Nuclear translocation contributes to regulation of DNA excision repair activities

DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927–939; M. Christmann, M.T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3–34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89–108]. Protein interactions are not only important for function, but also for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import, co-import of proteins, and alternative transport pathways. Most excision repair proteins appear to contain classical NLS sequences directing their nuclear import, however, additional import mechanisms add alternative regulatory levels to protein import, indirectly affecting protein function. Protein co-import appears to be a mechanism employed by the composite repair systems NER and MMR to enhance and regulate nuclear accumulation of repair proteins thereby ensuring faithful DNA repair.     

Proteomics reveals lowering oxygen alters cytoskeletal and endoplasmatic stress proteins in human endothelial cells

A proteomic approach was applied to explore the signalling pathways elicited by lowering O₂ in endothelial cells. Endothelial cells isolated from native umbilical cords were subjected to 21, 5, or 1% O₂ for 24 h. 2‐D PAGE was performed and candidate proteins were identified using LC‐MS/MS. Lowering of O₂ from 21 to 5% induced upregulation of cofilin‐1, cyclophilin A, tubulin and tubulin fragments, a fragment of glucose‐regulated protein 78 (Grp78) and calmodulin. The upregulation of Grp78 suggested that ER stress proteins were altered and indeed Grp94 and caspase 12 expression were increased in cells exposed to 5% O₂. The presence of ER stress is also supported by findings of blunted caffeine‐evoked ER calcium release in cells exposed to 5 and 1% O₂. Exposure to 1% O₂ caused increases in cofilin‐1, cyclophilin A, and caspase 12 as well as a decrease of β‐actin, but it did not alter the expression of calmodulin, tubulin, Grp78, and Grp94. Incubation with CoCl₂, a stabilizer of the hypoxia‐inducible factor, increased the expression of several of the proteins. The present investigations reveal that lowering O₂, probably in part through hypoxia‐inducible factor, alter the expression of a series of proteins mainly involved in cytoskeletal changes (e.g. cofilin‐1, tubulin, and β‐actin) and in ER stress/apoptosis (e.g. Grp78/94, caspase 12, and cyclophilin A).     

Ultrasonography of the metacarpophalangeal and proximal interphalangeal joints in rheumatoid arthritis: a comparison with magnetic resonance imaging, conventional radiography and clinical examination

Signs of inflammation and destruction in the finger joints are the principal features of rheumatoid arthritis (RA). There are few studies assessing the sensitivity and specificity of ultrasonography in detecting these signs. The objective of the present study was to investigate whether ultrasonography can provide information on signs of inflammation and destruction in RA finger joints that are not available with conventional radiography and clinical examination, and comparable to the information provided by magnetic resonance imaging (MRI). The second to fifth metacarpophalangeal and proximal interphalangeal joints of 40 RA patients and 20 control persons were assessed with ultrasonography, clinical examination, radiography and MRI. With MRI as the reference method, the sensitivity, specificity and accuracy of ultrasonography in detecting bone erosions in the finger joints were 0.59, 0.98 and 0.96, respectively; they were 0.42, 0.99 and 0.95 for radiography. The sensitivity, specificity and accuracy of ultrasonography, with signs of inflammation on T1-weighted MRI sequences as the reference method, were 0.70, 0.78 and 0.76, respectively; they were 0.40, 0.85 and 0.72 for the clinical examination. With MRI as the reference method, ultrasonography had higher sensitivity and accuracy in detecting signs of inflammation and destruction in RA finger joints than did clinical and radiographic examinations, without loss of specificity. This study shows that ultrasonography has the potential to improve assessment of patients with RA.     

