bluuebunny/crossref_metadata_2025 huggingface dataset:
Age-associated impairment of murine natural killer activity.
Authors: Albright J W , Albright J F
Published: May 2006

Natural cell-mediated cytotoxicity may be critical in the resistance displayed by animals and humans to tumors and various pathogenic microorganisms. Because the frequency of tumors and infections increases markedly in aging populations, we have compared the natural killer (NK) competence of lymphoid tissues (spleen, bone marrow) and of peritoneal cells of young adult and aged mice. Spontaneous NK activity was much lower, and the rate of target cell lysis was much less, in aged mice. The level of NK activity was only modestly increased in old, compared to young, mice when they were exposed to Trypanosoma musculi, an organism that provides strong stimulation of NK activity. Restricted NK activity of aged mice was not attributable to suppressor cells. The NK effector cells in old mice were characterized as being nonadherent to plastic, completely susceptible to lysis by complement plus an antiserum against specificity NK 1.2, and only slightly affected by treatment with antiserum against specificity Thy-1.2. Two indirect methods were employed to assess the relative frequency of splenic NK cells at the time of maximal stimulation by T. musculi: a cytotoxicity assay with antiserum against NK 1.2 and a binding assay involving monolayers of YAC-1 tumor target cells. Similar results were obtained in both assays, indicating that at maximal stimulation about 10% of the total spleen cells of both young and old mice were NK cells. We conclude (cautiously) that the functional efficiency of aged NK cells is impaired and that this defect may account, in part, for reduced ability of aged individuals to resist certain types of cancer and certain pathogenic microorganisms.
Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules.
Authors: Fleminger G , Ezra E , Kilpatrick D L , Udenfriend S
Published: May 2006

Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37 degrees C for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Met]enkephalin-Lys6 disappeared. All the above data indicate the presence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla.
Initiation of adenovirus DNA replication in vitro requires a specific DNA sequence.
Authors: Tamanoi F , Stillman B W
Published: May 2006

Initiation of adenovirus DNA replication in vitro occurs on a linearized plasmid DNA containing 3,327 base pairs of the adenovirus terminal sequence. Various deletions have been constructed in the plasmid DNA and their template activities examined. Deletions from an internal restriction enzyme cleavage site that retain only 20 base pairs or more of the adenovirus terminal sequence support initiation and limited chain elongation, whereas deletions that leave 14 base pairs or less of the terminal sequence do not. On the other hand, all deletions extending from the very terminus of the adenovirus DNA destroy the template activity. The terminal 20 base pairs of adenovirus DNA contain a sequence A-T-A-A-T-A-T-A-C-C, which is perfectly conserved in the DNAs from different serotypes of human adenovirus. Base changes within the conserved sequence greatly reduce the template activity. These results suggest that the terminal 20 base pairs constitute a functional origin for the initiation of adenovirus DNA replication in vitro.
Transcription in cloned spacers of Xenopus laevis ribosomal DNA.
Authors: Morgan G T , Reeder R H , Bakken A H
Published: May 2006

Rare individuals of Xenopus laevis exhibit frequent initiation of transcription in the spacers of oocyte ribosomal DNA (rDNA). Using electron microscopy we have characterized spacer transcription in such an individual and have confirmed that the sites of transcription initiation correspond to the imperfectly duplicated promoters ("Bam islands") present in the X. laevis rDNA spacer. We have cloned a repeat unit containing a gene and a spacer from this individual and have injected the recombinant plasmid, pXlr 164, into oocytes of other X. laevis individuals. In electron microscope preparations the spacers of some of the cloned repeats were transcribed by RNA polymerase I. This demonstrates that the ability to initiate transcription at the Bam islands is a property of the spacer DNA. On pXlr 164, initiation in the spacer occurred about 5% as frequently as transcription from the gene promoter. However, transcribed spacers were as closely packed with RNA polymerase as was the gene. We conclude that polymerase I promoters may vary over a wide range in the frequency with which they "activate" but that once activated all can load polymerases to maximal density. The presence or absence of spacer transcription had no observable effect on either the frequency of activation or the density of polymerase loading of the gene immediately downstream. A subclone, pXlr 264, containing only spacer DNA also showed regular initiation and termination, providing further evidence that there is an effective "fail-safe" termination signal 225 base pairs upstream from the rRNA gene initiation site.
Evaluation of ascorbic acid in protecting labile folic acid derivatives.
Authors: Wilson S D , Horne D W
Published: May 2006

The use of ascorbic acid as a reducing agent to protect labile, reduced derivatives of folic acid has been evaluated by high-performance liquid chromatographic separations and Lactobacillus casei microbiological assay of eluate fractions. Upon heating for 10 min at 100 degrees C, solutions of tetrahydropteroylglutamic acid (H4PteGlu) in 2% sodium ascorbate gave rise to 5,10-methylene-H4PteGlu and 5-methyl-H4PteGlu. H2PteGlu acid gave rise to 5-methyl-H4PteGlu and PteGlu. 10-Formyl-H4PteGlu gave rise to 5-formyl-H4PteGlu and 10-formyl-PteGlu. 5-Formyl-H4-PteGlu gave rise to a small amount of 10-formyl-PteGlu. 5-Methyl-H4PteGlu and PteGlu appeared stable to these conditions. These interconversions were not seen when solutions of these folate derivatives were kept at 0 degrees C in 1% ascorbate. These observations indicate that elevated temperatures are necessary for the interconversions of folates in ascorbate solutions. Assays of ascorbic acid solutions indicated the presence of formaldehyde (approximately equal to 6 mM). This was confirmed by the identification of 3,5-diacetyl-1,4-dihydrolutidine by UV, visible, and fluorescence spectroscopy and by thin-layer chromatography of chloroform extracts of the reaction mixture of ascorbic acid solutions, acetylacetone, and ammonium acetate. These results indicate that solutions of sodium ascorbate used at elevated temperatures are not suitable for extracting tissue for the subsequent assay of the individual folic acid derivatives.
Polymerization of actin: mechanism of the Mg2+-induced process at pH 8 and 20 degrees C.
Authors: Frieden C
Published: May 2006

