bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of capsid precursor processing and myristoylation in morphogenesis and infectivity of human immunodeficiency virus type 1.
Authors: Göttlinger H G , Sodroski J G , Haseltine W A
Published: May 2006

The effects on human immunodeficiency virus type 1 virion morphogenesis and on virus replication of mutations that affect posttranslational processing of the capsid precursor protein are described. A change in the glycine residue at position two from the N terminus abolishes the myristoylation of the precursor proteins and also prevents virus particle release. Mutations in the viral protease gene abolish proteolytic cleavage of the capsid precursor but do not prevent the formation and budding of virion particles of immature appearance. Mutations that alter the sequence of the sites normally used for cleavage of the major capsid protein p24 from the capsid precursor alter virion morphogenesis and prevent virus replication.
Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E-enriched lipoproteins.
Authors: Kowal R C , Herz J , Goldstein J L , Esser V , Brown M S
Published: May 2006

Low density lipoprotein receptor-related protein (LRP) is a recently described cell-surface protein of 4544 amino acids that contains reiterated sequences found in the 839-amino acid receptor for low density lipoprotein (LDL). In the current studies, we purified LRP from rat liver, prepared polyclonal antibodies that recognize the extracellular domain, and demonstrated an immunoreactive protein of approximately 600 kDa in human fibroblasts. The function of this LRP was studied in mutant human fibroblasts that do not produce LDL receptors. The mutant cells were incubated with beta-migrating very low density lipoprotein (beta-VLDL) that was isolated from cholesterol-fed rabbits and artificially enriched with apoprotein (apo) E by incubation in vitro with human apo E produced in a bacterial expression system. The apo E-enriched beta-VLDL, but not unincubated beta-VLDL, stimulated incorporation of [14C]-oleate into cholesteryl [14C]oleate 20- to 40-fold in the mutant cells. This stimulation was blocked by chloroquine, suggesting that such stimulation resulted from receptor-mediated uptake and lysosomal hydrolysis of the cholesteryl esters in apo E-enriched beta-VLDL. Stimulation of cholesterol esterification was blocked by the antibody against LRP, but not by an antibody against the LDL receptor. Unlike the LDL receptor, the amount of LRP was not reduced when cells were incubated with oxygenated sterols. We conclude that LRP can mediate the cellular uptake and lysosomal hydrolysis of cholesteryl esters contained in lipoproteins that are enriched in apo E.
Anion channel blockers cause apparent inhibition of exocytosis by reacting with agonist or secretory product, not with cell.
Authors: Vostal J G , Reid D M , Jones C E , Shulman N R
Published: May 2006

Agents that act as anion channel blockers (ACBs) and do not permeate cells appear to inhibit exocytosis in platelets, parathyroid cells, and neutrophils. Based in large part on these observations, anion influx through plasma membrane channels has been considered a factor controlling cellular secretion, but there have been no direct anion influx measurements in cells or granules to support this concept. We have found that ACBs inhibit only thrombin-induced platelet secretion, not secretion induced by ADP, collagen, or A23187. ACBs inhibit thrombin esterolytic activity, binding of thrombin to platelets, and thrombin-stimulated platelet production of malondialdehyde in proportion to the degree of inhibition of thrombin-induced platelet secretion. Thus inhibition of platelet secretion by ACBs is due to inactivation of the stimulatory agonist, thrombin, and not to interference with cellular secretion per se. We have also found that previously reported inhibition of secretion of parathyroid cells and neutrophils by ACBs can be explained by the ability of ACBs to interfere with detection of the cellular secretory products that were measured to assess exocytosis. Our measurements of parathyroid hormone and beta-glucuronidase in the presence of ACBs were reduced to the same degree as the reported reduction in apparent cellular secretion produced by these agents. We conclude that plasma membrane anion channels of the type that can be blocked by ACBs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, suramin, and probenecid do not participate in cellular secretory processes. Whether other types of anion channels exist that are not affected by these ACBs and whether there are mechanisms of anion flux during secretion not dependent on channels remain open questions.
Antidiabetic effects of interleukin 1.
Authors: del Rey A , Besedovsky H
Published: May 2006

Interleukin 1 (IL-1), a cytokine released mainly by activated macrophages-monocytes, affects glucose homeostasis and may mediate some of the metabolic derangements observed during certain inflammatory and infectious processes. In this report, it is shown that IL-1 acts as a hypoglycemic agent not only in normal animals but also in mice at early stages of alloxan-induced diabetes and in genetically diabetic, insulin-resistant C57BL/Ks db/db mice and C57BL/6J ob/ob mice. In these animal models, a single injection of a low dose of human recombinant IL-1 normalized glucose blood levels for several hours. This effect was not mediated by possible insulin secretagogue actions of the cytokine. Furthermore, IL-1 markedly reduced the levels of triglycerides in blood of streptozotocin-induced diabetic mice at later stages of the disease. Although the final mechanism of action is at present unknown, the results showed that IL-1 is a hormone with powerful antidiabetic properties. Defective production of this cytokine associated with diabetes could contribute to aggravate the course of the disease during infectious and inflammatory processes.
Coupling of nonpolymerizable monomeric actin to the F-actin binding region of the myosin head.
Authors: Bettache N , Bertrand R , Kassab R
Published: May 2006

