Showing papers in "Proceedings of the National Academy of Sciences of the United States of America in 1994"
TL;DR: In this article, a method for producing high-titer, helper-free infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line BOSC 23 and amphotropic line CAK 8.
Abstract: A method for producing high-titer, helper-free infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line BOSC 23 and amphotropic line CAK 8, both of which cell lines and their precursor cell lines are disclosed. Because of the advantages over stable packaging cell lines, the BOSC 23 and CAK 8 transient transfection systems greatly facilitate and extend the use of helper-free retroviral vectors. The cell lines and corresponding methods possess wide application in both the medical and biotechnical fields, including gene therapy. These potential applications are disclosed and illustrated.
2,587 citations
TL;DR: It is reported that glutamate, in addition to its receptor-mediated actions on neuronal excitability, stimulates glycolysis--i.e., glucose utilization and lactate production--in astrocytes and is consistent with data obtained from functional brain imaging studies indicating local nonoxidative glucose utilization during physiological activation.
Abstract: Glutamate, released at a majority of excitatory synapses in the central nervous system, depolarizes neurons by acting at specific receptors. Its action is terminated by removal from the synaptic cleft mostly via Na(+)-dependent uptake systems located on both neurons and astrocytes. Here we report that glutamate, in addition to its receptor-mediated actions on neuronal excitability, stimulates glycolysis--i.e., glucose utilization and lactate production--in astrocytes. This metabolic action is mediated by activation of a Na(+)-dependent uptake system and not by interaction with receptors. The mechanism involves the Na+/K(+)-ATPase, which is activated by an increase in the intracellular concentration of Na+ cotransported with glutamate by the electrogenic uptake system. Thus, when glutamate is released from active synapses and taken up by astrocytes, the newly identified signaling pathway described here would provide a simple and direct mechanism to tightly couple neuronal activity to glucose utilization. In addition, glutamate-stimulated glycolysis is consistent with data obtained from functional brain imaging studies indicating local nonoxidative glucose utilization during physiological activation.
2,521 citations
TL;DR: Electron microscopic examination of the corneal neovascularization of thalidomide-treated rabbits revealed specific ultrastructural changes similar to those seen in the deformed limb bud vasculature of Thalidomid-treated embryos.
Abstract: Thalidomide is a potent teratogen causing dysmelia (stunted limb growth) in humans. We have demonstrated that orally administered thalidomide is an inhibitor of angiogenesis induced by basic fibroblast growth factor in a rabbit cornea micropocket assay. Experiments including the analysis of thalidomide analogs revealed that the antiangiogenic activity correlated with the teratogenicity but not with the sedative or the mild immunosuppressive properties of thalidomide. Electron microscopic examination of the corneal neovascularization of thalidomide-treated rabbits revealed specific ultrastructural changes similar to those seen in the deformed limb bud vasculature of thalidomide-treated embryos. These experiments shed light on the mechanism of thalidomide's teratogenicity and hold promise for the potential use of thalidomide as an orally administered drug for the treatment of many diverse diseases dependent on angiogenesis.
2,364 citations
TL;DR: Evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.
Abstract: We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of aging. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Several mitochondrial functions decline with age. The contributing factors include the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Acetyl-L-carnitine, a high-energy mitochondrial substrate, appears to reverse many age-associated deficits in cellular function, in part by increasing cellular ATP production. Such evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.
1,977 citations
TL;DR: A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported, and mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology.
Abstract: Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone. Repeated cycles of point mutagenesis, recombination, and selection should allow in vitro molecular evolution of complex sequences, such as proteins. A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported. A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its original size and function. Similarly, a 2.7-kb plasmid could be efficiently reassembled. Complete recombination was obtained between two markers separated by 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and incorporated into the reassembled gene. Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology. As an example, a library of chimeras of the human and murine genes for interleukin 1 beta has been prepared. Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA. The advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution.
1,912 citations
TL;DR: The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.
Abstract: The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.
1,911 citations
TL;DR: It is reported here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes, which can then be applied to parallel DNA hybridization analysis, directly yielding sequence information.
Abstract: In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition.
1,753 citations
TL;DR: Approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes are suggested.
Abstract: An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes.
