Cholesterol 24-hydroxylase is a highly conserved cytochrome P450 that is responsible for the majority of cholesterol turnover in the vertebrate central nervous system. The enzyme is expressed in neurons, including hippocampal and cortical neurons that are important for learning and memory formation. Disruption of the cholesterol 24-hydroxylase gene in the mouse reduces both cholesterol turnover and synthesis in the brain but does not alter steady-state levels of cholesterol in the tissue. The decline in synthesis reduces the flow of metabolites through the cholesterol biosynthetic pathway, of which one, geranylgeraniol diphosphate, is required for learning in the whole animal and for synaptic plasticity in vitro. This review focuses on how the link between cholesterol metabolism and higher-order brain function was experimentally established.

Reactive oxygen species (ROS) are implicated in both cartilage aging and the pathogenesis of osteoarthritis. We developed an in vitro model to study the role of chondrocyte-derived ROS in cartilage matrix protein degradation. Matrix proteins in cultured primary articular chondrocytes were labeled with [ 3 H]proline, and the washed cell matrix was returned to a serum-free balanced salt solution. Exposure to hydrogen peroxide resulted in oxidative damage to the cell matrix as established by monitoring the release of labeled material into the medium. Calcium ionophore treatment of chondrocytes, in a dose-dependent manner, significantly enhanced the release of labeled matrix, suggesting a chondrocyte-dependent mechanism of matrix degradation. Antioxidant enzymes such as catalase or superoxide dismutase did not influence matrix release by the calcium ionophore-activated chondrocytes. However, vitamin E, at physiological concentrations, significantly diminished the release of labeled matrix by activated chondrocytes. The fact that vitamin E is a chain-breaking antioxidant indicates that the mechanism of matrix degradation and release is mediated by the lipid peroxidation process. Lipid peroxidation was measured in chondrocytes loaded with cis-parinaric acid. Both resting and activated cells showed constitutive and enhanced levels of lipid peroxidation activity, which were significantly reduced in the presence of vitamin E. In an immunoblot analysis, malondialdehyde and hydroxynonenal adducts were observed in chondrocyte-matrix extracts, and the amount of adducts increased with calcium ionophore treatment. Furthermore, vitamin E diminished aldehyde-protein adduct formation in activated extracts, which suggests that vitamin E has an antioxidant role in preventing protein oxidation. This study provides in vitro evidence linking chondrocyte lipid peroxidation to cartilage matrix protein (collagen) oxidation and degradation and suggests that vitamin E has a preventive role. These observations indicate that chondrocyte lipid peroxidation may have a role in the pathogenesis of cartilage aging and osteoarthritis.Cartilage degeneration is a hallmark of cartilage aging and osteoarthritis (1). Degeneration of articular cartilage in osteoarthritis is accompanied by chronic pain and significant disability. In a series of reports (2-7), we and others have documented that chondrocytes produce reactive oxygen species (ROS).1 The production of ROS by chondrocytes can contribute to degradation of the cartilage matrix. For example, ROS can mediate intracellular signaling and gene activation of cytokine and growth factor-induced products in chondrocytes (8, 9). In activated neutrophils and monocytes/macrophages, the cellspecific gene products of "NADPH-oxidase complex" physically come together and initiate single electron reduction of oxygen and the release of ROS outside the cells. Phagocytes use the toxic properties of ROS to eliminate pathogens (10, 11); in contrast, the biological role of secreted ROS in cartilage is not known.The ob...

Short-term BFMS placement is an effective therapy for pancreatic WON. The majority of recurrences developed in patients with ductal disconnection and did not require therapy. Additional pancreatic duct stents probably do not influence the recurrence rate.

