bluuebunny/crossref_metadata_2025 huggingface dataset:
Membranes from T and B lymphocytes have different patterns of tyrosine phosphorylation.
Authors: Earp H S , Austin K S , Buessow S C , Dy R , Gillespie G Y
Published: May 2006

Membrane fractions isolated from mouse and rat spleen expressed substantial tyrosine-specific protein kinase activity. Phosphotyrosine (P-Tyr) accumulation in endogenous membrane substrates was stimulated by vanadate or nonionic detergents. When in vitro phosphorylation was carried out at 0 degree C in the presence of 1 mM Mn2+ and Triton X-100, P-Tyr constituted up to 40-50% of the total phospho amino acid. Polyacrylamide gel electrophoresis showed that membranes from mixed lymphocyte populations have four major P-Tyr-containing proteins. Whereas nonionic detergents were potent stimuli for P-Tyr accumulation in all four substrates, tyrosine phosphorylation of two of these (p61 and p55) was markedly dependent on vanadate. These two substrates were present in membranes from surface Ig-bearing splenic lymphocytes purified by affinity chromatography and Raji, a human B-lymphoblastoid cell line. P-Tyr accumulation in the two other substrates observed in splenocyte membranes (p64 and p58) was much less dependent on vanadate. p64 and p58 were phosphorylated in membranes from mouse thymocytes and human and mouse T-lymphoma cell lines, while p61 and p55 were not. Thus it appears that in both murine and human lymphocytes, p64 and p58 served as T-cell-specific substrates, while p61 and p55 were specifically associated with B lymphocytes. Moreover, these distinct P-Tyr substrate patterns were conserved in some neoplastic cell lines derived from B and T cells.
Chromosome 20 deletion in human multiple endocrine neoplasia types 2A and 2B: a double-blind study.
Authors: Babu V R , Van Dyke D L , Jackson C E
Published: May 2006

Multiple endocrine neoplasia type 2A and 2B (MEN-2A and MEN-2B) are autosomal dominantly inherited syndromes in which medullary thyroid cancers are associated with adrenal pheochromocytomas. A double-blind analysis of high-resolution G-banded chromosomes was performed on blood specimens from patients in four MEN-2A families and five MEN-2B (mucosal neuroma phenotype) families and from control subjects. Excluding studies on duplicate blood specimens, 9 of 11 control subjects were scored as having normal chromosomes 20, and 11 of 14 MEN-2 patients were scored as having chromosomal deletion: del(20)(p12.2p12.2) (phi 2 = 9.00; P less than 0.001). Two new mutant MEN-2B patients had apparently normal chromosomes 20. These findings demonstrate that the dominant mutation in most MEN-2A and MEN-2B families is a visible chromosome deletion within band 20p12.2.
Classical conditioning of the rabbit eyelid response increases glutamate receptor binding in hippocampal synaptic membranes.
Authors: Mamounas L A , Thompson R F , Lynch G , Baudry M
Published: May 2006

Hippocampal pyramidal neurons exhibit a rapid within-trial increase in firing frequency during classical conditioning of the rabbit eyelid response. It has been proposed that the cellular mechanisms responsible for hippocampal long-term potentiation (LTP) may also mediate this learning-dependent increase in neuronal activity. The induction of LTP in rat hippocampal slices results in an increase in the number of [3H]glutamate-binding sites in the potentiated region. The present study investigates the kinetics of [3H]glutamate binding to hippocampal synaptic membranes after eyelid conditioning in the rabbit. We report that the regional distribution of [3H]glutamate binding across the layers of rabbit hippocampus is compatible with a dendritic localization. The pharmacological and ionic properties of the binding suggest that it is associated with an excitatory amino acid receptor. After eyelid conditioning, the maximal number of hippocampal [3H]glutamate-binding sites is increased in animals receiving paired presentations of the tone conditioned stimulus and corneal air-puff unconditioned stimulus relative to that found in naive or unpaired control animals. These results strengthen the hypothesis that an LTP-like mechanism underlies the increase in hippocampal firing frequency during rabbit eyelid conditioning.
Branch-specific heterosynaptic facilitation in
Aplysia
siphon sensory cells
Authors: Clark Gregory A. , Kandel Eric R.
Published: May 2006

Aplysia
siphon sensory cells exhibit heterosynaptic facilitation of transmitter release during both sensitization and classical conditioning of the siphon withdrawal response. In the present study, we asked whether facilitation must invariably enhance transmission at all terminals of a neuron or whether facilitation can instead occur at one set of terminals without also occurring at other terminals of the same cell. To examine this question, we compared effects of local application of serotonin and of connective stimulation on transmission at central and peripheral branches of single sensory cells. We found that heterosynaptic facilitation can be branch-specific and can occur at either central or peripheral synapses independently. We also found that siphon sensory cells exhibit homosynaptic post-tetanic potentiation, allowing us to compare effects of hetero- and homosynaptic facilitation in the same cells. By contrast to heterosynaptic facilitation, homosynaptic facilitation occurs concomitantly at both central and peripheral synapses of siphon sensory cells. Thus, while both heterosynaptic and homosynaptic facilitation involve increases in transmitter release from sensory neuron terminals, heterosynaptic facilitation provides a greater specificity and flexibility in the modification of synaptic connections.
Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor.
Authors: Conti-Tronconi B M , Hunkapiller M W , Raftery M A
Published: May 2006

