Showing papers in "Proceedings of the National Academy of Sciences of the United States of America in 1993"
TL;DR: It is argued that this damage to DNA, protein, and lipid is a major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular disease, immune-system decline, brain dysfunction, and cataracts.
Abstract: Metabolism, like other aspects of life, involves tradeoffs. Oxidant by-products of normal metabolism cause extensive damage to DNA, protein, and lipid. We argue that this damage (the same as that produced by radiation) is a major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular disease, immune-system decline, brain dysfunction, and cataracts. Antioxidant defenses against this damage include ascorbate, tocopherol, and carotenoids. Dietary fruits and vegetables are the principal source of ascorbate and carotenoids and are one source of tocopherol. Low dietary intake of fruits and vegetables doubles the risk of most types of cancer as compared to high intake and also markedly increases the risk of heart disease and cataracts. Since only 9% of Americans eat the recommended five servings of fruits and vegetables per day, the opportunity for improving health by improving diet is great.
6,007 citations
TL;DR: It is demonstrated that there was a highly significant association of apolipoprotein E type 4 allele (APOE-epsilon 4) and late-onset familial Alzheimer disease.
Abstract: Apolipoprotein E is immunochemically localized to the senile plaques, vascular amyloid, and neurofibrillary tangles of Alzheimer disease. In vitro, apolipoprotein E in cerebrospinal fluid binds to synthetic beta A4 peptide (the primary constituent of the senile plaque) with high avidity. Amino acids 12-28 of the beta A4 peptide are required. The gene for apolipoprotein E is located on chromosome 19q13.2, within the region previously associated with linkage of late-onset familial Alzheimer disease. Analysis of apolipoprotein E alleles in Alzheimer disease and controls demonstrated that there was a highly significant association of apolipoprotein E type 4 allele (APOE-epsilon 4) and late-onset familial Alzheimer disease. The allele frequency of the APOE-epsilon 4 in 30 random affected patients, each from a different Alzheimer disease family, was 0.50 +/- 0.06; the allele frequency of APOE-epsilon 4 in 91 age-matched unrelated controls was 0.16 +/- 0.03 (Z = 2.44, P = 0.014). A functional role of the apolipoprotein E-E4 isoform in the pathogenesis of late-onset familial Alzheimer disease is suggested.
4,179 citations
TL;DR: The results have important implications for the clinical use of genetically modified tumor cells as therapeutic cancer vaccines and the levels of anti-tumor immunity reported previously in cytokine gene transfer studies involving live, transduced cells could be achieved through the use of irradiated cells alone.
Abstract: To compare the ability of different cytokines and other molecules to enhance the immunogenicity of tumor cells, we generated 10 retroviruses encoding potential immunomodulators and studied the vaccination properties of murine tumor cells transduced by the viruses. Using a B16 melanoma model, in which irradiated tumor cells alone do not stimulate significant anti-tumor immunity, we found that irradiated tumor cells expressing murine granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated potent, long-lasting, and specific anti-tumor immunity, requiring both CD4+ and CD8+ cells. Irradiated cells expressing interleukins 4 and 6 also stimulated detectable, but weaker, activity. In contrast to the B16 system, we found that in a number of other tumor models, the levels of anti-tumor immunity reported previously in cytokine gene transfer studies involving live, transduced cells could be achieved through the use of irradiated cells alone. Nevertheless, manipulation of the vaccine or challenge doses made it possible to demonstrate the activity of murine GM-CSF in those systems as well. Overall, our results have important implications for the clinical use of genetically modified tumor cells as therapeutic cancer vaccines.
2,844 citations
TL;DR: Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.
Abstract: Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and after passage 14, ES cell-derived newborns died at birth. However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the original totipotency and gave rise to viable, completely ES cell-derived animals. The total in vitro culture time of the sublines at the time of testing was equivalent to passage 24 of the original line. Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.
2,430 citations
TL;DR: It is argued that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, and it is likely that this conformational transition is a fundamental event in the propagation of prions.
Abstract: Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.
2,230 citations
TL;DR: Pathological examination revealed an excessive inflammatory response with massive infiltration of lymphocytes and macrophages in many organs, but primarily in heart and lungs, which suggests a prominent role for TGF-beta 1 in homeostatic regulation of immune cell proliferation and extravasation into tissues.
