Robert Fujimura - Academia.edu (original) (raw)
Papers by Robert Fujimura
AIDS Research and Human Retroviruses, 1999
IV-1-associated brain pathology exhibits regional variability and we therefore studied the geneti... more IV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V 1± V5 dom ains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, m edial temporal lobe, and nonm edial temporal lobe) from three patients. W e found that in each separate brain region H IV-1 form s different quasispecies and that there is little gene flow among these regions. In further supp ort of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known am ino acid signatures associated with m acrophage tropism and the lack of syncytium form ation, we found 15 m ajority am ino acid signature patterns from the V1± V5 env sequences associated w ith the neuroanatom ical regions analyzed from the three individuals. Fu rtherm ore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be m uch sm aller than genetic distances between brain regions. Sp ecific strains of H IV-1 m ay be neurotropic or neuroinvasive (replication preference in brain tissue) and m ay contribute to pathology, cognitive loss, and neuropsychiatric disease.
AIDS, 2011
Diagnosis, neurology, and neuroimaging HIV-1 invades the central nervous system (CNS) early and c... more Diagnosis, neurology, and neuroimaging HIV-1 invades the central nervous system (CNS) early and can cause persistent infection and inflammation. Although early infection is typically asymptomatic, cerebrospinal fluid (CSF) analysis and magnetic resonance spectroscopy (MRS) imaging can detect CNS abnormalities during this period [8-11]. Chronic HIV-1 infection can result in neurodegenerative disease, overall termed NeuroAIDS. The clinical expression of this process includes neurocognitive impairments such as decreased attention/concentration, psychomotor speed, memory, learning, information processing, and executive function. There is also motor slowing, incoordination, and tremor, that may progress to disabling weakness, spasticity, extrapyramidal movement disorders, and paraparesis [12,13]. In addition, there may be behavioral effects such as apathy and irritability. Psychomotor retardation (associated with damage to the frontal-striatal systems) also may occur [14,15]. The clinical severity of this process ranges from asymptomatic neurocognitive impairment (ANI), to a mild neurocognitive disorder (MND), to full-blown HIV-associated
Proceedings of the National Academy of Sciences, 1966
CNS spectrums, 2000
Infection with human immunodeficiency virus type 1 (HIV-1) leads rapidly to infection of the brai... more Infection with human immunodeficiency virus type 1 (HIV-1) leads rapidly to infection of the brain and subsequent neuropsychological impairment, including subclinical impairment, minor cognitive-motor disorder, and HIV-1-associated dementia (HAD). This article reviews HAD and the factors involved in its pathogenesis; the effectiveness of antiretroviral therapy; the prevalence of HIV-1 and subtypes; and the role of chemokines and cytokines as the capstones associated with neuropathology due to inflammation.
Biochemistry Usa, 1971
Biochemical Analysis of the Naturally Repaired Sections of Bacteriophage T5 Deoxyribonucleic Acid... more Biochemical Analysis of the Naturally Repaired Sections of Bacteriophage T5 Deoxyribonucleic Acid. 111. Nucleotide
Journal of the Neurological Sciences, Oct 15, 2002
Macrophage/microglia (MF) are the principal immune cells in the central nervous system (CNS) conc... more Macrophage/microglia (MF) are the principal immune cells in the central nervous system (CNS) concomitant with inflammatory brain disease and play a significant role in the host defense against invading microorganisms. Astrocytes, as a significant component of the bloodbrain barrier, behave as one of the immune effecter cells in the CNS as well. However, both cell types may play a dual role, amplifying the effects of inflammation and mediating cellular damage as well as protecting the CNS. Interactions of the immune system, MF, and astrocytes result in altered production of neurotoxins and neurotrophins by these cells. These effects alter the neuronal structure and function during pathogenesis of HIV-1-associated dementia (HAD), Alzheimer disease (AD), and multiple sclerosis (MS). HAD primarily involves subcortical gray matter, and both HAD and MS affect sub-cortical white matter. AD is a cortical disease. The process of MF and astrocytes activation leading to neurotoxicity share similarities among the three diseases. Human Immunodeficiency Virus (HIV)-1-infected MF are involved in the pathogenesis of HAD and produce toxic molecules including cytokines, chemokines, and nitric oxide (NO). In AD, MFs produce these molecules and are activated by h-amyloid proteins and related oligopeptides. Demyelination in MS involves MF that become lipid laden, spurred by several possible antigens. In these three diseases, cytokine/chemokine communications between MF and astrocytes occur and are involved in the balance of protective and destructive actions by these cells. This review describes the role of MF and astrocytes in the pathogenesis of these three progressive neurological diseases, examining both beneficent and deleterious effects in each disease.
