Reduced levels of oxidized and glycoxidized proteins in human fibroblasts exposed to repeated mild heat shock during serial passaging in vitro.

Repeated mild heat shock (RMHS) has beneficial hormesis-like effects on various characteristics of human skin fibroblasts undergoing replicative senescence in vitro. We have tested whether RMHS could reduce the accumulation of oxidized and glycoxidized proteins, which is a major age-related change. Levels of carbonylated proteins, furosine, N(epsilon)-carboxymethyl-lysine-rich proteins and advanced glycation end products increased during serial passaging of fibroblasts in culture. However, the extent of accumulation of oxidized and glycoxidized proteins was significantly reduced in RMHS cells. The basal concentration of reduced glutathione was higher and that of oxidized glutathione was lower in RMHS cells. Whereas the basal level of heat shock protein HSP27 decreased in both RMHS and control cells during serial passaging, the increase of the basal level of HSP70 with increasing passage level was significantly higher in RMHS cells. These results show that the slower accumulation of damaged proteins in fibroblasts exposed to RMHS results partly from the increased ability of these cells to cope with oxidative stress, and to synthesize HSP responsible for protein capping and refolding.

Heat shock response and ageing: mechanisms and applications.

Ageing is associated with a decrease in the ability of cells to cope with environmental challenges. This is due partly to the attenuation of a primordial stress response, the so-called heat shock (HS) response, which induces the expression of heat shock proteins (HSPs), composed of chaperones and proteases. The attenuation of the HS response during ageing may be responsible for the accumulation of damaged proteins as well as abnormal regulation of cell death. Maintenance of the HS response by repeated mild heat stress causes anti-ageing hormetic effects on cells and organisms. Here, we describe the molecular mechanism and the state of the HS response as well as the role of specific HSPs during ageing, and discuss the possibility of hormetic modulation of ageing and longevity by repeated mild stress.

Generation and epitope mapping of high-affinity scFv to eukaryotic elongation factor 1A by dual application of phage display.

To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity-improve and map epitopes of two previously isolated, low-affinity single-chain Fv (scFv) G3 and D1. The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A-1 and eEF1A-2. By PCR-based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage-based affinity-selection, two 'families' of affinity-improved scFv were obtained. The scFv of highest affinity, A8, has a Kd of 9 nM to eEF1A-1. Interestingly, two affinity-improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv. Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen-binding. Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413-450 of eEF1A-1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage-antibody repertoire derived scFv were used to select phage-displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414-418) sequence found in hEF1A-1 were isolated. The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF-7 fibroblasts. In both cases a perinuclear localization of hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained.

De novo identification of cell-type specific antibody-antigen pairs by phage display subtraction. Isolation of a human single chain antibody fragment against human keratin 14.

The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology. A novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly non-differentially expressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.

Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture.

Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long-term subculturing. In order to study in vitro age-related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast-specific genes in early-passage (< 50% lifespan completed) with those of late-passage cells (> 90% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late-passage cells compared to early-passage cells, suggesting an in vitro age-related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic cells inhibited their proliferation by 57 +/- 1% and stimulated steady state mRNA levels of AP (1.7 +/- 0.1-fold) and OC (1.8 +/- 0.2-fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7 +/- 0.2-fold) and OC (3.0 +/- 0.3-fold) in late-passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age-related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment.

Regulation of elongation factor-1alpha expression by growth factors and anti-receptor blocking antibodies.

