bluuebunny/crossref_metadata_2025 huggingface dataset:
The type I human T-cell leukemia virus (HTLV-I) Rex trans-activator binds directly to the HTLV-I Rex and the type 1 human immunodeficiency virus Rev RNA response elements.
Authors: Bogerd H P , Huckaby G L , Ahmed Y F , Hanly S M , Greene W C
Published: May 2006

The Rex protein of the type I human T-cell leukemia virus (HTLV-I) is essential for the replication of this pathogenic retrovirus and, surprisingly, can also replace the function of the structurally distinct Rev protein of the type 1 human immunodeficiency virus (HIV-1). Rex action requires a 255-nucleotide viral RNA stem-loop structure termed the Rex RNA response element (RexRE) located in the 3' retroviral long terminal repeat. Rex function leads to the induced cytoplasmic expression of the incompletely spliced family of viral mRNAs that uniquely encode the HTLV-I structural and enzymatic proteins (Gag, Pol, and Env). Our studies now demonstrate that Rex acts by binding directly to the RexRE in a sequence-specific manner. These effects of Rex require the presence of a 10-nucleotide subregion of the RexRE that is essential for Rex function in vivo. Dominant-negative mutants of Rex also bind to the RexRE with high affinity, while a recessive-negative Rex mutant altered within its arginine-rich, positively charged domain fails to engage the RexRE. Analogously, both the wild-type and dominant-negative Rex proteins specifically bind to the structurally distinct HIV-1 Rev response element, a finding that likely underlies the respective stimulatory and inhibitory effects of these HTLV-I proteins in the heterologous HIV-1 system. However, consistent with their lack of amino acid homology, the binding sites for Rex and Rev within the HIV-1 Rev response element are distinct.
Metabolic heterogeneity in human calf muscle during maximal exercise.
Authors: Vandenborne K , McCully K , Kakihira H , Prammer M , Bolinger L , Detre J A , De Meirlier K , Walter G , Chance B , Leigh J S
Published: May 2006

Human skeletal muscle is composed of various muscle fiber types. We hypothesized that differences in metabolism between fiber types could be detected noninvasively with 31P nuclear magnetic resonance spectroscopy during maximal exercise. This assumes that during maximal exercise all fiber types are recruited and all vary in the amount of acidosis. The calf muscles of seven subjects were studied. Two different coils were applied: an 11-cm-diameter surface coil and a five-segment meander coil. The meander coil was used to localize the 31P signal to either the medial or the lateral gastrocnemius. Maximal exercise, consisting of rapid plantar flexions, resulted in an 83.7% +/- 7.8% decrease of the phosphocreatine pool and an 8-fold increase of the inorganic phosphate (Pi) pool. At rest the Pi pool was observed as a single resonance (pH 7.0). Toward the end of the first minute of exercise, three subjects showed three distinct Pi peaks. During the second minute of exercise the pH values stabilized at 7.12 +/- 0.12, 6.63 +/- 0.15, and 6.27 +/- 0.23. The same pattern was seen when the signal was collected from the medial or lateral gastrocnemius. In four subjects only two distinct Pi peaks were observed. The Pi peaks had differing relative areas in different subjects, but they were reproducible in each individual. This method allowed us to study the appearance and disappearance of the different Pi peaks, together with the changes in pH. Because multiple Pi peaks were seen in single muscles they most likely identify different muscle fiber types.
Glucose induces cAMP-independent growth-related changes in stationary-phase cells of Saccharomyces cerevisiae.
Authors: Granot D , Snyder M
Published: May 2006

Nutrients play a critical role in the decision to initiate a new cell cycle. Addition of nutrients to arrested cells such as stationary-phase cells and spores induces them to begin growth. We have analyzed the nutrients required to induce early cellular events in yeast. When stationary-phase cells or spores are incubated in the presence of only glucose, morphological and physiological changes characteristic of mitotically growing cells are induced and, in the absence of additional nutrients to support growth, the cells rapidly lose viability. Preincubation of stationary-phase cells in the presence of glucose decreases the time required to reach bud emergence upon the subsequent addition of rich medium. These processes are specifically induced by D-glucose and not by other components such as nitrogen source or L-glucose. The glucose-induced events are independent of the adenylate cyclase pathway, since strains with a temperature-sensitive mutation in either the adenylate cyclase gene (CDC35) or its regulator (CDC25) undergo glucose-induced cellular changes when incubated at the restrictive temperature. We suggest that glucose triggers events in the induction of a new mitotic cell cycle and that these events are either prior to the adenylate cyclase pathway or are in an alternative pathway.
DNA strand-specific mutations induced by (+/-)-3 alpha,4 beta-dihydroxy- 1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene in the dihydrofolate reductase gene.
Authors: Carothers A M , Mucha J , Grunberger D
Published: May 2006

