bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-dose interleukin 2 prevents the development of Epstein-Barr virus (EBV)-associated lymphoproliferative disease in scid/scid mice reconstituted i.p. with EBV-seropositive human peripheral blood lymphocytes.
Authors: Baiocchi R A , Caligiuri M A
Published: May 2006

When severe combined immune deficient (SCID) mice undergo i.p. injection with peripheral blood lymphocytes from normal human donors seropositive for EBV, a majority of these mice (hu-PBL-SCID mouse model) subsequently develop a fatal EBV+ lymphoproliferative disease (EBV-LPD) of human B-cell origin. Because T cells normally are critical in the control of EBV infection, we hypothesized that human T-cell dysfunction accounts for EBV-LPD in the hu-PBL-SCID mouse and that systemic administration of T-cell-derived cytokines would reestablish protective immunity against EBV-LPD. We show that the daily s.c. administration of a very low dose (500 international units) of polyethylene glycol-modified recombinant human interleukin 2 (PEG-IL-2) to hu-PBL-SCID mice can prevent the development of fatal EBV-LPD and significantly improves survival (78%), compared with the survival of hu-PBL-SCID mice treated with placebo (20%, P = 0.0008). Additional lymphocyte-depletion experiments showed that mouse natural killer cells and human CD8+ T cells provided cellular immunity necessary for the PEG-IL-2-mediated protective effect, whereas i.p. injection of human peripheral blood lymphocytes depleted of CD4+ T cells had no adverse effect when combined with PEG-IL-2 therapy and may have been beneficial. These data establish that very low-dose PEG-IL-2 therapy can overcome the immune deficiencies that lead to EBV-LPD in the hu-PBL-SCID mouse and point to the usefulness of this model for evaluating cytokine therapies in EBV-LPD. The use of low-dose IL-2 as a preventative immune therapy has potential application in immunocompromised individuals at high risk for EBV-LPD.
Specific T-cell tolerance may be preceded by a primary response.
Authors: Vidard L , Colarusso L J , Benacerraf B
Published: May 2006

We have evaluated the ability of ovalbumin to induce T-cell-specific tolerance in SJL mice. A significant decrease of interleukin 2 in lymph-node culture supernatants from tolerant mice upon antigen stimulation was seen. Oral tolerization was less effective than i.p.- or s.c.-tolerization protocols. Transfer experiments of either splenic or lymph-node T cells from tolerant mice to naive mice definitely ruled out suppression as a mechanism involved in tolerant mice. Surprisingly, we found that, before the establishment of specific T-cell tolerance to ovalbumin, T cells from mice that will display tolerance were responsive and synthesized interleukin 2 upon antigen challenge in vitro. Thus, we concluded that anergy cannot account solely for the T-cell unresponsiveness in tolerant mice. Furthermore, although we cannot rule out the hypothesis that the T-cell unresponsiveness in tolerant mice can be explained by programmed cell death of ovalbumin-specific T cells, these data led us to speculate that T-cell "refractoriness" could explain the drop of interleukin 2 production in lymph-node T-cell culture supernatant from tolerant mice.
The animal model of human amnesia: long-term memory impaired and short-term memory intact.
Authors: Alvarez P , Zola-Morgan S , Squire L R
Published: May 2006

Normal monkeys and monkeys with lesions of the hippocampal formation and adjacent cortex (the H+ lesion) were trained on the delayed nonmatching to sample (DNMS) task with a delay of 0.5 s between the sample and the choice. The animals with H+ lesions learned the task normally at this short delay and also exhibited the same pattern of response latencies as normal monkeys. This finding contrasts with previous observations that initial learning of the DNMS task with delays of 8-10 s is impaired after H+ lesions. The absence of an impairment at a delay of 0.5 s indicates that the H+ lesion does not affect short-term memory. In contrast, when monkeys with H+ lesions were tested at longer delays (> 30 s), an impairment was observed. This selective impairment occurred when the delays were presented sequentially (from 0.5 s to 10 min) and also when delays were presented in a mixed order (1 s, 1 min, and 10 min). The data indicate that the H+ lesion produces a selective impairment in long-term memory, in the absence of a detectable deficit in short-term memory or perception. Accordingly, the findings confirm the long-standing idea, based primarily on studies of humans, that short-term memory is independent of medial temporal lobe function. The findings thereby establish an important parallel between memory impairment in monkeys and humans and provide additional support for the validity of the animal model of human amnesia in the monkey.
Expansion of CREB's DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB.
Authors: Adya N , Zhao L J , Huang W , Boros I , Giam C Z
Published: May 2006