Journal of Biological Chemistry, Dec 25, 1982
Biochemistry Usa, 1971
Biochemical Analysis of the Naturally Repaired Sections of Bacteriophage T5 Deoxyribonucleic Acid... more Biochemical Analysis of the Naturally Repaired Sections of Bacteriophage T5 Deoxyribonucleic Acid. 111. Nucleotide
Http Dx Doi Org 10 1300 J128v02n03_03, Oct 20, 2008
To determine the heterogeneity of surface marker expression of macrophages in the temporal lobe o... more To determine the heterogeneity of surface marker expression of macrophages in the temporal lobe of patients who died with AIDS who were also Drug Abusers (DAs). We studied the expression of macrophage surface markers CD11c, CD14, CD68, and HLA-DR and T cell surface markers CD4, and CD8. The macrophage is the prime locus for HIV-1-associated pathology, is the most frequently infected cell in the brain, and has the highest virus load compared to other cells. We previously described the heterogeneity of macrophage surface marker expression and performed morphometric analysis in peripheral nerves of patients who died from AIDS compared to HIV-1 negative individuals. We showed that the HIV-related neuropathy in AIDS is a multifocal process. It is similarly important to determine the expression of macrophage surface markers in brain. Temporal lobe tissue was selected for this preliminary study because we previously found elevated HIV-1 proviral DNA load and inflammatory processes in this neuroanatomic location for subjects who died with AIDS. There is a high prevalence of Drug Abuse in Miami, Florida, associated with AIDS that may interactively affect HIV-associated pathology. Temporal lobe tissue was examined from 17 HIV-1-seropositive patients (4 with Drug Abuse and 13 without Drug Abuse) and 11 HIV-seronegative individuals (5 with Drug Abuse and 6 without Drug Abuse). Standard immunohistochemistry utilized alkaline phosphatase conjugate secondary antibody and fuchsin substrate. We found that HIV-1 infection and the interaction of HIV-1 infection and Drug Abuse produced changes in macrophage surface marker expression. Macrophage surface markers, CD11c, CD14, CD68, and HLA-DR, and T-cell marker CD4 were increased with statistical significance due to HIV-1 infection (all p < .001) whereas CD8 remained unchanged. Changes due to Drug Abuse alone were not significant. Interaction of Drug Abuse and HIV-infected individuals showed increased expression of CD68 (p = .011), HLA-DR (p = .001), CD4 (p = .027), and CD8 (p = .016). Drug Abuse and HIV-1 infection are factors that differentially and interactively result in multiple macrophages surface marker effects. In HIV-1 infected individuals, Drug Abuse stimulates surface marker expression. Since brain macrophage surface makers do not change uniformly as a result of Drug Abuse and HIV infection, these cells may be heterogeneous and contain sub-types (sub-sets). It remains to be determined which macrophage sub-types may be most pathognomic for pathology.
Clinical and Diagnostic Virology, 1994
The clinical hallmark of Alzheimer's disease (AD) is impairment of cognition associated with loss... more The clinical hallmark of Alzheimer's disease (AD) is impairment of cognition associated with loss of synapses, accumulation of amyloid β (Aβ) both within neurons and as extracellular deposits, and neurofibrillary degeneration composed of phospho-tau. Neurons in the hippocampus are among those that are most vulnerable. The purpose of this study was to investigate the expression of genes associated with cognition, synapse, and mitochondrial function in hippocampal neurons of AD compared to normal individuals. Neurons from the hippocampus with intraneuronal Aβ immunoreactivity were captured with laser microdissection; RNA was extracted; and levels of brain-derived neurotrophic factor (BDNF), TrkB (BDNF receptor), dynamin-1 (DYN), and cytochrome C oxidase subunit II (COX2) were assessed with quantitative real time-polymerase chain reaction. We found no significant differences in the expression of these genes in AD between neurons associated with Aβ compared to those lacking Aβ immunoreactivity. Double immunofluorescence microscopy showed the number of hippocampal neurons coexpressing Aβ or phospho-tau and either BDNF, TrkB, or DYN was similar in AD and controls. Our results suggest that intraneuronal Aβ or phospho-tau do not have obligatory effects on reducing the expression of genes important for memory and cognition in hippocampus of AD.
Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, 1997
A definitive relation between HIV-1 load and the clinical diagnosis of HIV-1-associated dementia ... more A definitive relation between HIV-1 load and the clinical diagnosis of HIV-1-associated dementia (HAD) has not yet been established. Knowledge of the neuroanatomic distribution of HIV-1 load in the brain of individuals with HAD and HIV-1 encephalitis may facilitate elucidation of this relation. Nine individuals with AIDS were analyzed postmortem by three independent methods with each assessment performed blinded to the others: 1) a neuropsychiatric review of clinical records for evidence of possible HAD, 2) HIV-1 DNA load determination by quantitative polymerase chain reaction (PCR) across several neuroanatomic regions, and 3) a pathologic examination for diagnosis of HIV-1 encephalitis by immunohistochemical techniques. Of eight AIDS cases with clinical records sufficient for neuropsychiatric review, seven were shown to have evidence for HAD. HIV-1 DNA was detected and quantified in specimens from all of the medial temporal lobe regions analyzed but was not detectable in the frontal lobe at the same level of sensitivity in two of these cases (<1 per 1000 cellular genomes). HIV-1 DNA load in the medial temporal lobe region was significantly larger than that in the frontal lobe. Only four of seven cases with evidence for HAD were also diagnosed with HIV-1 encephalitis.
We identified by immunobinding assay the polypeptides synthesized as the result of amber mutation... more We identified by immunobinding assay the polypeptides synthesized as the result of amber mutations in the DNA polymerase gene of bacteriophage T5. Comparison of the size of such polypeptides revealed the order of mutagenic loci of these mutations and the direction of transcription of the gene. Extracts of cells infected with wild-type T5 and with five amber mutants of the polymerase gene (D7, D8, D9, aml, and am6) were prepared, and the proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After transfer of the proteins to a nitrocellulose sheet, a radioimmunolabeling technique was used to identify the T5 DNA polymerase and its amber mutant polypeptides. Based on the relative sizes of the polypeptides, the transcription of the T5 DNA polymerase gene was determined to proceed in the order D7, D8, aml, D9, and am6. The molecular weights of the DNA polymerase polypeptides coded by D8, aml, D9, am6, and T5+ were 23,000, 45,000, 75,000, 83,000, and 96,000, respectively. The D7-coded polypeptide was not detectable. These data suggest that the carboxyl-terminal region of the enzyme is essential for the polymerase function.
Nucleic Acids Research, 1980
T5-induced DNA polymerase has an associated 3'-5' exonuclease activity. Both single-stranded and ... more T5-induced DNA polymerase has an associated 3'-5' exonuclease activity. Both single-stranded and duplex DNA are hydrolyzed by this enzyme in a quasi-processive manner. This is indicated by the results of polymer-challenge experiments utilizing product analysis techniques. Due to the quasi-processive mode of hydrolysis, the kinetics of label release from the 3'-terminally labeled oligonucleotide substrates, annealed to complementary homopolymers, show an initial high rate of hydrolysis. In the case of both single-stranded and duplex DNA substrates, hydrolysis seems to continue, at best, up to the point where the enzyme is five or six nucleotides away from the 5'end. The enzyme carries out mismatch repair, as evidenced by experiments with primer molecules containing improper base residues at the 3'-OH terminus. Control experiments with complementary base residues at the 3'-end indicate that extensive removal of terminal residue takes place in the presence of dNTP's only when such residues are "improper" in the Watson-Crick sense. In our previous publications we have noted that bacteriophage T5-induced DNA polymerase replicates DNA in a processive fashion (1,2). This imports biphasicity to the kinetics of DNA synthesis in vitro when short primer-templates are used as substrate. The enzyme continues to replicate a given primer-template until it is very close to the 5'-end of the template (1; unpublished observations). T5 polymerase possesses a 3'-5' exonuclease activity which utilizes single-stranded, duplex, denatured, and nicked DNA as substrates (3a,b); this enzyme is devoid of any 5'-3' exonuclease activity. Here we present evidence demonstrating that the mode of action of exonuclease activity is "quasi-processive, " as defined in (2). Both single-stranded and duplex DNA substrates are hydrolyzed in this manner. After hydrolysis of a given substrate molecule is initiated at the 3'-end, the enzyme continues to hydrolyze the molecule until it is five to six nucleotides away from the 5'-end. The oligonucleotides
