bluuebunny/crossref_metadata_2025 huggingface dataset:
Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase.
Authors: Rothenberg S P , Iqbal M P
Published: May 2006

A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of [3H]methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme.
Derangements in myocardial purine and pyrimidine nucleotide metabolism in patients with coronary artery disease and left ventricular hypertrophy.
Authors: Swain J L , Sabina R L , Peyton R B , Jones R N , Wechsler A S , Holmes E W
Published: May 2006

Studies in animal models of myocardial ischemia and left ventricular hypertrophy have demonstrated a number of derangements in purine and pyrimidine nucleotide content of myocardium that are postulated to play a role in the pathogenesis of muscle dysfunction in these disorders. The present study examined myocardium of patients with coronary artery disease, left ventricular hypertrophy, or neither of these two abnormalities, to determine whether derangements in purine and pyrimidine nucleotide metabolism occur in humans. In patients with coronary artery disease, endocardial content of ATP, GTP, UTP, CTP, and creatine phosphate was reduced and ranged between 60% and 86% of the amount found in the epicardium. In patients without coronary artery disease or ventricular hypertrophy, endocardial content of these nucleotides was equal to or greater than that of epicardium. Endocardial and epicardial content of inosine was increased in patients with coronary artery disease, and after vein bypass grafting inosine content fell to the levels observed in myocardium of patients with normal coronary arteries. In patients with left ventricular hypertrophy, endocardial content of ATP, GTP, UTP, CTP, and creatine phosphate was also reduced and ranged between 64% and 88% of the epicardial content. In contrast to results obtained in patients without left ventricular hypertrophy, epicardial content of GTP, UTP, and CTP was increased by 131%, 123%, and 132% in hypertrophied myocardium. Thus the changes noted in myocardial nucleotide content in patients are similar to those noted in animal models of occlusive coronary disease and ventricular hypertrophy. These results suggest that the pathophysiological abnormalities in nucleotide metabolism noted in animal models also occur in human myocardium.
Mechanism for shifting the phase of a circadian rhythm by serotonin: involvement of cAMP.
Authors: Eskin A , Corrent G , Lin C Y , McAdoo D J
Published: May 2006

Serotonin (5-hydroxytryptamine, 5-HT) shifts the phase of the circadian rhythm in the eye of Aplysia. We have examined the role of cAMP in mediating the effects of 5-HT on the rhythm. The phase shifts produced by 5-HT are mimicked by treatments that should increase intracellular levels of cAMP. An analogue of cAMP-8-benzylthio-cAMP, advanced and delayed the rhythm at phases in which 5-HT had similar effects on the rhythm. In addition, two phosphodiesterase inhibitors, Ro-20-1724 and papaverine, caused advance phase shifts where 5-HT advances the rhythm. The phosphodiesterase inhibitors Ro-20-1724 and 3-isobutyl-1-methylxanthine each potentiated the effect of subthreshold doses of 5-HT on the rhythm. The effects of 5-HT and 8-benzylthio-cAMP on the rhythm were nonadditive, indicating that 5-HT and 8-benzylthio-cAMP affect the rhythm through a common pathway. Finally, 5-HT produced large changes (13-fold) in the levels of cAMP in the eye. These results indicate that cAMP mediates the effect of 5-HT on the rhythm. There are two possible roles for cAMP in the circadian system. Either the cAMP system is an intracellular step in an entrainment pathway or it is part of the biological clock. Because 5-HT, 8-benzylthio-cAMP, and three phosphodiesterase inhibitors inhibit impulses from the eye, cAMP may also mediate the inhibition produced by 5-HT, or it might be involved in regulating the frequency of spontaneous impulses throughout the day.
Mapping of odor-related neuronal activity in the olfactory bulb by high-resolution 2-deoxyglucose autoradiography.
Authors: Lancet D , Greer C A , Kauer J S , Shepherd G M
Published: May 2006