The transactivator protein of human T-lymphotropic virus type I (HTLV-I), Tax, forms multiprotein complexes with the ubiquitous transcription factor CREB and the CREB/ATF-1 heterodimer. The interaction between Tax and CREB is highly specific and results in increased binding of the Tax/CREB complexes to the HTLV-I 21-bp repeats. Despite the extensive sequence similarities between CREB and ATF-1, Tax interacts with ATF-1 only marginally. Compared with CREB, Tax/CREB exhibits greatly increased DNA recognition specificity and preferentially assembles on a consensus binding site, GGGGG(T/A)TGACG(T/C)(A/C)TA(T/C)C-CCCC, homologous to the HTLV-I 21-bp repeats. Here we report that Tax affects CREB binding to the Tax-inducible DNA elements by interacting with the basic-leucine zipper (bZip) domain of CREB. We show by domain switching that the basic region in CREB bZip can confer on c-Jun and ATF-1 leucine zippers the ability to interact with Tax in vitro. Mutational analyses further demonstrate that the amino acid residues of CREB critical for Tax/CREB interaction are Ala-Ala-Arg at positions 282-284 (AAR284), immediately upstream of the highly conserved DNA-binding domain (R/K)XX(R/K) N(R/K)XAAXX(S/C)RX(R/K)(K/R) characteristic of all bZip proteins. Specific amino acid substitutions in AAR284 of CREB weakened or abolished Tax/CREB interaction, whereas reciprocal changes in ATF-1 allowed it to interact with Tax. These results support a model in which the specific interaction between Tax and the AAR284 residues near the DNA-binding domain of CREB results in a multiprotein complex with altered DNA recognition property. This protein complex assembles selectively on the viral Tax-responsive 21-bp repeats to augment transcription.
Macromolecular permeability across the blood-nerve and blood-brain barriers.
Authors: Poduslo J F , Curran G L , Berg C T
Published: May 2006

The permeability of insulin (Ins), nerve growth factor (NGF), albumin (Alb), transferrin (Trf), and IgG across the blood-nerve barrier (BNB) and blood-brain barrier (BBB) in normal adult rats was quantified by measuring the (permeability coefficient x surface area) product (PS) with the i.v. bolus-injection technique in the cannulated brachial vein and artery using radioiodinated proteins. The PS values of the BNB for IgG and Alb were low: 0.079 +/- 0.029 x 10(-6) and 0.101 +/- 0.088 x 10(-6) ml.g-1.s-1, (mean +/- SD, respectively). The PS values for NGF and Trf were 16.1-fold and 25.5-fold higher than for Alb. The PS for Ins across the BNB was 33.190 +/- 2.053 x 10(-6) ml.g-1.s-1--a remarkable 329-fold increase compared with Alb. The PS values of the BBB for IgG and Alb in different brain regions were all low, from 0.028 +/- 0.017 to 0.151 +/- 0.035 x 10(-6) ml.g-1.s-1 (mean +/- SD). NGF and Trf had comparable PS values from 13- to 32-fold higher than for Alb, except for the brain stem, where the PS for Trf was 66-fold higher than for Alb. The mean PS for Ins across the BBB ranged from 15.78 +/- 5.45 x 10(-6) ml.g-1.s-1 for the cortex to 22.62 +/- 7.50 x 10(-6) ml.g-1.s-1 for the brain stem--again a remarkable 105- to 390-fold increase relative to Alb. Because reliable PS measurements were obtained for all proteins tested, the BBB and BNB cannot be considered impermeable to proteins--a concept that has plagued brain- and nerve-barrier research. The low PS values for IgG and Alb indicate low rates of transfer; however, Alb, in particular, is the major protein of endoneurial and ventricular fluid, which suggests that these PS values may be significant. Ins had the highest PS values, which likely reflect the mechanism of transport across the barriers--that is, receptor-mediated transport. Because NGF and Trf had PS values 13- to 66-fold higher than for Alb, whether this reflects receptor-mediated uptake, adsorptive-mediated transcytosis, or some other mechanism is unclear. That the PS values for NGF and Trf differ from Alb and IgG clearly suggests, however, a different uptake mechanism. Finally, the remarkably high PS values for Ins across the BBB and BNB identify this protein and its putative receptor on capillary endothelial cells as a potential target for drug delivery into the central and peripheral nervous systems.
The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2.
Authors: Martens G J , Braks J A , Eib D W , Zhou Y , Lindberg I
Published: May 2006

The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells. Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2. Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors). Our findings indicate that 7B2 is a physiological inhibitor of PC2 and may provide alternative avenues for the manipulation of peptide hormone levels.
MecB of Bacillus subtilis, a member of the ClpC ATPase family, is a pleiotropic regulator controlling competence gene expression and growth at high temperature.
Authors: Msadek T , Kunst F , Rapoport G
Published: May 2006

