bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.
Authors: Sternberger L A , Sternberger N H
Published: May 2006

The immunocytochemical staining patterns of 37 neuron-specific monoclonal antibodies previously described fell into four groups: (i) anti-synapse-associated, (ii) anti-neurofibrillar, (iii) anti-perikaryonal-neurofibrillar, and (iv) a single antibody reactive with a widely distributed epitope that covered the patterns of groups ii and iii. Antibodies of groups ii, iii, and iv were shown to be specific to neurofilament triplet subunits, even though there was little overlap in staining patterns between groups ii and iii. We examined nine of these antibodies as to their ability to distinguish functional states of neurofilaments dependent upon phosphorylation. Upon digestion with phosphatase, electroblot staining of neurofilament components was abolished with the five antibodies from group ii, enhanced with the three antibodies from group iii, and unaffected with antibody iv. Immunocytochemical staining of Bouin-fixed paraffin sections of rat brain was unaffected by phosphatase pretreatment. With antibodies of group ii, digestion with trypsin also left staining unaffected, but when followed by digestion with phosphatase, staining was diminished with three out of five antibodies. In contrast, digestion with trypsin abolished all staining with each antibody from group iii. If followed by digestion with phosphatase, staining reappeared, but the group iii pattern was replaced by a group ii pattern. Staining of this pattern was again abolished upon a second treatment with trypsin. The antibody from group iv lost most of its groups ii and iii staining patterns when sections were digested with trypsin. The group ii pattern reappeared and, indeed, was enhanced upon a subsequent phosphatase treatment and was reduced again upon a second trypsin treatment. Staining by four out of five antibodies from group ii was inhibited by inorganic phosphate. The data indicate that certain nerve cell bodies, their dendrites, and at least proximal axons possess nonphosphorylated neurofilaments and that long fibers, including terminal axons, possess phosphorylated neurofilaments. We propose that phosphorylation may be a factor in stabilizing compacted forms of neurofilaments and that heterogeneity of the compacted structures may play a role in a possible multiplicity of function within individual nerve cells.
Topographic organization of certain tectal afferent and efferent connections can develop normally in the absence of retinal input.
Authors: O'Leary D D , Cowan W M
Published: May 2006

To test the hypothesis that the topographic organization of the connections of the optic tectum is determined during development by the "retinotopically" ordered input that it receives from the eye, we have mapped certain of the connections of the tectum in chicken embryos and chicks in which both eye rudiments were removed before the outgrowth of optic fibers. Because several of the connections of the tectum are normally organized retinotopically, we should expect, if this hypothesis were correct, that some or all of these connectional patterns would be significantly altered in such "eyeless" chickens. In fact, we have found that the connections formed between the optic tectum and two of the isthmic nuclei, with which it is reciprocally connected, show the same topographic organization in "eyeless" animals as in control chickens raised under the same conditions. This clearly indicates that the topographic organization of the chicken optic tectum is independently specified and is not contingent upon the input that it receives from the retina.
Physiological and x-ray studies of potential antisickling agents.
Authors: Abraham D J , Perutz M F , Phillips S E
Published: May 2006

Several aromatic compounds have been found to inhibit the gelling of sickle cell hemoglobin. We have tried to correlate the antigelling activity of such compounds with the stereo-chemistry of their binding sites in the hemoglobin molecule. This approach led to the discovery that two known antilipoproteinemia drugs, clofibrate and gemfibrozil, have antigelling activity. X-ray analysis showed that three pairs of molecules of clofibric acid, the active metabolite of clofibrate, bound to the walls of the internal cavity of deoxyhemoglobin A; only one pair bound to a quite different site, between helices A, E, and H of the alpha chains of carbon monoxide hemoglobin A. Unlike other antigelling agents, clofibric acid and related compounds decrease rather than increase the oxygen affinity of hemoglobin.
Distribution of receptors for transferrin and low density lipoprotein on the surface of giant HeLa cells.
Authors: Bretscher M S
Published: May 2006

Giant HeLa cells, having a spread diameter of about 200 micrometers, were briefly surface-labeled at 0 degree C with 125I-labeled transferrin, low density lipoprotein, anti-HeLa cell antibody, or concanavalin A. The cells were washed at 0 degree C, fixed, and autoradiographed. The distribution of grains when either anti-HeLa cell antibodies or concanavalin A was used was roughly as expected: the cell surfaces appeared uniformly labeled. When either transferrin or low density lipoprotein was used, about half the labeled cells had a nonuniform distribution of grains. On round cells, the cell periphery was more densely labeled than the middle of the cell; on elongated cells, cell protrusions were often more highly labeled than the rest of the cell. The simplest interpretation of these results is that, during their endocytic cycles through these cells, the transferrin and low density lipoprotein receptors are returned to the cell surface at the cell's leading edge.
Monoclonal antibody and an antibody-toxin conjugate to a cell surface proteoglycan of melanoma cells suppress in vivo tumor growth.
Authors: Bumol T F , Wang Q C , Reisfeld R A , Kaplan N O
Published: May 2006