A discrepancy of about 20% exists between the molecular weight of the alpha subunit of Torpedo californica electroplax acetylcholine receptor as determined by gel electrophoresis of the mature protein (Mr 40,000 +/- 2000) and by nucleotide sequence analysis of cDNA (Mr approximately equal to 50,000). We demonstrate by amino acid sequence analysis that post-translational processing does not occur and that the mature subunit has a Mr of approximately equal to 50,000. The functional acetylcholine receptor contains two copies of this alpha subunit in addition to one each of related beta, gamma, and delta subunits. The binding sites for cholinergic ligands that are located on the alpha subunits have been shown to be nonequivalent. Amino acid sequence analysis of peptides obtained by proteolytic cleavage of the alpha subunit reveals that N-asparagine glycosylation at a single site (residue 141) occurs to a different extent in the two copies of this polypeptide in the mature protein and provides an explanation for nonequivalence of their binding sites.
Human transferrin: cDNA characterization and chromosomal localization.
Authors: Yang F , Lum J B , McGill J R , Moore C M , Naylor S L , van Bragt P H , Baldwin W D , Bowman B H
Published: May 2006

Transferrin (Tf) is the major iron binding protein in vertebrate serum. It shares homologous amino acid sequences with four other proteins: lactotransferrin, ovotransferrin, melanoma antigen p97, and HuBlym-1. Antigen p97 and the Tf receptor genes have been mapped on human chromosome 3. The goal of the study described here was to initiate the characterization of the Tf gene by identifying and characterizing its cDNA and mapping its chromosomal location. Recombinant plasmids containing human cDNA encoding Tf have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. Within the 2.3 kilobase pairs of Tf cDNA analyzed, there is a probable leader sequence encoded by 57 nucleotides followed by 2037 nucleotides that encode the homologous amino and carboxyl domains. During evolution, three areas of the homologous amino and carboxyl domains have been strongly conserved, possibly reflecting functional constraints associated with iron binding. Chromosomal mapping by in situ hybridization and somatic cell hybrid analysis indicate that the Tf gene is located at q21-25 on human chromosome 3, consistent with linkage of the Tf, Tf receptor, and melanoma p97 loci.
Orientation of loci within the human major histocompatibility complex by chromosomal in situ hybridization.
Authors: Morton C C , Kirsch I R , Nance W E , Evans G A , Korman A J , Strominger J L
Published: May 2006

We have determined the localization and orientation of two genetic probes within the human major histocompatibility complex by chromosomal in situ hybridization. Our data indicate that a cloned genomic probe cross-hybridizing to HLA-A, -B, and -C heavy chain loci is homologous to sequences located on chromosome 6 at band p21.3 while a subclone of the genomic HLA-DR alpha-chain gene corresponding to the nonpolymorphic p34 protein is homologous to sequences in band 6p21.1. Our data suggest that this technique may permit the estimation of map distances between linked gene loci, assuming a uniform frequency of map units in the human genome. The relative positions of these genes was confirmed in a mother and son carrying a chromosome rearrangement involving 6p and 14p in which the sequences hybridizing to a DR alpha-chain genomic clone were found at the distal end of the 6p--chromosome [der(6)] while the sequences hybridizing to the HLA-A, -B, -C alpha-chain probe were found in the 14p+ chromosome [der(14)].
Monoclonal antibodies to native noncollagenous bone-specific proteins.
Authors: Stenner D D , Romberg R W , Tracy R P , Katzmann J A , Riggs B L , Mann K G
Published: May 2006

