bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in
Vicia faba
Authors: Grill L. K. , Garger S. J.
Published: May 2006

The cytoplasmic male sterility (CMS) trait of at least one line of
Vicia faba
plants is always associated with the presence of high molecular weight double-stranded RNA in the leaf tissue extracts. Subcellular fractions of leaf tissue from CMS and fertile maintainer plants were initially analyzed in an attempt to locate, identify, and characterize the genetic material involved with the sterility trait. This CMS-associated high molecular weight RNA was found only in the cytosol of the “447” male sterile line of
V.faba
plants and could not be isolated from the recurrent parent (maintainer), from lines that had been fertility-restored, or from lines that had reverted from the sterile condition. We have been able to move the CMS-associated RNA from donor to fertile host plants through a dodder bridge. These hots not only contain the RNA but now exhibit a male sterile phenotype, as detected by visual examination of the flower, the pollen, morphological characteristics, and pollen staining ability.
Inversions between ribosomal RNA genes of Escherichia coli.
Authors: Hill C W , Harnish B W
Published: May 2006

It might be anticipated that the presence of redundant but oppositely oriented sequences in a chromosome could allow inversion of the intervening material through homologous recombination. For example, the ribosomal RNA gene rrnD of Escherichia coli has the opposite orientation fro rrnB and rrnE and is separated from these genes by roughly 20% of the chromosome. Starting with a derivative of Cavalli Hfr, we have constructed mutants that have an inversion of the segment between rrnD and either rrnB or rrnE. These mutants are generally quite viable but do exhibit a slight reduction in growth rate relative to the parental strain. A major line of laboratory E. coli, W3110 and its derivatives, also has an inversion between rrnD and rrnE, probably created directly by a recombinational event between these highly homologous genes.
Anti-K562 cell monoclonal antibodies recognize hematopoietic progenitors.
Authors: Young N S , Hwang-Chen S P
Published: May 2006

The K562 leukemia cell has properties of self-renewal and pluripotency similar to those of the hematopoietic stem cell. Monoclonal antibodies to K562 cells have been produced by using hybridoma technology. By radioimmunoassay, some anti-K562 cell antibodies also bind to erythrocyte antigens or peripheral blood mononuclear cells; others are more specific for K562 cells. Antibody binding to hematopoietic progenitors was assayed by using the ability of these cells to form colonies in vitro. After exposure of human bone marrow cells to anti-K562 antibodies and complement, myeloid or erythroid colony formation was inhibited. Some of the inhibitory antibodies showed little binding to mature blood cells by radioimmunoassay, immunofluorescence, and complement cytotoxicity, suggesting that they recognize antigens specific to undifferentiated cells. With the fluorescence-activated cell sorter, one inhibitory antibody was shown to stain only 3% of bone marrow cells. Inhibitory anti-K562 antibodies also bind to myelogenous leukemia cells and virus-transformed lymphocytes. Thus, these antibodies appear to recognize antigens shared by normal hematopoietic progenitors, leukemic cells, and transformed lymphocytes.
Diazepam and (--)-pentobarbital: fluctuation analysis reveals different mechanisms for potentiation of gamma-aminobutyric acid responses in cultured central neurons.
Authors: Study R E , Barker J L
Published: May 2006

Diazepam and (--)-pentobarbital each potentiate the increase in chloride ion conductance produced by gamma-aminobutyric acid (GABA) i voltage-clamped mouse spinal neurons grown in culture. Fluctuation analysis was used to compare the properties of elementary ion-channel events underlying the chloride conductance produced by GABA alone and during potentiation by the two drugs. Neither drug altered the conductance of an open ion channel, but both drugs affected the kinetics of channel activity. Diazepam increased the frequency of channel openings and either did not affect or slightly increased the average open-channel lifetime, whereas (--)-pentobarbital decreased the frequency of channel openings and increased average open-channel lifetime. These changes in the kinetics of GABA-activated ion channels can quantitatively account for the potentiation of GABA responses observed with the drugs. Thus, the drugs each increase the response to GABA but do not act on channel kinetics in the same manner.
[3H]Methotrexate as a ligand for the folate receptor of Dictyostelium discoideum.
Authors: Nandini-Kishore S G , Frazier W A
Published: May 2006

Studies of the folate chemotactic receptor of vegetative Dictyostelium discoideum cells have been hampered by the presence of the degradative enzyme folate deaminase. The diaminopterin compounds aminopterin and methotrexate (MTX) are chemoattractants but are not attacked by the deaminase. [3',5',7,9-3H]methotrexate ([3H]MTX) is a nondegraded radioligand for the folate receptor. Binding to the receptor is rapid, reaching steady state in less than one min, and reversible in less than 15 s by an excess of unlabeled MTX. A single class of binding sites is found with a Kd of 2 x 10(-8) M, which correlates well with the concentration dependence of chemotaxis. Folate, aminopterin, and MTX all compete for [3H]MTX binding, whereas pterin, p-aminobenzoate, and nucleotides do not. Analysis of the receptor during differentiation indicates a decrease in site number by a factor of 3 with no change in affinity during the first 7 hr. During this time, the directional response (chemotaxis) to MTX and folate is lost, but a nondirectional stimulation of motility rate (chemokinesis) is retained. The response to cyclic AMP displays reciprocal behavior, first appearing as a chemokinetic response and then as a chemotactic response.
Human platelet-derived growth factor (PDGF): purification of PDGF-I and PDGF-II and separation of their reduced subunits.
Authors: Antoniades H N
Published: May 2006

