The effects of manganese doping on UVA absorption and free radical generation of micronised titanium dioxide and its consequences for the photostability of UVA absorbing organic sunscreen components.

The effect of manganese doping on the free radical generation rate, free radical scavenging and UVA absorption properties of micronised sunscreen grade titania has been studied with respect to enhancement of the UVA photostability of test sunscreen formulations containing the organic UVA absorber Parsol 1789. Manganese doping has been shown to increase the UVA:UVB absorption ratio of titania, reduce free radical generation rates by over 90%, and provide free radical scavenging behaviour. Adding manganese-doped titania to a test formulation incorporating Parsol 1789 shows that manganese doping increases UVA attenuation stability by up to 3 times the amount achieved by comparable commercial undoped titania materials. HPLC data shows this to be related to an improved stabilisation of the organic sunscreen components. Manganese doped titania shows improved efficacy over undoped titania in sunscreen formulations containing organic UV absorbers.     

Bone measurement by enhanced contrast image analysis: ovariectomized and intact Macaca fascicularis as a model for human postmenopausal osteoporosis

This paper presents an image enhancement and analysis system (DARWIN) based on an inexpensive microcomputer and applies the system to two bone morphometry problems relevant to postmenopausal osteoporosis. Using ovariectomized and intact female Macaca fascicularis as a model, we examined the radiodensity of the sixth lumbar vertebra and the cross-section area of the right femur. Significantly lower bone density was observed in the vertebral segments of the ovariectomized animals. No significant differences were observed in comparisons of the femoral cross sections. The reduction in radiographic density of the ovariectomized animals' vertebrae is similar to that observed in postmenopausal women, supporting the use of female cynomolgus macaques as models of bone loss in postmenopausal osteoporosis.     

Ubiquitous GFP expression in transgenic chickens using a lentiviral vector

We report the first ubiquitous green fluorescent protein expression in chicks using a lentiviral vector approach, with eGFP under the control of the phosphoglycerol kinase promoter. Several demonstrations of germline transmission in chicks have been reported previously, using markers that produce tissue-specific, but not ubiquitous, expression. Using embryos sired by a heterozygous male, we demonstrate germline transmission in the embryonic tissue that expresses eGFP uniformly, and that can be used in tissue transplants and processed by in situ hybridization and immunocytochemistry. Transgenic tissue is identifiable by both fluorescence microscopy and immunolabeling, resulting in a permanent marker identifying transgenic cells following processing of the tissue. Stable integration of the transgene has allowed breeding of homozygous males and females that will be used to produce transgenic embryos in 100% of eggs laid upon reaching sexual maturity. These results demonstrate that a transgenic approach in the chick model system is viable and useful even though a relatively long generation time is required. The transgenic chick model will benefit studies on embryonic development, as well as providing the pharmaceutical industry with an economical bioreactor.     

Effectiveness of the BT mite trap for detecting the storage mite pests, Acarus siro, Lepidoglyphus destructor and Tyrophagus longior

Traps have been used extensively to provide early warning of hidden pest infestations. To date, however, there is only one type of trap on the market in the U.K. for storage mites, namely the BT mite trap, or monitor. Laboratory studies have shown that under the test conditions (20 °C, 65% RH) the BT trap is effective at detecting mites for at least 10 days for all three species tested: Lepidoglyphus destructor, Tyrophagus longior and Acarus siro. Further tests showed that all three species reached a trap at a distance of approximately 80 cm in a 24 h period. In experiments using 100 mites of each species, and regardless of either temperature (15 or 20 °C) or relative humidity (65 or 80% RH), the most abundant species in the traps was T. longior, followed by A. siro then L. destructor. Trap catches were highest at 20 °C and 65% RH. Temperature had a greater effect on mite numbers than humidity. Tests using different densities of each mite species showed that the number of L. destructor found in/on the trap was significantly reduced when either of the other two species was dominant. It would appear that there is an interaction between L. destructor and the other two mite species which affects relative numbers found within the trap.The British Crowns right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.This revised version was published online in May 2005 with a corrected cover date.     