A detailed mechanism that fully accounts for the Mg2+-induced polymerization of actin in the presence or absence of Ca2+ at 20 degrees C and pH 8 is presented. In the absence of Ca2+, the mechanism of the Mg2+-induced polymerization is as follows: Mg2+ binds to a metal-binding site on G-actin and induces a conformational change, which is required for eventual polymerization. The overall dissociation constant for this binding is about 30 microM. This actin species then binds a second molecule of Mg2+ (Kd = 5 mM), which yields a species capable of polymerization. Dimer formation from this monomeric species is quite unfavorable, but trimer formation from dimer and monomer is much more favorable. The trimer may then elongate to give filaments. Ca2+, when present, binds at the same site as the tightly bound Mg2+ and must be displaced by Mg2+ before the conformational change can occur. The rate and dissociation constants for tight binding of Ca2+ and Mg2+ and for the conformational change are consistent with those observed previously by using a fluorescently labeled G-actin. With the mechanism proposed, it is possible to fit the full time course of polymerization over a wide range of actin concentrations, Mg2+ concentrations, and Ca2+ concentrations.
Involvement of a cell-surface glycoprotein in the cell-sorting process of Dictyostelium discoideum.
Authors: Siu C H , Des Roches B , Lam T Y
Published: May 2006

Cell sorting among prespore cells and prestalk cells in Dictyostelium discoideum was studied by using fluorochrome-labeled cells in an in vitro assay. Labeled prestalk cells first formed randomly mixed aggregates with unlabeled prespore cells. Then cells began to sort out from each other. About 3-4 hr later, prestalk cells became clustered at one pole of the aggregate. Aggregates deposited on an agar surface underwent morphogenesis and formed migrating slugs within 3 hr. The addition of Fab fragments directed against a cell-surface glycoprotein of Mr 150,000 (gp 150) to the cell mixture completely inhibited the cell-sorting phenomenon. Morphogenesis of such aggregates on agar was also delayed by 5 hr. However, inclusion of Fab fragments directed against the endogenous lectins, the contact site A glycoprotein, or vegetative cells had no detectable effect on cell sorting or morphogenesis of these reconstituted aggregates.
Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6.
Authors: Le Peuch C J , Ballester R , Rosen O M
Published: May 2006

The Ca2+-phospholipid-regulated protein kinase has been purified to homogeneity from a 100,000 X g supernatant fluid of rat brain homogenate by a procedure that includes DEAE-cellulose chromatography and successive filtrations on Ultrogel AcA 34 in EGTA and in phosphatidylserine and Ca2+. A more rapid purification consisting of DEAE-cellulose chromatography, Ultrogel AcA 34 gel filtration chromatography, and DEAE-trisacryl chromatography, all in the presence of EGTA, was also developed. Although the enzyme obtained by the latter procedure is not homogeneous, it exhibits properties similar to those of the pure enzyme and is more stable. In addition, the DEAE-trisacryl step permitted resolution of a contaminating Ca2+-inhibitable protein kinase that can interfere with studies of the Ca2+-phospholipid-stimulated enzyme. The homogeneous enzyme, purified about 300-fold, was estimated to have a Mr of 84,000. Its activity was 20- to 30-fold higher in the presence of phospholipid and Ca2+ than in the presence of phospholipid and EGTA, EGTA, or Ca2+ alone. The specific activity of the activated kinase was 852 nmol of P incorporated into histone per min/mg at 20 degrees C. The pure enzyme underwent autophosphorylation in a Ca2+- and phospholipid-dependent manner. This reaction was inhibited in the presence of histones without affecting the kinetic properties of the enzyme. Under optimal assay conditions, the homogeneous enzyme was activated 10-20% by either 10 microM diolein or 100 nM phorbol 12-myristate 13-acetate. Activation of the purified enzyme by diolein or the phorbol ester was far greater (3- to 4-fold) when aggregated instead of freshly sonicated phospholipids were used, suggesting that these compounds affect the interaction of the enzyme with phospholipids and Ca2+. The purified enzyme catalyzed the phosphorylation of the 40S ribosomal subunit protein S6. The Km for S6 was approximately equal to 1 microM and it was estimated that 2 mol of phosphate were incorporated per mol of S6. The observation that protein S6 can be phosphorylated by the purified Ca2+-phospholipid-dependent protein kinase may link recent reports that phorbol ester tumor promoters activate the Ca2+-phospholipid-dependent protein kinase in vitro and stimulate phosphorylation of the ribosomal protein S6 in vivo.
Polypeptides immunologically related to band 3 are present in nucleated somatic cells.
Authors: Kay M M , Tracey C M , Goodman J R , Cone J C , Bassel P S
Published: May 2006