Human platelet-derived growth factor (PDGF) was purified from lysates of clinically outdated human platelets by a direct procedure that allowed the recovery of active PDGF from stained sodium dodecyl sulfate/polyacrylamide gels. This technique enabled the identification and purification to homogeneity of two active PDGF polypeptides, and with a molecular weight of about 35,000 (PDGF-I) and the other with a molecular weight of about 32,000 (PDGF-II). Reduced PDGF-I produced two inactive subunits, with molecular weights of about 15,000 and 18,000. Reduced PDGF-II also produced two inactive subunits, with molecular weights of about 15,000 and 16,000. It is possible that PDGF-II derives from proteolytic cleavage of PDGF-I.
Catalytic mechanism of serine proteases: reexamination of the pH dependence of the histidyl 1J13C2-H coupling constant in the catalytic triad of alpha-lytic protease.
Authors: Bachovchin W W , Kaiser R , Richards J H , Roberts J D
Published: May 2006

L-Histidine, 90% 13C enriched at the C2 position, was incorporated into the catalytic triad of alpha-lytic protease (EC 3.4.21.12) with the aid of histidine-requiring mutant of Lysobacter enzymogenes (ATC 29487), and the pH dependence of the coupling constant between this carbon atom and its directly bonded proton was reinvestigated. The high degree of specific 13C isotopic enrichment attainable with the auxotroph permits direct observation and measurement of this coupling constant in proton-coupled 13C NMR spectra at 67.89 MHz and at 15.1 MHz. In contrast to the earlier study, the present study indicate that this coupling constant does respond to a microscopic ionization with pKa near 7.0; moreover, the magnitude of the values of 1JC-H observed are in accord with those expected for titration of the histidyl residue. We conclude that the original measurement must be in error and that this coupling constant now also supports a histidyl residue that titrates more or less normally as a component of the catalytic triad of serine proteases.
Inhibition of interferon-mediated induction of indoleamine 2,3-dioxygenase in mouse lung by inhibitors of prostaglandin biosynthesis.
Authors: Sayama S , Yoshida R , Oku T , Imanishi J , Kishida T , Hayaishi O
Published: May 2006

Inhibitors of fatty acid cyclooxygenase such as indomethacin (0.1 mM), phenylbutazone (0.3 mM), and aspirin (1 mM) were found to suppress almost completely the interferon-mediated induction of indoleamine 2,3-dioxygenase in mouse lung slices. However, phenacetin, an anti-inflammatory agent devoid of cyclooxygenase inhibitory activity, and sodium salicylate, a weak inhibitor of cyclooxygenase, were much less active under identical conditions. Glucocorticoids including dexamethasone, betamethasone, and cortisone, all of which are inhibitors of phospholipase A2, blocked induction of the dioxygenase by interferon in the nanomolar range, whereas other steroid hormones, such as aldosterone, testosterone, and 17 beta-estradiol, were all but ineffective. These results suggest that the enzymes phospholipase A2 and fatty acid cyclooxygenase, both of which are essential for the biosynthesis of prostaglandins, play an important role in the induction of indoleamine 2,3-dioxygenase by interferon.
Characterization of corticotropin receptors on adrenocortical cells.
Authors: Buckley D I , Ramachandran J
Published: May 2006

The binding of corticotropin (ACTH) to receptors on isolated rat adrenocortical cells was investigated with the aid of [[125I]ITyr23, Phe2, Nle4]ACTH-(1-38) (125I-ACTH analog) which retained full biological potency and had a specific radioactivity of 1800 +/- 75 Ci/mmol. Binding was highly specific to adrenocortical cells, and the radioactive peptide was displaced by low concentrations of ACTH but not by other basic peptides. Binding was rapid, reversible, and linearly related to the number of cells. 125I-ACTH analog was not significantly degraded by incubation with the cells at 23 degrees C for 1 hr. Scatchard analysis of the binding was compatible with a single class of binding sites with Kd = 1.41 +/- 0.21 nM, and the number of sites was estimated to be 3840 +/- 1045 per cell. The binding curve was superimposable on the concentration-response curve for cyclic AMP. Small, but significant amounts of 125I-ACTH analog were bound at concentrations sufficient for maximal stimulation of steroidogenesis. For a series of ACTH analogs, the concentrations of the peptides required for half-maximal stimulation of cyclic AMP production were in excellent agreement with the concentration required for half-maximal inhibition of binding. These results suggest that the adrenocortical cells contain only one class of ACTH receptors and that stimulation of a small fraction of these receptors (less than 3%) is sufficient for maximal steroidogenesis.
Repeated hexanucleotide C-C-C-C-A-A is present near free ends of macronuclear DNA of Tetrahymena.
Authors: Yao M C , Yao C H
Published: May 2006

The tandemly repeated hexanucleotide C-C-C-C-A-A has previously been found near the termini of extrachromosomal gene coding for ribosomal RNA as well as in many other locations of the genome of Tetrahymena. Moreover, the organization of these clusters of repeats in the somatic macronucleus is different from that in the germinal micronucleus. In this study we used the exonuclease Bal 31 to show that the repeats are located near free ends of DNA in the macronucleus. When whole cell DNA or macronuclear DNA was digested with Bal 31 to remove approximately 600 base pairs from free ends, 80% of the C4A2 repeats were removed, as judged by hybridization. Because no particular cluster was resistant to exonuclease digestion, we believe that essentially all the C4A2 repeats are located near free ends of DNA. The C4A2 repeats in the micronucleus, on the other hand, were not digested by Bal 31.
Fluoresceinated chemotactic peptide and high-affinity antifluorescein antibody as a probe of the temporal characteristics of neutrophil stimulation.
Authors: Sklar L A , Oades Z G , Jesaitis A J , Painter R G , Cochrane C G
Published: May 2006