The epidermal growth factor (EGF) family and its receptors regulate normal and cancerous epithelial cell proliferation, a process that could be suppressed by anti-receptor blocking antibodies. Polypeptide elongation factor-1alpha (EF-1alpha) is a multifunctional protein whose levels are positively correlated with the proliferative state of cells. To identify genes, whose expression may be modulated by anti-receptor blocking antibodies, we performed a differential display screening and isolated differentially expressed cDNAs. Isolates from one clone were 100% identical to human EF-1alpha. Both EGF and heregulin-beta1 (HRG) induced EF-1alpha promoter activity and mRNA and protein expression. Growth factor-mediated EF-1alpha expression was effectively blocked by pretreatment with humanized anti-EGF receptor antibody C225 or anti-human epidermal growth factor receptor-2 (HER2) antibody herceptin. Mutants and pharmacological inhibitors of p38(MAPK) and MEK, but not phosphatidylinositol 3-kinase, suppressed both constitutive and HRG-induced stimulation of EF-1alpha promoter activity in MCF-7 cells. Deletion analysis of the promoter suggested the requirement of the -393 to -204 region for growth factor-mediated transcription of EF-1alpha. Fine mapping and point mutation studies revealed a role of the SP1 site in the observed HRG-mediated regulation of the EF-1alpha promoter. In addition, we also provide new evidence to suggest that HRG stimulation of the EF-1alpha promoter involves increased physical interactions with acetylated histone H3 and histone H4. These results suggest that regulation of EF-1alpha expression by extracellular signals that function through human EGF receptor family members that are widely deregulated in human cancers and that growth factor regulation of EF-1alpha expression involve histone acetylation.

Identification of 6-furfuryladenine (kinetin) in human urine.

In contrast to the current view of kinetin (K, N(6)-furfuryladenine) as an unnatural and synthetic cytokinin, recently it has been identified in plant DNA and plant extract. Here we describe identification of K in human urine using chromatography/mass-spectrometry analysis for the first time. The amount of kinetin in urine taken from unhealthy patients lung carcinoma was established to be 0.5 ng in 20 ml and a 100-fold reduced amount in healthy subjects. Since this rare base is a potential source of structural constrains it has to be removed from DNA by enzymatic DNA-repair reactions. It seems that the presence of kinetin in human is linked to oxidative damage processes.

Modulating cellular aging in vitro: hormetic effects of repeated mild heat stress on protein oxidation and glycation.

Intracellular and extracellular proteins are subject to a variety of spontaneous non-enzymatic modifications which affect their structure, function and stability. Protein oxidation and glycation are tightly linked and are implicated in the development of many pathological consequences of aging. Although multiple endogenous pathways in the cell can prevent the formation of oxidized and glycated proteins, and repair and degrade abnormal proteins, such abnormal proteins do accumulate during aging. The heat shock response involving the family of stress-proteins or the so-called heat shock proteins (HSP), represents the quickest and highly conserved response to proteotoxic insults. Since repeated mild heat stress is able to prevent the onset of various age-related changes during cellular aging in vitro, we suggest that treatments which increase HSP expression should reduce the extent of accumulation of abnormal proteins during aging. Such modulation of aging is an example of hormesis, which is characterized by the beneficial effects resulting from the cellular responses to mild repeated stress.

Kinetin inhibits protein oxidation and glycoxidation in vitro.

We tested the ability of N(6)-furfuryladenine (kinetin) to protect against oxidative and glycoxidative protein damage generated in vitro by sugars and by an iron/ascorbate system. At 50 microM, kinetin was more efficient (82% inhibition) than adenine (49% inhibition) to inhibit the bovine serum albumin (BSA)-pentosidine formation in slow and fast glycation/glycoxidation models. Kinetin also inhibited the formation of BSA-carbonyls after oxidation significantly more than adenine did. However both compounds inhibited the advanced glycation end product (AGE) formation to the same extent (59-68% inhibition). At 200 microM, kinetin but not adenine, limited the aggregation of BSA during glycation. These data suggest that kinetin is a strong inhibitor of oxidative and glycoxidative protein-damage generated in vitro.

The human elongation factor 1 A-2 gene (EEF1A2): complete sequence and characterization of gene structure and promoter activity.

