bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypodense eosinophils and interleukin 5 activity in the blood of patients with the eosinophilia-myalgia syndrome.
Authors: Owen W F , Petersen J , Sheff D M , Folkerth R D , Anderson R J , Corson J M , Sheffer A L , Austen K F
Published: May 2006

The recent recognition of the eosinophilia-myalgia syndrome (EMS) associated with the ingestion of L-tryptophan prompted an analysis of the peripheral blood eosinophil phenotypes and of the serum eosinophil hematopoietins in this disorder. Five patients with an illness characterized by the abrupt onset of aching skeletal muscles, edema, thickening and induration of the skin, and marked blood eosinophilia associated with L-tryptophan ingestion provided eosinophils, serum, or both, for evaluation. Gradient sedimentation density analysis of the peripheral blood eosinophils from four of these patients revealed that 43 +/- 13% (mean +/- SEM) of the cells had converted to the abnormal (hypodense) sedimenting phenotype. When normodense eosinophils from the reference donors were cultured for 3 days in medium supplemented with increasing concentrations of serum from the patients with EMS, their viability increased in a dose-dependent manner to 45%, which was significantly augmented over the effect of normal serum. This eosinophil viability-sustaining activity was inhibited by 76 +/- 7% (mean +/- SEM; n = 3) by the addition of anti-interleukin 5 (IL-5) but not by neutralizing antibodies monospecific for either granulocyte/macrophage colony-stimulating factor (GM-CSF) or IL-3. IL-5, an eosinophilopoietic factor, converts normodense peripheral blood eosinophils in vitro to a hypodense sedimenting form with extended viability and augmented biologic responses to activating stimuli. Thus, the presence of IL-5 in the sera of patients with EMS may contribute to the development and maintenance of the eosinophilia and may regulate the conversion of the peripheral blood eosinophils to the hypodense phenotype with augmented pathobiologic potential.
An ancient, highly conserved family of cysteine-rich protein domains revealed by cloning type I and type II murine macrophage scavenger receptors.
Authors: Freeman M , Ashkenas J , Rees D J , Kingsley D M , Copeland N G , Jenkins N A , Krieger M
Published: May 2006

Scavenger receptors have been implicated in the development of atherosclerosis and other macrophage-associated functions. The bovine type I and type II scavenger receptors are multidomain transmembrane proteins that differ only by the presence in the type I receptor of an additional, extracellular cysteine-rich C-terminal domain. The isolation of type I and type II receptor cDNAs from a murine macrophage cell line, P388D1, establishes the presence of mRNAs encoding both receptor types in a single cell. Their sequences are highly similar to the bovine cDNAs. Receptor type-specific cDNA probes map to a common locus on murine chromosomes 8, suggesting that a single gene encodes both mRNAs. The type I-specific scavenger receptor cysteine-rich (SRCR) domain helps define a previously unrecognized family of remarkably well-conserved domains. Highly homologous SRCR domains (one, three, or four per polypeptide chain) are found in diverse secreted and cell-surface proteins from humans (e.g., CD5, complement factor I), mice (Ly-1), and sea urchins (speract receptor).
Cytokinin-induced mRNAs in cultured soybean cells.
Authors: Crowell D N , Kadlecek A T , John M C , Amasino R M
Published: May 2006

We have isolated 20 cDNA clones for which the corresponding mRNAs accumulate 2- to 20-fold within 4 hr of cytokinin addition to cytokinin-starved soybean suspension cultures. These changes in mRNA levels precede the greening and cell growth induced by cytokinin in this system. Treatment with cycloheximide before cytokinin addition enhances the accumulation of these messages. In addition, the abundance of these mRNAs is influenced by auxin. We demonstrate that the mRNAs corresponding to the cDNAs in this collection display a variety of patterns of accumulation in the tissues of an intact plant. We also show that different cytokinins induce similar increases in message levels. However, analogs of cytokinin that are much less active in bioassays induce lower levels of message accumulation. Sequence analyses indicate that two of the cDNAs correspond to ribosomal protein genes. The remaining cDNAs are likely to represent previously undiscovered genes that function early in the initiation of plant cell growth and/or chloroplast development.
Induction of specific clonal anergy in human T lymphocytes by Staphylococcus aureus enterotoxins.
Authors: O'Hehir R E , Lamb J R
Published: May 2006

The exotoxins produced by certain strains of Staphylococcus aureus are able to stimulate powerful polyclonal proliferative responses and to induce nonresponsiveness by clonal deletion of T lymphocytes expressing the appropriate T-cell antigen receptor V beta gene products. This paper examines the ability of S. aureus enterotoxins to modulate the responsiveness of human CD4+ T lymphocytes with defined antigen specificity. It was observed that certain S. aureus toxins were able to activate and induce anergy in hemagglutinin-reactive T cells expressing V beta 3+ elements. After exposure to S. aureus enterotoxins A, B, and D in the absence of antigen-presenting cells, the T cells failed to respond to their natural ligand presented in an immunogenic form, despite enhanced proliferation to exogenous interleukin 2. The S. aureus toxin-induced anergy was associated with modulation of T-cell membrane receptors; down-regulation of the T-cell antigen receptor was concomitant with enhanced expression of CD2 and CD25. Interestingly, CD28 was increased only on stimulation, suggesting this protein may be differentially expressed by activated and anergic T cells. These results indicate that bacterial toxins are able to induce antigen-specific nonresponsiveness in human T cells, the application of which may be relevant in the regulation of T cells expressing a particular family of V beta gene products.
A retroviral promoter is sufficient to convert proto-src to a transforming gene that is distinct from the src gene of Rous sarcoma virus.
Authors: Zhou H , Duesberg P H
Published: May 2006

