patients with vasospasm following aneurysmal SAH

Cytokines play a key role in mutual influence of the immunological, endocrine and CNS systems. It has been proven that proinflammatory ILs may intensify the cascade of biochemical changes in ischemic brain damage. Vasospasm, which may accompany SAH and often coexists with symptoms of DINDs, is the cause of ischemic changes in the brain. It is thought that immunological mechanisms may be one of the causes of degenerative-productive changes in vessel walls, in delayed vasospasm following SAH, which lead to substantial vasospasm and in consequence too cerebral ischemia. In the randomly selected group of patients, who underwent surgical treatment after aneurysmal SAH, we determined the concentration of IL-1 beta and IL-6 in CSF in the periods between Days 0 to 3; 4 to 7; and 8 to 15 after the occurrence of SAH. The presence and dynamics of development of vasospasm were assessed on the basis of increasing DINDs as well as CT and cerebral angiography. We examined the concentrations of ILs in CSF using radioimmunological methods, applying commercially available tests for their assessment. We found that in the period between 8 and 15 days after SAH, in increasing delayed vasospasm and DINDs, here is a statistically significant increase concentration of IL-1 beta in CSF (105.4 +/- 46.9 pg x ml-1; p<0.005), and no significant changes in patients without vasospasm and neurological deficits. On the other hand, we noted a statistically significant increase concentration of IL-6 in CSF (4802 +/- 1170 ng x ml-1; p<0.05) only in the acute phase after SAH (Days 0-3) in patients in poor clinical condition, in whom delayed vasospasm and cerebral ischemia developed later. This increase of ILs level in CSF is probably related to the intensity of the SAH, and secondarily aggravates the vasospasm and ischemic changes in the brain.     

Bovine mu-calpain (CAPN1) gene: new SNP within intron 14

The calpain system originally comprised molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. This proteolytic system plays a key role in the tenderisation process that occurs during post-mortem storage of meat under refrigerated conditioning. Their polymorphism is examined from the point of view of their effect on corresponding production traits. The calpain genes are investigated as potential candidate genes for a quantitative trait locus (QTL) affecting meat tenderness. In this study a new single nucleotide polymorphism (SNP) was found within intron 14 of the bovine CAPN1 gene, being transition C --> T at position 4685 nt (consensus sequence - GenBank No. AF 248054), as this mutation creates a new FokI restriction site detected with PCR-RFLP analysis. This sequence fragment of the SNP position has already been deposited in the GenBank database under accession No. AY639597. The RFLP-FokI polymorphism was studied in 141 bulls of seven breeds, including the native Polish Red (PR, preserved), and Polish Black-and White (BW) breed. The frequency of alleles T and C varied between the breeds considered, the mean reaching 0.38 and 0.62, respectively. Associations between CAPN1/FokI gene polymorphism and meat production traits were studied in BW (n = 84) young bulls. In the animals of the TT genotype the lean share in valuable cuts (%) was found more favourable than in CC animals.     

Why are diploid genomes widespread and dominant mutations rare?

We have used the sexual Penna ageing model to show that the relation between dominance and recessiveness could be a force which optimizes the genome size. While the possibility of complementation of the damaged allele by its functional counterparts (recessiveness) leads to the redundancy of genetic information, the dominant effect of defective genes tends to diminish the number of alleles fulfilling the same function. Playing with the fraction of dominant loci in the genome it is possible to obtain the condition where the diploid state of the genome is optimal. If the status of each bit position as dominant or recessive mutations is changed for each individual randomly and rarely, then after a long time a stationary equilibrium of many recessive and few dominant loci is established in the sexual Penna model. This effect vanishes if the same changing distribution of dominant loci applies to all individuals.     

Substituted benzoxazinones as potent positive allosteric AMPA receptor modulators: part II

AMPA receptors (AMPARs) are an important therapeutic target in the CNS. A series of substituted benzoxazinone derivatives with good to very good in vitro activity as positive allosteric AMPAR modulators was synthesized and evaluated. The appropriate substituent choice on the benzoxazinone fragment improved the affinity towards the AMPA receptor significantly in comparison to our lead molecule CX614.     