The eukaryotic elongation factor 1 A (eEF1A, formerly EF1alpha) is a key factor in protein synthesis, where it promotes the transfer of aminoacylated tRNAs to the A site of the ribosome. Two differentially expressed isoforms of eEF1A, designated eEF1A-1 and eEF1A-2, are found in mammals. Here we report the isolation and sequencing of the gene (HGMW-approved symbol EEF1A2) coding for the human eEF1A-2 isoform. Furthermore, we characterize the gene structure and the activity of the promoter. Isolation of overlapping clones from human libraries revealed that the human eEF1A-2 gene spans approximately 10 kb and consists of eight exons. The intron-exon boundaries of human EEF1A2 and EEF1A1 are conserved, yet the gene of the eEF1A-2 isoform is larger than the eEF1A-1 gene because of enlarged introns. Primer extension analysis identified the predominant transcription start site 166 bp upstream of the AUG codon. The start site maps to an adenine located within a consensus initiator element. Sequencing of a 2-kb 5'-flanking promoter region revealed no TATA element. However, several putative cis-regulatory elements were discovered. The 5'-promoter activity was characterized by transient transfection experiments. Progressive deletions of the upstream promoter region defined a minimal promoter region, ranging from -16 to +92, that is sufficient to drive transcription.

CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts.

In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). There is a progressive and significant reduction of the CBFA1 steady-state mRNA level down to 50% during cellular aging of human osteoblasts. In comparison to the normal cells, human osteosarcoma cell lines SaOS-2 and KHOS/NP, and the SV40-transformed human lung fibroblast cell line MRC5V2 have 20 to 40% higher levels of CBFA1 mRNA. Similar levels of CBFA1 mRNA are detectable in normal human skin fibroblasts, and these cells also exhibit an age-related decline to the same extent. In addition, the expression of topoisomerase I is reduced by 40% in senescent osteoblasts, and the mRNA levels are significantly higher (40-70%) in transformed osteoblasts and fibroblasts. These changes in gene expression may be among the causes of impaired osteoblast functions, resulting in reduced bone formation during aging.

Gene-specific DNA repair of pyrimidine dimers does not decline during cellular aging in vitro.

A large number of studies have demonstrated that various kinds of DNA damage accumulate during aging and one of the causes for this could be a decrease in DNA repair capacity. However, the level of total genomic repair has not been strongly correlated with aging. DNA repair of certain kinds of damage is known to be closely connected to the transcription process; thus, we chose to investigate the level of gene-specific repair of UV-induced damage using in vitro aging of human diploid skin fibroblasts and trabecular osteoblasts as model systems for aging. We find that the total genomic repair is not significantly affected during cellular aging of cultures of both human skin fibroblasts and trabecular osteoblasts. Gene-specific repair was analyzed during cellular aging in the dihydrofolate reductase housekeeping gene, the p53 tumor suppressor gene, and the inactive region X(754). There was no clear difference in the capacity of young and old cells to repair UV-induced pyrimidine dimers in any of the analyzed genes. Thus, in vitro senescent cells can sustain the ability to repair externally induced damage.

Identification of phage antibodies toward the Werner protein by selection on Western blots.

A procedure was established for selecting phage antibodies (phage-abs) from phage-displayed antibody repertoires by panning against proteins, separated by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Western blots). This immobilization strategy is applicable for secondary rounds of panning in selections against semipurified proteins, and directs the selection toward antibodies suitable as immunochemical reagents in Western blots. In model experiments, enrichment factors as high as 1.9x10(5) were obtained in a single round of panning. Furthermore, we demonstrate the application of this approach by selection of phage-abs recognizing the human Werner protein, which is defective in a premature aging syndrome.

Display of Ras on filamentous phage through cysteine replacement.

Phage display technology has been used in a variety of contexts to understand and manipulate biomolecular interactions between proteins and other biomolecules. In this paper we describe the establishment of a phage display system for elucidation of the interactions between the GTPase Ras and its panel of effectors. It is shown how technical problems associated with phage display of a protein with unpaired cysteines, likely to be caused by the oxidizing environment of the bacterial periplasm into which the protein is directed, can be overcome by cysteine replacement based on functional and structural studies. First, the catalytic domain (residues 1-166) of mammalian H-Ras (Ras) was observed to be displayed on phage in an incorrect conformation not detectable by antibodies recognizing conformational epitopes on Ras. Although truncation of the phage coat protein used as fusion partner (g3p) resulted in minor improvements in the display, Ras was tailored for phage display by cysteine replacement. By replacing the three cysteines at positions 51, 80 and 118 of Ras with the corresponding residues in Saccharomyces cerevisiae RAS1, the resulting fusion-phage is recognized by the conformation-dependent anti-Ras antibodies. Furthermore, display of cysteine-free Ras is demonstrated by GTP-analogue dependent binding to the Ras-binding domain of the Ras-effector Raf1. These data pave the way for analysis of Ras-effector interactions using phage display technology yet demonstrate that phage display of proteins with normally reduced cysteines should be approached with caution.

