Autosomal dominant retinitis pigmentosa (adRP; RP6): cosegregation of RP6 and the peripherin-RDS locus in a late-onset family of Irish origin.

We recently reported the localization of a gene for late-onset autosomal dominant retinitis pigmentosa (adRP; RP6), on the short arm of chromosome 6, by linkage analysis in a large family of Irish origin. It is notable that the gene encoding peripherin-RDS, a photoreceptor-specific protein, recently has been physically mapped on 6p. In our own analysis, an intrageneic marker derived from this gene cosegregated with the adRP disease locus with zero recombination (lod score 5.46 at q = .00). Using the CEPH reference panel, we now report the mapping of the peripherin-RDS gene relative to other 6p markers in the CEPH data base. Incorporation of these data into a multipoint analysis produced a lod score for adRP of 8.21, maximizing at the peripherin-RDS locus. This study provides strong evidence suggesting a role for peripherin-RDS in the etiology of one form of adRP.     

Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures

Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electrophoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 ± 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K⁺. When the enzyme is dialyzed in the presence of K⁺ and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K⁺. The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K⁺ concentrations increase. The change in threonine and K⁺ concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonine-insensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark.     

Targeting cancer stem cells with phytochemicals

Cancer, second only to heart disease, is the leading cause of death in the US. Although progress has been made in the early detection of cancer and in improvements of cancer therapies, the ability to provide long-term survival has been limited. Increasing evidence suggests that a minute, biologically unique population of cancer stem cells (SCs) exists in most neoplasms and may be responsible for tumor initiation, progression, metastasis, and relapse. Characterization of cancer SCs has led to the identification of key cellular activities that may make cancer SCs vulnerable to therapeutic interventions that target drug-effluxing capabilities, stem cell pathways, anti-apoptotic mechanisms, and induction of differentiation. Phytochemicals, compounds made from fruits, vegetables, and grains, possess anti-cancer properties and represent a promising therapeutic approach for the prevention and treatment of many cancers. This review summarizes the evidence for the cancer SC hypothesis and discusses the potential mechanisms by which phytochemicals might target cancer SCs.     

The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis

The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193-0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15-2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis.     

RNAi-mediated reversible opening of the blood-brain barrier

Background

The blood‐brain barrier (BBB) contains tight junctions (TJs) which reduce the space between adjacent endothelial cells lining the fine capillaries of the microvasculature of the brain to form a selective and regulatable barrier.

Methods

Using a hydrodynamic approach, we delivered siRNA targeting the TJ protein claudin‐5 to the endothelial cells of the BBB in mice.

Results

We have shown a significant decrease in claudin‐5 mRNA levels 24 and 48 hours post‐delivery of siRNA, with levels of protein expression decreasing up to 48 hours post‐injection compared to uninjected, phosphate‐buffered saline (PBS)‐injected and non‐targeting siRNA‐injected mice. We observed increased permeability at the BBB to molecules up to 742 Da, but not 4400 Da, using tracer molecule perfusion and MRI analysis. To illustrate the functional efficacy of size‐selective and transient barrier opening, we have shown that enhanced delivery of the small neuropeptide thyrotropin‐releasing hormone (TRH) (MW 360 Da) to the brains of mice 48 hours post‐injection of siRNA targeting claudin‐5 significantly modifies behavioural output.

Conclusions

These data demonstrate that it is now possible to transiently and size‐selectively open the BBB in mice, allowing in principle the delivery of a wide range of agents for the establishment and treatment of experimental mouse models of neurodegenerative, neuropsychiatric and malignant diseases. Copyright © 2008 John Wiley & Sons, Ltd.     

