Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy.

Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli.

A virulent double-stranded DNA bacteriophage, Phi K1-5, has been isolated and found to be capable of infecting Escherichia coli strains that possess either the K1 or the K5 polysaccharide capsule. Electron micrographs show that the virion consists of a small icosohedral head with short tail spikes, similar to members of the Podoviridae family. DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins. The first is the K5 lyase protein gene of Phi K5, which allows this phage to specifically infect K5 E. coli strains. A second open reading frame encodes a protein almost identical in amino acid sequence to the N-acetylneuraminidase (endosialidase) protein of Phi K1E, which allows this phage to specifically infect K1 strains of E. coli. We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated. A comparison of the tail gene regions of Phi K5, Phi K1E, and Phi K1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer.

Dopamine induces cell death, lipid peroxidation and DNA base damage in a catecholaminergic cell line derived from the central nervous system.

Dopamine can be autoxidized to superoxides and quinones. Superoxides can form hydroxyl radicals that are highly reactive with lipids, proteins and DNA leading to neuronal damage and cell death. We used a clonal catecholaminergic cell line (CATH.a) derived from the central nervous system to evaluate the effects of dopamine on cell death, lipid peroxidation and DNA base damage. Dopamine produces cell death in CATH.a cells and this is associated with an increase in annexin binding, which is an early indicator of apoptosis. Incubation of CATH.a cells with deferoximine, an iron chealator, partially antagonizes dopamine-induced cell death. In CATH.a cells, dopamine produces an increase in both lipid peroxidation, as measured by cis-parinaric acid fluorescence, and DNA oxidative base damage, as measured by 8-hydroxy-2'-deoxyguanosine formation. Cell death was inhibited 84-92% by the hydrophilic antioxidants, dithiothreitol, L-cysteine, and N-acetylcysteine. The lipophilic vitamins, retinol and vitamin E and the vitamin E analog, Trolox, inhibited dopamine-induced cell death by 18-33%. The lipophilic antioxidants probucol, propyl glycol and butylated hydroxyanisone had no inhibitory effect on dopamine-induced cell death. These data suggest that damage to DNA and lipids may be partially responsible for dopamine-induced cell death in CATH.a cells.

Localization by fluorescence in situ hybridization (FISH) of human mitochondrial polymerase gamma (POLG) to human chromosome band 15q24-->q26, and of mouse mitochondrial polymerase gamma (Polg) to mouse chromosome band 7E, with confirmation by direct sequence analysis of bacterial artificial chromosomes (BACs).

Cloned cDNAs for the human mitochondrial DNA polymerase gamma (POLG) were identified by homology with the yeast mitochondrial DNA polymerase catalytic subunit (MIP). Fluorescence in situ hybridization (FISH) of human and mouse bacterial artificial chromosomes (BACs), hybridized by radioactively labeled POLG cDNAs, mapped to human chromosome band 15q24-->q26, as well as to mouse chromosome band 7E. Direct sequencing of the BAC DNA without subcloning confirmed the presence of both human POLG and mouse mitochondrial DNA polymerase gamma (Polg) in the respective BACs.

Plasma protein variations in monozygotic twins discordant for schizophrenia.

Monozygotic twins discordant for schizophrenia were analyzed by two-dimensional (2-D) gel electrophoresis to identify extrahereditary factors important in the development of schizophrenia. Plasma protein patterns in 2-D gels of monozygotic twins discordant for schizophrenia were found to be significantly less alike than those of normal control monozygotic twins. Several polypeptide spots were found to be elevated in the plasma of the schizophrenic twin. One of these polypeptides, spot 782, was also found to be significantly (p < .001) elevated when schizophrenic patients were compared to unrelated normal control individuals. Spot 782 may be an isoform of haptoglobin. Quantitative variations in some plasma haptoglobin levels were seen between discordant twins, but not between unrelated schizophrenic and normal control individuals.

Long-circulating bacteriophage as antibacterial agents.

The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial-passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22. We demonstrated that the long-circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents.

Possible relationship between conditions associated with chronic hypoxia and brain mitochondrial DNA deletions.

