bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of stimulation of prostaglandin synthesis by a factor from rheumatoid synovial tissue.
Authors: Robinson D R , Bastian D , Hamer P J , Pichiarallo D M , Stephenson M L
Published: May 2006

Rheumatoid synovial cell monolayers, with [1-14C]arachidonic acid ([1-14C]AA) incorporated into cell lipids, are stimulated by a factor (RSF) produced by explant cultures of rheumatoid synovial tissue to produce up to 50-fold increases in [1-14C]prostaglandin E2 and [1-14C]prostaglandin I2. In contrast, levels of free [1-14C]AA released from RSF-stimulated cells are generally lower than [1-14C]AA levels in cultures of untreated cells. These observations are inconsistent with a mechanism of prostaglandin stimulation consisting of an increase in phospholipase activity, because this mechanism would increase free AA levels as well as prostaglandins. A mechanism is proposed in which free AA is maintained at low steady-state levels by reacylation of free AA into phospholipids at a rate more rapid than its reaction with cyclooxygenase to form prostaglandins. In this mechanism, stimulation of the rate of the cyclooxygenase step by RSF accounts for increased prostaglandin synthesis as well as the decreased release of AA. On the basis of data previously reported by others, it is suggested that this mechanism may also be applicable to the stimulation of prostaglandin synthesis by several other agents. Preliminary characterization of the RSF indicates that it is a protein, and molecular seive chromatography indicates that its molecular weight is about 18,000. The production of RSF by rheumatoid synovial tissue is suppressed to undetectable levels by 1 microM dexamethasone.
Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes.
Authors: Froehner S C , Gulbrandsen V , Hyman C , Jeng A Y , Neubig R R , Cohen J B
Published: May 2006

Highly purified cholinergic postsynaptic membranes from Torpedo electric tissue contain, in addition to the acetylcholine receptor (AcChoR), major proteins of Mr 43,000 and Mr approximately 90,000 and minor proteins that can be removed from the membranes by alkaline treatment. We have prepared an antiserum to these alkaline-extractable proteins that reacts with the Mr 43,000 protein but not with any of the other major membrane proteins, including the AcChoR subunits. Immunofluorescent staining of sections of Torpedo electric tissue shows that this antiserum binds to the innervated but not the uninnervated surface of the electrocytes. In rat diaphragm muscle, the antigens recognized by this antiserum are highly concentrated at the synapse. Synaptic staining of muscle is eliminated by prior incubation of the antiserum with the Mr 43,000 protein but not by incubation with affinity-purified AcChoR. This antiserum stains end plates of muscles denervated for 7 days. Antiserum to AcChoR binds to the subsynaptic membranes of electrocytes and muscle but does not react with the Mr 43,000 protein. Purified AcChoR blocks staining of synapses by anti-AcChoR but the Mr 43,000 protein does not. These results indicate that the Mr 43,000 protein is located in the innervated membrane of Torpedo electrocytes and that an immunologically similar component is highly concentrated in the postsynaptic membrane of mammalian muscle.
Chemical synthesis of acyl thioesters of acyl carrier protein with native structure.
Authors: Cronan J E , Klages A L
Published: May 2006

The acyl carrier protein (ACP) of Escherichia coli was converted to acyl-ACP by imidazole-catalyzed S-acylation with N-acylimidazole. The acylation was specific to the sulfhydryl group; no acylation of tyrosine or amino groups of the protein occurred. The acyl-ACP substrates synthesized had a native structure as determined by gel electrophoresis, hydrophobic chromatography, and enzymatic activity. N-Acylimidazoles are readily synthesized and permit preparation of those acyl-ACP substrates that cannot be produced enzymatically.
Stimulation of pancreatic islet metabolism and insulin release by a nonmetabolizable amino acid.
Authors: Sener A , Malaisse-Lagae F , Malaisse W J
Published: May 2006

The leucine analog beta-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) activates glutamate dehydrogenase [L-glutamate:NAD+ oxidoreductase (deaminating), EC 1.4.1.2] in pancreatic islet homogenates. In intact islets, BCH increased the islet content or output of NH4+, 2-ketoglutarate, malate, pyruvate, and alanine. BCH caused a dose-related increase in 14CO2 output from islets prelabeled with L-[U-14C]glutamine. BCH increased the islet content of ATP and stimulated both 45Ca net uptake and insulin release. The capacity of seven distinct amino acids to activate glutamate dehydrogenase tightly correlated with their ability to augment 14CO2 output from islets prelabeled with [U-14C]-glutamine and to stimulate insulin release in the presence of L-glutamine. The activation of glutamate dehydrogenase by BCH may thus account for the insulin-releasing capacity of the leucine analog.
Tumor promoters cause a rapid and reversible inhibition of the formation and maintenance of electrical cell coupling in culture.
Authors: Enomoto T , Sasaki Y , Shiba Y , Kanno Y , Yamasaki H
Published: May 2006