Polymerizations of skeletal G-actin induced by salt and myosin subfragment 1 (S-1) were suppressed by reaction of G-actin with m-maleimidobenzoyl-N-hydroxysuccinimide ester. The G-actin derivative, containing few intramolecular crosslinks and a free maleimide group, was covalently coupled in solution to the S-1 heavy chain. The resulting complex could no longer bind to F-actin. The SH-1 and SH-2 thiols of S-1 were not involved in the complexation and the covalent link was shown to be exclusively on the 50-kDa segment of the S-1 heavy chain. The specific conjugation of the two proteins followed formation of a reversibly associated pyrophosphate-sensitive binary complex which was characterized by different approaches. Potentially, these complexes may be useful in developing the crystallography of actin-bound S-1.
Molecular cloning, characterization, and expression of human ADP-ribosylation factors: two guanine nucleotide-dependent activators of cholera toxin.
Authors: Bobak D A , Nightingale M S , Murtagh J J , Price S R , Moss J , Vaughan M
Published: May 2006

ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A)+ RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A)+ RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFs also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs.
Rev-induced modulation of Nef protein underlies temporal regulation of human immunodeficiency virus replication.
Authors: Ahmad N , Maitra R K , Venkatesan S
Published: May 2006

The replication of human immunodeficiency virus type 1 (HIV-1) requires the concerted action of two virus-encoded transactivator proteins, Tat and Rev, and is in turn moderated by the viral transcriptional repressor Nef. We show here that the phenotype of a Rev- HIV-1 provirus was nonreplicating and was distinguished by accumulation of Nef protein and reduced Tat function. Provirus defective in both the rev and nef genes (Rev-Nef-) was also nonreplicating but had normal Tat function. Trans-complementation of the Rev- mutant with Rev caused a decrease of both the steady-state level and the rate of synthesis of Nef. This was accompanied by enhanced synthesis of viral structural proteins. Rev induced even greater levels of virus production from the Rev-Nef- double mutant. In contrast, exogenous Rev did not augment virus production from wild-type provirus. Virus production from Rev- and Rev-Nef- mutants induced by Rev was repressed by exogenous Nef. The repression induced by Nef could not be reversed by exogenous Rev. The ability of Rev to modulate Nef expression solely from the provirus, and thereby relieve the Nef-mediated inhibition of transcription from the viral long terminal repeat, reveals a delicate balance of the functions of these two proteins that might underlie the switch between latency and reactivation.
In-plane phase transition of an integral membrane protein: nucleation of the OmpF matrix porin rectangular polymorph.
Authors: Dorset D L , Massalski A K , Rosenbusch J P
Published: May 2006

A hexagonal polymorph (a = 79 A) of OmpF matrix porin from Escherichia coli spontaneously transforms to a rectangular form (a = 79 A, b = 137 A) after several months' storage in the refrigerator. Nucleation of this second polymorph is first disclosed by diffuse streaks in electron diffraction patterns or in computer-generated Fourier transforms of electron microscope images. With time, this streaking is resolved as an apparent superlattice, and eventually domains of orthorhombic polymorph are detected in the parent hexagonal lattice that can be oriented in either of three directions, depending on the polarity of the orthorhombic crystal growth. Models for this phenomenon based on protein trimer rotation successfully explain the progress of the phase transition and, if protein-protein interactions are the most important interactions between adjacent trimers in the lipid matrix, the transition is quite similar to what occurs with molecular crystals.
Large aqueous channels in membrane vesicles derived from the rough endoplasmic reticulum of canine pancreas or the plasma membrane of Escherichia coli.
Authors: Simon S M , Blobel G , Zimmerberg J
Published: May 2006

Voltage clamp conditions were used to study the membrane permeability properties of rough microsomes (RM) derived from the rough endoplasmic reticulum of canine pancreas and inverted vesicles (InV) derived from the plasma membrane of Escherichia coli. Membrane vesicles of RM or InV were fused to a planar lipid bilayer that was formed in a hole of a partition separating two chambers. Fusion of a single RM vesicle yielded a single-step conductance increase. Some preparations yielded unitary conductances of 20, 55, 80, and 115 pS in 45 mM potassium glutamate. These channels were largely open at negative membrane potential on the cytoplasmic side of the RM membrane, mostly closed at positive voltages, permeable to amino acids, and slightly more selective for anions than cations. There was a dramatic increase in the number of open channels when 100 microM GTP was added to the cytoplasmic side of the fused RM, whereas 100 microM guanosine 5'-[gamma-thio]triphosphate caused closing of channels. ATP had no effect. A large channel of 115 pS at 45 mM potassium glutamate was also detected after the fusion of InV. As both RM and InV share the ability to translocate secretory proteins, it is possible that the 115-pS channel in both membranes represents a protein-conducting channel.
Dictyostelium discoideum contains a gene encoding a myosin I heavy chain.
Authors: Jung G , Saxe C L , Kimmel A R , Hammer J A
Published: May 2006