Band 3, the major transmembrane polypeptide of erythrocytes, mediates the exchange of anions (chloride and bicarbonate) across the membrane. We suspected that band 3 was present on nucleated somatic cells as well as erythrocytes because the senescent cell antigen that is immunologically related to band 3 is present on lymphocytes, platelets, adult liver cells, and embryonic kidney cells; and antibodies prepared against the senescent cell antigen isolated from leukocytes react with erythrocyte band 3. For this reason, we examined human fibroblasts, lung cells, neutrophils, mononuclear leukocytes, squamous epithelial (mouth) cells, lung squamous epithelial carcinoma, mouse neuroblastoma cells, and rat hepatocytes for immunoreactive forms of band 3 by using monospecific antibodies to erythrocyte band 3. The results demonstrated that polypeptides sharing common antigenic determinants with erythrocyte band 3 are present in nucleated somatic cells as determined by immunofluorescence, immunoelectron microscopy, and immunoautoradiography. Peptide mapping revealed substantial sequence homology between erythrocyte band 3 and the band 3-like protein of leukocytes. Immunofluorescence studies indicate that the band 3-like proteins in nucleated cells participate in antibody-induced cell surface capping.
Molecular mechanisms of human hemoglobin switching: selective undermethylation and expression of globin genes in embryonic, fetal, and adult erythroblasts.
Authors: Mavilio F , Giampaolo A , Carè A , Migliaccio G , Calandrini M , Russo G , Pagliardi G L , Mastroberardino G , Marinucci M , Peschle C
Published: May 2006

The globin chain synthetic pattern and the extent of DNA methylation within embryonic, fetal, and adult beta-like globin gene domains were evaluated in greater than or equal to 90% purified human erythroblasts from yolk sacs and fetal livers in the 6- to 12-wk gestational period as well as from adult marrows. The 6-wk erythroblasts produce essentially embryonic epsilon chains, whereas the 12-wk erythroblasts synthesize largely fetal gamma globin and the adult marrow erythroblasts synthesize almost exclusively adult beta chains. In all phases of ontogenic development, a strong correlation exists between DNA hypomethylation in the close flanking sequences of globin genes and their expression. These results suggest that modulation of the methylation pattern may represent a key mechanism for regulating expression of human globin genes during embryonic leads to fetal and fetal leads to adult Hb switches in humans. In ontogenic development this mechanism might in turn correlate with a gradual modification of chromatin structure in the non-alpha gene cluster, thus leading to a 5' leads to 3' activation of globin genes in a balanced fashion.
Arylsulfatase in natural killer cells: its possible role in cytotoxicity.
Authors: Zucker-Franklin D , Grusky G , Yang J S
Published: May 2006

Ultrastructural cytochemistry of natural killer cells enriched by Percoll gradient centrifugation showed them to possess arylsulfatase (aryl-sulfate sulfohydrolase, EC 3.1.6.1). The enzyme was located in vesicles, granules, and the parallel tubular arrays, organelles characteristic for cytotoxic lymphocytes. Biochemically, peak enzyme activity correlated with the Percoll fractions containing cells with cytotoxicity for melanoma target cells. Treatment of natural killer cells with Na2SO4, a competitive inhibitor of arylsulfatase, suppressed cytotoxicity by almost 50%. Electron microscopy of effector-target cell conjugates, which had been permitted to incubate for only 30 min, disclosed numerous arylsulfatase-positive sites at the points of contact between the effector/target cell membranes. Thus, the enzyme was translocated to the surface before lysis of the target cell was morphologically evident. It is postulated that the parallel tubular arrays play a role in this translocation and that arylsulfatase may function in the degradation of cerebroside sulfate ester components of the target cell membrane to initiate the lytic event.
Glucocorticoids regulate proopiomelanocortin gene expression in vivo at the levels of transcription and secretion.
Authors: Birnberg N C , Lissitzky J C , Hinman M , Herbert E
Published: May 2006

After adrenalectomy, the plasma levels of adrenocorticotropic hormone (corticotropin, ACTH)/endorphin peptides in rats rise dramatically in the first 4 hr while pituitary peptide levels fall sharply. Eight hours after adrenalectomy, plasma levels are near control values again but they then increase continuously over the next 8 days. Proopiomelanocortin (POMC) mRNA levels in the anterior pituitary (quantitated by hybridization with cloned POMC cDNA) increase 2-fold in the first 24 hours, reaching 15- to 20-fold the control level 18 days after adrenalectomy. When dexamethasone is administered to rats 8 days after adrenalectomy, the above events are reversed. Plasma ACTH falls to control levels within 2 hr whereas anterior pituitary POMC mRNA requires 5 days of treatment for return to control levels. The levels of POMC mRNA in the neurointermediate lobe and the hypothalamus are not altered by either treatment. Adrenalectomy increases transcription of the POMC gene in the anterior pituitary approximately 20-fold and halves transcription of the growth hormone gene within 1 hr of operation. Administration of dexamethasone immediately after adrenalectomy suppresses the increase in transcription of the POMC gene and increases the transcription of the growth hormone gene. Transcription of the POMC gene(s) in the neurointermediate lobe is not altered by either of these treatments.
Oxygenation of hydrocarbons by cytochrome P-450 model compounds: modification of reactivity by axial ligands.
Authors: Collman J P , Kodadek T , Raybuck S A , Meunier B
Published: May 2006

The rate of olefin oxygenation catalyzed by synthetic metalloporphyrins is examined, employing sodium hypochlorite as the oxygen atom source. The rate of epoxidation and the stability of the catalyst are shown to be dependent on the nature of the axial ligand employed. A rationale for this effect is presented and analogy is made to the role of the thiolate ligand in cytochrome P-450.
Brain-blood barrier? Yes and no.
Authors: Broadwell R D , Balin B J , Salcman M , Kaplan R S
Published: May 2006