Hybridoma technology was used for preparation of murine monoclonal antibodies of high titer against bone-Gla protein and osteonectin. A procedure of immunization and hybridization similar to that already described [Katzmann, J.A., Nesheim, M.E., Hibbard, L.S. & Mann, K.G. (1981) Proc. Natl. Acad. Sci. USA 78, 162-166; and Foster, W.B., Katzmann, J.A., Miller, R.S., Nesheim, M.E. & Mann, K.G. (1982) Thromb. Res. 28, 649-661] was used. However, in contrast to earlier studies, mice were immunized with an unfractionated protein mixture that had been extracted from bone under nondenaturing conditions. The extract was labeled with 125I by the chloramine-T method. After fusion and initial hybrid growth, screening was accomplished by a solid-phase radioimmunoassay with total 125I-labeled bovine bone protein extract as the tracer. The identities of antibody-bound 125I-labeled proteins were assessed by dissolution of the solid-phase immune complex in NaDodSO4 and subsequent electrophoresis and autoradiography. Clones producing specific antibody to a single protein were selected by limiting dilution. The identity of the proteins against which the specific antibodies were produced was confirmed by immunoprecipitation, electrophoresis, and autoradiography. From two fusions, 30 positive hybrids to bone-Gla protein were identified; 7 of these were subcloned and 1 has been expanded as an ascites tumor. One hybrid population was positive for osteonectin, a Mr 15,000 peptide, and for bone-Gla protein. By limiting dilution, the osteonectin clone was selected and subsequently expanded as an ascites tumor. Titration curves made using the respective 125I-labeled purified proteins show the ascites tumors to be producing antibody of high titer (I50 = 10(-6) for anti-bone-Gla protein and (I50 = 10(-5) for antiosteonectin. Both of the antibovine antibodies are cross-reactive with the corresponding human protein. Immobilized specific anti-bone-Gla protein has been used to isolate human bone-Gla protein from an EDTA extract of human cortical bone. Thus, this method offers the possibility of developing a complete library of monoclonal antibodies against these and other bone-specific proteins.
Substance P modulation of acetylcholine-induced currents in embryonic chicken sympathetic and ciliary ganglion neurons.
Authors: Role L W
Published: May 2006

Substance P has been identified by combined immunohistochemical and radioimmunological techniques to be present in preganglionic cholinergic and sensory nerve fibers of amphibian, mammalian, and avian autonomic ganglia. The peptide has been shown to depolarize sympathetic neurons of frog and guinea pig and to decrease the cholinergic activation of Na+ influx and catecholamine release from chromaffin cells. The aim of this study was to examine the interaction of acetylcholine and substance P on autonomic neurons. This report demonstrates a direct effect of substance P on acetylcholine-induced inward currents in both sympathetic and parasympathetic neurons clamped near resting membrane potential. Under these conditions, substance P dramatically enhances the rate of decay of the inward current in the continued presence of agonist without substantially affecting peak inward current. This effect is consistent with an enhancement of acetylcholine-receptor desensitization. Since substance P-containing cell bodies have been demonstrated in the avian (preganglionic) column of Terni as well as in fibers from the nucleus of Edinger-Westphal, the observed peptide inhibition of cholinergic activation of the neurons may function physiologically to modulate synaptic function in autonomic ganglia.
All components required for the eventual activation of muscle-specific actin genes are localized in the subequatorial region of an uncleaved amphibian egg.
Authors: Gurdon J B , Mohun T J , Fairman S , Brennan S
Published: May 2006

Fertilized Xenopus eggs have been ligated with a hair loop into separate fragments before the first cleavage. The plane of the ligation was varied in relation to the animal-vegetal and dorso-ventral axes. The fragments that contained a nucleus were cultured for 24 hr until controls reached the neurula stage; they were then analyzed by S1 nuclease protection for their content of muscle-specific actin mRNA, using a gene-specific probe. We find that all egg components required for the eventual activation of these actin genes are localized, already at the 1-cell stage, in a region below the equator, and mostly on the dorsal (grey crescent) side. This material subsequently occupies the equivalent position in 8-cell and 32-cell embryos. We interpret our results, in combination with the previous work of others, to mean that mesoderm (including muscle) formation in Amphibia depends both on cytoplasmic substances already localized in the egg as well as on inductive cell interactions during cleavage.
RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates.
Authors: Reynolds P , Weber S , Prakash L
Published: May 2006

The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. We have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which we have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively, into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids.
Identification of a unique B-cell-stimulating factor produced by a cloned dendritic cell.
Authors: Clayberger C , DeKruyff R H , Fay R , Cantor H
Published: May 2006

We describe a cloned dendritic cell, clone Den-1, which is a potent accessory cell for some B-cell responses. Clone Den-1 produces a unique lymphokine that induces polyclonal B-cell proliferation in the absence of other costimulators. This clone or factors produced by it also stimulate purified B cells to develop plaque-forming cell responses to type 2 antigens. The effect of this factor(s) on various B-cell populations and its relationship to previously described B-cell-stimulating factors is discussed.
Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.
Authors: Mott J E , Grant R A , Ho Y S , Platt T
Published: May 2006

We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter. In p31-356, the normal rho promoter is deleted, but the remainder of the rho leader region, including the ribosome binding site, is present. In p39-AS, the rho leader is completely absent, and the lambda cII ribosome binding site replaces that of rho. Under noninducing conditions, expression of rho protein from these plasmids is repressed by the lambda cI protein in hosts carrying lambda cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of rho protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of approximately equal to 40% with p39-AS. The overproduced protein is functionally indistinguishable from the rho protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-AS in yields of up to 25 mg of rho per g of cells. In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid). Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.
Catalytic unit of adenlyate cyclase: purification and identification by affinity crosslinking.
Authors: Pfeuffer E , Dreher R M , Metzger H , Pfeuffer T
Published: May 2006

The guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG)-activated adenylate cyclase from rabbit myocardial membranes was purified approximately equal to 60,000-fold to a specific activity of 15 mumol X mg-1 X min-1 by Lubrol PX extraction, affinity chromatography, and gel permeation HPLC. The major purification (greater than 2000-fold) was achieved by affinity chromatography on forskolin-Sepharose, a method previously developed in this laboratory. The final product appeared as two major peptides of Mr 150,000 and 42,000 and one minor peptide of Mr 45,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis. It is suggested that the Mr 42,000 and 150,000 components represent the alpha-subunit of the stimulatory guanine nucleotide binding regulatory protein (GS) and the catalytic unit, respectively, because upon crosslinking of a reconstituted adenylate cyclase containing the [32P]ADP-ribosylated alpha-subunit of GS (Mr, 42,000), a single radiolabeled product of Mr 190,000 appeared on NaDodSO4/polyacrylamide gels. Further identification is based on the correlation of the Mr 150,000/42,000 bands with enzymatic activity when the purified enzyme was analyzed by various chromatographic procedures.
Respiratory burst oxidase from human neutrophils: purification and some properties.
Authors: Markert M , Glass G A , Babior B M
Published: May 2006

The respiratory burst oxidase of human neutrophils was purified by "dye-affinity" chromatography over a red agarose column. Electrophoresis of the purified enzyme on NaDodSO4 gel showed a single major band at 64,000-66,000 daltons, together with some minor contaminants. On a nondenaturing gel, the enzyme ran as two closely spaced bands, the faster of which contained flavin. When these two bands were rerun separately on a NaDodSO4 gel, they gave identical patterns, each showing a major band at ca. 65,000 daltons. The specific activity (mean +/- SEM) of the purified enzyme was 8.8 +/- 3.5 mumol of O-2 per min/mg of protein.
Molecular cloning and nucleotide sequence of the alpha and beta subunits of allophycocyanin from the cyanelle genome of Cyanophora paradoxa.
Authors: Bryant D A , de Lorimier R , Lambert D H , Dubbs J M , Stirewalt V L , Stevens S E , Porter R D , Tam J , Jay E
Published: May 2006

The genes for the alpha- and beta-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis. The AP beta-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP alpha- and beta-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning. The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined. The two open reading frames are in the same orientation and are separated by 39 base pairs. AP alpha is 5' to AP beta and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence. Sequences upstream from AP alpha closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP beta gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome.
Murine interferon-beta receptor-mediated endocytosis and nuclear membrane binding.
Authors: Kushnaryov V M , MacDonald H S , Sedmak J J , Grossberg S E
Published: May 2006

Radioiodinated mouse interferon-beta (125I-MuIFN-beta) bound with high affinity (Kd = 9.8 X 10(-10) M) to plasma membrane of L929 murine fibroblasts (4-6 X 10(3) receptor sites per cell). The binding was saturable and inhibited by a 100-fold excess of unlabeled MuIFN-beta but not by excess mouse IFN-gamma (MuIFN-gamma). MuIFN-beta bound at 4 degrees C was very rapidly internalized upon warming of the cells to 37 degrees C (t 1/2 = 1.5 min). Indirect immunoferritin labeling indicated that MuIFN-beta was initially located in coated pits and subsequently internalized by receptor-mediated endocytosis. Isolated L929 cell nuclei bound 125I-MuIFN-beta with a 7-fold higher affinity (Kd = 1.4 X 10(-10) M) and higher receptor density (about 10(4) per nucleus) than that for the plasma membrane. Binding to the nuclear membrane was inhibited by a 100-fold excess of unlabeled MuIFN-beta but not by excess MuIFN-gamma. Trypsin treatment of nuclei decreased IFN binding by 80%, suggesting that the putative nuclear receptors are protein. Specific binding of MuIFN-beta to nuclei was also shown by fluorescence and electron microscopy. We propose that the very rapid internalization of MuIFN-beta by receptor-mediated endocytosis is important in the cellular processing of IFN and that its high-affinity binding to the nuclear membrane suggests the nucleus as an intracellular site of IFN action.
Mitochondrial modulation of maternally transmitted antigen: analysis of cell hybrids.
Authors: Huston M M , Smith R , Hull R , Huston D P , Rich R R
Published: May 2006

Maternally transmitted antigen (Mta) is a murine cell surface class I-like antigen that is defined by specific cytotoxic lymphocyte reactivity. Mta is unique in that its expression requires cooperation between genetic elements both in the Qa/Tla region of chromosome 17 and in the cytoplasm. In view of the known cytoplasmic, and thus maternal, inheritance of mitochondria, we have directly assessed their potential involvement in Mta expression. The mitochondria-specific lethal dye rhodamine 6G (R6G) was used to control the input of mitochondria into cell hybrids. The parental lines, one of BALB/c and one of NZB origin, were known to differ in Mta and mtDNA phenotype. Our data show that most control BALB/c-NZB hybrids expressed the BALB/c Mta phenotype and likewise contained only BALB/c-type mtDNA. The NZB Mta phenotype was not coexpressed in the control hybrids. However, when the mitochondrial contribution from BALB/c was prevented by R6G treatment, the majority of the resultant hybrids expressed only the NZB Mta type and likewise contained only NZB mtDNA. The exceptional R6G-treated hybrids that continued to express the BALB/c Mta phenotype likewise contained only BALB/c mtDNA. Thus, in every case the mtDNA phenotype correlated with the Mta phenotype of the cells. Together, the data support the remarkable conclusion that mitochondria modulate the phenotypic expression of a cell surface molecule.
Efficient generation of antibodies to oncoproteins by using synthetic peptide antigens.
Authors: Tanaka T , Slamon D J , Cline M J
Published: May 2006

