bluuebunny/crossref_metadata_2025 huggingface dataset:
Affinity chromatography purification of cytochrome c binding enzymes.
Authors: Azzi A , Bill K , Broger C
Published: May 2006

An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.
Electron-electron double resonance and saturation-recovery studies of nitroxide electron and nuclear spin-lattice relaxation times and Heisenberg exchange rates: lateral diffusion in dimyristoyl phosphatidylcholine.
Authors: Popp C A , Hyde J S
Published: May 2006

Lateral diffusion constants of the stearic acid nitroxide radical spin label 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolindinyl oxide in dispersions of dimyristoyl phosphatidylcholine have been measured. Electron-electron double resonance methods were used to determine the product of the bimolecular collision frequency and T1e, the electron spin-lattice relaxation time. T1e in turn was measured by the technique of saturation recovery. The theoretical model of Träuble and Sackmann was then used to relate the bimolecular collision frequencies to the diffusion constants. Results are in agreement with other methods. Lower spin-label concentrations than were used in previous electron paramagnetic resonance studies are needed (label-to-lipid ratio less than 0.5 mol%). Analysis of the data also yields values of the nitrogen nuclear spin-lattice relaxation time of the nitroxide moiety. These values are indicative of membrane fluidity.
Immunological agnosis: a state that derives from T suppressor cell inhibition of antigen-presenting cells.
Authors: Ptak W , Gershon R K
Published: May 2006

The biologically active mediators of antigen-specific T suppressor cells can combine with antigen on cells that are specialized to present antigen (APC) and render these APC incapable of presenting not only the specific antigen that the product of the T suppressor cell sees but also any other antigen in or on the APC. Thus, antigen-bearing suppressed APC fail to activate either the helper or suppressor system involved in the regulation of contact sensitivity responses. These results demonstrate that APC are targets of T suppressor cells. They also imply that a metabolic event is required for functional antigen presentation and that T suppressor cells can block that metabolic pathway.
Multiple HLA-DR antigens: detection with monoclonal antibodies and translation in vitro.
Authors: Finn O J , Levy R
Published: May 2006

L203 and L227 are mouse monoclonal antibodies specific for human HLA-DR antigens. Their reactivity with multiple, structurally different, HLA-DR antigens was examined by using radioiodinated human lymphoblastoid cells and cell lines, heterozygous or homozygous at the HLA-D locus. Antibody L203 precipitates DR antigens composed of 34,000 Mr heavy chains (alpha), and 28,000-29,000 Mr light chains (beta). Antibody L227 recognizes two other species of DR antigens. Their heavy chains have similar molecular weights of 34,000 but their light chains are different, one (beta) being 28,000-29,000 Mr and the other (beta') 23,000-25,000 Mr. Whereas L203 is directed against a combinatorial determinant generated by both DR chains, L227 is able to recognize each of the two light chains alone and reprecipitate them after their separate elution from sodium dodecyl sulfate gels. Poly(A)-containing mRNA was isolated from Raji cells and translated in vitro by microinjection in Xenopus oocytes. The oocytes translated and fully assembled all the DR molecules recognized by L203 and L227 antibodies.
Abnormal humoral immune responses in peripheral blood lymphocyte cultures of bone marrow transplant recipients.
Authors: Pahwa S G , Pahwa R N , Friedrich W , O'Reilly R J , Good R A
Published: May 2006

The present study was aimed at investigating recovery of humoral immunity in vitro after bone marrow transplantation in patients with acute leukemia and severe aplastic anemia. Hemolytic plaque assays were utilized to quantitate pokeweed mitogen-stimulated polyclonal immunoglobulin production and sheep erythrocyte antigen-specific antibody responses in cultures of peripheral blood mononuclear cells of 39 patients beginning at 1 month, for variable periods up to a maximum of 4 years after marrow transplantation. Three phases were identified: an early period of primary B cell dysfunction with concomitant immunoregulatory T cell abnormalities--i.e., decreased helper and increased suppressor activities; an intermediate phase in which B cell dysfunction could be attributed in large measure to immunoregulatory T cell abnormalities; and a late phase of normal B and T lymphocyte functions. Patients with graft-versus-host disease differed from those without it in that they often did not manifest increased T cell suppressor activity in the early period, and they were noted to have prolonged and profound B and T cell abnormalities in the chronic phase of their disease. In selected patients, simultaneous assessment of ratios of Leu-2 to Leu-3 antigens on T cells by monoclonal antibodies and of immunoregulatory T cell functions revealed a correlation between the two only late in the post-transplant period. These studies provide an insight into the ontogeny of B cell function in the post-transplant period and indicate that in certain situations phenotypic alterations in T cell subsets cannot reliably be used to predict abnormalities in their function in recipients of marrow transplantation.
Reductive metabolism of carbon tetrachloride by human cytochromes P-450 reconstituted in phospholipid vesicles: mass spectral identification of trichloromethyl radical bound to dioleoyl phosphatidylcholine.
Authors: Trudell J R , Bösterling B , Trevor A J
Published: May 2006

