bluuebunny/crossref_metadata_2025 huggingface dataset:
Recruitment of a penicillin-binding protein gene from Neisseria flavescens during the emergence of penicillin resistance in Neisseria meningitidis.
Authors: Spratt B G , Zhang Q Y , Jones D M , Hutchison A , Brannigan J A , Dowson C G
Published: May 2006

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria meningitidis produce altered forms of penicillin-binding protein 2 that have decreased affinity for penicillin. The sequence of the penicillin-binding protein 2 gene (penA) from a penicillin-resistant strain of N. meningitidis was compared to the sequence of the same gene from penicillin-sensitive strains and from penicillin-sensitive and penicillin-resistant strains of Neisseria gonorrhoeae. The penA genes from penicillin-sensitive strains of N. gonorrhoeae and N. meningitidis were 98% identical. The gene from the penicillin-resistant strain of N. meningitidis consisted of regions that were almost identical to the corresponding regions in the penicillin-sensitive strains (less than 0.2% divergence) and two regions that were very different from them (approximately 22% divergence). The two blocks of altered sequence have arisen by the replacement of meningococcal sequences with the corresponding regions from the penA gene of Neisseria flavescens and result in an altered form of penicillin-binding protein 2 that contains 44 amino acid substitutions and 1 amino acid insertion compared to penicillin-binding protein 2 of penicillin-sensitive strains of N. meningitidis. A similar introduction of part of the penA gene of N. flavescens, or a very similar commensal Neisseria species, appears to have occurred independently during the development of altered penA genes in non-beta-lactamase-producing penicillin-resistant strains of N. gonorrhoeae.
Escherichia coli replication termination protein impedes the action of helicases.
Authors: Lee E H , Kornberg A , Hidaka M , Kobayashi T , Horiuchi T
Published: May 2006

Identification of the consensus sequence for termination of replication (ter) in Escherichia coli and the isolation of the ter-binding protein (TBP) allowed us to test their effects on replication forks initiated at the unique origin of the E. coli chromosome (oriC) in a purified enzyme system. Replication was severely impeded by ter in a unique orientation when purified TBP was supplied to bind it. The target for blockage within the replication complex can now be ascribed to the inability of dnaB helicase to separate the duplex strands when it encounters ter bound by TBP. Other helicases, such as rep and uvrD proteins, that translocate on DNA and displace strands in the direction opposite to that of dnaB protein are also blocked, but only when the TBP-bound ter is oriented in the other direction. From these results, we infer that the orientation of ter confers a particular polarity on the TBP seated on it, such that a helicase is blocked when it confronts TBP from one side, but can act, presumably by displacing TBP, when facing its other side. Thus, the intrinsic nature of the oriented TBP-ter complex is responsible for impeding the helicases, rather than any protein-protein interactions.
Overexpression and biosynthesis of CD4 in Chinese hamster ovary cells: coamplification using the multiple drug resistance gene.
Authors: König R , Ashwell G , Hanover J A
Published: May 2006

CD4 is a T-cell surface glycoprotein and serves as the receptor for the human immunodeficiency virus. Glycosylation of CD4 has been shown to be necessary for proper surface expression. To study the biosynthesis and assembly of CD4, wild-type and glycosylation-deficient mutant Chinese hamster ovary (CHO) cells were cotransfected with a cDNA encoding CD4 and a cDNA for the human multiple drug resistance gene, which allowed the amplification of the transfected CD4 cDNA sequences. Clones were isolated that exhibited high-level expression of CD4 resulting from the integration of several copies of CD4 cDNA. CD4 synthesized by these cells acquired resistance to endoglycosidase H after 20-30 min of chase, suggesting a rapid translocation of the glycoprotein from the rough endoplasmic reticulum to the medial Golgi apparatus. The sensitivity of CD4 to glycosidases suggested the presence of biantennary unsialylated complex-type oligosaccharides. Consistent with this, CD4 synthesized by the Lec2 mutant, which does not add sialic acid to oligosaccharides, was identical to the glycoprotein produced by wild-type CHO cells. The amplification strategy used to express CD4 at high levels in wild-type and mutant CHO cells will have general utility.
The kinetics of human granulopoiesis following treatment with granulocyte colony-stimulating factor in vivo.
Authors: Lord B I , Bronchud M H , Owens S , Chang J , Howell A , Souza L , Dexter T M
Published: May 2006

Cell proliferation in the bone marrow and blood of two patients with metastatic breast cancer who were treated with granulocyte colony-stimulating factor was studied by using [3H]thymidine labeling and autoradiography. Additionally, the fate of neutrophils labeled with 99mTc-hexamethylpropyleneamineoxime was observed following granulocyte colony-stimulating factor infusion. Proliferation increased in all stages of granulopoiesis, but a significant amount of the increased production stemmed from a greater input to the myeloblast compartment. Changes in the myelogram combined with the increased labeling indicated a faster throughput of cells, which resulted in labeled cells appearing in the circulation within 1 day compared to the normal 4 or 5 days. The 99mTc studies demonstrated no sequestration of circulating neutrophils by spleen, lungs, or liver. The half-life of the circulating neutrophils was not significantly changed, and calculations from the flow of labeled cells to the peripheral blood indicated an increase of 3.2 extra amplification divisions during neutrophil development. The dramatic neutrophil response to granulocyte colony-stimulating factor can therefore be accommodated by a relatively modest increase in granulopoietic activity.
Modulation of deferoxamine toxicity and clearance by covalent attachment to biocompatible polymers.
Authors: Hallaway P E , Eaton J W , Panter S S , Hedlund B E
Published: May 2006

