Mitochondria and degenerative disorders

In mammalian cells, mitochondria provide energy from aerobic metabolism. They play an important regulatory role in apoptosis, produce and detoxify free radicals, and serve as a cellular calcium buffer. Neurodegenerative disorders involving mitochondria can be divided into those caused by oxidative phosphorylation (OXPHOS) abnormalities either due to mitochondrial DNA (mtDNA) abnormalities, e.g., chronic external ophthalmoplegia, or due to nuclear mutations of OXPHOS proteins, e.g., complex I and II associated with Leigh syndrome. There are diseases caused by nuclear genes encoding non-OXPHOS mitochondrial proteins, such as frataxin in Friedreich ataxia (which is likely to play an important role in mitochondrial-cytosolic iron cycling), paraplegin (possibly a mitochondrial ATP-dependent zinc metalloprotease of the AAA-ATPases in hereditary spastic paraparesis), and possibly Wilson disease protein (an abnormal copper transporting ATP-dependent P-type ATPase associated with Wilson disease). Huntingon disease is an example of diseases with OXPHOS defects associated with mutations of nuclear genes encoding non-mitochondrial proteins such as huntingtin. There are also disorders with evidence of mitochondrial involvement that cannot as yet be assigned. These include Parkinson disease (where a complex I defect is described and free radicals are generated from dopamine metabolism), amyotrophic lateral sclerosis, and Alzheimer disease, where there is evidence to suggest mitochondrial involvement perhaps secondary to other abnormalities.     

A polyglutamine expansion disease protein sequesters PTIP to attenuate DNA repair and increase genomic instability

Glutamine (Q) expansion diseases are a family of degenerative disorders caused by the lengthening of CAG triplet repeats present in the coding sequences of seemingly unrelated genes whose mutant proteins drive pathogenesis. Despite all the molecular evidence for the genetic basis of these diseases, how mutant poly-Q proteins promote cell death and drive pathogenesis remains controversial. In this report, we show a specific interaction between the mutant androgen receptor (AR), a protein associated with spinal and bulbar muscular atrophy (SBMA), and the nuclear protein PTIP (Pax Transactivation-domain Interacting Protein), a protein with an unusually long Q-rich domain that functions in DNA repair. Upon exposure to ionizing radiation, PTIP localizes to nuclear foci that are sites of DNA damage and repair. However, the expression of poly-Q AR sequesters PTIP away from radiation-induced nuclear foci. This results in sensitivity to DNA-damaging agents and chromosomal instabilities. In a mouse model of SBMA, evidence for DNA damage is detected in muscle cell nuclei and muscular atrophy is accelerated when one copy of the gene encoding PTIP is removed. These data provide a new paradigm for understanding the mechanisms of cellular degeneration observed in poly-Q expansion diseases.     

Photoreactive DNA probes as a tool for studying the translesion synthesis system in Mammalian cell extracts

Translesion synthesis (TLS) is one of the DNA damage tolerance strategies that has evolved to enable orga-nisms to replicate their genome despite the presence of unrepaired damage. TLS complexes are dynamic systems composed of DNA polymerases and associated protein factors. Therefore, it is hard to study these assembles by X-ray analysis or other instrumental methods. Here, we have suggested applying the photoaffinity labeling technique for studying the TLS system in nuclear/cellular extracts. As a tool we proposed to use partial DNA duplexes containing base-substituted photoreactive deoxynucleotides at the 3' end of primer opposite to DNA damage at the template strand. We demonstrated that photoreactive dNTPs can be potentially used to synthesize photoreactive DNA probes mimicking the DNA intermediates of the first stage of translesion synthesis by specialized DNA polymerases. We used synthetic apurinic/apyrimidinic site (AP-site) - tetrahydrofuran (THF) and 8 oxoguanine as damages in +1 position of the template strand with respect to 3' end of primer. Activity of human DNA polymerases beta and lambda was exploited for construction of photoreactive DNAs using photo derivatives of dNTPs. The kinetic parameters of the elongation reaction in model systems were estimated. Using photoaffinity crosslinking we found that only a few proteins in the bovine testis nuclear extract were strongly labeled by TLS probes.     