The src genes of four natural isolates of avian sarcoma viruses differ from cellular proto-src in two genetic substitutions: the promoter of the cellular gene is replaced by a retroviral counterpart, and at least six codons from the 3' terminus are replaced by retroviral or heterologous cell-derived elements. Since virus constructs with a complete proto-src coding region failed to transform avian cells but acquired transforming function by point mutations of various codons, it has been proposed that point mutation is sufficient to convert proto-src to a transforming gene. However, promoter substitution is sufficient to convert two other proto-onc genes, proto-ras and proto-myc, to retroviral transforming genes. In view of this, we have reexamined whether promoter substitution, point mutation, or both are necessary to convert proto-src into a retroviral transforming gene. It was found that a recombinant virus (RpSV), in which the src gene of Rous sarcoma virus (RSV) was replaced by the complete coding region of proto-src, transformed quail and chicken embryo cells. The oncogene of RpSV differs from the src gene of RSV in three genetic properties: (i) it is weaker--e.g., transformed cells are flatter; (ii) it is slower--e.g., focus formation takes 9 to 12 days compared to 4 days for RSV; and (iii) its host range is narrower than that of RSV--e.g., only subsets of heterogeneous embryo cells are transformed by RpSV even after weeks or months. Replacement of the proto-src 3' terminus of RpSV by that of src from RSV generates a recombinant virus (RpvSV) that equals RSV in transforming function. It is concluded that a retroviral promoter, naturally substituted via illegitimate recombination with retroviruses, is sufficient to convert at least three proto-onc genes, src, myc, and ras, to retroviral transforming genes.
Multisurface method of pattern separation for medical diagnosis applied to breast cytology.
Authors: Wolberg W H , Mangasarian O L
Published: May 2006

Multisurface pattern separation is a mathematical method for distinguishing between elements of two pattern sets. Each element of the pattern sets is comprised of various scalar observations. In this paper, we use the diagnosis of breast cytology to demonstrate the applicability of this method to medical diagnosis and decision making. Each of 11 cytological characteristics of breast fine-needle aspirates reported to differ between benign and malignant samples was graded 1 to 10 at the time of sample collection. Nine characteristics were found to differ significantly between benign and malignant samples. Mathematically, these values for each sample were represented by a point in a nine-dimensional space of real variables. Benign points were separated from malignant ones by planes determined by linear programming. Correct separation was accomplished in 369 of 370 samples (201 benign and 169 malignant). In the one misclassified malignant case, the fine-needle aspirate cytology was so definitely benign and the cytology of the excised cancer so definitely malignant that we believe the tumor was missed on aspiration. Our mathematical method is applicable to other medical diagnostic and decision-making problems.
mutA and mutC: two mutator loci in Escherichia coli that stimulate transversions.
Authors: Michaels M L , Cruz C , Miller J H
Published: May 2006

Two transversion-specific mutator loci, mutA and mutC, were identified in Escherichia coli. Mutators with high rates of A.T----T.A transversions were identified using a screening technique that relied upon the reversion of an altered lacZ gene back to wild-type via a specific A.T----T.A transversion. Among the mutators collected, one class mapped to a previously unidentified locus that we designate mutA. Analysis of reverse mutations in lacZ and forward nonsense mutations in lacI showed that the mutA strain has higher levels of A.T----T.A and G.C----T.A transversions, and to a lesser degree A.T----C.G transversions. The mutA locus maps very near to, but is separable from, mutL, at about 95 min on the E. coli chromosome. Both its mutagenic specificity and complementation experiments confirmed that mutA is distinct from mutL and from a nearby mutator locus, miaA. The phenotype of a mutA mutL double mutator strain suggests that the mutA gene product prevents some replication errors. Another mutator, designated mutC, maps very near uvrC, at 42 min, but is distinguishable from uvrC, which has no mutator effects. The specificity of reversion of lacZ mutations in a mutC strain is identical to that in a mutA strain. Also, the behavior of a mutC mutS double mutant is identical to that of a mutA mutL double mutant. It is likely that mutA and mutC are components of the same error-avoidance system.
Schur maps, Young tableaux, and supersymmetric algebra.
Authors: Rota G C , Stein J A
Published: May 2006

It is shown that the Hopf algebra dual of a supersymmetric Hopf algebra admits two presentations, and a natural isomorphism between them is described.
Amplification by host cell factors of a sequence contained within the herpes simplex virus 1 genome.
Authors: Sears A E , Roizman B
Published: May 2006