A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.
Postsynaptic alpha-adrenergic receptors potentiate the beta-adrenergic stimulation of pineal serotonin N-acetyltransferase.
Authors: Klein D C , Sugden D , Weller J L
Published: May 2006

The role played by postsynaptic alpha-adrenergic receptors in the stimulation of pineal N-acetyltransferase (EC 2.3.1.5) and [3H]melatonin production was investigated in the rat. In vivo studies indicated that phenylephrine, an alpha-adrenergic agonist, potentiated and prolonged the effects of isoproterenol, a beta-adrenergic agonist. Similar observations were made in organ culture with glands devoid of functional nerve endings. In addition, a combination of 1 microM prazosin, an alpha 1-adrenergic blocking agent, and 1 microM propranolol, a beta-adrenergic blocking agent, was many times more potent then either agent alone in blocking the stimulatory effects of norepinephrine on N-acetyltransferase activity and [3H]melatonin production. These findings establish that norepinephrine acting through alpha- and beta-adrenergic receptors stimulates rat pineal N-acetyltransferase activity and, as a result, the production of melatonin. Apparently, beta-adrenergic activation is an absolute requirement, and an alpha-adrenergic receptor mechanism potentiates beta-adrenergic activation. These findings are significant because they demonstrate alpha-adrenergic potentiation of beta-adrenergic effects. In addition, they indicate that the widely held belief that melatonin production is regulated exclusively by a postsynaptic beta-adrenergic mechanism must be revised.
Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA.
Authors: Nagata K , Guggenheimer R A , Hurwitz J
Published: May 2006

Nuclear factor I, a 47-kilodalton protein, purified from nuclear extracts of uninfected HeLa cells, is involved in the initiation and possibly the elongation of replicating adenovirus (Ad) DNA in vitro. The binding of nuclear factor I to DNA has been monitored by a filter binding assay of nuclear factor I to DNA has been monitored by a filter binding assay using plasmid pLA1 DNA, which contains a 3,290 base-pair fragment derived from the left-hand terminus (coordinates, 0-9.4 map units) of Ad serotype 5 DNA. Nuclear factor I binds selectively to a double-stranded fragment spanning nucleotides 0-451 to the Ad genome. The retention of the 451-base-pair DNA fragment-nuclear factor I complex on nitrocellulose filters does not require Mg2+ or ATP and is resistant to high ionic strength. DNase I protection experiments revealed that nuclear factor I binds to a nucleotide sequence located at position 17-48, close to the terminus of Ad DNA. This 32-nucleotide sequence contains four "consensus" sequences present in various serotypes of Ad DNA and is capable of forming higher ordered structures. The role of nuclear factor I and this DNA sequence in the generation of Ad preterminal protein-dCMP initiation complex is discussed.
UC 729-6, a human lymphoblastoid B-cell line useful for generating antibody-secreting human-human hybridomas.
Authors: Glassy M C , Handley H H , Hagiwara H , Royston I
Published: May 2006

UC 729-6, a 6-thioguanine-resistant human lymphoblastoid B-cell line, was fused with normal and malignant human lymphocytes. Parent UC 729-6 cells were diploid with a 21p+ marker chromosome, expressed surface and cytoplasmic IgM kappa, and doubled every 17 hr. The resulting human-human hybridomas were pseudotetraploid containing the 21p+ marker, doubled every 20-30 hr, and secreted 3-9 micrograms of human Ig per ml per 10(6) hybrid cells for greater than 9 months in continuous culture. Human Ig-secreting hybridomas were generated in 88% (14/16) of the fusions carried out and were cloned by limiting dilution (one cell per three wells) without the use of feeder layers. The mean fusion frequencies (number of wells, plated at 10(5) cells per well, showing hybrid growth per 10(6) lymphocytes fused) of UC 729-6 with normal lymphocytes ranged from 0.45 to 2.9 and with malignant B lymphocytes, from 3 to 10. Analysis of the human-human hybridomas derived from lymphocytes isolated from regional draining lymph nodes of cancer patients revealed several that secreted human monoclonal antibody that reacted by an enzyme immunoassay with some carcinoma cell lines but not with normal fibroblast cell lines. These data suggest that (i) UC 729-6 can be fused with human lymphocytes to generate stable human-human hybridomas, some of which secrete antibody reactive to human cell surface antigens, and (ii) UC 729-6 can be used to rescue Ig from nonsecretory malignant B cells and thereby allow for the production of anti-idiotype antibodies.
Dissecting the antigenic mosaic of the Archaebacterium Methanobacterium thermoautotrophicum by monoclonal antibodies of defined molecular specificity.
Authors: Conway de Macario E , Macario A J , Magariños M C , König H , Kandler O
Published: May 2006

