Integrins uncouple Src-induced morphological and oncogenic transformation

Expression of activated mutants of c-Src in epithelial cells can induce tumorigenicity. In addition to such oncogenic transformation, the cells undergo a dramatic morphological transformation: cell-cell contacts are disrupted, spreading on extracellular matrix proteins is suppressed, actin stress fibers and focal contacts are lost, and podosomes are formed. We have previously shown that integrin alphavbeta3 strongly supports Src-mediated oncogenic transformation through an interaction at the beta3 cytoplasmic tail. Our current findings demonstrate that this interaction does not affect Src-mediated morphological alterations, thus separating oncogenic from morphological transformation. Moreover, beta1 and beta3 integrins differently affect the various aspects of Src-induced morphological transformation. High levels of beta3, but not beta1, integrins can prevent Src-induced cell rounding although stress fiber disassembly and podosome formation still occur. Studies using chimeric integrin subunits demonstrate that this protection requires the beta3 extracellular domain. Finally, like tumor formation, podosome assembly occurs independent of beta3 phosphorylation. Instead, phosphorylation of beta1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins regulate Src-mediated oncogenic transformation and various aspects of morphological transformation through dissociable pathways.     

GJB2 and mitochondrial A1555G gene mutations in nonsyndromic profound hearing loss and carrier frequencies in healthy individuals

This study aimed to assess mutations in GJB2 gene (connexin 26), as well as A1555G mitochondrial mutation in both the patients with profound genetic nonsyndromic hearing loss and healthy controls. Ninety-five patients with profound hearing loss (>90 dB) and 67 healthy controls were included. All patients had genetic nonsyndromic hearing loss. Molecular analyses were performed for connexin 26 (35delG, M34T, L90P, R184P, delE120, 167delT, 235delC and IVS1+1 A-->G) mutations, and for mitochondrial A1555G mutation. Twenty-two connexin 26 mutations were found in 14.7% of the patients, which were 35delG, R184P, del120E and IVS1+1 A-->G. Mitochondrial A1555G mutation was not encountered. The most common GJB2 gene mutation was 35delG, which was followed by del120E, IVS1+1 A-->G and R184P, and 14.3% of the patients segregated with DFNB1. In consanguineous marriages, the most common mutation was 35delG. The carrier frequency for 35delG mutation was 1.4% in the controls. 35delG and del120E populations, seems the most common connexin 26 mutations that cause genetic nonsyndromic hearing loss in this country. Nonsyndromic hearing loss mostly shows DFNB1 form of segregation.     

Toward chaperone-assisted crystallography: protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold

A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (V(H)H) as a crystallization chaperone. By using a yeast surface display system for V(H)H, we successfully introduced additional Met residues in the core of the V(H)H scaffold. We identified a set of SeMet-labeled V(H)H variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched V(H)H chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and V(H)H model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for V(H)H, the enhanced V(H)H domain described here will be an excellent scaffold for producing effective crystallization chaperones.     

A novel and short synthesis of (1,4/2)-cyclohex-5-ene-triol and its conversion to (+/-)-proto-quercitol

(1,4/2)-cyclohex-5-ene-triol was synthesized starting from cyclohexa-1,4-diene with two different approaches. Photooxygenation of cyclohexa-1,4-diene and epoxy-cyclohexene afforded anti-2,3-dioxabicyclo[2.2.2]oct-7-en-5-yl hydroperoxide and anti-7-oxabicyclo[4.1.0]hept-4-en-3-yl hydroperoxide, respectively. Hydroperoxy endoperoxide was reduced with aqueous sodium bisulfite; hydroperoxy-epoxide with dimethylsulfide-titanium tetraisopropoxide to give 7-oxabicyclo[4.1.0]hept-4-en-3-ol. Acidic hydrolysis of the epoxy-alcohol gave the (1,4/2)-cyclohex-3-ene-triol. Oxidation of the double bond with KMnO4 resulted in the formation of (+/-)-proto-quercitol.     