It has been proposed that covalent binding of reactive metabolites to liver membrane constituents may be responsible for the hepatoxicity of carbon tetrachloride. This study demonstrates that trichloromethyl free radical is the major reductive metabolite of carbon tetrachloride by cytochrome P-450 and that this free radical is capable of binding to double bonds of fatty acyl chains of the phospholipids in the membrane surrounding cytochrome P-450. The structural identification of the reactive free radical metabolite and the product of its addition to phospholipids was accomplished by use of a reconstituted system of human cytochromes P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5 in phospholipid vesicles. The reconstituted vesicles contained a mixture of dioleoyl phosphatidylcholine and egg phosphatidylethanolamine that served as both structural components and targets for trichloromethyl free radical binding. After incubation of these vesicles under a N2 atmosphere in the presence of NADPH with 14CCl4, the phospholipids were extracted and then separated by high-pressure liquid chromatography. The dioleoyl phosphatidylcholine fraction was transesterified and the resulting single 14C-labeled fatty acid methyl ester was purified by reverse-phase chromatography. Desorption chemical ionization mass spectrometry with ammonia as reagent gas as well as desorption electron-impact mass spectrometry permitted identification of the molecular structure as a mixture of 9- and 10-(trichloromethyl)stearate methyl esters.
Taurine in the mammalian cerebellum: Demonstration by autoradiography with [
3
H]taurine and immunocytochemistry with antibodies against the taurine-synthesizing enzyme, cysteine-sulfinic acid decarboxylase
Authors: Chan-Palay Victoria , Lin Chin-Tarng , Palay Sanford , Yamamoto Miyuki , Wu Jang-Yen
Published: May 2006

Taurine neurons and their dendrites and axons were visualized in the mammalian cerebellum by autoradiography, after
in vivo
injections of [
3
H]taurine directly into the cerebellar cortex or deep cerebellar nuclei, and by immunocytochemistry at the light- and electron-microscope levels with antibodies against cysteine-sulfinic acid decarboxylase (CSADCase; L-cysteine-sulfinate carboxylyase, EC 4.1.1.29). Uptake and sequestration of [
3
H]taurine labeled numerous Purkinje cell somata, primary dendrites, and axons; many granule cell somata, dendrites, and parallel fibers; stellate, basket, and Golgi cells; the larger neurons in all deep cerebellar nuclei; the largest neurons in the lateral vestibular nucleus; and, more rarely, Purkinje cell axonal terminals in the neuropil. The label at all sites was diminished by preinjection into the cerebellum of hypotaurine,
p
-chloromercuriphenylsulfonic acid, or β-alanine, and was virtually eliminated by strychnine. Immunocytochemical labeling with polyclonal antibodies directed against CSADCase, the enzyme responsible for the synthesis of hypotaurine from cysteine sulfinic acid and taurine from cysteic acid, had a similar distribution. In electron micrographs, immunoreactivity within Purkinje cell somata and dendrites was localized to the Golgi apparatus, the inner plasma membrane, and condensed nonmembranous foci (120 nm in diameter) marked by clumps of peroxidase reaction product. Large Nissl bodies were usually not CSADCase immunoreactive. Numerous immunoreactive granule cells, dendrites, and parallel fibers were recognized. Pretreatment of the animals with colchicine increased the intensity of CSADCase immunoreactivity but did not change the number or distribution of labeled cells. These experiments indicate that taurine is synthesized and involved in a specific uptake process by cerebellar neurons. Neuroglial cells do not synthesize taurine but some neuroglia take up [
3
H]taurine. These findings call for a reexamination of the physiological function of taurine in the cerebellum. A hypothesis is proposed that taurine may be involved in the regulation of calcium, in dendritic spike generation, and in the inhibition of impulse propagation in major Purkinje cell dendrites.
Secretory proteins induced in human fibroblasts under conditions used for the production of interferon beta.
Authors: Content J , De Wit L , Pierard D , Derynck R , De Clercq E , Fiers W
Published: May 2006

Human fibroblast cells treated with a combination of inhibitors of protein and RNA synthesis [cycloheximide and actinomycin D as used to superinduce interferon beta (IFN-beta)] secrete two proteins with molecular masses of 22000 and 27000 kilodaltons (called 22-kDal and 27-kDal) that are precipitable with an antiserum raised against impure IFN-beta but are antigenically distinct from IFN-beta 1. Translation in vitro of mRNA extracted from human fibroblast cells induced for the production of IFN-beta leads to the synthesis of a 26-kDal protein that is structurally closely related to the 22- and 27-kDal proteins. This 26-kDal protein mRNA is relatively abundant and also appears in human fibroblasts induced only with cycloheximide. It has been partially purified by sucrose gradient centrifugation and more extensively by diazobenzyloxymethyl-cellulose hybridization to plasmid DNA from a bacterial cDNA clone. When translated in an in vitro reticulocyte system supplemented with dog pancreas microsomes, the 26-kDal protein and two other intermediates corresponding presumably to its signal-cleaved (19-kDal) and partially glycosylated (24-kDal) forms were observed. Crude, partially purified, and highly purified 26-kDal mRNA failed to program the synthesis of antiviral or ppp(A2'p5')nA synthetase-inducing activity when translated in Xenopus laevis oocytes. Moreover, partially purified 22-kDal and 27-kDal (i.e., the in vivo equivalents of the 26-kDal protein) are also devoid of antiviral or ppp(A2'p5')nA synthetase-inducing activity. Hence, this 26-kDal mRNA, although presumably identical to the human IFN-beta 2 mRNA described by Weissenbach et al. [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, K., Soreq, H., Nir. U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl. Acad. Sci. USA 77, 7152-7156], cannot be considered to be a fibroblast interferon mRNA.
Simultaneous expression of immunoglobulin mu and delta heavy chains by a cloned B-cell lymphoma: a single copy of the VH gene is shared by two adjacent CH genes.
Authors: Knapp M R , Liu C P , Newell N , Ward R B , Tucker P W , Strober S , Blattner F
Published: May 2006

