bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient mapping of protein antigenic determinants.
Authors: Mehra V , Sweetser D , Young R A
Published: May 2006

A recombinant DNA expression strategy has been used to deduce the amino acid sequences of six different antigenic determinants in a single protein of Mycobacterium leprae, the etiologic agent of leprosy. The gene encoding the M. leprae 65-kDa antigen was sequenced and a lambda gt11 gene sublibrary was constructed with fragments of the gene. Recombinant DNA clones producing specific antigenic determinants were isolated by screening with monoclonal antibodies, and the sequences of their insert DNAs were determined with a rapid primer-extension method. The amino acid sequence of each determinant was deduced from the minimum overlap of insert DNAs from multiple antibody-positive DNA clones. Amino acid sequences for six different epitopes were elucidated. A peptide containing sequences for one of these epitopes was synthesized and shown to bind the appropriate monoclonal antibody; this antigenic determinant is unique to M. leprae. The approach described here can be used to rapidly elucidate protein epitopes that are recognized by antibodies or T cells.
Clonal diversity and T-cell receptor beta-chain variable gene expression in enlarged lymph nodes of MRL-lpr/lpr lupus mice.
Authors: Singer P A , McEvilly R J , Noonan D J , Dixon F J , Theofilopoulos A N
Published: May 2006

The autosomal recessive lpr gene accelerates a systemic lupus erythematosus-like disease in genetically predisposed mice and induces autoantibodies in mice of normal genetic background. The molecular mode(s) of action of the lpr gene and its chromosomal location remain unknown, but it is primarily expressed as a massive T-cell proliferation manifested only in the presence of a thymus. To define the clonal diversity and maturational stage of the abnormally proliferating T cells found in enlarged lymph nodes of MRL-lpr/lpr mice, and their possible role in autoreactive B-cell activation, we analyzed their T-cell receptor beta-chain variable region (V beta) gene sequences. Twenty-five VDJ-containing beta-chain cDNA sequences were examined, each of which was found to derive from a distinct rearrangement in the correct reading frame, yielding translatable beta-chain mRNAs. An additional 10 clones were derived from truncated nonfunctional mRNAs. D beta 1 and D beta 2 elements were used equally in the sequenced clones, and 10 of the possible 12 mouse J beta elements were represented. Remarkably, 60% of the functional beta-chain mRNAs expressed V beta 8.2 or V beta 8.3 genes, whereas the equally homologous V beta 8.1 gene was not represented at all. Other V beta genes were found at lower frequencies in the library, including one previously unidentified V beta gene. The results indicate that the clonal makeup of the abnormally proliferating lymph node T cells in MRL-lpr/lpr mice is heterogeneous, but V beta gene expression is significantly skewed in favor of V beta 8.2/8.3 genes. The preferential representation of V beta 8 genes might be caused by lpr gene-induced modification of T-cell thymic processing and relate to the lpr gene-associated autoimmunity.
Prospect for prevention of human immunodeficiency virus infection: purified 120-kDa envelope glycoprotein induces neutralizing antibody.
Authors: Robey W G , Arthur L O , Matthews T J , Langlois A , Copeland T D , Lerche N W , Oroszlan S , Bolognesi D P , Gilden R V , Fischinger P J
Published: May 2006

This study initiates an effort to develop a safe vaccine against the acquired immunodeficiency syndrome (AIDS) that is caused by infection with a retrovirus designated human immunodeficiency virus (HIV) [formerly human T-cell lymphotropic virus type III (HTLV-III)]. Other retrovirus models have shown that purified external glycoprotein subunits are immunogenic. The external envelope glycoprotein of HIV (gp120) has a molecular size of 120 kDa, is responsible for virus infectivity, and induces strong antibody response in humans. Purified HIV virus preparations contain relatively little gp120 so HIV-infected cells were used as the antigen source. The gp120 was localized on cell membranes and was solubilized with low levels of nonionic detergent. The glycoprotein was further purified by immunoaffinity chromatography over a resin prepared from IgGs isolated from patients. Homogeneity was achieved following extensive dialysis and polyacrylamide gel electrophoresis. The gp120 isolated from infected cells was shown to be structurally identical by peptide maps to virion gp120 and the amino-terminal amino acid sequence confirmed that the molecule was specified by the HIV genome. Goat, horse, and rhesus monkey (Macaca mulatta) immune sera to gp120 precipitated the homologous antigen and neutralized the in vitro infectivity of HIV. The induction of neutralizing antibody indicates that a gp120 subunit vaccine against HIV is theoretically possible.
HLA-DR4 in insulin-dependent diabetic parents and their diabetic offspring: a clue to dominant inheritance.
Authors: MacDonald M J , Gottschall J , Hunter J B , Winter K L
Published: May 2006