1,691 citations
TL;DR: Ample evidence is presented that silicon, when readily available to plants, plays a large role in their growth, mineral nutrition, mechanical strength, and resistance to fungal diseases, herbivory, and adverse chemical conditions of the medium.
Abstract: Silicon is the second most abundant element in soils, the mineral substrate for most of the world's plant life. The soil water, or the "soil solution," contains silicon, mainly as silicic acid, H4SiO4, at 0.1-0.6 mM--concentrations on the order of those of potassium, calcium, and other major plant nutrients, and well in excess of those of phosphate. Silicon is readily absorbed so that terrestrial plants contain it in appreciable concentrations, ranging from a fraction of 1% of the dry matter to several percent, and in some plants to 10% or even higher. In spite of this prominence of silicon as a mineral constituent of plants, it is not counted among the elements defined as "essential," or nutrients, for any terrestrial higher plants except members of the Equisitaceae. For that reason it is not included in the formulation of any of the commonly used nutrient solutions. The plant physiologist's solution-cultured plants are thus anomalous, containing only what silicon is derived as a contaminant of their environment. Ample evidence is presented that silicon, when readily available to plants, plays a large role in their growth, mineral nutrition, mechanical strength, and resistance to fungal diseases, herbivory, and adverse chemical conditions of the medium. Plants grown in conventional nutrient solutions are thus to an extent experimental artifacts. Omission of silicon from solution cultures may lead to distorted results in experiments on inorganic plant nutrition, growth and development, and responses to environmental stress.
1,558 citations
TL;DR: Computer simulations were used to determine expected values of statistics that reflect frequency distributions of allele size for the two-phase model and two alternatives, the one-step and geometric models, and found that estimates of interpopulation distances from SSRs were similar to those derived from analysis of mitochondrial DNA.
Abstract: Mutational processes of simple sequence repeats (SSRs) in complex genomes are poorly understood. We examined these processes by introducing a two-phase mutation model. In this model, most mutations are single-step changes, but infrequent large jumps in repeat number also occur. We used computer simulations to determine expected values of statistics that reflect frequency distributions of allele size for the two-phase model and two alternatives, the one-step and geometric models. The theoretical expectations for each model were tested by comparison with observed values for 10 SSR loci genotyped in the Sardinian population, whose genetic and demographic histories have been previously reconstructed. The two-phase model provided the best fit to the data for most of these loci in this population. In the analysis we assumed that the loci were neutral and that this population had undergone rapid population growth. Recent observations made for unstable trinucleotide repeats support our suggestion that frequent small changes and rare large changes in repeat number represent two distinct classes of mutation at SSR loci. We genotyped the same 10 loci in Egyptian and sub-Saharan African samples to assess the utility of SSRs for studying the divergence of populations and found that estimates of interpopulation distances from SSRs were similar to those derived from analysis of mitochondrial DNA.
1,547 citations
TL;DR: In an animal model of acute inflammation, a selective inhibitor of COX-2 inhibited edema at the inflammatory site and was analgesic but had no effect on PG production in the stomach and did not cause gastric toxicity.
Abstract: Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases, but significant side effects such as gastrointestinal erosion and renal damage limit their use. NSAIDs inhibit the enzyme cyclooxygenase (COX), which catalyzes the conversion of arachidonic acid to prostaglandins (PGs) and thromboxane. Two forms of COX have been identified--COX-1, which is constitutively expressed in most tissues and organs, and the inducible enzyme, COX-2, which has been localized primarily to inflammatory cells and tissues. In an animal model of acute inflammation (injection of carrageenan into the footpad), edema was produced that was associated with marked accumulation of COX-2 mRNA and thromboxane. A selective inhibitor of COX-2 (SC-58125) inhibited edema at the inflammatory site and was analgesic but had no effect on PG production in the stomach and did not cause gastric toxicity. These data suggest that selective inhibition of COX-2 may produce superior antiinflammatory drugs with substantial safety advantages over existing NSAIDs.
TL;DR: The findings suggest that aberrant methylation of CpG islands may participate in the tumor-suppressor gene inactivations which initiate or cause progression of common human cancers.