Abstract: To delineate specific developmental roles of transforming growth factor beta 1 (TGF-beta 1) we have disrupted its cognate gene in mouse embryonic stem cells by homologous recombination to generate TGF-beta 1 null mice. These mice do not produce detectable amounts of either TGF-beta 1 RNA or protein. After normal growth for the first 2 weeks they develop a rapid wasting syndrome and die by 3-4 weeks of age. Pathological examination revealed an excessive inflammatory response with massive infiltration of lymphocytes and macrophages in many organs, but primarily in heart and lungs. Many lesions resembled those found in autoimmune disorders, graft-vs.-host disease, or certain viral diseases. This phenotype suggests a prominent role for TGF-beta 1 in homeostatic regulation of immune cell proliferation and extravasation into tissues.
1,923 citations
TL;DR: A previously unrecognized mechanism for familial thromboembolic disease is described that is characterized by poor anticoagulant response to activated protein C (APC), and as two additional, unrelated cases with thrombosis and inherited poor antICOagulants response to APC are identified, this may constitute an important cause for familialThrombophilia.
Abstract: Although patients with thromboembolic disease frequently have family histories of thrombosis, well-defined defects such as inherited deficiencies of anticoagulant proteins are found only in a minority of cases. Based on the hypothesis that a poor anticoagulant response to activated protein C (APC) would predispose to thrombosis, a set of new coagulation assays was developed that measure the anticoagulant response in plasma to APC. A middle-aged man with a history of multiple thrombotic events was identified. The addition of APC to his plasma did not result in a normal anticoagulant response as measured by prolongation of clotting time in an activated partial thromboplastin time (APTT) assay. Four of the proband's relatives had medical histories of multiple thrombotic events, and they and several other family members responded poorly to APC in the APTT-based assay. Subnormal anticoagulant responses to APC were also found in factor IXa- and Xa-based assays. Several possible mechanisms for the observed phenomenon were ruled out, such as functional protein S deficiency, a protein C-inhibitory antibody, or a fast-acting protease inhibitor against APC. Moreover, restriction fragment-length polymorphism analysis excluded possible linkage of the underlying molecular defect to factor VIII and von Willebrand factor genes. We now describe a previously unrecognized mechanism for familial thromboembolic disease that is characterized by poor anticoagulant response to APC. This would appear to be explained best by a hypothesized inherited deficiency of a previously unrecognized cofactor to APC. As we have identified two additional, unrelated cases with thrombosis and inherited poor anticoagulant response to APC, this may constitute an important cause for familial thrombophilia.
1,915 citations
TL;DR: Prenatal male and female reproductive tract development can occur in the absence of estradiol receptor-mediated responsiveness, and the uteri and vagina do not respond in the animals with the estrogen receptor gene disruption.
Abstract: Estrogen receptor and its ligand, estradiol, have long been thought to be essential for survival, fertility, and female sexual differentiation and development. Consistent with this proposed crucial role, no human estrogen receptor gene mutations are known, unlike the androgen receptor, where many loss of function mutations have been found. We have generated mutant mice lacking responsiveness to estradiol by disrupting the estrogen receptor gene by gene targeting. Both male and female animals survive to adulthood with normal gross external phenotypes. Females are infertile; males have a decreased fertility. Females have hypoplastic uteri and hyperemic ovaries with no detectable corpora lutea. In adult wild-type and heterozygous females, 3-day estradiol treatment at 40 micrograms/kg stimulates a 3- to 4-fold increase in uterine wet weight and alters vaginal cornification, but the uteri and vagina do not respond in the animals with the estrogen receptor gene disruption. Prenatal male and female reproductive tract development can therefore occur in the absence of estradiol receptor-mediated responsiveness.
1,859 citations
TL;DR: The design of small antibody fragments with two antigen-binding sites, which comprise a heavy-chain variable domain connected to a light- Chain variable domain on the same polypeptide chain (VH-VL), are described.