Ventriculo-cisternal perfusion of horseradish peroxidase (HRP) in the mouse brain has demonstrated that a brain-blood barrier exists at the microvascular endothelium in brain parenchyma but not in the median eminence of the hypothalamus. The brain-blood barrier is similar to the blood-brain barrier in that: tight junctions prevent the movement of protein between endothelial cells, HRP taken into the endothelial cells is directed to lysosomal dense bodies, and, contrary to the literature, a vesicular transendothelial transport of HRP from brain to blood does not occur under normal conditions. The endocytosis of ventricular injected HRP from the abluminal side of the endothelium is demonstrably less than the endocytosis of intravenous injected HRP from the luminal side; hence, the cerebral endothelium expresses a degree of polarity regarding the internalization of its cell surface membrane and extracellular protein. The passage of cerebrospinal fluid-borne or blood-borne HRP between some ependymal cells of the median eminence is not precluded by tight junctions. These patent extracellular channels offer a direct pathway for the exchange of substances between cerebrospinal fluid in the third ventricle and fenestrated capillaries in the median eminence.
Characterization of a leukotriene D4 receptor in guinea pig lung.
Authors: Pong S S , DeHaven R N
Published: May 2006

[3H]Leukotriene D4 was found to bind, in a saturable manner and with exceedingly high affinity, to a membrane preparation from guinea pig lung. Measurement of saturation at equilibrium yielded Kd values of 5.46 +/- 0.31 X 10(-11) M at 20 degrees C and 2.12 +/- 0.37 X 10(-10) M at 0 degree C while the numbers of binding sites (Bmax) were 384 +/- 34 and 302 +/- 44 fmol/mg of protein at 20 and at 0 degree C, respectively. The time courses of both association and dissociation were slow but the rate of dissociation was accelerated by either NaCl or GTP. Binding was enhanced by Ca2+, Mg2+, and Mn2+ and inhibited by Na+ but not by Li+ or K+, suggesting that the binding of leukotriene D4 may be regulated by ions. Leukotriene E4, but not leukotriene C4, had a high affinity for the putative receptor, consistent with the pharmacological evidence that the actions of leukotrienes D4 and E4 are mediated by a receptor distinct from that for leukotriene C4. Affinities of stereoisomers and related compounds for the leukotriene D4 binding sites closely paralleled their contractile activities in guinea pig lung parenchymal strips. In addition, the antagonist of slow-reacting substance of anaphylaxis, FPL 55712, inhibited the binding of [3H]leukotriene D4 with a Ki value of 1 X 10(-7) M, which is in agreement with reported Kb values obtained in pharmacological studies.
AppppA, heat-shock stress, and cell oxidation.
Authors: Lee P C , Bochner B R , Ames B N
Published: May 2006

Salmonella typhimurium LT2 induces a set of heat-shock proteins analogous to those found previously in Escherichia coli. These are virtually the only proteins synthesized after a temperature shift from 28 degrees C to 50 degrees C. Using a two-dimensional thin-layer chromatographic system developed to resolve adenylylated nucleotides, we have found that S. typhimurium and E. coli accumulate P1,P4-diadenosine-5'-tetraphosphate (AppppA), P1-(adenosine-5')-P3-(guanosine-3'-diphosphate-5')-triphosphate (ApppGpp), P1-(adenosine-5')-P4-(guanosine-5')-tetraphosphate (AppppG), P1-(adenosine-5')-P3-(guanosine-5')-triphosphate (ApppG), and P1,P3-diadenosine-5'-triphosphate (ApppA) after heat shock. These same adenylylated nucleotides accumulate after exposure to ethanol, an agent also known to induce the heat-shock response in a variety of cells. AppppA, ApppGpp, AppppG, ApppG, and ApppA were previously shown to accumulate under conditions of oxidation stress. We proposed that these adenylylated nucleotides may be alarmones--i.e., regulatory molecules, alerting cells to the onset of oxidation stress. The finding that these dinucleotides accumulate in response to heat shock suggests that oxidation and heat shock have a common physiological effect on cells. We hypothesize that these dinucleotides signal the onset of these stresses and trigger the "heat-shock response."
Evidence for cross-bridge order in contraction of glycerinated skeletal muscle.
Authors: Burghardt T P , Ando T , Borejdo J
Published: May 2006

The linear dichroism of iodoacetylrhodamine labels attached to the single reactive thiol groups of myosin heads was measured to determine the spatial orientation of myosin cross-bridges in single glycerinated skeletal muscle fibers. We have shown previously that in rigor the chromophoric labels are well ordered and assume an orientation nearly perpendicular to the fiber axis; in the presence of MgADP, a large fraction of probe remains well ordered but the probe attitude assumes a more slanted orientation; in relaxed muscle, the probe order is largely lost, implying a high degree of cross-bridge disorder. In this paper, we report that during isometric contraction a large fraction of the probe shows a high degree of order, suggesting the attachment of approximately equal to 65% of the cross-bridges to actin. These ordered cross-bridges have a probe attitude similar to that of the MgADP-induced static state and hence are in a mechanical state quite distinct from rigor.
Increasing the intracellular Na+ concentration induces differentiation in a pre-B lymphocyte cell line.
Authors: Rosoff P M , Cantley L C
Published: May 2006