The spatial distribution of odor-induced neuronal activity in the olfactory bulb, the first relay station of the olfactory pathway, is believed to reflect important aspects of chemosensory coding. We report here the application of high-resolution 2-deoxyglucose autoradiography to the mapping of spatial patterns of metabolic activity at the level of single neurons in the olfactory bulb. It was found that glomeruli, which are synaptic complexes containing the first synaptic relay, tend to be uniformly active or inactive during odor exposure. Differential 2-deoxyglucose uptake was also observed in the somata of projection neurons (mitral cells) and interneurons (periglomerular and granule cells). This confirms and extends our previous studies in which odor-specific laminar and focal uptake patterns were revealed by the conventional x-ray film 2-deoxyglucose method due to Sokoloff and colleagues [Sokoloff, L., Reivich, M., Kennedy, C., DesRosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O. & Shinohara, M. (1977) J. Neurochem. 28, 897--916]. Based on results obtained by the two methods, it is suggested that the glomerulus as a whole serves as a functional unit of activity. The high-resolution results are interpreted in terms of the well-characterized synaptic organization of the olfactory bulb and also serve to illustrate the capability of the 2-deoxyglucose autoradiographic technique to map metabolic activity in single neurons of the vertebrate central nervous system.
Circulant orbitals and the electron-correlation problem
Authors: Pauncz Ruben , Chen Min-Bo , Parr Robert G.
Published: May 2006

Circulant, as well as canonical, orbitals are used in the different orbitals for different spins method for treating electron correlation. Circulant orbitals provide a theoretical justification for the use of a single parameter, even when the canonical orbitals have widely different orbital energies. Illustrative calculations on the ground state of the Be atom show the importance of choosing the correct “pairs” in the method. A two-parameter version of the conventional method gives 74% of the improvement obtained by a full configuration-interaction treatment using 20 linear parameters, while a one-parameter linear combination of two different coupling schemes of the circulant method gives ≈61%. The latter wavefunction provides a compact description of the electron correlation.
Left-handed helices for DNA: studies on poly[d(I-C)].
Authors: Ramaswamy N , Bansal M , Gupta G , Sasisekharan V
Published: May 2006

Earlier, we showed that, for the D form (n = 8 and h = 3.03 A, where n is number of nucleotide units per turn and h is height per nucleotide unit) of poly[d(A-T)], both right- and left-handed double helical models are stereochemically satisfactory and give good agreement with the observed fiber diffraction data. It was also noted that the conformations of the right- and left-handed D-DNA models are very similar to those of the right- and left-handed B-DNA models. This observation was consistent with the D leads to B transition in the solid phase. As a continuation of our earlier studies, we have carried out similar experiments with poly[d(I-C)]. We could obtain a crystalline D-form pattern (n = 8, h = 3.13 A) of the fiber at 75% relative humidity (r.h.); the hydrated (r.h. approximately equal to 95%) form of the same fiber gave the classical B-form pattern (n = 10, h = 3.40 A). In the present report, we show that both right- and left-handed double-helical models are consistent with the fiber diffraction data of poly[d(I-C)] in the D-form. Theoretical energy calculations also suggest that the right- and left-handed B- and D-DNA models are almost equally stable. Hence, we conclude that the right- and left-handed double-helical models of poly[d(I-C)] in a given form (B or D) are equally likely and that the fiber diffraction data do not permit discrimination.
Mouse EcoRI satellite DNA contains a sequence homologous to the long terminal repeat of the intracisternal A particle gene.
Authors: Brown A , Huang R C
Published: May 2006

EcoRI restriction endonuclease fragments from the mouse 1,350-base-pair EcoRI satellite sequence were cloned in pBR322 plasmid. One of these fragments, SAT-1, was hybridized to various restriction fragments from cloned mouse intracisternal type A particle (IAP) genes and to mouse total genomic DNA. Hybridization occurred between the SAT-1 sequence and each of the IAP clones. The common region of hybridization in each of the clones was the long terminal repeat region of the IAP genes.
Steady state of an ATP-driven calcium pump: limitations on kinetic and thermodynamic parameters.
Authors: Tanford C
Published: May 2006