We previously showed that the preferred mutation induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy- 1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPHDE) in the dihydrofolate reductase gene in Chinese hamster ovary cells was a purine to thymine transversion on the nontranscribed strand at the sequence 5'-RRR-3' (R is a purine and the mutated base is underlined). To determine whether the observed mutational strand specificity was due to bias in the phenotypic selection, we designed a nonsense-codon reversion assay in which a triple purine target was present on both strands and all R----T transversion mutations yielded amino acid substitutions that were compatible with dihydrofolate reductase enzyme activity. From the size of the targets, a 2:1 ratio of mutations at the purines on the nontranscribed strand was expected if the DNA strands were mutationally equivalent. We isolated a total of 66 BcPHDE-induced revertants of two mutants that carry point mutations at either the 5' or the 3' end of the gene. All reversions at the 5' end arose by substitution on the nontranscribed strand; those at the 3' end showed a strand bias that favored this strand by 7:1. For both mutants, R----T transversions accounted for 88% of all the induced base changes. Thus, in this system, mutational strand bias is independent of the selection for phenotype. The results are consistent with the model of preferential repair of the transcribed strand as proposed by others. The involvement of RNA polymerase in the selective repair recruitment is discussed.
Progression from meiosis I to meiosis II in Xenopus oocytes requires de novo translation of the mosxe protooncogene.
Authors: Kanki J P , Donoghue D J
Published: May 2006

The meiotic maturation of Xenopus oocytes exhibits an early requirement for expression of the mosxe protooncogene. The mosxe protein has also been shown to be a component of cytostatic factor (CSF), which is responsible for arrest at metaphase of meiosis II. In this study, we have assayed the appearance of CSF activity in oocytes induced to mature either by progesterone treatment or by overexpression of mosxe. Progesterone-stimulated oocytes did not exhibit CSF activity until 30-60 min after germinal vesicle breakdown (GVBD). Both the appearance of CSF activity and the progression from meiosis I to meiosis II were inhibited by microinjection of mosxe antisense oligonucleotides just prior to GVBD. These results demonstrate a translational requirement for mosxe, which is temporally distinct from the requirement for mosxe expression at the onset of meiotic maturation. In contrast to progesterone-treated oocytes, oocytes that were induced to mature by overexpression of mosxe exhibited CSF activity at least 3 hr prior to GVBD. Despite the early appearance of CSF, these oocytes were not arrested at meiosis I. These results indicate that, although CSF activity is capable of stabilizing maturation-promoting factor (MPF) at meiosis II and in cleaving embryos, it is incapable of stabilizing MPF prior to or at meiosis I. These studies show that the complex regulation of the cell cycle during meiosis differs significantly from the regulation of the cell cycle during mitosis.
Lactate rise detected by 1H NMR in human visual cortex during physiologic stimulation.
Authors: Prichard J , Rothman D , Novotny E , Petroff O , Kuwabara T , Avison M , Howseman A , Hanstock C , Shulman R
Published: May 2006

Brain lactate concentration is usually assumed to be stable except when pathologic conditions cause a mismatch between glycolysis and respiration. Using newly developed 1H NMR spectroscopic techniques that allow measurement of lactate in vivo, we detected lactate elevations of 0.3-0.9 mM in human visual cortex during physiologic photic stimulation. The maximum rise appeared in the first few minutes; thereafter lactate concentration declined while stimulation continued. The results are consistent with a transient excess of glycolysis over respiration in the visual cortex, occurring as a normal response to stimulation in the physiologic range.
Difference between the tau protein of Alzheimer paired helical filament core and normal tau revealed by epitope analysis of monoclonal antibodies 423 and 7.51.
Authors: Novak M , Jakes R , Edwards P C , Milstein C , Wischik C M
Published: May 2006

The microtubule-associated protein tau that is incorporated into paired helical filaments (PHFs) undergoes some form of aberrant posttranslational processing in Alzheimer disease. Difficulties in deciding which changes are critical for PHF formation stem in part from the lack of immunochemical markers specific for PHF tau. The only monoclonal antibody (mAb) that is known to react with PHF tau but not with the predominant normal adult tau species is mAb 423. Another mAb (7.51, described in this paper) recognizes a segment of tau that is included in the minimal recognition unit required by mAb 423. Unlike 423, which is PHF tau-specific, mAb 7.51 recognizes all PHF core-derived tau as well as native soluble tau and recombinant tau expressed in bacteria and so serves as a generic tau marker. Both epitopes are in the 12-kDa fragment released from the Pronase-resistant core of the PHF (which encompasses the tandem repeat region). The mAb 7.51 epitope requires segments located in the last two repeats, which are common to all tau isoforms. The mAb 423 epitope requires sequences located near both the N and the C terminus of the 12-kDa fragment common to three- and four-repeat tau isoforms. Fragments denatured by concentrated formic acid and SDS regain 423 reactivity when denaturing agents are removed. Since the primary amino acid sequences of PHF tau and normal tau are identical in the repeat region, we conclude that 423 reactivity also requires a modification(s) occurring within an approximately 90-residue segment that are not present in tau proteins so far described in the human brain.
Tyrosine kinase activity coupled to the high-affinity nerve growth factor-receptor complex.
Authors: Meakin S O , Shooter E M
Published: May 2006