We have cloned and completely sequenced a gene encoding the heavy chain of Dictyostelium myosin I. Like the myosin I molecules from Acanthamoeba, the Dictyostelium myosin I heavy chain is composed of a globular head domain fused to a 45-kDa glycine-, proline-, and alanine-rich carboxyl-terminal domain, rather than the coiled-coil rod domain of conventional myosins. Comparisons of the Dictyostelium myosin I heavy-chain amino acid sequence with those of the Acanthamoeba myosins I reveal that they are highly similar throughout, including the unconventional carboxyl-terminal domains. The Dictyostelium myosin I gene is expressed in growing cells as a 3600-nucleotide mRNA. Measurements of the steady-state level of this mRNA at different times during starvation-induced aggregation and development are consistent with a role for myosin I in chemotaxis and aggregation. Generation of Dictyostelium cells lacking myosin I by gene disruption and/or antisense RNA production should provide a way to test directly the role of this nonfilamentous myosin in cell motility. These experiments will be simplified by the fact that Southern blot analyses of Dictyostelium genomic DNA are consistent with there being a single myosin I heavy-chain gene.
Specific HLA-DQB and HLA-DRB1 alleles confer susceptibility to pemphigus vulgaris.
Authors: Scharf S J , Freidmann A , Steinman L , Brautbar C , Erlich H A
Published: May 2006

The autoimmune dermatologic disease pemphigus vulgaris (PV) is associated with the serotypes HLA-DR4 and HLA-DRw6. Based on nucleotide sequence and oligonucleotide probe analysis of enzymatically amplified DNA encoding HLA-DR beta chain (HLA-DRB) and HLA-DQ beta chain (HLA-DQB; henceforth HLA is omitted from designations), we showed previously that the DR4 susceptibility was associated with the Dw10 DRB1 allele [encoding the mixed lymphocyte culture (MLC)-defined Dw10 specificity]. The DRw6 susceptibility similarly was shown to be associated with a rare DQB allele (DQB1.3), which differed from another nonsusceptible allele by only a valine-to-aspartic acid substitution at position 57. Given the linkage disequilibrium that characterizes HLA haplotypes, it is difficult to assign disease susceptibility to a specific locus rather than to a closely linked gene(s) on the same haplotype. To address this problem, we have analyzed all of the polymorphic loci of the class II HLA region (DRB1, DRB3, DQA, DQB, and DPB) on the DRw6 haplotypes in patients and controls. In 22 PV patients, 4 different DRw6 haplotypes were found that encode the same DQ beta chain (DQB1.3) but contained silent nucleotide differences at the DQB locus as well as coding sequence differences in the DQA and DRB loci. These results, obtained by using a method for allele-specific polymerase chain reaction amplification, strongly support the hypothesis that the allele DQB1.3 confers susceptibility. This DQB allele is correlated with the MLC-defined Dw9 specificity and is associated with two different DRB1 alleles (the common "6A" associated with DRw13 and the rare "6B" associated with DRw14). Since 86% (19 of 22) of DRw6+ patients contain the DQB1.3 allele (vs. 3% of controls), whereas 64% (14 of 22) contain the DRB1 allele 6B (vs. 6% of the controls), we conclude that most of the DRw6 susceptibility to PV can be accounted for by the DQ beta chain.
Regulation of the complex pattern of sevenless expression in the developing Drosophila eye.
Authors: Bowtell D D , Kimmel B E , Simon M A , Rubin G M
Published: May 2006

The sevenless gene encodes a cell surface receptor that has protein-tyrosine kinase activity and is expressed in a highly specific and complex pattern in the developing Drosophila eye. We have coupled the sevenless promoter to the reporter gene lacZ and have examined the pattern of beta-galactosidase expression in the developing eyes of transgenic larvae. Our results indicate that the dynamic pattern of sevenless protein expression is regulated transcriptionally. Promoter sequences located 5' of the coding region are insufficient for the wild-type level of gene expression but appear to be able to confer the correct pattern of expression. In contrast, enhancer sequences within the body of the gene can confer both the correct pattern and a normal level of expression on either the sevenless promoter or heterologous promoters. Thus the complex pattern of sevenless expression is redundantly encoded within proximal promoter sequences and enhancer elements internal to the gene but relies on these enhancer sequences for correct quantitative expression.
Growth hormone activates phospholipase C in proximal tubular basolateral membranes from canine kidney.
Authors: Rogers S A , Hammerman M R
Published: May 2006