The Bacillus subtilis DegS-DegU histidine kinase-response regulator pair controls the expression of genes encoding degradative enzymes such as levansucrase (sacB) and of genes involved in genetic competence. The mecA and mecB mutations were previously isolated as allowing competence gene expression in complex media. We have shown that the mec mutations also lead to overexpression of sacB, bypassing the DegS-DegU requirement. This expression was shown to be entirely dependent upon ComK, a positive regulator of competence gene expression. The mecB gene was cloned and its nucleotide sequence was determined. The predicted MecB protein show very high similarity over its entire length with members of the ClpC family of ATPases (60% identity). MecB is essential for growth of B. subtilis at high temperature. MecB also acts as a negative regulator of ComK synthesis, thus preventing late competence gene expression. We suggest that under these conditions MecB may interact with MecA to sequester or otherwise inactivate ComK. In response to an unknown signal, active ComK would accumulate through a positive feedback loop, leading to expression of competence genes allowing DNA uptake.
Specific human granulocyte-macrophage colony-stimulating factor antagonists.
Authors: Hercus T R , Bagley C J , Cambareri B , Dottore M , Woodcock J M , Vadas M A , Shannon M F , Lopez A F
Published: May 2006

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function. We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding. We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activities. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor alpha chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor alpha-chain-common beta-chain complex. From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity. Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor alpha-mediated stimulation of neutrophil superoxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.
Resistance-associated point mutations in insecticide-insensitive acetylcholinesterase.
Authors: Mutero A , Pralavorio M , Bride J M , Fournier D
Published: May 2006

Extensive utilization of pesticides against insects provides us with a good model for studying the adaptation of a eukaryotic genome to a strong selective pressure. One mechanism of resistance is the alteration of acetylcholinesterase (EC 3.1.1.7), the molecular target for organophosphates and carbamates. Here, we report the sequence analysis of the Ace gene in several resistant field strains of Drosophila melanogaster. This analysis resulted in the identification of five point mutations associated with reduced sensitivities to insecticides. In some cases, several of these mutations were found to be combined in the same protein, leading to different resistance patterns. Our results suggest that recombination between resistant alleles preexisting in natural populations is a mechanism by which insects rapidly adapt to new selective pressures.
A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function.
Authors: Fabian J R , Vojtek A B , Cooper J A , Morrison D K
Published: May 2006

Ras and Raf-1 are key proteins involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Genetic and biochemical studies demonstrate that Raf-1 functions downstream of Ras in many signaling pathways. Although Raf-1 directly associates with GTP-bound Ras, an effect of this interaction on Raf-1 activity in vivo has not been established. To examine the biological consequence of the Ras/Raf-1 interaction in vivo, we set out to identify key residues of Raf-1 required for Ras binding. In this report, we show that a single amino acid mutation in Raf-1 (Arg89 to Leu) disrupted the interaction with Ras in vitro and in the yeast two-hybrid system. This mutation prevented Ras-mediated but not tyrosine kinase-mediated enzymatic activation of Raf-1 in the baculovirus/Sf9 expression system. Furthermore, kinase-defective Raf-1 proteins containing the Arg89-->Leu mutation were no longer dominant-inhibitory or capable of blocking Ras-mediated signal transduction in Xenopus laevis oocytes. These results demonstrate that the association of Raf-1 and Ras modulates both the kinase activity and the biological function of Raf-1 and identify Arg89 as a critical residue involved in this interaction. In addition, the finding that tyrosine kinases can stimulate the enzymatic activity of Raf-1 proteins containing a mutation at the Ras-interaction site suggests that Raf-1 can be activated by Ras-independent pathways.
RNase inhibition of human immunodeficiency virus infection of H9 cells.
Authors: Youle R J , Wu Y N , Mikulski S M , Shogen K , Hamilton R S , Newton D , D'Alessio G , Gravell M
Published: May 2006

Onconase and bovine seminal RNase, two members of the RNase A superfamily, inhibit human immunodeficiency virus type 1 replication in H9 leukemia cells 90-99.9% over a 4-day incubation at concentrations not toxic to uninfected H9 cells. Two other members of the same protein family, bovine pancreatic RNase A and human eosinophil-derived neurotoxin, have no detectable antiviral activity, demonstrating a strikingly selective antiviral activity among homologous ribonucleases. The antiviral RNases do not appear to affect viral particles directly but inhibit replication in host cell cultures. Onconase, already in clinical trials for cancer therapy, and bovine seminal RNase have potential as antiviral therapeutics.
Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization.
Authors: Boggs B A , Chinault A C
Published: May 2006

We have used fluorescence in situ hybridization on interphase nuclei of normal female cells to compare the replication timing patterns of genes on the human X chromosome that are known to escape X inactivation with those that are inactivated. By this procedure it was possible not only to determine the relative time of replication of the earlier-replicating allele for different loci but also to estimate the degree of asynchrony of replication of the two alleles for each individual locus. Loci such as HPRT and FRAXA, which are normally inactivated, displayed a high degree of replication asynchrony, whereas loci that are not inactivated (ZFX and RPS4X) were found to replicate very synchronously. Interestingly, examination of XIST, which is expressed only from the inactive X chromosome, by this procedure revealed that it also replicated asynchronously, with the expressed copy apparently replicating first. Therefore, by examining different loci from the X chromosome it was determined that there is a strict correlation between the expression and relative time of replication of individual genes.
Emergence of a replicating species from an in vitro RNA evolution reaction.
Authors: Breaker R R , Joyce G F
Published: May 2006