A numerical analysis was carried out to explore limitations on kinetic and thermodynamic parameters for an ATP-driven Ca pump. A conventional pump reaction cycle was employed, with a transport stoichiometry of two Ca ions per cycle. Rigid requirements were imposed to represent the needs of physiological function, defined as the ability to maintain the cytoplasmic Ca concentration below 10(-7) M against a 3 mM concentration on the opposite side of the membrane. Realistic physical limits were placed on the magnitudes of rate constants for individual reaction steps. Reversibility under laboratory conditions was assumed. The results show that these requirements can be satisfied simultaneously only if the equilibrium constant for binding Ca from the cytoplasmic (uptake) side of the membrane is much larger than the binding constant on the discharge side. More generally, the results demonstrate that limitations on rate constants make it possible for the pump to maintain an adequate rate only if steady-state levels of kinetically important (slowly reacting) reaction intermediates do not become too disparate. Experimental data for the sarcoplasmic reticulum calcium pump support these theoretical conclusions.
Identification of a cholesterol-regulated 53,000-dalton cytosolic protein in UT-1 cells and cloning of its cDNA.
Authors: Luskey K L , Chin D J , MacDonald R J , Liscum L , Goldstein J L , Brown M S
Published: May 2006

UT-1 cells, a clone of Chinese hamster ovary (CHO) cells, have a 100- to 1,000-fold elevation in the amount of 3-hydroxy-3-methylglutaryl CoA reductase and therefore grow in the presence of compactin, an inhibitor of reductase. In this paper, we report that UT-1 cells also have a markedly increased amount of another protein with a Mr of 53,000 and an isoelectric point of approximately equal to 6. Whereas the reductase is an enzyme of the endoplasmic reticulum, the 53,000-dalton protein (termed the "53k" protein) is in the cytosol. It is not precipitated by an antireductase antibody. Synthesis of the 53k protein, like that of the reductase, is suppressed when UT-1 cells are incubated with plasma low density lipoprotein (LDL). We prepared a library of recombinant plasmids containing double-stranded cDNAs from UT-1 cells. Using differential colony hybridization, we identified recombinant plasmids containing double-stranded cDNA inserts encoding mRNAs expressed at high levels in UT-1 cells as compared with CHO cells. One of the plasmids, designated p53k-3, contained a 0.97-kilobase double-stranded cDNA that hybridized to a 3.8-kilobase mRNA. When translated in vitro, this 3.8-kilobase mRNA directed the synthesis of a protein identical to the cellular 53k protein as determined by two-dimensional gel electrophoresis. Hybridization studies showed that the mRNA for the 53k protein was present in much larger amounts in UT-1 cells than in parental CHO cells. In both cell types, the content of this mRNA decreased markedly when the cells were incubated with LDL. Although the function of the 53k protein is not known, circumstantial evidence suggests that it may represent cytosolic 3-hydroxy-3-methylglutaryl CoA synthase, the enzyme preceeding the reductase in the cholesterol biosynthetic pathway. The current data indicate that the synthesis of at least two proteins, the reductase and the 53k protein, are induced to high levels in compactin-resistant UT-1 cells and that the synthesis of both is suppressed coordinately by LDL.
Translation of mRNAs for subunits of chloroplast coupling factor 1 in spinach
Authors: Watanabe Akira , Price Carl A.
Published: May 2006

The chloroplast coupling factor 1 consists of five nonidentical subunits, three of which (α, β, and ε subunits) have been shown in several laboratories to be synthesized within chloroplasts. The site of synthesis of the remaining two (γ and δ subunits) was investigated by analyzing products directed by spinach leaf RNAs in wheat germ and reticulocyte translation systems
in vitro
. It was found that poly(A)
+
RNA directs the synthesis of two distinct polypeptides, one of which is immunochemically related to the γ subunit but is 4,000 daltons larger. The other shares antigenic sites with the δ subunit but is 8,000 daltons larger. When wheat germ or reticulocyte translation systems were programmed with RNAs from purified chloroplasts, the only products related to CF
1
that we could detect were a putative precursor of β, 2,000 daltons larger than the mature subunit, and some smaller polypeptides, which appear to be incomplete translation products of β. From these results it appears likely that the γ and δ subunits are synthesized in the cytoplasm as larger precursors and that β is synthesized within the chloroplast as a precursor.
Extracts from the brains of hibernating and alert ground squirrels: effects on cells in culture.
Authors: Amorese D A , Swan H , Bamburg J R
Published: May 2006