PCR-LIS-SSCP快速分析非缺失型α-地中海贫血点突变

目的建立一种简便快速的筛查非缺失型α－地中海贫血点突变的ＳＳＣＰ分析方法。方法在选择性扩增α２珠蛋白基因的基础上,巢式ＰＣＲ扩增突变热点区域,低离子强度（ＬＩＳ）条件下将ＰＣＲ产物热变性,用非变性胶电泳分析其ＳＳＣＰ。结果ＬＩＳ条件下处理样品,ＰＣＲ产物变性效率明显提高,电泳结果可检出区别于野生型对照的３种含基因突变的电泳带型,且电泳时间仅需３小时。结论ＰＣＲ－ＬＩＳ－ＳＳ－ＣＰ可用于快速筛查非缺失型α－地中海贫血点突变。     

Integrative recombination between adenovirus type 12 DNA and mammalian DNA in a cell-free system: joining by short sequence homologies

A cell-free system was developed to investigate the mechanism of how junctions are formed between viral and cellular DNAs during adenoviral DNA integration into the hamster cell genome. Recombination between the segment of adenovirus type 12 (Ad12) DNA, that comprises sequence coordinates 20 885–24 053, subsequently termed PstI-D fragment and the hamster preinsertion DNA sequence p7 was studied in a cell-free system. The p7 DNA segment had served as viral DNA integration site in the Ad12-induced tumor CLAC1. Nuclear extracts initially from uninfected BHK21 hamster cells were fractionated by a series of chromatographic steps. DNAs of the in vitro generated recombinants were analyzed in detail. In the course of the recombination reaction, the two linear molecules were joined. The reaction took place between two short homologous sequences one of which was always at or very close to a DNA terminus, the second one could be several kilobase pairs remote from a DNA terminus. Apparently, the nucleotide sequence at the terminus of one recombining molecule determined the point of junction by searching for short homologies in the partner molecule. The recombination reaction was not conservative, the sequences in-between the short sequence homologies and one of the short sequence homologies were deleted in the in vitro recombinants. Two main criteria influenced the choice of interacting short sequence homologies: perfect homologies of 8–9 bp were most frequently found, they were preferred over more extended, but less perfect homologies. Comparing different short sequence homologies with similar stabilities, those combinations seemed to be chosen in the reaction which led to a minimal loss of nucleotides in the recombinants. The in vitro activity was found in nuclear extracts from both hamster and human cells. The activity was, hence, available for Ad12 DNA in productively infected human and abortively infected hamster cells. The specific recombination activity was increased in nuclear extracts of hamster cells abortively infected with Ad12. The junction sites in the recombinants, which were generated by the cell-free system, were very similar to junctions between adenoviral and cellular DNAs cloned from Ad12-induced tumor cells and Ad12-transformed cell lines.     

Cell surface proteins from Shigella dysenteriae type 1

A simple extraction procedure was used for preparing cell surface proteins (CSPs) from Shigella dysenteriae type 1. The preparations obtained using either buffer or water extractions were free from lipopolysaccharide (LPS), as well as cytoplasmic and periplasmic proteins. By SDS-PAGE, about 25 polypeptides were detected, and Western-blot analysis recognised 15 polypeptide antigens. When analysed by crossed immunoelectrophoresis, using anti-Shigella dysenteriae type 1 rabbit sera, 18 antigenic bands were identified. Proteins obtained by this method were found to be highly immunogenic in rabbits. The cell-surface proteins were compared to outer membrane proteins (OMPs) obtained from the S. dysenteriae type 1 strain by a standard procedure involving lysozyme-EDTA extraction, sucrose density centrifugation, and detergent treatment. They were found to contain periplasmic, cytoplasmic, and lipopolysaccharide contaminants. Thus, the procedure described here offers a quick and simple alternative for obtaining relatively pure cell surface proteins from Shigella dysenteriae type 1. This method will be useful when immunogenically active proteins free from other cellular components are required for studies.     