The ability of lipopolysaccharide (LPS), a polyclonal B-cell mitogen, to induce differentiation in the pre-B cell tumor line 70Z/3 can be mimicked by drugs that increase the intracellular Na+ concentration. Pharmacologically increasing the cellular Na+ content with monensin, a sodium ionophore, or ouabain, a specific inhibitor of Na+, K+-ATPase, induces surface IgM expression in these cells. We have shown that LPS stimulates uptake of Na+ through an amiloride-sensitive pathway. These results show that the essential action of LPS to induce surface IgM expression is activation of a Na+ uptake system.
Biochemical consequences of the insertion of a suppressor-mutator (
Spm
) receptor at the bronze-1 locus in maize
Authors: Klein Anita S. , Nelson Oliver E.
Published: May 2006

Transposition of a receptor element (
Rs
) for the suppressor-mutator (
Spm
) has generated the first
Spm
-controlled mutable allele of the bronze-1 (
bz
) locus in maize (
Zea mays
L.). In the absence of
Spm, bz-m13
conditions full anthocyanin pigmentation in the aleurone but has a markedly decreased level compared to wild type of the gene product, UDPglucose:flavonol 3-
O
-glucosyltransferase (UFGT; EC 2.4.1.91). This UFGT activity appears to be qualitatively similar to the wild-type enzyme. Throughout endosperm development, the amount of a crossreactive material far exceeds the amount of detectable UFGT activity. This and other observations suggest that this insertion of
Rs
leads to the production of two polypeptides from the
bz-m13
allele.
Genetic control of levels of murine kidney glucuronidase mRNA in response to androgen.
Authors: Palmer R , Gallagher P M , Boyko W L , Ganschow R E
Published: May 2006

A cis-acting genetic element, designated Gus-r, regulates the androgen-induced rates of murine glucuronidase (EC 3.2.1.31) synthesis in kidney tubule cells and is tightly linked to the glucuronidase structural gene, Gus-s. To investigate the molecular mechanism underlying this regulation, we have cloned a glucuronidase-specific cDNA sequence in plasmid pBR322. This cloned DNA has been utilized as a probe in blot hybridization analyses to determine whether the control of androgen responsiveness of kidney glucuronidase synthesis by Gus-r is exerted over the level or the translatability of glucuronidase mRNA. Three important observations emerged from these studies: (i) glucuronidase mRNA exists as a single size class of approximately 2,800 nucleotides; (ii) androgen stimulation of glucuronidase synthesis is directly related to the level of glucuronidase mRNA; and (iii) strain differences in levels of kidney glucuronidase mRNA accumulated in response to androgen are controlled by alleles of Gus-r. Thus, Gus-r regulates the androgen responsiveness of glucuronidase synthesis by controlling the amount of glucuronidase mRNA available for translation and is a cis-acting genetic element that regulates the hormonal responsiveness of a specific mRNA.
Plasma clearance of sulfobromophthalein and its interaction with hepatic binding proteins in normal and analbuminemic rats: is plasma albumin essential for vectorial transport of organic anions in the liver?
Authors: Inoue M , Okajima K , Nagase S , Morino Y
Published: May 2006

To investigate a possible function of plasma albumin in the vectorial transport of organic anions by the liver, the plasma disappearance of sulfobromophthalein (BSP) and its interaction with plasma and liver cytosolic proteins were studied in normal rats and mutant Nagase analbuminemic rats (NAR). After intravenous administration of BSP, plasma BSP decreased rapidly in both NAR and control animals: plasma clearance values of BSP in NAR and controls were 12.45 and 7.40 ml/min per kg, respectively. Gel exclusion Sephadex G-100 chromatography of BSP with control rat serum revealed a protein peak in the void volume and another in the albumin fraction. BSP chromatographed exclusively with the albumin fraction; binding of BSP to plasma albumin occurred stoichiometrically. Similar studies with NAR serum revealed a single protein peak, in the void volume; a small amount of BSP chromatographed with this protein peak. The amount of BSP that chromatographed with NAR serum protein(s) was 8% of that with control rat serum albumin. Sephadex G-100 chromatography of BSP with control rat liver cytosol revealed four peaks of protein-bound BSP in fractions corresponding to the void volume (fraction X), albumin, glutathione S-transferases (fraction Y, Mr 45,000), and fraction Z (Mr 12,000); fraction Y was the major component of BSP binding. Gel chromatography of NAR liver cytosol with BSP revealed three BSP peaks, fractions X, Y, and Z; fraction X was the major component of BSP binding. Total BSP binding by 30 mg of hepatic cytosolic proteins was 4.5 nmol for controls and 10.4 nmol for NAR. Isoelectric focusing of liver cytosol revealed no quantitative or qualitative differences in glutathione S-transferase isozymes between control and mutant animals. Intravenously administered BSP (5 mumol/kg) rapidly appeared in bile as the free form and the glutathione conjugate in normal rats and NAR; 41% and 57% of injected BSP was excreted within 60 min in NAR and control rat bile, respectively. These results indicate that binding of BSP to plasma albumin is not indispensable to transhepatocyte transport of BSP in vivo.
Relative contents and concomitant release of prodynorphin/neoendorphin-derived peptides in rat hippocampus.
Authors: Chavkin C , Bakhit C , Weber E , Bloom F E
Published: May 2006