The antigenic mosaic of the Archaebacterium Methanobacterium thermoautotrophicum, strain delta H, was analyzed with a panel of six monoclonal antibodies. Five antigenic determinants were identified. One contains N-acetyl-D-glucosamine, another contains N-acetyl-D-galactosamine, and a third contains gamma-glutamylalanine (gamma-Glu-Ala). These residues are not involved, at least as immunodominant epitopes, in the other two determinants, one of which contains L-talosaminuronic acid, a component of pseudomurein found only in Methanobacteriaceae. Each of the five determinants was recognized by one monoclonal antibody exclusively. A sixth antibody recognized a structure containing gamma-Glu-Ala that could be either a sixth determinant or a subdeterminant within the site already recognized as containing gamma-Glu-Ala. We postulate that two of the determinants are strain specific, three are species specific, and one is a common antigen.
Characterization of human hybridomas secreting antibody to tetanus toxoid.
Authors: Larrick J W , Truitt K E , Raubitschek A A , Senyk G , Wang J C
Published: May 2006

We have selected a thioguanine-resistant lymphoblastoid cell line (LTR228) that forms human-human hybrids with high efficiency. Fusions with peripheral B cells consistently yield one colony per 10(5) cells plated. To produce antitetanus monoclonal antibodies, we withdrew blood from persons who had recently received booster injections of tetanus toxoid. T cells were separated from peripheral mononuclear cells by 2-aminoethylisothiouronium bromide-induced rosette formation, given 1,500 rads (1 rad = 0.01 gray), and cultured in a 1:1 ratio with nonrosetting cells. After 3 days of pokeweed mitogen stimulation, heterokaryons were produced by a plate-fusion technique and cultured in Iscove's Dulbecco's minimal essential medium for 24 hr prior to hybrid selection. Colonies appeared after 10-14 days in hypoxanthine/azaserine supplemented medium. A direct binding enzyme-linked immunosorbent assay with specific tetanus toxoid inhibition identified positive wells. The hybridomas were cloned twice in soft agarose and by limiting dilution. The subcloned hybridomas double every 26 hr (vs. every 16 hr for LTR228) and produce 1-5 micrograms of specific IgG, kappa antibody per 10(6) cells per ml per 24 hr. All subclones (almost 200) continue to secrete antibody after 11 months of continuous culture. Twelve representative subclones have near tetraploid amounts of DNA. From hybridomas grown in 5-liter spinner flasks, milligram quantities of the IgG, kappa antibody were purified by staphylococcus protein A affinity chromatography. Specific antibody from hybridoma cultures protected mice injected with 1,000 times the LD50 of tetanus toxin. Our cell line and associated techniques should permit the production of therapeutically important human monoclonal antibodies.
Trapping of the beta-adrenergic receptor in the hormone-induced state.
Authors: Neufeld G , Steiner S , Korner M , Schramm M
Published: May 2006

Isoproterenol and other agonists readily dissociate from the beta-adrenergic receptor in turkey erythrocyte membranes. However, when a low concentration of deoxycholate is added, the receptor locks the prebound agonist; i.e., the rate of dissociation of the prebound agonist decreases drastically. The dissociation of prebound antagonists is slightly increased by deoxycholate. Locking, which is thus agonist specific, occurs in the cold, is reversed when detergent is removed from the membranes, and appears not to require the guanyl nucleotide binding protein of the adenylate cyclase system. It is suggested that this induced fit of a receptor to an agonist represents the specific conformational response that normally propagates in the receptor molecule in its interaction with the next component along the pathway of signal transmission.
Primary structure of the essential replicon of the plasmid pSC101.
Authors: Vocke C , Bastia D
Published: May 2006

The replicon of the low copy number plasmid pSC101 has an obligatory requirement for the dnaA initiator protein of Escherichia coli as well as a plasmid-encoded initiator protein. We have identified the cistron of the plasmid-encoded initiator by DNA sequence analysis. Fusion of the initiator cistron with the lacZ gene of E. coli yielded a fusion protein of approximately equal to 150 kilodaltons, thus confirming that the open reading frame detected by DNA sequence analysis actually encoded a 37.5-kilodalton protein. Deletion of 26 amino acid residues from the COOH terminus of the plasmid initiator abolished autonomous replication from pSC101 origin. By in vitro deletion analysis we have shown that, although sequences downstream from the initiator cistron are dispensable, a maximum of 400 base pairs immediately upstream from the NH2-terminal region of the initiator is necessary for plasmid replication. These upstream sequences contain an A + T-rich region and three tandem repeats of a 21-base pair sequence; these features are characteristics of other replication origins.
Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization.
Authors: Blum H E , Stowring L , Figus A , Montgomery C K , Haase A T , Vyas G N
Published: May 2006

A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefulness of in situ hybridization in the study of pathogenesis of HBV infection.
Anticodon loop size and sequence requirements for recognition of formylmethionine tRNA by methionyl-tRNA synthetase.
Authors: Schulman L H , Pelka H
Published: May 2006