Diagnostic immunohistochemistry in neuromuscular disorders

Most neuromuscular disorders display only non-specific myopathological features in routine histological preparations. However, a number of proteins, including sarcolemmal, sarcomeric, and nuclear proteins as well as enzymes with defects responsible for neuromuscular disorders, have been identified during the past two decades, allowing a more specific and firm diagnosis of muscle diseases. Identification of protein defects relies predominantly on immunohistochemical preparations and on Western blot analysis. While immunohistochemistry is very useful in identifying abnormal expression of primary protein abnormalities in recessive conditions, it is less helpful in detecting primary defects in dominantly inherited disorders. Abnormal immunohistochemical expression patterns can be confirmed by Western blot analysis which may also be informative in dominant disorders, although its role has yet to be established. Besides identification of specific protein defects, immunohistochemistry is also helpful in the differentiation of inflammatory myopathies by subtyping cellular infiltrates and demonstrating up-regulation of subtle immunological parameters such as cell adhesion molecules. The role of immunohistochemistry in denervating disorders, however, remains controversial in the absence of a reliable marker of muscle fibre denervation. Nevertheless, as well as the diagnostic value of immunocytochemical analysis it may also widen understanding of muscle fibre pathology as well as help in the development of therapeutic strategies.     

Mitochondrial Encephalomyopathies: Defects of Nuclear DNA

The term "mitochondrial diseases" encompasses a heterogeneous group of disorders in which a primary mitochondrial dysfunction is suspected or proven by morphologic, genetic, or biochemical criteria. Clinically, these progressive disorders usually affect muscle, either alone (mitochondrial myopathies) or in combination with other systems, most often brain (encephalomyopathies). Mitochondria are unique among intracellular organelles in that mitochondrial proteins are encoded by two genomes, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). The vast majority of mitochondrial proteins are encoded by the nuclear genome, whereas mtDNA (a circular, double stranded 16.5 kb molecule) encodes only 13 polypeptides, all of them subunits of respiratory chain complexes. In addition to structural genes, mtDNA also codes for 22 transfer RNAs and two ribosomal RNAs. Our understanding of mitochondrial diseases has grown at an impressive rate in the past few years, and most of the progress has been in the area of mtDNA genetics, where several mtDNA mutations have been associated with specific diseases (reviewed in this issue by Zeviani et al.). In comparison, our understanding of mitochondrial disorders due to nDNA lesions has lagged behind and, to date, molecular defects of nuclear genes have been documented in only a few patients. We will review which alterations in the nuclear genome can cause mitochondrial disorders and which criteria are useful in identifying such mutations. While several examples will be provided, this is not intended as a complete review of the subject.     

Interference in SV40 DNA infections: a possible basis for cellular competence.

At the restrictive temperature, DNA from temperature-sensitive mutants of simian virus 40 (SV40) (in the A or B/C genes) interferes with plaque formation by wild-type SV40 DNA. This interference occurs early in the first cycle of infection. Nonhomologous DNA also can interfere with SV40 DNA infection. The mode of interference by ts SV40 DNAs and nonhomologous DNAs appears to be the same. In conjunction with other observations the interference by nonhomologous DNA and the similarity of interference by tsA DNA and tsB DNA suggest that interference does not require expression of any viral gene product. Since interference is observed even when the infections by interfering DNA and wild-type DNA are separated in time, interference must occur after the DNAs interact with cells. Furthermore, the different efficiencies with which structurally similar DNAs (SV40, PM2 and φX174 replicative form) interfere indicate that interference depends at least in part on nucleotide sequence. Those two observations suggest that interference is an intracellular, perhaps nuclear, phenomenon. The possible relevance of this interference phenomenon to observations on cellular competence for SV40 DNA infections is discussed.     

Affinity partitioning of a poly(histidine)-tagged integral membrane protein, cytochrome bo3 ubiquinol oxidase, in a detergent--polymer aqueous two-phase system containing metal-chelating polymer

A system has been developed for selective partitioning of membrane proteins. For the first time, an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli, has been affinity partitioned in an aqueous two-phase system. The systems used were different detergent/polymer aqueous two-phase systems containing a metal-chelating polymer, such as poly(ethyleneglycol)-iminodiacetic acid-Cu(II) as well as dextran-iminodiacetic acid-Cu(II). Many non-ionic detergents, such as alkyl(polyethyleneoxide) (CmEOn), Triton, Tween and alkylglucosides, form two-phase systems in mixture with polymers, such as dextran and poly(ethyleneglycol), i.e., a micelle-enriched phase in equilibrium with a polymer-enriched phase are formed. In general, membrane proteins partition strongly to the micelle phase. We show that it is possible to selectively partition a poly(histidine)-tagged integral membrane protein into the polymer phase by metal affinity partitioning, with a shift in the partitioning coefficient from 0.015 to 4.8 (300-fold). The affinity partitioning was characterized and the effects of ligand concentration, pH, time, salts, buffer type, imidazole and charged detergent are discussed. Thus, a fast and mild affinity procedure for the purification of integral membrane proteins can be developed in affinity detergent/polymer aqueous two-phase systems, and the method is especially suitable for the purification of labile integral membrane proteins, such as receptors.     