Towards Miscanthus combustion quality improvement: the role of flowering and senescence

In commercially grown Miscanthus × giganteus, despite imposing a yield penalty, postwinter harvests improve quality criteria for thermal conversion and crop sustainability through remobilization of nutrients to the underground rhizome. We examined 16 Miscanthus genotypes with different flowering and senescence times for variation in N, P, K, moisture, ash, Cl and Si contents, hypothesizing that early flowering and senescence could result in improved biomass quality and/or enable an earlier harvest of biomass (in autumn at peak yield). Ideal crop characteristics at harvest are low N and P to reduce future fertilizer inputs, low K and Cl to reduce corrosion in boilers, low moisture to reduce spoilage and transportation costs, and low Si and ash to reduce slagging and consequent operational downtime. Stems and leaves were harvested during summer, autumn and then the following spring after overwinter ripening. In spring, stem contents of N were 30–60 mg kg^?1, P were 203–1132 mg kg^?1, K were 290–4098 mg kg^?1, Cl were 10–23 mg kg^?1 and moisture were 12–38%. Notably, late senescence resulted in increased N, P, K, Cl, moisture and ash contents, and should therefore be avoided for thermochemical conversion. Flowering and senescence led to overall improved combustion quality, where flowered genotypes tended towards lower P, K, Cl and moisture contents; marginally less, or similar, N, Si and ash contents; and a similar higher heating value, compared to those that had not flowered. Such genotypes could potentially be harvested in the autumn. However, one genotype that did not flower in our trial exhibited sufficiently low N and K content in autumn to meet the ENplus wood pellet standards for those traits, and some of the lowest P, moisture and ash contents in our trial, and is thus a target for future research and breeding.     

Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan

AIMS: The aim of this work was to engineer a gut commensal bacterium, Bacteroidesovatus, to produce and secrete a biologically active cytokine in a regulated manner as a basis for novel immunotherapies for chronic gut disorders. METHODS AND RESULTS: Bacteroides ovatus was engineered to produce murine interleukin-2 (MuIL2) intracellularly in response to xylan in culture media by inserting the MuIL2 gene into the xylanase operon of the organism. A second strain was engineered to secrete MuIL2 by adding Bacteroides fragilis enterotoxin secretion signal sequence to the protein. The recombinant strains produced MuIL2 only in the presence of xylan as determined by ELISA of cell lysates and culture supernatants. The IL2-dependent cell line CTLL-2 was used to demonstrate that MuIL2 produced by both B. ovatus strains was biologically active. This activity could be blocked by an anti-IL2 neutralizing antibody. The xylan-inducible nature of this system was demonstrated by RT-PCR. CONCLUSIONS: Bacteroides ovatus was successfully engineered to produce and secrete biologically active MuIL2 in a xylan-inducible manner. SIGNIFICANCE AND IMPACT OF THE STUDY: The production and secretion of a biologically active mammalian protein by a member of the gut microflora could lead to the development of new long-term immunotherapies for inflammatory gut diseases.     

The influence of design, materials and kinematics on the in vitro wear of total knee replacements

Debris-induced osteolysis due to surface wear of ultra high molecular weight polyethylene (UHMWPE) bearings is a potential long-term failure mechanism of total knee replacements (TKR). This study investigated the effect of prosthesis design, kinematics and bearing material on the wear of UHMWPE bearings using a physiological knee simulator. The use of a curved fixed bearing design with stabilised polyethylene bearings reduced wear in comparison to more flat-on-flat components which were sterilised by gamma irradiation in air. Medium levels of crosslinking further improved the wear resistance of fixed bearing TKR due to resistance to strain softening when subjected to multidirectional motion at the femoral-insert articulating interface. Backside motion was shown to be a contributing factor to the overall rate of UHMWPE wear in fixed bearing components. Wear of fixed bearing prostheses was reduced significantly when anterior-posterior displacement and internal-external rotation kinematics were reduced due to decreased cross shear on the articulating surface and a reduction in AP displacement. Rotating platform mobile bearing prostheses exhibited reduced wear rates in comparison to fixed bearing components in these simulator studies due to redistribution of knee motion to two articulating interfaces with more linear motions at each interface. This was observed in two rotating platform designs with different UHMWPE bearing materials. In knee simulator studies, wear of TKR bearings was dependent on kinematics at the articulating surfaces and the prosthesis design, as well as the type of material.