Abstract: Bivalent and bispecific antibodies and their fragments have immense potential for practical application. Here we describe the design of small antibody fragments with two antigen-binding sites. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. As indicated by a computer graphic model of the dimers, the two pairs of domains can pack together with the antigen-binding sites pointing in opposite directions. The dimeric antibody fragments, or "diabodies," can be designed for bivalent or bispecific interactions. Starting from the monoclonal antibodies NQ11.7.22 (NQ11) and D1.3 directed against the hapten phenyloxazolone and hen egg lysozyme, respectively, we built bivalent fragments (VHNQ11-VLNQ11)2 and (VHD1.3-VLD1.3)2 and bispecific fragments VHNQ11-VLD1.3 and VHD1.3-VLNQ11. The fragments were expressed by secretion from bacteria and shown to bind specifically to the hapten and/or antigen. Those with 5- and 15-residue linkers had similar binding affinities to the parent antibodies, but a fragment with the VH domain joined directly to the VL domain was found to have slower dissociation kinetics and an improved affinity for hapten. Diabodies offer a ready means of constructing small bivalent and bispecific antibody fragments in bacteria.
1,788 citations
TL;DR: Data from multilocus enzyme electrophoresis of bacterial populations were analyzed using a statistical test designed to detect associations between genes at different loci, and found panmictic, epidemic, and clonal population structures.
Abstract: Data from multilocus enzyme electrophoresis of bacterial populations were analyzed using a statistical test designed to detect associations between genes at different loci. Some species (e.g., Salmonella) were found to be clonal at all levels of analysis. At the other extreme, Neisseria gonorrhoeae is panmictic, with random association between loci. Two intermediate types of population structure were also found. Neisseria meningitidis displays what we have called an "epidemic" structure. There is significant association between loci, but this arises only because of the recent, explosive, increase in particular electrophoretic types; when this effect is eliminated the population is found to be effectively panmictic. In contrast, linkage disequilibrium in a population of Rhizobium meliloti exists because the sample consisted of two genetically isolated divisions, often fixed for different alleles: within each division association between loci was almost random. The method of analysis is appropriate whenever there is doubt about the extent of genetic recombination between members of a population. To illustrate this we analyzed data on protozoan parasites and again found panmictic, epidemic, and clonal population structures.
1,767 citations
TL;DR: BF 389, an experimental drug currently being tested in humans, was the most potent and most selective inhibitor of COX-2 in intact cells, indicating there are clear pharmacological differences between the two enzymes.
Abstract: Constitutive cyclooxygenase (COX-1; prostaglandin-endoperoxide synthase, EC 1.14.99.1) is present in cells under physiological conditions, whereas COX-2 is induced by some cytokines, mitogens, and endotoxin presumably in pathological conditions, such as inflammation. Therefore, we have assessed the relative inhibitory effects of some nonsteroidal antiinflammatory drugs on the activities of COX-1 (in bovine aortic endothelial cells) and COX-2 (in endotoxin-activated J774.2 macrophages) in intact cells, broken cells, and purified enzyme preparations (COX-1 in sheep seminal vesicles; COX-2 in sheep placenta). Similar potencies of aspirin, indomethacin, and ibuprofen against the broken cell and purified enzyme preparations indicated no influence of species. Aspirin, indomethacin, and ibuprofen were more potent inhibitors of COX-1 than COX-2 in all models used. The relative potencies of aspirin and indomethacin varied only slightly between models, although the IC50 values were different. Ibuprofen was more potent as an inhibitor of COX-2 in intact cells than in either broken cells or purified enzymes. Sodium salicylate was a weak inhibitor of both COX isoforms in intact cells and was inactive against COX in either broken cells or purified enzyme preparations. Diclofenac, BW 755C, acetaminophen, and naproxen were approximately equipotent inhibitors of COX-1 and COX-2 in intact cells. BF 389, an experimental drug currently being tested in humans, was the most potent and most selective inhibitor of COX-2 in intact cells. Thus, there are clear pharmacological differences between the two enzymes. The use of such models of COX-1 and COX-2 activity will lead to the identification of selective inhibitors of COX-2 with presumably less side effects than present therapies. Some inhibitors had higher activity in intact cells than against purified enzymes, suggesting that pure enzyme preparations may not be predictive of therapeutic action.