Previous work from our laboratory identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. To further investigate the structural requirements for recognition in this region, we have synthesized a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with pancreatic RNase. This step also removes two nucleotides from the 3' CpCpA end. T4 RNA ligase is used to join oligonucleotides of defined length and sequence to the 5' half-molecule and subsequently to link the 3' and modified 5' fragment to regenerate the anticodon loop. The final step of the synthesis involves repair of the 3' terminus with tRNA nucleotidyltransferase. The synthetic derivative containing the anticodon CAU is aminoacylated with the same kinetics as intact tRNAfMet. Base substitutions in the wobble position reduce aminoacylation rates by at least five orders of magnitude. The rates of aminoacylation of derivatives having base substitutions in the other two positions of the anticodon are 1/55 to 1/18,500 times normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, indicating that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of methionyl-tRNA synthetase with functional groups on the nucleotide bases of the anticodon sequence.
Genetic recombination of bacteriophage lambda DNAs in Xenopus oocytes.
Authors: Carroll D
Published: May 2006

Pairs of genetically marked bacteriophage lambda DNAs have been injected into Xenopus laevis oocyte nuclei. After suitable incubation, DNA was recovered and packaged into phage particles in vitro. When these were plated onto a selective host, phage recombinant for parental markers were observed. Recombination was dependent on both parents being present in the same oocyte nucleus and was roughly proportional to the physical separation of the markers. Thus, the oocytes appear to contain the machinery necessary for performing typical genetic recombination. This system offers a great deal of scope and flexibility for future studies of recombination mechanisms at the molecular level in vertebrates.
Restriction fragment length polymorphisms associated with immunoglobulin C gamma genes reveal linkage disequilibrium and genomic organization.
Authors: Bech-Hansen N T , Linsley P S , Cox D W
Published: May 2006

We have demonstrated that restriction fragment length polymorphisms (RFLPs) produced by BamHI can be used as markers for constant (C) region heavy chain genes C psi gamma (C gamma pseudogene), C gamma 2, and C gamma 4. These RFLPs were found nonrandomly associated in the population sample studied. Of the eight combinations (haplotypes) of RFLPs theoretically possible, only two accounted for a total of 88% of the 116 chromosomes examined, a value greater than the total of 25% expected from random segregation of alleles. This indicates considerable linkage disequilibrium between C psi gamma, C gamma 2, and C gamma 4. Quantitative assessment of the degree of association between C gamma gene RFLPs, Gm markers, and switch region RFLPs adjacent to C mu and C alpha 1 revealed that C psi gamma is most tightly associated with C gamma 2 (r = 0.81 and 0.95 for the two common haplotypes), suggesting that C psi gamma maps to a position lying between C alpha 1 and C gamma 2. The association analysis used here should have general applicability for studying the genomic organization of other multigene families.
Characterization of the human receptor for T-cell growth factor.
Authors: Leonard W J , Depper J M , Robb R J , Waldmann T A , Greene W C
Published: May 2006

Anti-Tac monoclonal antibody has been identified as a putative antibody against the receptor for T-cell growth factor (TCGF). We now show that: (i) TCGF blocks 85% of 3H-labeled anti-Tac binding to phytohemagglutinin-activated lymphoblasts and (ii) both anti-Tac and anti-TCGF immunoprecipitate a protein band that appears to represent TCGF crosslinked to its receptor on HUT-102B2 cells. In HUT-102B2 cells, the TCGF receptor is a Mr 50,000 glycoprotein with internal disulfide bond(s) and a pI of 5.5-6.0, and it represents approximately equal to 0.05% of total cellular de novo protein synthesis. It contains a peptide of Mr 33,000 that is processed to a mature form that includes N-linked and O-linked sugars and sialic acid.
Carbon starvation and growth rate-dependent regulation of the Escherichia coli ribosomal RNA promoters: differential control of dual promoters.
Authors: Sarmientos P , Cashel M
Published: May 2006

We studied the effects of carbon starvation and of varying the growth rate on the activity of each of the two tandem ribosomal RNA promoters from the rrnA operon of Escherichia coli. The cellular abundance of plasmid-encoded transcripts arising at promoters P1 and P2 and terminating at the ribosomal RNA terminator in promoter-terminator fusions, together with transcript turnover rates, was used to estimate promoter activities. The rate of synthesis of the P1-promoted transcript was found to increase exponentially with growth rate and predominate at fast growth rates. The activity of the downstream promoter (P2) changed only slightly at different growth rates. Upon carbon starvation, little or no activity of the upstream promoter was detectable, while P2 activity persisted. We interpret this to mean that the dual promoters are differentially regulated so as to have separate adaptive and maintenance functions. This model simplifies most features of rRNA regulation known in E. coli.
Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics.
Authors: Franke W W , Schiller D L , Hatzfeld M , Winter S
Published: May 2006