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.
A mitochondrial DNA mutation at nucleotide pair 14459 of the NADH dehydrogenase subunit 6 gene associated with maternally inherited Leber hereditary optic neuropathy and dystonia.
Authors: Jun A S , Brown M D , Wallace D C
Published: May 2006

A five-generation Hispanic family expressing maternally transmitted Leber hereditary optic neuropathy and/or early-onset dystonia associated with bilateral basal ganglia lesions was studied. Buffy coat mitochondrial DNA (mtDNA) from a severely affected child was amplified by the polymerase chain reaction and greater than 90% sequenced. The mtDNA proved to be a Native American haplogroup D genotype and differed from the standard "Cambridge" sequence at 40 nucleotide positions. One of these variants, a G-to-A transition at nucleotide pair (np) 14459, changed a moderately conserved alanine to a valine at NADH dehydrogenase subunit 6 (ND6) residue 72. The np 14459 variant was not found in any of 38 Native American haplogroup D mtDNAs, nor was it detected in 108 Asian, 103 Caucasian, or 99 African mtDNAs. Six maternal relatives in three generations were tested and were found to harbor the mutation, with one female affected with Leber hereditary optic neuropathy being heteroplasmic. Thus, the np 14459 G-to-A missense mutation is specific to this family, alters a moderately conserved amino acid in a complex I gene, is a unique mtDNA variant in Native American haplogroup D, and is heteroplasmic, suggesting that it is the disease-causing mutation.
Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3' enhancer.
Authors: Firth J D , Ebert B L , Pugh C W , Ratcliffe P J
Published: May 2006

Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet the transcriptional enhancer lying 3' to the Epo gene shows activity inducible by hypoxia after transfection into a wide variety of cultured cells. The implication of this finding is that many cells which do not produce Epo contain a similar, if not identical, oxygen-regulated control system, suggesting that the same system is involved in the regulation of other genes. We report that the human phosphoglycerate kinase 1 and mouse lactate dehydrogenase A genes are induced by hypoxia with characteristics which resemble induction of the Epo gene. In each case expression is induced by cobalt, but not by cyanide, and hypoxic induction is blocked by the protein-synthesis inhibitor cycloheximide. We show that the relevant cis-acting control sequences are located in the 5' flanking regions of the two genes, and we define an 18-bp element in the 5' flanking sequence of the phosphoglycerate kinase 1 gene which is both necessary and sufficient for the hypoxic response, and which has sequence and protein-binding similarities to the hypoxia-inducible factor 1 binding site within the Epo 3' enhancer.
Inference of human evolution through cladistic analysis of nuclear DNA restriction polymorphisms.
Authors: Mountain J L , Cavalli-Sforza L L
Published: May 2006

Testing of nuclear DNA polymorphisms in human populations has been extended to closely related primates. For many polymorphisms, one allele is shared by two or more species: such shared alleles are likely to be ancestral and provide insight not only into the relationships among the primates but also into the evolutionary history of modern humans. Humans from among eight worldwide populations share an allele with chimpanzees for 62 out of 79 polymorphisms examined. Frequencies of these ancestral alleles strengthen the conclusion that the earliest major separation of modern humans was between Africans and non-Africans. The average time since mutation of the ancestral alleles producing the current set of polymorphisms is estimated to be 700,000 years. While differences among ancestral allele frequencies in human populations suggest that natural selection may have played a role in the evolution of a subset of these polymorphisms, simulations indicate that a European bias in the ascertainment of polymorphisms may be at least partially responsible for observed differences. Simulations also suggest that observed heterozygosity levels in African populations, for classical polymorphisms and restriction fragment length polymorphisms, are artificially low due to the same bias. Observed patterns of mean heterozygosity and mean ancestral allele frequency provide support for the hypothesis that Europeans and northeast Asians are closely related. This work suggests that polymorphisms should be selected by testing a random sample of extant humans.
Phospholipase C-gamma 1 can induce DNA synthesis by a mechanism independent of its lipase activity.
Authors: Smith M R , Liu Y L , Matthews N T , Rhee S G , Sung W K , Kung H F
Published: May 2006