A class of high molecular weight iron chelators has been prepared by covalently attaching deferoxamine (DFO), by its amino group, to a variety of biocompatible polymers such as dextran and hydroxyethyl-starch. The iron-binding properties of DFO are virtually unchanged after the attachment procedure, but the toxicity and circulatory half-life are profoundly altered. Competitive iron-binding experiments indicate that the conjugates retain a high affinity for ferric iron. In addition, the derivatives inhibit iron-driven lipid peroxidation as effectively as the parent drug. However, the LD50 in mice (based on DFO equivalents) is approximately 4000 mg/kg for dextran-DFO as compared to 250 mg/kg for free DFO. Consistent with the greatly decreased LD50, intravenous administration of the conjugates in dogs at a dose of 100 mg/kg (body weight) does not cause the severe hypotension associated with intravenous administration of DFO. The plasma half-lives of these adducts are increased greater than 10-fold for dextran-DFO and hydroxyethyl-starch-DFO compared to the free drug. Finally, and most importantly, the conjugates are effective in mediating in vivo iron mobilization and excretion. Because recent evidence implicates iron as an important component of tissue injury in many disease states, these high molecular weight iron chelators may have potential for improved therapy, allowing higher sustained plasma concentrations of the active drug.
Gonadotropin stimulates oocyte translation by increasing magnesium activity through intracellular potassium-magnesium exchange.
Authors: Horowitz S B , Tluczek L J
Published: May 2006

We previously showed that gonadotropin increases the K+ activity in Xenopus oocytes and that this is a signal for increased translation. However, K+ need not act to control synthesis directly but may act through an unidentified downstream effector. Using microinjection to vary the salt content of oocytes and concomitantly measuring [3H]leucine incorporation, we found that small changes in Mg2+ greatly affect translation rates. (Ca2+ had little influence.) By measuring intracellular ion activities, we found that oocyte cations existed in a buffer-like (ion-exchange) equilibrium in which K+ and Mg2+ are the preponderant monovalent and divalent cations. Hence, increasing cellular K+ activity might increase translation by causing Mg2+ activity to rise. If so, the increased translation rates produced by hormone treatment or K+ injection would be prevented by EDTA, a Mg2+ chelating agent. This prediction was tested and confirmed. We conclude that, when gonadotropin increases K+ activity, the cell's internal ion-exchange equilibrium is altered thereby increasing Mg2+ activity and this up-regulates translation.
Echinomycin-induced hypersensitivity to osmium tetroxide of DNA fragments incapable of forming Hoogsteen base pairs.
Authors: McLean M J , Seela F , Waring M J
Published: May 2006

To show conclusively that the critical structural deformation of double-helical DNA that is induced by the binding of quinoxaline antibiotics does not involve the formation of Hoogsteen base pairs, we have prepared a DNA fragment containing the nucleoside analog 7-deaza-2'-deoxyadenosine in one of the two strands. This DNA fragment was subjected to treatment with the thymidine-specific reagent osmium tetroxide and to DNase I "footprinting" in the presence or absence of micromolar concentrations of echinomycin. We report that this anti-tumor antibiotic binds to DNA containing the nucleoside analog as well as to natural DNA and that the previously reported hypersensitivity to osmium tetroxide of certain thymidine residues adjacent to echinomycin binding sites is maintained in analog-containing DNA. Since these thymidines are rendered incapable of participating in Hoogsteen base pairs by the incorporation of 7-deaza-2'-deoxyadenosine, we conclude that this unusual base-pairing scheme is not the cause of the observed hypersensitivity to osmium tetroxide and that it therefore results from a large local unwinding of the DNA in the presence of the antibiotic. Moreover, preventing the possibility of Hoogsteen base pairing does not preclude echinomycin binding.
Isolation of a candidate cDNA for the gene causing retinal degeneration in the rd mouse.
Authors: Bowes C , Danciger M , Kozak C A , Farber D B
Published: May 2006

The inherited retinal degeneration of the rd mouse results in the exclusive loss of one cell type, the photoreceptors. We took advantage of this visual-cell loss to devise a strategy for the isolation of photoreceptor-specific cDNAs based on the use of subtractive and differential hybridizations. The resulting pool of photoreceptor-specific cDNAs was screened for a candidate cDNA for the rd gene, and a putative rd cDNA that maps to mouse chromosome 5, the chromosome to which the rd gene has been assigned, was identified. On Northern blots the candidate rd cDNA hybridizes a 3.3-kilobase RNA species from 9- to 11-day-old developing normal retina and, much more faintly, a 3.6-kb RNA species from age-matched rd retina. The 0.3-kilobase difference in the size of the mRNAs hybridized suggests that a structural alteration in the gene corresponding to the candidate rd cDNA has occurred in the rd mouse. This was further supported by the detection of polymorphisms between rd/rd and +/+ mouse genomic DNA after digestion with restriction endonucleases and probing with the candidate rd cDNA. Expression of mRNAs hybridized by the candidate rd cDNA is detected in normal and diseased retinas at postnatal day 1 but the signal intensity is considerably lower in the rd retina. To our knowledge, this is the earliest molecular defect reported in the rd retina that is observed prior to any phenotypic signs of photoreceptor degeneration.
Abscisic acid is involved in the wound-induced expression of the proteinase inhibitor II gene in potato and tomato
Authors: Pēna-Cortés Hugo , Sánchez-Serrano José J. , Mertens Rüdiger , Willmitzer Lothar , Prat Salomé
Published: May 2006

