Testing of promising dressing materials in cultures of fibroblasts and keratinocytes of human skin]

Cell cultures of epidermal keratinocytes and dermal fibroblasts were used to test collagen dressings in vitro. It was shown that effects of the dressings on skin major cell types might be differentially evaluated.     

Ultrastructure of the eye of a euphausiid crustacean

The compound eye of the Antarctic euphausiid Euphausia superba is a spherical clear zone eye. The dioptric system consists of a hexagonally-faceted cornea, two corneagenous cells, two crystalline cone cells which form the bipartite crystalline cone, and two accessory cone cells. The dioptric system of each ommatidium is separated from that of adjacent ommatidia by six distal pigment cells and a basement membrane. The proximal tip of the crystalline cone is cupped by the distal ends of the seven retinula cells whose nuclei are arranged in a staggered array slightly distal to the middle of the clear zone. In the distal half of the clear zone, each narrow retinula cell column is surrounded by large proximal extensions of the six distal pigment cells. The pigment cells narrow more proximally and terminate at the proximal basement membrane. A specialized axial channel complex extends from the crystalline cone through the clear zone, and is continuous with a conical refractive element which caps the distal end of the rhabdom. The rhabdom is fused, and made up of alternating highly birefringent layers of orthogonally-oriented microvilli. It is surrounded by a narrow extra-cellular space which is continuous with the distal refractive element and a second conical refractive element at the proximal end of the rhabdom.     

Prediction of buried helices in multispan alpha helical membrane proteins

Analysis of a database of structures of membrane proteins shows that membrane proteins composed of 10 or more transmembrane (TM) helices often contain buried helices that are inaccessible to phospholipids. We introduce a method for identifying TM helices that are least phospholipid accessible and for prediction of fully buried TM helices in membrane proteins from sequence information alone. Our method is based on the calculation of residue lipophilicity and evolutionary conservation. Given that the number of buried helices in a membrane protein is known, our method achieves an accuracy of 78% and a Matthew's correlation coefficient of 0.68. A server for this tool (RANTS) is available online at http://gila.bioengr.uic.edu/lab/.     

Lipid-binding surfaces of membrane proteins: evidence from evolutionary and structural analysis

Membrane proteins function in the diverse environment of the lipid bilayer. Experimental evidence suggests that some lipid molecules bind tightly to specific sites on the membrane protein surface. These lipid molecules often act as co-factors and play important functional roles. In this study, we have assessed the evolutionary selection pressure experienced at lipid-binding sites in a set of α-helical and β-barrel membrane proteins using posterior probability analysis of the ratio of synonymous vs. nonsynonymous substitutions (ω-ratio). We have also carried out a geometric analysis of the membrane protein structures to identify residues in close contact with co-crystallized lipids. We found that residues forming cholesterol-binding sites in both β(2)-adrenergic receptor and Na(+)-K(+)-ATPase exhibit strong conservation, which can be characterized by an expanded cholesterol consensus motif for GPCRs. Our results suggest the functional importance of aromatic stacking interactions and interhelical hydrogen bonds in facilitating protein-cholesterol interactions, which is now reflected in the expanded motif. We also find that residues forming the cardiolipin-binding site in formate dehydrogenase-N γ-subunit and the phosphatidylglycerol binding site in KcsA are under strong purifying selection pressure. Although the lipopolysaccharide (LPS)-binding site in ferric hydroxamate uptake receptor (FhuA) is only weakly conserved, we show using a statistical mechanical model that LPS binds to the least stable FhuA β-strand and protects it from the bulk lipid. Our results suggest that specific lipid binding may be a general mechanism employed by β-barrel membrane proteins to stabilize weakly stable regions. Overall, we find that the residues forming specific lipid binding sites on the surfaces of membrane proteins often experience strong purifying selection pressure.     

Increased Light Exposure Alleviates One Form of Photoreceptor Degeneration Marked by Elevated Calcium in the Dark

Background

In one group of gene mutations that cause photoreceptor degeneration in human patients, guanylyl cyclase is overactive in the dark. The ensuing excess opening of cGMP-gated cation channels causes intracellular calcium to rise to toxic levels. The Y99C mutation in guanylate cyclase-activating protein 1 (GCAP1) has been shown to act this way. We determined whether prolonged light exposure, which lowers cGMP levels through activation of phototransduction, might protect photoreceptors in a line of transgenic mice carrying the GCAP1-Y99C.