Subunit complexes of cytokeratin polypeptides from intermediate-sized filaments (IF) of various tissues and cultured cells from rat, cow, and man were solubilized in low-salt buffer containing 4 M urea and exposed to increasing concentrations of urea, followed by urea gradient electrophoresis or two-dimensional gel electrophoresis at different urea concentrations. Correspondingly, cytokeratin polypeptides dissociated in 9.5 or 10 M urea were dialyzed into lower concentrations of urea and allowed to reassociate into specific complexes. It was found that the polypeptide constituents of a given cytokeratin complex dissociate in the form of a rather sharp "melting curve" and that dissociated polypeptides reassociate in the same mode of dependence on urea concentration. The midpoint of melting in urea (Um) is a characteristic property of a given complex of cytokeratin polypeptides. Um values differ markedly between different cytokeratin complexes, ranging from 5.9 to 9.0 M urea. The results also show that cytokeratins do not form complexes with vimentin, another type of IF protein. The data suggest that certain cytokeratin polypeptides are complementary and contain sequences that direct their association into specific complexes forming IF subunits.
beta-Adrenergic receptor regulation of N-linked protein glycosylation in rat parotid acinar cells.
Authors: Kousvelari E E , Grant S R , Baum B J
Published: May 2006

We have investigated the relationship between beta-adrenergic receptor stimulation and protein glycosylation and secretion in rat parotid gland cells in vitro. The potent beta-adrenergic agonist (-)-isoproterenol increases [3H]mannose incorporation into newly synthesized glycoproteins. This effect is enhanced if cells are first preincubated with dolichyl phosphate and is not observed after muscarinic-cholinergic or alpha-adrenergic stimulation of cells. The increase in [3H]mannose incorporation is abolished by incubation of cells with tunicamycin, suggesting that the glycosylation events being studied involved asparagine-linked oligosaccharides. The extent of increase in glycosylation is dependent on the concentration of (-)-isoproterenol to which cells are exposed. (+/-)-Propanolol totally abolishes the (-)-isoproterenol-induced increase in [3H]mannose incorporation, in a manner similar to its effects on exocrine secretion. Our findings suggest that beta-adrenergic receptor activation has a profound influence on N-linked protein glycosylation in rat parotid cells in addition to eliciting exocrine protein release.
Beta-adrenergic stimulation of Ca2+ fluxes, endocytosis, hexose transport, and amino acid transport in mouse kidney cortex is mediated by polyamine synthesis.
Authors: Koenig H , Goldstone A D , Lu C Y
Published: May 2006

We recently found that the beta-adrenergic agonist 1-isoproterenol evokes a rapid (less than 5 min) Ca2+- and receptor-dependent stimulation of endocytosis, hexose transport, and amino acid transport in mouse renal cortex involving proximal tubule cells. This response is associated with increased Ca2+ fluxes and a mobilization of mitochondrial calcium, suggesting that stimulus-response (stimulus-"transport") coupling is mediated by cytosolic Ca2+. We show here that 1 microM isoproterenol evokes a rapid (less than 60 sec) transient increase in the activity of ornithine decarboxylase followed by an early (less than 2 min) sustained increase in putrescine, spermidine, and spermine concentrations in mouse kidney cortex slices in vitro. Small doses of isoproterenol (down to 24 nmol/kg) elicited a rapid (less than 2 min) increase in polyamines in vivo. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine (5 mM) suppressed the testosterone-induced increase in polyamine levels and rates of endocytosis, hexose transport, and amino acid transport, measured by horseradish peroxidase, [14C]aminoisobutyric acid, and deoxy[3H]glucose uptake. alpha-Difluoromethylornithine also blocked the isoproterenol-induced increase in 45Ca influx and efflux and 45Ca redistribution; 0.5 mM putrescine nullified alpha-difluoromethylornithine inhibition and restored the increment in polyamines, 45Ca fluxes, endocytosis, hexose transport, and amino acid transport. These data implicate polyamine synthesis in isoproterenol stimulation of Ca2+ fluxes and membrane transport processes and support a model for signal transduction and stimulus-response coupling in which ornithine decarboxylase activation and polyamine synthesis play a pivotal role in regulating Ca2+ fluxes. In this model the polyamines generate local Ca2+ signals by stimulating Ca2+ influx or mobilizing intracellular calcium (or both) through a cation exchange reaction.
Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell--cell contact and cell membranes.
Authors: Nakamura T , Yoshimoto K , Nakayama Y , Tomita Y , Ichihara A
Published: May 2006

In primary monolayer cultures of rat mature hepatocytes, many metabolic functions as well as cell growth are regulated by cell density. There are two types of regulatory response of these functions to change of cell density. Growth-related functions, such as DNA synthesis, induction of glucose-6-phosphate dehydrogenase, 2-aminoisobutyric acid transport, synthesis of cellular protein, and cholesterogenesis, are stimulated by low cell density. In contrast, functions related to hepatocyte-specific characters, such as the inductions of tyrosine aminotransferase, serine dehydratase, and malic enzyme and synthesis of triglycerides, are stimulated by high cell density. The reciprocal responses of these cellular activities to cell density were mimicked by addition of plasma membranes purified from adult rat liver to hepatocytes cultured at low cell density. The modulator activity was heat labile and trypsin sensitive. The activity was also found in plasma membranes from kidney, brain, and erythrocytes, although the specific activities of these preparations seemed to be different. These results suggest that the reciprocal regulations of cell growth and hepatocyte-specific functions are mediated by some surface components via cell-cell contact.
Transformation of retinal glia cells into lens phenotype: expression of MP26, a lens plasma membrane antigen.
Authors: Moscona A A , Brown M , Degenstein L , Fox L , Soh B M
Published: May 2006