Plants respond to wounding or pathogen attack by a variety of biochemical reactions, involving in some instances gene activation in tissues far apart from the actual site of wounding or pathogen invasion. One of the best analyzed examples for such a systemic reaction is the wound-induced expression of proteinase inhibitor genes in tomato and potato leaves. Local wounding of potato or tomato plants results in the accumulation of proteinase inhibitors I and II throughout the aerial part of the plant. In contrast to wild-type plants, abscisic acid-deficient mutants of potato (droopy) and tomato (
sit
) show a drastically reduced induction of these genes in response to plant wounding. High levels of proteinase inhibitor II gene expression are obtained in mutant and wild-type plants upon exogenous application of abscisic acid. Measurements of the endogenous abscisic acid levels in wild-type plants show that wounding results in increased levels of this phytohormone in wounded and nonwounded systemically induced leaves. Thus these results show that the plant hormone abscisic acid is involved in the wound-induced activation of the proteinase inhibitor II gene. Furthermore, they are compatible with a model assuming this hormone to be the actual mediator of the systemic wound response.
Phytochrome induces changes in the immunodetectable level of a wall peroxidase that precede growth changes in maize seedlings
Authors: Kim Sung-Ha , Shinkle James R. , Roux Stanley J.
Published: May 2006

The regulatory pigment phytochrome induces rapid and opposite growth changes in different regions of etiolated maize seedling: it stimulates the elongation rate of coleoptiles and inhibits that of mesocotyls. As measured by a quantitative immunoassay, phytochrome also promotes rapid and opposite changes in the extractable content of a
M
r
98,000 anionic isoperoxidase in the cell walls of these same organs: it induces a decrease of this peroxidase in coleoptiles and an increase in mesocotyls. The peroxidase changes precede the growth changes. As measured by video stereomicroscopy or a position transducer, red light (R), which photoactivates phytochrome, stimulates coleoptile elongation with a lag of about 15-20 min and suppresses mesocotyl growth with a lag of 45-50 min. R also induces a 50% reduction in the extractable level of the anionic peroxidase in coleoptile walls in less than 10 min and a 40% increase in the level of this peroxidase in mesocotyl walls within 30 min. Ascorbic acid, an inhibitor of peroxidase activity, blocks the effects of R on mesocotyl section growth. These results are relevant to hypotheses that postulate that certain wall peroxidases can participate in light-induced changes in growth rate by their effects on wall extensibility.
Cytochemical observation of regulated bacterial beta-galactosidase gene expression in mammalian cells.
Authors: Liu H S , Feliciano E S , Stambrook P J
Published: May 2006

Bacterial beta-galactosidase, encoded by the lacZ gene, serves as a sensitive cytochemical marker in eukaryotic cells and tissues. In transient expression experiments, human and simian cells stain blue 48 hr after transfection with a plasmid containing a lacZ gene, whose expression is directed by a simian virus 40 promoter containing a synthetic lactose operator sequence. Transfection efficiency was about 0.6%. Incorporation of an operator sequence within the promoter permits regulation of beta-galactosidase gene expression by the lacI gene product, the lac repressor. When cells were cotransfected with the lacZ plasmid and a second plasmid containing the lacI gene, beta-galactosidase activity was extinguished. Its activity could be reestablished to original levels upon application of isopropyl beta-D-thiogalactoside to transfected cells. A cell line that stably carries both the lacI and lacZ genes was efficiently induced to synthesize beta-galactosidase after isopropyl beta-D-thiogalactoside administration. In transient expression experiments and in stably transfected lines, repression and induction of beta-galactosidase activity were predominantly at the transcriptional level.
Evolution of immunoglobulin light chains: cDNA clones specifying sandbar shark constant regions.
Authors: Schluter S F , Hohman V S , Edmundson A B , Marchalonis J J
Published: May 2006