The cloned murine B-cell lymphoma line (BCL1) that expresses surface IgM and IgD is considered to be a model for the immunoglobulin gene expression of the mature virgin B cell. Of particular interest is the mechanism by which a single VH gene is shared by two CH genes. We examined the organization of the immunoglobulin heavy chain genes in BCL1 DNA. A single arrangement of CH genes was found with the expressed VHDJH gene complex just 5' to the Cmu gene. The complete DNA sequence of the VH gene was determined. No rearrangement occurred in the intervening DNA between the JH and C mu genes or between the C mu and C delta genes. We conclude that dual expression of mu and delta heavy chains using a single VH gene is accomplished by alternate processing of a primary transcript that encompasses the the VHDJH complex and both CH genes.
Purification and initial characterization of rat interleukin 2.
Authors: Di Sabato G
Published: May 2006

With the sequential use of dialysis, chromatography on Sephadex G-100, reactive red 120-agarose, p-hydroxymercuribenzoate-agarose, phenyl-Sepharose, poly(L-lysine)-agarose, and isoelectrofocusing, the thymocyte stimulatory factor activity of interleukin 2 from rat spleen was purified about 8,000-fold. By the same procedures, the T cell growth factor activity of interleukin 2 was purified about 6,000-fold. The major peaks of thymocyte stimulatory factor activity and T cell growth factor activity cochromatographed in the various systems used, but T cell growth factor activity without significant thymocyte stimulatory factor activity was sometimes present. Both activities were found to have a pI between pH 5.50 and 6.30. Detectable thymocyte stimulatory factor activity was found at concentrations as low as 0.1-0.2 ng of protein per 0.2 ml. Dose--response plots were linear up to at least 1 ng of protein. Preparations of interleukin 2 labeled with 125I-containing Bolton--Hunter reagent and purified by the procedure mentioned above were electrophoresed on a polyacrylamide gel under denaturing and reducing conditions. The 125I-labeled material migrated in one major band with a molecular weight under 20,000 and three smaller bands with molecular weights of about 20,000, 60,000, and 90,000. Material with thymocyte stimulatory factor activity did not bind to a number of lectin-gels.
An 18,200-dalton adrenal protein that contains four [Met]enkephalin sequences.
Authors: Kilpatrick D L , Jones B N , Lewis R V , Stern A S , Kojima K , Shively J E , Udenfriend S
Published: May 2006

An 18,200-dalton adrenal enkephalin-containing polypeptide was purified to homogeneity and shown to contain four internal [Met]enkephalin sequences. Protein sequence analysis and mapping of trypsin-derived peptides demonstrated that the previously described 12,600-dalton and 5,300-dalton adrenal enkephalin-containing polypeptides compose the amino-terminal and carboxyl-terminal sequences, respectively, of this larger protein. They are linked by two basic amino acid residues to form the entire sequence of the 18,200-dalton protein. This protein is the largest enkephalin-containing polypeptide yet isolated from beef adrenal medulla and serves as the precursor for several other previously characterized adrenal enkephalin-containing proteins and peptides.
Partial primary structure of bovine plasma fibronectin: Three types of internal homology
Authors: Petersen Torben E. , Thøgersen Hans C. , Skorstengaard Karna , Vibe-Pedersen Karen , Sahl Preben , Sottrup-Jensen Lars , Magnusson Staffan
Published: May 2006

Approximately one-half of the amino acid sequence (911 amino acid residues out of 1,880 expected) for bovine plasma fibronectin (cold-insoluble globulin) has been determined. Three types of internal homology were identified, showing that a number of partial gene duplications (multiplications) have occurred during the evolution of this protein. Digestion of fibronectin with plasmin results in major fragments with molecular masses of 29, 170, 23, and 6 kilodaltons (kDal). The NH
2
-terminal 29-kDal fragment consists of 259 residues ordered as five mutually homologous domains (type I homology) with two disulfide bonds in each domain. The 170-kDal fragment shows two to three bands after NaDodSO
4
gel electrophoresis, indicating heterogeneity. This fragment contains the gelatin binding site and the strong heparin binding site present in fibronectin. Digestion of the 170-kDal fragment with chymotrypsin liberates a 45-kDal fragment that also binds to gelatin. This fragment contains at least one domain of type I homology and two domains of type II homology. Further digestion of the 170-kDal fragment with chymotrypsin results in the formation of a 30-kDal fragment that retains the heparin binding activity. This fragment contains sequences constituting type III homology. The 23-kDal fragment consists of 178 residues having three domains of type I homology. The 6-kDal fragment consists of two identical peptides of 26 residues, and these two peptides are linked to each other by two disulfide bonds that form the interchain bridges. Another one of the peptides for which the sequence was determined links the COOH-terminus of the 29-kDal fragment to the NH
2
-terminus of the 170-kDal fragment. This and the fact that the COOH-terminal residue of the 6-kDal fragment is a glutamic acid residue order the four plasmin-digestion fragments as 29-, 170-, 23-, and 6-kDal in the intact fibronectin molecule.
Metal and sulfur composition of iron-molybdenum cofactor of nitrogenase.
Authors: Nelson M J , Levy M A , Orme-Johnson W H
Published: May 2006