Gene arrays at Pneumocystis carinii telomeres

In the fungus Pneumocystis carinii, at least three gene families (PRT1, MSR, and MSG) have the potential to generate high-frequency antigenic variation, which is likely to be a strategy by which this parasitic fungus is able to prolong its survival in the rat lung. Members of these gene families are clustered at chromosome termini, a location that fosters recombination, which has been implicated in selective expression of MSG genes. To gain insight into the architecture, evolution, and regulation of these gene clusters, six telomeric segments of the genome were sequenced. Each of the segments began with one or more unique genes, after which were members of different gene families, arranged in a head-to-tail array. The three-gene repeat PRT1-MSR-MSG was common, suggesting that duplications of these repeats have contributed to expansion of all three families. However, members of a gene family in an array were no more similar to one another than to members in other arrays, indicating rapid divergence after duplication. The intergenic spacers were more conserved than the genes and contained sequence motifs also present in subtelomeres, which in other species have been implicated in gene expression and recombination. Long mononucleotide tracts were present in some MSR genes. These unstable sequences can be expected to suffer frequent frameshift mutations, providing P. carinii with another mechanism to generate antigen variation.     

Dynamic lateral organization of opioid receptors (kappa,muwt and muN40D) in the plasma membrane at the nanoscale level

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOP_wt), and its naturally occurring isoform (MOP_N40D) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble‐averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside‐enriched domains and partial association with cholesterol‐enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor‐specific. KOP‐containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOP_N40D. Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOP_wt, whereas this effect was not observed for MOP_N40D.      

Expression of a dominant-negative mutant TGF-beta type II receptor in transgenic mice reveals essential roles for TGF-beta in regulation of growth and differentiation in the exocrine pancreas.

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-beta (TGF-beta) signaling in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocked signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments.     

Establishment and characterization of a novel polarized MDCK epithelial cellular model for CFTR studies.

F508del is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsible for the genetic disease Cystic Fibrosis (CF). It results in a major failure of CFTR to traffic to the apical membrane of epithelial cells, where it should function as a chloride (Cl-) channel. Most studies on localization, processing and cellular trafficking of wild-type (wt) and F508del-CFTR have been performed in non-epithelial cells. Notwithstanding, polarized epithelial cells possess distinctly organized and regulated membrane trafficking pathways. We have used Madin-Darby canine kidney (MDCK) type II cells (proximal tubular cells which do not express endogenous CFTR) to generate novel epithelial, polarized cellular models stably expressing wt- or F508del-CFTR through transduction with recombinant lentiviral vectors. Characterization of these cell lines shows that wt-CFTR is correctly processed and apically localized, producing a cAMP-activated Cl- conductance. In contrast, F508del-CFTR is mostly detected in itsimmature form, localized intracellularly and producing only residual Cl- conductance. These novel cell lines constitute bona fide models and significantly improved resources to investigate the molecular mechanisms of polarized membrane traffic of wt- and F508del-CFTR in the same cellular background. They are also useful to identify/validate novel therapeutic compounds for CF.     

Functional coupling between enzymes of the chromaffin granule membrane

The reactions of cytochrome b561 with other redox-active components of the adrenal chromaffin granule were examined using optical difference spectroscopy. It was shown that there is no direct electron transfer between the cytochrome and dopamine beta-hydroxylase, but that in the presence of ascorbate, turnover of dopamine beta-hydroxylase causes an oxidation of the cytochrome, which is partially reversed by the action of the mitochondrial NADH:A-. oxidoreductase. Thus, these three proteins may be functionally coupled via ascorbate. A quantitative study of the relationship between the redox state of the cytochrome and the ascorbate radical concentration measured by EPR showed that ascorbate reduces the cytochrome in a one-electron transfer reaction. Generation of a proton electrochemical gradient across the granule membrane causes only a small (20 mV) increase in the cytochrome midpoint potential suggesting the cytochrome is not a proton pump. The data are consistent with a model in which cytochrome b561, by reacting with ascorbate or ascorbate free radical on either side of the granule membrane, could couple the ascorbate-consuming reaction of the dopamine beta-hydroxylase inside the chromaffin granule to the ascorbate-regenerating reaction of the NADH:A-. oxidoreductase on the outer mitochondrial membrane. The H+-ATPase of the granule membrane could both drive the flow of electrons in the direction from cytosol to granule and replenish protons consumed by the turnover of dopamine beta-hydroxylase inside the granule.