We report that a cloned 1620-base-pair (bp) DNA fragment mapping in the BamHI O fragment of herpes simplex virus 1 DNA is amplified after transfection into uninfected cells. The DNA fragment maps entirely within a portion of the open reading frame encoding the large subunit of the viral ribonucleotide reductase and does not contain any of the known lytic origins of viral DNA synthesis. Amplification of this sequence in transfected cells results in accumulation of full-sized Dpn I-resistant plasmids containing the sequence in Hirt extracts of low molecular weight DNA. Subfragments of the 1620-bp fragment were not amplified, whereas larger fragments containing the intact 1620-bp fragment were amplified. The amplification of the fragment in MCF7 cells, which express steroid receptors, was stimulated by the addition of estrogen to the medium. Addition of progesterone, dexamethasone, or testosterone was ineffective. The viral genome therefore contains at least one origin of DNA synthesis capable of supporting replication of viral DNA by cellular factors. The existence of such a host origin of DNA replication in the viral genome was predicted by the hypothesis that viral DNA is amplified by cellular enzymes in sensory neurons harboring latent virus; the link between these sequences and amplification of viral DNA during latency remains to be proven.
A different cytochrome P450 form is induced in primary cultures of rat hepatocytes.
Authors: Emi Y , Chijiiwa C , Omura T
Published: May 2006

A 49-kDa protein (P49) was discovered in the primary cultures of rat hepatocytes. P49 cross-reacted with the antibodies against purified P450IIC11 [formerly P-450(M-1)]. P49 was located in microsomes and highly induced after plating of isolated hepatocytes on collagen-coated culture dishes. To characterize P49, cDNA clones were screened from a rat liver lambda gt11 expression library. From sequence analysis of the cloned cDNAs, the amino acid sequence of P49 was deduced, and the protein was identified as a previously uncharacterized form of cytochrome P450. P49 consists of 489 amino acids and shows approximately 60% similarity with the members of class IIC subfamily of rat cytochrome P450, such as P450IIC11 and P450IIC12 [formerly P-450(F-1)]. RNA blot analysis indicates that the mRNA translating P49 was induced approximately 20- to 30-fold at 70 hr in the primary cultures compared with the liver of adult rats. Induction of P49 was not affected by density of the plated cells and the presence or absence of several hormones, serum, or antibiotics in the culture medium. On the other hand, lower induction of P49 was seen when the hepatocytes were cultured on Matrigel-coated plates. Expression of P49 mRNA was low in the liver of adult rats and was not detectable in the livers of 1- and 2-week-old male and female rats. P49 is an additional form of cytochrome P450, which is induced in the primary culture of rat hepatocytes.
Characterization of the steroid-metabolizing capacity of the hepatic cytochrome P450IIC5 expressed in COS-1 cells: 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase type activity.
Authors: Trant J M , Lorence M C , Johnson E F , Shackleton C H , Mason J I , Estabrook R W
Published: May 2006

Cytochrome P450IIC5 (rabbit liver 21-hydroxylase) is unusual among hepatic forms of cytochromes P450 because it catalyzes the conversion of one active steroid hormone (progesterone) to another active hormone (deoxycorticosterone). Another interesting aspect of this steroid-hydroxylating enzyme is the ability to convert delta 5-3 beta-hydroxysteroids to the delta 4-3-ketosteroid configuration. The delta 5-3-beta-hydroxysteroid, pregnenolone, was readily 21-hydroxylated, and this product was further metabolized to the delta 4-3-ketosteroid, deoxycorticosterone. It is suggested that the mechanism of this cytochrome P450-mediated, 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase-like reaction is through a gem-diol formation. In this study, COS-1 cells were transfected with the plasmid encoding cytochrome P450IIC5 to express a functional enzyme within the cell milieu. Transfected COS cells preferentially metabolize pregnenolone compared with all other steroids tested. Progesterone and 17 alpha-hydroxypregnenolone are also 21-hydroxylated, whereas 17 alpha-hydroxyprogesterone is a poor substrate. Substrate preference of this 21-hydroxylase differs from that seen with bovine adrenal P450XXIA1 (formerly P450C21) hydroxylase. Additionally, this study demonstrated that C19 steroids, like dehydroepiandrosterone and androstenedione, are hydroxylated at the 16 alpha position. Contrary to previous reports, no metabolite of estradiol-17 beta was detected, presumably due to the unstable nature of catechol estrogens (2-hydroxyestradiol).
Interferon gamma regulates binding of two nuclear protein complexes in a macrophage cell line.
Authors: Finn P W , Kara C J , Douhan J , Van T T , Folsom V , Glimcher L H
Published: May 2006