The sulfur content of N-methylformamide solutions of cofactor from Clostridium pasteurianum nitrogenase has been determined to be 11.9 (+/- 0.9) mol per mol of molybdenum. This number was determined radiochemically, using iron-molybdenum cofactor isolated from molybdenum-iron protein from bacteria grown on 35SO4. A total of 3.2 (+/- 0.2) mol of sulfur per mol of molybdenum was found to be present in cysteine and methionine, probably arising from contaminating proteins not intrinsic to the cofactor. Combined with accumulated evidence that is discussed, these results lead to an updated stoichiometry of MoFe6S8 or 9, not MoFe6S4 as previously thought, for this cluster.
Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides.
Authors: Conner B J , Reyes A A , Morin C , Itakura K , Teplitz R L , Wallace R B
Published: May 2006

Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human beta-globin gene (beta A) and one complementary to the sickle cell beta-globin gene (beta S). The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the beta A gene from the beta S allele. The DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) only hybridized with the beta A specific probe; the DNA from those homozygous for the sickle cell beta-globin gene (beta S beta S) only hybridized with the beta S specific probe. The DNA from heterozygous individuals (beta A beta S) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.
Primary structures of bovine liver low molecular weight kininogen precursors and their two mRNAs.
Authors: Nawa H , Kitamura N , Hirose T , Asai M , Inayama S , Nakanishi S
Published: May 2006

By using a mixture of synthetic oligodeoxyribonucleotides as a probe, cloned cDNA sequences specific for low molecular weight (LMW) kininogen have been isolated from a cDNA library of bovine liver mRNA sequences. Nucleotide sequence analyses of cloned cDNA inserts have revealed that bovine liver LMW kininogens are encoded by at least two very similar but distinct mRNAs. The corresponding amino acid sequences show that the LMW kininogen precursors of the two types, composed of 436 and 434 amino acid residues, both contain two internally homologous sequences in the amino-terminal portion between a signal peptide and a bradykinin moiety. The two mRNAs exhibit 15 nucleotide substitutions and 6 nucleotide deletions/additions in their protein-coding regions. The replacement of 13 amino acid residues and the deletions/additions of 2 amino acid residues in the two LMW kininogen precursors are all localized within the internally homologous regions, implying that these regions may be biologically significant in relation to the existence of two LMW kininogens. The nucleotide changes in the two mRNAs also occur in the limited portions that principally encode the internally homologous amino acid sequences. This suggests that the mRNAs are transcribed from the same gene to generate two LMW kininogen precursors differing only in the internally homologous sequences.
Decreased DNA-binding ability of purified transformation-specific proteins from deletion mutants of the acute avian leukemia virus MC29.
Authors: Donner P , Bunte T , Greiser-Wilke I , Moelling K
Published: May 2006

Avian myelocytomatosis virus MC29 is a highly oncogenic replication-defective retrovirus that predominantly affects hematopoietic cells and causes acute leukemia in vivo and that transforms hematopoietic cells as well as fibroblasts in vitro. The transformation-specific sequence, v-myc, is expressed as part of a fusion protein that contains the viral structural protein p19. By use of monoclonal antibodies against p19 we showed that the v-myc-encoded protein is located in the nucleus of MC29-transformed fibroblasts and that after purification over an immunoaffinity column the protein binds to double-stranded DNA. In this report we describe the analysis of the v-myc gene product from MC29-transformed bone marrow cells. The immunoaffinity column-purified protein from these cells also bound to DNA and was indistinguishable from the purified protein from MC29-transformed fibroblasts. In addition, the v-myc gene products from fibroblasts transformed by three nonconditional mutants of MC29--which transform hematopoietic cells with a markedly decreased efficiency in vivo and in vitro but still transform fibroblasts in vitro, expressing deleted v-myc proteins--were analyzed. In contrast to the wild-type protein, the purified mutant proteins had decreased DNA-binding abilities. Furthermore, a preferential binding of the wild-type protein to poly(dG) . poly(dC) duplexes was observed. Such a specificity was lost with a mutant protein. These results provide evidence that the interaction of the v-myc protein with DNA may be directly involved in transformation of the hematopoietic target cells. Further, the transformation-specific fusion proteins purified from cells transformed by avian erythroblastosis virus, which belongs to a different class of acute leukemia viruses, and by Fujinami sarcoma virus were found not to be DNA-binding proteins, suggesting the existence of different transformation mechanisms.
Enzymic synthesis and cofactor activity of 3'-pyrophosphocoenzyme A.
Authors: Mukai J I , Sy J , Lipmann F
Published: May 2006