Abstract: Mutational inactivation and allelic loss of the von Hippel-Lindau (VHL) gene appear to be causal events for the majority of spontaneous clear-cell renal carcinomas. We now show that hypermethylation of a normally unmethylated CpG island in the 5' region provides another potentially important mechanism for inactivation of the VHL gene in a significant portion of these cancers. This hypermethylation was found in 5 of 26 (19%) tumors examined. Four of these had lost one copy of VHL while one retained two heavily methylated alleles. Four of the tumors with VHL hypermethylation had no detectable mutations, whereas one had a missense mutation in addition to hypermethylation of the single retained allele. As would be predicted for the consequence of methylation in this 5' CpG island, none of the 5 tumors expressed the VHL gene. In contrast, normal kidney and all tumors examined with inactivating VHL gene mutations but no CpG island methylation had expression. In a renal cell culture line, treatment with 5-aza-2'-deoxycytidine resulted in reexpression of the VHL gene. These findings suggest that aberrant methylation of CpG islands may participate in the tumor-suppressor gene inactivations which initiate or cause progression of common human cancers.
TL;DR: A hemispheric encoding/retrieval asymmetry model of prefrontal involvement in encoding and retrieval of episodic memory is proposed, which suggests that left and right prefrontal lobes are part of an extensive neuronal network that subserves episodic remembering, but the two prefrontal hemispheres play different roles.
Abstract: Data are reviewed from positron emission tomography studies of encoding and retrieval processes in episodic memory. These data suggest a hemispheric encoding/retrieval asymmetry model of prefrontal involvement in encoding and retrieval of episodic memory. According to this model, the left and right prefrontal lobes are part of an extensive neuronal network that subserves episodic remembering, but the two prefrontal hemispheres play different roles. Left prefrontal cortical regions are differentially more involved in retrieval of information from semantic memory and in simultaneously encoding novel aspects of the retrieved information into episodic memory. Right prefrontal cortical regions, on the other hand, are differentially more involved in episodic memory retrieval.
TL;DR: A phylogenetic hypothesis based on mitochondrial DNA sequences for 14 divergent races of Heliconius erato reveals that similar wing patterns have evolved rapidly and convergently within the species, suggesting a simultaneous radiation of races within the last 200,000 years.
Abstract: The neotropical Heliconius butterflies are famous examples of Mullerian mimicry, due to the diverse array of shared, brightly colored wing patterns that advertise the butterflies' unpalatability. The parallel geographical variation in these patterns within several widespread species has been invoked to support the controversial Pleistocene refugium hypothesis of tropical diversification. However, in no Heliconius species have either evolutionary rates or relationships among geographical races been explicitly examined. I present a phylogenetic hypothesis based on mitochondrial DNA sequences for 14 divergent races of Heliconius erato, which reveals that similar wing patterns have evolved rapidly and convergently within the species. There is a basal split between groups of races from east and west of the Andes, reflecting a vicariant separation at the base of the Pleistocene. Within each of these clades, sequence divergence is very low, and some haplotypes are shared between allopatric races with radically different wing patterns. The topology implies a simultaneous radiation of races in these two areas within the last 200,000 years. Ages for the clades are estimated by comparing sequence divergence to a plot of mitochondrial divergence in several arthropod taxa with independently dated divergence times. This plot is linear and suggests that mitochondrial DNA in arthropods evolves in a clocklike manner, at least initially, when sequence divergence is low.
TL;DR: A measure, called neural complexity (CN), that captures the interplay between functional segregation and functional integration in brains of higher vertebrates and may prove useful in analyzing complexity in other biological domains such as gene regulation and embryogenesis.
Abstract: In brains of higher vertebrates, the functional segregation of local areas that differ in their anatomy and physiology contrasts sharply with their global integration during perception and behavior. In this paper, we introduce a measure, called neural complexity (CN), that captures the interplay between these two fundamental aspects of brain organization. We express functional segregation within a neural system in terms of the relative statistical independence of small subsets of the system and functional integration in terms of significant deviations from independence of large subsets. CN is then obtained from estimates of the average deviation from statistical independence for subsets of increasing size. CN is shown to be high when functional segregation coexists with integration and to be low when the components of a system are either completely independent (segregated) or completely dependent (integrated). We apply this complexity measure in computer simulations of cortical areas to examine how some basic principles of neuroanatomical organization constrain brain dynamics. We show that the connectivity patterns of the cerebral cortex, such as a high density of connections, strong local connectivity organizing cells into neuronal groups, patchiness in the connectivity among neuronal groups, and prevalent reciprocal connections, are associated with high values of CN. The approach outlined here may prove useful in analyzing complexity in other biological domains such as gene regulation and embryogenesis.