The brain relies heavily on aerobic metabolism which requires functional mitochondria. Mitochondria are subcellular organelles with their own genome which codes for 13 essential protein subunits. By employing PCR assays to examine brain tissue from 43 age-comparable individuals (between ages 34 and 73), we found a correlation between mitochondrial DNA deletion mutations, mtDNA4977 deletions, and conditions associated with chronic hypoxia. In prior studies, utilizing only 6 to 12 clinical samples, mtDNA4977 deletions were reported to increase in specific regions of the brain with aging. However, we found 12-fold and 5-fold higher levels of mtDNA4977 deletions in the putamen and the superior frontal gyrus of the cortex, respectively, from individuals who had conditions associated with chronic hypoxia when compared with individuals without evidence of such conditions. These findings suggest that chronic hypoxia should be more closely examined in the pathophysiology of central nervous system diseases.

Preliminary study of an increase of a plasma apolipoprotein E variant associated with peripheral nerve damage. A finding in patients with chronic spinal pain.

STUDY DESIGN: This two-dimensional gel electrophoretic study analyzed the plasma of six groups of patients to determine the association of an elevated apolipoprotein E variant with peripheral nerve damage (PND). OBJECTIVES: To find a statistically significant plasma protein alteration in patients with PND including chronic spinal pain. SUMMARY OF BACKGROUND DATA: A twofold to fivefold increase in human plasma apolipoprotein E may be a physiologic response to PND as a 250-fold local increase in apolipoprotein E was reported in experimental PND studies in mammals. METHODS: A total of 36 patients with chronic lumbar pain, 28 normal control subjects, and 33 patients with other conditions were studied. Venipuncture was performed and plasma was studied using the technique of two-dimensional gel electrophoresis. Chi-square analysis was used to evaluate results. RESULTS: A statistically significant (P < 0.005) elevation of the plasma apolipoprotein E variant was found in patients with chronic lumbar pain. It also was elevated in patients with chronic cervical pain, extraspinal pain with PND, and chronic inflammatory diseases; but not in extraspinal pain without PND, or asymptomatic biomechanically deficient lumbar spines. CONCLUSIONS: This quantitative protein alteration, although not specific for PND, may prove useful in the treatment of conditions with this disorder, including chronic spinal pain.

The protein disease database of human body fluids: I. Rationale for the development of this database.

We are developing a relational database to facilitate quantitative and qualitative comparisons of proteins in human body fluids in normal and disease states. For decades researchers and clinicians have been studying proteins in body fluids such as serum, plasma, cerebrospinal fluid and urine. Currently, most clinicians evaluate only a few specific proteins in a body fluid such as plasma when they suspect that a patient has a disease. Now, however, high resolution two-dimensional protein electrophoresis allows the simultaneous evaluation of 1,500 to 3,000 proteins in complex solutions, such as the body fluids. This and other high resolution methods have encouraged us to collect the clinical data for the body fluid proteins into an easily accessed database. For this reason, it has been constructed on the Internet World Wide Web (WWW) under the title Protein Disease Database (PDD). In addition, this database will provide a linkage between the disease-associated protein alterations and images of the appropriate proteins on high-resolution electrophoretic gels of the body fluids. This effort requires the normalization of data to account for variations in methods of measurement. Initial efforts in the establishment of the PDD have been concentrated on alterations in the acute-phase proteins in individuals with acute and chronic diseases. Even at this early stage in the development of our database, it has proven to be useful as we have found that there appear to be several common acute-phase protein alterations in the plasma and cerebrospinal fluid from patients with Alzheimer's disease, schizophrenia and major depression. Our goal is to provide access to the PDD so that systematic correlations and relationships between disease states can be examined and extended.

The Protein Disease Database of human body fluids: II. Computer methods and data issues.

