DFNB44, a novel autosomal recessive non-syndromic hearing impairment locus, maps to chromosome 7p14.1-q11.22

The genetic etiology for many forms of hearing impairment (HI) is very diverse. Non-syndromic HI (NSHI) is one of the most heterogeneous traits known. Autosomal recessive forms of prelingual HI account for approximately 75% of hereditary cases. A novel autosomal recessive NSHI locus, DFNB44, was mapped to a 20.9 cM genetic interval on chromosome 7p14.1-q11.22, according to the Marshfield genetic map, in a consanguineous Pakistani family. Multipoint linkage analysis resulted in a maximum LOD score of 5.0 at marker D7S1818. The 3-unit support interval ranged from marker D7S2209 to marker D7S2435, spanning a 30.1 Mb region on the sequence-based physical map.     

Curtisians E-H: four p-terphenyl derivatives from the inedible mushroom Paxillus curtisii

Four p-terphenyl derivatives named curtisians E-H (1-4) were isolated from the methanolic extract of fruit bodies of the Basidiomycete Paxillus curtisii by a combination of Sephadex LH-20, silica gel column chromatography and preparative reversed phase HPLC. The relative stereochemistries of the curtisians were elucidated by 2D NMR, MS, IR and UV spectroscopy with the absolute configurations at C(3a-3d) of the side chains being established as S by GC-MS on a chiral column previously used for separation of authentic standards.     

Thelephantins A,B and C: three benzoyl p-terphenyl derivatives from the inedible mushroom Thelephora aurantiotincta

Three benzoyl p-terphenyl derivatives named thelephantins A, B and C were isolated from the ethyl acetate extract of fruit bodies of the Thelephoraceous Basidiomycete Thelephora aurantiotincta. Their structures were elucidated by analysis of high-resolution 2D NMR, MS, IR and UV spectra.     

Cadherin-mediated cell-cell adhesion: sticking together as a family

The cadherins comprise a family of single-pass transmembrane proteins critical for cell-cell adhesion in vertebrates and invertebrates. The recently determined structure of the whole ectodomain from C-cadherin suggests that the adhesion of cadherins presented by juxtaposed cells is mediated by a strand-swapped dimer in which core hydrophobic elements are exchanged between the partner molecules. Sequence analysis suggests that several cadherin subfamilies share this adhesive mechanism. Recent work has shed new light on the molecular basis of cadherin adhesion, although understanding the specificity of these interactions remains a major challenge.     

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO. Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. We have conducted a modular P-element based EP (enhancer/promoter) misexpression screen to look for genes involved in Apterous activity. We have found Osa, a member of the Brahma chromatin-remodeling complex, as a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. We propose that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression.     

Hemoglobin Einstein: semisynthetic deletion in the B-helix of the alpha-chain

The influence of the deletion of the tetra peptide segment alpha(23-26) of the B-helix of the alpha-chain of hemoglobin-A on its assembly, structure, and functional properties has been investigated. The hemoglobin with the deletion, ss-Hemoglobin-Einstein, is readily assembled from semisynthetic alpha(1-141) des(23-26) globin and human betaA-chain. The deletion of alpha(23-26) modulates the O2 affinity of hemoglobin in a buffer/allosteric effector specific fashion, but has little influence on the Bohr effect. The deletion has no influence on the thermodynamic stability of the alpha1beta1 and the alpha1beta2 interface. The semisynthetic hemoglobin exhibits normal intersubunit interactions at the alpha1beta1 and alpha1beta2 interfaces as reflected by 1H-NMR spectroscopy. Molecular modeling studies of ss-Hemoglobin-Einstein suggest that the segment alpha(28-35) is in a helical conformation, while the segment alpha(19-22) is the nonhelical AB region. The shortened B-helix conserves the interactions of alpha1beta1 interface. The results demonstrate a high degree of plasticity in the hemoglobin structure that accommodates the deletion of alpha(23-26) without perturbing its overall global conformation.     

Effects of cell tension on the small GTPase Rac

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.     

Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.