The effect of tumor promoters on electrical coupling between human FL cells was investigated with a microelectrode technique. When a low concentration (100 ng/ml) of 12-O-tetradecanoylphorbol 13-acetate (TPA) was added to culture medium, only 6% of the cells showed electrical coupling after 5 hr, whereas in control medium more than 90% of the cells were coupled. In the presence of TPA, cell coupling remained suppressed for at least another 19 hr. When TPA was washed out from the culture medium, the cells commenced electrical coupling: 90% of the cells were coupled within 4 hr of the removal of TPA, a rate very similar to that of nontreated control cells. Therefore, TPA-mediated inhibition of cell coupling is reversible. When TPA was added to a culture in which more than 90% of the cells had already established electrical coupling, the percentage of coupled cells decreased to 6% within 8 hr, indicating that TPA can also diminish already established cell coupling. Inhibition of cell coupling also was achieved with other mouse skin tumor promoters--phorbol 12,13-didecanoate, ingenol dibenzoate, and mezerein--whereas nonpromoting derivatives--phorbol and 4 alpha-phorbol 12,13-didecanoate-showed no effect. None of these compounds changed the membrane potential, membrane resistance, or growth rate of FL cells. Thus, it appears that TPA and structurally-related tumor promoters specifically disturb the formation or function, or both, of cell-cell junctions, without significantly affecting the general properties of the surface membrane or the growth of FL cells.
Initiation of nuclear DNA replication: evidence for formation of committed prereplicative cellular state.
Authors: Das M
Published: May 2006

This paper explores the transitional states that bridge the gap between nuclear quiescence and mitogenesis. It presents evidence for the formation of a committed but prereplicative state. Quiescent murine Swiss 3T3 cells were exposed to an external mitogenic stimulant (epidermal growth factor or excess serum) and simultaneously to a synchronizer which inhibits entry into the S phase. Thus, the cells were stimulated to synthesize DNA, but the normal replicative response to this stimulus was blocked. The block to DNA replication was removed at varying times after removal of the stimulant. Experiments were done to monitor the decay of commitment to DNA synthesis after removal of the external stimulant. This decay turned out to be a first-order process. The half-life (time required for loss of the commitment to DNA synthesis in half of the initially sensitized cells) was found to be approximately 5 hr. The same result was found whether total DNA synthesis or individually replicating cells were measured and was independent of the type of external growth stimulant or blocker used. These results point to the existence, on the mitogenic pathway, of a committed but prereplicative state. The committed state appears to represent a unit or global property of the whole cell rather than, for example, a critical concentration of some active inducer molecule because the latter would display multi-hit decay kinetics rather than the single-step lability actually observed.
Lysosomal compartment of macrophages: extending the definition of GERL.
Authors: Novikoff P M , Yam A , Novikoff A B
Published: May 2006

"Professional phagocytes" such as Kupffer cells show an extensive network of interconnected residual bodies with cytochemically demonstrable acid phosphatase [orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2]. In such cells and in other (hepatocytes, etc.) this compartment may be considered a part of GERL [a hydrolase-rich region of endoplasmic reticulum (ER) situated at the trans face of the Golgi apparatus from which various Lysosomes appear to arise].
Hyperoxia and hypoxia in pregnancy: simple experimental manipulation alters the incidence of cleft lip and palate in CL/Fr mice.
Authors: Millicovsky G , Johnston M C
Published: May 2006

This paper describes alterations in the incidence of cleft lip and palate in CL/Fr mice subsequent to experimental manipulation of maternal respiratory oxygen levels during a critical period of pregnancy. Only a few previous studies have shown that the incidence of some "genetically determined" malformations in mammals can be decreased by environmental procedures. In addition to demonstrating a decreased incidence of cleft lip and palate subsequent to maternal hyperoxia on gestational days 10 and 11 in a genetically susceptible strain, the results of the present study show that hypoxia at this time increases the incidence of cleft lip and palate.
Possibility of extensive neutral evolution under stabilizing selection with special reference to nonrandom usage of synonymous codons.
Authors: Kimura M
Published: May 2006

The rate of evolution in terms of the number of mutant substitutions in a finite population is investigated assuming a quantitative character subject to stabilizing selection, which is known to be the most prevalent type of natural selection. It is shown that, if a large number of segregating loci (or sites) are involved, the average selection coefficient per mutant under stabilizing selection may be exceedingly small. These mutants are very slightly deleterious but nearly neutral, so that mutant substitutions are mainly controlled by random drift, although the rate of evolution may be lower as compared with the situation in which all the mutations are strictly neutral. This is treated quantitatively by using the diffusion equation method in population genetics. A model of random drift under stabilizing selection is then applied to the problem of "nonrandom" or unequal usage of synonymous codons, and it is shown that such nonrandomness can readily be understood within the framework of the neutral mutation--random drift hypothesis (the neutral theory, for short) of molecular evolution.
Selective growth of a population of human basophil cells in vitro.
Authors: Razin E , Rifkind A B , Cordon-Cardo C , Good R A
Published: May 2006

An initially homogeneous population of basophilic polymorphonuclear leukocytes was derived from human fetal liver cells grown in culture for 5-7 days. The cells were characterized as basophils by their morphology, histologic staining characteristics, and histamine content, and by the presence of IgE receptors on their surface. In this culture system the basophils were viable for up to 10 days.
Spontaneous and evoked release of prothoracicotropin from multiple neurohemal organs of the tobacco hornworm
Authors: Carrow Grant M. , Calabrese Ronald L. , Williams Carroll M.
Published: May 2006