Sharks are living fossils that are indistinguishable morphologically from their Devonian ancestors of approximately equal to 400 million years ago. If parallel conservatism characterizes their biochemical evolution, characterization of their immunoglobulin chains could provide information regarding the primordial features of these essential defense molecules. Shark immunoglobulins are polydisperse like those of mammals, but these species lack homogeneous myeloma proteins. This heterogeneity has precluded direct determination of the sequence of elasmobranch light-chain proteins. We have sequenced four cDNA clones that contain the constant-region sequence as well as varying degrees of variable- or joining-region segments. The sandbar shark (Carcharhinus plumbeus) has at least four distinct light-chain constant regions, and these can be considered homologs of mammalian lambda chains. Approximately 40% identity was found in comparison from sharks to mammals. Certain stretches of sequence were remarkably conserved, whereas others varied in a manner consistent with accepted concepts of speciation. One hexapeptide (Ala-Thr-Leu-Val-Cys-Leu) occurred in lambda constant regions of all vertebrate species. There was a universal conservation of certain cysteines, phenylalanines, tryptophans, and glycines and strong identities in the block of residues from Ser-176 to Trp-186. Comparison of the shark sequence with that of the characterized human lambda myeloma protein Mcg indicates a strong conservation of three-dimensional structure in this light-chain domain representing species whose ancestors diverged early in vertebrate evolution. The shark light-chain sequence contains primordial features shared by mammalian kappa and lambda chains and by T-cell receptor beta chains.
Mutational analysis of the HLA-DQ3.2 insulin-dependent diabetes mellitus susceptibility gene.
Authors: Kwok W W , Lotshaw C , Milner E C , Knitter-Jack N , Nepom G T
Published: May 2006

The human major histocompatibility complex includes approximately 14 class II HLA genes within the HLA-D region, most of which exist in multiple allelic forms. One of these genes, the DQ3.2 beta gene, accounts for the well-documented association of HLA-DR4 with insulin-dependent diabetes mellitus and is the single allele most highly correlated with this disease. We analyzed the amino acid substitutions that lead to the structural differences distinguishing DQ3.2 beta from its nondiabetogenic, but closely related allele, DQ3.1 beta. Site-directed mutagenesis of the DQ3.2 beta gene was used to convert key nucleotides into DQ3.1 beta codons. Subsequent expression studies of these mutated DQ3.2 beta clones using retroviral vectors defined amino acid 45 as critical for generating serologic epitopes characterizing the DQw3.1 beta and DQw3.2 beta molecules.
Species distribution of a phosphoprotein (parafusin) involved in exocytosis.
Authors: Satir B H , Hamasaki T , Reichman M , Murtaugh T J
Published: May 2006

A cytosolic phosphoprotein that appears to function in membrane fusion during exocytosis of secretory products has previously been isolated from Paramecium tetraurelia. This phosphoprotein, parafusin, with Mr 63,000, is rapidly dephosphorylated via a Ca2+-dependent process when secretagogues induce exocytosis in competent cells. Dephosphorylation does not occur in exocytosis-incompetent cells. Polyclonal antibodies against purified parafusin have now been used to show that this protein is present in unicellular organisms and cells of metazoan groups of wide evolutionary divergence, such as yeast, insects, and mammals, including humans. These results suggest that parafusin was present early in the history of eukaryotes and that it is of functional importance in the general mechanism of exocytosis and membrane fusion.
Human immunodeficiency virus type 1 negative factor is a transcriptional silencer.
Authors: Niederman T M , Thielan B J , Ratner L
Published: May 2006

The negative factor (nef) of human immunodeficiency virus (HIV) type 1 acts to down-regulate virus replication. To decipher the step in the virus life cycle affected by nef, functional proviral clones with (pHIV F-) or without (pHIV F+) a deletion mutation in the nef gene were constructed. In CD4+ cells, 30- to 50-fold more virus was produced over the course of 18-20 days with cultures infected with F- compared to F+ virus. In CD4- cell lines, 2- to 10-fold greater virus production was found from cultures transfected with pHIV F- than those transfected with pHIV F+. The negative regulatory effects of nef on pHIV F- could be supplied in trans with a plasmid expressing only the nef gene product. Virus produced by COS-1 cells transfected with pHIV F- or pHIV F+ showed similar binding, uptake, uncoating, and reverse transcription. Analysis of HIV-1 RNA and structural protein levels and rates of viral RNA synthesis in CD4- cells also showed 2- to 10-fold higher levels in cells transfected with pHIV F- compared to pHIV F+. The activity of a HIV-1-chloramphenicol acetyltransferase (CAT) plasmid was also suppressed by nef, whereas other CAT plasmids were unaffected. These findings demonstrate that nef acts as a specific silencer of HIV-1 transcription. This activity may be critical for maintenance of HIV-1 latency in vivo.
Rapid activation by interferon alpha of a latent DNA-binding protein present in the cytoplasm of untreated cells.
Authors: Dale T C , Imam A M , Kerr I M , Stark G R
Published: May 2006

The highly conserved interferon (IFN)-stimulated regulatory elements of the human genes 6-16 and 9-27 bind to one or more proteins (E factor) detected in extracts of human Bristol 8 B cells or human foreskin fibroblast cells treated with IFN-alpha. E factor is not detectable in extracts of untreated cells and appears in IFN-treated cells within less than 1 min in a form extractable with low salt and thus presumably not bound to DNA. After a few more minutes, the level of this form decreases in parallel with the increase of a form extractable only with high salt and thus presumably bound to DNA. Induction of E factor by IFN-alpha can occur in nuclei-free cytoplasts, whereas no E factor was detected in IFN-treated nucleoplasts. Together, these results suggest a model for signal transduction in which latent E factor, located in the cytoplasm, is activated or released from an inhibitor very rapidly upon binding of IFN-alpha to its receptor. Active E factor can then migrate to the nucleus, where it binds to the IFN-stimulated regulatory elements of IFN-regulated genes, activating their transcription.
Fourier analysis of cell motility: correlation of motility with metastatic potential.
Authors: Partin A W , Schoeniger J S , Mohler J L , Coffey D S
Published: May 2006