Insulin-dependent diabetes mellitus (IDDM) susceptibility determinants are known to be associated with both HLA-DR3 and -DR4. We monitored the inheritance of HLA-DR alleles in 37 families in which IDDM affected one parent and at least one offspring in order to try to learn more about the modes of inheritance of IDDM determinants. Ninety-seven insulin-dependent diabetics whose parents did not have diabetes and 158 nondiabetics were used as control groups for estimates of DR allele frequencies in the overall diabetic and general populations. The proportion of diabetic parents who transmitted DR4 to diabetic offspring (78%) was significantly higher (P less than 0.001) than the gene frequency of DR4 in the overall diabetic population (43%). The proportion of nondiabetic parents who transmitted DR4 to diabetic offspring (22%) was not significantly different from the gene frequency of DR4 in the nondiabetic population (16%), but it was significantly lower (P less than 0.05) than the gene frequency in the overall IDDM population. These proportions suggest that inheritance of the DR4-associated IDDM susceptibility determinant is not recessive, because in recessive inheritance expression of a trait depends on each parent contributing a susceptibility determinant. The proportions of diabetic and nondiabetic parents who transmitted the DR allele associated with the susceptibility determinant would then equal one another. The transmission of predominantly DR4 from affected parents to affected offspring suggests that susceptibility to IDDM is inherited primarily via a single dose of a potent determinant associated with DR4, as in dominant inheritance. When DR3 was transmitted at all it was usually by the nondiabetic parent. Only 8% of diabetic parents transmitted DR3 but 35% of nondiabetic parents transmitted DR3. The proportion of nondiabetic parents who transmitted DR3 was similar to the gene frequency of DR3 in the overall diabetic population (29%), but it was significantly higher than the gene frequency of DR3 in the nondiabetic population (15%; P less than 0.005). The percentage of diabetic offspring with the genotype DR3DR4 (35%) was identical to the percentage of individuals in the overall IDDM population with this genotype (35%). Numerous population data indicate that the DR3DR4 genotype carries a higher relative risk for IDDM than any other genotype, which suggests synergism between the DR3- and DR4-associated determinants.The family data reported here support this synergism but suggest that the DR4-associated determinant can give substantial susceptibility independent of the DR3-associated determinant and that the DR3-associated determinant is often expressed as enhancing susceptibility in the presence of the dominant DR4- associated determinant.
The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist.
Authors: Wong E H , Kemp J A , Priestley T , Knight A R , Woodruff G N , Iversen L L
Published: May 2006

The compound MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate)] is a potent anticonvulsant that is active after oral administration and whose mechanism of action is unknown. We have detected high-affinity (Kd = 37.2 +/- 2.7 nM) binding sites for [3H]MK-801 in rat brain membranes. These sites are heat-labile, stereoselective, and regionally specific, with the hippocampus showing the highest density of sites, followed by cerebral cortex, corpus striatum, and medulla-pons. There was no detectable binding in the cerebellum. MK-801 binding sites exhibited a novel pharmacological profile, since none of the major neurotransmitter candidates were active at these sites. The only compounds that were able to compete for [3H]MK-801 binding sites were substances known to block the responses of excitatory amino acids mediated by the N-methyl-D-aspartate (N-Me-D-Asp) receptor subtype. These comprised the dissociative anesthetics phencyclidine and ketamine and the sigma-type opioid N-allylnormetazocine (SKF 10,047). Neurophysiological studies in vitro, using a rat cortical-slice preparation, demonstrated a potent, selective, and noncompetitive antagonistic action of MK-801 on depolarizing responses to N-Me-D-Asp but not to kainate or quisqualate. The potencies of phencyclidine, ketamine, SKF 10,047, and the enantiomers of MK-801 as N-Me-D-Asp antagonists correlated closely (r = 0.99) with their potencies as inhibitors of [3H]MK-801 binding. This suggests that the MK-801 binding sites are associated with N-Me-D-Asp receptors and provides an explanation for the mechanism of action of MK-801 as an anticonvulsant.
Directly observed relations in complete galaxy samples and the predictions of redshift-distance power laws
Authors: Segal I. E.
Published: May 2006

Directly observed relations in complete galaxy samples (apparent magnitude or diameter vs. redshift) are compared with the predictions of redshift-distance power laws. The predictions are obtained by an objective, nonparametric, statistically uniform, and fully reproducible procedure. In all cases the linear law fits even more poorly than a cubic law, and the optimal law is approximately quadratic. Even a 1.2 power law is conspicuously better-fitting than a linear law. The results of the present study in terms of directly measured quantities are consistent with and confirm earlier studies in terms of theoretical quantities such as absolute magnitudes and diameters. They show that there is no positive evidence for the Hubble law in manifestly fair galaxy samples and that the law can be reconciled with the data in complete samples only, if at all, by the adjunction of a tissue of ancillary hypotheses, none of which is capable of direct observational substantiation.
Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin.
Authors: Wewer U M , Liotta L A , Jaye M , Ricca G A , Drohan W N , Claysmith A P , Rao C N , Wirth P , Coligan J E , Albrechtsen R
Published: May 2006