Peptide nucleic acids (PNAs) are a powerful tool for recognition of double-stranded DNA. Strand invasion is most efficient when pyrimidine PNAs are linked to form a bisPNA in which one strand binds by Watson-Crick base pairing while the other binds by Hoogsteen base pairing to the newly formed PNA-DNA duplex. Within many genes, however, polypyrimidine target sequences may not be located in optimal positions relative to transcription factor binding sites, and this deficiency may complicate attempts to identify potent antigene PNAs. To increase the versatility of strand invasion by PNAs, we have synthesized bisPNAs and bisPNA-peptide conjugates containing a mixed base extension of the Watson-Crick polypyrimidine strand. We find that these tail-clamp PNAs (TC-PNAs) bind duplex DNA and inhibit transcription. DNA recognition occurs with single-stranded or TC-bisPNAs and requires attachment of positively charged amino acids. Association rate constants, k(a), for binding to DNA by TC-PNAs are as high as 35000 M(-1) s(-1) and are usually only a fewfold lower than for analogous PNAs that lack mixed base extensions. The ability to bind duplex DNA is not always necessary for inhibition of transcription, possibly because PNAs can bind to accessible DNA within the transcription bubble created by RNA polymerase. These results, together with similar findings independently obtained by Nielsen and colleagues [Bentin, T., Larsen, H. J., and Nielsen, P. E. (2003) Biochemistry 42, 13987-13995], expand the range of sequences within duplex DNA that are accessible to PNAs and suggest that TC-PNA-peptide conjugates are good candidates for further testing as antigene agents.

Objective. To investigate the role of oxidative functions in human osteoarthritic (OA) chondrocytes and to investigate the presence of in vivo molecular markers of lipoxidation in OA cartilage.Methods. An in vitro model of cartilage collagen degradation was used. Lipid peroxidation activity and overall oxidative function in OA chondrocytes were monitored by cis-parinaric acid and dichlorofluorescein assays, respectively. In vivo molecular markers of lipoxidation in normal and OA cartilage were studied using immunohistochemistry to detect the presence of malondialdehyde and hydroxynonenal adducts.Results. Human OA chondrocytes showed a robust amount of 3 H-proline-labeled collagen degradation upon stimulation with lipopolysaccharide and calcium ionophore A21387, as compared with that in untreated OA chondrocytes. Primary OA chondrocytes showed both spontaneous and inducible levels of lipid peroxidation activity. However, lipid peroxidation activity was already maximally elevated in more than 50% of the OA chondrocyte samples. Overall, spontaneous and inducible oxidative activities were observed in all OA samples. Immunohistochemical analysis of control OA tissue sections that were not treated with monoclonal antibody showed little immunoreactivity. OA cartilage sections treated with monoclonal antibodies showed specific immunoreactivity on the cartilage surface, at sites of OA lesions, at the pericellular matrix, and at intra-and intercellular matrices. Normal cartilage sections showed faint surface reactivity.Conclusion. Our observations suggest that human OA chondrocytes demonstrate spontaneous and inducible cell-associated lipoxidative and nonlipoxidative activity. Lipoxidative activity appears to be enhanced in OA chondrocytes. The presence of molecular markers of in vivo lipid peroxidation was demonstrated in OA cartilage, suggesting its role in the pathogenesis of the disease.Osteoarthritis (OA) is the most common form of joint disease that affects humans. The incidence of OA increases during every decade of life, and by the age of 65 years, almost one-third of the population has OA of the knee joints. The economic burden attributed to the joint pain and disability of OA amounts to billions of dollars each year (1). As the population demographic in the US changes to a predominantly older generation, the increasing prevalence of OA will be a major public health problem.There is currently no treatment available that will prevent or cure OA. Pharmacologic and nonpharmacologic agents used for OA provide only symptomatic relief of pain. The lack of specific therapy for this disease is perhaps due to our limited understanding of its pathogenesis. Understanding the molecular mechanisms involved in the development of OA will help us to develop ways to prevent or reverse the degenerative process of the disease.Current concepts of the pathogenic mechanisms of OA suggest that there is a shift in the homeostatic balance between the destruction and synthesis of bone and cartilage, with a net progressive destruction of t...