We report the development of a computerized, mathematical system for quantitating the various types of cell motility. This Fourier analysis method simultaneously quantifies for individual cells (i) temporal changes in cell shape represented by cell ruffling, undulation, and pseudopodal extension, (ii) cell translation, and (iii) average cell size and shape. This spatial-temporal Fourier analysis was tested on a series of well-characterized animal tumor cell lines of rat prostatic cancer to study in a quantitative manner the correlation of cell motility with increasing in vivo metastatic potential. Fourier motility coefficients measuring pseudopodal extension correlated best with metastatic potential in the cell lines studied. This study demonstrated that Fourier analysis provides quantitative measurement of cell motility that may be applied to the study of biological processes. This analysis should aid in the study of the motility of individual cells in various areas of cellular and tumor biology.
Conservation of structural and functional domains in complement component C3 of Xenopus and mammals.
Authors: Grossberger D , Marcuz A , Du Pasquier L , Lambris J D
Published: May 2006

The cDNA sequence and the deduced amino acid sequence of the Mr 34,000 C-terminal fragment of Xenopus laevis complement component C3 are presented. The sequence of Xenopus C3 has 57% nucleotide identity to the corresponding sequence of human C3 and approximately 49% amino acid identity to C3 from human, mouse, and rabbit. The Xenopus C3 sequence shows clusters of high and of low similarity to the mammalian C3 sequences. One of these regions of high similarity represents the domain of mammalian C3b involved in the binding of properdin, a regulator of the alternative pathway of complement activation. It is not clear whether the other highly conserved regions are involved in binding to other C3 ligands. The Xenopus C3 sequence completely lacks the Arg-Gly-Asp sequence, which has been suggested to be the recognition site of the human complement receptor type 3 on the iC3b fragment of human C3. The Xenopus C3 gene is shown not to be linked to the Xenopus major histocompatibility complex, as is also the case in mammals. Since the gene of the related molecule C4 is MHC-linked in both mammals and Xenopus, the C3 and C4 genes may have separated before Xenopus and mammals speciated.
Amino acid polymorphisms for esterase-6 in Drosophila melanogaster.
Authors: Cooke P H , Oakeshott J G
Published: May 2006

High-resolution electrophoresis has revealed 10 allozymes of esterase-6 (EC 3.1.1.1) in Drosophila melanogaster. The sequences of 13 isolates of the Est6 gene covering all 10 allozymes were obtained and 52 nucleotide differences were found. Sixteen of these cause amino acid replacements, of which three result in charge differences whose size and direction are consistent with the electrophoretic mobilities of the allozymes in which they occur. The smeared electrophoretic phenotype of one allozyme can be explained by the loss of a cysteine residue involved in a disulfide bridge. Several minor mobility variants within the major F and S electrophoretic phenotypes differ by amino acid substitutions that are generally conservative for charge but not for some other properties (size, polarity, or hydrophobicity). Four amino acid differences are found among different isolates of the same allozymes and, overall, 12 amino acid haplotypes occur among the 13 isolates sequenced. Nevertheless, the most common variants within F and S are distinguished by only two amino acids (Asn/Asp at 237 and Thr/Ala at 247), and these are the most likely targets for the selection underlying complementary latitudinal clines in F and S frequencies.
Coordinate viral induction of tumor necrosis factor alpha and interferon beta in human B cells and monocytes.
Authors: Goldfeld A E , Maniatis T
Published: May 2006

Human tumor necrosis factor alpha (TNF-alpha) gene expression can be induced primarily in cells of the monocyte/macrophage lineage by a variety of inducers, including lipopolysaccharide, phorbol esters such as phorbol 12-myristate 13-acetate, and virus or synthetic double-stranded RNA [poly(I).poly(C)]. In this paper we show that the TNF-alpha gene also responds to virus and phorbol 12-myristate 13-acetate in B lymphocytes and that virus is the most potent inducer of TNF-alpha mRNA in both monocyte and B-cell lines. In addition, we show that viral infection coinduces the expression of TNF-alpha and interferon beta mRNA and that viral induction of both genes is blocked by the kinase inhibitor 2-aminopurine. Inhibition of protein synthesis with cycloheximide had no effect on mRNA expression of the genes in one of three cell lines tested (U937) but blocked the viral induction of both genes in another (Namalwa). Thus, the regulatory factors required for mRNA induction of both genes are present prior to the addition of virus in U937 but not in Namalwa cells. However, in a third cell line (JY), cycloheximide blocked viral induction of the interferon beta gene but not the TNF-alpha gene. Taken together, these observations suggest that viral induction of TNF-alpha and interferon beta gene expression may involve overlapping pathways with both common and distinct regulatory factors.
Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium.
Authors: Wahl M I , Nishibe S , Suh P G , Rhee S G , Carpenter G
Published: May 2006