To examine the efficiency of generating protein-reactive antipeptide antibodies, 35 peptides encoded by retroviral or cellular oncogenes were used to immunize rabbits. Thirty-two peptides elicited antipeptide antibodies, of which 56% reacted with their respective oncoproteins. The length of the immunizing peptide was an important factor in generating antibodies reactive with native protein. Similar peptides differing in a single or a few amino acids could elicite antisera of markedly different reactivities.
Expression of a second Epstein-Barr virus-determined nuclear antigen in mouse cells after gene transfer with a cloned fragment of the viral genome.
Authors: Rymo L , Klein G , Ricksten A
Published: May 2006

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned in a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analyzed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed the EBV-determined nuclear antigen (EBNA) as expected. Cells transfected with recombinant vectors containing the BamHI W, Y, and H fragment part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. The BamHI WYH-induced EBNA polypeptide is similar in size to the EBNA2 polypeptide in Raji cells, as shown by gel electrophoresis and immunoblotting. The antigen is not detected in cells transfected with EcoRI A-derived vectors in which the BamHI H fragment has been deleted or in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA-transfected fibroblast clones.
Production of plasminogen activator in cultures of superior cervical ganglia and isolated Schwann cells.
Authors: Alvarez-Buylla A , Valinsky J E
Published: May 2006

Plasminogen activator has been implicated in tissue remodeling and cell migration during embryogenesis. In the developing nervous system, these processes are evident in the migration of neurons, axonal extension, Schwann cell migration, and the ensheathment and myelination of nerves. We have studied the production of plasminogen activator in cultures of superior cervical ganglia under conditions in which both neurons and glia are present. We have found that a principal source of the enzyme in these cultures is the glial cells and that the enzyme could not be detected at the growing tips of neurites. Plasminogen activator is also produced by Schwann cells isolated from neonatal rat sciatic nerve. The production of the enzyme by these cells is stimulated 6- to 10-fold by cholera toxin. Isolated Schwann cells and glial cells in the ganglion explant cultures produce the tissue form of plasminogen activator, a form of the enzyme not often found in nonmalignant cells. Preliminary experiments suggest that neuronal-glial interactions may regulate enzyme production by Schwann cells.
Characterization of Lewis antigens in normal colon and gastrointestinal adenocarcinomas.
Authors: Blaszczyk M , Pak K Y , Herlyn M , Sears H F , Steplewski Z
Published: May 2006

The Lewis a antigen (Lea)- and Lewis b antigen (Leb)-active glycolipids and glycoproteins in normal colon mucosa, colon carcinoma tumors, and gastrointestinal tumor cell lines were studied by using monoclonal antibodies against Lewis determinants in solid-phase radioimmunoassay, chromatogram binding assay, and immunoprecipitation/PAGE. Lea-5 was a major component of the purified non-acid glycolipid fraction from normal colon mucosas from four patients examined and Lea-7 was a minor component in three of these four cases. Lea in colon carcinoma of the same patients was expressed as Lea-5 in all four cases and as both Lea-5 and Lea-7 in one of the four cases. All normal colon mucosa samples expressed Lea but not Leb. Leb-6 was expressed in all tumors obtained from the same patients as normal colon mucosa (Leb negative) samples and in the gastrointestinal tumor cell lines. These findings indicate that Leb is a colon adenocarcinoma-associated antigen and that Lea and Leb are coexpressed in tumor tissue. Evidence is presented for the coexistence of Lea and Leb carbohydrate determinants in both glycolipid and glycoprotein forms in the same cells.
Insulin-stimulated glucose uptake in rat diaphragm during postnatal development: lack of correlation with the number of insulin receptors and of intracellular glucose transporters.
Authors: Wang C
Published: May 2006