Interferon gamma (IFN-gamma) is a potent inducer of major histocompatibility complex (MHC) antigens during normal immune responses and in abnormal responses in autoimmune disease. In this report we identify two nuclear factors whose binding to the murine E beta class II MHC beta-chain gene is regulated by this cytokine. IFN-gamma stimulation of murine macrophages results in the appearance of increased binding of one protein complex, complex A, and decreased binding of a second, faster migrating protein complex, complex B. Although the contact residues for both of these proteins lie within the highly conserved Y-box transcriptional element, their binding specificity differs. The protein in complex B is a CCAAT-box-binding protein that may be similar or identical to NF-Y or YB1, previously identified class II Y-box-binding proteins. The DNA sequence requirements for the binding of the slower migrating complex, complex A, are not limited to CCAAT-box sequences but include sequences upstream of the Y box. These upstream sequences are required both for IFN-gamma-induced gene transcription and for IFN-gamma-induced modulation of binding activity. These data suggest a model in which upstream sequences contribute to formation of a lymphokine-regulated complex downstream. The IFN-gamma-induced binding protein described as complex A in this report differs from the IFN-gamma, -alpha, or -beta-induced nuclear factors previously identified.
Suppression of T-cell proliferation by Mycobacterium leprae and its products: the role of lipopolysaccharide.
Authors: Molloy A , Gaudernack G , Levis W R , Cohn Z A , Kaplan G
Published: May 2006

Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed the proliferative response to the mitogen anti-CD3 of peripheral blood mononuclear cells and isolated T cells. Suppression was nonspecific and occurred with cells from lepromatous and tuberculoid leprosy patients as well as control donors. The purified lipoarabinomannans from M. leprae and Mycobacterium tuberculosis had a similar spectrum of inhibition whereas their deacylated derivatives were without effect. All mycobacterial preparations of either a crude or purified state, which suppressed cellular responses, contained appreciable quantities of bacterial lipopolysaccharide by the Limulus amebocyte assay. Contamination with lipopolysaccharide could account for the extent and nonselectivity of the T-cell suppression. Suppression was also monocyte-dependent and in part due to the release of arachidonate metabolites of the cyclooxygenase pathway.
Binding of cystatin C to C4: the importance of sense-antisense peptides in their interaction.
Authors: Ghiso J , Saball E , Leoni J , Rostagno A , Frangione B
Published: May 2006

Hydropathic anticomplementarity of amino acids indicates that peptides derived from complementary DNA strands may form amphiphilic structures and bind one another. By using this concept, we have found that the antisense peptide Ser-Tyr-Asp-Leu complementary to the segment Gln-Ile-Val-Ala-Gly (residues 55-59) in cystatin C (an inhibitor of cysteine proteases) is located at positions 611-614 of the beta chain of human C4, the fourth component of complement. Here we describe and characterize the specific interaction between cystatin C and C4 by ligand affinity chromatography and ELISA. Interaction between the two native proteins was mimicked on replacement of one of them with the corresponding sense-antisense peptide coupled to a carrier protein, and the binding was inhibited by these synthetic peptides in solution. Through the interaction with C4, cystatin C may play a regulatory role in complement activation that might be of particular importance at tissue sites where both proteins are produced by macrophages.
Clusters of intragenic Alu repeats predispose the human C1 inhibitor locus to deleterious rearrangements.
Authors: Stoppa-Lyonnet D , Carter P E , Meo T , Tosi M
Published: May 2006

Frequent alterations in the structure of the complement component C1 inhibitor gene have been found in patients affected by the common variant of hereditary angioedema, characterized by low plasma levels of C1 inhibitor. This control protein limits the enzymic activity of the first component of complement and of other plasma serine proteases. Sequence comparisons of a 4.6-kilobase-long segment of the normal gene and the corresponding gene segments isolated from two patients carrying family-specific DNA deletions point to unusually long clusters of tandem repeats of the Alu sequence family as a source of genetic instability in this locus. Unequal crossovers, in a variety of registers, among Alu sequences of the clusters result in deletions of variable length that encompass exon 4. In a third family, exon 4 was instead found to be duplicated along with the same tracts of flanking introns lost in one of the deletions. In addition to undergoing Alu-mediated partial deletions and duplications, the gene is also a target for more recent retroposition events. Gross alterations in the C1 inhibitor gene account for about 20% of the hereditary angioedema chromosomes and consequently make this gene a prime example of the mutagenic liability of Alu repeats.
Membrane sidedness and the interaction of H+ and K+ on Ca2(+)-activated K+ transport in human red blood cells.
Authors: Heinz A , Hoffman J F
Published: May 2006

The sided effects of H+ on Ca2(+)-stimulated K+ transport (the Gardos channel) were studied in human red blood cells. Cells were loaded with Ca2+ during energy depletion with the internal pH adjusted to desired levels prior to treatment with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which inhibits pH equilibration across the membrane. This treatment provides a "pH clamp" whereby the internal and external H+ (H+i and H+o) concentrations can be varied separately. Channel activity was evaluated by measuring either net K+ loss or unidirectional 42K+ efflux from cells where SO2(-4) replaced Cl- on both sides of the membrane. When pHi was set at 7.4, decreasing pHo from values of 8.0 to 5.0 inhibited K+ efflux. This effect of H+o could be overcome by increasing K+o at all values of pHo. In addition, this effect of K+o could be separated from its effects on altering the membrane potential, indicating an interaction between K+o and H+o on the channel. A similar interaction was shown to occur between H+i and K+i. K+o is known to be required for activation of Ca2(+)-stimulated K+ transport, since the channel in cells preincubated in the absence of K+o (prior to exposure to Ca+i) becomes refractory to subsequent activation by Ca2+i and K+o. We found that H+o would not substitute for K+o in this regard nor would H+o inhibit the protective effect of K+o; in addition, H+ was not transported inward in exchange for K+i. Thus it would appear that there are two external sites where K+o interacts with the channel. One site is antagonized by H+o, whereas the second site is required for channel activation independent of H+ in the range studied. The inside of the channel would have, by an analogous argument, at least one site where K+i and H+i interact.
Two-locus autosomal sex determination: on the evolutionary genetic stability of the even sex ratio.
Authors: Liberman U , Feldman M W , Eshel I , Otto S P
Published: May 2006