Epidermal growth factor (EGF) rapidly stimulates the formation of inositol 1,4,5-trisphosphate in a variety of cell types. Previously we have found that in intact cells stimulation of phospholipase C (PLC) activity by EGF is correlated with the retention of increased amounts of PLC activity by a phosphotyrosine immunoaffinity matrix, suggesting that the EGF-receptor tyrosine kinase phosphorylates PLC. We now define parameters of the mechanism by which EGF addition to A-431 cells stimulates phosphotyrosine immunoisolation of PLC activity and demonstrate that EGF addition to A-431 cells increases tyrosine phosphorylation of PLC. EGF rapidly and reversibly stimulated the anti-phosphotyrosine recovery of increased PLC activity when cells were treated with growth factor at 3 degrees C, indicating that receptor internalization is not required and that the phosphorylation event occurs prior to formation of inositol 1,4,5-trisphosphate. Also, the EGF stimulation of anti-phosphotyrosine recovery of PLC activity occurred in the absence of extracellular Ca2+. Stimulation of PLC activity in intact cells by other agonists, such as bradykinin or ATP, did not result in increased anti-phosphotyrosine recovery of PLC activity, suggesting two separate mechanisms exist in A-431 cells for hormone-stimulated formation of inositol phosphates. Finally, using monoclonal antibodies that specifically recognize three distinct PLC isozymes, we show that an approximately 145-kDa PLC isozyme (PLC-II) is present in A-431 cells and that EGF treatment of A-431 cells stimulates phosphorylation of PLC-II on both tyrosine and serine residues.
A murine early thymocyte developmental sequence is marked by transient expression of the interleukin 2 receptor.
Authors: Pearse M , Wu L , Egerton M , Wilson A , Shortman K , Scollay R
Published: May 2006

Precursors of all T-lineage cells are found in the population of thymocytes that lacks the CD4 and CD8 surface markers. These "double-negative" thymocytes are heterogeneous and can be divided into discrete subpopulations based on their expression of other surface markers. We have determined the relative maturity of these subpopulations based on the extent of rearrangement and expression of their T-cell receptor genes, their cell cycle status, and their thymus reconstitution capacity. Within the subpopulation of double negatives expressing high levels of the heat-stable antigen, the additional markers phagocytic glycoprotein 1 (Pgp-1) and interleukin 2 receptor (IL-2R) can be used to define the sequence IL-2R- Pgp-1+----IL-2R+ Pgp-1-----IL-2R- Pgp-1-, which occurs before the expression of CD4 and CD8. Transient expression of the IL-2R marks an important developmental point in the sequence just prior to a burst of cell proliferation and a loss of thymus reconstitution ability. The earliest cells in this sequence are already partially rearranged for genes in the C beta 1 region. IL-2R expression marks a second wave of T-cell antigen receptor of beta-chain gene rearrangement and the initiation of T-cell antigen receptor alpha- and beta-chain gene expression.
Energy metabolism of monocytic Ehrlichia.
Authors: Weiss E , Williams J C , Dasch G A , Kang Y H
Published: May 2006

We investigated if the monocytic Ehrlichia are totally dependent on their host cells for energy, or, as Rickettsia, are capable of some ATP synthesis in vitro. The Miyayama strain of Ehrlichia sennetsu and the Maryland and Illinois strains of Ehrlichia risticii were cultivated in a mouse macrophage cell line, separated from host cell constituents by procedures that included Renografin or Percoll gradient centrifugation, and tested after cryopreservation. Cells incubated without a metabolizing substrate contained little, if any, ATP. When the Ehrlichia cells were incubated for 1 hr at 34 degrees C with glutamine, significant amounts of ATP were detected. The amounts of ATP attained with glutamine were decreased in some instances by the addition of atractyloside, an inhibitor of adenine nucleotide translocase in mitochondria, and were decreased consistently and to a greater extent by 2,4-dinitrophenol. When ATP, instead of glutamine, was added to the ehrlichiae, upon incubation the amount of ATP was markedly decreased. Comparable responses under all these conditions were obtained with Rickettsia typhi, although the final ATP levels were higher. Control preparations derived from uninfected mouse macrophages or from the discards of the Ehrlichia purification procedures contained negligible amounts of ATP, which were not increased by incubation with glutamine. We conclude that with respect to ATP metabolism, the monocytic Ehrlichia resemble Rickettsia more closely than Chlamydia, even though Ehrlichia resemble Chlamydia in their intracellular location in the phagosomes and in possibly having a developmental cycle.
Spatially and temporally resolved calcium concentration changes in oscillating neurons of crab stomatogastric ganglion.
Authors: Ross W N , Graubard K
Published: May 2006

Calcium concentration changes during oscillations of the membrane potential of crab (Cancer irroratus or Cancer borealis) stomatogastric neurons were monitored at many positions by using the calcium indicator dye arsenazo III and a photodiode array. Data analysis algorithms using signal averaging techniques were developed to improve the time resolution of the measured calcium changes. As previously reported, calcium oscillations were detected from all regions of the neuropil but not from the soma or axon. In some cells step increases in intracellular neuropil calcium were correlated with each of the action potentials in the burst (on the peak of the voltage oscillation). In other cells we observed calcium oscillations phase-locked to the membrane potential with no spike-related component. A few cells had both spike-evoked and graded potential components to the calcium oscillations. In those cells, the spatial distribution of the spike-correlated calcium influx differed from that of the voltage-oscillation-correlated calcium influx, suggesting that different neurites might interact with their postsynaptic targets with different mixtures of graded and spike-correlated transmitter release.
Noise in neural networks: thresholds, hysteresis, and neuromodulation of signal-to-noise.
Authors: Keeler J D , Pichler E E , Ross J
Published: May 2006