The insulin responsiveness of the membrane transport system for glucose (2-deoxy-D-glucose) in diaphragm was measured during postnatal development of the rat. At birth, the basal rate of 2-deoxy-D-glucose transport is 3 nmol/min X g and it gradually decreases to 1 nmol/min X g over a period of 40 days. On the other hand, the insulin-stimulated rate of transport is 6 nmol/min X g at birth, it increases to 9 nmol/min X g in 16- to 20-day-old rats, and it decreases again to approximately 4 nmol/min X g in the 40-day-old rats. The stimulation of 2-deoxy-D-glucose transport by insulin is 2-fold at birth and increases to 4- to 5-fold 20 days after birth. The number of insulin receptors in the plasma membrane and the number of intracellular glucose transporters was also measured as a function of age to determine if there might be a correlation between these components of the insulin responsive system and the development of the increased insulin stimulation of 2-deoxy-D-glucose transport. The number of insulin receptors per g of wet weight decreased continuously with increasing age; the diaphragm of 40-day-old rats had about 50% of the receptors present in the diaphragm of the newborn rat. Similarly, the number of intracellular D-glucose transporters per g of wet weight decreased with increasing age; for adult rats, the number of transporters per g of diaphragm was 60% of that of newborn rats. The results indicate that the extent of insulin stimulation of glucose (2-deoxy-D-glucose) transport in the diaphragm during the first 20 days of life is not directly or simply related to the number of insulin receptors or the number of intracellular glucose transporters. The extent of the insulin response depends on some other factor that activates or is part of the machinery for translocation of the transporter.
Eubacteria, halobacteria, and the origin of photosynthesis: the photocytes.
Authors: Lake J A , Clark M W , Henderson E , Fay S P , Oakes M , Scheinman A , Thornber J P , Mah R A
Published: May 2006

The halobacteria and the photosynthetic members of the eubacteria have previously been classified in two separate urkingdoms--the archaebacteria and the eubacteria, respectively. They were thought to be no more closely related to each other than they each were to the eukaryotes. In accord with this earlier classification, photosynthesis was thought to have originated twice by independent events--once within the eubacteria and once within the archaebacteria. In this paper, however, using three-dimensional ribosome structure as a probe of evolutionary divergences, we show that the eubacteria and the halobacteria are more closely related to each other than they are to any other known organisms. The simplest interpretation of our data is that all extant photosynthetic cells are descended from a single common ancestor that possessed a primeval photosynthetic mechanism. Numerous data on the occurrence of related biochemical processes in halobacteria and eubacteria support this theory. Essential components of the photosynthetic apparatus, such as carotenoids, are present in both halobacteria and in eubacteria, including the nonphotosynthetic eubacteria, suggesting that photosynthesis could be a primitive property of both groups. Our data indicate that together the eubacteria and the halobacteria form a monophyletic group for which we propose the name "photocytes." If other techniques of phylogenetic analysis confirm this evolutionary tree, we propose that the photocytes be given urkingdom status.
Activation of the c-myc oncogene by the immunoglobulin heavy-chain gene enhancer after multiple switch region-mediated chromosome rearrangements in a murine plasmacytoma.
Authors: Fahrlander P D , Sümegi J , Yang J Q , Wiener F , Marcu K B , Klein G
Published: May 2006

Presented is a detailed molecular analysis of the rearranged c-myc oncogene from ABPC45, an unusual plasmacytoma that was originally classified as translocation-negative. Previous data obtained by high-resolution chromosome banding suggested that this tumor was a member of a small group distinguished by the absence of rcpt (12;15) or (6;15) and further characterized by a band deletion near the c-myc locus on chromosome 15. However, genomic Southern blotting and analysis of the cloned oncogene in the present study reveal that (i) chromosome 12 sequences lie 365 base pairs 5' of the rearranged c-myc; (ii) this DNA consists of immunoglobulin alpha switch region and 5' immunoglobulin mu switch region sequences that are rearranged in an aberrant fashion; and (iii) the immunoglobulin heavy-chain gene enhancer element now resides approximately 2.5 kilobase pairs 5' of c-myc. We infer from these and other data that the rearrangement of c-myc in ABPC45 occurred via a multistep switch region-mediated process and that a reciprocal translocation has indeed taken place. Unlike many other plasmacytomas, this event did not interrupt the normal c-myc transcription unit. Rather, disruption of gene regulation is manifested in part by a change in relative usage of the two promoters normally used by the unrearranged gene. In contrast to several of its counterparts in Burkitt lymphomas, DNA sequence analysis of the translocated c-myc gene of ABPC45 reveals that it has not acquired point mutations in the noncoding first exon. These results strongly imply that a cis-acting regulatory element normally located 5' of exon 1 is lost and that heavy-chain constant region or enhancer sequences exert similar cis effects on translocated c-myc loci.
Differential amplification, assembly, and relocation of multiple DNA sequences in human neuroblastomas and neuroblastoma cell lines.
Authors: Shiloh Y , Shipley J , Brodeur G M , Bruns G , Korf B , Donlon T , Schreck R R , Seeger R , Sakai K , Latt S A
Published: May 2006