The 3'-pyrophosphate derivative of CoA was synthesized by using the excreted 5'-to-3' pyrophosphoryl-transferring enzyme from Streptomyces adephospholyticus and ATP as donor and dephospho-CoA as acceptor. Cofactor activity of this new coenzyme A derivative was tested with Clostridium kluyveri phosphotransacetylase and hog heart succinic thiokinase. With the phosphotransacetylase, 3'-pyrophospho-CoA was found to be twice as active as CoA whereas dephospho-CoA was inactive. However, succinic thiokinase utilized all three types of CoA equally well. Adenosine 5'-monophosphate 3'-pyrophosphate also was synthesized and used as an analog of adenosine 5'-monophosphate 3'-monophosphate in the dog liver's sulfotransferase-catalyzed sulfate transfer from p-nitrophenyl sulfate to phenol. In contrast to the pyrophospho derivative of coenzyme A, adenosine 5'-monophosphate 3'-pyrophosphate was inactive as a cofactor.
Opposing effects of interferon produced in bacteria and of tumor promoters on myogenesis in human myoblast cultures.
Authors: Fisher P B , Miranda A F , Babiss L E , Pestka S , Weinstein I B
Published: May 2006

We have studied the effects of human leukocyte interferon produced in bacteria and diterpene phorbol ester tumor promoters on differentiation of normal human myoblast cultures derived from mature skeletal muscle. Interferon (100-5,000 units/ml) induced an acceleration of myotube formation and creatine kinase (CK; EC 2.7.3.2) isoenzyme transition from CK-BB to CK-MM. Heat-inactivated or trypsin-treated interferon did not affect the differentiation process. In contrast, the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), but not its inactive structural analogues phorbol and 4 alpha-phorbol 12,13-didecanoate, caused a dose-dependent (0.01-100 ng/ml) inhibition of myotube formation and CK isoenzyme transition. Neither interferon nor TPA had a significant effect on myoblast proliferation prior to fusion, and the cloning efficiencies were similar as well. Opposing effects of interferon and TPA were also demonstrated by simultaneous application of these agents to the cultures. These studies suggest that some of the antitumor effects of interferon may relate to its capacity to modulate cellular differentiation.
Surface marker for hemopoietic and endothelial cell lineages in quail that is defined by a monoclonal antibody.
Authors: Peault B M , Thiery J P , Le Douarin N M
Published: May 2006

A mouse monoclonal antibody raised in response to quail immunoglobulin mu chain was found to exhibit a broad reactivity towards hemopoietic and endothelial cells in the quail (Coturnix coturnix japonica). Indirect immunofluorescence assays were performed at several stages of embryonic development and until 3 weeks after hatching, on either isolated cells or tissue sections. They revealed that the defined surface marker, referred to as MB1, (i) is expressed early on both intra- and extraembryonic hemopoietic stem cells and is transmitted to the whole progeny of these precursors, with the exception of mature erythrocytes, and (ii) is a constant feature of the endothelial cell surface throughout ontogenesis and adult life. In addition, this epitope is included in several soluble plasma components. MB1 expression was not detected in chicken tissues, and this characteristic was used to confirm its lineage restriction in quail-chicken chimeras. We stress the value of this species- and lineage-specific marker in study of the development of the hemopoietic and endothelial cell families, with special reference to their possible early common embryonic origin.
Differential methylation of chloroplast DNA regulates maternal inheritance in a methylated mutant of
Chlamydomonas
Authors: Sager Ruth , Grabowy Constance
Published: May 2006

In
Chlamydomonas
, the maternal inheritance of chloroplast genes correlates with the differential methylation of chloroplast DNA (chlDNA) in females (
mt
+
) but not in males (
mt
-
). Our previous studies have supported our methylation-restriction model in which the maternal transmission is accounted for by the differential methylation in gametes which protects female but not male chlDNA from degradation during zygote formation. In the mutant
me-1
[Bolen, P. L., Grant, D. M., Swinton, D., Boynton, J. E. & Gillham, N. W. (1982)
Cell
28, 335-343], chlDNA of vegetative cells of both mating types is heavily methylated even before gametogenesis; nonetheless, maternal inheritance occurs in mutants as in wild type. To investigate the mechanism of maternal inheritance in the
me-1
mutant, we have compared restriction fragment patterns after agarose gel electrophoresis of chlDNAs from mutant vegetative cells and gametes with those from wild type, by using a set of 32 restriction enzymes of which 17 were methylation-sensitive in this system. We find that additional methylation occurs during gametogenesis in the mutant female (
mt
+
) but not in the corresponding male (
mt
-
). Thus, gamete-specific, mating-type-specific methylation occurs in the
me-1
mutant as in the wild type, consistent with our methylation-restriction model. In the
me-1
mutant, gametic methylation occurs on a background of vegetative cell methylation not present in wild-type cells and irrelevant to the regulation of chloroplast inheritance. Comparison of the
me-1
mutation with the
mat-1
mutation [Sager, R., Grabowy, C. & Sano, H. (1981)
Cell
24, 41-47] provides evidence for the existence of two different chlDNA methylation control systems:
mat-1
, linked to the mating type locus and regulating the mating-type-specific methylation that correlates with maternal inheritance, and
me-1
, unlinked to the mating type locus and unrelated to the regulation of maternal inheritance.
Developmental regulation of Ca2+ and K+ currents during hormone-induced maturation of starfish oocytes.
Authors: Moody W J , Lansman J B
Published: May 2006