TL;DR: It is reported here that stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules and may prove useful as a tool for biomedical science and biotechnology.
Abstract: In the adult male, a population of diploid stem-cell spermatogonia continuously undergoes self-renewal and produces progeny cells, which initiate the complex process of cellular differentiation that results in mature spermatozoa. We report here that stem cells isolated from testes of donor male mice will repopulate sterile testes when injected into seminiferous tubules. Donor cell spermatogenesis in recipient testes showed normal morpholigical characteristics and produced mature spermatozoa. This methodology, besides opening new avenues of basic research into spermatogenesis and stem-cell self-renewal, may prove useful as a tool for biomedical science and biotechnology.
TL;DR: It is demonstrated that HSP participation in multimolecular complex formation is required for src-mediated transformation and can provide a target for drug modulation.
Abstract: The molecular mechanisms by which oncogenic tyrosine kinases induce cellular transformation are unclear. Herbimycin A, geldanamycin, and certain other benzoquinone ansamycins display an unusual capacity to revert tyrosine kinase-induced oncogenic transformation. As an approach to the study of v-src-mediated transformation, we examined ansamycin action in transformed cells and found that drug-induced reversion could be achieved without direct inhibition of src phosphorylating activity. To identify mechanisms other than kinase inhibition for drug-mediated reversion, we prepared a solid phase-immobilized geldanamycin derivative and affinity precipitated the molecular targets with which the drug interacted. In a range of cell lines, immobilized geldanamycin bound elements of a major class of heat shock protein (HSP90) in a stable and pharmacologically specific manner. Consistent with these binding data, we found that soluble geldanamycin and herbimycin A inhibited specifically the formation of a previously described src-HSP90 heteroprotein complex. A related benzoquinone ansamycin that failed to revert transformed cells did not inhibit the formation of this complex. These results demonstrate that HSP participation in multimolecular complex formation is required for src-mediated transformation and can provide a target for drug modulation.
TL;DR: Although Nrf2 is expressed ubiquitously, a role of this protein in mediating enhancer activity of hypersensitive site 2 in erythroid cells cannot be excluded and NRF2 contains a powerful acidic activation domain that may participate in the transcriptional stimulation of beta-globin genes.
Abstract: Hypersensitive site 2 located in the beta-globin locus control region confers high levels of expression to the genes of the beta-globin cluster. A tandem repeat of the consensus sequence for the transcription factors AP1 and NF-E2 (activating protein 1 and nuclear factor erythroid 2, respectively) is present within hypersensitive site 2 and is absolutely required for strong enhancer activity. This sequence binds, in vitro and in vivo, to ubiquitous proteins of the AP1 family and to the recently cloned erythroid-specific transcription factor NF-E2. Using the tandem repeat as a recognition site probe to screen a lambda gt11 cDNA expression library from K562 cells, we isolated several DNA binding proteins. Here, we report the characterization of one of the clones isolated. The gene, which we named Nrf2 (NF-E2-related factor 2), is encoded within a 2.2-kb transcript and predicts a 66-kDa protein with a basic leucine zipper DNA binding domain highly homologous to that of NF-E2. Although Nrf2 is expressed ubiquitously, a role of this protein in mediating enhancer activity of hypersensitive site 2 in erythroid cells cannot be excluded. In this respect, Nrf2 contains a powerful acidic activation domain that may participate in the transcriptional stimulation of beta-globin genes.
TL;DR: By using convection to supplement simple diffusion, enhanced distribution of large and small molecules can be obtained in the brain while achieving drug concentrations orders of magnitude greater than systemic levels.