In two-locus models of sex determination, there are two kinds of interior (polymorphic) equilibria. One class has the even sex ratio, and the other has equal allele frequencies in the two sexes. Equilibria of the second class may exhibit linkage disequilibrium. The condition for external stability of these second-class equilibria to invasion by a new allele is that the appropriately averaged sex ratio near the equilibrium be moved closer to the even sex ratio than the average among the resident genotypes. However, invasion by a new chromosome depends on the recombination fraction in a way that appears to preclude general results about the evolutionary genetic stability of the even sex ratio in this situation.
An epitope in human immunodeficiency virus 1 reverse transcriptase recognized by both mouse and human cytotoxic T lymphocytes.
Authors: Hosmalin A , Clerici M , Houghten R , Pendleton C D , Flexner C , Lucey D R , Moss B , Germain R N , Shearer G M , Berzofsky J A
Published: May 2006

T-cell-mediated cytotoxicity may play an important role in control of infection by the human immunodeficiency virus (HIV). In this study, we have identified and characterized a relatively conserved epitope in the HIV-1 reverse transcriptase recognized by murine and human cytotoxic T cells. This epitope was identified using a murine antigen-specific CD8+ class I major histocompatibility complex-restricted cytotoxic T-cell (CTL) line, a transfected fibroblast cell line expressing the HIV-1 pol gene, recombinant vaccinia viruses containing different truncated versions of the pol gene, and overlapping synthetic peptides. The optimal antigenic site was identified as residues 203-219 by synthesizing extended or truncated peptide analogs of the antigenic fragment. The optimal peptide was then tested for sensitization of autologous Epstein-Barr virus-transformed B-cell targets for killing by fresh human peripheral blood mononuclear cells. It was recognized by CTLs from several HIV-seropositive patients but not from any seronegative donor. Therefore, this peptide is a good candidate for inclusion in an AIDS vaccine. This study demonstrates that the same CTL epitope can be seen by murine and human CD8+ CTLs, as previously demonstrated for epitopes recognized by CD4+ helper T cells, and suggests the utility of screening for immunodominant CTL epitopes in mice prior to carrying out studies in humans.
Free electron laser irradiation at 200 microns affects DNA synthesis in living cells.
Authors: Berns M W , Bewley W , Sun C H , Templin P
Published: May 2006

We describe the effect of a 200-microns wavelength free electron laser beam on the ability of asynchronized and synchronized mammalian tissue culture cells to incorporate tritiated thymidine. Compared to controls (unexposed cells), a significant proportion of exposed cells exhibited a reduction in isotope incorporation. The results suggest that this wavelength may affect DNA synthesis.
Sequence-specific and polarized replication termination in vitro: complementation of extracts of tus- Escherichia coli by purified Ter protein and analysis of termination intermediates.
Authors: MacAllister T , Khatri G S , Bastia D
Published: May 2006

We have developed an in vitro replication system in which purified replication termination protein (Ter) elicits specific and polarized termination of DNA replication at terminator sites (tau) in a cell extract of tus- Escherichia coli that does not encode Ter protein. Using this system and two-dimensional agarose gel electrophoresis, we have identified intermediates with stalled replication forks. The replication bubbles contain both arrested leading strands and lagging strands that were initiated at the ColE1 origin of replication and that had progressed unidirectionally until arrested at tau. To dissect the system further, we have analyzed the kinetics of the formation of the termination intermediates and have discovered that the earliest termination intermediate had properties consistent with an arrested D loop. The D loop contained an arrested leading strand. Thus, in this test system, there appears to be a transient uncoupling of leading- and lagging-strand synthesis during termination of replication at tau sites.
Restoration of normal lysosomal function in mucopolysaccharidosis type VII cells by retroviral vector-mediated gene transfer.
Authors: Wolfe J H , Schuchman E H , Stramm L E , Concaugh E A , Haskins M E , Aguirre G D , Patterson D F , Desnick R J , Gilboa E
Published: May 2006

Retroviral vectors were constructed containing a rat beta-glucuronidase cDNA driven by heterologous promoters. Vector-mediated gene transfer into human and canine beta-glucuronidase-deficient mucopolysaccharidosis type VII fibroblasts completely corrected the deficiency in beta-glucuronidase enzymatic activity. In primary cultures of canine mucopolysaccharidosis type VII retinal pigment epithelial cells, which contain large amounts of undegraded glycosaminoglycan substrates, vector correction restored normal processing of specific glycosaminoglycans in the lysosomal compartment. In canine mucopolysaccharidosis type VII bone marrow cells, beta-glucuronidase was expressed at high levels in transduced cells. Thus, the vector-encoded beta-glucuronidase was expressed at therapeutic levels in the appropriate organelle and corrected the metabolic defect in cells exhibiting the characteristic pathology of this lysosomal storage disorder.
Early appearance and long-term persistence of the submicroscopic extrachromosomal elements (amplisomes) containing the amplified DHFR genes in human cell lines.
Authors: Pauletti G , Lai E , Attardi G
Published: May 2006