TL;DR: The recombinant soluble human receptor binds vascular endothelial cell growth factor with high affinity and inhibits its mitogenic activity forascular endothelial cells; thus this soluble receptor could act as an efficient specific antagonist of vascular endothelium cell growthFactor in vivo.
Abstract: Vascular endothelial cell growth factor, a mitogen selective for vascular endothelial cells in vitro that promotes angiogenesis in vivo, functions through distinct membrane-spanning tyrosine kinase receptors. The cDNA encoding a soluble truncated form of one such receptor, fms-like tyrosine kinase receptor, has been cloned from a human vascular endothelial cell library. The mRNA coding region distinctive to this cDNA has been confirmed to be present in vascular endothelial cells. Soluble fms-like tyrosine kinase receptor mRNA, generated by alternative splicing of the same pre-mRNA used to produce the full-length membrane-spanning receptor, encodes the six N-terminal immunoglobulin-like extracellular ligand-binding domains but does not encode the last such domain, transmembrane-spanning region, and intracellular tyrosine kinase domains. The recombinant soluble human receptor binds vascular endothelial cell growth factor with high affinity and inhibits its mitogenic activity for vascular endothelial cells; thus this soluble receptor could act as an efficient specific antagonist of vascular endothelial cell growth factor in vivo.
TL;DR: The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo, and facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system.
Abstract: The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.
TL;DR: In an autopsy series of brains of late-onset AD patients, a strong association of APOE4 allele with increased vascular and plaque A beta deposits is found.
Abstract: Amyloid beta-peptide (A beta) deposition in senile plaques and cerebral vessels is a neuropathological feature of Alzheimer disease (AD). We examined the possibility that commonly observed variability in A beta deposition in late-onset AD might be related to apolipoprotein E genotype (APOE gene; the two most common alleles are 3 and 4), since APOE4 is a susceptibility gene for late-onset AD and apolipoprotein E interacts strongly with A beta in vitro. In an autopsy series of brains of late-onset AD patients, we found a strong association of APOE4 allele with increased vascular and plaque A beta deposits. Late-onset AD patients with one or two APOE4 alleles have a distinct neuropathological phenotype compared with patients homozygous for APOE3.
TL;DR: Primary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid, and the availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidsosis in AD.
Abstract: A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major beta/A4-protein (A beta) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences of these peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-A beta component of AD amyloid) and its precursor NACP. NAC is the second component, after A beta, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid. NACP is detected as a M(r) 19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD.
TL;DR: It is demonstrated that NO enhances COX activity through a mechanism independent of cGMP and suggested that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response.
Abstract: We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-1 and COX-2, respectively). Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (1 microgram/ml, 18 h) caused an increase in the release of nitrite (NO2-) and prostaglandin E2 (PGE2), products of NOS and COX, respectively. Production of both NO2- and PGE2 was blocked by the NOS inhibitors NG-monomethyl-L-arginine or aminoguanidine. The effects of NG-monomethyl-L-arginine or aminoguanidine were reversed by coincubation with L-Arg, the precursor for NO synthesis, but not by D-Arg. RAW264.7 cells stimulated for 18 h with lipopolysaccharide in L-Arg-free medium (to reduce NO generation by the endogenous NOS pathway) failed to release NO2- and accumulated at least 4-fold less PGE2 when compared to cells in the presence of L-Arg. PGE2 production elicited by a 15-min arachidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the release obtained with cells induced in medium containing L-Arg. To examine the NO activation of the induced form of COX in the absence of an endogenous L-Arg, human fetal fibroblasts were first stimulated for 18 h with interleukin 1 beta. These cells released PGE2 but not NO2-, consistent with the induction of COX but not NOS in the fibroblast. Exogenous NO either as a gaseous solution or released by a NO donor, sodium nitroprusside or glyceryl trinitrate, increased COX activity in the interleukin 1 beta-stimulated fibroblasts by 5-fold; these effects were abolished by coincubation with hemoglobin (10 microM), which binds and inactivates NO, but not by methylene blue, an inhibitor of the soluble guanylate cyclase. Furthermore, sodium nitroprusside (0.25-1 mM) increased arachidonic acid-stimulated PGE2 production by murine recombinant COX-1 and COX-2. These results demonstrate that NO enhances COX activity through a mechanism independent of cGMP and suggest that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response. The data suggest that NO directly interacts with COX to cause an increase in the enzymatic activity.