Inositol phospholipid-specific phospholipase C (PLC) is involved in several signaling pathways leading to cellular growth and differentiation. Our previous studies reported the induction of DNA synthesis in quiescent NIH 3T3 cells after microinjection of PLC and the inhibition of serum- or Ras-stimulated DNA synthesis by a mixture of monoclonal antibodies to PLC-gamma 1. In the course of our investigation of anti-PLC-gamma 1 monoclonal antibodies, we found that each antibody exerts different inhibitory effects on the phosphatidylinositol-hydrolyzing activity of PLC-gamma 1 and that the inhibition of enzymatic activity does not correlate with the inhibition of DNA synthesis observed in the microinjection assay. PLC-gamma 1 with defective enzymatic activity was synthesized by substituting phenylalanine for histidine within the PLC-gamma 1 catalytic domain at amino acids 335 and 380, and mutant enzymes were expressed using a vaccinia expression system. The mutant enzymes were purified and microinjected into quiescent NIH 3T3 cells to evaluate their mitogenic activity. A moderate induction of DNA synthesis occurred after injection of mutant PLC-gamma 1. This mitogenic activity was inhibited by an antibody (alpha E 8-4) that does not significantly inhibit PLC-gamma 1 enzyme activity, which indicates that something else has to be inhibited. Furthermore, the partial induction of DNA synthesis observed with mutant PLC-gamma 1 was increased to levels seen with wild-type PLC-gamma 1 by coinjection of mutant PLC-gamma 1 with two second messengers, diacylglycerol and inositol trisphosphate. These results suggest that the mitogenic activity of PLC-gamma 1 does not exclusively result from the enzymatic activity of the lipase and that another activity inherent to the PLC-gamma 1 molecule can also induce DNA synthesis in quiescent cells.
Paradoxical fate and biological action of peroxynitrite on human platelets.
Authors: Moro M A , Darley-Usmar V M , Goodwin D A , Read N G , Zamora-Pino R , Feelisch M , Radomski M W , Moncada S
Published: May 2006

Peroxynitrite (ONOO-), which is formed from the reaction of nitric oxide (NO) and superoxide (O2-), has been suggested to be responsible for some of the cytotoxic effects of these molecules. When protonated, ONOO- gives rise to hydroxyl (OH.) and nitrogen dioxide (NO2) radicals, which are capable of inducing tissue damage. We have investigated the effects of ONOO- on human platelets in vitro in order to explore the potential of this oxidant to contribute to tissue damage. ONOO- caused aggregation of washed platelets and reversed the inhibition of aggregation induced by S-nitroso-N-acetyl-DL-penicillamine (SNAP), prostacyclin, and indomethacin. However, in platelet-rich plasma, ONOO- not only did not possess proaggregatory properties but acted as an inhibitor of platelet aggregation. This reversal of the aggregatory effect of ONOO- could also be achieved in washed platelets by adding low concentrations of plasma, human serum albumin, or glutathione and was inhibited by hemoglobin. An analysis of the reaction products of ONOO- and glutathione revealed the presence of both NO and S-nitrosoglutathione in quantities sufficient to account for the antiaggregatory effects observed. Thus the fate and therefore the actions of ONOO- in biological systems are critically dependent on the biological environment in which this oxidant is present.
T-cell tolerance toward a transgenic beta-cell antigen and transcription of endogenous pancreatic genes in thymus.
Authors: Jolicoeur C , Hanahan D , Smith K M
Published: May 2006

Transgenic mice expressing T antigen (Tag) in pancreatic beta cells establish systemic tolerance toward this self-protein. The self-tolerance in two families of rat insulin promoter (RIP)-Tag mice, expressing different levels of Tag protein, has been characterized. These mice have impaired antibody responses to Tag, show diminished Tag-specific T-cell proliferation, and evidence an inability to generate Tag-specific cytotoxic T cells. The existence of systemic tolerance toward a beta-cell-specific protein motivated examination of transgene expression in the thymus. Indeed, low levels of Tag mRNA were detected intrathymically. Remarkably, this expression is a valid property of the insulin gene regulatory region, since insulin RNA was also expressed in the thymus of nontransgenic mice. RNA for other pancreatic genes was also detected in the thymus, thus raising the possibility that many tissue-specific genes could be expressed intrathymically during immunological development and induction of self-tolerance. These results raise important questions for future research into the role of the thymus in tolerance induction toward so-called tissue-specific antigens.
Rates and patterns of chloroplast DNA evolution.
Authors: Clegg M T , Gaut B S , Learn G H , Morton B R
Published: May 2006

The chloroplast genome (cpDNA) of plants has been a focus of research in plant molecular evolution and systematics. Several features of this genome have facilitated molecular evolutionary analyses. First, the genome is small and constitutes an abundant component of cellular DNA. Second, the chloroplast genome has been extensively characterized at the molecular level providing the basic information to support comparative evolutionary research. And third, rates of nucleotide substitution are relatively slow and therefore provide the appropriate window of resolution to study plant phylogeny at deep levels of evolution. Despite a conservative rate of evolution and a relatively stable gene content, comparative molecular analyses reveal complex patterns of mutational changes. Non-coding regions of cpDNA diverge through insertion/deletion changes that are sometimes site dependent. Coding genes exhibit different patterns of codon bias that appear to violate the equilibrium assumptions of some evolutionary models. Rates of molecular change often vary among plant families and orders in a manner that violates the assumption of a simple molecular clock. Finally, protein-coding genes exhibit patterns of amino acid change that appear to depend on protein structure, and these patterns may reveal subtle aspects of structure/function relationships. Only comparative studies of molecular sequences have the resolution to reveal this underlying complexity. A complete description of the complexity of molecular change is essential to a full understanding of the mechanisms of evolutionary change and in the formulation of realistic models of mutational processes.
Human growth hormone (hGH) secretion in milk of goats after direct transfer of the hGH gene into the mammary gland by using replication-defective retrovirus vectors.
Authors: Archer J S , Kennan W S , Gould M N , Bremel R D
Published: May 2006