Structure of cytomatrix and nuclear matrix revealed by embedment-free electron microscopy

Embedment-free electron microscopy (EFEM) is a new method which allows the visualisation of cytoskeleton in whole-mounted cells. In this study we employed EFEM to investigate the structure of cellular scaffolds in glioma C6 cell line. The cells were extracted with Triton X-100 that dissolves phospholipids in the membranes and removes most of cytoplasmic soluble proteins. The DNA and nuclear histones were removed with DNase I and high-salt buffer, respectively. The remaining cellular frameworks were temporary embedded in diethylene glycol distearate (DGD), sectioned and observed in transmission and scanning electron microscope after the removal of DGD. The predominant structure was the extensive meshwork of 10-20 nm filaments in the cytoplasm (cytomatrix) and 15-30 nm filaments in the nucleus (nuclear matrix). The 5 nm filaments, presumably corresponding to the actin filaments, were present in the cytomatrix, but not in the nuclear matrix. Moreover, the ultrathin (3 nm) filaments, connecting other cytoskeletal components were detected. Those are possibly identical with the previously described plectin filaments. For the first time we report the occurrence of ultrathin filaments in the nuclear matrix. Thus, in a addition to the well known cytoskeletal components (microtubules, intermediate filaments, actin microfilaments) EFEM showed a new type of filaments (the ultrathin filaments) in the cytomatrix and nuclear matrix. Further immunocytochemical studies are needed to determine the biochemical identity of the filaments observed in EFEM.     

Heat-mediated,ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes

Here, we report an ultra-rapid method for the transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, the electro-transfer was performed with heated (70-75 degrees C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight proteins (a purified protein, molecular weight protein standards and proteins from a human tissue extract) could be carried out in 10 min for a 0.75-mm, 7% SDS-PAGE gel. For 10% and 12.5% gels (0.75 mm), the corresponding time was 15 min. In the case of 1.5-mm gels, a complete transfer could be carried out in 20 min for 7%, 10% and 12.5% gels. The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. When the heat-mediated transfer method was compared with a conventional transfer protocol, under similar conditions, we found that the latter method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is very rapid, avoids the use of methanol and is particularly useful for the transfer of high molecular weight proteins.     

Characterization of a versatile in vitro DNA-packaging system based on hybrid lambda/phi 29 proheads.

We have studied the assembly of bacteriophage lambda head proteins on the phage phi 29 connector to produce in vitro chimeric proheads, whose ability to package different types of DNA depends on the physical integrity of the phi 29 connector. Terminal protein-free phi 29 as well as nonviral DNAs have been shown to be efficiently packaged by this hybrid system. An RNA, that can be provided by any of the extracts used in the complementation mixture, was required for DNA packaging, both by the hybrid system as well as by the homologous lambda system. The DNA-packaging activity of RNase-treated proheads can be restored by adding a mixture of ribosomal RNAs. There is also a requirement for a minimal length of DNA to be stably packaged. The packaging protein p16 of phi 29 can replace the lambda terminase complex in the in vitro packaging system, both with the chimeric as well as genuine lambda proheads.     

Relationship between abnormalities of genes involved in DNA damage responses and malignant tumors/autoimmune diseases]