We describe experiments in which dissociated cells from differentiated, post-mitotic neural retina of late chicken embryos (13 and 16 days) rapidly and consistently transform (transdifferentiate) in vitro into lens-like phenotype and form spherical lentoids. Using immunohistochemical and other tests, we have established that the lentoids arise from the progeny of definitive retinal glia cells (Müller cells). An early event in their transformation is the appearance in the cell surface of MP26, a plasma membrane protein characteristic of lens but not found in the retina. The results support the hypothesis that the phenotype of definitive glia cells in the retina is stabilized by contact-mediated interactions with neurons; disruption of cell contacts and cell separation alter surface properties of the glia cells, decontrol their phenotype, and predispose them to phenotype transformation.
Directional transport and integration of donor DNA in Haemophilus influenzae transformation.
Authors: Barany F , Kahn M E , Smith H O
Published: May 2006

DNA transport and integration in Haemophilus influenzae transformation was studied with a plasmid clone of homologous DNA (pCML6). Our results indicate that: (i) donor DNA enters specialized membranous extensions on the cell surface, which we have termed "transformasomes"; (ii) linear DNA undergoes degradation upon exiting transformasomes; and (iii) DNA without a free end remains within transformasomes and is not degraded. By comparing the fate of label from uniformly labeled versus middle-labeled DNA, it appears that donor DNA undergoes degradation from an end prior to recombining with the chromosome. Using donor DNA with covalently closed termini (hairpin ends) prevents efficient exit from transformasomes. When one hairpin is removed, exit of donor DNA is shown to be directional from the free end, with preferential homologous integration of the 3' strand from that end.
Pseudo arylsulfatase-A deficiency in healthy individuals: genetic and biochemical relationship to metachromatic leukodystrophy.
Authors: Chang P L , Davidson R G
Published: May 2006

Metachromatic leukodystrophy is a hereditary neurodegenerative disorder in man associated with deficient arylsulfatase-A activity (aryl-sulfate sulfohydrolase, EC 3.1.6.1). The same enzyme deficiency has been noted in clinically normal individuals, a condition known as pseudo arylsulfatase-A deficiency. With a nonselective method, somatic cell hybrids were obtained from cultured fibroblasts of these two types of individuals; the hybrids showed no restoration of arylsulfatase-A activity. Thus, metachromatic leukodystrophy and pseudo arylsulfatase-A deficiency are allelic conditions. Although these conditions cannot be distinguished by simple quantitative arylsulfatase-A activity assays, they can be differentiated with sucrose density gradient centrifugation, Cellogel electrophoresis, or isoelectric focusing in polyacrylamide gels. In each case, a small amount of activity with characteristics of arylsulfatase-A was found only from fibroblasts of pseudo arylsulfatase-A-deficient individuals and not from those of metachromatic leukodystrophy patients. This residual enzyme has the same pH optimum, heat stability, inhibitor sensitivity, and Km as the normal enzyme but slightly different isoelectric points. In conclusion, although pseudo arylsulfatase-A deficiency and metachromatic leukodystrophy have very different clinical outcomes, they are due to mutations of the same structural gene, coding for arylsulfatase-A. These two conditions can be differentiated now by simple electrophoretic analysis of the residual arylsulfatase-A activity.
Experimental tumoricidal effects of monoclonal antibody against solid breast tumors.
Authors: Capone P M , Papsidero L D , Croghan G A , Chu T M
Published: May 2006

Two distinct monoclonal antibodies (mAbs) were effective in the therapy of breast carcinomas of human origin established and growing in nude mice. Passive administration of either of the antibodies produced very rapid (less than 1 week) and significant reduction of in vivo tumor volume. Each of the mAbs showed in vivo targeting of the tumors. Histological analysis of mAb-treated tumors revealed extensive cellular necrosis. Each of the antibodies in vitro was effective in complement-mediated cytolysis at a concentration less than 1 ng/ml. The tumoricidal responses show that this is a useful model for passive human immunotherapy using mAbs.
Antibodies to histones in systemic lupus erythematosus: localization of prominent autoantigens on histones H1 and H2B.
Authors: Hardin J A , Thomas J O
Published: May 2006

By the technique of immunoblotting we have assessed the ability of sera from 24 patients with systemic lupus erythematosus to bind nuclear proteins. Of the 11 patients who had antibodies to histones, 10 had antibodies to histone H1 and 9 of these also had antibodies to histone H2B. Antibodies to the other histones (H2A, H3, and H4) were less apparent. Five of the 11 patients (and two others in the remainder of the sample of 24) also had antibodies to a small number of nonhistone proteins that are probably components of ribonucleoprotein particles, but there was no obvious correlation between the presence of antihistone antibodies and the known antiribonucleoprotein activity of these sera. Separate determinants on H1 and H2B were demonstrated by immunoblotting with affinity-purified anti-H1 and anti-H2B antibodies derived from serum that showed both specificities. The localization of the determinants within the histone polypeptide chains was shown by immunoblotting with large fragments produced by specific proteolytic or chemical cleavage of the histones. The strongest determinant on H1 was located within the COOH-terminal half, with a weaker determinant being present within the NH2-terminal half; the H2B determinant(s) was located entirely within the NH2-terminal half of the molecule. The selectivity with which the antihistone antibodies in systemic lupus erythematosus are produced against the more exposed histones in the nucleosome (and perhaps against the most exposed regions of these histones) is consistent with the involvement of intact chromatin structures as immunogens in this disease.
Structure of the human interleukin 2 gene.
Authors: Fujita T , Takaoka C , Matsui H , Taniguchi T
Published: May 2006