Genetic control of methyl halide production in Arabidopsis

Methyl chloride (CH(3)Cl) and methyl bromide (CH(3)Br) are the primary carriers of natural chlorine and bromine, respectively, to the stratosphere, where they catalyze the destruction of ozone, whereas methyl iodide (CH(3)I) influences aerosol formation and ozone loss in the boundary layer. CH(3)Br is also an agricultural pesticide whose use is regulated by international agreement. Despite the economic and environmental importance of these methyl halides, their natural sources and biological production mechanisms are poorly understood. Besides CH(3)Br fumigation, important sources include oceans, biomass burning, tropical plants, salt marshes, and certain crops and fungi. Here, we demonstrate that the model plant Arabidopsis thaliana produces and emits methyl halides and that the enzyme primarily responsible for the production is encoded by the HARMLESS TO OZONE LAYER (HOL) gene. The encoded protein belongs to a group of methyltransferases capable of catalyzing the S-adenosyl-L-methionine (SAM)-dependent methylation of chloride (Cl(-)), bromide (Br(-)), and iodide (I(-)) to produce methyl halides. In mutant plants with the HOL gene disrupted, methyl halide production is largely eliminated. A phylogenetic analysis with the HOL gene suggests that the ability to produce methyl halides is widespread among vascular plants. This approach provides a genetic basis for understanding and predicting patterns of methyl halide production by plants.     

Protein profiling of the human epidermis from the elderly reveals up-regulation of a signature of interferon-gamma-induced polypeptides that includes manganese-superoxide dismutase and the p85beta subunit of phosphatidylinositol 3-kinase

Aging of the human skin is a complex process that consists of chronological and extrinsic aging, the latter caused mainly by exposure to ultraviolet radiation (photoaging). Here we present studies in which we have used proteomic profiling technologies and two-dimensional (2D) PAGE database resources to identify proteins whose expression is deregulated in the epidermis of the elderly. Fresh punch biopsies from the forearm of 20 pairs of young and old donors (21-30 and 75-92 years old, respectively) were dissected to yield an epidermal fraction that consisted mainly of differentiated cells. One- to two-mm3 epidermal pieces were labeled with [35S]methionine for 18 h, lysed, and subjected to 2D PAGE (isoelectric focusing and non-equilibrium pH gradient electrophoresis) and phosphorimage autoradiography. Proteins were identified by matching the gels with the master 2D gel image of human keratinocytes (proteomics.cancer.dk). In selected cases 2D PAGE immunoblotting and/or mass spectrometry confirmed the identity. Quantitative analysis of 172 well focused and abundant polypeptides showed that the level of most proteins (148) remains unaffected by the aging process. Twenty-two proteins were consistently deregulated by a factor of 1.5 or more across the 20 sample pairs. Among these we identified a group of six polypeptides (Mx-A, manganese-superoxide dismutase, tryptophanyl-tRNA synthetase, the p85beta subunit of phosphatidylinositol 3-kinase, and proteasomal proteins PA28-alpha and SSP 0107) that is induced by interferon-gamma in primary human keratinocytes and that represents a specific protein signature for the effect of this cytokine. Changes in the expression of the eukaryotic initiation factor 5A, NM23 H2, cyclophilin A, HSP60, annexin I, and plasminogen activator inhibitor 2 were also observed. Two proteins exhibited irregular behavior from individual to individual. Besides arguing for a role of interferon-gamma in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo.     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Technical and economic effects of an inline progesterone indicator in a dairy herd estimated by stochastic simulation

For several years progesterone in milk or blood has been recognized as an indicator of different cow states related to reproduction. For this study, an existing simulation model was modified in order to analyze the technical and economic effects of including information on progesterone status in an automatic inline monitoring system. Implementation of an inline progesterone indicator was assumed to directly affect the estrus detection rate, the period until treatment for post-partum anestrus and the number of mistimed AIs. Different implementations of an inline progesterone indicator were simulated in a typical Danish dairy herd with 120 cows and in other herd situations represented by: a herd with poor reproduction efficiency, a herd with a high estrus detection rate and a herd using a 9 week postponed AI period for primiparous cows. It was concluded that implementation of an inline progesterone indicator in a dairy herd previously using visual estrus detection has a break-even price of 3-81 euros per cow-year depending on differences in implementation type and herd reproduction management. The highest break-even price was found using the assumptions that simulated a herd with initially poor reproductive efficiency. With the assumptions that simulated a typical Danish herd the break-even price was 46 euros per cow-year. Attributable effects of using the indicator, including effects of labor time, are discussed.