Submicroscopic extrachromosomal elements (amplisomes) containing amplified dihydrofolate reductase (DHFR) genes have been investigated in a methotrexate-resistant derivative of the human cell line HeLa BU25, 10B3, by field-inversion gel electrophoresis. The amount and kinetics of formation of these elements have been correlated with the level and time course of overall DHFR gene amplification. The amplisomes account for the great majority and possibly the totality of the amplified DHFR genes in 10B3 cells. They appear very early during the development of methotrexate resistance and increase in parallel with the amplified genes. These observations suggest that these elements are involved in an early event, possibly the first event, of gene amplification in this system. Amplisomes tend to be lost from 10B3 cells in the absence of selective pressure, although much more slowly than expected from simple dilution of nonreplicating elements. Surprisingly, under selective pressure, these elements have shown no tendency to become integrated into chromosomes or to generate minute chromosomes over a period of almost 1 year, in contrast to what has been described in other systems.
Thermodynamic extent of counterion release upon binding oligolysines to single-stranded nucleic acids.
Authors: Mascotti D P , Lohman T M
Published: May 2006

A major contribution to the binding free energy associated with most protein-nucleic acid complexes is the increase in entropy due to counterion release from the nucleic acid that results from electrostatic interactions. To examine this quantitatively, we have measured the thermodynamic extent of counterion release that results from the interaction between single-stranded homopolynucleotides and a series of oligolysines, possessing net charges z = 2-6, 8, and 10. This was accomplished by measuring the salt dependence of the intrinsic equilibrium binding constants--i.e., (delta log Kobs/delta log[K+])--over the range from 6 mM to 0.5 M potassium acetate. These data provide a rigorous test of linear polyelectrolyte theories that have been used to interpret the effects of changes in bulk salt concentration on protein-DNA binding equilibria, since single-stranded nucleic acids have a lower axial charge density than duplex DNA. Upon binding to poly(U), the thermodynamic extent of counterion release per oligolysine charge, z, is 0.71 +/- 0.03, which is significantly less than unity and less than that measured upon binding duplex DNA. These results are most simply interpreted using the limiting law predictions of counterion condensation and cylindrical Poisson-Boltzmann theories, even at the high salt concentrations used in our experiments. Accurate estimates of the thermodynamic extent of counterion binding and release for model systems such as these facilitate our understanding of the energetics of protein-nucleic acid interactions. These data indicate that for simple oligovalent cations, the number of ionic interactions formed in a complex with a linear nucleic acid can be accurately estimated from a measure of the salt dependence of the equilibrium binding constant, if the thermodynamic extent of ion release is known.
Calcitonin gene-related peptide stimulates proliferation of human endothelial cells.
Authors: Haegerstrand A , Dalsgaard C J , Jonzon B , Larsson O , Nilsson J
Published: May 2006

The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. 125I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.
Polyoma virus small tumor antigen pre-mRNA splicing requires cooperation between two 3' splice sites.
Authors: Ge H , Noble J , Colgan J , Manley J L
Published: May 2006

We have studied splicing of the polyoma virus early region pre-mRNA in vitro. This RNA is alternatively spliced in vivo to produce mRNA encoding the large, middle-sized (MTAg), and small (StAg) tumor antigens. Our primary interest was to learn how the 48-nucleotide StAg intron is excised, because the length of this intron is significantly less than the apparent minimum established for mammalian introns. Although the products of all three splices are detected in vitro, characterization of the pathway and sequence requirements of StAg splicing suggests that splicing factors interact with the precursor RNA in an unexpected way to catalyze removal of this intron. Specifically, StAg splicing uses either of two lariat branch points, one of which is located only 4 nucleotides from the 3' splice site. Furthermore, the StAg splice absolutely requires that the alternative MTAg 3' splice site, located 14 nucleotides downstream of the StAg 3' splice site, be intact. Insertion mutations that increase or decrease the quality of the MTAg pyrimidine stretch enhance or repress StAg as well as MTAg splicing, and a single-base change in the MTAg AG splice acceptor totally blocks both splices. These results demonstrate the ability of two 3' splice sites to cooperate with each other to bring about removal of a single intron.
Strands hybridize in postreplicative adenovirus overlap recombination.
Authors: Ahern K G , Wang K , Xu F Y , Mathews C Z , Pearson G D
Published: May 2006