Changes in the electrical properties of starfish oocytes during hormone-induced maturation (the reinitiation of meiosis prior to fertilization) were studied by using the voltage-clamp technique. Three voltage-dependent ionic currents dominate the current-voltage relation of the immature oocyte: an inward Ca2+ current, a fast transient K+ current similar to the "A current" of molluscan neurons, and an inwardly rectifying K+ current. During in vitro maturation stimulated by the natural maturing hormone 1-methyladenine, gradual changes in the amplitudes of all three currents were seen: the Ca2+ currents became larger, and both K+ currents became smaller. The kinetics of the currents were not significantly altered during maturation. As a result of these changes, action potentials in the mature egg had lower thresholds, faster rates of rise, and larger overshoots than those of the immature oocyte. We also found that the total membrane capacitance decreased substantially during maturation, perhaps indicating a decrease in membrane surface area triggered by the hormone. The significance of these results is discussed in terms of the preparation of the immature oocyte for fertilization and the mechanisms of modification of ion channel properties during development.
ADP-ribosylation in inner membrane of rat liver mitochondria.
Authors: Richter C , Winterhalter K H , Baumhüter S , Lötscher H R , Moser B
Published: May 2006

NAD+ glycohydrolase activity is found at high levels in submitochondrial particles. It leads to the reaction products ADP-ribose, nicotinamide, and small amounts of 5'-AMP. Furthermore, submitochondrial particles catalyze the exchange reaction: [adenosine-14C]ADP-ribose + NAD+ in equilibrium [adenosine-14C]-NAD+ + ADP-ribose. When submitochondrial particles are incubated with NAD+, mono(ADP-ribosyl)ation of protein molecules migrating with an apparent molecular weight of 30,000 in sodium dodecyl sulfate/polyacrylamide gel electrophoresis is demonstrable. Inhibitor studies suggest attachment of ADP-ribose to arginine residues. ADP-ribose bound to submitochondrial particles is rapidly turning over. The release of ADP-ribose from the protein is probably enzyme catalyzed. The rapid turnover, the specificity of the modification, and the inhibition of ADP-ribosylation by ATP and nicotinamide suggest a regulatory role of mono(ADP-ribosyl)ation of a protein in the inner mitochondrial membrane.
Modification of T-cell proliferation and interleukin 2 production in mice infected with Trypanosoma cruzi.
Authors: Harel-Bellan A , Joskowicz M , Fradelizi D , Eisen H
Published: May 2006

Acute infection of mice with Trypanosoma cruzi results in severe immunodepression and the appearance of autoimmune symptoms. In vitro, concanavalin A-stimulated T cells from spleens of infected animals could neither produce nor respond to interleukin 2. Interleukin 2 production was not restored by addition of exogenous interleukin 1, and proliferative response to concanavalin A was not restored by exogenous interleukin 2. A population of Thy-1-negative cells in the spleen of infected animals was shown to suppress the concanavalin A proliferative response and, to a lesser extent, the production of interleukin 2. These and other symptoms of T. cruzi-infected mice are similar to the immune dysfunction of autoimmune lpr/lpr mice. These findings are discussed in relationship to the pathology of Chagas disease.
Permanent cell line expressing human factor VIII-related antigen established by hybridization.
Authors: Edgell C J , McDonald C C , Graham J B
Published: May 2006

A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line.
Hepatic sequestration and biliary secretion of epidermal growth factor: evidence for a high-capacity uptake system.
Authors: St Hilaire R J , Hradek G T , Jones A L
Published: May 2006

Epidermal growth factor (EGF) promotes hepatocyte growth and is bound in the liver by specific receptors. We have determined hepatic uptake of EGF in intact rats after an intravenous or intraportal injection of a bolus of 125I-labeled EGF. Ninety-nine percent of the intraportal dose was taken up by the liver in 3 min, whereas only 58% of the intravenous dose appeared in the liver in 10 min. Uptake was inhibited by simultaneous treatment with an excess of unlabeled EGF. At time zero, uptake appeared to be complete. Disappearance from the liver followed first-order kinetics. Within 90 min of an intraportal injection, an average of 19% of the injected radioisotope appeared in bile, of which approximately one-fifth was shown to be immunoprecipitable with a specific anti-EGF antiserum. Light microscopic autoradiography demonstrated a very steep portal-to-central lobular concentration gradient consistent with a high-capacity uptake system. After intraportal injection or after incubation with cultured hepatocytes, labeled EGF was shown to be bound to its hepatic receptors. The main receptor-ligand complex had a Mr of approximately equal to 160,000-170,000, determined by NaDodSO4/polyacrylamide gel electrophoresis.
Quantitative removal of hepatitis B viral antigens from serum by a monoclonal IgM coupled to a biocompatible solid-phase support.
Authors: Marciniak R A , Wands J R , Bruns R R , Malchesky P S , Nose Y , Haber E
Published: May 2006