Methodology/Principal Findings

We reared cohorts of GCAP1-Y99C transgenic mice under standard cyclic, constant dark and constant light conditions. Mouse eyes were analyzed by histology and by immunofluorescence for GFAP upregulation, a non-specific marker for photoreceptor degeneration. Full-field electroretinograms (ERGs) were recorded to assess retinal function. Consistent with our hypothesis, constant darkness accelerated disease, while continuous lighting arrested photoreceptor degeneration.

Conclusions/Significance

In contrast to most forms of retinal degeneration, which are exacerbated by increased exposure to ambient light, a subset with mutations that cause overly active guanylyl cyclase and high intracellular calcium benefitted from prolonged light exposure. These findings may have therapeutic implications for patients with these types of genetic defects.     

Helix-helix packing and interfacial pairwise interactions of residues in membrane proteins

Helix-helix packing plays a critical role in maintaining the tertiary structures of helical membrane proteins. By examining the overall distribution of voids and pockets in the transmembrane (TM) regions of helical membrane proteins, we found that bacteriorhodopsin and halorhodopsin are the most tightly packed, whereas mechanosensitive channel is the least tightly packed. Large residues F, W, and H have the highest propensity to be in a TM void or a pocket, whereas small residues such as S, G, A, and T are least likely to be found in a void or a pocket. The coordination number for non-bonded interactions for each of the residue types is found to correlate with the size of the residue. To assess specific interhelical interactions between residues, we have developed a new computational method to characterize nearest neighboring atoms that are in physical contact. Using an atom-based probabilistic model, we estimate the membrane helical interfacial pairwise (MHIP) propensity. We found that there are many residue pairs that have high propensity for interhelical interactions, but disulfide bonds are rarely found in the TM regions. The high propensity pairs include residue pairs between an aromatic residue and a basic residue (W-R, W-H, and Y-K). In addition, many residue pairs have high propensity to form interhelical polar-polar atomic contacts, for example, residue pairs between two ionizable residues, between one ionizable residue and one N or Q. Soluble proteins do not share this pattern of diverse polar-polar interhelical interaction. Exploratory analysis by clustering of the MHIP values suggests that residues similar in side-chain branchness, cyclic structures, and size tend to have correlated behavior in participating interhelical interactions. A chi-square test rejects the null hypothesis that membrane protein and soluble protein have the same distribution of interhelical pairwise propensity. This observation may help us to understand the folding mechanism of membrane proteins.     

Localization of acid organelles in frog skeletal muscle fibers

By means of fluorescent and phase-contrast microscopy the distribution of acid membrane organelles in normal and vacuolated frog skeletal muscle fibers has been studied. The vacuolation of the T-system was produced by loading and subsequent removal of glycerol (80-110 mM), or it appeared as a result of Zenker's necrosis. Acridine orange (AO) was used as a marker for acid intracellular compartments. AO accumulated in granules localized near the nuclear poles (more seldom around the nucleus)' and in the intermyofibrillar spaces. Typically the AO granules make up short longitudinal chains or regular pairs, where the distances between neighboring granules are short-dated to sarcomere lengths. Almost all granules emit in red, but about one third of them simultaneously emit in green, which is characteristic of AO monomers. In the vicinity of necrotic boundary or under the influence of brefeldin A, a green component of fluorescence appears in most granules. Treatment with monensin leads to granule disappearance. Vacuoles accompanying the glycerol treatment or developing of necrosis do not accumulate AO and exert no effect on the localization of AO-granules. The nature of cellular organelles accumulating AO in skeletal muscle fibers is discussed.     

Response of respiratory neurons of the medulla oblongata to the septum stimulation in hypoxia

In the oxygen deficiency conditions, we studied influence of irritation of ventral (BNST), lateral (LSN) and medial (MSN) nuclei of the septum on the impulse activity of the bulbar respiratory neurons and on respiration. Phases of hypoxia were the model of experiment. In conditions of normal atmospheric pressure, the electrical stimulation of BNST, LSN and MSN nuclei of the septum exerted inhibiting as well as activating influence with the inhibiting influence prevailing. In difficult conditions of hypoxia (7.5-8 thousand meters) on the reduction of the impulse activity of neurons, stimulation of septum nucleuses induced uncharacteristic reactions of those neurons.