The contents and molecular forms of five different prodynorphin-derived opioid peptides were compared in extracts of rat hippocampus by radioimmunoassay after C18-HPLC resolution. Dynorphin (Dyn) A(1-17) immunoreactivity (ir) and Dyn B-ir were heterogeneous in form; Dyn A(1-8)-ir, alpha-neoendorphin (alpha neo)-ir and beta-neoendorphin (beta neo)-ir each eluted as single homogeneous peaks of immunoreactivity. The fraction of immunoreactivity having the same retention as the appropriate synthetic standard was used to estimate the actual hippocampal content of each peptide. Comparison of these values showed that the concentrations of Dyn B, alpha neo, and Dyn A(1-8) were nearly equal, whereas both Dyn A(1-17) and beta neo were 1/5th to 1/10th the value of the other three. Calcium-dependent K+-stimulated release of these prodynorphin-derived opioids from hippocampal slices was detected. The stimulated rates of release were highest for Dyn B-ir followed by alpha neo-ir, then beta neo-ir and Dyn A(1-8)-ir with Dyn A(1-17)-ir lowest. The relative rates of stimulated release were in agreement with the relative proportions of peptide present within the tissue. This evidence of the presence and release of these opioid peptides considerably strengthens the hypothesis that this family of endogenous opioids plays a neurotransmitter role in the hippocampus.
Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli.
Authors: Konigsberg W , Godson G N
Published: May 2006

Amino acid sequence and composition data of Escherichia coli dnaG primase protein and its tryptic peptides have confirmed that the dnaG gene contains an unusually high number of codons that are not frequently used in most E. coli genes. In 25 E. coli proteins analyzed the codons AUA, UCG, CCU, CCC, ACG, CAA, AAT, and AGG are infrequently used, occurring as 4% of the total codons in the reading frame and 11% and 10% in the nonreading frames. In dnaG they occur as 11% in the reading frame and 12% in the nonreading frames. The rpsU and rpoD genes, which flank the dnaG gene [Smiley, B. L., Lupski, J. R., Svec, P. S., McMacken, R. & Godson, G. N. (1982) Proc. Natl. Acad. Sci. USA 79, 4550-4554], however, have normal codon usage. Translational modulation using isoaccepting tRNA availability may therefore be part of the mechanism of keeping the dnaG gene expression low, while expression of the adjacent rpsU and rpoD genes on the same mRNA transcript is high.
Identification and characterization of cells deficient in the mannose 6-phosphate receptor: evidence for an alternate pathway for lysosomal enzyme targeting.
Authors: Gabel C A , Goldberg D E , Kornfeld S
Published: May 2006

Newly synthesized lysosomal enzymes acquire phosphomannosyl units, which allow binding of the enzymes to the mannose 6-phosphate receptor and subsequent translocation to lysosomes. In some cell types, this sequence of events is necessary for the delivery of these enzymes to lysosomes. Using a slime mold lysosomal hydrolase as a probe, we have identified three murine cell lines that lack the receptor and one line that contains very low (3%) receptor activity. Each of these lines synthesizes the mannose 6-phosphate recognition marker on its lysosomal enzymes, but, unlike cell lines with high levels of receptor, the cells accumulate oligosaccharides containing phosphomonoesters. The receptor-deficient lines possess high levels of intracellular acid hydrolase activity, which is contained in dense granules characteristic of lysosomes. The data suggest that intracellular mechanisms independent of the mannose 6-phosphate receptor must exist in some cells for the delivery of acid hydrolases to lysosomal organelles.
Isolation of a developmental gene of Bacillus subtilis and its expression in Escherichia coli.
Authors: Vasantha N , Uratani B , Ramaley R F , Freese E
Published: May 2006

Glucose dehydrogenase of Bacillus subtilis is a developmental enzyme that is not found in growing (vegetative) cells but is synthesized after the differentiation process that leads to the production of endospores has started. We have isolated the gene coding for this enzyme from a lambda Charon 4A phage library of B. subtilis DNA. It is transcribed and translated in vegetative cells of the nondifferentiating organism Escherichia coli into enzymatically active glucose dehydrogenase that has the same physicochemical properties as the enzyme produced in B. subtilis during sporulation. Subcloning of the lambda DNA insert into pBR322 plasmid derivatives showed that the glucose dehydrogenase gene was transcribed in E. coli from a promoter within the B. subtilis genome.
A substance P antagonist inhibits vagally induced increase in vascular permeability and bronchial smooth muscle contraction in the guinea pig
Authors: Lundberg J. M. , Saria A. , Brodin E. , Rosell S. , Folkers K.
Published: May 2006

Electrical stimulation of the cervical vagus nerve in anesthetized guinea pigs induced a rapid increase in respiratory insufflation pressure, suggesting increased airway resistance. After intravenous administration of a substance P (SP) antagonist, [D-Arg
1
,D-Pro
2
,D-Trp
7,9
,Leu
11
]SP, the insufflation pressure response to vagal stimulation was reduced by 78% while the cardiovascular effects were unchanged. Histamine receptor-blocking agents were used to inhibit the effects of histamine release induced by the SP-antagonist. [D-Arg
1
,D-Pro
2
,D-Trp
7,9
,Leu
11
]SP also reduced the increase in insufflation pressure caused by intravenous SP or capsaicin. The long-lasting noncholinergic contraction of the main and hilus bronchi induced by field stimulation
in vitro
, as well as the contractile effects of SP and capsaicin, were also blocked by the SP antagonist. The cholinergic contractions and the noncholinergic tracheal relaxation on field stimulation
in vitro
were, however, not blocked by the antagonist. Vagal stimulation
in vivo
also increased vascular permeability in the respiratory tract and esophagus, causing a subepithelial edema as indicated by Evans blue extravasation. Previous treatment with [D-Arg
1
,D-Pro
2
,D-Trp
7,9
,Leu
11
]SP inhibited the permeability increase induced by both vagus nerve stimulation and exogenous SP. SP release from vagal sensory nerves was indirectly shown by reduction in the bronchial levels of SP after nerve stimulation
in vivo
. The data suggest that a major portion of the vagally or capsaicin-induced increase in smooth muscle tone is caused by SP release from sensory neurons. In addition, activation of vagal SP-containing sensory nerves induces local edema. Tracheobronchial afferent SP-containing C fibers may thus exert local control of smooth muscle tone and vascular permeability in normal and pathophysiological conditions.
Separate sites for binding and nicking of bacteriophage lambda DNA by terminase.
Authors: Feiss M , Kobayashi I , Widner W
Published: May 2006