To delineate pathways for "signal" transduction by growth hormone (GH) in proximal tubule, we incubated basolateral membranes isolated from canine kidney with human growth hormone (hGH) or human prolactin (hPrl) and measured levels of inositol trisphosphate (InsP3) in suspensions and of diacylglycerol extractable from the membranes. Incubation with hGH, but not hPrl, increased levels of InsP3 and diacylglycerol in a concentration-dependent manner. Half-maximal effects occurred between 0.1 and 1 nM hGH. Increased levels of InsP3 were measured after as little as 5 sec of incubation with 1 nM hGH, and increase was maximal after 15 sec. Increases were no longer detectable after 60 sec because of dephosphorylation of InsP3 in membrane suspensions. hGH did not affect rates of dephosphorylation. hGH-stimulated increases in InsP3 were detectable in membranes suspended in 0, 0.1, and 0.2 mM calcium but not in 0.3 or 1.0 microMs calcium. 125I-labeled hGH-receptor complexes with Mr values of 66,000 and 140,000 were identified in isolated basolateral membranes. Our findings establish that GH activates phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) in isolated canine renal proximal tubular basolateral membranes, potentially after binding to a specific receptor. This process could mediate "signal" transmission by GH across the plasma membrane of the proximal tubular cell and elsewhere.
Cellular proteins specifically bind single- and double-stranded DNA and RNA from the initiation site of a transcript that crosses the origin of DNA replication of herpes simplex virus 1.
Authors: Roller R J , McCormick A L , Roizman B
Published: May 2006

The small-component origins of herpes simplex virus 1 DNA synthesis are transcribed late in infection by an RNA with heterogeneous initiation sites approximately 290-360 base pairs from the origins. We report that cellular proteins react with a labeled RNA probe representing the 5' terminus of a subset of this RNA but not with the complementary strand of this RNA. The proteins form two complexes. Complex 2 was formed by all nuclear extracts tested, whereas complex 1 was invariably formed by proteins present only in nuclear extracts of mock-infected cells. Complex 1 protects a contiguous stretch of 40 nucleotides of the labeled RNA probe from nuclease degradation. Formation of complex 1 was competitively inhibited in a sequence-specific fashion by single-stranded RNA and DNA and by double-stranded RNA and DNA. The protein(s) forming complex 1 is, thus, quite distinct from known nucleic acid-binding proteins in that they recognize a specific nucleotide sequence, irrespective of the nature (single- and double-stranded RNA and DNA) of the nucleic acid. We conclude the following: (i) the proteins forming complex 1 and 2 are probably different, (ii) complex 1 is neither required throughout infection for viral replication nor able to hinder viral replication in cells in culture, and (iii) cells susceptible to infection encode one or more proteins that recognize specific sequences in single-stranded nucleic acids; either these proteins impart a compatible conformation on single-stranded nucleic acids with the conformation of the same strand in the double-stranded nucleic acid, or these proteins confer a specific, distinct conformation to both single-stranded and double-stranded nucleic acids.
Transcript encoded on the opposite strand of the human steroid 21-hydroxylase/complement component C4 gene locus.
Authors: Morel Y , Bristow J , Gitelman S E , Miller W L
Published: May 2006

The gene encoding human adrenal steroid 21-hydroxylase (P450c21) and its highly similar pseudogene are duplicated in tandem with the two genes encoding the fourth component of human serum hemolytic complement (C4). This 60-kilobase gene complex, which lies within the major histocompatibility complex on the short arm of human chromosome 6, has been studied in considerable detail because genetic disorders in steroid 21-hydroxylation and in C4 are common. We have cloned a cDNA encoded by a previously unidentified gene in this region. This gene lies on the strand of DNA opposite from the strand containing the P450c21 and C4 genes, and it overlaps the last exon of P450c21. The newly identified gene encodes mRNAs of 3.5 and 1.8 kilobases that are expressed in the adrenal and in a Leydig cell tumor but are not expressed in nonsteroidogenic tissues. The sequence of the longest cDNA (2.7 kilobases) shows no similarity to known sequences available in two computerized data bases. The 5' end of this sequence is characterized by three repeats, each encoding about 100 amino acids flanked by potential sites for proteolytic cleavage. Although numerous studies have shown that gene deletions causing congenital adrenal hyperplasia occur in this region, none of these gene deletions extends into this newly identified gene, suggesting that it encodes an essential function.
Cloning the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants.
Authors: Sato T , Theologis A
Published: May 2006

Ethylene is the plant hormone that controls several features of plant growth and development. The rate-limiting step in its synthesis is the formation of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) from S-adenosylmethionine (AdoMet), catalyzed by ACC synthase. We have isolated a complementary DNA sequence encoding ACC synthase from zucchini (Cucurbita) fruits. The biological activity of the clone was confirmed by the ability of the cloned sequence to direct ACC synthase activity in Escherichia coli and yeast. In vivo studies using the ACC cDNA as probe showed that the ACC synthase gene is induced by a diverse group of inducers, including wounding, Li+ ions, and the plant hormone auxin.
Immunological demonstration of the accumulation of insulin, but not insulin receptors, in nuclei of insulin-treated cells.
Authors: Soler A P , Thompson K A , Smith R M , Jarett L
Published: May 2006