We describe a postreplicative mechanism for adenovirus overlap recombination. An adenovirus minichromosome system was used to study overlap recombination driven by adenovirus DNA replication. Crossing-over appeared to occur equally at, but not within, the borders of the overlap between partner molecules. We propose that recombination in the minichromosome system proceeds through an intermediate formed by direct hybridization of complementary sequences on displaced strands generated by adenovirus-specific DNA replication. Some, but not all, heterologous regions in the intermediate are susceptible to mismatch correction. This pathway is intrinsically nonreciprocal and differs significantly from other adenovirus recombinational mechanisms that have been described previously.
Expression and subcellular location of the tetrapyrrole synthesis enzyme porphobilinogen deaminase in light-grown Euglena gracilis and three nonchlorophyllous cell lines.
Authors: Shashidhara L S , Smith A G
Published: May 2006

The expression and subcellular location of porphobilinogen deaminase (PBGD, also known as hydroxymethylbilane synthase; EC 4.3.1.8), one of the early enzymes of porphyrin synthesis, was investigated in light-grown Euglena and in three cell lines that do not contain chlorophyll: dark-grown Euglena, a streptomycin-bleached mutant, and Astasia longa. In wild-type Euglena, immunogold electron microscopy demonstrated that all the immunodetectable enzyme protein was in the chloroplast. PBGD was shown to be photoregulated, and like many other nuclear-encoded proteins in Euglena, the regulation was at the posttranscriptional level. In the three nonchlorophyllous cell lines, as in light-grown Euglena, a single protein of 40 kDa was detected with antiserum to PBGD. This same antiserum immunoprecipitated a larger precursor protein from the total translation products of poly(A)+ RNA, and a single transcript, which was large enough to encode the precursor, was detected on Northern blots of all four cell types. Therefore, in cells that make chlorophyll and those that do not (or cannot), PBGD is located in the plastid. No evidence was obtained for another form of the enzyme, which suggests that in Euglena there is only one pathway for the synthesis of the tetrapyrrole moiety of chlorophyll and heme.
Low temperature and peptides favor the formation of class I heterodimers on RMA-S cells at the cell surface.
Authors: Rock K L , Gramm C , Benacerraf B
Published: May 2006

RMA-S murine cells have a mutation that interferes with the assembly of class I major histocompatibility complex (MHC) heterodimers and are deficient in the expression of class I molecules on the cell surface. The mutant phenotype has been reported to be normalized upon incubation of RMA-S cells at 25 degrees C. We find that much of the increased expression of class I heterodimers is dependent on culturing RMA-S cells in bovine serum or with purified bovine beta 2-microglobulin. Furthermore, epitopes that are associated with class I MHC molecules that have bound xenogeneic beta 2-microglobulin are preferentially formed on RMA-S cells cultured at 25 degrees C. These heterologous class I molecules are thermolabile. Increased expression of class I molecules has also been observed on RMA-S cells incubated at 37 degrees C in the presence of class I-restricted peptides. We find that the increased expression of Db molecules induced by influenza virus nucleoprotein residues 365-380 is similarly dependent on culturing RMA-S cells in bovine serum or with purified bovine beta 2-microglobulin.
Barrel construction in rodent neocortex: role of thalamic afferents versus extracellular matrix molecules.
Authors: Jhaveri S , Erzurumlu R S , Crossin K
Published: May 2006

The rodent primary somatosensory cortex is characterized by aggregates of cellular and axonal elements that replicate the distribution of mystacial vibrissae on the face. The periphery-related cortical pattern ("barrels") is influenced by an amalgam of elements extrinsic (i.e., afferents) and intrinsic (i.e., neurons, glia, and their substrate) to the developing neocortex. To assign the role of some of these elements in cortical pattern formation, we have examined the temporal correlation between periphery-related patterns formed by thalamocortical axons and by extracellular matrix (ECM) molecules associated with neurons and glia in the cortex. Thalamocortical axons were labeled with the lipophilic tracer 1,1'-dioctydecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in aldehyde-fixed neonatal rat brains, and the same brains were also prepared for immunohistochemical localization of ECM molecules cytotactin and cytotactin-binding proteoglycan. We present evidence that thalamocortical axons form a periphery-related pattern well before such an organization is detectable in the distribution of ECM molecules. Furthermore, a patterned distribution of ECM molecules results from the down-regulation of these molecules from barrel centers, where thalamic axons have established vibrissa-specific patches. We conclude that thalamic axons convey the blueprint of the sensory periphery onto the neocortex and that ECM molecules do not participate in the initial formation of this pattern.
Photosynthetic electron transport controls nitrogen assimilation in cyanobacteria by means of posttranslational modification of the glnB gene product.
Authors: Tsinoremas N F , Castets A M , Harrison M A , Allen J F , Tandeau de Marsac N
Published: May 2006

A glnB gene is identified in the cyanobacterium Synechococcus sp. PCC 7942, and its gene product is found to be covalently modified as a result of imbalance in electron transfer in photosynthesis, where photosystem II is favored over photosystem I. The gene was cloned and sequenced and found to encode a polypeptide of 112 amino acid residues, whose sequence shows a high degree of similarity to the Escherichia coli regulatory protein, PII. In E. coli, PII is involved in signal transduction in transcriptional and post-translational regulation of nitrogen assimilation. Increase in ammonium ion concentration is shown to decrease covalent modification of the Synechococcus PII protein, as in enteric bacteria. We therefore propose that the photosynthetic electron transport chain may regulate the pathway of nitrogen assimilation in cyanobacteria by means of posttranslational, covalent modification of the glnB gene product. The existence of the glnB gene in different strains of cyanobacteria is demonstrated and its implications are discussed.
Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes.
Authors: Van Blerkom J
Published: May 2006