DNA amplification, manifested by homogeneously staining regions in chromosomes and by extrachromosomal, double minute bodies, is characteristic of many neuroblastoma cell lines. Sequences recruited from a specific domain on the short arm of chromosome 2 (2p) are amplified in advanced-stage primary neuroblastomas, whereas sequences from distinctly different regions of 2p are amplified in the neuroblastoma cell line IMR-32. Five different DNA segments, which include the oncogene N-myc, three other fragments derived from the homogeneously staining region of the neuroblastoma cell line IMR-32, and a fifth fragment, derived from the neuroblastoma cell line NB-9, showed differential and variable amplification in 24 advanced-stage neuroblastoma tumors out of 112 tested specimens. All five fragments were mapped within the chromosomal region 2p23-2p25 by three different approaches. However, eight other fragments cloned from the homogeneously staining region of IMR-32 cells, which were not amplified in the tumor tissues examined, were mapped to two more proximal domains of 2p, thousands of kilobases apart from each other and from the chromosomal domain that is amplified in the tumors. These results establish the amplification, to different degrees, of a variable-sized segment of one domain near the terminus of 2p in advanced neuroblastomas. These tumors might ultimately be distinguished according to the pattern of amplification of DNA segments within this domain. The data presented also indicate the existence of a new and complex amplification mechanism in at least one neuroblastoma cell line (IMR-32), which involves not only relocation of DNA from specific genomic domains but also the formation of novel units by splicing together very distant DNA segments.
Site-specific DNA inversion is enhanced by a DNA sequence element in
cis
Authors: Huber Hans E. , Iida Shigeru , Arber Werner , Bickle Thomas A.
Published: May 2006

A segment of the bacteriophage P1 genome, called the C segment, can be inverted by site-specific recombination; the two different orientations of the invertible segment confer different host ranges to the phage. Inversion is catalyzed by the product of the
cin
gene which is adjacent to one of the crossover sites flanking the C segment. The Cin-catalyzed recombination can be measured in
trans
by using tester plasmids in which inversion switches on antibiotic-resistance genes. We show here that an additional sequence, distinct from the two crossover sites, is needed in
cis
for efficient inversion. This sequence is part of the
cin
structural gene and stimulates recombination more than 100-fold. We have localized the major enhancer sequence on a 72-base-pair fragment and found its activity to be largely independent of the orientation or position of the sequence with respect to the crossover sites.
Molecular characterization of MT3 antigens by two-dimensional gel electrophoresis, NH2-terminal amino acid sequence analysis, and southern blot analysis.
Authors: Sorrentino R , Lillie J , Strominger J L
Published: May 2006

The monoclonal antibody 109d6, which recognizes major histocompatibility antigen MT3-like serologic determinants, has been used to characterize the molecules bearing this determinant in HLA-DR4 and -DR7 homozygous cell lines by two-dimensional gel and sequencing analyses. By these two criteria, these molecules are identical to each other. Southern blot analysis of genomic DNA from HLA-DR1 through -DR7 homozygous cell lines with DR beta-chain gene probes reveals a striking similarity in the pattern of hybridizing fragments between DR4 and DR7 haplotypes and among DR3, DR5, and DRw6 haplotypes reminiscent of the MT3/MT2 allodeterminant distribution. The sharing of the MT2 determinant between DR3, DR5, and DRw6 haplotypes and of the MT3 determinant between DR4 and DR7 haplotypes is part of a broader "homology," which may be a consequence of more recent separation of the haplotypes sharing the MT2 determinant on the one hand and the haplotypes sharing the MT3 determinant on the other hand.
The C1q subunit of the first component of complement binds to laminin: a mechanism for the deposition and retention of immune complexes in basement membrane.
Authors: Bohnsack J F , Tenner A J , Laurie G W , Kleinman H K , Martin G R , Brown E J
Published: May 2006

The C1q subunit of complement component C1 is known to bind to immune complexes, which often are deposited in basement membrane. We investigated the possibility that this deposition is a result of binding to laminin, a large basement membrane glycoprotein. C1q showed saturable binding to immobilized laminin; this binding was increased at reduced ionic strength. Intact C1 did not bind laminin. A ternary complex was formed by laminin, C1q, and aggregated IgG. This complex formation was dependent on and proportional to the amount of C1q bound to the aggregated IgG. Binding of laminin to C1q occurred with a Kd of 2 nM and was stronger than the binding of C1q to fibronectin. Preliminary data, including electron micrographs of rotary-shadowed preparations, suggest that laminin binds to the collagen-like tail of C1q. Electron microscopy localized the site of interaction with C1q to a short arm of laminin. Since laminin is found only in basement membranes, the interaction between laminin and C1q could be involved in the deposition and retention of immune complexes in these structures.
Role of hepatic tetrahydrofolate in the species difference in methanol toxicity.
Authors: Black K A , Eells J T , Noker P E , Hawtrey C A , Tephly T R
Published: May 2006