Antibodies directed against nerve growth factor (NGF) immunoprecipitate a tyrosine kinase activity from NGF-treated PC12 rat pheochromocytoma cells. Based on several criteria, this activity has been correlated with the high-affinity and not the low-affinity NGF-receptor complex. The in vitro kinase activity and the tyrosine phosphorylation of the high-affinity complex can be blocked by an agent that inhibits NGF (and not epidermal growth factor)-induced tyrosine phosphorylation in PC12 cells, as well as NGF-induced neuronal differentiation of PC12 cells. These observations suggest that the high-affinity NGF-receptor complex is a substrate of tyrosine kinase activity. Phosphorylation reactions by the complex, performed in the absence of added substrate, label a single phosphopeptide of 130-135 kDa. This observation suggests that this phosphopeptide may represent the phosphorylation of the receptor kinase or the phosphorylation of a coimmunoprecipitating substrate, and possible signal-transducing molecule, of the high-affinity NGF-receptor complex.
Proteinase trapping: screening for viral proteinase mutants by alpha complementation.
Authors: Liebig H D , Skern T , Luderer M , Sommergruber W , Blaas D , Kuechler E
Published: May 2006

Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus. This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18. When a fusion protein of the alpha fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha complementation was not affected, as the 2A proteinase cleaved itself off the alpha fragment. However, fusion of an inactive 2A prevented alpha complementation, as the 2A polypeptide remained fused to the alpha fragment. After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in alpha complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes. Intermolecular cleavage was then examined by expressing an alpha fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector. alpha complementation indicated intermolecular processing of the 2A cleavage site on the alpha fragment-inactive proteinase fusion protein. This versatile system thus allows the high-density screening of both active and inactive proteinase mutants, cleaving either intramolecularly or intermolecularly, and should be applicable to other proteinases of high specificity.
A role for microtubule bundles in the morphogenesis of chicken erythrocytes.
Authors: Winckler B , Solomon F
Published: May 2006

The acquisition of cellular asymmetry is one of the post-mitotic events in development. The cells of the avian erythropoietic lineage acquire a simple invariant asymmetry as they mature. Erythrocytes develop in suspension from spherical through discoid to lentil-shaped (lentiform) cells, with a single rigorously specified microtubule bundle, the marginal band. We show here that developing erythrocytes can express highly asymmetric morphologies, including elongated cell bodies and long processes in response to two stimuli: mechanical manipulation (repeated washing) and exposure to cytochalasin D. These experiments suggest that erythrocytes pass through a developmental stage during which microtubules are able to exert elongating forces on the cell. That stage is one in which these cells normally change shape, from spherical to discoid. The results suggest that microtubules may both guide and drive the formation of the marginal band and the characteristic morphology of these cells.
A cell cycle-responsive transcriptional control element and a negative control element in the gene encoding DNA polymerase alpha in Saccharomyces cerevisiae.
Authors: Gordon C B , Campbell J L
Published: May 2006

Transcription of the POL1 gene of Saccharomyces cerevisiae, which encodes DNA polymerase alpha, the DNA polymerase required for the initiation of DNA replication, has previously been shown to be cell cycle regulated. To understand how the POL1 gene senses cell cycle position, we have investigated the cis-acting elements that respond to the factors that govern cell cycle progression. In this report we demonstrate that a region of 54 nucleotides containing the repeated element ACGCGT, which conforms to an Mlu I restriction endonuclease recognition site, contains all information necessary for transcriptional activation and cell cycle responsiveness. Although oligonucleotides lacking either one or both of the repeated Mlu I sites can function as an upstream activating sequence, the presence of at least one Mlu I site stimulates expression and, moreover, is absolutely essential for cell cycle regulation. A synthetic oligonucleotide corresponding to a 19-base-pair sequence in the POL1 promoter containing one Mlu I site can function as an autonomous cell cycle-responsive upstream element (upstream activation sequence) with temporal regulation indistinguishable from that previously described for the POL1 gene. Thus, the Mlu I site is an essential part of a cis-acting element responsible for the observed periodic activation. This sequence differs from previously defined cell cycle-responsive transcriptional control elements in the yeast HO endonuclease and histone genes. We also present evidence for a negative regulatory element in the 5' flanking region of the Mlu I upstream activation sequence.
Nucleotide sequence analysis of Adh genes estimates the time of geographic isolation of the Bogota population of Drosophila pseudoobscura.
Authors: Schaeffer S W , Miller E L
Published: May 2006

The population of Drosophila pseudoobscura at Bogota, Columbia, is geographically and partially reproductively isolated from populations in the main body of the species in North America. The degree of genetic differentiation and time of divergence between populations at Bogota and Apple Hill, CA, were estimated by comparison of 3388 nucleotides in the alcohol dehydrogenase region (Adh and Adh-Dup genes) of 18 strains. Of the 146 polymorphic nucleotide sites detected, 68 and 31 were unique to the Apple Hill and Bogota samples, respectively, and 53 were shared. On the basis of an observed net divergence per nucleotide site of 0.264% between the two samples, the Bogota and North American populations were estimated to have been separated for at least 155,000 years. This divergence time suggests that D. pseudoobscura extended its range from North America to South America in a period of Pleistocene glaciation, when habitat suitable for the species presumably existed in lowland Central America.
Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation.
Authors: Seger R , Ahn N G , Boulton T G , Yancopoulos G D , Panayotatos N , Radziejewska E , Ericsson L , Bratlien R L , Cobb M H , Krebs E G
Published: May 2006