The cohesive end site (cos) is the site of action of bacteriophage lambda terminase, the enzyme that introduces staggered nicks to generate the 12-base cohesive ends of mature lambda DNA. Deletion mutations that remove the lambda cohesive end sequence have been isolated after in vitro mutagenesis. The deletions were obtained by digesting the DNA of a cos duplication phage with S1 nuclease to remove the cohesive ends and adjacent base pairs, followed by blunt end ligation and DNA packaging into phage particles. cos2 is the result of a 22-base-pair deletion that exactly removes the segment of rotational symmetry that includes the cohesive end sequence. The cos2 mutation abolishes nicking by terminase but does not affect terminase binding. We conclude that cos contains two sites that interact with terminase: cosN, the nicking site; and cosB, a binding site for terminase.
Follicle-stimulating hormone-dependent phosphorylation of vimentin in cultures of rat Sertoli cells.
Authors: Spruill W A , Steiner A L , Tres L L , Kierszenbaum A L
Published: May 2006

Endogenous protein phosphorylation was investigated in cultured rat Sertoli cells after treatment with follicle-stimulating hormone (FSH) and pharmacological agents that activate cAMP-dependent protein kinases. In intact Sertoli cells, both phosphorylation and dephosphorylation of proteins occurred in response to treatment with these agents. Studies using cell-free preparations suggest that four phosphoproteins phosphorylated by cAMP or the catalytic subunit of cAMP-dependent protein kinase were also phosphorylated in a FSH-dependent manner in intact cells. These data suggest that FSH-dependent phosphorylation in Sertoli cells occurs through activation of a cAMP-dependent protein kinase. A FSH-dependent phosphoprotein with a molecular weight of 58,000 was identified as the intermediate filament protein vimentin, based on its migration in two-dimensional gels and its peptide map. The cellular distribution of vimentin was monitored by immunofluorescence in Sertoli cells after treatment with FSH. Results of this study support a role for intermediate filaments in FSH-dependent events in Sertoli cells.
The periseptal annulus: An organelle associated with cell division in Gram-negative bacteria
Authors: MacAlister T. J. , Macdonald B. , Rothfield L. I.
Published: May 2006

Evidence is presented that the site of cell division in
Salmonella typhimurium
is flanked by two circumferential zones of cell envelope differentiation, the periseptal annuli, which separate the division site from the remainder of the cell envelope. Each annulus is composed of a continuous structure in which the membranous elements of the cell envelope are closely associated with the murein cytoskeleton. The paired annuli appear early in the division process and the region between them defines a new cellular domain, the periseptal compartment, within which the division septum is formed.
Isolation and characterization of a temperature-sensitive mutant of avian myeloblastosis virus.
Authors: Moscovici M G , Moscovici C
Published: May 2006

A temperature-sensitive (ts) mutant, GA 907/7, was isolated after mutagen treatment of avian myeloblastosis virus. When bone marrow cells or secondary yolk sac macrophages were infected with GA 907/7, the expression of transformation was greatly reduced at 41 degrees C. The results of temperature-shift experiments suggest that in GA 907/7 the putative v-myb gene product is functional only at 35.5 degrees C. Moreover, when ts-induced transformed cells were shifted to 41 degrees C, a partial morphological conversion to macrophage-like cells was obtained, while the majority of the cells underwent senescence and lysis. No leukemia was obtained when GA 907/7 was injected in 1-day-old chickens. Finally, a continuous cell line releasing genetically stable mutant virus was obtained after transformation of secondary yolk sac cells.
Peptide antibody specific for the amino terminus of skeletal muscle alpha-actin.
Authors: Bulinski J C , Kumar S , Titani K , Hauschka S D
Published: May 2006

The NH2-terminal peptide of skeletal muscle alpha-actin (S alpha N peptide), which contains a primary sequence unique to this actin isozyme, was used to prepare an isozyme-specific peptide antibody. S alpha N peptide was purified from chicken breast muscle actin by preparative reverse-phase HPLC and was coupled to hemocyanin. This complex was used to immunize rabbits in order to elicit actin antibodies specific for the skeletal muscle alpha-actin isozyme. The antibody obtained, called S alpha N antibody, was reactive with S alpha N peptide and with skeletal muscle alpha-actin as well as with cardiac muscle alpha-actin. S alpha N antibody did not react with either of the actin isozymes present in smooth muscle (smooth muscle alpha and gamma) or in brain (nonmuscle beta and gamma). S alpha N antibody was used to detect muscle-specific actin in differentiating mouse and human myoblasts by using immunoblots of myoblast extracts and immunofluorescent staining of fixed cells.
Total synthesis of human beta-lipotropin.
Authors: Blake J , Li C H
Published: May 2006