The human laminin receptor was purified and molecularly cloned to investigate its biosynthetic regulation. Laminin receptor from normal and neoplastic tissue was preparatively affinity purified to homogeneity based on the high affinity of the receptor for laminin. The apparent molecular weight of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were isolated after screening a human endothelial lambda gt11 cDNA library with a monoclonal antibody directed against a domain of the laminin receptor involved in ligand binding. Definitive identification of the cDNA clones was based on comparison of cDNA sequence with the amino acid sequence of a cyanogen bromide-generated octapeptide of purified placental laminin receptor. The laminin receptor mRNA is approximately 1700 bases long. The level of laminin receptor mRNA in a variety of human carcinoma-derived cell lines correlated with the number of laminin receptors on the cell surfaces of those cells. This suggests that the amount of laminin receptor mRNA may be a rate-limiting control step in the biosynthesis of the laminin receptor, and hence in the regulation of cellular attachment to basement membranes via laminin.
Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.
Authors: Rein A , McClure M R , Rice N R , Luftig R B , Schultz A M
Published: May 2006

It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.
Nucleotide sequence of rat preputial gland beta-glucuronidase cDNA and in vitro insertion of its encoded polypeptide into microsomal membranes.
Authors: Nishimura Y , Rosenfeld M G , Kreibich G , Gubler U , Sabatini D D , Adesnik M , Andy R
Published: May 2006

We have selected the rat preputial gland beta-glucuronidase as a model protein to study the sorting of newly synthesized lysosomal hydrolases to the lysosome. The complete coding sequence of beta-glucuronidase messenger RNA was determined from the sequences of a group of overlapping cDNA clones isolated from preputial gland cDNA libraries. The beta-glucuronidase mRNA primary translation product contains 648 amino acids, including an amino-terminal signal sequence of 22 residues. The polypeptide has four potential sites for the addition of asparagine-linked core oligosaccharides. A 376-residue segment of beta-glucuronidase shows extensive homology (23% sequence identity) to a portion of Escherichia coli beta-galactosidase. This homology most likely reflects an evolutionary relationship between the bacterial and eukaryotic enzymes and the conservation of structural features necessary for the glycosidase activity of both proteins. Translation of mRNA synthesized in vitro by transcription of a cDNA containing the entire beta-glucuronidase coding region yielded a polypeptide that was immunoprecipitated with anti-beta-glucuronidase antiserum and had the same electrophoretic mobility as the primary translation product of natural beta-glucuronidase mRNA. In the presence of microsomal membranes, the in vitro-synthesized beta-glucuronidase underwent cotranslational incorporation into the microsomes, as indicated by removal of the signal sequence and the addition of several oligosaccharide chains. The beta-glucuronidase cDNA will provide a useful tool to study the mechanism of mannose phosphorylation and other aspects of the sorting of lysosomal enzymes to lysosomes.
Stable integration of mouse DNA into Ia-negative human B-lymphoma cells causes reexpression of the human Ia-positive phenotype.
Authors: Guardiola J , Scarpellino L , Carra G , Accolla R S
Published: May 2006

RJ 2.2.5, a variant of the human B-lymphoma cell line Raji, does not express the HLA-DR, -DQ, and -DP class II (or Ia) histocompatibility antigens, as a result of a defect in the transcription of the corresponding genes. This defect is corrected after fusion of RJ 2.2.5 cells with mouse Ia-positive cells. Previous work showed that the trans-acting transcriptional activator supplied by the mouse cells is encoded by a locus on mouse chromosome 16. We show here that reexpression of human major histocompatibility complex class II genes by RJ 2.2.5 cells can also be achieved by stable integration of mouse genomic sequences into the RJ 2.2.5 genome after DNA-mediated gene transfer.
Suppression of type II collagen-induced arthritis by intragastric administration of soluble type II collagen.
Authors: Nagler-Anderson C , Bober L A , Robinson M E , Siskind G W , Thorbecke G J
Published: May 2006

Although oral administration of protein antigens may lead to specific immunologic unresponsiveness, this method of immunoregulation has not been applied to models of autoimmune disease. Type II collagen-induced arthritis is an animal model of polyarthritis induced in susceptible mice and rats by immunization with type II collagen, a major component of cartilage. Intragastric administration of soluble type II collagen, prior to immunization with type II collagen in adjuvant, suppresses the incidence of collagen-induced arthritis. Administration of denatured type II collagen has no observable effect on the incidence or severity of the disease. The overall magnitude of the antibody response is not significantly reduced in collagen-fed mice as compared to controls. While the isotype distribution of the anti-collagen antibodies is similar in the two groups, there is a tendency toward reduced IgG2 responses in the collagen-fed mice.
Identification and properties of N-methyl-D-aspartate receptors in rat brain synaptic plasma membranes.
Authors: Monaghan D T , Cotman C W
Published: May 2006