Although insulin is known to regulate nuclear-related processes, such as cell growth and gene transcription, the mechanisms involved are poorly understood. Previous studies suggested that translocation of insulin or its receptor to cell nuclei might be involved in some of these processes. The present investigation demonstrated that intact insulin, but not the insulin receptor, accumulated in nuclei of insulin-treated cells. Cell fractionation studies demonstrated that the nuclear accumulation of 125I-labeled insulin was time-, temperature-, and insulin-concentration-dependent. Electron microscopic immunocytochemistry demonstrated that the insulin that accumulated in the nucleus was immunologically intact and associated with the heterochromatin. Only 1% of the 125I-labeled insulin extracted from isolated nuclei was eluted from a Sephadex G-50 column as 125I-labeled tyrosine. Plasma membrane insulin receptors were not detected in the nucleus by immuno electron microscopy or when wheat germ agglutinin-purified extracts of the nuclei were subjected to PAGE, electrotransfer, and immunoblotting with anti-insulin receptor antibodies. These results suggested that internalized insulin dissociated from its receptor and accumulated in the nucleus without its membrane receptor. We propose that some of insulin's effects on nuclear function may be caused by the translocation of the intact and biologically active hormone to the nucleus and its binding to nuclear components in the heterochromatin.
Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins.
Authors: Aricó B , Miller J F , Roy C , Stibitz S , Monack D , Falkow S , Gross R , Rappuoli R
Published: May 2006

The bvg locus of Bordetella pertussis is required for coordinate regulation of several factors associated with virulence. The control system is modulated by various environmental signals, including low temperature, MgSO4, and nicotinic acid. The nucleotide sequence of the bvg region has been determined and three open reading frames, bvgA, bvgB, and bvgC, are present. Twelve-base-pair linker insertion mutations in any of these open reading frames result in a Bvg- phenotype. The predicted protein products of bvgA and bvgC share homology with a family of prokaryotic regulatory proteins that respond to environmental stimuli and are members of two-component sensory transduction systems. We propose a model in which BvgB and the N-terminal portion of BvgC are localized in the periplasm. Environmental signals are recognized, transduced to the cytoplasmic portion of BvgC, and then transmitted to BvgA, a positive regulator of transcription.
Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte beta-adrenergic responsiveness.
Authors: Gulick T , Chung M K , Pieper S J , Lange L G , Schreiner G F
Published: May 2006

Reversible congestive heart failure can accompany cardiac allograft rejection and inflammatory myocarditis, conditions associated with an immune cell infiltrate of the myocardium. To determine whether immune cell secretory products alter cardiac muscle metabolism without cytotoxicity, we cultured cardiac myocytes in the presence of culture supernatants from activated immune cells. We observed that these culture supernatants inhibit beta-adrenergic agonist-mediated increases in cultured cardiac myocyte contractility and intracellular cAMP accumulation. The myocyte contractile response to increased extracellular Ca2+ concentration is unaltered by prior exposure to these culture supernatants, as is the increase in myocyte intracellular cAMP concentration in response to stimulation with forskolin, a direct adenyl cyclase activator. Inhibition occurs in the absence of alteration in beta-adrenergic receptor density or ligand binding affinity. Suppressive activity is attributable to the macrophage-derived cytokines interleukin 1 and tumor necrosis factor. Thus, these observations describe a role for defined cytokines in regulating the hormonal responsiveness and function of contractile cells. The effects of interleukin 1 and tumor necrosis factor on intracellular cAMP accumulation may be a model for immune modulation of other cellular functions dependent upon cyclic nucleotide metabolism. The uncoupling of agonist-occupied receptors from adenyl cyclase suggests that beta-receptor or guanine nucleotide binding protein function is altered by the direct or indirect action of cytokines on cardiac muscle cells.
Alterations in the p53 gene and the clonal evolution of the blast crisis of chronic myelocytic leukemia.
Authors: Ahuja H , Bar-Eli M , Advani S H , Benchimol S , Cline M J
Published: May 2006

Molecular mechanisms responsible for the clinical progression of chronic myelocytic leukemia to its accelerated phase or to blast crisis have not been defined. We found alterations of the p53 gene (p53 is a 53-kDa nuclear protein) including deletions and rearrangements in 8 of 34 patients in blast crisis and 1 of 4 patients in the accelerated phase, but in only 1 of 38 patients in the chronic phase of chronic myelocytic leukemia. Only two other examples of p53 gene alterations were found among 203 patients with hematologic malignancies and solid tumors. Transcripts of the p53 gene were uniformly found in chronic-phase cells, but gene expression was variable in blast crisis, and transcripts were reduced or undetectable in 10 of 16 patients. Heterogeneous alterations in the structure and expression of the p53 gene appear to be relatively frequent in blast crisis and may be involved in the evolution of disease.
Critical size and curvature of wave formation in an excitable chemical medium
Authors: Foerster P. , Müller S. C. , Hess B.
Published: May 2006

The critical radius for the outward propagation of waves in an excitable solution of the Belousov—Zhabotinskii reaction was experimentally analyzed and found to be ≈20 μm, being in a range predicted by theory. Thus, the wave initiation depends on the critical radius in an all-or-none fashion. For waves having high positive curvature of wave fronts, a linear relationship between the curvature and their normal velocity was established, allowing computation of a diffusion coefficient of 1.9 × 10
-5
cm
2
/s for the autocatalytic species, which agrees well with results previously obtained for negatively curved wave fronts. The analysis of the dispersion of wave velocity yielded the decrease of wave velocity for small initiation periods as predicted theoretically.
Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific γ-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase)
Authors: Grill Erwin , Löffler Susanne , Winnacker Ernst-L. , Zenk Meinhart H.
Published: May 2006