Functional properties of threefold and fourfold channels in ferritin deduced from electrostatic calculations

The iron storage protein ferritin contains threefold and fourfold symmetric channels that are thought to provide pathways for the transfer of Fe(2+) ions in and out of the protein. Using the known crystal structure of the ferritin protein, we perform electrostatic potential energy calculations to elucidate the functional properties of these channels. The threefold channel is shown to be responsible for the transit of Fe(2+) ions. Monovalent ions can also diffuse through the threefold channel but presence of divalent ions in the pore retards this process leading to a selectivity mechanism similar to the one observed in calcium channels. The fourfold channel is found to be impermeant to all cations with the possible exception of protons. Because proton transfer is essential to maintain the electroneutrality of the protein during iron deposition, we suggest that the function of the fourfold channel is to form a "proton wire" that facilitates their transfer in and out of ferritin.     

Plasma soluble intercellular adhesion molecule 1 levels are increased in type 2 diabetic patients with nephropathy

BACKGROUND/AIM: Intercellular adhesion molecule 1 (ICAM-1) is a mediator in the recruitment of leukocytes in the glomerular cells. The role of ICAM-1 in diabetic complications is still a matter of debate. This study was performed to investigate the relation of plasma soluble ICAM-1 (sICAM-1) to nephropathy in patients with type 2 diabetes mellitus. METHODS: Ninety-three patients (24 males and 69 females) with type 2 diabetes mellitus were included into the study. Fifty patients had nephropathy, and 43 were free from nephropathy. Fifty healthy subjects (14 males and 36 females) served as the control group (group 1). Twenty-five of the diabetic patients had microalbuminuria (group 2), 25 had macroalbuminuria (group 3), and 43 had neither micro- nor macroalbuminuria (group 4). The plasma sICAM-1 levels were measured in blood samples drawn after fasting. RESULTS: The mean plasma sICAM-1 levels were not different in the 93 diabetic patients as compared with the healthy controls (392.7 +/- 119.5 vs. 350.1 +/- 90.2 ng/ml, p > 0.05). The mean sICAM-1 level was significantly higher in the diabetic patients with nephropathy than in those without nephropathy (430.3 +/- 78.2 vs. 368.2 +/- 122.5 ng/ml, p = 0.03) and in the controls (430.3 +/- 78.2 vs. 350.1 +/- 90.2 ng/ml, p = 0.016). The difference in sICAM-1 levels between groups 2 and 3 was not significant (p > 0.05). The plasma sICAM-1 levels were significantly higher in both groups 2 and 3 than in both groups 1 and 4 (434.5 +/- 129.2 vs. 427.2 +/- 113.7 ng/ml and 368.2 +/- 122.5 vs. 350.1 +/- 90.2 ng/ml, respectively). CONCLUSIONS: The plasma sICAM-1 levels in patients with type 2 diabetes mellitus are not significantly different from those in nondiabetic subjects. High levels of sICAM-1 suggest that sICAM-1 may play a role in the development of nephropathy in patients with type 2 diabetes mellitus.     

Role of the dielectric constants of membrane proteins and channel water in ion permeation

Using both analytical solutions obtained from simplified systems and numerical results from more realistic cases, we investigate the role played by the dielectric constant of membrane proteins epsilon(p) and pore water epsilon(w) in permeation of ions across channels. We show that the boundary and its curvature are the crucial factors in determining how an ion's potential energy depends on the dielectric constants near an interface. The potential energy of an ion outside a globular protein has a dominant 1/epsilon(w) dependence, but this becomes 1/epsilon(p) for an ion inside a cavity. For channels, where the boundaries are in between these two extremes, the situation is more complex. In general, we find that variations in epsilon(w) have a much larger impact on the potential energy of an ion compared to those in epsilon(p). Therefore a better understanding of the effective epsilon(w) values employed in channel models is desirable. Although the precise value of epsilon(p) is not a crucial determinant of ion permeation properties, it still needs to be chosen carefully when quantitative comparisons with data are made.