Journal of Virological Methods, 1995
A PCR method was developed to compare HIV-l DNA loads in brain tissue samples. The method determi... more A PCR method was developed to compare HIV-l DNA loads in brain tissue samples. The method determines the ratio of the amplified product of an HIV DNA sequence to that of a host cellular DNA sequence using standard DNAs as reference. The standards include DNA from a line of human cells that harbor one HIV-l provirus per cellular genome, and DNA from non-infected human cells. The standard DNAs were mixed in varying proportions and used to establish conditions of amplification under which the ratios of their PCR-amplified products corresponded with the ratios of the amounts of the DNAs themselves. The method was evaluated using known mixtures of the standard DNAs. Using the conditions thus obtained, ratios of HIV proviral DNA to cellular genomic DNA were obtained for tissue DNA samples taken from several different locations within the brain of two deceased HIV-infected patients. Results showed that HIV DNA was non-uniformly distributed within each brain (lo-250 per lo3 cellular genomes); the highest ratios were found in the hippocampus for each patient, independent of postmortem neuropathological findings. The criteria for quantitative PCR have general applicability to comparative studies of any proviral DNA loads in different tissue samples.
Journal of Molecular Biology, 1966
Journal of Molecular Biology, 1965
The oligonucleotides resulting from ribonuclease digestion of 32P•labeled R17 RNA were separated ... more The oligonucleotides resulting from ribonuclease digestion of 32P•labeled R17 RNA were separated on the basis oftheir chain length. The observed distribution was in agreement with statistical expectation. Three kinds of octanucleotides occurred. Knowing their specific radioactivity and the total radioactivity in all of the fractions, it was concluded that RI7 RNA contains approximately 3342 nucleotide residues. The molecular weight of RI7 RNA is thus approximately 1•1 X 10 6 •
Journal of Molecular Biology, 1962
ABSTRACT Infection of E. coli with phage T4 causes a sudden inhibition of the synthesis of host D... more ABSTRACT Infection of E. coli with phage T4 causes a sudden inhibition of the synthesis of host DNA, host RNA, and host protein. Experiments were performed to test the hypothesis that the physical destruction of the host chromosome is a primary cause of this inhibition. Physical destruction of host DNA after infection was studied using sucrose density gradient sedimentation and CsCl equilibrium density gradient centrifugation techniques. No destruction of host DNA was detected after 5 min. However, a definite destruction was observed after 10 min. The addition of Chloromycetin or streptomycin before infection prevented this destruction. Nevertheless, host DNA synthesis and host messenger RNA synthesis are inhibited under such conditions. Therefore, physical destruction of the host chromosome does not seem to be the primary cause of the inhibition. Functional integrity of the host chromosome after infection was studied. During infection in the presence of streptomycin, its functional integrity was demonstrated by introducing the inhibited host chromosome into phage-resistant female cells and observing the subsequent expression of its function to synthesize β-galactosidase. Thus, the primary inhibitory process seems to be reversible in its inherent nature. Some features of such an inhibitory mechanism are briefly discussed.
Biophysical Journal, 1962
The adsorption of purified bX174 to E. coli C and to E. coli C cell walls was investigated. Adsor... more The adsorption of purified bX174 to E. coli C and to E. coli C cell walls was investigated. Adsorption was measured by assaying for unadsorbed plaque formers. The amount of irreversible and reversible adsorption depends upon pH and divalent ion concentration. Maximum irreversible adsorption occurs in 0.1 M CaCI. at 360C. There is no detectable reversible adsorption at conditions of pH and CaCl, concentration optimum for irreversible adsorption. Under these optimum conditions, diffusion is not the rate-limiting factor, and the encounter efficiency appears to be low. The rate constant is 1.0 X 10' ml/ sec. Phages adsorbed irreversibly to live cells cause infection and to the isolated cell walls apparently cause release of DNA. There is a specific 4X174 receptor site on the mucocomplex portion of the cell wall.