An enzyme has been discovered and characterized from
Silene cucubalus
cell suspension cultures that catalyzes the transfer of the γ-glutamylcysteine dipeptide moiety of glutathione to an acceptor glutathione molecule or a growing chain of [Glu(-Cys)]

n

-Gly oligomers, thus synthesizing phytochelatins, the metal-binding peptides of higher plants and select fungi. The enzyme was named γ-glutamylcysteine dipeptidyl transpeptidase and given the trivial name phytochelatin synthase. The primary reaction catalyzed is [Glu(-Cys)]-Gly + [Glu(-Cys)]

n

-Gly → [Glu(-Cys)]

n
+1

-Gly + Gly. The enzyme is isoelectric near pH 4.8 and has temperature and pH optima at 35°C and 7.9, respectively. Phytochelatin synthase is constitutively present in cell cultures of various plant species and its formation is not noticeably induced by heavy metal ions in the growth medium. The enzyme (
M
r
95,000) seems to be composed of four subunits, the dimer (
M
r
50,000) being also catalytically active. Cd
2+
is by far the best metal activator of the enzyme followed by Ag
+
, Bi
3+
, Pb
2+
, Zn
2+
, Cu
2+
, Hg
2+
, and Au
+
. The
K
m
for glutathione is 6.7 mM. The enzyme activity seems to be self-regulated in that the product of the reaction (the phytochelatins) chelates the enzyme-activating metal, thus terminating the enzyme reaction. The molar ratio of the γ-glutamylcysteine dipeptide in phytochelatin to Cd
2+
in the newly formed complex was 2:1.
Reaction path study of conformational transitions and helix formation in a tetrapeptide.
Authors: Czerminski R , Elber R
Published: May 2006

Conformational transitions between the 112 stable states of the tetrapeptide isobutyryl-Ala3-NH-methyl (IAN) are studied theoretically. The objective of the investigation is to advance the understanding of helix formation and of conformational transitions in polypeptides. The possible reaction paths between extended chain and helical configurations are examined in detail. The study of the multiple reaction paths in this 48-atom molecule became possible due to development of a new computational algorithm. It is shown that the helix-coil transitions in IAN follow a sequence of local dihedral flips and that the number of the available routes for the transition is significantly lower than in a random search. A quasi-melting point is obtained at 5 +/- 1 kcal (1 cal = 4.18 J)/mol above the lowest energy minimum. Below this point the molecule is trapped in one or very few minima, and above it the molecule hops between a large number of configurations.
Assay of vacuolar pH in yeast and identification of acidification-defective mutants.
Authors: Preston R A , Murphy R F , Jones E W
Published: May 2006

As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification. Vacuoles were labeled with 6-carboxyfluorescein with the membrane-permeant precursor 6-carboxyfluorescein diacetate. Dual-excitation flow cytometry was used to calibrate the pH-dependence of 6-carboxyfluorescein fluorescence in vivo. Vacuoles in wild-type yeast were mildly acidic, pH 6.2, in cells grown under several different conditions. Cultures labeled with 6-carboxyfluorescein were screened by fluorescence-ratio microscopy to detect mutants that had defects related to vacuolar acidification. A recessive nuclear mutation, vph1-1, caused an abnormally high vacuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine. Acidification in a pep12::LEU2 mutant appeared defective by fluorescence-ratio microscopy and quinacrine-uptake assays, but the vacuolar pH in the pep12::LEU2 mutant was nearly normal (pH 6.3) in flow cytometric assays.
Specific, major histocompatibility complex-unrestricted recognition of tumor-associated mucins by human cytotoxic T cells.
Authors: Barnd D L , Lan M S , Metzgar R S , Finn O J
Published: May 2006

We have previously reported the establishment of cytotoxic T-cell lines from pancreatic cancer patients, by continuously stimulating tumor-draining lymph node cells with allogeneic pancreatic tumor cell lines. After the preliminary characterization of their phenotype and tumor specificity, detailed studies performed with one of the cell lines, W.D., show that it recognizes a specific antigen, a large and heavily glycosylated mucin molecule, expressed on pancreatic and breast tumors and tumor cell lines. Although this recognition appears major histocompatibility complex (MHC)-unrestricted, the antigen receptor used by the cytotoxic T cell is the alpha/beta heterodimer, typically found on MHC-restricted T cells. The target antigen is atypical, however, in its ability to directly bind and activate the T cells in the absence of self MHC, presumably by abundant and regularly repeated antigenic epitopes. These findings are important because they demonstrate a specific T-cell response against a human tumor-associated antigen. In addition to pancreatic and breast tumors, various mucin molecules are known to be produced by other tumors of epithelial cell origin and could be expected to stimulate similar T-cell-mediated immune responses.
In vitro activity of the nitrogen fixation regulatory protein NIFA.
Authors: Santero E , Hoover T , Keener J , Kustu S
Published: May 2006