Release of neurohormone from putative cephalic neurohemal organs was directly demonstrated in an insect. The prothoracicotropic hormone (PTTH) of the tobacco hornworm,
Manduca sexta
, was measured indirectly by its ability to stimulate the secretion of α-ecdysone by inactive prothoracic glands; the ecdysone was measured by radioimmunoassay. The PTTH released spontaneously from intact brain-retrocerebral complexes was localized to the retrocerebral complex by placing a waxy barrier across the nerves connecting the corpora cardiaca to the brain. Isolated corpora allata spontaneously released much more PTTH than did either isolated corpora cardiaca or isolated brains. Media containing 100 mM potassium stimulated PTTH release from both isolated corpora allata and isolated corpora cardiaca. In calcium-free media, spontaneous PTTH release was diminished and release could not be stimulated by high potassium. These results indicate that depolarization of the neurosecretory cells is correlated with calcium-dependent neurohormone release and that there are multiple neurohemal organs for PTTH. The biological activities of stored and circulating PTTH are compared.
Phencyclidine (angel dust)/sigma "opiate" receptor: visualization by tritium-sensitive film.
Authors: Quirion R , Hammer R P , Herkenham M , Pert C B
Published: May 2006

[3H]Phencyclidine ([3H]PCP) binds specifically to an apparently single class of binding sites on slide-mounted sections of rat olfactory bulb (Kd = 46 nM; Bmax = 10.5 fmol per slice). Bound [3H]PCP can be displaced by nonradioactive PCP and a series of its analogs with relative potencies that correlate closely (P less than 0.001) with values determined in a rat discrimination test that utilized PCP as a cue. Although morphine, naloxone, and opiate peptides do not displace bound [3H]PCP, psychotomimetic benzomorphans, classed as "sigma opiates," are quite potent displacers in vitro and have PCP-like behavioral properties in vivo. These results suggest that phencyclidine and the sigma opiates act at the same sites. [3H]PCP binding sites were visualized by using tritium-sensitive LKB film analyzed by computerized densitometry and color coding. The [3H]PCP binds most densely to cortical areas, diffusely in neocortex, and somewhat heterogeneously in the laminae of the hippocampal formation and dentate gyrus. Most of the brainstem and spinal cord show low specific [3H]PCP binding, with gray matter generally showing more binding than white.
Neuropeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH): immunological detection and neuronal localization in insect central nervous system.
Authors: Bishop C A , O'Shea M , Miller R J
Published: May 2006

Proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) is a pentapeptide first extracted from cockroaches. It is known to have many neurohormonal effects and has been associated with specific, identified cockroach neurons. We have produced proctolin antisera and report here on their application in detecting proctolin-like immunoreactivity (PLI) in the cockroach central nervous system. Radioimmunoassay, capable of detectable 50 fmol of proctolin, was used to quantify the distribution of PLI. Highest concentrations were detected in the genital ganglia and lowest in the cerebral ganglia. Immunohistochemistry on the cockroach central nervous system demonstrated that PLI is localized to neurons. Neurons stained by using immunohistochemistry were widespread in the ganglia. Cell bodies were found to be in constant positions from animal to animal and to occur in bilaterally symmetrical pairs. These neurons are potentially identifiable.
Specific cell—cell contacts are essential for induction of gene expression during differentiation of
Dictyostelium discoideum
Authors: Blumberg Daphne D. , Margolskee Jeanne P. , Barklis Eric , Chung Stephen N. , Cohen Naomi S. , Lodish Harvey F.
Published: May 2006

Postaggregation
Dictyostelium discoideum
cells contain 2000-3000 mRNA species that are absent from pre-aggregation cells. These aggregation-dependent sequences compose 30% of the mass of the late mRNA and represent the transcription products of an additional 11% of the single-copy genome. By analysis of mutants that are blocked at different stages of differentiation, we show that induction of expression of these genes is correlated with the formation of tight cell—cell contacts that resist EDTA. In particular, mutants that exhibit chemotaxis and aggregate to form loose mounds but do not form cell—cell contacts that resist EDTA fail to induce these late mRNA and protein species. By contrast, mutants that form normal contacts but progress no further through development do express the late mRNA species. Thus, interactions at the cell surface are involved in developmental induction of a large group of coregulated mRNAs. We have employed two independent assays for these developmentally regulated mRNAs: hybridization of gel-separated RNAs to cloned nuclear DNAs and hybridization of mRNA to a cDNA probe specific for the population of 2000-3000 regulated sequences.
Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies.
Authors: Tzartos S J , Seybold M E , Lindstrom J M
Published: May 2006

The pattern of antibody specificities in sera from patients with myasthenia gravis (MG) was determined by the ability of monoclonal antibodies against defined determinants on the acetylcholine receptor molecule to inhibit binding of the serum antibodies to receptor from human muscle. We found that MG patients produce fundamentally the same pattern of specificities as that produced by animals immunized with receptor purified from fish electric organs or mammalian muscle. Most of the antibodies are directed at the "main immunogenic region' which is located on the extracellular surface of the alpha subunit and is distinct from the acetylcholine binding site. Regions on the beta and gamma subunits near the main immunogenic region are also significantly immunogenic. In one patient the proportions of antibodies to various regions are constant over time despite changes in total antibody amount and clinical state. Between patients there is no obvious correlation between antibody specificities and clinical state. These data suggest that the autoimmune response in MG is stimulated by human receptor rather than a crossreacting (e.g., viral) antigen and that in both MG and experimental autoimmune MG the pattern of specificities produced is determined by the inherently immunogenic structural features of the receptor molecule. They also suggest that the wide differences in clinical state sometimes observed between patients with the same total concentration of antireceptor antibody are due primarily to differences in endogenous factors which affect the safety factor for neuromuscular transmission rather than to the presence of especially pathogenic antireceptor specificities.
Interferon-dependent induction of mRNA for the major histocompatibility antigens in human fibroblasts and lymphoblastoid cells.
Authors: Fellous M , Nir U , Wallach D , Merlin G , Rubinstein M , Revel M
Published: May 2006