AIDS Research and Human Retroviruses, 1999
IV-1-associated brain pathology exhibits regional variability and we therefore studied the geneti... more IV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V 1± V5 dom ains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, m edial temporal lobe, and nonm edial temporal lobe) from three patients. W e found that in each separate brain region H IV-1 form s different quasispecies and that there is little gene flow among these regions. In further supp ort of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known am ino acid signatures associated with m acrophage tropism and the lack of syncytium form ation, we found 15 m ajority am ino acid signature patterns from the V1± V5 env sequences associated w ith the neuroanatom ical regions analyzed from the three individuals. Fu rtherm ore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be m uch sm aller than genetic distances between brain regions. Sp ecific strains of H IV-1 m ay be neurotropic or neuroinvasive (replication preference in brain tissue) and m ay contribute to pathology, cognitive loss, and neuropsychiatric disease.
AIDS, 2011
Diagnosis, neurology, and neuroimaging HIV-1 invades the central nervous system (CNS) early and c... more Diagnosis, neurology, and neuroimaging HIV-1 invades the central nervous system (CNS) early and can cause persistent infection and inflammation. Although early infection is typically asymptomatic, cerebrospinal fluid (CSF) analysis and magnetic resonance spectroscopy (MRS) imaging can detect CNS abnormalities during this period [8-11]. Chronic HIV-1 infection can result in neurodegenerative disease, overall termed NeuroAIDS. The clinical expression of this process includes neurocognitive impairments such as decreased attention/concentration, psychomotor speed, memory, learning, information processing, and executive function. There is also motor slowing, incoordination, and tremor, that may progress to disabling weakness, spasticity, extrapyramidal movement disorders, and paraparesis [12,13]. In addition, there may be behavioral effects such as apathy and irritability. Psychomotor retardation (associated with damage to the frontal-striatal systems) also may occur [14,15]. The clinical severity of this process ranges from asymptomatic neurocognitive impairment (ANI), to a mild neurocognitive disorder (MND), to full-blown HIV-associated
Proceedings of the National Academy of Sciences, 1966
CNS spectrums, 2000
Infection with human immunodeficiency virus type 1 (HIV-1) leads rapidly to infection of the brai... more Infection with human immunodeficiency virus type 1 (HIV-1) leads rapidly to infection of the brain and subsequent neuropsychological impairment, including subclinical impairment, minor cognitive-motor disorder, and HIV-1-associated dementia (HAD). This article reviews HAD and the factors involved in its pathogenesis; the effectiveness of antiretroviral therapy; the prevalence of HIV-1 and subtypes; and the role of chemokines and cytokines as the capstones associated with neuropathology due to inflammation.
Biochemistry Usa, 1971
Biochemical Analysis of the Naturally Repaired Sections of Bacteriophage T5 Deoxyribonucleic Acid... more Biochemical Analysis of the Naturally Repaired Sections of Bacteriophage T5 Deoxyribonucleic Acid. 111. Nucleotide
Journal of the Neurological Sciences, Oct 15, 2002
Macrophage/microglia (MF) are the principal immune cells in the central nervous system (CNS) conc... more Macrophage/microglia (MF) are the principal immune cells in the central nervous system (CNS) concomitant with inflammatory brain disease and play a significant role in the host defense against invading microorganisms. Astrocytes, as a significant component of the bloodbrain barrier, behave as one of the immune effecter cells in the CNS as well. However, both cell types may play a dual role, amplifying the effects of inflammation and mediating cellular damage as well as protecting the CNS. Interactions of the immune system, MF, and astrocytes result in altered production of neurotoxins and neurotrophins by these cells. These effects alter the neuronal structure and function during pathogenesis of HIV-1-associated dementia (HAD), Alzheimer disease (AD), and multiple sclerosis (MS). HAD primarily involves subcortical gray matter, and both HAD and MS affect sub-cortical white matter. AD is a cortical disease. The process of MF and astrocytes activation leading to neurotoxicity share similarities among the three diseases. Human Immunodeficiency Virus (HIV)-1-infected MF are involved in the pathogenesis of HAD and produce toxic molecules including cytokines, chemokines, and nitric oxide (NO). In AD, MFs produce these molecules and are activated by h-amyloid proteins and related oligopeptides. Demyelination in MS involves MF that become lipid laden, spurred by several possible antigens. In these three diseases, cytokine/chemokine communications between MF and astrocytes occur and are involved in the balance of protective and destructive actions by these cells. This review describes the role of MF and astrocytes in the pathogenesis of these three progressive neurological diseases, examining both beneficent and deleterious effects in each disease.
