The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

Background  

SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3).     

Control of glycolysis in magnesium deficiency: studies on intact red cells and hemolysates]

The behaviour of glycolytic flux and glycolytic metabolic concentrations was studied under conditions of magnesium deficiency. The Mg-deficiency was produced in whole animals (rats) by feeding a diet almost completely free of Mg and in hemolysates of men by the addition of a chelating agent. The results show that the decrease of the free Mg-level is diminished by partial destruction of ATP and 2,3-DPG. The analysis of the control strength of the overall flux leads to the conclusion that the decrease of the glycolytic rate is caused by an inhibition of the hexokinase-phosphofructokinase-control system. The decrease of the MgATP-Complex and free Mg++-level explains the diminished phosphorylation of glucose by the hexokinase. The ATP-inhibition of the phosphofructokinase is amplified by a small increase of free ATP-concentration and a simultaneous decrease of the Fru-6P-level. The increase of the PEP-level is caused by the diminished free Mg++ and MgATP-complex and does not demonstrate a larger control strength of the pyruvate kinase.     

Energy metabolism of erythrocytes in pyruvate kinase enzymopathies

Until now pyruvate kinase enzymopathies have been described only for red blood cells. On the basis of these results special structural properties of the erythrocyte PK was assumed, which are not yet totally established. PK defects may cause a nonspherocytic hemolytic anemia. This enzymopathy is characterized by a polymorphism, which is expressed in more than 5 different pathological variants. Up to now 16 cases of PK deficiency have ben diagnozed in the GDR. The following parameters are used for the characterization of the PK: the PEP-dependance, the inhibition by ATP and alanine, the specificity to nucleotides, the stability to temperature and urea and the maturation dependence. Two pathological variants of the PK with a decreased PEP-affinity are described. Furthermore the differences in the energy metabolism of the red blood cells of these two patients under aerobic and anaerobic conditions are discussed.     

Elasticity and adhesion of resting and lipopolysaccharide-stimulated macrophages

Colloidal Force Microscopy was employed to study the viscoelastic and adhesive properties of macrophages upon stimulation with lipopolysaccharide (LPS). Force vs. distance measurements were performed. The adhesion of LPS-stimulated cells (separation force=37+/-3 nN) was almost twice as high as that of resting macrophages (16+/-1 nN). Upon retraction pulling of membrane tethers was observed. Tether lengths and forces at which rupture take place did not depend on stimulation. The reduced Young's modulus K, a measure of cytoskeleton elasticity, was three times lower than that of the control. The data show that LPS has profound effects on cytomechanical and adhesion properties of macrophages.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

Insights into the biodiversity of the Succulent Karoo hotspot of South Africa: the population genetics of a rare and endemic halictid bee,Patellapis doleritica

Population genetic analyses are especially relevant for species considered threatened or highly endemic and for which other forms of biological information are lacking. Patellapis doleritica is a recently described communally nesting halictid bee of conservation concern because it is rare and endemic to the Succulent Karoo of South Africa. Moreover, its dispersal is considered to be restricted by its specialised nesting requirements and inclement weather conditions during its limited annual flight period, traits which may be common to other bee species of the region. We hypothesised that gene flow in P. doleritica was low, leading to marked genetic differentiation. Using 7 microsatellites, we investigated its mating and population genetic structure in 258 individuals (171 females and 87 males) from 7 populations spanning most of its known range. Deviation from Hardy–Weinberg equilibrium (F_IS = + 0.254) suggested P. doleritica to be inbred, as in many other communal nesting bee species. Global F_ST (0.028) and global G′_ST (0.216) revealed modest but significant differentiation between most populations, even across the very limited range of the species (ca. 25 km), with one genetically extreme outlier population. Despite inbreeding, we detected a surprisingly low frequency of diploid males (2 %). Patellapis doleritica nevertheless deserves special conservation attention since it is an endemic species with a low overall abundance and therefore possibly prone to environmental change and local extinction.     

Increased cardenolides production by elicitation of Digitalis lanata shoots cultured in temporary immersion systems

Digitalis lanata is an important source of cardenolides such as digoxin and lanatoside C, which have been widely applied in the treatment of cardiac insufficiencies. Elicitation is one of the most effective methods to enhance the biosynthesis of several secondary metabolites in medicinal plants. We studied the effect of elicitation with Chitoplant^?, Silioplant^? and methyl jasmonate on biomass and cardenolides accumulation in shoots of D. lanata cultivated in temporary immersion systems. Morphological response of the shoots was influenced by elicitors. A reduction in length and number of shoots was evident with all MJ concentrations. Regarding biomass production, Chitoplant^? (0.1?g?l^?1) was found to impact significantly on fresh and dry weight of the shoots. HPLC analysis revealed a higher content of lanatoside C compared to digoxin in all treatments. The highest accumulation of lanatoside C was achieved with Chitoplant^? (0.1?g?l^?1), which resulted in 316???g?g-DW^?1 and with Silioplant^? (0.01?g?l^?1; 310???g?g-DW^?1), which accounted for a 2.2-fold increase in lanatoside C content compared to non-elicited shoot cultures. Additionally, elicitation of D. lanata shoots in temporary immersion systems resulted in an oxidative stress characterized by hydrogen peroxide and malondialdehyde accumulation. These observations point to a connection between hydrogen peroxide generation, lipid peroxidation and cardenolide accumulation. The optimization of elicitor treatment and culture conditions for cardenolide production as well as the advantages of TIS for this purpose are discussed.     

Enemy at the Gates: Interaction-Specific Stomatal Responses to Pathogenic Challenge

Stomata regulate gas exchange and their closure in response to pathogens may, in some cases, contribute to resistance. However, in the cereal mildew and rust systems, stomatal closure follows establishment of compatible infections. In incompatible systems, expression of major (R) gene controlled hypersensitive responses (HR), causes drastic, permanent stomatal dysfunction: stomata become locked open following powdery mildew attack and locked shut following rust attack. Thus, stomatal locking can be a hitherto unsuspected negative consequence of R gene resistance that carries a physiological cost affecting plant performance.Key Words: stomata, rust, mildew, hypersensitive response, stomatal lock-up