Abstract: For many compounds (neurotrophic factors, antibodies, growth factors, genetic vectors, enzymes) slow diffusion in the brain severely limits drug distribution and effect after direct drug administration into brain parenchyma. We investigated convection as a means to enhance the distribution of the large and small molecules 111In-labeled transferrin (111In-Tf; M(r), 80,000) and [14C]sucrose (M(r), 359) over centimeter distances by maintaining a pressure gradient during interstitial infusion into white matter to generate bulk flow through the brain interstitium. The volume of distribution (Vd) containing > or = 1% concentration of infusion solution increased linearly with the infusion volume (Vi) for 111In-Tf(Vd/Vi, 6:1) and [14C]sucrose (Vd/Vi, 13:1). Twenty-four hours after infusion, the distribution of 111In-Tf was increased and more homogeneous, and penetration into gray matter had occurred. By using convection to supplement simple diffusion, enhanced distribution of large and small molecules can be obtained in the brain while achieving drug concentrations orders of magnitude greater than systemic levels.
TL;DR: The data suggest that tissue-specific responsiveness to peroxisome proliferators, including certain fatty acids, is in part a consequence of differential expression of multiple, pharmacologically distinct PPAR isoforms.
Abstract: To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in mammals, we have cloned and characterized two PPAR alpha-related cDNAs (designated PPAR gamma and -delta, respectively) from mouse. The three PPAR isoforms display widely divergent patterns of expression during embryogenesis and in the adult. Surprisingly, PPAR gamma and -delta are not activated by pirinixic acid (Wy 14,643), a potent peroxisome proliferator and activator of PPAR alpha. However, PPAR gamma and -delta are activated by the structurally distinct peroxisome proliferator LY-171883 and linoleic acid, respectively, indicating that each of the isoforms can act as a regulated activator of transcription. These data suggest that tissue-specific responsiveness to peroxisome proliferators, including certain fatty acids, is in part a consequence of differential expression of multiple, pharmacologically distinct PPAR isoforms.
TL;DR: The results indicate that beta A neurotoxicity requires fibril formation, and suggest that a common cytopathic effect of amyloid fibrils may contribute to the pathogenesis of Alzheimer disease and other amyloidsoses.
Abstract: beta-Amyloid (beta A) is normally produced as a nontoxic soluble peptide. In Alzheimer disease, beta A aggregates and accumulates in the brain as inert diffuse plaques or compact plaques associated with neurodegenerative changes. To determine the relationship of neurotoxicity to the physical state of beta A, we created (i) nonamyloidogenic amorphous aggregates of beta A [amorphous beta A (Am-beta A)] analogous to diffuse plaques and (ii) amyloidogenic fibrils of beta A [fibrillar beta A (Fib-beta A)] analogous to compact plaques. In primary rat hippocampal culture, Fib-beta A was neurotoxic, whereas Am-beta A was not toxic. Fib-beta A caused significant loss of synapses in viable neurons, while Am-beta A had no effect on synapse number. The amyloid fibril-binding dye Congo red inhibited Fib-beta A neurotoxicity by inhibiting fibril formation or by binding to preformed fibrils. Congo red also inhibited the pancreatic islet cell toxicity of diabetes-associated amylin, another type of amyloid fibril. These results indicate that beta A neurotoxicity requires fibril formation. These findings and our previous demonstration that amylin fibrils are toxic suggest that a common cytopathic effect of amyloid fibrils may contribute to the pathogenesis of Alzheimer disease and other amyloidoses.
TL;DR: Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells to be delivered to tissue culture.
Abstract: The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to beta-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-beta-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages. Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.
TL;DR: Results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects associated with the nonselective,COX-1-directed antiinflammatory drugs.
Abstract: We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammation in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by high levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-directed antiinflammatory drugs.
TL;DR: The base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased and improvements were achieved by the combination of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1.
Abstract: A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.
TL;DR: Results show that TNF-alpha directly interferes with the signaling of insulin through its receptor and consequently blocks biological actions of insulin.
Abstract: Insulin resistance is a common problem associated with infections and cancer and, most importantly, is the central component of non-insulin-dependent diabetes mellitus. We have recently shown that tumor necrosis factor (TNF) alpha is a key mediator of insulin resistance in animal models of non-insulin-dependent diabetes mellitus. Here, we investigate how TNF-alpha interferes with insulin action. Chronic exposure of adipocytes to low concentrations of TNF-alpha strongly inhibits insulin-stimulated glucose uptake. Concurrently, TNF-alpha treatment causes a moderate decrease in the insulin-stimulated autophosphorylation of the insulin receptor (IR) and a dramatic decrease in the phosphorylation of IR substrate 1, the major substrate of the IR in vivo. The IR isolated from TNF-alpha-treated cells is also defective in the ability to autophosphorylate and phosphorylate IR substrate 1 in vitro. These results show that TNF-alpha directly interferes with the signaling of insulin through its receptor and consequently blocks biological actions of insulin.