In human cells treated with interferons, there is an increase in the amount of HLA-A,B,C and beta 2-microglobulin exposed on the cell surface. We have used a cloned HLA-A,B,C cDNA probe to demonstrate by molecular hybridization that this effect of interferon is preceded by a large increase in the amount of HLA mRNA in the cell. This effect was found in five different human cell lines, with purified leukocyte and fibroblast interferons. The increase in HLA mRNA is comparable in its kinetics and dose-response to the induction of (2'-5') oligo(A) synthetase mRNA by interferons. Therefore, interferons seem to activate at least two cellular genes which have different biochemical functions.
Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins.
Authors: Steitz T A , Ohlendorf D H , McKay D B , Anderson W F , Matthews B W
Published: May 2006

It is shown that there is a structural similarity between the presumed DNA-binding regions of the Escherichia coli catabolite gene activator protein ("CAP") and the cro repressor protein ("cro") from bacteriophage lambda. The correspondence between the two proteins is particularly striking for a structural unit consisting of two consecutive alpha-helices. The 24 alpha-carbon atoms that constitute the two-helical structural units in the two proteins can be superimposed with a root-mean-square disagreement of 1.1 A. It is shown that this agreement is very unlikely to be due to a chance correspondence. For both CAP activator and cro repressor proteins it is the second alpha-helix of the two-helical unit that has been proposed to bind within the major groove of left-handed or right-handed B DNA, respectively [McKay, D. B. & Steitz, T. A. (1981) Nature (London) 290, 744-749; Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B. W. (1981) Nature (London) 290, 754-758]. The structural correspondence between CAP and cro seen here, together with other recent evidence of sequence homologies between cro, CAP, and other proteins that bind double-stranded DNA, suggests that the two-helical unit is likely to be a common feature of many DNA-binding proteins. The results also suggest that some principles of specific protein-double-stranded DNA interaction may be general and include recognition via alpha-helices fitting into the major groove of the DNA.
Identification of cytokeratin antigens in Novikoff ascites hepatoma.
Authors: Schmidt W N , Pardue R L , Tutt M C , Briggs R C , Brinkley B R , Hnilica L S
Published: May 2006

Further characterization of three polypeptide antigens present in Novikoff ascites hepatoma (molecular weights 39,000, 49,000, and 56,000--p39, p49, and p56, respectively), has established the identity of these proteins as cytokeratins. Two-dimensional gel electrophoresis revealed that each of the three proteins exists in several isoelectric forms, all characteristic of the keratins. The antigens p39, and p56 had amino acid compositions compatible with this family of proteins, and immunofluorescence microscopy localized the antigens on elaborate filament arrays indicative of the cytokeratins. Antibodies monospecific for the p39 antigen, purified by using affinity chromatography, were used to demonstrate that protein p39 was specific for Novikoff hepatoma cells. These results suggest that the cytokeratins contain cell-specific that can be useful as markers for differentiation and neoplasia.
Structure and function of peptide initiation factor 2: differential loss of activities during proteolysis and generation of a terminal fragment containing the phosphorylation sites of the alpha subunit.
Authors: Zardeneta G , Kramer G , Hardesty B
Published: May 2006

A procedure is described by which the 38,000-dalton alpha subunit of native eukaryotic peptide initiation factor 2 (eIF-2) can be cleaved by trypsin to yield a 34,000-dalton fragment and a peptide of about 4,000 daltons after elimination of the beta subunit. Under nondenaturing conditions the 4,000-dalton peptide remains bound to the modified eIF-2 and still can be phosphorylated by the heme-controlled eIF-2 alpha kinase from reticulocytes. All of the phosphorylation sites for this protein kinase are located on the 4,000-dalton peptide. The ability of eIF-2 to form a ternary complex with GTP and Met-tRNAf and the ability to promote binding of Met-tRNAf to 40S ribosomal subunits are lost differentially during the proteolysis. Loss of te latter activity occurs rapidly and appears to be correlated with loss of the beta subunit. Loss of activity for ternary complex formation is correlated with the appearance of the 4,000-dalton peptide.
Matching molecules in the catch mechanism.
Authors: Cohen C
Published: May 2006

A new structural model is advanced to account for the specialized "catch" contraction of molluscan smooth muscles. The myosin of the thick filaments of these muscles is pictured as comprising a single layer of molecules whose assembly and activity are controlled by the underlying core of paramyosin. This organization differs from that in other muscles in which the myosin is grouped into bundles. The catch state--in which the lifetime of the actin-myosin linkage is extended--would depend directly on this special organization that allows an interaction of the entire rod region of myosin with the paramyosin core. This interaction might be modulated by phosphorylation of paramyosin.
Human platelet-derived growth factor stimulates amino acid transport and protein synthesis by human diploid fibroblasts in plasma-free media.
Authors: Owen A J , Geyer R P , Antoniades H N
Published: May 2006