N(6)-Furfuryladenine, kinetin, protects against Fenton reaction-mediated oxidative damage to DNA.

N(6)-Furfuryladenine (kinetin) has been shown to have anti-ageing effects on several different systems including plants, human cells in culture, and fruitflies. Since most of the experimental data point toward kinetin acting as an antioxidant both in vitro and in vivo, and since much evidence supporting a causal role of oxidative damage in ageing is accumulating, we tested the antioxidant properties of kinetin directly. Using 8-oxo-2'deoxyguanosine (8-oxo-dG) in calf thymus DNA as a marker for oxidative damage, we demonstrate that kinetin significantly (P < 0.005) protects the DNA against oxidative damage mediated by the Fenton reaction. Kinetin inhibited 8-oxo-dG formation in a dose-dependent manner with a maximum of 50% protection observed at 100 microM kinetin.

Rapid identification of DNA-binding proteins by mass spectrometry.

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.

Structural information for explaining the molecular mechanism of protein biosynthesis.

Protein biosynthesis is controlled by a number of proteins external to the ribosome. Of these, extensive structural investigations have been performed on elongation factor-Tu and elongation factor-G. This now gives a rather complete structural picture of the functional cycle of elongation factor-Tu and especially of the elongation phase of protein biosynthesis. The discovery that three domains of elongation factor-G are structurally mimicking the amino-acylated tRNA in the ternary complex of elongation factor-Tu has been the basis of much discussion of the functional similarities and functional differences of elongation factor-Tu and elongation factor-G in their interactions with the ribosome. Elongation factor-G:GDP is now thought to leave the ribosome in a state ready for checking the codon-anticodon interaction of the aminoacyl-tRNA contained in the ternary complex of elongation factor-Tu. Elongation factor-G does this by mimicking the shape of the ternary complex. Other translation factors such as the initiation factor-2 and the release factor 1 or 2 are also thought to mimic tRNA. These observations raise questions concerning the possible evolution of G-proteins involved in protein biosynthesis.

Telomere shortening during aging of human osteoblasts in vitro and leukocytes in vivo: lack of excessive telomere loss in osteoporotic patients.

We have compared the telomere length, as assessed by Southern analysis, of telomere restriction fragments (TRFs) generated by RsaI/HinfI digestion of genomic DNA in: (i) in vitro cultured human trabecular osteoblasts undergoing cellular aging; and (ii) peripheral blood leukocytes (PBL) obtained from three groups of women: young (aged 20-26 years, n = 15), elderly (aged 48-85 years, n = 15) and osteoporotic (aged 52-81 years, n = 14). The mean TRF length in human osteoblasts undergoing aging in vitro decreased from an average of 9.32 kilobasepairs (kb) in middle-aged cells to an average of 7.80 kb in old cells. The rate of TRF shortening was about 100 bp per population doubling, which is similar to what has been reported for other cell types, such as human fibroblasts. Furthermore, there was a 30% decline in the total amount of telomeric DNA in senescent osteoblasts as compared with young cells. In the case of PBL, TRF length in the DNA extracted from young women was slightly longer (6.76 +/- 0.64 kb) than that from a group of elderly women (6.42 +/- 0.71 kb). A comparison of TRFs in the DNA extracted from the PBL from osteoporotic patients and from age-matched controls did not show any significant differences (6.47 +/- 0.94 versus 6.42 +/- 0.71 kb, respectively). Therefore, using TRF length as a marker for cellular aging in vitro and in vivo, our data comparing TRFs from osteoporotic patients and age-matched controls do not support the notion of the occurrence of a generalized premature cellular aging in osteoporotic patients.