TL;DR: MCP-1 is the major lymphocyte chemoattractant secreted by mitogen-stimulated peripheral blood mononuclear cells and is capable of acting as a potent T-lymphocyte, as well as monocyte, chemoATTractant.
Abstract: We have utilized a transendothelial lymphocyte chemotaxis assay to identify and purify a lymphocyte chemoattractant in supernatants of mitogen-stimulated peripheral blood mononuclear cells. Amino acid sequence analysis revealed identity with monocyte chemoattractant protein 1 (MCP-1), a chemoattractant previously thought to be specific for monocytes. Recombinant MCP-1 is chemoattractive for purified T lymphocytes and for CD3+ lymphocytes in peripheral blood lymphocyte preparations. The T-cell response to MCP-1 is dose-dependent and chemotactic, rather than chemokinetic. Phenotyping of chemoattracted T lymphocytes shows they are an activated memory subset. The response to MCP-1 by T lymphocytes can be duplicated in the absence of an endothelial monolayer and the majority of T-lymphocyte chemotactic activity in mitogen-stimulated peripheral blood mononuclear cell supernatants can be neutralized by antibody to MCP-1. Thus, MCP-1 is the major lymphocyte chemoattractant secreted by mitogen-stimulated peripheral blood mononuclear cells and is capable of acting as a potent T-lymphocyte, as well as monocyte, chemoattractant. This may help explain why monocytes and T lymphocytes of the memory subset are always found together at sites of antigen-induced inflammation.
TL;DR: It is shown that candidate human stem cells with a CD34+CD38lo phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilical cord blood and that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA.
Abstract: The proliferative life-span of the stem cells that sustain hematopoiesis throughout life is not known. It has been proposed that the sequential loss of telomeric DNA from the ends of human chromosomes with each somatic cell division eventually reaches a critical point that triggers cellular senescence. We now show that candidate human stem cells with a CD34+CD38lo phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilical cord blood. We also found that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA. These findings strongly suggest that the proliferative potential of most, if not all, hematopoietic stem cells is limited and decreases with age, a concept that has widespread implications for models of normal and abnormal hematopoiesis as well as gene therapy.
TL;DR: A gene encoding a melanocyte lineage-specific protein (MART-1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy is identified and opens possibilities for the development of immunotherapies for patients with melanoma.
Abstract: By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma.
TL;DR: Evidence is presented that beta-amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes and generate free radical peptides.
Abstract: beta-Amyloid is a 39- to 43-amino-acid neurotoxic peptide that aggregates to form the core of Alzheimer disease-associated senile (amyloid) plaques. No satisfactory hypothesis has yet been proposed to explain the mechanism of beta-amyloid aggregation and toxicity. We present mass spectrometric and electron paramagnetic resonance spin trapping evidence that beta-amyloid, in aqueous solution, fragments and generates free radical peptides. beta-Amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes. Also, salicylate hydroxylation assays indicate that reactive oxygen species are generated by the beta-amyloid-(25-35) fragment during cell-free incubation. These results are formulated into a free radical-based unifying hypothesis for neurotoxicity of beta-amyloid and are discussed with reference to membrane molecular alterations in Alzheimer disease.
TL;DR: The discovery of arbuscules in Aglaophyton major, an Early Devonian land plant, provides unequivocal evidence that mycorrhizae were established >400 million years ago and indicates that nutrient transfer mutualism may have been in existence when plants invaded the land.
Abstract: The discovery of arbuscules in Aglaophyton major, an Early Devonian land plant, provides unequivocal evidence that mycorrhizae were established >400 million years ago. Nonseptate hyphac and arbuscules occur in a specialized meristematic region of the cortex that continually provided new cells for fungal infection. Arbuscules are morphologically identical to those of living arbuscular mycorrhizae in consisting of a basal trunk and repeatedly branched bush-like tuft within the plant cell. Although interpretations of the evolution of mycorrhizal mutualisms continue to be speculative, the existence of arbuscules in the Early Devonian indicates that nutrient transfer mutualism may have been in existence when plants invaded the land.