. (1999) 'The cardiomyopathy and lens cataract mutation in B-crystallin alters its protein structure, chaperone activity, and interaction withintermediate laments in vitro.', Journal of biological chemistry., 274 (47). pp. 33235-33243. Further information on publisher's website: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details.

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance. (Cancer Res 2005; 65(20): 9510-6)

The array CGH technique (Array Comparative Genome Hybridization) has been developed to detect chromosomal copy number changes on a genome-wide and/or high-resolution scale. It is used in human genetics and oncology, with great promise for clinical application. Until recently primarily PCR amplified bacterial artificial chromosomes (BACs) or cDNAs have been spotted as elements on the array. The large-scale DNA isolations or PCR amplifications of the large-insert clones necessary for manufacturing the arrays are elaborate and time-consuming. Lack of a high-resolution highly sensitive (commercial) alternative has undoubtedly hindered the implementation of array CGH in research and diagnostics. Recently, synthetic oligonucleotides as arrayed elements have been introduced as an alternative substrate for array CGH, both by academic institutions as well as by commercial providers. Oligonucleotide libraries or ready-made arrays can be bought off-the-shelf saving considerable time and efforts. For RNA expression profiling, we have seen a gradual transition from in-house printed cDNA-based expression arrays to oligonucleotide arrays and we expect a similar transition for array CGH. This review compares the different platforms and will attempt to shine a light on the ‘BAC to the future’ of the array CGH technique.

The R120G mutation in ␣B-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of ␣B-crystallin for desmin filaments. The apparent dissociation constant of R120G ␣B-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G ␣B-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type ␣B-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G ␣B-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in ␣B-crystallin. INTRODUCTIONThe study of many different human diseases caused by mutations in intermediate filament (IF) proteins (McLean and Lane, 1995;Fuchs and Cleveland, 1998;Coulombe and Omary, 2002; has shown that filament aggregation is a common feature (van den IJssel et al., 1999), implying that 10-nm filaments need to be arranged in networks to be functional. What then determines the distribution and location of IF networks in cells? IFs are dynamic structures, individually and collectively (Helfand et al., 2003). The composition of the IFs themselves is clearly a very important factor because that can influence the distribution of filaments (Cary and Klymkowsky, 1994a,b) and their dynamics (Chou et al., 2003) and the effect of some intermediate filament mutations (Zhou et al., 2003). The filament composition can also determine the spacing between filaments, an important part of organizing individual filaments into an ordered arrangement or network. So vimentin, but not nestin, facilitates the correct spacing of glial fibrillary acidic protein filaments (Eliasson et al., 1999) and, of course, altering the proportion of the different neurofilament proteins changes the packing density of neurofilaments (Xu et al., 1996). Then, the provision of appropriate docking sites, for instance on the plasma membrane (Borradori and Sonnenberg, 1999;Green and Gaudry, 2000;Garrod et al., 2002), as provided by members of the spectraplakin protein family (Leung et al., 2001;Roper and Brown, 2003) and also by some IF proteins themselves, such as syncoilin (Poon et al., 2002). IF composition and available attachment sites are therefore important factors in network formation.This point has been firmly made for des...

Stress-activated protein kinase 2a, also called p38, is inhibited by SB 203580 and this drug has been used widely to implicate this enzyme in the regulation of many physiological processes. Here, we introduce a novel method of general application, which can be used to establish whether the effects of SB 203580 are mediated via inhibition of stress-activated protein kinase 2a/p38 or whether they result from`non-specific' effects. Four events thought to occur upon activation of stressactivated protein kinase 2a/p38 have been established unequivocally. These are the activation of mitogen-activated protein kinase-activated protein kinase-2 and mitogen-and stressactivated protein kinase-1 and the phosphorylation of their presumed substrates, heat shock protein 27 and the transcription factor cyclic AMP response element binding protein, respectively. In contrast, the SB 203580-induced activation of c-Raf is independent of stress-activated protein kinase 2a/p38 inhibition.z 1999 Federation of European Biochemical Societies.