Mammary-specific promoters have been used in transgenic animals to limit transgene expression to the mammary gland. Gene therapy techniques to target just one organ for introduction of a foreign gene have also been demonstrated. We have directly infused replication-defective retroviruses encoding hGH into the mammary gland of goats via the teat canal during a period of hormone-induced mammogenesis. This resulted in the secretion of hGH into the milk when lactation commenced on day 14 of the regime. Levels of hGH in the milk were highest on the first day of lactation, averaging approximately 60 ng/ml, and declined to a plateau of 12 ng/ml from day 9 to day 15 of lactation. Thus we report targeting of replication-defective retroviruses to the mammary secretory epithelial cells to produce foreign proteins in the milk of ruminants.
Receptor to nucleus signaling by prolactin and interleukin 2 via activation of latent DNA-binding factors.
Authors: Gilmour K C , Reich N C
Published: May 2006

The mechanism of action of prolactin (PRL), a lactogenic and immunoregulatory hormone, has remained undetermined despite its critical role in development. This study identifies a DNA-binding factor induced by PRL that appears to mediate a signal from the cell surface receptor to specific gene expression in the nucleus. PRL stimulates the proliferation of Nb2 T-lymphoma cells and activates transcription of the interferon-regulatory factor 1 (IRF-1) gene. Within minutes of PRL stimulation, a PRL-induced factor (PRLIF) is activated and binds to a target site in the promoter of the IRF-1 gene. The PRLIF-binding site contains an inverted GAAA repeat that is also functional in the hormone-responsive beta-casein gene. The PRL-receptor complex signals tyrosine phosphorylation of JAK2, a nonreceptor tyrosine kinase, which may lead to activation of PRLIF. T-cell proliferation and transcriptional activation of the IRF-1 gene is also induced by the cytokine interleukin 2 (IL-2). This report demonstrates the rapid activation of an IL-2 nuclear-activated factor that recognizes the same GAAA inverted repeat in the IRF-1 promoter. PRLIF and IL-2 nuclear-activated factor are newly identified factors that appear to serve fundamental roles in the signal transduction pathways of PRL and IL-2, respectively, leading to the transcriptional regulation of responsive genes.
A circularly permuted recombinant interleukin 4 toxin with increased activity.
Authors: Kreitman R J , Puri R K , Pastan I
Published: May 2006

Fusion of ligands such as growth factors to other proteins often dramatically reduces the affinity of the ligand for its receptor. With recombinant DNA techniques, the attachment point between the two proteins has until now been restricted to either the amino or the carboxyl terminus of the ligand. However, binding may be greatly compromised if both ends are close to the site at which the ligand binds to its receptor. To construct a single-chain growth factor fusion protein with the connection at a new site on the growth factor, we constructed a DNA fragment encoding circularly permuted interleukin 4 (IL4), termed IL4(38-37). This was accomplished by placing a start codon before position 38, connecting codons 1 and 129 with a sequence encoding a peptide linker, and placing a stop codon after codon 37 of IL4. IL4(38-37) was fused via its new carboxyl terminus, Lys37, to a truncated form of Pseudomonas exotoxin. The purified circularly permuted IL4-toxin bound to the IL4 receptor with 10-fold higher affinity than an IL4-toxin in which the toxin was fused to the carboxyl terminus of IL4. Circular permuteins of growth factors can improve the effectiveness of recombinant fusion proteins, because the junction can be moved to a site on the growth factor which allows it to bind with higher affinity.
DNA-dependent protein kinase (Ku protein-p350 complex) assembles on double-stranded DNA.
Authors: Suwa A , Hirakata M , Takeda Y , Jesch S A , Mimori T , Hardin J A
Published: May 2006

The Ku protein is an autoantigen that consists of 70- and 80-kDa polypeptides. It associates with double-stranded DNA at free ends. In the present study, we examined the ability of anti-Ku antibodies to immunoprecipitate various structures from extracts of HeLa cells prepared at different salt concentrations. Under physiological conditions, these antibodies identified a complex containing the Ku protein and the 350-kDa component (p350) of DNA-dependent protein kinase (DNA-PK), which appeared to be closely associated on the DNA strand. In reconstitution experiments with cell extracts and biochemically purified components, the Ku protein-p350 complex formed only in the presence of double-stranded DNA. The reconstituted complex was catalytically active. Together with previous studies, these results indicate that the Ku protein interacts with DNA to create a binding site for p350 as the DNA-PK holoenzyme assembles.
Early discordant binocular vision disrupts signal transfer in the lateral geniculate nucleus.
Authors: Chino Y M , Cheng H , Smith E L , Garraghty P E , Roe A W , Sur M
Published: May 2006