The total synthesis of human beta-lipotropin has been accomplished by the new segment-coupling method in aqueous solution. The peptides Ac-Arg-beta-lipotropin-(61-89) (I) and [GlyS60]-beta-lipotropin-(1-60) (II) were synthesized by the solid-phase method. Reaction of peptide I with citraconic anhydride followed by brief digestion with trypsin to remove the acetylarginyl group, gave Ia. Reaction of peptide II with citraconic anhydride gave the citraconyl peptide IIa. Ia and IIa were coupled together in 50% dimethylformamide by reaction with silver nitrate/N-hydroxysuccinimide. After removal of the citraconyl groups in 25% acetic acid, a 10% yield of synthetic beta-lipotropin could be isolated. The synthetic product was shown to be identical to native human beta-lipotropin by paper electrophoresis, isoelectric focusing, HPLC, lipolytic activity in isolated rabbit fat cells, and radioimmunoassay.
Fibrinogen and fibrin structure and fibrin formation measured by using magnetic orientation.
Authors: Freyssinet J M , Torbet J , Hudry-Clergeon G , Maret G
Published: May 2006

Accurate birefringence measurements show that fibrinogen orients to a small degree in high magnetic fields. This effect can be explained as due to the molecule having about 30% (by weight) alpha-helix oriented relatively parallel to the long axis. Birefringence measurements on fully oriented fibrin suggest that aligned alpha-helical content is less than that estimated for fibrinogen. But because of limitations in the analysis this difference must be viewed with caution. Highly oriented fibrin results when polymerization takes place slowly in a strong magnetic field. Low-angle neutron diffraction patterns from oriented fibrin made in the presence of EDTA, made in the presence of calcium, or stabilized with factor XIIIa are very similar, showing that the packing of the molecules within the fibers is the same or very similar in these different preparations. The induced magnetic birefringence was used to follow fibrin formation under conditions in which thrombin was rate limiting. The fiber network formed by approximately the gelation point constitutes a kind of matrix or frame that is largely built upon during the remaining approximately 85% of the reaction. After gelation the reaction is pseudo-first order.
Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli.
Authors: Joyce C M , Grindley N D
Published: May 2006

Using currently available gene fusion techniques, we have constructed plasmids that direct the overproduction of the carboxyl-terminal two-thirds of DNA polymerase I, corresponding to the proteolytically derived "Klenow fragment." We have obtained overproduction amounting to several percent of the cellular protein using constructs in which expression is directed either from the lac promoter or from the leftward promoter of phage lambda. The polymerase fragment has been purified to homogeneity from such overproducing strains by a rapid three-stage purification procedure, yielding material capable of carrying out the same reactions (polymerization, 3' labeling, DNA sequence analysis) as the proteolytically derived fragment. The availability of such overproducing strains should greatly facilitate structural and mechanistic studies of DNA polymerase I. Moreover, the techniques we have described for the cloning and expression of a gene fragment should be generally applicable for the study of protein structure and function in other systems.
Characterization of the Escherichia coli thyA gene and its amplified thymidylate synthetase product.
Authors: Belfort M , Maley G F , Maley F
Published: May 2006

The 7.8-kilobase HindIII insert in phage lambda NM589thyA [Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S. & Murray, N. (1976) Mol. Gen. Genet. 146, 199] was confirmed as originating from Escherichia coli by hybridization analysis and was shown to encode the thymidylate synthetase (5,-10-methylenetetrahydrofolate:dUMP C-methyltransferase EC 2.1.1.45) of E. coli K-12 by using biochemical, structural, and immunologic criteria. The 7.8-kilobase insert was reduced in size to a quasi-random population of DNA subfragments by partial digestion with the 4-base-pair recognition enzymes Alu I and Hae III. A clone containing a 1.1- to 1.2-kilobase fragment that encompassed the gene was obtained from this mixture by selecting for Thy+ recombinants. Fusion of this DNA fragment to the phage lambda rho L promoter in plasmid pKC30 revealed the direction of transcription of the thyA gene, and, in a phage lambda lysogen containing a thermolabile repressor, intracellular synthetase levels were increased about 700-fold. The enzyme was purified to homogeneity from this source by affinity chromatography, and some of its properties are described.
Complete nucleotide sequence of a French bean storage protein gene: Phaseolin
Authors: Slightom Jerry L. , Sun Samuel M. , Hall Timothy C.
Published: May 2006

The complete nucleotide sequences of the gene and the mRNA coding for a specific phaseolin type French bean major storage protein have been determined. Comparison of these sequences reveals a phaseolin gene structure consisting of 80 base pairs (bp) of 5′ untranslated DNA, 1,263 bp of protein-encoding DNA which is interrupted by five intervening sequences (IV
S1
, 72 bp; IV
S2
, 88 bp; IV
S3
, 124 bp; IV
S4
, 128 bp; and IV
S5
, 103 bp), and 135 bp of 3′ untranslated DNA. Sequences characteristic of eukaryotic promoters “CCAAT” and “TATA” are present in the 5′ flanking DNA, and the eukaryotic poly(A) addition signal A-A-T-A-A-A occurs 16 bp before the first nucleotide of poly(A). The derived amino acid sequence yields an amino acid composition and a molecular weight compatible with those found for the β-type phaseolin protein. Two regions that probably serve as carbohydrate-peptide linkage recognition sites have been identified. A region of highly hydrophobic amino acids at the NH
2
terminus of the protein suggests the presence of a signal peptide in the newly synthesized phaseolin protein.