Microtubule-associated protein 2 kinase (MAP kinase), which exists in several forms, is a protein serine/threonine kinase that participates in a growth factor-activated protein kinase cascade in which it activates a ribosomal protein S6 kinase (pp90rsk) while being regulated itself by a cytoplasmic factor (MAP kinase activator). Experiments with recombinant MAP kinase, ERK2, purified from Escherichia coli in a nonactivated form revealed a self-catalyzed phosphate incorporation into both tyrosine and threonine residues. Another MAP kinase, ERK1, purified from insulin-stimulated cells also autophosphorylated on tyrosine and threonine residues. Autophosphorylation of ERK2 correlated with its autoactivation, although both autophosphorylation and autoactivation were slow compared to that occurring in the presence of MAP kinase activator. Therefore, we propose that autophosphorylation is probably involved in the MAP kinase activation process in vitro, but it may not be sufficient for full activation. The specificity toward tyrosine and threonine residues indicates that the MAP kinases ERK1 and ERK2 are members of a group of kinases with specificity for tyrosine as well as serine and threonine residues.
Loss of both CSF1R (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion.
Authors: Boultwood J , Rack K , Kelly S , Madden J , Sakaguchi A Y , Wang L M , Oscier D G , Buckle V J , Wainscoat J S
Published: May 2006

A high proportion of patients with myelodysplasia show characteristic karyotypic abnormalities in bone marrow cells. The most distinctive of the myelodysplastic syndromes is the 5q- syndrome characterized by refractory anemia, poorly lobulated megakaryocytes, and an interstitial deletion of the long arm of chromosome 5 (5q deletion) as the sole karyotypic abnormality. Recently, several genes encoding hemopoietic growth factors and receptors, comprising the interleukins 3, 4, and 5, macrophage colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, and the receptor for macrophage-colony-stimulating factor [the CSF1R (formerly FMS) gene product], have been localized to the long arm of chromosome 5, and there has been much speculation that deletion of one or more of these genes may be critical to the pathogenesis of the associated myeloid disorders. One candidate gene is CSF1R, which is required for normal proliferation and differentiation of hemopoietic cells of the myeloid lineage. We have carried out a molecular examination of the CSF1R, both on the 5q- chromosome and on the apparently normal homologous chromosome 5, in 10 patients with myelodysplasia and a 5q deletion. We have found, using restriction fragment length polymorphism analysis and gene dosage experiments, that all 10 patients showed deletion of CSF1R; 6 of 10 were hemizygous and 4 of 10 homozygous for CSF1R loss. The homozygous CSF1R loss has been confirmed in 2 patients by an in situ hybridization technique comparing the signal in affected cells to that in control sex-mismatched cells on the same slides. In those patients considered to have homozygous CSF1R loss by DNA experiments the gene was deleted from the 5q chromosome in all cells and from the apparently normal chromosome 5 in a subset of cells. This loss of one CSF1R allele, together with loss in some cells of the remaining allele on the homologous chromosome 5, in patients with myelodysplasia indicates that this is a region of critical gene loss on 5q. The loss of the hemopoietic growth factor receptor gene CSF1R may be important in the pathogenesis of human myeloid leukemia.
Purification and initial characterization of the lymphoid-cell protein-tyrosine kinase p56lck from a baculovirus expression system.
Authors: Ramer S E , Winkler D G , Carrera A , Roberts T M , Walsh C T
Published: May 2006

The lymphocyte-specific protein-tyrosine kinase p56lck has been purified 90-fold to approximately 30% purity in 30% yield from a baculovirus expression system by a two-column purification procedure. At least two forms of p56lck were isolated, differing in the extent of phosphorylation and migrating as 56- and 59-kDa species on SDS/PAGE but as a single 56-kDa band after treatment with potato acid phosphatase. Autophosphorylation of purified p56lck occurred at a rate of 25 fmol/min to a maximum incorporation of approximately 2 mol of phosphate per mol of p56lck with tyrosine-394 (but not tyrosine-505) and other, unidentified tyrosine residue(s) being the major sites of phosphorylation in vitro. Phosphorylation of tyrosine-containing peptides was monitored using an automated HPLC system. Although peptide substrate Km values were in the 1-5 mM range, the Vmax for the 13-amino acid peptide RRLIEDAEYAARG (modified p60src autophosphorylation site) was 120 min-1 (350 min-1 when adjusted for p56lck purity), suggesting that the enzyme purified from recombinant baculovirus-infected Sf9 cells has a high catalytic turnover compared with other tyrosine kinases.
Purification of parathyroid hypertensive factor from plasma of spontaneously hypertensive rats.
Authors: Benishin C G , Lewanczuk R Z , Pang P K
Published: May 2006

Parathyroid hypertensive factor (PHF) is a newly described hypertensive factor that may be related to elevation of blood pressure in 30-40% of North American essential hypertensive patients. PHF is also found in several animal models of hypertension, including spontaneously hypertensive rats, and deoxycorticosterone acetate salt hypertensive rats. Plasma collected from spontaneously hypertensive rats (SHR) was used in the present study for purification of PHF. Plasma was dialyzed at a molecular mass cutoff of 1 kDa, and then ultrafiltered at a molecular mass cutoff of 5 kDa. PHF activity, as determined by bioassay (characteristic delayed hypertensive response in normotensive rat) was retained in the fraction that was greater than 1 kDa and less than 5 kDa. Dialyzed and ultrafiltered SHR plasma was fractionated by molecular-exclusion chromatography, either with Bio-Gel P-6 liquid chromatography, or TSK 2000 SW HPLC. The biological activity was detected in a discrete region corresponding to a molecular mass of 2.5-3 kDa. When the molecular-exclusion fraction was subsequently fractionated by reverse-phase HPLC, biological activity was located in a single discrete peak, which did not occur in plasma from normotensive rats prepared in a similar manner. The biologically active fraction of PHF was inactivated by trypsin; this and its UV spectrum indicate the presence of a peptide structure.
A transduction pathway associated with receptors coupled to the inhibitory guanine nucleotide binding protein Gi that amplifies ATP-mediated arachidonic acid release.
Authors: Felder C C , Williams H L , Axelrod J
Published: May 2006