Aqueous extracts were prepared from pulverized, acetone-dehydrated brains of hibernating and alert ground squirrels. Addition of these extracts to Chinese hamster ovary cells in suspension culture resulted in a decrease in the amount of [3H]thymidine incorporated into acid-precipitable material without affecting the transport or phosphorylation of the nucleoside. The inhibition was time- and dose-dependent and full recovery occurred about 2 hr after exposure of the cells to the active extract. The active factor is readily oxidized during storage at -70 degrees C but full activity can be restored by treatment with 2-mercaptoethanol. The peptide nature of the active material is indicated by its susceptibility to proteases and by loss of activity after alkylation. Fasted or cold-acclimated rats also develop increased levels of active substance in their brains; however, brains of hibernating squirrels contain 10- to 50-fold more of the active substance than brains from either alert squirrels or rats. A time-dependent increase in activity of extracts from hibernating brain incubated with a homogenate from alert brain suggests that the peptide is activated or generated in the mixture.
Antibodies elicited against cis-diamminedichloroplatinum(II)-modified DNA are specific for cis-diamminedichloroplatinum(II)-DNA adducts formed in vivo and in vitro.
Authors: Poirier M C , Lippard S J , Zwelling L A , Ushay H M , Kerrigan D , Thill C C , Santella R M , Grunberger D , Yuspa S H
Published: May 2006

Rabbit antiserum elicited against calf thymus DNA modified to 4.4% (Pt drug/nucleotide ratio = 0.044) with the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) contains antibodies specific for the Pt-modified DNA immunogen as well as for Pt-DNA adducts formed in both cultured mouse leukemia L1210 cells and in L1210 cells from the ascites fluid of tumor-bearing mice exposed to cis-DDP. Pt-modified DNA was electrostatically complexed to methylated bovine serum albumin and injected into rabbits. Early bleedings of the derived antiserum were used to establish a competitive enzyme-linked immunosorbent assay (ELISA), which demonstrated specificity for the Pt-modified DNA but not for DNA or the Pt drug alone. In the ELISA, 50% inhibition occurred at a concentration of 0.5 nM Pt (on DNA) as determined by atomic absorption spectroscopy. This value corresponds to a lower limit of detectability of one adduct in 10(7) nucleotides, with 50 micrograms of sample DNA added per microtiter well. DNA isolated from cultured mouse L1210 cells exposed to increasing doses of the Pt drug was found by ELISA to contain from 0.2 to 10.0 fmol of Pt adduct per microgram of DNA. These levels remained stable for up to 4 hr after a 1-hr drug treatment, during which time DNA interstrand crosslinks developed. Thus, the antiserum appears not to be specific for DNA interstrand crosslinks. DNAs from L1210 cells exposed to trans-diamminedichloroplatinum(II) and L-phenylalanine mustard were not recognized in the ELISA. DNA prepared from the ascites cells of mice bearing the L1210 tumor 5 hr after injection of cis-DDP was found to contain about 2 fmol of Pt per microgram of DNA. This work establishes that cis-DDP-DNA adducts prepared in vitro are relevant to the in vivo binding of the Pt drug to its biological target, DNA, and opens new avenues for studying the mechanism of action of the Pt anticancer drugs.
Achievement of high rates of in vitro synthesis of 1,4-beta-D-glucan: activation by cooperative interaction of the Acetobacter xylinum enzyme system with GTP, polyethylene glycol, and a protein factor.
Authors: Aloni Y , Delmer D P , Benziman M
Published: May 2006

Regulatory properties of a cellulose synthase (UDP-forming)(UDPglucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase, EC 2.4.1.12) have been demonstrated by using enzyme preparations derived from cells of Acetobacter xylinum. Preparation of a particulate fraction in the presence of 20% (wt/vol) polyethylene glycol-4000 (PEG-4000) yields enzyme with activity 3- to 10-fold higher than that previously reported. The enzyme prepared in this fashion also shows a further marked, specific activation by GTP. The Ka for GTP is 34 microM. Guanosine 5'-[gamma-thio]triphosphate, an analog of GTP, is even more effective than GTP (Ka for guanosine 5'-[gamma-thio]triphosphate = 17 microM). A large number of other nucleotides and nucleotide derivatives were tested with no effect. Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor necessary for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself has no synthase activity nor does it stimulate activity of the enzyme in the absence of GTP. In the presence of GTP, protein factor, and PEG-4000, initial rates of enzyme activity 200 times greater than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose.
Host-specific activation of transcription by tandem repeats from simian virus 40 and Moloney murine sarcoma virus.
Authors: Laimins L A , Khoury G , Gorman C , Howard B , Gruss P
Published: May 2006