The Protein Disease Database (PDD) is a relational database of proteins and diseases. With this database it is possible to screen for quantitative protein abnormalities associated with disease states. These quantitative relationships use data drawn from the peer-reviewed biomedical literature. Assays may also include those observed in high-resolution electrophoretic gels that offer the potential to quantitate many proteins in a single test as well as data gathered by enzymatic or immunologic assays. We are using the Internet World Wide Web (WWW) and the Web browser paradigm as an access method for wide distribution and querying of the Protein Disease Database. The WWW hypertext transfer protocol and its Common Gateway Interface make it possible to build powerful graphical user interfaces that can support easy-to-use data retrieval using query specification forms or images. The details of these interactions are totally transparent to the users of these forms. Using a client-server SQL relational database, user query access, initial data entry and database maintenance are all performed over the Internet with a Web browser. We discuss the underlying design issues, mapping mechanisms and assumptions that we used in constructing the system, data entry, access to the database server, security, and synthesis of derived two-dimensional gel image maps and hypertext documents resulting from SQL database searches.

Protein alterations in olfactory neuroblasts from Alzheimer donors.

Definitive diagnosis of Alzheimer's disease (AD) is made by pathologic examination of postmortem brain tissue in conjunction with a clinical history of dementia. To date, there are no good biological markers for a positive diagnosis of AD in the living patient. In an effort to identify biological markers useful both in the clinical and pathologic diagnosis of AD, we have investigated disease-specific protein alterations in cultured olfactory neurons. Olfactory neurons are readily accessible by biopsy, can be propagated in primary cell culture as olfactory neuroblasts (ONs), and exhibit several elements of AD brain pathophysiology making them powerful tools for the study of AD. Two-dimensional gel analysis of ON proteins from neuropsychologically evaluated AD donors revealed a set of five proteins (Mr 17-50 kD, pI 4.8-6.7) that were significantly altered in concentration when compared to cells from age-matched controls. Further characterization and microsequence analysis could lead to the identification of proteins that may have important diagnostic or therapeutic value in the treatment of AD.

Heat shock proteins protect against stress-related phosphorylation of tau in neuronal PC12 cells that have acquired thermotolerance.

A68, or PHF-tau, is an abnormally phosphorylated form of the microtubule-associated protein tau, which is a primary protein constituent of paired helical filaments (PHFs) and, ultimately, of Alzheimer's disease-associated neurofibrillary tangles (NFTs). Previously, we have shown that in heat-shocked neuronal PC12 cells, tau is hyperphosphorylated and transformed to an A68-like state as determined by immunologic and biochemical criteria. In the present study, we investigated the role of heat shock protein of 72 kDa (hsp72) in the protection of tau against hyperphosphorylation during heat shock. Neuronal PC12 cells were exposed either directly to a heat shock (45 degrees C for 30 min) or to a conditioning heat stress (43 degrees C for 90 min followed by a 4 hr recovery at 37 degrees C) followed by the heat shock. Hsp72 was maximally induced immediately after heat shock in conditioned (acquired thermotolerant, ATT) cells, while unconditioned (nonacquired thermotolerant, non-ATT) cells required 9 hr of recovery to exhibit maximal hsp72 induction. The differential time course of hsp72 induction during recovery of ATT and non-ATT cells correlated with the presence of normal tau. Immediately after the heat shock, when hsps were maximally induced, ATT cells exhibited the normal form of tau. With longer recovery times, the levels of hsp72 were reduced and tau was hyperphosphorylated. A similar correlation was observed in non-ATT cells. In the presence of L-azetidyl 2-carboxylic acid, ATT cells synthesized nonfunctional hsp72, as exhibited by the inability of the cells to recover from the effects of heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of gene expression in the cerebral cortices of rat brains containing subcortical lesions.

Neurotoxic lesion of the nucleus basalis of Meynert in the rat brain, which results in the loss of subcortical cholinergic innervation to the cerebral cortex, is an animal model for the cortical cholinergic deficits that are characteristic of Alzheimer's disease. Previously, we have shown that amyloid precursor protein is induced in the cortex in response to this disrupted innervation. We have investigated the synthesis and accumulation of proteins in lesioned versus control cortices. Total proteins from cortices were separated by high resolution two-dimensional gel electrophoresis and visualized by silver stain. Of the greater than 1,000 polypeptides examined, only one exhibited a consistent alteration in the lesioned sample. This unidentified protein (Mr 34 kD, pI 5.5) was normally present in scant amounts but was virtually absent in the lesioned cortex (0.056% total integrated density (TID) and 0.008% TID, respectively; p < 0.04). To investigate gene expression more directly, polysomes purified from lesioned and control cortices were assayed in vitro. Examination of [35S] incorporation into translation products by two-dimensional gels and autoradiography revealed three newly synthesized polypeptide differences in the lesioned samples. One protein (M(r) 47 kD, pI 6.1) exhibited elevated levels with the lesion (0.05% to 0.16%; p = 0.02) while two other proteins (M(r) 34 kD, pI 5.5, and M(r) 33 kD, pI 5.7) exhibited reduced levels (0.20% to 0.04%, p < 0.02, and 0.34% to 0.12%, p = 0.04, respectively).