Journal of Biological Chemistry, Dec 25, 1982
Biochemistry Usa, 1971
Biochemical Analysis of the Naturally Repaired Sections of Bacteriophage T5 Deoxyribonucleic Acid... more Biochemical Analysis of the Naturally Repaired Sections of Bacteriophage T5 Deoxyribonucleic Acid. 111. Nucleotide
Http Dx Doi Org 10 1300 J128v02n03_03, Oct 20, 2008
To determine the heterogeneity of surface marker expression of macrophages in the temporal lobe o... more To determine the heterogeneity of surface marker expression of macrophages in the temporal lobe of patients who died with AIDS who were also Drug Abusers (DAs). We studied the expression of macrophage surface markers CD11c, CD14, CD68, and HLA-DR and T cell surface markers CD4, and CD8. The macrophage is the prime locus for HIV-1-associated pathology, is the most frequently infected cell in the brain, and has the highest virus load compared to other cells. We previously described the heterogeneity of macrophage surface marker expression and performed morphometric analysis in peripheral nerves of patients who died from AIDS compared to HIV-1 negative individuals. We showed that the HIV-related neuropathy in AIDS is a multifocal process. It is similarly important to determine the expression of macrophage surface markers in brain. Temporal lobe tissue was selected for this preliminary study because we previously found elevated HIV-1 proviral DNA load and inflammatory processes in this neuroanatomic location for subjects who died with AIDS. There is a high prevalence of Drug Abuse in Miami, Florida, associated with AIDS that may interactively affect HIV-associated pathology. Temporal lobe tissue was examined from 17 HIV-1-seropositive patients (4 with Drug Abuse and 13 without Drug Abuse) and 11 HIV-seronegative individuals (5 with Drug Abuse and 6 without Drug Abuse). Standard immunohistochemistry utilized alkaline phosphatase conjugate secondary antibody and fuchsin substrate. We found that HIV-1 infection and the interaction of HIV-1 infection and Drug Abuse produced changes in macrophage surface marker expression. Macrophage surface markers, CD11c, CD14, CD68, and HLA-DR, and T-cell marker CD4 were increased with statistical significance due to HIV-1 infection (all p < .001) whereas CD8 remained unchanged. Changes due to Drug Abuse alone were not significant. Interaction of Drug Abuse and HIV-infected individuals showed increased expression of CD68 (p = .011), HLA-DR (p = .001), CD4 (p = .027), and CD8 (p = .016). Drug Abuse and HIV-1 infection are factors that differentially and interactively result in multiple macrophages surface marker effects. In HIV-1 infected individuals, Drug Abuse stimulates surface marker expression. Since brain macrophage surface makers do not change uniformly as a result of Drug Abuse and HIV infection, these cells may be heterogeneous and contain sub-types (sub-sets). It remains to be determined which macrophage sub-types may be most pathognomic for pathology.
Clinical and Diagnostic Virology, 1994
The clinical hallmark of Alzheimer's disease (AD) is impairment of cognition associated with loss... more The clinical hallmark of Alzheimer's disease (AD) is impairment of cognition associated with loss of synapses, accumulation of amyloid β (Aβ) both within neurons and as extracellular deposits, and neurofibrillary degeneration composed of phospho-tau. Neurons in the hippocampus are among those that are most vulnerable. The purpose of this study was to investigate the expression of genes associated with cognition, synapse, and mitochondrial function in hippocampal neurons of AD compared to normal individuals. Neurons from the hippocampus with intraneuronal Aβ immunoreactivity were captured with laser microdissection; RNA was extracted; and levels of brain-derived neurotrophic factor (BDNF), TrkB (BDNF receptor), dynamin-1 (DYN), and cytochrome C oxidase subunit II (COX2) were assessed with quantitative real time-polymerase chain reaction. We found no significant differences in the expression of these genes in AD between neurons associated with Aβ compared to those lacking Aβ immunoreactivity. Double immunofluorescence microscopy showed the number of hippocampal neurons coexpressing Aβ or phospho-tau and either BDNF, TrkB, or DYN was similar in AD and controls. Our results suggest that intraneuronal Aβ or phospho-tau do not have obligatory effects on reducing the expression of genes important for memory and cognition in hippocampus of AD.
Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, 1997
A definitive relation between HIV-1 load and the clinical diagnosis of HIV-1-associated dementia ... more A definitive relation between HIV-1 load and the clinical diagnosis of HIV-1-associated dementia (HAD) has not yet been established. Knowledge of the neuroanatomic distribution of HIV-1 load in the brain of individuals with HAD and HIV-1 encephalitis may facilitate elucidation of this relation. Nine individuals with AIDS were analyzed postmortem by three independent methods with each assessment performed blinded to the others: 1) a neuropsychiatric review of clinical records for evidence of possible HAD, 2) HIV-1 DNA load determination by quantitative polymerase chain reaction (PCR) across several neuroanatomic regions, and 3) a pathologic examination for diagnosis of HIV-1 encephalitis by immunohistochemical techniques. Of eight AIDS cases with clinical records sufficient for neuropsychiatric review, seven were shown to have evidence for HAD. HIV-1 DNA was detected and quantified in specimens from all of the medial temporal lobe regions analyzed but was not detectable in the frontal lobe at the same level of sensitivity in two of these cases (<1 per 1000 cellular genomes). HIV-1 DNA load in the medial temporal lobe region was significantly larger than that in the frontal lobe. Only four of seven cases with evidence for HAD were also diagnosed with HIV-1 encephalitis.
We identified by immunobinding assay the polypeptides synthesized as the result of amber mutation... more We identified by immunobinding assay the polypeptides synthesized as the result of amber mutations in the DNA polymerase gene of bacteriophage T5. Comparison of the size of such polypeptides revealed the order of mutagenic loci of these mutations and the direction of transcription of the gene. Extracts of cells infected with wild-type T5 and with five amber mutants of the polymerase gene (D7, D8, D9, aml, and am6) were prepared, and the proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After transfer of the proteins to a nitrocellulose sheet, a radioimmunolabeling technique was used to identify the T5 DNA polymerase and its amber mutant polypeptides. Based on the relative sizes of the polypeptides, the transcription of the T5 DNA polymerase gene was determined to proceed in the order D7, D8, aml, D9, and am6. The molecular weights of the DNA polymerase polypeptides coded by D8, aml, D9, am6, and T5+ were 23,000, 45,000, 75,000, 83,000, and 96,000, respectively. The D7-coded polypeptide was not detectable. These data suggest that the carboxyl-terminal region of the enzyme is essential for the polymerase function.
Nucleic Acids Research, 1980
T5-induced DNA polymerase has an associated 3'-5' exonuclease activity. Both single-stranded and ... more T5-induced DNA polymerase has an associated 3'-5' exonuclease activity. Both single-stranded and duplex DNA are hydrolyzed by this enzyme in a quasi-processive manner. This is indicated by the results of polymer-challenge experiments utilizing product analysis techniques. Due to the quasi-processive mode of hydrolysis, the kinetics of label release from the 3'-terminally labeled oligonucleotide substrates, annealed to complementary homopolymers, show an initial high rate of hydrolysis. In the case of both single-stranded and duplex DNA substrates, hydrolysis seems to continue, at best, up to the point where the enzyme is five or six nucleotides away from the 5'end. The enzyme carries out mismatch repair, as evidenced by experiments with primer molecules containing improper base residues at the 3'-OH terminus. Control experiments with complementary base residues at the 3'-end indicate that extensive removal of terminal residue takes place in the presence of dNTP's only when such residues are "improper" in the Watson-Crick sense. In our previous publications we have noted that bacteriophage T5-induced DNA polymerase replicates DNA in a processive fashion (1,2). This imports biphasicity to the kinetics of DNA synthesis in vitro when short primer-templates are used as substrate. The enzyme continues to replicate a given primer-template until it is very close to the 5'-end of the template (1; unpublished observations). T5 polymerase possesses a 3'-5' exonuclease activity which utilizes single-stranded, duplex, denatured, and nicked DNA as substrates (3a,b); this enzyme is devoid of any 5'-3' exonuclease activity. Here we present evidence demonstrating that the mode of action of exonuclease activity is "quasi-processive, " as defined in (2). Both single-stranded and duplex DNA substrates are hydrolyzed in this manner. After hydrolysis of a given substrate molecule is initiated at the 3'-end, the enzyme continues to hydrolyze the molecule until it is five to six nucleotides away from the 5'-end. The oligonucleotides