The excitatory amino acid receptors selectively activated by N-methyl-D-aspartate (N-Me-D-Asp) (also known as NMDA) are a major determinant of central nervous system neuronal excitability. We report here that rat brain synaptic plasma membranes contain a distinct population of L-[3H]glutamate binding sites with pharmacological properties indicative of the N-Me-D-Asp receptor. The N-Me-D-Asp sites are readily distinguished from other L-[3H]glutamate binding and uptake sites by their sharp pH optimum, more rapid association rate, preferential localization in synaptic structures, and lack of dependence on temperature and inorganic ions. As with other receptor systems, ligand binding at the N-Me-D-Asp site is reduced by guanine nucleotides but not by adenosine nucleotides. Binding is insensitive to ketamine and cyclazocine, indicating that sigma opiates inhibit N-Me-D-Asp excitation at a site different from that of the N-Me-D-Asp binding site. The quantitative pharmacological properties of N-Me-D-Asp-sensitive L-[3H]glutamate binding sites determined in a well-defined dendritic field (stratum radiatum of CA1) by quantitative autoradiography closely correlate to those of both the electrophysiologically identified N-Me-D-Asp receptors in the same dendritic field and the N-Me-D-Asp sites studied in membrane preparations. Under conditions that selectively reveal N-Me-D-Asp receptors, these sites are found to exhibit considerable anatomical specificity as evidenced by variations within cortical, striatal, and thalamic regions. Autoradiography also showed that regions in rodent and primate brain that are especially sensitive to anoxic and excitotoxic neuronal damage (e.g., Sommer's sector or CA1) have a high level of N-Me-D-Asp sites. Since N-Me-D-Asp receptors are known to contribute to these causes of neuronal loss, their selective distribution partially accounts for the pattern of selective damage seen in these pathological conditions.
Recombination within the myelin basic protein gene created the dysmyelinating shiverer mouse mutation.
Authors: Molineaux S M , Engh H , de Ferra F , Hudson L , Lazzarini R A
Published: May 2006

Shiverer (shi) is an autosomal recessive mutation in the mouse characterized by an almost total lack of central nervous system myelin. While small amounts of other myelin components are present in the brain of the shi mouse, the four forms of myelin basic protein (MBP) are not detectable. Previous investigations by us and others indicate that the MBP gene has undergone a major rearrangement in the shi mutant. Herein, we report in detail the nature and extent of the rearrangement: a 20-kilobase region within the MBP gene is missing in the mutant. We map the 5' breakpoint of the deletion to the second intron and the 3' breakpoint to a site 2 kilobases beyond the last MBP exon. The junction of the upstream and downstream portions of the gene contains only one nucleotide not accounted for by the wild-type MBP gene sequence. The 3' side of the deletion occurs in the 3rd of 11 tandem repeats of a 31-base-pair sequence. This region is rich in alternating purine and pyrimidine stretches, sequences that have been associated with both Z-DNA structures and gene rearrangements. The recombination junction shares several features with the junctions characterized by Anderson et al. [Anderson, R., Kato, S. & Camerini-Otero, D. (1984) Proc. Natl. Acad. Sci. USA 81, 206-210] in mouse L cells and is consistent with their model for a partially homologous recombination event. The structure of the shi recombination junction suggests that the donor DNA molecules were aligned in a partially homologous region before staggered cutting and joining occurred.
Inhibition of the in vivo parathyroid hormone-mediated calcemic response in rats by a synthetic hormone antagonist.
Authors: Doppelt S H , Neer R M , Nussbaum S R , Federico P , Potts J T , Rosenblatt M
Published: May 2006

The parathyroid hormone (PTH) analog, [Tyr34]bovine PTH-(7-34)-amide, can inhibit the PTH-mediated elevation of plasma calcium in thyroparathyroidectomized rats in vivo. The analog is devoid of PTH-like agonist activity in this system. Repeated doses of analog inhibit the animal's calcemic response to PTH. The elevation in serum calcium levels mediated by PTH in this assay reflects PTH action (calcium mobilization) on bone. Earlier studies demonstrated antagonist properties of the analog in a renal-based assay; PTH-stimulated increases in urinary phosphate and cyclic AMP excretion were completely inhibited by the synthetic analog. Along with previous studies, this report indicates that [Tyr34]bovine PTH-(7-34)-amide is an effective in vivo antagonist for several major parameters of PTH action in both kidney and bone.
Deconvolution of non-Gaussian linear processes with vanishing spectral values
Authors: Lii Keh-Shin , Rosenblatt Murray
Published: May 2006

We consider the problem of estimating the filter generating a non-Gaussian linear process and the deconvolution of that process when the spectral density of the process has zeros. Without using a minimum phase assumption we show that often if there are only finitely many zeros there are procedures to effect such an estimation and deconvolution.
Nucleotides flanking the promoter sequence influence the transcription of the yeast mitochondrial gene coding for ATPase subunit 9.
Authors: Biswas T K , Getz G S
Published: May 2006

The conserved nonanucleotide sequence functions as a promoter in the yeast (Saccharomyces cerevisiae) mitochondrial genome. A mitochondrial gene, Oli 1, which codes for ATPase subunit 9, has two identical nonanucleotide promoter sequences separated by 78 nucleotides, but they initiate transcription with very different efficiencies in vivo and in vitro. Deletion analysis has revealed that the nucleotide at position +2 of the weak downstream promoter accounts for its poor in vitro transcriptional activity. This finding was confirmed with site-specific mutations at +2 and +3 positions of a consensus synthetic promoter. The nonanucleotide mitochondrial promoter with a pyrimidine at position +2 acts as a weak promoter, whereas the same sequence with a purine at the +2 position functions as a strong promoter. The nucleotide at the +3 position further contributes to the relative promoter strength. These results suggest not only that the conserved nine-nucleotide sequence is required for the correct transcriptional initiation but also that other neighboring nucleotides influence the efficiency of promoter function.
Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells.
Authors: Ledley F D , Grenett H E , McGinnis-Shelnutt M , Woo S L
Published: May 2006

Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into psi 2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected psi 2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylalanine hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normally synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.
Eosinophil differentiation factor also has B-cell growth factor activity: proposed name interleukin 4.
Authors: Sanderson C J , O'Garra A , Warren D J , Klaus G G
Published: May 2006

A mouse lymphokine that stimulates the production of functional eosinophils in liquid bone marrow cultures has recently been described [Sanderson, C.J., Warren, D.J. & Strath, M. (1985) J. Exp. Med. 162, 60-74]. This factor appears to be specific for the eosinophil lineage in hemopoietic differentiation and is analogous to colony-stimulating factors described for other hemopoietic lineages. In this paper we report that this factor appears to be identical to the B-cell growth factor II described by Swain and Dutton [Swain, S.L. & Dutton, R.W. (1982) J. Exp. Med. 156, 1821-1834]. This conclusion is based on the coordinate expression of the two activities by a panel of alloreactive T-cell clones and lines and on copurification through a series of protein separation techniques. The reason for a single lymphokine's having these widely differing biological activities is unclear, and its duality presents problems in using terminology based on either assay system. For this reason we propose the name "interleukin 4" for this molecule, and we suggest the defining property should be its eosinophil-differentiating activity.
In vitro development of B lymphocytes from long-term cultured precursor cells.
Authors: Denis K A , Witte O N
Published: May 2006

An in vitro system for the study of B-lymphocyte differentiation from precursor elements has been established. Two separate sets of culture conditions are utilized to control B-lymphocyte precursor growth versus differentiation. The first, modeled after the bone marrow culture system of Dexter and colleagues [Dexter, T. M. & Testa, N. G. (1976) in Methods in Cell Biology, ed. Prescott, D. M. (Academic, New York), Vol. 14, pp. 387-395], allows precursor replication whereas the second set of culture conditions is permissive for differentiation to more mature members of the B-lymphocyte lineage. The shift from one set of conditions to the other allows us to follow the kinetics of this differentiation from precursor cells to pre-B and B lymphocytes over a 5-week period. The resulting population of B-lineage cells synthesizes heterogeneous immunoglobulin molecules as analyzed by two-dimensional gel electrophoresis and has a heterogeneous array of immunoglobulin gene rearrangements, suggesting an origin from very early uncommitted precursors. This in vitro system will allow the study of genes that regulate B-lymphocyte differentiation and will aid in the isolation of the cellular intermediates in the B-cell developmental pathway.
Central action of dendrotoxin: selective reduction of a transient K conductance in hippocampus and binding to localized acceptors.
Authors: Halliwell J V , Othman I B , Pelchen-Matthews A , Dolly J O
Published: May 2006

Dendrotoxin, a small single-chain protein from the venom of Dendroaspis angusticeps, is highly toxic following intracerebroventricular injection into rats. Voltage-clamp analysis of CA1 neurons in hippocampal slices, treated with tetrodotoxin, revealed that nanomolar concentrations of dendrotoxin reduce selectively a transient, voltage-dependent K conductance. Epileptiform activity known to be induced by dendrotoxin can be attributed to such an action. Membrane currents not affected directly by the toxin include (i) Ca-activated K conductance; (ii) noninactivating voltage-dependent K conductance; (iii) inactivating and noninactivating Ca conductances; (iv) persistent inward (anomalous) rectifier current. Persistence of the effects of the toxin when Cd was included to suppress spontaneous transmitter release indicates a direct action on the neuronal membrane. Using biologically active, 125I-labeled dendrotoxin, protein acceptor sites of high affinity were detected on cerebrocortical synaptosomal membranes and sections of rat brain. In hippocampus, toxin binding was shown autoradiographically to reside in synapse-rich and white matter regions, with lower levels in cell body layers. This acceptor is implicated in the action of toxin because its affinities for dendrotoxin congeners are proportional to their central neurotoxicities and potencies in reducing the transient, voltage-dependent K conductance.
Endogenous inhibitor of nonlysosomal high molecular weight protease and calcium-dependent protease.
Authors: Murakami K , Etlinger J D
Published: May 2006