Antifluorescein antibody molecules were used to interrupt the stimulation of neutrophils by a fluoresceinated chemotactic peptide. From the results we construct a semiquantitative relationship among ligand-receptor interaction, the time course of cell triggering and response, and aspects of cellular adaptation. The interaction of the antibody with the free fluoresceinated peptide is complete within a few seconds and the peptide-antibody complex neither stimulates the cells nor inhibits subsequent stimulation by unlabeled peptide. When antibody is added to a cell suspension that has been stimulated with the fluoresceinated peptide, we observe that: (i) the apparent membrane depolarization response monitored by a fluorescent dye can be inhibited only if antibody is added within 30 sec of stimulation; (ii) the superoxide response can be inhibited even if antibody is added more than 1 min after stimulation and decays with an intrinsic half-life of about 12 sec; (iii) responses to a second dose of nonfluoresceinated peptide are enhanced if the antibody is added within 2 min of stimulation by the fluoresceinated peptide. These results suggest that different neutrophil responses depend in individual ways on the time course and extent of ligand binding to its receptor. A comparison of these data with the time course of binding permits an estimate of the number of receptors involved in these responses.
Enhanced graphic matrix analysis of nucleic acid and protein sequences.
Authors: Maizel J V , Lenk R P
Published: May 2006

The enhanced graphic matrix procedure analyzes nucleic acid and amino acid sequences for features of possible biological interest and reveals the spatial patterns of such features. When a sequence is compared to itself the technique shows regions of self-complementarity, direct repeats, and palindromic subsequences. Comparison of two different sequences, exemplified by immunoglobulin kappa light chain genes, by using colored graphic matrices showed domains of similarity, regions of divergence, and features explainable by transpositions. Analysis of mouse constant domain immunoglobulin sequences revealed self-complementary regions that can be used to fold the molecule into a structure consistent with electron microscopic observations. Computer translation of nucleic acid sequences into all possible amino acid sequences followed by graphic matrix analysis provides a way to detect the most likely protein encoding regions and can predict the correct reading frames in sequences in which splicing patterns are not defined. Application of this technique to regions of simian virus 40 and polyoma virus demonstrates the frames of translation and shows the agreement of sequences determined in separate laboratories with different virus isolates. The graphic matrix technique can also be used to assemble fragmentary sequences during determination, to display local variations in base composition, to detect distant evolutionary relationships, and to display intragenic variation in rates of evolution.
Albumin-IgG complexes in human serum and plasma that inhibit blood platelet adhesion.
Authors: Sharma N C , Mohammad S F , Chuang H Y , Mason R G
Published: May 2006

Macromolecular albumin-IgG complexes have been isolated from human serum and characterized immunologically. These complexes are shown to be present in plasma also and appear to be normal constituents of blood. The complexes can be demonstrated in normal serum and plasma by immunoelectrophoresis and rocket immunoelectrophoresis, as well as by use of a two-step radioimmunologic binding test. The albumin-IgG complexes inhibit adhesion of human platelets to glass and account for 12-16% of the total protein in serum.
Evidence for a higher molecular weight precursor of cholesterol side-chain-cleavage cytochrome P-450 and induction of mitochondrial and cytosolic proteins by corticotropin in adult bovine adrenal cells.
Authors: DuBois R N , Simpson E R , Tuckey J , Lambeth J D , Waterman M R
Published: May 2006

Adult bovine adrenal cortical cells in monolayer culture were used to study the induction of cholesterol side-chain-cleavage cytochrome P-450 by corticotropin (ACTH). In the presence of 1 microM ACTH, there was a 4-fold increase in cortisol production by these cells over a 72-hr period and a corresponding increase in total cytochrome P-450 content. The incorporation of [35S]methionine into a number of cellular proteins was stimulated by the presence of ACTH in the culture medium, whereas the incorporation into other proteins was decreased. The temporal profile of these changes varied from one protein to another. Examination of the incorporation of [35S]methionine into mitochondrial protein showed an increased production of a radiolabeled protein that comigrated with the form of cytochrome P-450 known as side-chain-cleavage cytochrome upon incubation with ACTH. Thus, it appears that the cytochrome P-450scc content is increased in bovine adrenal cortical cells exposed to ACTH. Cytochrome P-450scc, synthesized in a cell-free translation system directed by RNA isolated from bovine adrenal cortical tissue or from cells, had a molecular weight of 54,500. Cytochrome P-450scc isolated from bovine adrenal mitochondria had a molecular weight of 49,000. Thus, cytochrome P-450scc is synthesized as a larger precursor that must be processed by proteolytic cleavage before or upon insertion into the mitochondrion.
Enzymatic hydrolysis of cellulose: Visual characterization of the process
Authors: White Alan R. , Brown R. Malcolm
Published: May 2006