We have detected activity of the nitrogen fixation regulatory protein NIFA of Klebsiella pneumoniae in vitro. To do so we directed synthesis of NIFA in a coupled transcription-translation system and detected its ability to activate expression of a translational fusion between the nifH and lacZ genes. We infer that NIFA stimulates initiation of transcription by sigma 54 holoenzyme from the nifHDK promoter. The activity of NIFA was lost rapidly under both aerobic and anaerobic conditions at 30 degrees C and was lost somewhat less rapidly at 0 degrees C. Loss of activity was not accompanied by degradation of NIFA polypeptide. Loss of activity was approximately exponential and was not affected by NIFA concentration over a 5-fold range. Therefore, NIFA inactivation does not appear to be due to self-association. We found that the factor in crude extracts previously demonstrated to bind to the nifHDK promoter-regulatory region [Beynon, J., Cannon, M., Buchanan-Wollaston, V., and Cannon, F. (1983) Cell 34, 665-671] is the integration host factor, which is known to bend DNA. Since the binding site for integration host factor lies between the upstream binding site for NIFA and the nifHDK promoter, integration host factor may bend the DNA between these two sites to facilitate productive interactions between NIFA and sigma 54 holoenzyme.
A model for embryonal rhabdomyosarcoma tumorigenesis that involves genome imprinting.
Authors: Scrable H , Cavenee W , Ghavimi F , Lovell M , Morgan K , Sapienza C
Published: May 2006

Embryonal rhabdomyosarcomas (malignant pediatric tumors of striated muscle origin) have been shown to arise from cells that are clonally isodisomic for loci on chromosome 11p. We determined the parental origin of alleles in this genomic region in familial and sporadic cases of this disease and found that isodisomic chromosome 11p alleles in each tumor were of paternal origin. We have developed a modification of Knudson's two-hit model from these data that is capable of explaining the preferential allele retention and of resolving the apparent contradiction between such specific and early events in several embryonal tumors and discrepancies in the inheritance of predisposition in some of these diseases.
Inheritance and expression of foreign genes in transgenic soybean plants
Authors: Christou Paul , Swain William F. , Yang Ning-Sun , McCabe Dennis E.
Published: May 2006

DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants were followed in the first (R
1
) and second (R
2
) generation of self-pollinated progeny. One plant (4615) was transformed with the
Escherichia coli
genes for β-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for β-glucuronidase. Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R
1
generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover, all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R
2
generation. R
1
plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R
2
generation, demonstrating recovery of both homozygous and heterozygous R
1
plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from single transformed cells by a
de novo
organogenic pathway.
A genetically engineered cell line that produces empty capsids of B19 (human) parvovirus.
Authors: Kajigaya S , Shimada T , Fujita S , Young N S
Published: May 2006

B19 parvovirus is pathogenic in humans, causing the common childhood exanthem fifth disease and bone-marrow failure, both acute (transient aplastic crisis of hemolysis) and chronic (pure erythrocyte aplasia in immunodeficiency). The virus is tropic for a human red cell progenitor cell, and failure to culture B19 in a cell line has limited its clinical study. We cotransfected the right half of the cloned B19 genome and a minigene derived from the human dihydrofolate reductase gene (DHFR) into dhfr--Chinese hamster ovary cells and screened selected clones by RNA analysis; after amplification in methotrexate, clones were tested for capsid protein expression. A cell line, designated 3-11-5, stably expressed nearly full-length transcripts for the two capsid proteins. These cells produced the major and minor structural protein species in natural proportions that self-assembled into virion capsids. Capsids from 3-11-5 cells could be separated from virions by sucrose gradient sedimentation and had the density on cesium chloride isopycnic sedimentation of empty parvovirus capsids. Capsid protein was present in both nuclei and cytoplasm on immunofluorescence study but fractionated with the cytosol on purification. Empty capsid production was equal to or greater than virion production by infected bone-marrow cells, 1000-2000 capsids per cell, but cell growth was not diminished by capsid production. This cell line will be useful in developing practical assays for B19 parvovirus antibody and a vaccine for the virus, as well as potentially serving as a packaging cell line for gene therapy.
Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia.
Authors: Giaid A , Gibson S J , Ibrahim B N , Legon S , Bloom S R , Yanagisawa M , Masaki T , Varndell I M , Polak J M
Published: May 2006