An endogenous inhibitor of high molecular weight protease was purified from human erythrocytes and partially characterized. The inhibitor was isolated by DEAE-Sephacel ion-exchange chromatography followed by separation on a Bio-Gel A-0.5m column. The inhibitor displayed a native Mr of 240,000 and contained a single subunit of Mr 40,000 after NaDodSO4/polyacrylamide gel electrophoresis. The Mr 240,000 hexamer inhibited high molecular weight protease noncompetitively (Ki = 8.3 X 10(-8) M) and showed marked susceptibility to proteolytic digestion and heat treatment. The purified factor was also a potent inhibitor of calcium-dependent protease (Ki = 2.8 X 10(-8) M), whereas it had no effect on trypsin, chymotrypsin, or papain. Heat treatment (50-70 degrees C X 10 min) caused loss of inhibition against high molecular weight protease; however, inhibition of calcium-dependent protease was stable under the same conditions. This result is consistent with different domains on the inhibitor that interact with high molecular weight protease and calcium-dependent protease. Together with earlier studies in which repression of inhibitor by an ATP-ubiquitin-dependent process was proposed, the present results suggest a general mechanism for regulation of multiple nonlysosomal proteases that are complexed with endogenous inhibitors.
Identification of a pituitary factor responsible for enhancement of plasminogen activator activity in breast tumor cells.
Authors: Mira-y-Lopez R , Joseph-Silverstein J , Rifkin D B , Ossowski L
Published: May 2006

Sheep pituitary glands contain a protein that stimulates plasminogen activator (PA) activity 3- to 20-fold in serum-free cultures of T47D, MTW9/PL, and SC115 breast tumor cells. This protein was found to be similar to basic fibroblast growth factor (bFGF) in size, cationic nature, and affinity for heparin. Purified human placental bFGF, a homologue of human and bovine pituitary bFGF, was effective in stimulating mammary tumor cell PA at a concentration of 1 ng/ml. Antibodies to placental bFGF blocked the PA stimulatory activity of sheep pituitary extracts. Because of these properties, the active protein in sheep pituitary glands was identified as bFGF. This represents another function of bFGF, indicating that its spectrum of target cells is wider than previously thought. Because of previously established correlations between PA production in vitro and tumor growth in vivo, we suggest that bFGF may contribute to the growth of breast carcinomas in vivo. Other types of carcinomas may also be affected by bFGF.
Codon insertion mutants of the adenovirus terminal protein.
Authors: Freimuth P I , Ginsberg H S
Published: May 2006

A series of codon insertion mutants was isolated following restriction site-directed linker insertion mutagenesis of the open reading frame for the type 5 adenovirus terminal protein precursor. The conditionally lethal mutant H5sub100 bears an insertion mutation upstream of the first AUG in the reading frame, fails to replicate its DNA under nonpermissive conditions, and was assigned to the terminal protein complementation group. These data establish that terminal protein is an essential polypeptide required for DNA replication in vivo and indicate that the NH2-terminal region of the precursor is encoded in an upstream mRNA leader. The extended eclipse period of the viral replication cycle in H5in179-infected cells is probably a consequence of delayed onset of DNA replication. Analysis of DNA replication in coinfections with wild-type virus shows that the in179 mutation has cis and trans effects. The trans-dominant, negative-complementing in179 terminal protein precursor inhibits wild-type DNA replication in a dose-dependent manner. Replication of parental in179 templates is not stimulated by an excess of coinfecting wild-type virus, indicating that the mutant terminal protein covalently bound to the in179 template in some way interferes with the replication of that template. The implications of these results for the structure and function of the terminal protein are discussed.
Trans-acting elements modulate expression of the human c-myc gene in Burkitt lymphoma cells.
Authors: Chung J , Sinn E , Reed R R , Leder P
Published: May 2006

We have used a competition assay to identify the targets of trans-acting elements that modulate the expression of the human c-myc gene (designated MYC in human gene nomenclature). For this purpose, a c-myc hybrid indicator gene was formed by joining the c-myc promoter region, first noncoding exon, and intron to the bacterial gene for chloramphenicol acetyltransferase (CAT). The test assay consisted of cotransfecting the indicator gene with competing fragments of DNA derived from suspected control regions of the c-myc gene. Such experiments test the hypothesis that control regions are often targets for the binding of trans-acting regulatory factors that can be diverted to competing fragments of DNA. A negatively acting element will be diverted from the indicator gene, allowing the gene's enhanced expression, whereas a positively acting element will behave oppositely. Control indicator genes driven by non-myc promoters assess the specificity of the effect. Using this approach, we find three c-myc regions that are capable of enhancing the expression of the indicator gene in competition assays (i.e., putative sites of negative modulation). In addition, we find sequences near the c-myc promoters that suppress expression in competition assays (i.e., putative binding sites of positively acting factors). These results, with appropriate controls, suggest the existence of target sites near the c-myc gene that specifically modulate its expression both positively and negatively. Their locations fit well with regions damaged or lost in many Burkitt lymphoma and murine plasmacytoma translocations.
Transforming gene in human atherosclerotic plaque DNA.
Authors: Penn A , Garte S J , Warren L , Nesta D , Mindich B
Published: May 2006