Purified human platelet-derived growth factor (PDGF) induces an increase in amino acid uptake via system A in quiescent human diploid fibroblasts. Cells must be exposed to PDGF for 45 min to obtain maximum transport stimulation. Transport stimulation requires protein synthesis. Transient exposure to PDGF, alone, in the absence of plasma components can stimulate transport. Acid-insoluble [3H]leucine incorporation is also stimulated by PDGF treatment, and this event also does not require the presence of plasma components. Finally, antiserum to PDGF that blocks PDGF-stimulated DNA synthesis in these cells also blocks PDGF-stimulated amino acid uptake and protein synthesis. Increased amino acid uptake and protein synthesis that occur soon after addition of fresh serum to quiescent cells can be attributed to the action of PDGF acting alone and should be useful as markers for the investigation of early cellular events caused by PDGF.
Somatic cell cloning in polyester stacks.
Authors: Raetz C R , Wermuth M M , McIntyre T M , Esko J D , Wing D C
Published: May 2006

Single somatic cells, including fibroblasts, myelomas, and hybridomas, proliferate normally when trapped between a plastic dish and a disc of polyester cloth. Contact between the overlay and the plastic for 8-16 days results in identical colony patterns on the cloth and the plate. When several cloth discs are simultaneously stacked over Chinese hamster ovary cells, three or four-high resolution colony copies can be generated from a single master dish. The colonies on the cloth can be analyzed by radiochemical methods [Esko, J. D. & Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 1190-1193] or by "replica plating" to a new disc. The use of polyester cloth, singly or in stacks, has several major advantages over previous techniques for somatic cell replica plating, including: (i) broad applicability to diverse cell lines such as fragile membrane mutants of Chinese hamster ovary cells and relatively nonadherent myelomas or hybridomas; (ii) the possibility of generating multiple copies of the same colony population, allowing simultaneous analysis for several enzymes or cellular components; and (iii) superior resolution and transfer efficiency in copying colony patterns from one surface to another. The remarkable capacity of animal cell colonies to proliferate upward through "polyester stacks" may reflect chemotropic movement of individual cells and opens new approaches to somatic cell genetics.
Inverted terminal repeats are added to genes during macronuclear development in Oxytricha nova.
Authors: Boswell R E , Klobutcher L A , Prescott D M
Published: May 2006

In the hypotrichous ciliate Oxytricha nova all of the macronuclear DNA is in the form of low molecular weight, gene-sized molecules with an average size of 2,200 base pairs. These molecules are produced during macronuclear development by excision from micronuclear chromosomes. All, or nearly all, of the small macronuclear DNA molecules possess an inverted terminal repeat sequence consisting of 5' C4A4 3' repeats. The hypothesis that this terminal sequence serves as a recognition signal for excision of gene-sized molecules from chromosomes has been tested. A sequence containing the C4A4 repeat has been isolated and used to screen clones of micronuclear DNA for the presence of the repeat sequence. The results show that the intact repeated C4A4 sequence is not present at the ends of macronuclear sequences as they exist in the micronuclear chromosomes. Therefore, the entire terminal repeat is not a recognition sequence for gene excision but must be added to the ends of gene-sized molecules during or after the excision process.
Complement activation by isolated myelin: activation of the classical pathway in the absence of myelin-specific antibodies.
Authors: Vanguri P , Koski C L , Silverman B , Shin M L
Published: May 2006

Many pathological conditions of the central nervous system involve damage to and removal of myelin membrane. Very little is known about initiation of this membrane damage and the mechanisms of disposal of the damaged tissue. We are interested in the interaction between complement (the components of complement are designated C1, C2, C3, etc.) and myelin membranes and the possible role of complement in amplifying myelin damage and in the disposal of damaged myelin in vivo, because activation of complement generates both membrane-attack complexes and opsonin(s). In this study, we found that isolated rat or human myelin consumes complement in the absence of specific antibodies. Activation of complement was demonstrated by showing C3 cleavage in fresh serum incubated with myelin. Incubation of central nervous system myelin with C2-deficient serum produced no C3 consumption and only minor factor B conversion, thus excluding the alternative pathway of activation. Involvement of the classical pathway was shown directly by the C1 fixation and transfer assay. Myelin incubated with C2-deficient serum or with purified C1 and then washed contained C1 activity that could lyse sheep erythrocytes sensitized with anti-Forssman IgM antibody and carrying C4, together with C2 and C3-C9. Membranes in brain tissues other than myelin (heavy membrane fraction obtained on sucrose density gradient centrifugation) were unable to activate C1.
A dynorphin-like opioid in the central nervous system of an amphibian.
Authors: Cone R I , Goldstein A
Published: May 2006