Cellulose from the Gram-negative bacterium
Acetobacter xylinum
has been used as a model substrate for visualizing the action of cellulase enzymes from the fungus
Trichoderma reesei
. High-resolution electron microscopy reveals that
A. xylinum
normally produces a ribbon of cellulose that is a composite of bundles of crystalline microfibrils. Visual patterns of the process of cellulose degradation have been established. Enzymes are initially observed bound to the cellulose ribbon. Within 10 min, the ribbon is split along its long axis into bundles of microfibrils which are subsequently thinned until they are completely dissolved within 30 min. Incubations with purified components of the cellulase enzyme system produced less dramatic changes in ribbon structure. Purified 1,4-β-D-glucan cellobiohydrolase I (D) (EC 3.2.1.91) produced no visible change in cellulose structure. Purified endo-1,4-β-D-glucanase IV (EC 3.2.1.4) produced some splaying of ribbons into microfibril bundles. In both cases, whole ribbons were present even after 60 min of incubation, visually confirming the synergistic mode of action of these enzymes.
Inhibition of the mobility of mouse lymphocyte surface immunoglobulins by locally bound concanavalin A.
Authors: Henis Y I , Elson E L
Published: May 2006

Fluorescence photobleaching recovery was used to study directly and quantitatively the inhibition of the lateral mobility of surface immunoglobulins (sIg) on mouse lymphocytes by localized binding of concanavalin A (Con A) coupled to platelets. Up to a threshold occupancy of about 10% of the upper cell surface by Con A-platelets, the diffusion coefficient and mobile fraction of sIg remained as in untreated cells (5.3 X 10(-10) cm2/sec and 0.65, respectively). At higher surface occupancy, these values decreased to 8 X 10(-11) cm2/sec and 0.11. The magnitude of the effect was independent of the percentage occupancy above the threshold and of the distance from the bound Con A-platelets, indicating a cooperative and propagated phenomenon. Treatment with colchicine or cytochalasin B separately induced only partial reversal of the Con A-induced modulation. Treatment with both reversal of the Con A-induced modulation. Treatment with both drugs together was synergistic and fully reversed the mobility inhibition. The modulation was unaffected by NaN3 and 2-deoxyglucose, suggesting no dependence on metabolic energy. Con A-platelets did not affect the mobility of a lipid probe. Models for the Con A-induced modulation and the relationship between the effects of Con A on sIg mobility and patch formation are discussed.
Coevolution of species in competition: a theoretical study.
Authors: Matessi C , Jayakar S D
Published: May 2006

A model is presented for the coevolution of several species in competition, each species being genetically variable at one locus with respect to this competition. A function was fund that is maximized during coevolution. This maximization principle was used in order to study the result of competition for a single resource on the utilization functions of the species competing for that resource. In particular, the conditions under which ecological character displacement can evolve were examined.
Detection of enzyme polymorphism by using monoclonal antibodies.
Authors: Slaughter C A , Coseo M C , Cancro M P , Harris H
Published: May 2006

Six monoclonal antibodies against human placental alkaline phosphate [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1], a highly polymorphic enzyme, were tested for reactivity against a panel of 295 placental extracts that had been typed electrophoretically. The products of the three common alleles as well as several rare alleles could be discriminated by the various antibodies. In some cases differences between allelic products were reflected by essentially "all-or-none" reactions, but in other cases the differences were smaller and demonstrable only by quantitative analysis of the binding. Evidence for allelic differences not detectable electrophoretically was also obtained.
Specific receptors for phorbol esters in lymphoid cell populations: role in enhanced production of T-cell growth factor.
Authors: Sando J J , Hilfiker M L , Salomon D S , Farrar J J
Published: May 2006

Phorbol ester tumor promoters act synergistically with concanavalin A to cause production of T-cell growth factor by normal human peripheral blood lymphocytes. A specific, saturable, binding component which may mediate the phorbol ester effect has been identified by using [20-3H]phorbol 12,13-dibutyrate in a whole-cell binding assay. Specific binding is maximal with 5 min at 37 or 23 degrees C but the level of bound ligand rapidly decreases to about 50% within 1 hr. At 4 degrees C, 2 hr are required to reach maximal binding, and the binding is stable for at least 20 hr. Binding is reversible at 37 and 4 degrees C with time courses similar to those for initial binding at the respective temperatures. Saturation of the specific binding occurs at a concentration (approximately 30 nM) consistent with that producing maximal T-cell growth factor activity. Scatchard analysis of the binding after 30 min at 37 degrees C demonstrates a lower Kd (9 nM) than that determined after 2 hr at 4 degrees C (22 nM). The median number of sites per cell for six donors was 2 X 10(5) (range, 1.3-4 X 10(5). Other tumor-promoting phorbol esters compete for [20-3H]phorbol 12,13-dibutyrate binding in approximate proportion to their activity in stimulating T-cell growth factor production. Phorbol, 4-alpha-phorbol didecanoate, dexamethasone, retinoic acid, butyric acid, and dimethyl sulfoxide do not compete for specific binding.
Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment.
Authors: Straley S C , Brubaker R R
Published: May 2006

A procedure is described for fractionating Yersinia grown in small cultures into inner and outer membranes and soluble cytoplasmic proteins. The procedure was applied to the three recognized species of the genus grown under conditions simulating mammalian intracellular fluid with respect to Ca2+ and Mg2+. These conditions are known to elicit the production of the plague virulence antigen V. Isolates capable of making this antigen were compared with virulence-antigen-negative derivatives by two-dimensional electrophoresis. The V antigen was localized to the soluble protein fraction as a peptide that comigrates with the major component of a specific immunoprecipitate. This peptide had an apparent molecular weight of 38,000 and was not found in either apparent molecular weight of 38,000 and was not found in either membrane fraction. The comparison of virulence antigen-producers and nonproducers of Y. pseudotuberculosis and Y. enterocolitica revealed large qualitative and quantitative differences in outer membrane protein patterns, whereas the same comparison for Y. pestis showed only minor differences. The complexity of changes in the various protein fractions corroborate data in the literature indicating that extensive physiological changes occur in virulent organisms cultivated under simulated intracellular conditions.
Trade-off between
r
-selection and
K
-selection in
Drosophila
populations
Authors: Mueller Laurence D. , Ayala Francisco J.
Published: May 2006