The susceptibility of various species to methanol toxicity is inversely related to the rate of tetrahydrofolate (H4folate)-dependent formate oxidation to carbon dioxide. Thus, the levels of various folate derivatives and folate-dependent enzyme activities present in the livers of monkeys, which are sensitive to methanol, and rats, which are not, were compared in order to investigate the biochemical basis of this species difference. Hepatic H4folate levels in monkeys were 60% of those in rats, and formylated-H4folate derivatives were 2-fold higher in monkeys than in rats. No significant difference between monkeys and rats in the levels of total hepatic folate or 5-methyl-H4folate was observed. The activities of formyl-H4folate synthetase (EC 6.3.4.3) and formyl-H4folate dehydrogenase (EC 1.5.1.6) were 4- and 2-fold higher, respectively, in monkeys than in rats. There was no significant difference between monkeys and rats in methionine synthetase activity (EC 2.1.1.13). Dihydrofolate reductase activity (EC 1.5.1.3) in monkeys was 20% of that in rats. 5,10-Methylene-H4folate reductase (NADPH) activity (EC 1.1.1.171) in monkeys was 40% and 25% of that in rats when the rates of the forward and reverse reactions, respectively, were compared. Serine hydroxymethyltransferase activity (EC 2.1.2.1) was 2-fold higher in monkeys than in rats. The differences in the activities of methylene-H4folate reductase and serine hydroxymethyl-transferase between monkeys and rats may have contributed to the difference in hepatic H4folate levels. The 40% lower level of hepatic H4folate in monkeys, as compared to rats, relates well to the 50% lower maximal rate of formate oxidation in monkeys. Thus, the species difference in susceptibility to methanol may be explained by the difference in the level of hepatic H4folate.
Homology of the rat basophilic leukemia cell and the rat mucosal mast cell.
Authors: Seldin D C , Adelman S , Austen K F , Stevens R L , Hein A , Caulfield J P , Woodbury R G
Published: May 2006

Secretory granules of the rat basophilic leukemia (RBL-1) cell, a chemically generated tumor cell line maintained in tissue culture, were shown to stain with alcian blue but not with safranin counterstain and to have sparse, small, electron-dense granules. A Mr 25,000 protein was the major [3H]diisopropyl fluorophosphate-binding protein in extracts of RBL-1 cells. Double-immunodiffusion analysis of extracts revealed immunoreactivity for rat mast cell protease (RMCP)-II, a Mr 25,000 neutral protease present in the secretory granules of rat mucosal mast cells and cultured rat bone marrow-derived mast cells, but no immunoreactivity for RMCP-I, the predominant neutral protease of rat connective tissue mast cells. By radial immunodiffusion, there was 66.8 ng of RMCP-II per 10(6) cells. Whereas rat connective tissue mast cells stain with alcian blue and safranin and contain heparin proteoglycan, rat mucosal and rat bone marrow-derived mast cells stain with alcian blue only and contain a non-heparin proteoglycan and lesser amounts of histamine. Proliferation of rat mucosal mast cells in vivo and rat bone marrow-derived mast cells in vitro requires T-cell factors, whereas no comparable requirement has been observed for connective tissue mast cells. The transformed RBL-1 tumor cells, whose growth is independent of factors other than those present in standard tissue culture medium, has previously been shown to contain predominantly chondroitin sulfate di-B proteoglycans and low amounts of histamine. The similar histology and secretory granule biochemistry of the rat mucosal mast cells, rat culture-derived mast cell, and RBL-1 cell suggest that they comprise a single mast cell subclass distinct from the rat connective tissue mast cell.
6-Phosphogluconolactonase deficiency, a hereditary erythrocyte enzyme deficiency: possible interaction with glucose-6-phosphate dehydrogenase deficiency.
Authors: Beutler E , Kuhl W , Gelbart T
Published: May 2006

Partial deficiency of 6-phosphogluconolactonase (EC 3.1.1.31) of the erythrocytes was discovered as an autosomal dominant disorder. Hemolytic anemia occurred in an individual who had inherited both the gene for 6-phosphogluconolactonase deficiency and that for deficiency of a nonhemolytic variant of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). It is proposed that the interaction of this hereditary erythrocyte abnormality with glucose-6-phosphate dehydrogenase deficiency may explain hemolysis in some other patients who have inherited polymorphic variants of glucose-6-phosphate dehydrogenase.
The small cardioactive peptides A and B of Aplysia are derived from a common precursor molecule.
Authors: Mahon A C , Lloyd P E , Weiss K R , Kupfermann I , Scheller R H
Published: May 2006

We have identified cells in the central nervous system of the marine mollusc Aplysia that react with antibody raised against the small cardioactive peptide B (SCPB). Antisera to this neuropeptide stained a subset of central neurons that include the large identified buccal neurons, B1 and B2. The distribution of SCP-containing neurons was used in a strategy to isolate a cDNA clone encoding the precursor protein for the peptide. RNA from neurons B1 and B2 and from cells that did not stain with SCPB antisera was used to direct the synthesis of radiolabeled cDNA probes. A cDNA clone complimentary to mRNA specifically expressed in the B1 and B2 cells was isolated by differentially screening a buccal cDNA library with these probes. The cloned cDNA segment is 1394 nucleotides in length and contains a 408-base-pair open reading frame. The predicted precursor protein is composed of 136 amino acids and has a characteristic hydrophobic leader sequence. The sizes of the precursor protein with and without this leader sequence agree with in vivo and in vitro labeling studies. The amino acid sequences for SCPB and a related peptide, SCPA, are present and are flanked by known proteolytic processing sites.