ATP is copackaged and coreleased with adrenergic, serotonergic, and cholinergic neurotransmitters, suggesting a possible interaction between the signaling pathways for ATP and these coreleased neurotransmitters. Muscarinic m2 and m4, alpha 2-adrenergic, and D2-dopaminergic neurotransmitter receptors, which have in common their ability to inhibit adenylate cyclase through the inhibitory guanine nucleotide binding protein Gi, were transfected and expressed in Chinese hamster ovary (CHO) cells that contain endogenous ATP receptors coupled to the release of arachidonic acid. Normal functional coupling of m2, m4, alpha 2, and D2 receptors was demonstrated by their ability to inhibit forskolin-stimulated cAMP accumulation with dose-response activities consistent with previous reports for these Gi-coupled receptors. Stimulation of m2, m4, alpha 2, and D2 receptors resulted in an augmentation of ATP-stimulated arachidonic acid release. With the exception of the m4 receptor, none of the receptors tested was able to stimulate arachidonic acid release in the absence of ATP. Potentiation of ATP-stimulated arachidonic acid release was independent of changes in cAMP. The augmentation of ATP-stimulated arachidonic acid release and the inhibition of cAMP accumulation were both blocked by pertussis toxin, an inhibitor of Gi, but with different dose-response characteristics. Inhibition of protein kinase C with staurosporine or long-term pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the augmentation response. This demonstrates that Gi-coupled inhibitory receptors can amplify ATP-receptor-stimulated arachidonic acid release through a pertussis-toxin-sensitive G protein, independent of their ability to inhibit adenylate cyclase activity.
Inhibition of promoter activity by methylation: possible involvement of protein mediators.
Authors: Levine A , Cantoni G L , Razin A
Published: May 2006

To study the relationship between DNA methylation and promoter activity we have methylated in vitro the promoters of the mouse metallothionein I gene and the herpes simplex virus thymidine kinase gene. We have transiently transfected these promoters fused to the human growth hormone in their methylated or unmethylated state into mouse L or F9 cells. Promoters methylated by methylase (M.) Hpa II and M.Hha I caused inhibition of reporter gene expression in L cells but not in F9 cells, while methylation of all CpGs by M.Sss I caused inhibition in both cell lines. Repression of promoter activity by M.Hpa II and M.Hha I methylation, but not by M.Sss I methylation, could be alleviated by cotransfection with an excess of untranscribable DNA methylated with M.Sss I. The methylated sites in nuclei isolated from the transfected L cells, but not F9 cells, were found to be protected from Msp I digestion. Taken together these results suggest that a factor present in L cells and missing in F9 cells mediates the methylation-directed inhibition of promoter activity. The ability of methylated DNA to overcome the inhibition seems to reflect competition for the mediator factor. Interestingly, treatment with Zn2+ ions brought about activation of the methylated promoter of the metallothionein gene. Similarly, butyrate could override the repression of the thymidine kinase methylated promoter. These activations were not accompanied by demethylation of the promoter or displacement of the mediator factor.
Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering.
Authors: Jessen T H , Weber R E , Fermi G , Tame J , Braunitzer G
Published: May 2006

Of two closely related species of geese, one, the greylag goose, lives in the Indian plains all year round, while the other, the bar-headed goose, lives at the Tibetan lakes and migrates across the Himalayas to winter in India. Another species, the Andean goose, lives in the High Andes all year round. Possession of a Hb with high oxygen affinity helps to adapt bar-headed and Andean geese to high altitudes. The Hb amino acid sequences of the bar-headed and the greylag geese differ by four substitutions, of which only one is unique among bird sequences: Pro-119 alpha (H2)----Ala. Perutz proposed that the two-carbon gap left by this substitution at the alpha 1 beta 1 contact raises the oxygen affinity, because it relaxes the tension in the deoxy or T structure [Perutz, M. F. (1983) Mol. Biol. Evol. 1, 1-28]. It was later found that the Hb of the Andean goose has a gap in the same position, due to the complementary substitution Leu-55 beta (D6)----Ser. We have tested Perutz's hypothesis by introducing each of these substitutions into human globin synthesized in Escherichia coli. The reconstituted Hbs combine cooperatively with oxygen. Their oxygen affinities exceed that of normal human Hb by an even larger factor than that found between the high-flying geese and the greylag goose. The mutant Hb Met-55 beta (D6)----Ser was crystallized. Its structure is the same as that of HbA, except in the immediate environment of the gap left by the substitution of the serine for the methionine side chain, which evidently causes the increased oxygen affinity of this Hb.
Rapid medium perfusion rate significantly increases the productivity and longevity of human bone marrow cultures.
Authors: Schwartz R M , Palsson B O , Emerson S G
Published: May 2006