Influence of proteolytic enzymes and calcium-binding agents on nuclear and cell surface topography

Summary Transmission electron microscopy was used to study the effects of proteolytic enzymes (collagenase, trypsin, clostripain), the calcium chelator ethyleneglycol-bis-(-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), and the calcium ionophore A 23187 on substrate adhesion and fine structure of chondrocytes and fibroblasts. Monolayer cultured cells responded to treatment with the proteolytic enzymes followed by EGTA or A 23187 by rounding and detaching from the substrate. This was accompanied by the formation of a microvillous surface, deep nuclear folds, and numerous cytoplasmic vacuoles. Labeling experiments with colloidal thorium dioxide indicated that the vacuoles were formed by endocytosis and fusion of endocytic vesicles with preexisting lysosomes. To a variable extent, similar changes were produced by trypsin or EGTA alone. The cells regained their normal fine structure after withdrawal of the reagents and when seeded onto a substrate. In suspension culture, recovery was incomplete; the cells retained a rounded shape and an increased number of cytoplasmic vacuoles.The results suggest that changes in plasma membrane composition and its permeability to calcium represent the primary signal for cell rounding and detachment. The cellular mechanisms responsible for the associated folding of the nuclear envelope and the cell surface remain unidentified. Nevertheless, this is believed to represent a means of handling of excess membrane during sudden transition from a flattened to a rounded shape. Membrane stored in folds and vacuoles is reutilized when the cells reattach and spread out on a substrate.Expert technical assistance was provided by Karin Blomgren and Anne-Marie Motakefi. Financial support was obtained from the Swedish Medical Research Council (06537), the King Gustaf V 80th Birthday Fund and from the Funds of Leiden University     

Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis

Sudden exposure of plants to high light (HL) leads to metabolic and physiological disruption of the photosynthetic cells. Changes in ROS content, adjustment of photosynthetic processes and the antioxidant pools and, ultimately, gene induction are essential components for a successful acclimation to the new light conditions. The influence of salicylic acid (SA) on plant growth, short-term acclimation to HL, and on the redox homeostasis of Arabidopsis thaliana leaves was assessed here. The dwarf phenotype displayed by mutants with high SA content (cpr1-1, cpr5-1, cpr6-1, and dnd1-1) was less pronounced when these plants were grown in HL, suggesting that the inhibitory effect of SA on growth was partly overcome at higher light intensities. Moreover, higher SA content affected energy conversion processes in low light, but did not impair short-term acclimation to HL. On the other hand, mutants with low foliar SA content (NahG and sid2-2) were impaired in acclimation to transient exposure to HL and thus predisposed to oxidative stress. Low and high SA levels were strictly correlated to a lower and higher foliar H(2)O(2) content, respectively. Furthermore high SA was also associated with higher GSH contents, suggesting a tight correlation between SA, H(2)O(2) and GSH contents in plants. These observations implied an essential role of SA in the acclimation processes and in regulating the redox homeostasis of the cell. Implications for the role of SA in pathogen defence signalling are also discussed.     

A macrokinetic modelling of the biosynthesis of lovastatin by Aspergillus terreus

In this work a simple kinetic model to describe the biosynthesis of lovastatin by Aspergillus terreus ATCC 20542 was proposed. Several series of experiments were conducted at different media compositions. The concentrations of C- and N-sources were changed over a wide range and so were the initial biomass concentrations. From these runs the relationships ruling the substrates uptake, biomass and product formation were learnt. Lovastatin biosynthesis appeared to be partly growth associated. The inhibitive effect of organic nitrogen on lovastatin biosynthesis was found and lactose appeared to be an important limiting substrate in the formation of lovastatin. The parameters of the model were evaluated on the basis of the kinetic data obtained in the separate experiments made in triplicate at two chosen media compositions. Other results obtained at different media compositions were independent of the ones mentioned above and used for the verification of the model. The validity of the model was also examined for the lactose-fed fed-batch run. Finally, a sensitivity analysis of the model parameters was performed. The formulated model, although relatively simplified, described the experimental data quite well and could be regarded as the background for further attempts to mathematically describe the process of lovastatin biosynthesis.