We study a neural-network model including Gaussian noise, higher-order neuronal interactions, and neuromodulation. For a first-order network, there is a threshold in the noise level (phase transition) above which the network displays only disorganized behavior and critical slowing down near the noise threshold. The network can tolerate more noise if it has higher-order feedback interactions, which also lead to hysteresis and multistability in the network dynamics. The signal-to-noise ratio can be adjusted in a biological neural network by neuromodulators such as norepinephrine. Comparisons are made to experimental results and further investigations are suggested to test the effects of hysteresis and neuromodulation in pattern recognition and learning. We propose that norepinephrine may "quench" the neural patterns of activity to enhance the ability to learn details.
Human immunodeficiency virus-infected T cells and monocytes are killed by monoclonal human anti-gp41 antibodies coupled to ricin A chain.
Authors: Till M A , Zolla-Pazner S , Gorny M K , Patton J S , Uhr J W , Vitetta E S
Published: May 2006

Two human monoclonal antibodies specific for the envelope glycoprotein (gp), gp41, of the human immunodeficiency virus were conjugated to deglycosylated ricin A chain. These immunotoxins killed human immunodeficiency virus-infected H9 (T cell) and U937 (monocyte) cell lines but were nontoxic to the uninfected cell lines or to class II-positive Daudi cells. Specific killing of infected H9 cells could be completely blocked by recombinant gp160 and partially blocked by unconjugated anti-gp41 antibody but was not blocked by recombinant gp120 or human IgG demonstrating specificity for gp41. The specific toxicity of the immunotoxins for infected U937 cells was markedly potentiated by chloroquine.
A general native-state method for determination of proliferation capacity of human normal and tumor tissues in vitro.
Authors: Hoffman R M , Connors K M , Meerson-Monosov A Z , Herrera H , Price J H
Published: May 2006

An important need in cancer research and treatment is a physiological means in vitro by which to assess the proliferation capacity of human tumors and corresponding normal tissue for comparison. We have recently developed a native-state, three-dimensional, gel-supported primary culture system that allows every type of human cancer to grow in vitro at more than 90% frequency, with maintenance of tissue architecture, tumor-stromal interaction, and differentiated functions. Here we demonstrate that the native-state culture system allows proliferation indices to be determined for all solid cancer types explanted directly from surgery into long-term culture. Normal tissues also proliferate readily in this system. The degree of resolution of measurement of cell proliferation by histological autoradiography within the cultured tissues is greatly enhanced with the use of epi-illumination polarization microscopy. The histological status of the cultured tissues can be assessed simultaneously with the proliferation status. Carcinomas generally have areas of high epithelial proliferation with quiescent stromal cells. Sarcomas have high proliferation of cells of mesenchymal organ. Normal tissues can also proliferate at high rates. An image analysis system has been developed to automate proliferation determination. The high-resolution physiological means described here to measure the proliferation capacity of tissues will be important in further understanding of the deregulation of cell proliferation in cancer as well as in cancer prognosis and treatment.
Identification and sequence analysis of a second form of prolactin receptor by molecular cloning of complementary DNA from rabbit mammary gland.
Authors: Edery M , Jolicoeur C , Levi-Meyrueis C , Dusanter-Fourt I , Pétridou B , Boutin J M , Lesueur L , Kelly P A , Djiane J
Published: May 2006

Two lambda gt11 clones containing fragments of cDNA encoding the prolactin receptor from rabbit mammary gland were isolated using a rat liver prolactin receptor cDNA probe. An 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular, amino-terminal, prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular, carboxyl-terminal domain of 358 residues. This latter domain is much longer than the cytoplasmic domain (57 residues) previously described for the rat liver prolactin receptor. In addition, the sequence identity of this form of prolactin receptor with the growth hormone receptor is extended in the cytoplasmic domain.
Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.
Authors: Fontecave M , Eliasson R , Reichard P
Published: May 2006

Ribonucleoside diphosphate reductase from Escherichia coli and mammalian cells provides the deoxyribonucleoside triphosphates for DNA synthesis. The active enzyme contains a tyrosyl free radical whose formation requires oxygen. Earlier genetic evidence suggested that the enzyme is not required for anaerobic growth of E. coli, implicating the activity of a different enzyme or enzyme system for deoxyribonucleotide synthesis in the absence of oxygen. We now conclude from isotope incorporation experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. Extracts from anaerobically grown bacteria contain a different enzyme activity capable of reducing CTP to dCTP. To obtain an active enzyme, strict anaerobiosis must be maintained during extract preparation and during assay of the enzyme. The reaction is stimulated by NADPH, Mg2+, and ATP. Inhibition by deoxyribonucleoside triphosphates suggests that the anaerobic enzyme has allosteric properties. Antibodies raised against the aerobic enzyme do not inhibit the new activity, and hydroxyurea, a potent scavenger of the tyrosyl radical of the aerobic enzyme, only weakly inhibits the anaerobic enzyme. The anaerobic enzyme has interesting evolutionary aspects since it might reflect on an activity that in the absence of oxygen made possible the transition from an "RNA world" into a "DNA world."
Human immunodeficiency viral long terminal repeat is functional and can be trans-activated in Escherichia coli.
Authors: Kashanchi F , Wood C
Published: May 2006