We have cloned two species of EcoRI-cleaved DNA segments that together cover the entire sequence for the human interleukin 2 gene and have determined the nucleotide sequence of the gene and its flanking regions. The gene contains three introns and the exon sequences can be aligned with the previously reported cDNA sequence almost perfectly except for a few nucleotides in the 3' nontranslated region. The promoter region contains a prototype "TATA" sequence as well as a notable palindromic sequence. Particularly interesting is the presence of sequences in this region that are homologous to the promoter region of the human interferon-gamma gene. In addition, a sequence that closely resembles the core sequence for the viral enhancer elements has been found in the second intron. Such sequences may play a role in the expression of the interleukin 2 gene in lectin- or antigen-stimulated T lymphocytes.
Genome-linked protein VPg of poliovirus is present as free VPg and VPg-pUpU in poliovirus-infected cells.
Authors: Crawford N M , Baltimore D
Published: May 2006

VPg is a virus-encoded protein covalently attached to the 5' end of poliovirus virion RNA. We have used antibody prepared against chemically synthesized VPg to detect two forms of VPg in infected cells. Both forms were specifically immunoprecipitated from lysates of infected cells labeled with [3H]-leucine. One appears to be unmodified VPg because it had the same electrophoretic mobility as synthetic VPg. The other had a larger apparent molecular weight than VPg and could be labeled in vivo with 32Pi. Its structure is VPg-pUpU, the UMP dinucleotide being attached to VPg via a phosphodiester bond to tyrosine, the third amino acid from the NH2 terminus of VPg. This structure is identical to that found at the 5' end of virion and minus-strand RNA.
Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast.
Authors: Urdea M S , Merryweather J P , Mullenbach G T , Coit D , Heberlein U , Valenzuela P , Barr P J
Published: May 2006

We have chemically synthesized and expressed in yeast a gene coding for human epidermal growth factor (urogastrone), a 53-amino-acid polypeptide that has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion. The synthetic gene, consisting of 170 base pairs, was designed with yeast-preferred codons and assembled by enzymatic ligation of synthetic fragments produced by phosphoramidite chemistry. The DNA synthesis protocol used allows for facile synthesis of oligonucleotides larger than 50 bases. Yeast cells were transformed with plasmids containing the synthetic gene under control of a yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter and were shown to synthesize a biologically active human epidermal growth factor.
Direct hybridization of sorted human chromosomes: localization of the Y chromosome on the flow karyotype.
Authors: Bernheim A , Metezeau P , Guellaën G , Fellous M , Goldberg M E , Berger R
Published: May 2006

A method is described for directly hybridizing a small number of sorted chromosomes with specific DNA probes. The chromosomes are analyzed by flow cytometry and sorted by deflecting the droplets containing the desired chromosomes onto a nitrocellulose filter. By using probes specific for the human Y chromosome, it has been possible to unambiguously identify the peak corresponding to the Y chromosome in the flow karyotypes of a variety of male cell lines. The position of this peak was found to vary significantly from individual to individual, correlating with the heterochromatin chromosomal polymorphism of the human Y chromosome. The sensitivity of the hybridization was such that, with a probe for a male-specific repetitive sequence, only 2,500 sorted chromosomes were enough to obtain a clear, positive signal; 10,000 were needed with a probe specific for a weakly repeated (maximum, 3-fold) sequence of Y chromosome. With this new method, chromosome sorting may be a rapid and efficient way to assign DNA sequences to chromosomes.
Antibodies against synthetic peptides of the B subunit of cholera toxin: crossreaction and neutralization of the toxin.
Authors: Jacob C O , Sela M , Arnon R
Published: May 2006

Six peptides corresponding to various segments of the B subunit of cholera toxin have been synthesized and covalently linked to tetanus toxoid. Of the antibodies raised in rabbits against these conjugates, four crossreacted to different extents with the intact B subunit and whole native cholera toxin. Antisera to the peptide of sequence 75-85 were not crossreactive, whereas elongation by six amino acid residues resulted in a peptide (69-85) leading to antibodies crossreactive with the cholera toxin. Of most interest was peptide CTP3 (50-64), which was the only one that reacted with antisera to cholera toxin and which led to antibodies reacting with the cholera toxin to a similar level as its homologous peptide-antipeptide reaction. Indeed, antisera to CTP3 neutralized significantly the biological activity of cholera toxin, as followed by skin vascular permeability and by fluid accumulation in ligated small intestinal loops of rabbits.
Evidence for extensive polymorphism of class I genes in the rat major histocompatibility complex (RT1).
Authors: Palmer M , Wettstein P J , Frelinger J A
Published: May 2006