We have provided evidence for the existence of a biologically active opioid in toad (Bufo marinus) brain that is immunoreactive with antiserum raised against dynorphin (1-13). Compared with porcine dynorphin, this opioid is similar in apparent molecular weight on the basis of gel permeation chromatography and is more hydrophobic on the basis of high-performance liquid chromatography. After purification, its opioid biological activity was demonstrated on the guinea pig ileum myenteric plexus-longitudinal muscle preparation. It was found to be less potent, and to have a similar sensitivity to antagonism by naloxone, in comparison with porcine dynorphin. Because it is immunoreactive with antiserum specific for porcine dynorphin, it probably has considerable sequence homology. Generally, the tissue distribution of immunoreactive dynorphin in the toad is similar to that in the rat, with highest concentrations in the neurointermediate lobe of the pituitary. However, the anterior lobe of the toad pituitary contains considerably lower concentrations than are found in the rat anterior lobe. There appear to be three size classes of immunoreactive dynorphin in toad neural tissue, each with apparent molecular weight below 12,000, similar to the size classes of immunoreactive dynorphin found in pig and rat. However, in toad spinal cord (and possibly in brain) there is immunoreactive dynorphin of greater apparent molecular weight, which has not been reported in mammalian tissue. The contribution of each molecular size to the total immunoreactivity varies from tissue to tissue and is different from that observed in the rat.
Synthesis and biological activity of a profluorescent analogue of coenzyme B12.
Authors: Rosendahl M S , Omann G M , Leonard N J
Published: May 2006

We describe here the synthesis and chemical properties of linear(lin)-benzoadenosylcobalamin, a coenzyme B12 analogue that has a laterally extended nucleoside in the upper axial position. It is an effective competitive inhibitor of ribonucleotide reductase from Lactobacillus leichmannii. lin-Benzoadenosylcobalamin is nonfluorescent in solution but, on homolytic (light) or heterolytic (acid, cyanide) cleavage of the carbon-cobalt bond, forms fluorescent products. In addition, fluorescence is detectable on binding of the coenzyme analogue to ribonucleotide reductase, and the observed fluorescence polarization of the lin-benzoadenosyl moiety indicates that it is bound loosely to the enzyme when the coenzyme is partially dissociated.
Estimation of phylogenetic relationships from DNA restriction patterns and selection of endonuclease cleavage sites.
Authors: Adams J , Rothman E D
Published: May 2006

The distribution of cleavage sites and their related sequences have been analyzed for 54 restriction endonucleases in the genome of human mitochondrial DNA; in three papova viruses, BK, simian virus 40, and polyoma; and in three bacteriophages, phi X174, fd, and G4. The results show that the cleavage sites and related sequences for most of the restriction enzymes tested are distributed nonrandomly. These results (i) constitute prima facie evidence for the action of natural selection, either direct or indirect on the restriction sites, and (ii) suggest that estimates of phylogenetic relationship, based on a phenetic approach using restriction enzyme data, will be biased.
Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome.
Authors: Wong E M , Muesing M A , Polisky B
Published: May 2006

We have isolated two mutant plasmid derivatives of ColE1 that exhibit temperature-sensitive replication properties. Both mutants have a normal copy number at 30 degrees C but increase their copy number 30- to 40-fold after a shift in temperature to 42 degrees C. A plasmid-encoded enzyme, beta-lactamase (penicillinase, EC 3.5.2.6), undergoes a 30- to 40-fold increase in specific activity concomitant with the increase in plasmid copy number. The copts phenotype of these mutants is not due to the synthesis of a temperature-sensitive polypeptide. Both mutations are located in an untranslated region of the plasmid genome encoding two overlapping transcripts involved in plasmid replication: a small transcript known as RNA I that acts as a negative control element in replication and a large transcript that has been characterized as the replication primer in vitro. The mutations alter the sequence encoding the primer but lie immediately 5' to the initiating nucleotide of RNA I, in the RNA I promoter region. The possibility that the temperature-dependent plasmid DNA amplification is a consequence of a temperature-sensitive RNA I promoter was tested by inserting the RNA I promoters from the wild-type and mutant plasmids into a plasmid in which galactokinase expression is dependent upon an exogenous promoter. These experiments demonstrate that the mutant promoters are not temperature-sensitive. Rather, the mutations may affect the secondary structure of the replication primer in a region important for RNA I interaction.
Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation.
Authors: Carmichael G G , Schaffhausen B S , Dorsky D I , Oliver D B , Benjamin T L
Published: May 2006

We have constructed a transformation-defective polyoma virus mutant (Py 1387-T) that directs the synthesis of a normal small tumor antigen, a functional large tumor antigen, and a truncated (51,000-dalton) middle-sized tumor (mT) antigen that lacks 37 amino acids at its COOH terminus. The shortened mT polypeptide is missing the hydrophobic "tail" thought to be responsible for the anchorage of this protein into the plasma membrane and is in fact in cytosol fractions. This truncated mT polypeptide is inactive in an in vitro protein kinase assay and is altered in its phosphorylation in vivo. Mutant 1387-T differs from wild-type virus in having a T.A base pair instead of a C.G base at nucleotide position 1387. This change was introduced into viral DNA by using a synthetic undecanucleotide as a specific mutagen. Wild-type polyoma DNA was rendered single stranded by molecular cloning into coliphage M13. The oligonucleotide, which hybridizes with a mismatch at the site to be altered, was used to prime the synthesis of double-stranded closed circular DNA. Progeny recombinant phage were screened by DNA sequence analysis for the desired base change. The polyoma mutant was reconstructed from recombinant phage replicative form DNA molecules containing the mutation.
Murine T cell-specific Ia antigens: monoclonal antibodies define an I-A-encoded T lymphocyte structure.
Authors: Hayes C E , Hullett D A
Published: May 2006