The mammalian lateral geniculate nucleus (LGN) is known to regulate signal transfer from the retina to the brain neocortex in a highly complex manner. Besides inputs from the brainstem, extraretinal inputs via corticogeniculate projections and local inhibitory neurons modulate signal transfer in the LGN. However, very little is known about whether the postnatal development of LGN signal-transfer mechanisms is influenced by early discordant binocular vision. By intraunit comparisons of responses between individual X-LGN cells and their direct retinal inputs, the efficiency of signal transfer was found permanently reduced due to an early interocular misalignment (strabismus). The contrast sensitivity and spatial resolution of cat LGN cells were significantly lower relative to their retinal inputs, and there was substantial decrease in signal-transfer speed. The observed physiological deficits were associated with immature X-retinogeniculate axon arbors. Thus, contrary to previous ideas, conflicting binocular inputs can produce neural deficits in subcortical visual structures.
Two highly homologous members of the ClC chloride channel family in both rat and human kidney.
Authors: Kieferle S , Fong P , Bens M , Vandewalle A , Jentsch T J
Published: May 2006

We have cloned two closely related putative Cl- channels from both rat kidney (designated rClC-K1 and rClC-K2) and human kidney (hClC-Ka and hClC-Kb) by sequence homology to the ClC family of voltage-gated Cl- channels. While rClC-K1 is nearly identical to ClC-K1, a channel recently isolated by a similar strategy, rClC-K2 is 80% identical to rClC-K1 and is encoded by a different gene. hClC-Ka and hClC-Kb show approximately 90% identity, while being approximately 80% identical to the rat proteins. All ClC-K gene products are expressed predominantly in the kidney. While rClC-K1 is expressed strongly in the cortical thick ascending limb and the distal convoluted tubule, with minor expression in the S3 segment of the proximal tubule and the cortical collecting tubule, rClC-K2 is expressed in all segments of the nephron examined, including the glomerulus. Since they are related more closely to each other than to the rat proteins, hClC-Ka and hClC-Kb cannot be regarded as strict homologs of rClC-K1 or rClC-K2. After injection of ClC-K cRNAs into oocytes, corresponding proteins were made and glycosylated, though no additional Cl- currents were detectable. Glycosylation occurs between domains D8 and D9, leading to a revision of the transmembrane topology model for ClC channels.
A kinase-negative epidermal growth factor receptor that retains the capacity to stimulate DNA synthesis.
Authors: Coker K J , Staros J V , Guyer C A
Published: May 2006

The residue proposed to serve as the catalytic base for phosphoryl transfer, Asp-813, of the human epidermal growth factor receptor (EGFR) was mutated to Ala, and the mutant receptor (D813A) was expressed in Chinese hamster ovary (CHO) cells. Partially purified D813A exhibited no detectable kinase activity in the absence or presence of EGF. A low level of EGF-stimulable phosphorylation of D813A was detectable in intact cells, apparently due to the activity of an associated Tyr kinase(s). As previously observed for kinase-inactive Lys-721 mutants, EGF binding to D813A stimulates mitogen-activated protein kinase activity. Surprisingly, and unlike results reported for Lys-721 mutants, D813A is capable of stimulating both 86Rb+ uptake and DNA synthesis in response to EGF. These data suggest not only that Asp-813 is critical to the catalytic activity of the EGFR but also that differences may exist in the signaling properties of kinase-negative Lys-721 and kinase-negative Asp-813 EGFR mutants.
Renal epithelial cells rapidly bind and internalize calcium oxalate monohydrate crystals.
Authors: Lieske J C , Swift H , Martin T , Patterson B , Toback F G
Published: May 2006

Renal tubular fluid is supersaturated with calcium and oxalate ions, which can nucleate to form crystals of calcium oxalate monohydrate (COM), the most abundant constituent of kidney stones. However, the mechanisms by which nascent crystals are retained in the nephron and then grow into kidney stones are unclear. An interaction of COM crystals with the surface of renal epithelial cells could be a critical initiating event in nephrolithiasis. To investigate this possibility we used cultures of monkey kidney epithelial cells (BSC-1 line) as a model system and found that [14C]COM crystals bound to the cell surface within seconds. Scanning electron microscopy revealed that crystals bind first to apical microvilli, which subsequently migrate over the crystalline surface. When visualized by transmission electron microscopy, intracellular crystals were located within vesicles. Cytoskeletal responses to crystal uptake were sought by immunofluorescence microscopy, which revealed concentration of F-actin at sites of crystal contact as well as a generalized reorganization of the intermediate filament network containing cytokeratin 8. Uptake of COM crystals did not adversely affect renal epithelial cell growth, and internalized crystals were apparently distributed to daughter cells during division. Rapid adherence of COM crystals to the apical surface of tubular epithelial cells could promote crystal retention in the kidney. Elucidation of factors that regulate this process may provide insight into the pathogenesis of nephrolithiasis.
Memory consolidation and the medial temporal lobe: a simple network model.
Authors: Alvarez P , Squire L R
Published: May 2006