Long-term in vitro human hematopoietic (Dexter) cultures are limited both in their longevity (8-12 weeks) and in their cell production over time. Hematopoiesis in vivo continues unabated throughout life, suggesting that failure to obtain continuous hematopoiesis in vitro is probably due to the failure of culture conditions to accurately reproduce the in vivo state. Since bone marrow cells in vivo exist at high density and are rapidly perfused by plasma components, we examined the effects of the culture medium perfusion rate and inoculum density on longevity and productivity of human bone marrow cultures. Culture efficiency and longevity were relatively independent of the variation in inoculum density from 10(6) to 5 x 10(6) cells per ml but were significantly altered by the medium perfusion rate. Increased culture perfusion was superior to traditional Dexter schedules, with 0.5 medium volume exchange per day (3.5 volume per week) being optimal. The cultures under these conditions demonstrated an increasing rate of cell production from weeks 4 to 10, with the cell production rate doubling approximately every 2 weeks. Following weeks 10-12 in culture, the cell production rate of all cultures decayed. Production of nonadherent progenitor cells was also highest in cultures perfused at 3.5 medium volume exchanges per week, where progenitor cell production was stable from weeks 6 to 18. The nonadherent cells collected were predominantly macrophages by week 19, except for the cultures perfused at a rate of 3.5 volumes per week and seeded at 5 x 10(6) cells per ml, in which production of granulocytes remained high through week 19. Establishment of more optimal perfusion conditions supports the continuous stable generation of progenitor cells over 5 months in culture, suggesting that primitive stem cells are surviving and proliferating in these cultures.
Transforming growth factor beta 1, a potent chemoattractant for human neutrophils, bypasses classic signal-transduction pathways.
Authors: Reibman J , Meixler S , Lee T C , Gold L I , Cronstein B N , Haines K A , Kolasinski S L , Weissmann G
Published: May 2006

Transforming growth factor beta 1 (TGF-beta 1), a homodimeric polypeptide (Mr 25,000), derives from inflammatory cells and acts as a chemoattractant for monocytes and fibroblasts. We report here that TGF-beta 1 is also the most potent chemoattractant yet described for human peripheral blood neutrophils. Recombinant TGF-beta 1 elicited dose-dependent directed migration of neutrophils under agarose that was inhibited in the presence of a neutralizing antibody to TGF-beta 1. Maximal chemotaxis was evoked by TGF-beta 1 at femtomolar concentrations, whereas conventional chemoattractants act at nanomolar concentrations: on a molar basis, TGF-beta 1 was 150,000 times more potent than fMet-Leu-Phe. In contrast, TGF-beta 1 provoked neither exocytosis nor the production of superoxide by neutrophils. We further analyzed the mechanism by which TGF-beta 1 elicits chemotaxis (GTPase activity, [Ca2+], and actin polymerization). In contrast to the conventional chemoattractant fMet-Leu-Phe, TGF-beta neither activated classic heterotrimeric guanine nucleotide-binding proteins nor provoked global mobilization of intracellular Ca2+. Chemoattraction by both fMet-Leu-Phe and TGF-beta 1 was inhibited by cycloheximide and actinomycin D. Moreover, chemotaxis in response to TGF-beta 1 was associated with the polymerization of actin. The selectivity and potency of TGF-beta 1 as a chemoattractant suggest that it elicits directed cell migration by means of a pathway that depends not on classic intracellular signals but on protein synthesis.
Mutations in the D strand of the human CD4 V1 domain affect CD4 interactions with the human immunodeficiency virus envelope glycoprotein gp120 and HLA class II antigens similarly.
Authors: Piatier-Tonneau D , Gastinel L N , Moussy G , Bénichou B , Amblard F , Vaigot P , Auffray C
Published: May 2006

CD4, a cell surface glycoprotein expressed primarily by T lymphocytes and monocytes, interacts with HLA class II antigens to regulate the immune response. In AIDS, CD4 is the receptor for the human immunodeficiency virus, which binds to CD4 through envelope glycoprotein gp120. Delineation of the ligand-binding sites of CD4 is necessary for the development of immunomodulators and antiviral agents. Although the gp120 binding site has been characterized in detail, much less is known about the class II binding site, and it is as yet uncertain whether they partially or fully overlap. To investigate CD4 binding sites, a cellular adhesion assay between COS cells transiently transfected with CD4 and B lymphocytes expressing HLA class II antigens has been developed that is strictly dependent on the CD4--class II interaction, quantitative, and highly reproducible. Mutants of CD4 expressing amino acids with distinct physicochemical properties at positions Arg-54, Ala-55, Asp-56, and Ser-57 in V1, the first extracellular immunoglobulin-like domain, have been generated and studied qualitatively and quantitatively for interaction with HLA class II antigens, for membrane expression, for the integrity of CD4 epitopes recognized by a panel of monoclonal antibodies, and for gp120 binding. The results obtained show that the mutations in this tetrapeptide, which forms the core of a synthetic peptide previously shown to have immunosuppressive properties, affect the two binding functions of CD4 similarly, lending support to the hypothesis that the human immunodeficiency virus mimicks HLA class II binding to CD4.
Pyridinone derivatives: specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity.
Authors: Goldman M E , Nunberg J H , O'Brien J A , Quintero J C , Schleif W A , Freund K F , Gaul S L , Saari W S , Wai J S , Hoffman J M
Published: May 2006