The monoclonal hypothesis equates atherosclerotic plaques with benign smooth muscle cell tumors and proposes that plaques can arise via mutational or viral events. Here, we provide direct evidence that molecular events, heretofore associated only with tumor cells, are common to plaque cells as well. Three distinct groups of human coronary artery plaque (hCAP) DNA samples transfected into NIH 3T3 cells gave rise to transformed foci. DNA samples from a panel of normal noncancerous human tissues, including coronary artery, were negative in the assay. Southern-blotted focus DNA yielded positive signals when hybridized to the 32P-labeled nick-translated repetitive human "Alu" DNA sequence. The DNA from cloned foci was used successfully in a second round of transfection. Focus DNA hybridized to nick-translated v-Ki-ras, v-Ha-ras, or N-ras probes failed to detect human fragments of these genes. Primary focus cells from each of five clones elicited tumors after injection into nude mice (6/42). Several distinct high molecular weight (greater than 6.6 kilobases) bands were detected after BamHI-digested tumor DNA was hybridized to Alu. Preliminary characterization of these hCAP DNA-associated tumors indicates that they are similar to the fibrosarcomas that arise after injection of ras-transformed cells into nude mice. We propose that transforming genes in plaque cells behave in a manner analogous to the way in which oncogenes behave in cancer cells.
Cytosolic free calcium and cell spreading decrease in fibroblasts from aged and Alzheimer donors.
Authors: Peterson C , Ratan R R , Shelanski M L , Goldman J E
Published: May 2006

Aging and Alzheimer disease lead to alterations in calcium homeostasis. The concentration of cytosolic free calcium in cultured skin fibroblasts during aging and Alzheimer disease was determined with the calcium-sensitive fluorescent dyes quin-2 and fura-2. The Alzheimer donors showed a decline of 70% when compared to age-matched controls (P less than 0.001) and 81% when compared to cells from young adult donors (P less than 0.001). This reduction in quin-2-calcium fluorescence does not appear to be due to quenching by heavy metals or alterations in intracellular pH. Similar decreases in free cytosolic calcium were observed with fura-2. In addition, cells from aged and Alzheimer donors spread more slowly than those from young donors, and this deficit can be partially reversed by treatment with the calcium ionophore A23187. These studies agree with accumulating evidence that, at the cellular level, Alzheimer disease is a systemic, as well as cerebral, disease. The precise molecular basis of the decreased cytosolic calcium in fibroblasts is unknown, but there is evidence that it may be pathophysiologically important.
Ultrasonic absorption evidence for enhanced volume fluctuations in the tobacco mosaic virus protein helical aggregate
Authors: Cerf R. , Dormoy Y.
Published: May 2006

The increased ultrasonic absorption brought about by self-assembly of biomolecules is analyzed for the assembly process from the 20S aggregate to the helical rod of tobacco mosaic virus protein in solution, designated here as the 20S → P-helix transition. The analysis is based on theoretical developments in ultrasonic relaxation spectrometry presented previously and illustrates the possibility that this technique can be used for characterizing fluctuations. The analysis makes use of NMR data for the system in solution and of x-ray diffraction data for the closely related transition from the two-ring disk to the virion. These x-ray data comprise the high-resolution structures and the Debye-Waller temperature factors of the main chain atoms of both the two-ring disk in crystals and the virion in oriented gel form. First, reduced ultrasonic spectra are obtained for the 4S, 20S, and helical rod aggregates. The fluctuation-enhancement factor for the helical rod is determined independently of any deconvolution into normal modes of relaxation and is shown not to depend on the particular procedure of reduction employed. The increase of ultrasonic absorption in the 20S → P-helix transition primarily reveals enhancement of the relaxing system's normal-mode volume fluctuations. The observed relaxations probably involve one conformational process per subunit. The normal-mode volume fluctuations are then estimated from a bimodal least-squares best fit to the data, and a lower bound for the reaction volume associated with the fast steps is obtained. Two mechanisms are considered as follows: (
i
) a destabilization process in which the free-energy difference between two states is reduced and (
ii
) an increase in reaction volumes of local conformation changes in the helical aggregate, resulting from the formation of a “carboxyl cage-like” structure and from the change in environment produced inside the cage. Increased reaction volumes would not be detected with x-ray diffraction. The possible occurrence of fluctuations at the RNA binding site raises the question of whether a quaternary structure that exhibits significant conformational fluctuations must be present for the binding of the nucleic acid.
Transformation-dependent increases in endogenous cytochalasin-like activity in chicken embryo fibroblasts infected by Rous sarcoma virus.
Authors: Magargal W W , Lin S
Published: May 2006

Transformation of chicken embryo fibroblasts by infection with Rous sarcoma virus has been shown to cause disruption of actin filament organization as seen with fluorescence staining techniques. This study is an attempt to use quantitative biochemical techniques to compare actin-related parameters in normal and transformed cells. Normal cells and cells infected with a temperature-sensitive mutant virus (NY68) and grown at the restrictive temperature of 41.5 degrees C have normal bundles of actin filaments, or F-actin; these cells also have about the same number of high-affinity cytochalasin binding sites at the ends of F-actin (approximately 5 pmol of sites per mg of cellular protein; Kd, 20 nM). In contrast, infected cells grown at the permissive temperature of 37 degrees C have a more diffuse pattern of actin filaments, and the number of cytochalasin binding sites in these transformed cells was below the level of detection. DNase I inhibition assays showed that the percent of unpolymerized actin, or G-actin, in cell extracts was not significantly different between normal and transformed cells (approximately 50%). In assays of cell extracts for endogenous cytochalasin-like activity on actin filaments (i.e., retardation of filament assembly at the fast-growing end, inhibition of cytochalasin binding to actin "nuclei," and decrease of low-shear viscosity of solutions of actin filaments), infected cells at 37 degrees C showed a higher level of activity per mg of protein than did uninfected cells or infected cells at 41.5 degrees C. These results suggest that the increase in endogenous cytochalasin-like activity in transformed cells may relate to the decrease in measurable cytochalasin binding sites and the abnormal distribution of actin filaments previously seen by fluorescence staining techniques.
Fine specificity, idiotypy, and nature of cloned heavy-chain variable region genes of murine monoclonal rheumatoid factor antibodies.
Authors: Manheimer-Lory A J , Monestier M , Bellon B , Alt F W , Bona C A
Published: May 2006

