Ten mutants of bacteriorhodopsin, each containing a single cysteine residue regularly spaced along helix D and facing the lipid bilayer, were derivatized with a nitroxide spin label. Collision rates of the nitroxide with apolar oxygen increased with distance from the membrane/solution interface. Collision rates with polar metal ion complexes decreased over the same distance. Although the collision rates depend on steric constraints imposed by the local protein structure and on the depth in the membrane, the ratio of the collision rate of oxygen to those of a polar metal ion complex is independent of structural features of the protein. The logarithm of the ratio is a linear function of depth within the membrane. Calibration of this ratio parameter with spin-labeled phospholipids allows localization of the individual nitroxides, and hence the bacteriorhodopsin molecule, relative to the plane of the phosphate groups of the bilayer. The spacing between residues is consistent with the pitch ofan a-helix. These results provide a general strategy for determining the immersion depth of nitroxides in bilayers.Site-directed spin labeling has become a powerful tool for determination of membrane protein structures and their disposition with respect to the bilayer (1-3). In previous studies, information on the region where transmembrane helices intersect with the membrane/solution interface has been obtained by analysis of a consecutive series of mutants that traverse the interface (1). If it were possible to determine the vertical distance of a spin-labeled side chain from the plane of the phosphates of the lipid headgroups, analysis of only one or two spin-labeled mutants would be sufficient to determine the position ofa transmembrane domain relative to the membrane.EPR methods for estimation of the depth of immersion of a nitroxide in the membrane have been reported (4-6). These methods are based upon the dipolar interactions of the nitroxide with paramagnetic reagents constrained to the aqueous phase and require knowledge of the spacial distribution of the paramagnetic reagents in solution. This distribution may be readily deduced for a pure bilayer with a nitroxide on a lipid chain, but not for a nitroxide attached to a protein in a bilayer. This is because the protein has an unknown excluded volume for the paramagnetic reagent in the aqueous phase.In this report, we make use of site-directed spin labeling to introduce nitroxides along the entire length of helix D of bacteriorhodopsin (bR). The collision frequencies of the nitroxides with paramagnetic reagents are dependent upon position, and this effect is shown to provide an approach for localization of nitroxides in the membrane interior. MATERIALS AND METHODSEgg yolk phosphatidylcholine (PC) and 1-palmitoyl-2-(ndoxylpalmitoyl) PC spin-labeled isomers with n = 5, 7, 10, 12, and 16 were obtained from Avanti Polar Lipids. Ethylenediamine-N,N'-diacetic acid (EDDA), nickel(II) acetylacetonate (NiAA), and Ni(OH)2 were obtained from Aldrich. bR mutants were prepare...

In this paper we extend the classical SIS epidemic model from a deterministic framework to a stochastic one, and formulate it as a stochastic differential equation (SDE) for the number of infectious individuals I(t). We then prove that this SDE has a unique global positive solution I(t) and establish conditions for extinction and persistence of I(t). We discuss perturbation by stochastic noise. In the case of persistence we show the existence of a stationary distribution and derive expressions for its mean and variance. The results are illustrated by computer simulations, including two examples based on real life diseases.

Epidermolytic hyperkeratosis is a hereditary skin disorder characterized by blistering and a marked thickening of the stratum corneum. In one family, affected individuals exhibited a mutation in the highly conserved carboxyl terminal of the rod domain of keratin 1. In two other families, affected individuals had mutations in the highly conserved amino terminal of the rod domain of keratin 10. Structural analysis of these mutations predicts that heterodimer formation would be unaffected, although filament assembly and elongation would be severely compromised. These data imply that an intact keratin intermediate filament network is required for the maintenance of both cellular and tissue integrity.

Assembly of proteins within lipid bilayers is essential for the biogenesis and function of biological membranes. Little is known, however, about the underlying mechanism of assembly, and it is not clear whether it is possible to observe individual folding steps for integral membrane proteins either in vivo or in vitro. Fluorescence spectroscopy is used here to follow the time course of folding events for bacteriorhodopsin in mixed detergent/lipid micelles. Transient folding-intermediates are detected and binding of the retinal chromophore occurs at a late stage, when it binds to an apoprotein intermediate.

To determine whether a functional type II receptor of transforming growth factor ␤ (TGF-␤) is required to mediate the growth inhibitory effect of TGF-␤ on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-␤ receptor (⌬␤RII) in the epidermis. The ⌬␤RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F 1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from ⌬␤RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-␤-induced growth inhibition. These data document the role of the type II TGF-␤ receptor in mediating TGF-␤-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.

In this paper we analyse a stochastic model representing HIV internal virus dynamics. The stochasticity in the model is introduced by parameter perturbation which is a standard technique in stochastic population modelling. We show that the model established in this paper possesses non-negative solutions as this is essential in any population dynamics model. We also carry out analysis on the asymptotic behaviour of the model. We approximate one of the variables by a mean reverting process and nd out the mean and variance of this process. Numerical simulations conclude the paper

The regulatory elements of the human keratin K1 gene have been used to target expression of the v-Ha-ras oncogene exclusively in the epidermis of transgenic mice. We developed 12 transgenic mouse lines that express the HK1.ras transgene, producing epidermal hyperplasia in neonates and hyperkeratosis in juveniles. Eventually this skin phenotype diminished but with time adult animals developed papillomas that could persist or regress. The rate and frequency of tumorigenesis appeared to be limited, which suggests that v-Ha-ras requires a second or even third event to elicit and maintain a benign phenotype in transgenic mice. Since in certain transgenic lines papillomas appeared at wound sites, it appears that the promotion stimulus from wounding may be a second event. We envision that such transgenic mice that express v-Ha-ras in the epidermis will become a powerful model for assessing how environmental and molecular factors affect the process of multistage skin carcinogenesis in vivo, as well as a model for evaluating novel therapeutic protocols.

We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by -100-fold relative to wild-type bacteriorhodopsin. Time-resolved visible absorption spectroscopy and resonance Raman experimnents, respectively, show the presence of long-lived O4ike and N-like intermediates in the photocycles of the above mutants. We infer the existence of an equilibrium between the N and 0 intermediates in the photocycles of these mutants. The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or 0 intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon. Since the 13-cis --all-trans reisomerization of retinal is completed during the decay of the N and 0 intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle. This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal. intermediate (17). Both isomerization steps are generally believed to be associated with changes in protein conformation. To understand how changes in retinal geometry are coupled to conformational changes in the protein, we are carrying out systematic replacements of amino acids that line the retinal binding pocket. We report here that the interaction of Leu-93, a residue in helix C (Fig. 1), with retinal has an important role in the photocycle and in light/dark adaptation. Time-resolved visible spectroscopic studies show that the half-time for the decay of the 0 intermediate of the photocycle is increased to -800 ms in the mutants where Leu-93 was replaced by Ala (L93A) or Thr (L93T) as compared to a rate of <5 ms for wild-type bR. Resonance Raman (RR) experiments provide evidence for the presence ofa long-lived N intermediate in the photocycles of these mutants. We conclude that Leu-93 in bR is involved in coupling protein conformational changes that occur during the decay of the N and 0 intermediates of the photocycle to 13-cis -+ all-trans reisomerization of the chromophore.