Derivatives of pyridinones were found to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevent the spread of HIV-1 infection in cell culture without an appreciable effect on other retroviral or cellular polymerases. 3-[( (4,7-Dimethyl-1,3-benzoxazol-2-yl) methyl]amino ]-5-ethyl-6-methylpyridin-2(1H)-one (L-697,639) and 3-[[ (4,7-dichloro-1,3-benzoxazol-2-yl) methyl]amino]-5-ethyl-6-methylpyridin-2(1H)-one (L-697,661), two compounds within this series, had HIV-1 RT IC50 values in the range of 20-800 nM, depending upon the template-primer used. The most potent inhibition was obtained with rC.dG and dA.dT as template--primers. With rC.dG, reversible slow-binding non-competitive inhibition was observed. [3H]L-697,639 bound preferentially to enzyme-template-primer complexes. This binding was magnesium-dependent and saturable with a stoichiometry of 1 mol of [3H]L-697,639 per mol of RT heterodimer. Displacement of [3H]L-697,639 was seen with phosphonoformate. In human T-lymphoid-cell culture, L-697,639 and L-697,661 inhibited the spread of HIV-1 infection by at least 95% at concentrations of 12-200 nM. Synergism between 3'-azido-3'-deoxythymidine or dideoxyinosine and either of these compounds was also demonstrated in cell culture. Based upon their specificity for HIV-1 RT activity, template-primer dependence on potency and ability to displace [3H]L-697,639; a tetrahydroimidazo [4,5,1-jk] [1,4]-benzodiazepin-2(1H)-thione derivative R82150 and the dipyridodiazepinone BI-RG-587 appear to inhibit RT activity by the same mechanism as the pyridinones.
Isolation of a cDNA for HSF2: evidence for two heat shock factor genes in humans.
Authors: Schuetz T J , Gallo G J , Sheldon L , Tempst P , Kingston R E
Published: May 2006

The heat shock response is transcriptionally regulated by an evolutionarily conserved protein termed heat shock factor (HSF). We report the purification to homogeneity and the partial peptide sequence of HSF from HeLa cells. The peptide sequence was used to isolate a human cDNA with a predicted open reading frame that has homology to the DNA binding domains of both Saccharomyces cerevisiae and Drosophila HSFs. The cDNA directs the synthesis of a protein that binds to the heat shock element with specificity identical to HeLa HSF and stimulates transcription from a heat shock promoter. The expressed protein cross-reacts with anti-HSF antibodies. Surprisingly, however, this cDNA does not encode all of the peptides obtained from purified HeLa HSF. These peptides are encoded by a distinct human cDNA, HSF1, described by Rabindran et al. [Rabindran, S. K., Giorgi, G., Clos, J. & Wu, C. (1991) Proc. Natl. Acad. Sci. USA 88, 6906-6910.] It therefore appears that there is a human heat shock factor gene family and that at least two separate but related HSF proteins regulate the stress response in humans.
Transfer of genetic epilepsy by embryonic brain grafts in the chicken.
Authors: Teillet M A , Naquet R , Le Gal La Salle G , Merat P , Schuler B , Le Douarin N M
Published: May 2006

In the Fayoumi chicken, a spontaneous recessive autosomal mutation (F.Epi) is responsible for high susceptibility to seizures that are especially inducible by intermittent light stimulation. Substitution of defined areas of the encephalic neuroepithelium in normal chicken embryos at 2 days of incubation by their counterparts from homozygous F.Epi embryos generates the epileptic phenotype in the chimeras. It was found that grafting primordia of both prosencephalon and mesencephalon of homozygous F.Epi birds is necessary and sufficient for transfer of the full disease. When grafted alone, the homozygous F.Epi prosencephalon, although showing the typical epileptic interictal electroencephalogram, does not allow the complete epileptic seizures to occur in the hosts. Grafts of mesencephalon and/or rhombencephalon modify neither the behavior nor the electroencephalographic pattern of the recipient chickens. Cooperation of forebrain and midbrain activities is therefore required to yield epileptic seizures in this model.
Identification and purification of a factor that binds to the Mlu I cell cycle box of yeast DNA replication genes.
Authors: Verma R , Patapoutian A , Gordon C B , Campbell J L
Published: May 2006

In Saccharomyces cerevisiae, the genes encoding at least 10 enzymes involved in DNA replication are periodically expressed in the late G1 and S phases of the cell cycle. All of these genes have one copy or more of the sequence ACGCGT, which conforms to the recognition site for the Mlu I restriction endonuclease. For the CDC21, CDC9, and POL1 genes, the Mlu I site has been shown to be absolutely required for periodic transcription. Using nuclear extracts fractionated by conventional and oligonucleotide affinity chromatography, we have purified a 17-kDa protein that recognizes the Mlu I motif. Synthetic oligonucleotides containing mutated Mlu I sites do not bind the protein. In contrast, synthetic oligonucleotides derived from the CDC2, CDC6, and CDC21 genes, which are expressed with the same timing as POL1, bind purified protein efficiently.
Use of site-directed mutagenesis to enhance the epitope-shielding effect of covalent modification of proteins with polyethylene glycol.
Authors: Hershfield M S , Chaffee S , Koro-Johnson L , Mary A , Smith A A , Short S A
Published: May 2006

