Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair ...intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cardiovascular disease (CVD) is the leading cause of global mortality, with an increasing burden on the populations of low and middle income countries as identified by the World Health Organization. ...Rapid, noninvasive screening methods for CVD using low-cost technology would allow for better healthcare. Pulse Wave Velocity (PWV) as measured by noninvasive methods such as photoplethysmograph and pressure sensors has been validated as a method for screening for CVD. PWV correlates closely with arterial stiffness, which is a strong indicator of CVD and is calculated using artery length and Pulse Transit Time (PTT) using multiple simultaneous recordings at two superficial arterial sites. While PTT is generally agreed to correlate with CVD, there is still ambiguity concerning the best way to analyze the arterial pulse contour to ascertain patient health. Proper pulse wave analysis could prove to be useful diagnosis of arterial stiffness in patients with hypertension. This analysis studied the efficacy of comparing the foot-to-foot (PTTf) and peak-to-peak (PTTp) delays as a means of differentiating between healthy control patients and hypertensive patients. Wave reflection is thought to affect the PTTp while leaving the PTTf unaffected, so that a comparison should reveal a ratio close to one in healthy patients but a ratio deviated from one in hypertensive patients. The results of this analysis reveal that increased arterial stiffness is consistently associated with reduced PTTf and improved upon anti-hypertensive drug treatment. However the results did not show a statistically significant difference in comparing some of the control and test groups using the ratio of PTT f and PTTp. These results should be further pursued and validated with more detailed analysis, rigid and consistent parameters, and better accounting for wave reflection.
Translated from the Original Italian Edition of 1510, with a Preface, by John Winter Jones, Esq., F.S.A. And Edited, with Notes and an Introduction, by George Percy Badger. This is a new ...print-on-demand hardback edition of the volume first published in 1863.
Water Structure in Cubic Insulin Crystals Badger, John; Caspar, D. L. D.
Proceedings of the National Academy of Sciences - PNAS,
01/1991, Letnik:
88, Številka:
2
Journal Article
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The electron density distribution of the solvent in the cubic insulin crystal structure, which occupies 65% of the volume, has been mapped from 1.7-$\overset{^\circ}{A}$resolution diffraction data by ...an iterative difference Fourier method, using the previously determined protein structure as the refinement restraint. Starting with phases from the protein and a flat solvent model, the difference map calculated from the data was added outside the protein envelope, and the modified map was then used to recalculate phases for the iterative refinement. Tests of the method with model data, with the experimental data and a variant protein model, and by carrying out a partial refinement of the solvent map demonstrate that the refinement algorithm produces reliable values for the solvent density within the noise level of the data. Fluctuations in density are observed throughout the solvent space, demonstrating that nonrandom arrangements of the water molecules extend several layers from the well-ordered hydration shell in contact with the protein surface. Such ordering may account for the hydration force opposing close approach of hydrophilic surfaces and other long-range water-dependent interactions in living structures.
We have developed new software for locating heavy‐atom sites for phase determination from single isomorphous replacement (SIR) and multiwavelength anomalous diffraction (MAD) data. The first ...component of the software is a program for finding heavy‐atom sites when presented with SIR (i.e. native and derivative) structure‐factor amplitudes or MAD‐FA structure‐factor amplitudes (i.e. the derived structure factors corresponding to the anomalously scattering atoms). The principle on which this program is based is a reciprocal‐space maximization of a Patterson correlation coefficient between the SIR or MAD‐FA data and the calculated intensities from search atoms placed at trial positions. The program has been successfully tested on two sets of SIR data for the cytochrome c′ protein and on MAD‐FA data from the DnaK protein. The second component of the software is a three‐dimensional viewer which may be used to assess difference Patterson maps and which provides peak‐picking and atom‐editing facilities for interpreting cross‐difference Fourier maps.
The binding to human rhinovirus 14 of a series of eight antiviral agents that inhibit picornaviral uncoating after entry into host cells has been characterized crystallographically. All of these bind ...into the same hydrophobic pocket within the viral protein VP1 β -barrel structure, although the orientation and position of each compound within the pocket was found to differ. The compounds cause the protein shell to be less flexible, thereby inhibiting disassembly. Although the antiviral potency of these compounds varies by 120-fold, they all induce the same conformational changes on the virion. The interactions of these compounds with the viral capsid are consistent with their observed antiviral activities against human rhinovirus 14 drug-resistant mutants and other rhinovirus serotypes. Crystallographic studies of one of these mutants confirm the partial sequencing data and support the finding that this is a single mutation that occurs within the binding pocket.
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