TL;DR: It is demonstrated that dystrophin-deficient muscle fibers of the mdx mouse exhibit an increased susceptibility to contraction-induced sarcolemmal rupture, which strongly support the proposition that the primary function of dyStrophin is to provide mechanical reinforcement to the sarcolemma and thereby protect it from the membrane stresses developed during muscle contraction.
Abstract: The protein dystrophin, normally found on the cytoplasmic surface of skeletal muscle cell membranes, is absent in patients with Duchenne muscular dystrophy as well as mdx (X-linked muscular dystrophy) mice. Although its primary structure has been determined, the precise functional role of dystrophin remains the subject of speculation. In the present study, we demonstrate that dystrophin-deficient muscle fibers of the mdx mouse exhibit an increased susceptibility to contraction-induced sarcolemmal rupture. The level of sarcolemmal damage is directly correlated with the magnitude of mechanical stress placed upon the membrane during contraction rather than the number of activations of the muscle. These findings strongly support the proposition that the primary function of dystrophin is to provide mechanical reinforcement to the sarcolemma and thereby protect it from the membrane stresses developed during muscle contraction. Furthermore, the methodology used in this study should prove useful in assessing the efficacy of dystrophin gene therapy in the mdx mouse.
TL;DR: It is demonstrated that Hif-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed, providing evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to Hypoxia.
Abstract: Transcription of the human erythropoietin (EPO) gene is activated in Hep3B cells exposed to hypoxia. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor whose DNA binding activity is induced by hypoxia in Hep3B cells, and HIF-1 binds at a site in the EPO gene enhancer that is required for hypoxic activation of transcription. In this paper, we demonstrate that HIF-1 DNA binding activity is also induced by hypoxia in a variety of mammalian cell lines in which the EPO gene is not transcribed. The composition of the HIF-1 DNA binding complex and its isolated DNA binding subunit and the mechanism of HIF-1 activation appear to be similar or identical in EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells and mutations that eliminate HIF-1 binding eliminate inducibility. These results provide evidence that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia.
TL;DR: This report shows that SVZ cells labeled in the brains of adult mice with [3H]thymidine differentiate directly into neurons and glia in explant cultures, and shows that 98% of the neurons that differentiate from the SVZ explants are derived from precursor cells that underwent their last division in vivo.
Abstract: Subventricular zone (SVZ) cells proliferate spontaneously in vivo in the telencephalon of adult mammals. Several studies suggest that SVZ cells do not differentiate after mitosis into neurons or glia but die. In the present work, we show that SVZ cells labeled in the brains of adult mice with [3H]thymidine differentiate directly into neurons and glia in explant cultures. In vitro labeling with [3H]thymidine shows that 98% of the neurons that differentiate from the SVZ explants are derived from precursor cells that underwent their last division in vivo. This report identifies the SVZ cells as neuronal precursors in an adult mammalian brain.
TL;DR: Differences in the two isoforms in complexing with the beta/A4 peptide may be involved in the pathogenesis of the intra- and extracellular lesions of Alzheimer disease.
Abstract: Apolipoprotein E (apoE), a plasma apolipoprotein that plays a central role in lipoprotein metabolism, is localized in the senile plaques, congophilic angiopathy, and neurofibrillary tangles of Alzheimer disease. Late-onset familial and sporadic Alzheimer disease patients have an increased frequency of one of the three common apoE alleles, epsilon 4, suggesting apoE4 is associated with increased susceptibility to disease. To follow up on this suggestion, we compared the binding of synthetic amyloid beta (beta/A4) peptide to purified apoE4 and apoE3, the most common isoform. Both isoforms bound synthetic beta/A4 peptide, the primary constituent of the plaque and angiopathy, forming a complex that resisted dissociation by boiling in SDS. Oxygen-mediated complex formation was implicated because binding was increased in oxygenated buffer, reduced in nitrogen-purged buffer, and prevented by reduction with dithiothreitol or 2-mercaptoethanol. Binding of beta/A4 peptide was saturable at 10(-4) M peptide and required residues 12-28. Examination of apoE fragments revealed that residues 244-272 are critical for complex formation. Both oxidized apoE4 and apoE3 bound beta/A4 peptide; however, binding to apoE4 was observed in minutes, whereas binding to apoE3 required hours. In addition, apoE4 did not bind beta/A4 peptide at pH < 6.6, whereas apoE3 bound beta/A4 peptide from pH 7.6 to 4.6. Together these results indicate differences in the two isoforms in complexing with the beta/A4 peptide. Binding of beta/A4 peptide by oxidized apoE may determine the sequestration or targeting of either apoE or beta/A4 peptide, and isoform-specific differences in apoE binding or oxidation may be involved in the pathogenesis of the intra- and extracellular lesions of Alzheimer disease.