Density-dependent genetic evolution was tested in experimental populations of
Drosophila melanogaster
subject for eight generations to natural selection under high (
K
-selection) or low (
r
-selection) population density regimes. The test consisted of determining at high and at low densities the per capita rate of population growth of the selected populations. At high densities, the
K
-selected populations showed a higher per capita rate of population growth than did
r
-selected populations, but the reverse was true at low densities. These results corroborate the predictions derived from formal models of density-dependent selection. However, no evidence of a trade-off in per capita rate of growth was observed in 25 populations of
D. melanogaster
, each homozygous for a different second chromosome sampled from a natural population.
Conformation of cyclo(Gly-L-Pro-L-Pro-Gly-L-Pro-L-Pro)
2
Mg
2+
complex crystallized from C
2
H
3
CN solution
Authors: Karle Isabella L. , Karle Jerome
Published: May 2006

The cyclic hexapeptides (Gly-L-Pro-L-Pro-Gly-L-Pro-L-Pro) in the (peptide—Mg—peptide)
2+
complex have nearly identical asymmetric conformations. Each has two
cis
Pro-Pro linkages and lacks any intraring hydrogen bonds. The Mg
2+
ion forms six ligands in a regular octahedral array with the carbonyl oxygen atoms of the two Gly residues and one Pro residue of each peptide. The “sandwich” complex has an approximate 2-fold rotation axis through the Mg
2+
relating the two peptide moieties. Cyclo(Gly-Pro-Pro-Gly-Pro-Pro)
2
Mg(ClO
4
)
2
· 4C
2
H
3
CN crystallizes in space group P3
1
with
a
=
b
= 15.744(4) Å, c = 24.002(6) Å, γ = 120°, and Z = 3. A highlight of the structure determination is the ready location of the Mg self-vector in a Harker section and the development of the entire structure by use of the tangent formula starting with the known position of the Mg atom.
Comparison of [3H]glucosamine-labeled glycoproteins from human renal cancer and normal kidney epithelial cell cultures by two-dimensional polyacrylamide gel electrophoresis.
Authors: Ogata S , Ueda R , Lloyd K O
Published: May 2006

Two-dimensional polyacrylamide gel electrophoresis has been used to compare the Nonidet P-40 soluble, [3H]glucosamine-labeled glycoproteins of human kidney cancer cell lines and short-term cultures of normal kidney epithelia. Two pairs of autologous combinations of tumor and normal cells and also six other cancer lines and four normal cultures were examined. Because autologous cells could be compared, possible differences due to allogeneic variations could be eliminated. The electrophoretic patterns given by tumor and normal cells were extremely similar. Nevertheless, more than 10 differences could be recognized when randomly selected combinations of cancer and normal cells were examined in detail. Of these differences, however, only one was consistently found in all possible combinations of cancer and normal cells. Cancer cells had three prominent components of molecular weight 27,500 and pIs of 5.7, 5.3, and 4.9, respectively. Normal cells have a fainter and different constellation of spots in this region, with the more acid component (pI 4.9) being absent (or barely detectable). The pattern of fetal kidney cells resembled that given by normal adult kidney.
Local anesthetics, mepacrine, and propranolol are antagonists of calmodulin.
Authors: Volpi M , Sha'afi R I , Epstein P M , Andrenyak D M , Feinstein M B
Published: May 2006

Local anesthetics such as dibucaine, QX572, tetracaine, and phenacaine, as well as other drugs with local anesthetic-like properties (e.g., mepacrine, propranolol, and SKF 525A) inhibit the specific calmodulin-dependent stimulation of erythrocyte Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and cyclic nucleotide phosphodiesterases (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) from brain and heart. Basal activities of these enzymes in the absence of calmodulin are relatively unaffected by concentrations of local anesthetics that strongly inhibit the specific stimulation by calmodulin. Increasing calmodulin, but not Ca2+, overcomes the inhibitory action of the local anesthetics on brain phosphodiesterase. However, excess calmodulin does not fully restore activity of erythrocyte CA2+-stimulated ATPase. Although the mechanism(s) by which the local anesthetics act is unclear, they inhibit binding of 125I-labeled calmodulin to the erythrocyte membrane. Antagonism of calmodulin provides a molecular mechanism that may explain the inhibition of many Ca2+-dependent cellular processes by local anesthetics--e.g., Ca2+ transport, exocytosis, excitation-contraction coupling, non-muscle-cell motility, and aggregation.
Relationship between phosphorylation and activity of heme-regulated eukaryotic initiation factor 2 alpha kinase.
Authors: Fagard R , London I M
Published: May 2006

In heme-deficient reticulocytes and their lysates, a heme-regulated inhibitor of protein synthesis is activated; this inhibitor is a cyclic AMP-independent protein kinase that specifically phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF-2 alpha). Heme regulates this kinase by inhibiting its activation and activity. The purified heme-regulated kinase (HRI) undergoes autophosphorylation; at least 3 mol of phosphate can be incorporated per HRI subunit (Mr 80,000). The phosphorylation of HRI, its eIF-2 alpha kinase activity, and its ability to inhibit protein synthesis are diminished by hemin (5 microM) and increased by N-ethylmaleimide (MalNEt). Treatment of MalNEt-activated HRI with hemin reduces its autophosphorylation and its ability to inhibit protein synthesis . These findings demonstrate a correlation of the phosphorylation of HRI, its eIF-2 alpha kinase activity, and its inhibition of protein synthesis. The mechanism of hemin regulation of HRI activity was studied by examining the binding of hemin to purified HRI. Significant binding was demonstrable by difference spectroscopy which revealed a pronounced shift in the absorption spectrum of hemin with the appearance of a peak at 418 nm, a shift similar to that observed with proteins known to bind hemin. These findings are consistent with a direct effect of hemin on HRI.
Mechanism of activation of cyclic nucleotide phosphodiesterase: requirement of the binding of four Ca2+ to calmodulin for activation.
Authors: Huang C Y , Chau V , Chock P B , Wang J H , Sharma R K
Published: May 2006

Kinetic studies on the activation of cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) as a function of calmodulin and Ca2+ concentrations have been carried out. A general approach to analyzing the mechanism of activation, which takes into consideration the various interactions among phosphodiesterase and calmodulin liganded with Ca2+ to differing degrees, is presented. The method is applicable to other calmodulin-regulated enzyme systems. Our kinetic analysis reveals that all four Ca2+ must be bound to calmodulin for the protein to form an activated complex with phosphodiesterase. The mechanistic and regulatory advantages of having four Ca2+ sites on calmodulin can be briefly stated as follows. (i) With the enzyme--calmodulin--Ca4(2+) complex as the dominant active species, the activation of phosphodiesterase as a function of Ca2+ concentration is highly cooperative. This phenomenon serves as an effective on/off switch for phosphodiesterase activation. (ii) At normal cellular levels of Ca2+ (less than 0.1 microM), phosphodiesterase and calmodulin do not form a complex. Thus, the distribution of calmodulin among its various target enzymes is reshuffled for each Ca2+ surge. (iii) The affinity between the enzyme and the fully liganded calmodulin (0.1-1 mM) is 10(4)-10(5) times better than that in the absence of Ca2+ (greater than or equal to 10 microM). The tremendous increase in affinity can be achieved rather easily through a 10- to 20-fold increase in the affinity of Ca2+ for the enzyme-calmodulin complex in each of the four binding steps.
Enumeration of structure-sensitive graphical subsets: Calculations
Authors: Marrifield R. E. , Simmons H. E.
Published: May 2006

Numerical calculations are presented, for all connected graphs on six and fewer vertices, of the numbers of independent sets, connected sets, point and line covers, externally stable sets, kernels, and irredundant sets. With the exception of the number of kernels, the numbers of such sets are all highly structure-sensitive quantities.
Human insulin prepared by recombinant DNA techniques and native human insulin interact identically with insulin receptors.
Authors: Keefer L M , Piron M A , De Meyts P
Published: May 2006

Human insulin synthesized from A and B chains separately produced in Escherichia coli from cloned synthetic genes (prepared by the Eli Lilly Research Laboratories, Indianapolis, IN) was characterized by examining its interaction with human cultured lymphocytes, human circulating erythrocytes in vitro, and isolated rat fat cells. The binding behavior of the biosynthetic insulin with human cells was indistinguishable from that of native human or porcine insulins, with respect to affinity, association and dissociation kinetics, negative cooperativity, and the down-regulation of lymphocyte receptors. Similarly, the biosynthetic insulin was as potent as the native insulins in stimulating lipogenesis in isolated rat fat cells. We also examined the receptor binding characteristics of 125I-labeled human and porcine insulins monoiodinated solely at Tyr-A14, which were obtained by means of high-performance liquid chromatography of the iodination reaction mixture (this material was prepared by B. Frank, Eli Lilly Research Laboratories). In all aspects studied, the pure [TyrA14-125I]iodoinsulins were superior as tracers to the monoiodoinsulin purified by the more conventional method of gel filtration.
ATP-induced formation of an associated complex between microtubules and neurofilaments.
Authors: Runge M S , Laue T M , Yphantis D A , Lifsics M R , Saito A , Altin M , Reinke K , Williams R C
Published: May 2006

Neurofilaments (also called 10-nm filaments or intermediate filaments) from bovine brain were incubated with microtubule protein at 37 degrees C in the presence or absence of 1 mM ATP and in a buffer that allowed microtubule assembly. Falling-ball viscometry revealed that the (non-Newtonian) apparent viscosity of the ATP-containing mixtures is 5-20 times greater than that of the mixtures prepared without ATP. A larger ATP-dependent increase in viscosity (approximately 100-fold) was seen when purified tubulin replaced microtubule protein. The magnitude of the increase depended on the concentrations of both neurofilaments and tubulin. The presence of both neurofilaments and assembled microtubules was necessary for the increase to occur. The viscosity was drastically reduced by stirring or by cooling of the mixtures to 0 degrees C. Sedimentation velocity experiments, conducted at 35 degrees C on mixtures previously incubated at 35 degrees C, revealed the presence of a fraction of very rapidly sedimenting material (sedimentation coefficient greater than 1000 S) in the ATP-containing solutions but not in those prepared without ATP. It is concluded that an ATP-induced complex is formed between microtubules and neurofilaments. The observed complex may reflect interactions between microtubules and neurofilaments that are significant in vivo.
Structure of chromatin and the linking number of DNA.
Authors: Worcel A , Strogatz S , Riley D
Published: May 2006