T lymphocytes express a unique I-A subregion-controlled surface molecule that is not expressed on B lymphocytes. We produced antisera and monoclonal antibodies recognizing this structure. Exhaustive B cell adsorption (A.TH X B10.HTT)F antiserum, produced against activated A.TL T cells, left antibodies that bind an I-Ak specificity on some B10.A(4R) T lymphocytes (11 +/- 2%, mean +/- SEM). Similarly, exhaustive B cell adsorption of (A/J X B10.MBR)F antiserum, produced against activated B10.A(5R) cells, left antibodies specific for an I-Ab determinant on B10.A(5R)T cells (17 +/- 2%). We fused A.TL-immune (A.TH X B10.HTT)F splenocytes with NS-1 myeloma cells and identified antibody-producing hybrid cells by a fluorescent enzyme-linked immunosorbent microassay described herein. Eight monoclonal antibodies were selected; these lyse 7--26% of peripheral T cells from I-Ak strains. Thymocytes and bone marrow cells do not express the I-Ak T cells determinant. Exhaustive B cell adsorption did not remove I-Ak T cell-specific monoclonal antibodies.
Production and characterization of monoclonal antibodies to rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
Authors: Clark R E , Martin G G , Barton M C , Shapiro D J
Published: May 2006

Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the key regulatory enzyme in cholesterol biosynthesis, has been purified to apparent homogeneity. Purified HMG-CoA reductase yields a single diffuse band when NaDodSO4/polyacrylamide gels are stained with Coomassie blue and yields two adjacent bands when gels are stained with silver. Purified reductase was used to elicit the production of monoclonal antibodies. Spleen cells from BALB/c mice immunized with purified HMG-CoA reductase were fused with Sp-2/0 myeloma cells. Clones producing monoclonal antibodies to HMG-CoA reductase were identified by using a solid-phase radioimmunoassay and were subcloned in soft agar. The three relatively stable hybridoma lines isolated secrete different Igs as judged by their antibody subclasses and differing abilities to inhibit HMG-CoA reductase in solution. Efficient precipitation of solubilized HMG-CoA reductase was achieved with the two IgG antibodies but not with the IgM. A mixture of all three monoclonal antibodies immunoprecipitates more than 90% of the HMG-CoA reductase activity in solubilized rat liver extracts. These monoclonal antibodies should be useful probes for investigation of the regulation of HMG-CoA reductase and cholesterol synthesis.
Infrared spectroscopy of photodissociated carboxymyoglobin at low temperatures.
Authors: Alben J O , Beece D , Bowne S F , Doster W , Eisenstein L , Frauenfelder H , Good D , McDonald J D , Marden M C , Moh P P , Reinisch L , Reynolds A H , Shyamsunder E , Yue K T
Published: May 2006

We have studied the infrared spectra of the bound and photodissociated states of Mb-12CO and Mb-13CO from 5.2 to 300 K. The absorbance peaks seen between 1800 and 2200 cm-1 correspond to CO stretching vibrations. In the bound state of Mb-12CO, the known lines A0 at 1969, A1 at 1945, and A2 at 1927 cm-1, have center frequencies, widths, and absorbances that are independent of temperature between 5.2 and 160 K. Above 160 K, A2 gradually shifts to 1933 cm-1. The low-temperature photodissociated state (Mb) shows three lines (B0, B1, B2) at 2144, 2131, and 2119 cm-1 for 12CO. The absorbances of the three lines depend on temperature. B0 is tentatively assigned to free CO in the heme pocket and B1 and B2, to CO weakly bound to the heme or heme pocket wall. The data are consistent with a model in which photodissociation of MbCO leads to B1 and B2. B2 decays thermally to B1 above 13 K; rebinding to A occurs from B1. The barriers between B2 and B1 and between B1 and A are described by activation enthalpy spectra. Heme and the central metal atom in state Mb have near-infrared, EPR, and Mössbauer spectra that differ slightly from those of deoxyMb. The observation of essentially free CO in state B implies that the difference between Mb and deoxyMb is not due to an interaction of the flashed-off ligand with the protein but is caused by an incomplete relaxation of the protein structure at low temperatures.
Responses of corn root protoplasts to exogenous reduced nicotinamide adenine dinucleotide: Oxygen consumption, ion uptake, and membrane potential
Authors: Lin Willy
Published: May 2006

Addition of 1.5 mM NADH tripled the O
2
consumption in corn root protoplasts. The stimulation was temperature and pH dependent, specific to NADH, and accompanied by a 2- to 3-fold increase in K
+
and P
i
uptake into protoplasts. The increase in ion uptake was not due to the accumulation of NADH into protoplasts. The effect of exogenous NADH on O
2
consumption and ion uptake was also evident in corn root segments but to a lesser extent. A 20-mV hyperpolarization of protoplast membrane potential occurred on addition of NADH and was abolished by the uncoupler carbonyl cyanide
p
-trifluoromethoxyphenylhydrazone. Increases in cell volume of 30% and 40% were detected in response to NADH/H
+
and NADH/H
+
/K
+
, respectively. The data are discussed in terms of a transmembrane redox reaction and the possibility that some part of the energy-linked ion transport may be driven by a NADH → O
2
electron-transport system in the plasmalemma.
Photosensitized production of hydrogen by hydrogenase in reversed micelles
Authors: Hilhorst Riet , Laane Colja , Veeger Cees
Published: May 2006

