Identification of mRNAs differentially expressed in quiescence or in late G1 phase of the cell cycle in human breast cancer cells by using the differential display method.

BACKGROUND: The decision for a cell to enter the DNA synthesis (S) phase of the cell cycle or to arrest in quiescence is likely to be determined by genes expressed in the late G1 phase, at the restriction point. Loss of restriction point control is associated with malignant cellular transformation and cancer. For this reason, identifying genes that are differentially expressed in late G1 phase versus quiescence is important for understanding the molecular basis of normal and malignant growth. MATERIALS AND METHODS: The differential display (DD) method detects mRNA species that are different between sets of mammalian cells, allowing their recovery and cloning of the corresponding cDNAs. Using this technique, we compared mRNAs from synchronized human breast cancer cells (21 PT) in quiescence and in late G1. RESULTS: Six mRNAs differentially expressed in late G1 or in quiescence were identified. One mRNA expressed 10 hr after serum induction showed 99% homology to a peptide transporter involved in antigen presentation of the class I major histocompatibility complex (TAP-1) mRNA. Another mRNA expressed specifically in quiescence and down-regulated 2 hr following serum induction showed 98% homology to human NADP+ -dependent cytoplasmic malic enzyme (EC1.1.1.40) mRNA, which is an important enzyme in fatty acid synthesis and lipogenesis. Three others showed high homology to different mRNAs in the GeneBank, corresponding to genes having unknown functions. Finally, one mRNA revealed no significant homology to known genes in the GeneBank. CONCLUSIONS: We conclude that DD is an efficient and powerful method for the identification of growth-related genes which may have a role in cancer development.     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

A role for Bach1 and HO-2 in suppression of basal and UVA-induced HO-1 expression in human keratinocytes

Ultraviolet A (UVA) radiation is an oxidizing agent that strongly induces the heme oxygenase 1 (HO-1) gene and expression of the protein in cultured human skin fibroblasts but weakly induces it in skin keratinocytes. Lower basal levels of HO-1 and much higher basal levels of HO-2 protein are observed in keratinocytes compared with fibroblasts. Using both overexpression and knockdown approaches, we demonstrate that HO-2 modulates basal and UVA-induced HO-1 protein levels, whereas HO-1 levels do not affect HO-2 levels in skin fibroblasts and keratinocytes. Silencing of Bach1 strongly increases HO-1 levels in transformed HaCaT keratinocytes and these HO-1 levels are not further increased by either UVA irradiation or silencing of HO-2. This is consistent with the conclusion that high constitutive levels of HO-2 expression in keratinocytes are responsible for the resistance of these cells to HO-1 induction by UVA radiation and that Bach1 plays a predominant role in influencing the lack of HO-1 expression in keratinocytes. Bach1 inhibition leading to HO-1 induction reduced UVA-irradiation-induced damage as monitored both by the extent of LDH release and by nuclear condensation, so that Bach1 inhibition seems to protect against UVA-irradiation-induced damage in keratinocytes.     

Germline Allele-Specific Expression of DAPK1 in Chronic Lymphocytic Leukemia

We previously reported a rare germline variant (c.1-6531) that resulted in allele–specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5′ upstream regulatory region, within distinct exons or in the 3′-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.     

Thermodynamics of neutral protein evolution

Naturally evolving proteins gradually accumulate mutations while continuing to fold to stable structures. This process of neutral evolution is an important mode of genetic change and forms the basis for the molecular clock. We present a mathematical theory that predicts the number of accumulated mutations, the index of dispersion, and the distribution of stabilities in an evolving protein population from knowledge of the stability effects (delta deltaG values) for single mutations. Our theory quantitatively describes how neutral evolution leads to marginally stable proteins and provides formulas for calculating how fluctuations in stability can overdisperse the molecular clock. It also shows that the structural influences on the rate of sequence evolution observed in earlier simulations can be calculated using just the single-mutation delta deltaG values. We consider both the case when the product of the population size and mutation rate is small and the case when this product is large, and show that in the latter case the proteins evolve excess mutational robustness that is manifested by extra stability and an increase in the rate of sequence evolution. All our theoretical predictions are confirmed by simulations with lattice proteins. Our work provides a mathematical foundation for understanding how protein biophysics shapes the process of evolution.     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.     

Modelling a binuclear metal binding site in the photosynthetic reaction centre II

Based on the experimental data and homologous sites in Protein Data Bank (PDB) a model for metal binding sites in D1/D2 heterodimer has been proposed. On searching for tetranuclear and binuclear Mn binding sites in the PDB, a suitable sequence homology in thermolysin and D1 could be observed. From the homology and site-directed mutagenesis data, a model for binuclear Mn-Ca or Mn-Mn has been built and it is extended to a tetranuclear Mn centre.     

The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin

Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.     

PathSys: integrating molecular interaction graphs for systems biology

Background  

The goal of information integration in systems biology is to combine information from a number of databases and data sets, which are obtained from both high and low throughput experiments, under one data management scheme such that the cumulative information provides greater biological insight than is possible with individual information sources considered separately.