We investigated the immunochemical and molecular characteristics of murine monoclonal rheumatoid factors. Study of the fine specificity of 20 monoclonal rheumatoid factor antibodies shows a wide degree of heterogeneity. However, many express an interstrain cross-reactive idiotype. We show that our rheumatoid factors utilize a restricted set of the heavy-chain variable region (VH) repertoire representing the more 3' VH families. Preferential expression of 3' VH families is known to occur early in development. We report the nucleotide sequence of two cloned rheumatoid factor VH genes, Y19-10 (VH J558) and 129-48 (VH 7183) in which no major differences are observed between VH genes encoding the heavy chain of autoantibodies and antibodies against foreign antigens.
Structure-function analysis of murine interleukin 1: biologically active polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of a 270-amino acid precursor.
Authors: DeChiara T M , Young D , Semionow R , Stern A S , Batula-Bernardo C , Fiedler-Nagy C , Kaffka K L , Kilian P L , Yamazaki S , Mizel S B
Published: May 2006

Murine interleukin 1 (IL-1) is initially synthesized as a 270-amino acid precursor protein. Guided by amino-terminal end sequence analyses of mouse macrophage-derived IL-1, it was shown that expression of the carboxyl-terminal 156 amino acids (i.e., amino acids 115-270) of this precursor in Escherichia coli yields biologically active recombinant IL-1 (rIL-1) protein. To answer questions about precursor processing and the size of the smallest biologically active IL-1 fragment, we have engineered deletions of the rIL-1 (115-270) gene to encode two amino-terminal deletion analogs, rIL-1 (131-270) and rIL-1 (144-270), and a carboxyl-terminal deletion analog, rIL-1 (131-257, 270). The analogs were produced in E. coli, purified to homogeneity, and assayed for biological activity on murine thymocytes, human rheumatoid synovial cells, and human dermal fibroblasts and for their ability to bind to IL-1 receptors on murine EL-4 thymoma cells. The amino-terminal deletion analog rIL-1 (131-270) possessed a specific activity in the murine thymocyte proliferation assay equivalent to that of the 115-270 parent protein and exhibited significant biological activity in stimulating the production of collagenase and prostaglandin E2 by synovial cells and fibroblasts. The more extensive amino-terminal deletion analog rIL-1 (144-270) was inactive in all biological assays and failed to compete in the receptor binding assay. The carboxyl-terminal deletion analog rIL-1 (131-257, 270) competed less efficiently (by a factor of 100) in the receptor binding assay, retained weak biological activity on synovial cells and fibroblasts, and only demonstrated full intrinsic activity in the thymocyte proliferation assay when 100-200 times more protein was assayed. These results suggest that biologically active murine IL-1 polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of the 270-amino acid precursor. Furthermore, it appears that the integrity of the carboxyl terminus of the 270-amino acid precursor is important for activity but that different amino termini can be utilized to generate molecules with equivalent specific activities. This amino-terminal end flexibility supports a processing model for IL-1 maturation that partially explains IL-1 polypeptide heterogeneity.
Cardiac myocyte hypertrophy is associated with c-myc protooncogene expression.
Authors: Starksen N F , Simpson P C , Bishopric N , Coughlin S R , Lee W M , Escobedo J A , Williams L T
Published: May 2006

The mechanism of hormonally induced cell hypertrophy is unknown. Stimulation of cardiac myocytes by alpha 1-adrenergic agents, phorbol esters, and serum induces an increase in the cell size of nondividing cardiac myocytes in primary culture. Expression of the c-myc gene, known to be increased in growth factor-induced cell division, was studied in this model of cell hypertrophy. The alpha-adrenergic agonist norepinephrine (0.002-20 microM) increased levels of c-myc-encoded mRNA to 10-fold over control levels. This increase was detectable at 30 min, peaked at 2 hr, and returned to baseline by 6 hr after stimulation. The norepinephrine response was abolished by the alpha 1-antagonist terazosin (2 microM) but was not affected by the beta-adrenergic antagonist propranolol (2 microM) and was only slightly (25%) attenuated by the alpha 2-adrenergic antagonist yohimbine (2 microM). Serum and the phorbol ester tumor promoter phorbol 12-myristate 13-acetate also enhanced c-myc expression in cardiac myocyte cultures. These findings show that the induction of cardiac myocyte hypertrophy is associated with enhanced expression of the c-myc gene and suggest that hormonally induced cell hypertrophy and cell division share common mechanistic pathways.