Recent observations suggest that the basic supranucleosomal structure of chromatin is a zigzag helical ribbon with a repeat unit made of two nucleosomes connected by a relaxed spacer DNA. A remarkable feature of one particular ribbon is that it solves the apparent paradox between the number of DNA turns per nucleosome and the total linking number of a nucleosome-containing closed circular DNA molecule. We show here that the repeat unit of the proposed structure, which contains two nucleosomes with -1 3/4 DNA turns per nucleosome and one spacer crossover per repeat, contributes -2 to the linking number of closed circular DNA. Space-filling models show that the cylindrical 250-A chromatin fiber can be generated by twisting the ribbon.
Field desorption mass spectrometry of oligosaccharides
Authors: Linscheid Michael , D'Angona Jay , Burlingame Alma L. , Dell Anne , Ballou Clinton E.
Published: May 2006

Field desorption mass spectrometry has been used to analyze carbohydrate polymers with 5 to 14 hexose units without prior derivatization. In all examples, the molecular weight of the oligosaccharide could be determined by means of the abundant quasimolecular ions of the type MNa
+
, MH
+
, MNa
2
2+
, and MNa
3
3+
. Fragmentation at glycosidic linkages was observed in varying extents. The reduced oligosaccharide Man
8
GlcNAcH
2
, obtained from IgM [Cohen, R. E. & Ballou, C. E. (1980)
Biochemistry
19, 4345-4358], gave quasimolecular ion signals MNa
+
at
m
/
z
1544, MH
+
at
m
/
z
1522, MNa
2
2+
at
m
/
z
784, and MNa
3
3+
at
m
/
z
530, all corresponding to its assumed molecular weight of 1519.5. Mycobacterial methylmannose polysaccharides with the general structure Man
x
MeMan
y
-OCH
3
[Yamada, H., Cohen, R. E. & Ballou, C. E. (1979)
J. Biol. Chem.
254, 1972-1979] were also successfully analyzed. Man
1
MeMan
13
-OCH
3
, the largest homolog, gave the expected signal of the quasimolecular ion MNa
+
at
m
/
z
2506. The larger polysaccharides were analyzed by using a KRATOS MS-50 mass spectrometer with a high-field magnet enabling full sensitivity to be maintained up to 3000 atomic mass units. Polysaccharides up to
m
/
z
1978 were analyzed by using a KRATOS MS-9 mass spectrometer operated at 4 Kv. The signal-to-noise ratio, which becomes a serious problem in field desorption mass spectrometry at low accelerating voltages, and the low instrument sensitivity were improved considerably by our use of a method of adding scans with low total ion currents obtained over a longer desorption time. In this way, we obtained complete sequence information on methylmannose polysaccharides up to Man
1
MeMan
9
-OCH
3
(MNa
+
at
m
/
z
1802). Analysis of a presumed Man
1
MeMan
7
-OCH
3
, gave a spectrum consistent only with the structure Man
2
MeMan
6
-OCH
3
, revealing the existence of a methylmannose homolog with 2 unmethylated mannoses at the nonreducing end of the chain.
Presence of a highly repetitive and widely dispersed DNA sequence in the human genome.
Authors: Tashima M , Calabretta B , Torelli G , Scofield M , Maizel A , Saunders G F
Published: May 2006

A genomic DNA library consisting of human DNA fragments about 18 kilobases long cloned in a bacteriophage lambda vector was found to contain a specific repeated DNA segment. The repeated sequence is present in greater than 95% of the genomic library, and selected clones contain at least two copies of the sequence. Our experiments indicate that this highly repetitive sequence (approximately 400,000 copies per haploid genome) is widely distributed in the human genome and is represented in the cytoplasmic polysomal mRNA. This sequence is homologous to the 300-base-pair Alu repeat family, the predominant repeat sequence in man.
Selective inhibition of
Escherichia coli
protein synthesis and growth by nonionic oligonucleotides complementary to the 3′ end of 16S rRNA
Authors: Jayaraman Krishna , McParland Kevin , Miller Paul , Ts'o Paul O. P.
Published: May 2006

A series of nonionic oligonucleotide analogues, the deoxyribooligonucleoside methylphosphonates, were synthesized. The base sequences of these compounds, d(A
p
G
p
G
p
), d(A
p
G
p
G
p
)
2
, and d[(A
p
G
p
G
p
)
2
T], are complementary to the Shine-Dalgarno sequence (-A-C-C-U-C-C-U-) found at the 3′ end of bacterial 16S rRNA. These nonionic oligonucleotide analogues were tested for their ability to inhibit the
in vitro
translation of mRNAs in cell-free systems of
Escherichia coli
and rabbit reticulocyte. In the
E. coli
system, both d(A
p
G
p
G
p
)
2
and d[(A
p
G
p
G
p
)
2
T] effectively inhibited MS-2 RNA-directed protein synthesis but they had much less effect on either poly(U)- or poly(A)-directed polypeptide synthesis. In the reticulocyte system, these compounds had no significant effect on the translation of globin mRNA. The observation that d[(A
p
G
p
G
p
)
2
[
3
H]T)] binds to 70S ribosomes (association constant, 2.0 × 10
4
M
-1
, 37°C) together with the specificity of the inhibitory action of these compounds on protein synthesis strongly suggests that inhibition of translation is a consequence of analogue binding to Shine-Dalgarno sequence of 16S rRNA. The oligonucleoside methylphosphonates inhibited both protein synthesis (without concurrent inhibition of RNA synthesis) and colony formation by
E. coli
ML 308-225 (a permeable mutant) whose cell wall contains negligible quantities of lipopolysaccharide but had no effect on wild-type
E. coli
B. Our preliminary results on the uptake of oligodeoxyribonucleoside methylphosphonates by
E. coli
B show that these cells are not permeable to oligomers longer than 4 nucleotidyl units. Although oligodeoxyribonucleoside methylphosphonates are taken up by mammalian cells in culture, this series of analogues had negligible inhibitory effects on colony formation by transformed human cells. This study indicates that this class of nonionic oligonucleotide analogues can be used to probe and regulate the function and structure of nucleic acids of defined sequence within living cells.
Method to determine the reading frame of a protein from the purine/pyrimidine genome sequence and its possible evolutionary justification.
Authors: Shepherd J C
Published: May 2006