The major histocompatibility complex of the rat (RT1) has been poorly characterized with respect to the number, linkage, and polymorphism of class I genes. To estimate the number of class I RT1 genes and the relative extent of their polymorphism, we performed Southern blot analysis with liver DNA from rat strains expressing eight RT1 haplotypes. After digestion with EcoRI and BamHI, the DNA was separated on agarose gels, blotted onto nitrocellulose, and hybridized with mouse H-2 cDNA probes, pH-2III and pH-2IIa. Ten to 20 EcoRI and 13 to 20 BamHI bands hybridized with pH-2III and pH-2IIa; restriction fragment length patterns were observed to be highly polymorphic. The restriction fragments associated with different RT1 haplotypes differed by 17-70%; this range is similar to the differences observed between mouse H-2 haplotypes. The same restriction fragment pattern was observed in DNA from three different rat strains sharing the same RT1 allele, confirming that the patterns were RT1-associated. Further, the RT1l and RT1lvl haplotypes, which differ at a single previously identified RT1-linked locus, were associated with EcoRI restriction pattern differences of 39-50%, confirming the supposition that RT1 class I genes identified by previous serological and T-cell-mediated assays have identified only a minority of the actual number of RT1-linked class I genes. In summary, the results reported in this communication demonstrate that the RT1 complex encompasses a large family of highly polymorphic class I genes similar to the H-2 and HL-A complexes of mouse and man.
Rapid similarity searches of nucleic acid and protein data banks.
Authors: Wilbur W J , Lipman D J
Published: May 2006

With the development of large data banks of protein and nucleic acid sequences, the need for efficient methods of searching such banks for sequences similar to a given sequence has become evident. We present an algorithm for the global comparison of sequences based on matching k-tuples of sequence elements for a fixed k. The method results in substantial reduction in the time required to search a data bank when compared with prior techniques of similarity analysis, with minimal loss in sensitivity. The algorithm has also been adapted, in a separate implementation, to produce rigorous sequence alignments. Currently, using the DEC KL-10 system, we can compare all sequences in the entire Protein Data Bank of the National Biomedical Research Foundation with a 350-residue query sequence in less than 3 min and carry out a similar analysis with a 500-base query sequence against all eukaryotic sequences in the Los Alamos Nucleic Acid Data Base in less than 2 min.
Nucleotide sequence of the simian sarcoma virus genome: demonstration that its acquired cellular sequences encode the transforming gene product p28sis.
Authors: Devare S G , Reddy E P , Law J D , Robbins K C , Aaronson S A
Published: May 2006

The complete nucleotide sequence of the proviral genome of simian sarcoma virus (SSV), an acute transforming retrovirus of primate origin, has been determined. Like other transforming viruses, SSV contains sequences derived from its helper virus, simian sarcoma-associated virus (SSAV), and a cell-derived (v-sis) insertion sequence. By comparison with the sequence of Moloney murine leukemia virus, it was possible to precisely localize and define sequences contributed by SSAV during the generation of SSV. Comparative sequence analysis of SSV and SSAV showed that SSAV provides regulatory sequences for initiation and termination of transcription of the SSV transforming gene. Moreover, coding sequences for the putative protein product of this gene appear to initiate from the amino terminus of the SSAV env gene. Antibodies to synthetic peptides derived from the carboxy and amino termini of the putative protein predicted by the open reading frame identified within v-sis specifically detect a Mr 28,000 protein, p28sis, in SSV-transformed cells. These and other findings confirm the predicted amino acid sequence of this protein and localize it to the coding region of the SSV transforming gene.
Structural requirement for IS50-mediated gene transposition.
Authors: Berg D E
Published: May 2006

Replicative transposition is signaled by the formation of cointegrates in which donor and target replicons are joined by direct repeats of a transposable element. Elements not generating such cointegrates may move by a conservative breaking and joining process. The IS50 elements forming the terminal repeats of Tn5 [which carries the determinant for kanamycin resistance (Kanr)] contain genes and sites necessary for transposition and mediate the movement of any DNA segment they bracket. To determine if IS50 generates cointegrates, the products of transposition from pBR322::Tn5 plasmids to an F factor in recA-Escherichia coli were examined. With monomeric pBR322::Tn5 plasmids, transposition of Kanr (from Tn5) was generally not accompanied by movement of the determinant for ampicillin resistance (Ampr) (from the pBR322 vector). With dimeric pBR322::Tn5 plasmids, by contrast, half of the transpositions of kanr were accompanied by transposition of ampr. Restriction endonuclease analyses indicated that these F-Kanr Ampr chimeras contained inserts of a single copy of the pBR322 vector sequence bracketed by one Tn5 element and one IS50 element or by a pair of Tn5 elements. None of 79 chimeras tested was a true cointegrate. Because IS50 seems to move only a segment of the donor replicon it is proposed that IS50 transposition is conservative.