TL;DR: This work designed and constructed chimeric genes composed of a single-chain Fv domain of an antibody linked with gamma or zeta chains, the common signal-transducing subunits of the immunoglobulin receptor and the TCR, which could be expressed as functional surface receptors in a cytolytic T-cell hybridoma.
Abstract: The generation of tumor-specific lymphocytes and their use in adoptive immunotherapy is limited to a few malignancies because most spontaneous tumors are very weak or not at all immunogenic. On the other hand, many anti-tumor antibodies have been described which bind tumor-associated antigens shared among tumors of the same histology. Combining the variable regions (Fv) of an antibody with the constant regions of the T-cell receptor (TCR) chains results in chimeric genes endowing T lymphocytes with antibody-type specificity, potentially allowing cellular adoptive immunotherapy against types of tumors not previously possible. To generalize and extend this approach to additional lymphocyte-activating molecules, we designed and constructed chimeric genes composed of a single-chain Fv domain (scFv) of an antibody linked with gamma or zeta chains, the common signal-transducing subunits of the immunoglobulin receptor and the TCR. Such chimeric genes containing the Fv region of an anti-trinitophenyl antibody could be expressed as functional surface receptors in a cytolytic T-cell hybridoma. They triggered interleukin 2 secretion upon encountering antigen and mediated non-major-histocompatibility-complex-restricted hapten-specific target cell lysis. Such chimeric receptors can be exploited to provide T cells and other effector lymphocytes, such as natural killer cells, with antibody-type recognition directly coupled to cellular activation.
TL;DR: A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database and each protein was uniquely identified from over 91,000 protein sequences.
Abstract: A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.
TL;DR: By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis, and 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA.
Abstract: Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.
TL;DR: The nucleotide sequence and expression of an immunodominant antigen of H. pylori and the immune response to the antigen during disease are reported and it is suggested that only bacteria harboring this protein are associated with disease.
Abstract: Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma. We report the nucleotide sequence and expression of an immunodominant antigen of H. pylori and the immune response to the antigen during disease. The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates. The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene. Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin. An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseases and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease.
TL;DR: Although this intracellular signal transduction pathway is extremely complex, conservation of many of its components has been observed in yeast, nematodes, Drosophila, and mammals, Thus, these signaling proteins may participate in the regulation of a variety of cellular processes.
Abstract: An explosion of new information linking activation of cell surface signal initiators to changes in gene expression has recently emerged. The focus of much of this information has centered around the agonist-dependent activation of the mitogen-activated protein (MAP) kinases. Although this intracellular signal transduction pathway is extremely complex, conservation of many of its components has been observed in yeast, nematodes, Drosophila, and mammals. Thus, these signaling proteins may participate in the regulation of a variety of cellular processes.
TL;DR: This work has shown that a 16-residue peptide has a characteristic beta-sheet circular dichroism spectrum in water and spontaneously assembles to form a macroscopic membrane, which may be a model for studying the insoluble peptides found in certain neurological disorders.
Abstract: A 16-residue peptide [(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2] has a characteristic beta-sheet circular dichroism spectrum in water. Upon the addition of salt, the peptide spontaneously assembles to form a macroscopic membrane. The membrane does not dissolve in heat or in acidic or alkaline solutions, nor does it dissolve upon addition of guanidine hydrochloride, SDS/urea, or a variety of proteolytic enzymes. Scanning EM reveals a network of interwoven filaments approximately 10-20 nm in diameter. An important component of the stability is probably due to formation of complementary ionic bonds between glutamic and lysine side chains. This phenomenon may be a model for studying the insoluble peptides found in certain neurological disorders. It may also have implications for biomaterials and origin-of-life research.