The periodic variations obtained by correlating the relative positions of purines and pyrimidines (and of the four bases thymine, cytosine, adenine, and guanine) in a wide variety of genomes of wholly or partly known sequence suggest that there may be enough of an earlier comma-free coding system (i.e., only readable in one frame) still present to permit determination of the reading frame and approximate extent of the present protein coding stretches. The characteristics of these variations support the hypothesis that these primitive messages were formed of coding triplets having the form RNY (R = purine; Y = pyrimidine; and N = purine or pyrimidine). The base sequences and reading frames that have a minimal deviation from such a message are still good predictors of actual coding regions and reading frames in spite of the many mutations that have occurred since such a genetic code was last in use. In fact, the right frame for almost all the proteins in a number of viruses and various prokaryotes and eukaryotes is deduced purely from purine/pyrimidine information and not by using the normal start and stop signals.
delta-Aminolevulinic acid synthase from Euglena gracilis.
Authors: Beale S I , Foley T , Dzelzkalns V
Published: May 2006

delta-Aminolevulinic acid (ALA) synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] activity was detected in cell extracts of the unicellular green flagellate alga Euglena gracilis. The enzyme was identified by substrate and cofactor requirements, and activity was proportional to number of cells extracted and duration of incubation. The incubation product was spectrophotometrically and chromatographically identical to ALA. ALA synthase activity is present in two wild-type strains, Z and bacillaris, and in nongreening, aplastidic strains derived from them. When grown in the dark, wild-type strains have amounts of ALA synthase activity equal to the amounts in their aplastidic derivative strains. Growth in the light or dark does not affect the level of ALA synthase activity in the aplastidic strains, but the wild-type strains have only one-third as much activity when grown in the light. We propose that ALA synthase is responsible for nonplastid tetrapyrrole biosynthesis in Euglena.
Eradication of spontaneous metastases and activation of alveolar macrophages by intravenous injection of liposomes containing muramyl dipeptide.
Authors: Fidler I J , Sone S , Fogler W E , Barnes Z L
Published: May 2006

The multiple systemic administration of multilamellar liposomes composed of phosphatidylserine and phosphatidylcholine (molar ratio 3:7) that contained water-soluble muramyl dipeptide (MDP) activated alveolar macrophages to become tumoricidal and eradicated established spontaneous pulmonary and lymph node metastases. Spontaneously metastasizing melanoma cells were injected into the footpads of mice. After 4-5 weeks, the tumors were resected by a midfemoral amputation; 3 days later, twice-weekly injections of liposomes were initiated and continued for 4 weeks. In some experiments the mice were killed 2 weeks after the final treatment. Seventy-four percent of animals injected with liposomes containing MDP were free of visible metastases. In a separate life-span experiment, 60% of mice treated with liposome-encapsulated MDP were tumor-free 120 days after the last liposome treatment or 110 days after all control mice treated with free MDP or control liposome preparations had died of disseminated cancer. These data suggest that the systemic administration of liposomes containing MDP, or similar compounds that produce macrophage activation, may provide an additional useful approach to the therapeutic regimens currently used to eradicate cancer metastases.
A mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein.
Authors: Russel M , Model P
Published: May 2006

Morphogenesis of filamentous phage includes synthesis of the phage major coat protein in precursor form, its insertion into the host cell plasma membrane, its cleavage to the mature form of the protein, and its assembly there into virions. The M13 mutant am8H1R6 encodes a coat protein in which leucine replaces glutamic acid as residue 2 of the mature protein [Boeke, J. D., Russel, M. & Model, P. (1980) J. Mol. Biol. 144, 103-116]. The coat protein precursor produced by this variant is a poor substrate for the Escherichia coli signal peptidase both in vivo and in vitro. This pre-coat protein, which is eventually processed and assembled into viable phage particles, is associated with the membrane fraction of the infected cell. We conclude that the domain recognized by the signal peptidase extends beyond the signal peptide itself. Furthermore, membrane association and signal peptide cleavage can be separated temporally under conditions that permit membrane insertion, cleavage, and phage assembly.
Malate dehydrogenase: viability of cytosolic nulls and lethality of mitochondrial nulls in maize.
Authors: Goodman M M , Newton K J , Stuber C W
Published: May 2006

Five independently inherited loci on five distinct chromosomes encode the mitochondrial and cytosolic isozymes of NAD-dependent malate dehydrogenase (MDH; L-malate:NAD+ oxidoreductase, EC 1.1.1.37). Multiple alleles, including electrophoretic nulls, occur for each locus. However, a single allele of normal activity at one of the three loci encoding the mitochondrial MDHs is sufficient for normal development, whereas plants with essentially no cytosolic MDH activity function normally. The requirement of a normal activity allele at one of the three structural loci encoding the mitochondrial MDHs demonstrates in plants that a commonly studied dehydrogenase enzyme is essential for normal embryogenesis.