Modification by covalent attachment of polyethylene glycol (PEG) can reduce the immunogenicity and prolong the circulating life of proteins, but the utility of this approach for any protein is restricted by the number and distribution of PEG attachment sites (e.g., epsilon-amino groups of lysine residues). We have developed a strategy for introducing additional sites for PEG attachment by using site-directed mutagenesis to selectively replace arginine with lysine codons and tested it with purine nucleoside phosphorylase (PNP) from Escherichia coli, an extremely stable but immunogenic enzyme, that could potentially be used to treat an inherited deficiency of PNP. A triple mutant, RK3, possessing three Arg----Lys substitutions was constructed that increased the number of lysines per PNP subunit from 14 to 17, providing an additional 18 potential PEG attachment sites per hexameric enzyme molecule. The wild-type and RK3 enzymes had similar catalytic activity, antigenicity, and immunogenicity. After PEG modification, both enzymes retained catalytic activity, the plasma half-life of both enzymes in mice increased from approximately 4 hr to 4 days, and the binding of both enzymes by antisera raised against each unmodified enzyme was markedly diminished. However, antibody raised against wild-type PEG-PNP did not bind the PEG-RK3 enzyme. PEG-RK3 PNP was also substantially less immunogenic than wild-type PEG-PNP. Accelerated antibody-mediated clearance of PEG-PNP occurred in 2 of 12 mice treated with PEG-RK3 PNP, compared with 10 of 16 mice treated with the modified wild-type enzyme. This combined use of directed mutagenesis and PEG modification is aimed at permitting the widest choice of proteins, including products of genetic and chemical "engineering," to be used for therapy of inherited and acquired disorders.
Insulin regulation of the glucagon gene is mediated by an insulin-responsive DNA element.
Authors: Philippe J
Published: May 2006

Diabetes mellitus is characterized by insulin deficiency and high plasma glucagon levels, which can be normalized by insulin replacement. It has previously been reported that glucagon gene expression is negatively regulated by insulin at the transcriptional level. By transfection studies, I have now localized a DNA control element that mediates insulin effects on glucagon gene transcription. This element also confers insulin responsiveness to a heterologous promoter. DNA-binding proteins that specifically interact with this insulin-responsive element are found in both glucagon- and non-glucagon-producing cells; and the pattern of binding, as assessed by the gel retardation assay, is not modified by prior insulin treatment.
Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.
Authors: Holland P M , Abramson R D , Watson R , Gelfand D H
Published: May 2006

The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'----3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.
Tension responses to rapid pressure release in glycerinated rabbit muscle fibers.
Authors: Fortune N S , Geeves M A , Ranatunga K W
Published: May 2006

We have previously shown that the isometric tension of a fully calcium-activated skinned rabbit psoas muscle fiber is reversibly depressed by increased hydrostatic pressure. We report here the characterization of tension transients induced by a rapid (less than 1-ms) release of increased pressure at 12 degrees C. The tension transient consists of three clear phases, an initial further decrease of tension in phase with pressure change followed by two phases of tension increase back to the level recorded at ambient pressure. The mean reciprocal relaxation time for phase 2 (1/tau 2) was approximately 17 s-1 and that for phase 3 (1/tau 3) was 3 s-1. The presence of 20 mM inorganic phosphate markedly increased 1/tau 2 to approximately 52 s-1 and decreased 1/tau 3 to approximately 1.7 s-1. These observations are interpreted in terms of a pressure-sensitive transition between two attached crossbridge states of low (or zero) and higher force. This is compatible with the pressure-sensitive isomerization of actomyosin previously observed in solution. The results presented allow us to propose a coupling between a specific pressure-sensitive isomerization of purified actomyosin, the phosphate release step of the ATPase pathway, and the force-generating event of the cross-bridge cycle.
Import of simian virus 40 virions through nuclear pore complexes.
Authors: Clever J , Yamada M , Kasamatsu H
Published: May 2006

How the DNA tumor virus, simian virus 40, reaches the nucleus is unknown. In this report we have tested the affinity of simian virus 40 toward the nucleus by microinjecting virion particles into the cytoplasm under conditions in which cell-surface-mediated viral infection was blocked. Subcellular localization of viral structural proteins Vp1, Vp2, and Vp3, large tumor antigen, and virion particles was followed immunocytochemically and ultrastructurally. Both virion particles and viral structural proteins localized in the nucleus within 1-2 hr after cytoplasmic injection and subsequently expressed large tumor antigen, which was detected in the nucleus as early as 3 hr after cytoplasmic injection. Vp1 and large tumor antigen nuclear accumulation, as well as virion nuclear entry, were blocked by wheat germ agglutinin and an anti-nucleoporin monoclonal antibody, mAb 414. Virion particles were visualized in the vicinity of nuclear pores and in the cytoplasm with this agent. We conclude that virion particles are karyophilic and enter through nuclear pores. This study suggests that virion structural proteins facilitate virion import into the nucleus and viral gene expression.