A perinuclear accumulation of mitochondria characterizes the premetaphase I stages of reinitiated meiosis in the laboratory mouse oocyte. The cellular basis of this organelle-specific translocation was examined by fluorescent probe analysis, immunostaining, and immunoelectron microscopy in oocytes cultured at specific stages of meiotic maturation in the presence and absence of drugs that influence the stability of microfilaments and microtubules. The results suggest that a temporal, spatial, and developmental relationship exists between the location of microtubule organizing centers and the progressive translocation of mitochondria to the nuclear region. The findings indicate that mitochondrial translocations are mediated by microtubules and that individual microtubule organizing centers are not only foci for mitochondrial aggregation but may also facilitate the establishment of the circular bivalent configuration.
A cluster of transcribed sequences between the Pb and Ob genes of the murine major histocompatibility complex.
Authors: Cho S , Attaya M , Brown M G , Monaco J J
Published: May 2006

The region of the murine major histocompatibility complex (MHC) between the Pb (A beta 3) and Ob (A beta 2) genes controls the expression of an intracellular complex named the LMP (low molecular weight polypeptide) complex. DNA probes for at least seven different genes mapping to this region were isolated. These hybridize to a minimum of eight different transcripts ranging from approximately 1.3 to 3.7 kilobases (kb). The deduced amino acid sequences of the corresponding cDNAs indicate that three of these genes are new members of the MHC class II gene family. These genes are transcribed in a tissue-specific pattern similar to that of the traditional class II genes. Two of the remaining four genes, HAM1 and HAM2, are homologous to one another and to a family of eukaryotic and prokaryotic transport proteins and may be involved in antigen processing. The tissue distribution of HAM1 transcripts is consistent with its proposed role in class I-restricted antigen processing, whereas HAM2 transcription appears more restricted and may be involved in antigen processing for class II-restricted T cells. The HAM2 gene may produce two differentially spliced transcripts. The identity of the remaining two genes is not known. Analyses of transcript sizes, tissue distribution, sequence, and genetic mapping data suggest that none of these genes code for LMP antigens.
Identification and characterization of a cis-acting element that interferes with glucocorticoid-inducible activation of the mouse mammary tumor virus promoter.
Authors: Tanaka H , Dong Y , Li Q , Okret S , Gustafsson J A
Published: May 2006

The rat hepatoma cell line M1.19 is stably infected by the mouse mammary tumor virus (MMTV), and the expression of the virus is induced by glucocorticoid treatment. However, in the 6.10.2 variant of M1.19, an increase in MMTV transcription is hardly detectable upon exposure to hormone. The mechanism of hormone-unresponsiveness in these cells has been unclear. In this study, we show that nuclear extract from 6.10.2 cells contains a specific DNA-binding activity that recognizes a sequence motif extending from positions -163 to -147 on the MMTV promoter. An oligonucleotide probe spanning this region binds a nuclear factor distinct from the glucocorticoid receptor. In vivo competition experiments, where increased amounts of a plasmid containing this element were transfected into 6.10.2 cells, showed a dose-dependent increase in hormonal inducibility of MMTV expression. Together, these results indicate that this sequence motif negatively modulates glucocorticoid-inducible activation of the MMTV promoter. Moreover, we have characterized a nuclear factor that preferentially binds to the coding strand of this element.
Molecular structure of Bacillus subtilis aspartate transcarbamoylase at 3.0 A resolution.
Authors: Stevens R C , Reinisch K M , Lipscomb W N
Published: May 2006

The three-dimensional structure of Bacillus subtilis aspartate transcarbamoylase (ATCase; aspartate carbamoyltransferase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) has been solved by the molecular replacement method at 3.0 A resolution and refined to a crystallographic R factor of 0.19. The enzyme crystallizes in the space group C2 with unit cell dimensions a = 258.5, b = 153.2, and c = 51.9 A and beta = 97.7 degrees. The asymmetric unit is composed of three monomers related by noncrystallographic threefold symmetry. A total of 295 of 304 amino acid residues have been built into the monomer. The last 9 residues in the C terminus were not included in the final model. Each monomer consists of 34% alpha-helix and 18% beta-strand. Three solvent-exposed loop regions (residues 69-84, 178-191, and 212-229) are not well defined in terms of electron density. The catalytic trimer of ATCase from B. subtilis shows great similarity to the catalytic trimer in Escherichia coli ATCase, which was used in constructing the model for molecular replacement. The unliganded trimer from B. subtilis, which is not cooperative, resembles the T (inactive) state slightly more than the R (active)-state form of the E. coli trimer. However, certain regions in the B. subtilis trimer exhibit shifts toward the E. coli R-state conformation.
Phonon Green's function.
Authors: Schwinger J
Published: May 2006

The concepts of source and quantum action principle are used to produce the phonon Green's function appropriate for an initial phonon vacuum state. An application to the Mossbauer effect is presented.