Some forms of memory have been shown to depend on a system of medial temporal lobe structures that includes the hippocampus and the adjacent cortical areas (entorhinal, perirhinal, and parahippocampal cortex). The role of this system is only temporary, however, as indicated by the fact that, after damage to the medial temporal lobe, recent memories are impaired but very remote memories are intact. Here we review the evidence that the medial temporal lobe memory system is involved in a process of consolidation: memories are initially dependent on this system but gradually become established in other areas of the brain. We then review some of the ideas that have been proposed about the phenomenon of consolidation and suggest a synthesis of these views. Finally, we describe a simple neural network model that captures some key features of consolidation.
Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia.
Authors: Banner L R , Patterson P H
Published: May 2006

The neuropoietic cytokine cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) acts as a trophic factor, enhancing neuronal survival, and as a differentiation factor, altering neuronal gene expression. There is also evidence that its plays a role in the response of adult neural tissue to injury. We have examined this possibility further in rats by analyzing changes in the levels of mRNAs for CDF/LIF and its two receptor subunits in response to peripheral nerve damage in culture and in vivo. Using a quantitative RNase protection assay, we find that CDF/LIF mRNA increases dramatically (176-fold) in adult, but not neonatal, sympathetic ganglia and in adult dorsal root ganglia and sciatic nerve after organ culture for 24 hr. This mRNA is clearly detectable by in situ hybridization only in the nonneuronal cells of these structures. When the sciatic nerve is transected in vivo, CDF/LIF mRNA increases significantly in the regions immediately proximal and distal to the lesion site. The mRNA for the ligand binding subunit of the CDF/LIF receptor complex decreases somewhat upon culture and nerve section. The dramatic rise in CDF/LIF mRNA after nerve injury is further evidence that this cytokine is involved in the response to damage, a function that overlaps with its postulated role in wounding or infection in several nonneural tissues.
Piebald lethal (sl) acts early to disrupt the development of neural crest-derived melanocytes.
Authors: Pavan W J , Tilghman S M
Published: May 2006

Mice homozygous for the piebald lethal (sl) mutation have a predominantly white coat due to the absence of neural crest-derived melanocytes in the hair follicles. To investigate the time in embryonic development when the s1 gene affects the melanocyte lineage, we compared the distribution of melanocyte precursors in wild-type and mutant embryos, using an antibody specific for tyrosinase-related protein 2 (TRP-2). TRP-2 positive cells were first observed adjacent to the anterior cardinal vein in 10.5-day postcoitem wild-type embryos. From 11.5 to 13.5 days postcoitem, there was a nonuniform distribution of TRP-2 positive cells along the anterior-posterior axis, with the highest density of cells in the head and tail regions. Along the dorsal-ventral axis, the cells were restricted to positions lateral, but never dorsal, to the neural tube. In homozygous sl/sl embryos TRP-2 staining was restricted to the non-neural crest-derived melanocytes of the pigmented retinal epithelium and the telencephalon. Few positive cells were seen in areas that will form neural crest-derived melanocytes in the inner ear, skin, hair follicles, leg musculature, or heart. We conclude that the piebald lethal mutation acts prior to the onset of TRP-2 expression to disrupt the development of neural crest-derived melanocytes. The non-uniform distribution of melanoblasts in wild-type mice suggests that piebald acts stochastically to affect melanocyte development.
Hot spots of retinoic acid synthesis in the developing spinal cord.
Authors: McCaffery P , Dräger U C
Published: May 2006

The embryonic spinal cord is known to be rich in retinoic acid, and several indirect lines of evidence point to a dorsoventral concentration difference of this compound. Previous measurements of dorsoventral retinoic acid levels, however, showed only minor differences. By a combination of microdissection and bioassay techniques, we compared retinoic acid levels with retinaldehyde dehydrogenase levels along spinal cords from early embryonic to postnatal mice. Both parameters vary in parallel, indicating that the principal reason for regional retinoic acid differences in the developing spinal cord is different levels of retinoic acid-generating enzyme. Consistent with previous reports, we observed overall quite high synthesis, decreasing with age, and no dorsoventral difference throughout much of the spinal cord length. In two locations, however, ventral synthesis exceeds dorsal synthesis by several orders of magnitude. These hot spots colocalize with the origins of the limb innervations. They are highest during early stages of limb innervation and disappear slowly postnatally. The synthesis hot spots are likely to create local retinoic acid diffusion halos, which may influence the survival of neurons in the limb regions of the spinal cord and which probably promote innervation of the developing limbs.