Hydrogenase (hydrogen:ferricytochrome
c
3
oxidoreductase, EC 1.12.2.1) from
Desulfovibrio vulgaris
was encapsulated in reversed micelles with cetyltrimethylammonium bromide as surfactant and a chloroform/octane mixture as solvent. Reducing equivalents for hydrogenase-catalyzed hydrogen production were provided by vectorial photosensitized electron transfer from a donor (thiophenol) in the organic phase through a surfactant-Ru
2+
sensitizer located in the interphase to methyl viologen concentrated in the aqueous core of the reversed micelle. The results show that reversed micelles provide a microenvironment that (
i
) stabilizes hydrogenase against inactivation and (
ii
) allows an efficient vectorial photosensitized electron and proton flow from the organic phase to hydrogenase in the aqueous phase.
Proteins specified by avian erythroblastosis virus: coding region localization and identification of a previously undetected erb-B polypeptide.
Authors: Privalsky M L , Bishop J M
Published: May 2006

Avian erythroblastosis virus (AEV) induces erythroblastosis and sarcomas in chickens. Two domains within the viral genome, erb-A and erb-B, have been implicated in AEV-mediated oncogenesis. By use of hybridization-arrested translations and hybridization-selections of mRNA, we have mapped on the viral genome the polypeptides specified by the erb domains. The results of hybridization-arrest with DNA representing the spliced 5' leader region of the AEV mRNA suggested that the authentic product of the erb-B domain was a 61,000 molecular weight protein, not a 41,000 molecular weight polypeptide previously identified.
Infrared evidence that the Schiff base of bacteriorhodopsin is protonated: bR570 and K intermediates.
Authors: Rothschild K J , Marrero H
Published: May 2006

It is possible, by using Fourier-transform infrared (FTIR) difference spectroscopy, to detect the conformational changes occurring in both the protein and the chromophore of bacteriorhodopsin during the photocycle. In contrast to Raman spectroscopy, a laser is unnecessary and hence the problem of a perturbing probe beam is eliminated. Furthermore, the relatively high signal-to-noise ratio obtainable with FTIR enables measurements to be made in minutes over a large spectral range. In the study reported in this paper, we used this method to examine the state of protonation of the retinylidene Schiff base in light-adapted bR570 and in K, the first intermediate in the photocycle. Resonance Raman spectroscopy provides evidence that bR570 is protonated, but these results have been questioned on the basis of theoretical and experimental grounds. FTIR difference spectral changes in the bR570-to-K transition clearly indicate that bR570 contains a protonated Schiff base. In contrast, the K intermediate displays a Schiff base that is altered but still is associated to some degree with a proton. Because the low-temperature FTIR difference spectrum of bR570 and K is similar to the recently reported low-temperature resonance Raman spectra of bR570 and K [Braiman, M. & Mathies, R. (1982) Proc. Natl. Acad. Sci. USA 79, 403-407], we can assign most, but not all, vibrational changes in the bR570-to-K transition to the chromophore. These results are consistent with a simple model of the first step in the photocycle which involves a movement of the Schiff base proton away from a counterion.
sigma, a repetitive element found adjacent to tRNA genes of yeast.
Authors: del Rey F J , Donahue T F , Fink G R
Published: May 2006

sigma is a DNA element of about 340 base pairs (bp) that is repeated many times in the yeast genome. The element has 8-bp inverted repeats at its ends and is flanked by 5-bp direct repeats. The 5-bp repeats are different for each sigma and have no homology with the ends of the sigma sequence. sigma is located 16 or 18 bp from the 5' end of several tRNA genes. Southern analysis of different yeast strains shows that the pattern of hybridization is different even for closely related strains.
Platelets of pseudohypoparathyroid patients: Evidence that distinct receptor-cyclase coupling proteins mediate stimulation and inhibition of adenylate cyclase
Authors: Motulsky Harvey J. , Hughes Richard J. , Brickman Arnold S. , Farfel Zvi , Bourne Henry R. , Insel Paul A.
Published: May 2006

We studied platelets of patients with the genetic disorder pseudohypoparathyroidism (PHP) to test whether the nucleotide-binding proteins mediating stimulation of adenylate cyclase (termed N
s
) are identical to those mediating inhibition of cyclase (termed N
i
). Functional responses to hormones that work through stimulation of adenylate cyclase are blunted in PHP patients. The erythrocytes of many of these patients (PHP-Ia) have previously been shown to have decreased N
s
activity whereas those of other PHP patients (PHP-Ib) have normal N
s
activity. We find that this decreased N
s
activity (measured by the ability to restore adenylate cyclase activity to membranes prepared from S49 cyc
-
cells) also occurs in the platelets of PHP-Ia but not of PHP-Ib patients. Platelets from both groups of patients accumulate less cAMP in response to prostacyclin than do platelets from control subjects. In contrast to the decreased N
s
function in patients with PHP-Ia, we find that N
i
function in platelets is similar in these patients and control subjects in several types of experiments: (
i
) epinephrine-mediated inhibition of prostacyclin-stimulated cAMP production in intact platelets; (
ii
) the affinity of platelet α
2
-adrenergic receptors for epinephrine, as determined by competition for [
3
H]yohimbine binding; (
iii
) the decrease in receptor affinity for epinephrine produced by Na
+
and GTP; and (
iv
) the concentration dependence of GTP for decreasing the affinity of these receptors for epinephrine. Because N
i
is expressed normally in platelets from patients that are genetically deficient in N
s
, we conclude that N
s
and N
i
are likely to be distinct gene products.