The simian virus (SV40) 72-base pair (bp) tandem repeated sequences have recently been shown to function as activators or enhancers of early viral transcription. A recombinant viral genome was recently constructed by inserting 72-bp tandem repeats from the Moloney murine sarcoma virus (MSV) in place of the 72-bp repeats of SV40. Although this genome replicates in monkey kidney cells, its rate of large tumor antigen expression and replication is considerably slower than that of wild-type SV40. In mouse cells, however, equivalent levels of large tumor antigen appear to be expressed from both wild-type and recombinant genomes, suggesting a relationship between the level of enhancer activity and the host cell. To confirm this observation, we have applied a sensitive quantitative assay for gene expression based on the conversion of chloramphenicol to its acetylated forms. The gene encoding the enzymatic function chloramphenicol acetyltransferase was inserted into two vectors in which the enhancer sequences from SV40 or MSV were placed adjacent to the early SV40 promoter. The SV40 tandem repeats appear to activate gene expression to significantly higher levels in monkey kidney cells, but the MSV repeats are more active in two lines of mouse cells. These findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.
Protein synthesis in rabbit reticulocytes: characteristics of the protein factor RF that reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates.
Authors: Grace M , Ralston R O , Banerjee A C , Gupta N K
Published: May 2006

During heme deficiency in reticulocyte lysates, the heme-regulated translational inhibitor of protein synthesis (HRI) is activated and shuts off protein synthesis. In partial reactions, HRI phosphorylates the Mr 38,000 subunit (alpha subunit) of eukaryotic initiation factor 2 (eIF-2), which forms a ternary complex, Met-tRNAf X eIF-2 X GTP. The eIF-2 alpha (P) thus formed is not recognized by two eIF-2 ancillary factors, Co-eIF-2B (which promotes the dissociation of the ternary complex at high Mg2+) and Co-eIF-2C (which reverses the inhibition of ternary complex formation), and thus, is presumably inactive in peptide chain initiation. A protein factor, designated RF, which reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates, has been purified from reticulocyte cell supernatant. RF is a high molecular weight (Mr approximately equal to 450,000) protein complex composed of multiple polypeptides. An active RF preparation contains Co-eIF-2B and Co-eIF-2C activities, and these two activities in RF preparation are not inhibited by HRI and ATP--i.e., eIF-2 alpha (P) is recognized. During purification, RF remains associated with eIF-2 activity (eIF-2 X RF) and can be freed of this eIF-2 activity by CM-Sephadex chromatography. Both eIF-2 X RF and RF contain a Mr 38,000 polypeptide component that is indistinguishable from the Mr 38,000 subunit of eIF-2 by two-dimensional gel electrophoresis. It has been observed that a significant part of this Mr 38,000 polypeptide component in eIF-2 X RF and almost the entire Mr 38,000 polypeptide component in RF remain unphosphorylated after prolonged incubation with HRI and ATP. A possible role of this free Mr 38,000 polypeptide in RF action is discussed.
Hormone changes during the menstrual cycle in abetalipoproteinemia: reduced luteal phase progesterone in a patient with homozygous hypobetalipoproteinemia.
Authors: Illingworth D R , Corbin D K , Kemp E D , Keenan E J
Published: May 2006