We covalently linked to regenerated cellulose filters a high-affinity monoclonal IgM produced against epitopes that reside on hepatitis B viral surface antigen (HBsAg). Conditions were established whereby as much as 250 micrograms of anti-HBsAg IgM could be linked to 2-4 mg of regenerated cellulose acetate by using cyanogen bromide and trichloro-s-triazine coupling agents. The immunoreactivity of the monoclonal anti-HBsAg IgM was preserved, and quantitative binding studies with HBsAg suggests that more than one functional binding site on the IgM molecule was operative. The specificity of the monoclonal anti-HBsAg IgM was established by demonstrating that a nonspecific monoclonal IgM (against influenza hemagglutinin), when coupled to the filters under identical conditions, had no effect on removal of HBsAg from serum. Most importantly, the monoclonal anti-HBsAg IgM-coupled filters quantitatively removed low levels of HBsAg from serum; after the third pass through the filter, HBsAg was undetectable in the perfusate. Further, the stability of the covalent bond between the anti-HBsAg IgM and regenerated cellulose acetate was shown by the lack of detectable murine monoclonal anti-HBsAg IgM in filtered serum despite 50 passages through the filter. Thus, we have demonstrated that monoclonal IgM antibodies with predefined specificity, when coupled to a biocompatible solid-phase support, may serve as a high-affinity and specific immunoabsorbant for quantitative removal and recovery of viral antigens from human serum. By using this approach, specific removal and recovery of many other substances from serum or plasma would seem possible.
Dibutyryl cAMP-inducible alkaline phosphatase in animal cell plasma membranes: fluorescence detection of mutant clones on polyester cloth.
Authors: Gum J R , Raetz C R
Published: May 2006

We have developed a rapid screening assay that allows us to estimate the alkaline phosphatase content of mouse L-M cell colonies immobilized on polyester cloth. This permitted the identification and isolation of two mutant clones with increased constitutive alkaline phosphatase activity and six clones that fail to express this activity when treated with dibutyryl cyclic AMP. Both of the strains with increased constitutive activity have basal enzymatic activities that are 6- to 7-fold higher than the activity of the parental strain. The extents to which the cyclic nucleotide further induces alkaline phosphatase in these two strains are different, however, indicating that they represent two classes of mutants. Studies using amino acids and synthetic peptides as alkaline phosphatase inhibitors suggest that only one alkaline phosphatase isoenzyme predominates, in both the parental and the mutant cell lines, with or without induction by cyclic nucleotide. Comparison to mouse tissues indicates that our cell lines express an isozyme resembling that found in kidney and bone. The six clones that fail to express alkaline phosphatase activity when treated with dibutyryl cyclic AMP also have extremely low basal levels of the enzyme. All of these mutant strains continue to synthesize protein when treated with dibutyryl cyclic AMP and undergo growth cessation and morphological changes in the presence of this agent. Thus, the mutations all appear to affect factors specific to the expression of alkaline phosphatase activity rather than factors that affect general cellular responsiveness or permeability to dibutyryl cyclic AMP. The characterization of these strains may help elucidate mechanisms of eukaryotic membrane protein biogenesis, enzyme induction, and regulation of gene expression by cyclic nucleotides.
Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: facilitation by fluorescence-activated flow sorting of metaphase chromosomes.
Authors: Kanda N , Schreck R , Alt F , Bruns G , Baltimore D , Latt S
Published: May 2006

Human neuroblastoma IMR-32 cells have large homogeneously staining regions (HSRs), primarily in the short arms of chromosome 1. We have constructed a recombinant phage library that is enriched for DNA present in the HSR of this chromosome by using fluorescence-activated flow sorting for initial chromosome purification. Eleven distinct cloned DNA segments were identified that showed significantly greater hybridization to IMR-32 genomic DNA, detected by Southern blotting, than to normal human genomic DNA. These sequences have also been localized to the HSR of chromosome 1 by in situ hybridization. Based on an approximate 50-fold sequence amplification for each cloned segment and a total HSR size of 150,000 kilobases, the amplified unit in the HSR is estimated to be 3,000 kilobases. Sequences homologous to all cloned HSR DNA segments were mapped to human chromosome 2 by using human-mouse hybrid cells. Further work using in situ hybridization demonstrated that cloned HSR segments were localized in the short arm of chromosome 2 in both normal and IMR-32 cells. Thus, the amplification of these sequences in IMR-32 cells may have involved transposition from chromosome 2 to chromosome I.
Population genetics of multigene families that are dispersed into two or more chromosomes.
Authors: Ohta T , Dover G A
Published: May 2006

The evolution of multigene families whose members are dispersed into two or more nonhomologous chromosomes is studied from the standpoint of population genetics. By using a simple model of gene conversion, equilibrium and transient properties of the probability of identity of genes belonging to the family are investigated. Also, the time until fixation of a mutant belonging to a subdivided multigene family is studied by an approximation method. It is shown that dispersion of the family into two or more chromosomes has a relatively minor effect on the extent of identity between genes, unless the conversion rate between genes on nonhomologous chromosomes is extremely low, or unless the number of nonhomologous chromosomes on which gene members are dispersed is large.
Monoclonal antibody to the message sequence Tyr-Gly-Gly-Phe of opioid peptides exhibits the specificity requirements of mammalian opioid receptors.
Authors: Meo T , Gramsch C , Inan R , Höllt V , Weber E , Herz A , Riethmüller G
Published: May 2006