The localization of endothelin 1 mRNA and endothelin-like immunoreactivity was investigated in samples of neurologically normal nervous system tissue from 10 adults by using in situ hybridization and immunocytochemistry. Tissue sections of spinal cord and dorsal root ganglia were hybridized with an 35S-radiolabeled endothelin 1 complementary RNA probe. Autoradiograms showed labeled neurons in the spinal cord (laminae IV-VI and many motoneurons) and numerous small and large neurons in the dorsal root ganglia. Endothelin 1 transcripts were also found in association with the endothelial layer of some blood vessels in the white matter of the spinal cord. A similar distribution of immunoreactivity was seen using three antisera to endothelin 1, but fewer cells were immunostained than were labeled after the hybridization. Two other endothelin 1 antisera immunostained the endothelial lining of blood vessels in the spinal cord white matter but not neurons. In the ganglia, endothelin 1 transcripts were localized to all cells expressing beta-preprotachykinin and most expressing calcitonin gene-related peptide mRNAs; in the majority of the motoneurons, coexistence of endothelin 1 and calcitonin gene-related peptide mRNAs was noted. There was a similar pattern of coexistence for respective peptide immunoreactivities in the ganglia and spinal cord. The expression of endothelin 1 mRNA in distinct neuronal cell types suggests that this peptide plays a part in neural transmission/modulation and adds a further dimension to the known vascular-related actions of endothelin 1.
Analysis of the sequence-specific interactions between Cro repressor and operator DNA by systematic base substitution experiments.
Authors: Takeda Y , Sarai A , Rivera V M
Published: May 2006

We measured quantitatively the binding affinities of purified Cro repressor to the chemically synthesized wild-type and mutant OR1 operators, consisting of all three possible base-pair substitutions and of thymine to uracil substitutions at each base-pair position of the 17-base-pair operator sequence. The sequence-specific interactions between Cro repressor and the operator DNA occur at the symmetrically disposed outer 7-base-pair positions of each half operator and at the central base-pair position. The binding of Cro is almost symmetrical with respect to the pseudo-twofold symmetry of the binding site. The binding free energy changes calculated from the affinity changes are mostly additive for specific Cro binding. Also the binding affinities of Cro to the operators or any other DNA sequences can be predicted by simple addition of free energy changes of single base substitutions. We isolated cro mutants by site-directed mutagenesis and studied their DNA binding to the wild-type and base-substituted mutant operators. The sequence-specific contacts derived from such studies are significantly different from the models proposed by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Takeda, Y. & Matthews, B. W. (1982) Nature (London) 298, 719-723] and by Hochschild et al. [Hochschild, A., Douhan, J., III, & Ptashne, M. (1986) Cell 47, 807-816].
Regulation of endocytic pH by the Na+,K+-ATPase in living cells.
Authors: Cain C C , Sipe D M , Murphy R F
Published: May 2006

Acidification of endocytosed ligands destined for lysosomes is biphasic, with a rapid drop to pH 6, followed by a slow decrease to pH 5. Continuous measurements of transferrin acidification have confirmed that the pH minimum in early (presorting) endosomes is approximately pH 6. On the basis of measurements of endosomal acidification in vitro, it has been proposed that the pH in the early endosome is limited by the internalization of the Na+,K+-ATPase, which generates an interior-positive membrane potential in this compartment [Fuchs, R., Schmid, S. & Mellman, I. (1989) Proc. Natl. Acad. Sci. USA 86, 539-543]. We present two lines of evidence that strongly implicate the Na+,K+-ATPase as a major regulatory element of endocytic pH in vivo. First, ouabain, a specific inhibitor of the Na+,K+-ATPase, interferes with the regulation of acidification in early endocytic compartments. Transferrin is normally rapidly acidified to pH 6.0-6.2, followed by alkalinization during recycling. In the presence of ouabain, the minimum pH of transferrin-containing endosomes decreases from 6.0-6.2 to less than 5.3. Second, ouabain eliminates the resistance to both the growth inhibitory and vacuologenic effects of chloroquine in the lysosomal acidification defective cell line CHL60-64. The phenotype of this cell line is consistent with a defect in the removal or inactivation of the early acidification regulatory elements from the late endocytic compartments. The ouabain data suggest that the defect in this cell line is due to improper localization of the Na+,K+-ATPase. A model for pH regulation and vacuolation by weak bases is discussed.
Construction of functional artificial minichromosomes in the fission yeast Schizosaccharomyces pombe.
Authors: Hahnenberger K M , Baum M P , Polizzi C M , Carbon J , Clarke L
Published: May 2006

The centromere DNAs from chromosomes I and III of Schizosaccharomyces pombe have been cloned in an artificial chromosome vector in both budding and fission yeasts. In S. pombe, synthetic linear and circular minichromosomes containing an intact centromere are stable mitotically and behave as independent genetic linkage groups that segregate properly through meiosis. These experiments present a general strategy for the isolation of centromeres from other organisms.
The interleukin 2 gene is expressed in the syncytiotrophoblast of the human placenta.
Authors: Boehm K D , Kelley M F , Ilan J , Ilan J
Published: May 2006

The lymphokine interleukin 2 is an important immune system regulatory glycopolypeptide. It is produced by antigen- or mitogen-stimulated T lymphocytes and is required for the proliferation or clonal expansion of activated T lymphocytes. In this report, it is demonstrated by RNA transfer blot hybridization that the poly(A)+ RNA population of the human placenta contains a 0.85-kilobase RNA transcript that specifically hybridizes to a human interleukin 2 cDNA probe. By using hybridization histochemistry in situ, it is further shown that interleukin 2 RNA transcripts are localized, primarily, to the syncytial (syncytiotrophoblast) layer of the human placenta. Possible roles for syncytiotrophoblast-produced interleukin 2 are suggested and discussed.