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) contains the viral promoter, which is responsible for viral gene expression in eukaryotic cells. We have demonstrated that HIV LTR can also function as a promoter in Escherichia coli. A recombinant plasmid containing the HIV LTR linked to the chloramphenicol acetyltransferase gene can express the enzyme efficiently upon transformation into bacteria. Mung bean nuclease analysis mapped the bacterial transcriptional start site of the promoter to the U3 region of the LTR, in contrast to transcription in eukaryotic cells, which initiates in the U3-R boundary of the LTR. The HIV LTR, besides being fully functional in E. coli, can also be specifically trans-activated by the HIV tat gene product. Trans-activation is demonstrated by an increase in chloramphenicol acetyltransferase activity as well as an increase in the mRNA level of the enzyme. This trans-activation of HIV LTR by tat protein in bacteria offers a useful system to investigate further the specific interaction between tat protein with HIV LTR and the mechanisms of trans-activation.
Perfection of a synaptic receptor: kinetics and energetics of the acetylcholine receptor.
Authors: Jackson M B
Published: May 2006

The energetics and kinetics of activation of the acetylcholine receptor are evaluated in the context of optimizing rapid synaptic transmission. Physiological needs are used as the basis for estimating optimal values for the closed-to-open channel equilibrium constants of the liganded and unliganded receptor. An estimate is made of the maximum energy that can be derived from the binding of acetylcholine to a perfectly designed receptor binding site. Application of the principle of detailed balance shows that with only one ligand binding site the receptor will not be able to derive enough energy from acetylcholine binding to drive a sufficiently large change in the channel conformational equilibrium. This then provides a rationale for the existence of a second binding site, rather than the often invoked advantage of cooperativity. With two binding sites there is a considerable excess of binding energy and consequently considerable flexibility in how binding energy can be utilized. It is shown that the receptor must have at least one binding site that binds acetylcholine weakly when the channel is closed. This is essential to rapid response termination. However, making the other binding site bind more tightly can enhance and accelerate the activation of the receptor. To optimize both response activation and termination the best solution is to make the two binding sites different in their binding affinities. This qualitatively reproduces an experimental observation.
31P nuclear magnetic resonance study of the metabolic pools of adenosine triphosphate in cultured bovine adrenal medullary chromaffin cells.
Authors: Painter G R , Diliberto E J , Knoth J
Published: May 2006

31P NMR was used to resolve and determine the relative quantity and mobility of ATP in the cytosolic and vesicular compartments of isolated adrenomedullary chromaffin cells. The cells were cultured on microcarrier beads and superfused with an oxygenated medium--thereby permitting dense suspensions of viable cells to be maintained in the NMR probe for extended time periods. Under these conditions, distinct 31P signals could be seen for ATP within the vesicular and the cytosolic pools. Comparison of the integrated areas of the beta-phosphate resonances from the two ATP pools indicated 77% of the endogenous ATP was in the vesicular pool. From this observation and the assumption that the concentration of ATP in the vesicle is 87.5 mM, the concentration of ATP calculated to be in the cytoplasmic pool was approximately 4 mM. The pH in the vesicle determined from the chemical shift of the gamma-phosphate resonance of vesicular ATP was 5.84 +/- 0.17 (n = 6), slightly higher than the intragranular pH measured in hypoxic cells (5.57 +/- 0.15, n = 8). Spin-lattice relaxation times of ATP 31P resonances in the vesicular pool were from 12 to 14 times shorter than the ATP resonances in the cytosol, corresponding to a decrease in molecular mobility due to incorporation of ATP within a catecholamine-storage complex.
Muscle coenzyme Q deficiency in familial mitochondrial encephalomyopathy.
Authors: Ogasahara S , Engel A G , Frens D , Mack D
Published: May 2006

The electron transport system of muscle mitochondria was examined in a familial syndrome of lactacidemia, mitochondrial myopathy, and encephalopathy. The propositus, a 14-year-old female, and her 12-year-old sister had suffered from progressive muscle weakness, abnormal fatigability, and central nervous system dysfunction since early childhood. In the propositus, the state 3 respiratory rate of muscle mitochondria with NADH-linked substrates and with succinate was markedly reduced. The levels of cytochromes a + a3, b, and c + c1 were normal. The activities of complexes I, II, III, and IV of the electron transport chain were normal or increased. By contrast, the activities of complex I-III and of complex II-III, both of which need coenzyme Q10 (CoQ10), were abnormally low. On direct measurement, the mitochondrial CoQ10 content was 3.7% of the mean value observed in 10 controls. Serum and cultured fibroblasts of the propositus had normal CoQ10 contents. In the younger sister, the respiratory activities and CoQ10 level of muscle mitochondria were similar to those observed in the propositus. The findings establish CoQ10 deficiency as a cause of a familial mitochondrial cytopathy and suggest that the disease results from a tissue-specific defect of CoQ10 biosynthesis.