Chromosomal distribution of 320 genes from a brain cDNA library.

We have determined the chromosomal assignment of 320 brain expressed genes by studying the segregation of polymerase chain reaction (PCR) products in human rodent somatic cell hybrids and by genetically mapping polymorphic cDNAs using the CEPH (Centre d'Etude du Polymophisme Humaine) reference pedigrees and database. These mapped genes can function as markers on the physical map of the human genome, as well as serve as candidate disease gene loci. Distribution of these genes to the human chromosomes correlates well with the GC content of the chromosomes. However, the distribution of these genes does not correlate well with the cytogenetic length of each chromosome.

Eliminating mitochondrial DNA competition for nuclear DNA primers.

Mitochondrial DNA (mtDNA) sequences were synthesized with nuclear DNA (nucDNA) sequence-tagged site (STS) primers by mismatch priming in three independent studies of the human nuclear genome. Mismatch primer binding sites on the mtDNA were identified with from 6- to 10-bp identity at the 3' ends of the primers. In two of three cases, single-stranded mtDNA copies were gel-isolated with intended nucDNA PCR products. During routine screening of the STSs, the radiolabeled gel-isolated products hybridized to polymorphic mtDNA restriction fragments. Intense signals after overnight exposure of radiolabeled PCR probes on Southern blots suggest contaminating mtDNA PCR products. The theoretical annealing temperatures of the mismatches were well below the annealing temperatures of the PCR primers, demonstrating annealing reactions driven by the molar surplus of the primers, that is, mass action. The probability that two primers (either one of a pair or both), designed to amplify nucDNA, will bind to and amplify mtDNA may be as high as 1 in 64, assuming that an identical match with only the 3' hexanucleotide is sufficient for amplification. To circumvent this problem we have developed OLIGFIND, a program that has identified the 104 of 4096 possible hexamers that are not present in human mtDNA. Our results suggest that time could be saved by designing STS primers with one of these 104 hexamers at the 3' end. OLIGFIND can also evaluate primer 3' ends for potential PCR products from mtDNA.

Altered expression and phosphorylation of amyloid precursor protein in heat shocked neuronal PC12 cells.

The pathology of the Alzheimer's disease (AD) brain, including amyloid plaques, neurofibrillary tangles and neuronal degeneration, indicates that neurons affected by AD exist under conditions of stress. In fact, the brains of AD patients undergo many changes classically associated with the heat shock response, which is one form of a stress response. These changes include reduced protein synthesis, disrupted cytoskeleton, increased number of proteins associated with ubiquitin, and the induction of heat shock proteins. To investigate the response of neurons to stress, we examined neuronal PC12 cells incubated at either 37 degrees C (control cells) or 45 degrees C (heat-shocked cells). After a 30 min exposure at 45 degrees C, the heat-shocked cells exhibited several features characteristic of the classical heat shock response including a 45% reduction in total protein synthesis, the induction of heat shock protein 72, and an increased phosphorylation of the protein synthesis initiation factor eIF-2 alpha. We used this cellular model system to study the neuronal response to stress specifically focusing on protein synthesis elongation factor 2 (EF-2) and the Alzheimer's amyloid precursor protein (APP), the precursor form of beta-amyloid peptide. Hyperphosphorylation of EF-2 has been observed in the neocortex and hippocampus of AD brain. However, in our system, we find no hyperphosphorylation of EF-2 in response to heat shock. Heat-shocked neuronal PC12 cells exhibited two additional APP-like polypeptides not present in controls. We also found a significant decrease in the phosphorylation state of APP in response to heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)