TL;DR: Reconsideration of the generation time hypothesis to include physiological effects such as metabolic rate improves the theoretical underpinnings of molecular evolution.
Abstract: There is increasing evidence for variation in rates of nucleotide substitution among divergent taxonomic groups. Here, we summarize published rate data and show a strong relationship between substitution rate and body size. For instance, rates of nuclear and mtDNA evolution are slow in whales, intermediate in primates, and fast in rodents. A similar relationship exists for poikilothermic vertebrates. However, these taxa have slower mtDNA substitution rates overall than do homeotherms of similar size. A number of physiological and life history variables are highly correlated with body size. Of these, generation time and metabolic rate explain some patterns of rate heterogeneity equally well. In many cases, however, differences in metabolic rate explain important exceptions to the generation time model. Correlation between metabolic rate and nucleotide substitution may be mediated by (i) the mutagenic effects of oxygen radicals that are abundant by-products of aerobic respiration, and (ii) increased rates of DNA synthesis and nucleotide replacement in organisms with higher metabolic rates. Both of these factors increase mutation rate by decreasing the "nucleotide generation time," the average length of time before a nucleotide is copied either through replication or repair. Reconsideration of the generation time hypothesis to include physiological effects such as metabolic rate improves the theoretical underpinnings of molecular evolution.
TL;DR: The statistical distribution for the sum of the scores of multiple high-scoring segments is described and its application to the identification of possible transmembrane segments and the evaluation of sequence similarity is illustrated.
Abstract: Score-based measures of molecular-sequence features provide versatile aids for the study of proteins and DNA They are used by many sequence data base search programs, as well as for identifying distinctive properties of single sequences For any such measure, it is important to know what can be expected to occur purely by chance The statistical distribution of high-scoring segments has been described elsewhere However, molecular sequences will frequently yield several high-scoring segments for which some combined assessment is in order This paper describes the statistical distribution for the sum of the scores of multiple high-scoring segments and illustrates its application to the identification of possible transmembrane segments and the evaluation of sequence similarity
TL;DR: In this paper, the authors explored the idea of a conditional test via multiple regression analysis for quantitative trait loci (QTL) mapping and showed that such a test can potentially increase the precision of QTL mapping substantially.
Abstract: It is now possible to use complete genetic linkage maps to locate major quantitative trait loci (QTLs) on chromosome regions. The current methods of QTL mapping (e.g., interval mapping, which uses a pair or two pairs of flanking markers at a time for mapping) can be subject to the effects of other linked QTLs on a chromosome because the genetic background is not controlled. As a result, mapping of QTLs can be biased, and the resolution of mapping is not very high. Ideally when we test a marker interval for a QTL, we would like our test statistic to be independent of the effects of possible QTLs at other regions of the chromosome so that the effects of QTLs can be separated. This test statistic can be constructed by using a pair of markers to locate the testing position and at the same time using other markers to control the genetic background through a multiple regression analysis. Theory is developed in this paper to explore the idea of a conditional test via multiple regression analysis. Various properties of multiple regression analysis in relation to QTL mapping are examined. Theoretical analysis indicates that it is advantageous to construct such a testing procedure for mapping QTLs and that such a test can potentially increase the precision of QTL mapping substantially.
TL;DR: The structure of the HIV-1 RT/DNA/Fab complex should aid the understanding of general mechanisms of nucleic acid polymerization and AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies.
Abstract: The crystal structure of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p51), a 19-base/18-base double-stranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. The four individual subdomains of RT that make up the polymerase domains of p66 and p51 are named fingers, palm, thumb, and connection [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790]. The overall folding of the subdomains is similar in p66 and p51 but the spatial arrangements of the subdomains are dramatically different. The template-primer has A-form and B-form regions separated by a significant bend (40-45 degrees). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and fingers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two alpha-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nucleophilic attack on the alpha-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies.