The Processing Pathway of Prelamin A

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subsequent to prenylation observed in other CAAX box proteins-the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine. To characterize the maturation of prelamin A further, we have developed a CHO-K1 cell line that possesses a dexamethasone-inducible human prelamin A against a genetic background of high mevalonate uptake. Utilizing this cell line in association with antibodies specific to the transgenic prelamin A, we have been able to demonstrate directly in vivo that prelamin A undergoes farnesylation and carboxymethylation prior to conversion to lamin A, as is the case for other prenylated proteins. We have demonstrated previously that in the absence of isoprenylation, conversion of prelamin A to lamin A is blocked, but that unprocessed prelamin A is transported to the nucleus where it can still undergo maturation. Consistent with the implications of these prior studies, we now demonstrate the presence of both subunits of farnesyl-protein transferase in the nucleus

Reduced Macrophage Apoptosis is Associated with Accelerated Atherosclerosis in Low-Denstiy Lipoprotein Receptor-Null Mice

Objective— The majority of apoptotic cells in atherosclerotic lesions are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains unclear. Elevated expression of Bax, one of the pivotal proapoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol-treated mouse macrophages. In this study, we examined the effect of Bax deficiency in bone marrow-derived leukocytes on the development of atherosclerosis in low-density lipoprotein receptor-null (LDLR−/−) mice. Methods and Results— Fourteen 8-week-old male LDLR−/− mice were lethally irradiated and reconstituted with either wild-type (WT) C57BL6 or Bax-null (Bax−/−) bone marrow. Three weeks later, the mice were challenged with a Western diet for 10 weeks. No differences were found in the plasma cholesterol level between the WT and Bax−/− group. However, quantitation of cross sections from proximal aorta revealed a 49.2% increase (P=0.0259) in the mean lesion area of the Bax−/− group compared with the WT group. A 53% decrease in apoptotic macrophages in the Bax−/− group was found by TUNEL staining (P\u3c0.05). Conclusions— The reduction of apoptotic activity in macrophages stimulates atherosclerosis in LDLR−/− mice, which is consistent with the hypothesis that macrophage apoptosis suppresses the development of atherosclerosis

Melting of Single Lipid Components in Binary Lipid Mixtures: A Comparison between FTIR Spectroscopy, DSC and Monte Carlo Simulations

Monte Carlo (MC) Simulations, Differential Scanning Calorimetry (DSC) and Fourier Transform InfraRed (FTIR) spectroscopy were used to study the melting behavior of single lipid components in two-component membranes of 1,2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-d54) and 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC). Microscopic information on the temperature dependent melting of the single lipid species could be investigated using FTIR. The microscopic behavior measured could be well described by the results from the MC simulations. These simulations also allowed to calculate heat capacity profiles as determined with DSC. These ones provide macroscopic information about melting enthalpies and entropy changes which are not accessible with FTIR. Therefore, the MC simulations allowed us to link the two different experimental approaches of FTIR and DSC.Comment: 12 pages, 5 figures, corrected typo in table 1 in which previously it said Tm,1 instead of Tm,

Au-Ag template stripped pattern for scanning probe investigations of DNA arrays produced by Dip Pen Nanolithography

We report on DNA arrays produced by Dip Pen Nanolithography (DPN) on a novel Au-Ag micro patterned template stripped surface. DNA arrays have been investigated by atomic force microscopy (AFM) and scanning tunnelling microscopy (STM) showing that the patterned template stripped substrate enables easy retrieval of the DPN-functionalized zone with a standard optical microscope permitting a multi-instrument and multi-technique local detection and analysis. Moreover the smooth surface of the Au squares (abput 5-10 angstrom roughness) allows to be sensitive to the hybridization of the oligonucleotide array with label-free target DNA. Our Au-Ag substrates, combining the retrieving capabilities of the patterned surface with the smoothness of the template stripped technique, are candidates for the investigation of DPN nanostructures and for the development of label free detection methods for DNA nanoarrays based on the use of scanning probes.Comment: Langmuir (accepted

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs

Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli

Now that many genomes have been sequenced and the products of newly identified genes have been annotated, the next goal is to engineer the desired phenotypes in organisms of interest. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA-binding proteins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than 6.4 × 104 ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in E. coli and selected for industrially important traits, such as resistance to heat shock, osmotic pressure and cold shock. Genes associated with the heat-shock resistance phenotype were then characterized. These results and the general applicability of this platform clearly indicate that novel ATFs are powerful tools for the phenotypic engineering of microorganisms and can facilitate microbial functional genomic studies

Reduced expression of lamin A/C correlates with poor histological differentiation and prognosis in primary gastric carcinoma

<p>Abstract</p> <p>Background</p> <p>Lamin A/C is very important in DNA replication, RNA dependent transcription and nuclear stabilization. Reduced or absent lamin A/C expression has been found to be a common feature of a variety of different cancers. To investigate the role of lamin A/C in gastric carcinoma (GC) pathogenesis, we analyzed the correlations between the lamin A/C expression level and clinicopathological factors and studied its prognostic role in primary GC.</p> <p>Methods</p> <p>The expression of lamin A/C at mRNA level was detected by the reverse transcription-polymerase chain reaction (RT-PCR) and real time RT-PCR, and western blot was used to examine the protein expression. Lamin A/C expression and its prognostic significance were investigated by performing immunohistochemical analysis on a total of 126 GC clinical tissue samples.</p> <p>Results</p> <p>Both lamin A/C mRNA and protein expression were downregulated in the majority of tumours compared with corresponding normal gastric tissues (<it>p </it>= 0.011 and <it>p </it>= 0.036, respectively). Real time RT-PCR further validated that downregulation of lamin A/C is associated with poor histological differentiation (r = 0.438, <it>p </it>= 0.025). The immunohistochemical staining showed an evident decrease of lamin A/C expression in 55.6% (70/126) GC cases. Importantly, the negative lamin A/C expression correlated strongly with histological classification (r = 0.361, <it>p </it>= 0.034). Survival analysis revealed that patients with lamin A/C downregulation have a poorer prognosis (<it>p </it>= 0.034). In addition, lamin A/C expression was found to be an independent prognostic factor by multivariate analysis.</p> <p>Conclusion</p> <p>Data of this study suggest that lamin A/C is involved in the pathogenesis of GC, and it may serve as a valuable biomarker for assessing the prognosis for primary GC.</p

Proteome and Membrane Fatty Acid Analyses on Oligotropha carboxidovorans OM5 Grown under Chemolithoautotrophic and Heterotrophic Conditions

Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium able to utilize CO and H2 to derive energy for fixation of CO2. Thus, it is capable of growth using syngas, which is a mixture of varying amounts of CO and H2 generated by organic waste gasification. O. carboxidovorans is capable also of heterotrophic growth in standard bacteriologic media. Here we characterize how the O. carboxidovorans proteome adapts to different lifestyles of chemolithoautotrophy and heterotrophy. Fatty acid methyl ester (FAME) analysis of O. carboxidovorans grown with acetate or with syngas showed that the bacterium changes membrane fatty acid composition. Quantitative shotgun proteomic analysis of O. carboxidovorans grown in the presence of acetate and syngas showed production of proteins encoded on the megaplasmid for assimilating CO and H2 as well as proteins encoded on the chromosome that might have contributed to fatty acid and acetate metabolism. We found that adaptation to chemolithoautotrophic growth involved adaptations in cell envelope, oxidative homeostasis, and metabolic pathways such as glyoxylate shunt and amino acid/cofactor biosynthetic enzymes

The Mutant Form of Lamin A that Causes Hutchinson-Gilford Progeria Is a Biomarker of Cellular Aging in Human Skin

Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by accelerated aging and early death, frequently from stroke or coronary artery disease. 90% of HGPS cases carry the LMNA G608G (GGC>GGT) mutation within exon 11 of LMNA, activating a splice donor site that results in production of a dominant negative form of lamin A protein, denoted progerin. Screening 150 skin biopsies from unaffected individuals (newborn to 97 years) showed that a similar splicing event occurs in vivo at a low level in the skin at all ages. While progerin mRNA remains low, the protein accumulates in the skin with age in a subset of dermal fibroblasts and in a few terminally differentiated keratinocytes. Progerin-positive fibroblasts localize near the basement membrane and in the papillary dermis of young adult skin; however, their numbers increase and their distribution reaches the deep reticular dermis in elderly skin. Our findings demonstrate that progerin expression is a biomarker of normal cellular aging and may potentially be linked to terminal differentiation and senescence in elderly individuals