Journal of Virological Methods, 1995
A PCR method was developed to compare HIV-l DNA loads in brain tissue samples. The method determi... more A PCR method was developed to compare HIV-l DNA loads in brain tissue samples. The method determines the ratio of the amplified product of an HIV DNA sequence to that of a host cellular DNA sequence using standard DNAs as reference. The standards include DNA from a line of human cells that harbor one HIV-l provirus per cellular genome, and DNA from non-infected human cells. The standard DNAs were mixed in varying proportions and used to establish conditions of amplification under which the ratios of their PCR-amplified products corresponded with the ratios of the amounts of the DNAs themselves. The method was evaluated using known mixtures of the standard DNAs. Using the conditions thus obtained, ratios of HIV proviral DNA to cellular genomic DNA were obtained for tissue DNA samples taken from several different locations within the brain of two deceased HIV-infected patients. Results showed that HIV DNA was non-uniformly distributed within each brain (lo-250 per lo3 cellular genomes); the highest ratios were found in the hippocampus for each patient, independent of postmortem neuropathological findings. The criteria for quantitative PCR have general applicability to comparative studies of any proviral DNA loads in different tissue samples.
Journal of Molecular Biology, 1966
Journal of Molecular Biology, 1965
The oligonucleotides resulting from ribonuclease digestion of 32P•labeled R17 RNA were separated ... more The oligonucleotides resulting from ribonuclease digestion of 32P•labeled R17 RNA were separated on the basis oftheir chain length. The observed distribution was in agreement with statistical expectation. Three kinds of octanucleotides occurred. Knowing their specific radioactivity and the total radioactivity in all of the fractions, it was concluded that RI7 RNA contains approximately 3342 nucleotide residues. The molecular weight of RI7 RNA is thus approximately 1•1 X 10 6 •
Journal of Molecular Biology, 1962
ABSTRACT Infection of E. coli with phage T4 causes a sudden inhibition of the synthesis of host D... more ABSTRACT Infection of E. coli with phage T4 causes a sudden inhibition of the synthesis of host DNA, host RNA, and host protein. Experiments were performed to test the hypothesis that the physical destruction of the host chromosome is a primary cause of this inhibition. Physical destruction of host DNA after infection was studied using sucrose density gradient sedimentation and CsCl equilibrium density gradient centrifugation techniques. No destruction of host DNA was detected after 5 min. However, a definite destruction was observed after 10 min. The addition of Chloromycetin or streptomycin before infection prevented this destruction. Nevertheless, host DNA synthesis and host messenger RNA synthesis are inhibited under such conditions. Therefore, physical destruction of the host chromosome does not seem to be the primary cause of the inhibition. Functional integrity of the host chromosome after infection was studied. During infection in the presence of streptomycin, its functional integrity was demonstrated by introducing the inhibited host chromosome into phage-resistant female cells and observing the subsequent expression of its function to synthesize β-galactosidase. Thus, the primary inhibitory process seems to be reversible in its inherent nature. Some features of such an inhibitory mechanism are briefly discussed.
Biophysical Journal, 1962
The adsorption of purified bX174 to E. coli C and to E. coli C cell walls was investigated. Adsor... more The adsorption of purified bX174 to E. coli C and to E. coli C cell walls was investigated. Adsorption was measured by assaying for unadsorbed plaque formers. The amount of irreversible and reversible adsorption depends upon pH and divalent ion concentration. Maximum irreversible adsorption occurs in 0.1 M CaCI. at 360C. There is no detectable reversible adsorption at conditions of pH and CaCl, concentration optimum for irreversible adsorption. Under these optimum conditions, diffusion is not the rate-limiting factor, and the encounter efficiency appears to be low. The rate constant is 1.0 X 10' ml/ sec. Phages adsorbed irreversibly to live cells cause infection and to the isolated cell walls apparently cause release of DNA. There is a specific 4X174 receptor site on the mucocomplex portion of the cell wall.