The maintenance of genomic stability is an essential cellular function for a variety of well-coordinated regulation of biological activities of organisms, and a failure in its function results in the accumulation of mutations and/or abnormality in the induction of apoptosis, eventually leading to onsets of various diseases, including malignant tumors. DNA damage responses, in particular cell-cycle checkpoint regulation, play important roles in maintaining genomic integrity. In response to DNA damages induced by gamma-irradiation, ultraviolet irradiation, various chemicals, or reactive oxygen species (ROS), intrinsic cell-cycle checkpoint machinery is rapidly activated to arrest cells at particular cell-cycle points, and during cell-cycle checkpoint arrest cells may try to repair damaged DNAs, and then re-start cell-cycle upon the completion of DNA repair. Alternatively, if the extents of DNA damage overwhelm the capacity of the cellular repair machinery, cells may undergo apoptosis to prevent the accumulation of mutations within the organisms. In this article, we will first explain about our current view of DNA damage responses, in particular cell-cycle checkpoint regulation, and summarize our knowledge of the relationships between abnormalities of genes involved in DNA damage responses and malignant tumors, including hematopoietic malignancies. We will also discuss a possible implication of DNA damage responses in autoimmune diseases, such as rheumatoid arthritis.     

A chemical mismatch cleavage method useful for the detection of point mutations in the p53 gene in lung cancer

Point mutations in genes can be etiologic of pulmonary diseases, as in the case of the inherited disorders alpha-1-antitrypsin deficiency and cystic fibrosis or in the context of dominant and recessive oncogenes in lung cancer. Various methodologies have been developed to screen for single-base mutations. These techniques include direct DNA sequencing, RNase protection, denaturing gradient gel electrophoresis, and chemical mismatch cleavage. The latter method offers the advantages of rapid and efficient analysis of genomic or cDNA and is thus ideally suited to screening applications. Furthermore, all possible single-base changes can theoretically be detected. In the present work, chemical mismatch cleavage was utilized to detect mutations in the p53 gene in small cell and non-small cell lung cancer. This technique was modified by using a two-step, hemi-nested PCR procedure for preparation of target genomic DNAs permitting an expanded target size for analysis. Evaluation by chemical mismatch cleavage of eight p53 cDNAs derived from lung tumors shown to have different mutations by DNA sequencing correctly detected the presence of a point mutation in all instances. Analysis of six additional tumor genomic DNAs with defined mutations in the corresponding p53 cDNAs accurately confirmed the mutation at the level of the genome. The technique also identified codon 72 and intron 6 polymorphisms. Using the intron 6 polymorphism, loss of heterozygosity at the p53 locus in tumor DNA was readily detected by chemical mismatch cleavage. Finally, utilizing this technique for scanning analysis of the p53 gene of uncharacterized lung tumor DNAs, additional mutations were identified in a prospective manner which were confirmed by sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gadd45 proteins: relevance to aging, longevity and age-related pathologies

The Gadd45 proteins have been intensively studied, in view of their important role in key cellular processes. Indeed, the Gadd45 proteins stand at the crossroad of the cell fates by controlling the balance between cell (DNA) repair, eliminating (apoptosis) or preventing the expansion of potentially dangerous cells (cell cycle arrest, cellular senescence), and maintaining the stem cell pool. However, the biogerontological aspects have not thus far received sufficient attention. Here we analyzed the pathways and modes of action by which Gadd45 members are involved in aging, longevity and age-related diseases. Because of their pleiotropic action, a decreased inducibility of Gadd45 members may have far-reaching consequences including genome instability, accumulation of DNA damage, and disorders in cellular homeostasis - all of which may eventually contribute to the aging process and age-related disorders (promotion of tumorigenesis, immune disorders, insulin resistance and reduced responsiveness to stress). Most recently, the dGadd45 gene has been identified as a longevity regulator in Drosophila. Although further wide-scale research is warranted, it is becoming increasingly clear that Gadd45s are highly relevant to aging, age-related diseases (ARDs) and to the control of life span, suggesting them as potential therapeutic targets in ARDs and pro-longevity interventions.     

Effective construction of DNA vaccines against variable influenza genes by homologous recombination

We demonstrate the potential of cloning by homologous recombination as a rapid method to construct DNA molecules encoding newly developing hemagglutinins (HA) of influenza A virus. The variable parts of the HA genes were cloned into a basic construct containing the HA gene from an H3N2 strain. The recombinant DNAs thus created encode different variable domains with neutralising epitopes from four recently circulating influenza A H3 strains. The technology allows rapid production of DNA constructs for vaccines that can induce antibody and, particularly, cellular immune responses. These new constructs were also capable of conferring protection to challenge in mice. The technology may hence be a valuable tool for rapid adaptation of influenza vaccines to changes in the circulating influenza strains.