Six myeloma cell hybrids producing antibodies to human beta-endorphin were isolated from a single mouse spleen. The monoclonal antibodies displayed different binding patterns with the antigen. We report the characterization of one antibody which recognizes the tetrapeptide Tyr-Gly-Gly-Phe representing the message sequence found at the NH2 terminus of all naturally occurring mammalian opioid peptides. Competition experiments in radioimmunoassay and immunohistochemistry show that the antibody fails to bind the beta-endorphin precursor beta-lipotrophin, does not discriminate among opioid peptides that share the same message sequence but have different COOH-terminal extensions, and does not react with pharmacologically inactive derivatives of beta-endorphin. The antibody recognition of the message sequence of natural opioid peptides is sensitive to those molecular changes that affect their receptor binding competence.
Comparison of T3-associated 49- and 43-kilodalton cell surface molecules on individual human T-cell clones: evidence for peptide variability in T-cell receptor structures.
Authors: Reinherz E L , Meuer S C , Fitzgerald K A , Hussey R E , Hodgdon J C , Acuto O , Schlossman S F
Published: May 2006

Two monoclonal antibodies, anti-Ti1A and anti-Ti1B, were shown to define the clonally unique surface receptor on CT8III, a human cytolytic T lymphocyte specific for a class I major histocompatibility gene product. In the present report, this surface structure was characterized and related to the 20-kilodalton (kDa) T3 glycoprotein present on all mature human T lymphocytes. The results demonstrated that the anti-clonotypic antibodies react with an epitope on a disulfide-linked heterodimer of 49- and 43-kDa subunits exclusively expressed by CT8III. This structure is associated with T3 in the cell membrane. Similar T3-associated 49/43-kDa molecules were detected on eight additional clones, although these did not express the determinant defined by anti-Ti1A or anti-Ti1B. By probing clones of differing specificities derived from the same donor with anti-T3, it was possible to compare these T3-associated heterodimers. Biochemical analysis indicated that the 49/43-kDa structures, but not the T3 molecules themselves, had isoelectric point variability and unique peptide maps after digestion with chymotrypsin or staphylococcal protease V8. These findings support the idea that the 49/43-kDa heterodimer contains the variable region of the T cell's antigen receptor structure.
Inhibition by estradiol of the lactogenic effect of prolactin in primate mammary tissue: reversal by antiestrogens LY 156758 and tamoxifen.
Authors: Kleinberg D L , Todd J , Babitsky G
Published: May 2006

Increasing concentrations of estradiol (E2) ranging from 0.01 to 10 nM were found to inhibit partially but significantly the lactogenic effect of ovine prolactin (oPRL) on alpha-lactalbumin production in primate mammary tissues maintained in organ culture for 9 days. E2 at 10 nM inhibited by 38% (mean) PRL-stimulated alpha-lactalbumin production measured by radioimmunoassay. E2 antagonized the effect of oPRL by reducing new alpha-lactalbumin synthesis as determined by specific immunoprecipitation of alpha-lactalbumin and by analysis with NaDodSO4 gel electrophoresis. In immunoprecipitation studies, the mean inhibition of alpha-lactalbumin production was 57.6%. E2 in the absence of oPRL had no effect on alpha-lactalbumin production. In contrast to previous observations in rodents, progesterone was found to be a much weaker inhibitor of PRL-induced alpha-lactalbumin production than was E2 in primate breast tissues. Mean inhibition of oPRL-stimulated alpha-lactalbumin production was 32.3% with 10 microM progesterone and 8.3% with 10 nM. The inhibitory effect of E2 on oPRL-stimulated alpha-lactalbumin production was significantly reversed by both tamoxifen and a new antiestrogen, LY 156758. Although exact comparison of the effects of these two antiestrogens was not possible, it was apparent that LY 156758 was more potent in blocking the E2 inhibitory effect. In summary, these studies provide evidence that physiologic concentrations of estradiol partially block the lactogenic effect of PRL in primate mammary glands, suggesting a new role for estrogen in mammary physiology. The inhibitory effect of estrogen treatment on milk production in women after parturition may possibly be explained by this direct antagonism between E2 and PRL.
Growth-related changes in specific mRNAs of cultured mouse cells.
Authors: Linzer D I , Nathans D
Published: May 2006

A cDNA plasmid library has been constructed from the poly(A)+ RNA present in BALB/c3T3 cells after serum stimulation. Of 3,500 clones tested, approximately 0.5% contained inserts corresponding to mRNAs present at higher levels in serum-stimulated BALB/c 3T3 cell cultures than in quiescent cultures. Most of these RNA species increased 2- to 5-fold, and the kinetics of increase for various RNAs differed. One clone (28H6) hybridized to a 1-kilobase RNA species that is present at barely detectable levels in resting cells but is increased at least 15- to 20-fold after serum stimulation, reaching a maximal level coincident with the onset of DNA synthesis. This RNA was at a high level in proliferating cells but decreased rapidly as cells reached confluence. 28H6 RNA was also increased in resting cells infected with simian virus 40 or stimulated with platelet-derived growth factor.