Progesterone synthesis by the human corpus luteum requires a source of cholesterol, which can be derived from both local synthesis and uptake of low density lipoproteins (LDL). When the corpus luteum is maintained in organ culture, progesterone synthesis is primarily dependent on LDL and the rate of progesterone production during growth in a LDL-free media is suboptimal. An in vivo situation analogous to that of corpus luteum grown in LDL-depleted media exists naturally in patients with abetalipoproteinemia. To determine whether a complete deficiency of plasma LDL affects serum concentrations of progesterone (particularly during the luteal phase) or those of other hormones, we have measured the serum concentrations of luteinizing hormone, follicle-stimulating hormone, prolactin, estradiol, estrone, and progesterone during the menstrual cycle in a patient with phenotypic abetalipoproteinemia (on the basis of homozygous hypobetalipoproteinemia). Our results show a normal cyclical pattern with midcycle increases in the concentrations of luteinizing and follicle-stimulating hormones, prolactin, and estrogens but a distinctly subnormal increase in the luteal phase concentrations of progesterone. These results suggest that, in patients with phenotypic abetalipoproteinemia, the absence of LDL leads to an impairment in the maximal rates of production of progesterone by the corpus luteum.
Monoclonal antibodies distinguish antigenically discrete neuronal types in the vertebrate central nervous system.
Authors: McKay R D , Hockfield S J
Published: May 2006

Eight hundred hybridoma lines were generated from mice immunized with the fixed gray matter of cat spinal cord. Of these lines, 47 were positive when screened immunohistochemically against sections of the cat spinal cord. Twenty-nine lines secreted antibodies that bound to neuronal antigens. Of these, 16 bound to axons only, 8 bound to axons and cell bodies, and 5 bound to cell bodies only. Eighteen lines secreted antibodies that bound to glial cells. Five lines that secreted antibodies that intensely stained spinal cord sections were cloned and screened against other parts of the central nervous system. Each of these five antibodies bound to specific subsets of neurons. For example, in the spinal cord, one antibody (Cat-301) recognized a surface determinant on the dendrites and cell bodies of neurons that, in morphology and location, resemble long-distance projection neurons. A second antibody (Cat-201) recognized an antigen in axons and in the cytoplasm of neuronal cell bodies that may be a subset of those recognized by Cat-301. A third antibody (Cat-101) recognized only axons. The subcellular localization of the antigen recognized by each antibody is the same in all areas of the central nervous system we have examined. The fact that each of the antibodies described here has a restricted distribution in the central nervous system shows that there is a high degree of molecular diversity among vertebrate neurons and that hybridoma technology can be used to explore this diversity. This class of reagents should be a useful addition to the many established techniques for studying the organization of the vertebrate central nervous system.
Epoxidation reactions catalyzed by rat liver cytochromes P-450 and P-448 occur at different faces of the 8,9-double bond of 8-methylbenz[a]anthracene.
Authors: Yang S K , Chou M W , Fu P P , Wislocki P G , Lu A H
Published: May 2006

8-Methylbenz[a]anthracene (8-MeBaA) transdihydrodiol metabolites were isolated by reversed-phase and normal-phase HPLCs from incubations of 8-MeBaA with liver microsomes or a reconstituted system containing purified cytochrome P-448 and epoxide hydrolase. Regardless of the enzyme source, the metabolically formed 8-MeBaA trans-3,4- and -5,6-dihydrodiols were found to be enriched in one enantiomeric isomer and differed only in the degree of optical purity. The 8-MeBaA trans-8,9-dihydrodiol formed by liver microsomes from either untreated or phenobarbital-treated rats was enriched with the (+)-enantiomer. In contrast, the 8-MeBaA trans-8,9-dihydrodiol formed either by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted rat liver enzyme system containing cytochrome P-448 and epoxide hydrolase was enriched with the (-)enantiomer. These results indicate that, in catalyzing the formation of 3,4- and 5,6-epoxide intermediates, the interaction with the unsubstituted 3,4- and 5,6-double bonds of 8-MeBaA by the different forms of cytochrome P-450 occur preferentially on the same face of the aromatic plane and they differ only in the degree of stereoselectivity. However, different forms of cytochrome P-450 may interact with different faces of the aromatic plane at the methyl-substituted 8,9-double bond of 8-MeBaA, resulting in the formation of trans-8,9-dihydrodiols enriched in different enantiomeric forms. This demonstrates that different forms of cytochrome P-450 may catalyze the epoxidation reaction preferentially at different sides of the methyl-substituted double bond of a planar polycyclic hydrocarbon molecule. These properties may be used to further classify and to understand the enzyme-substrate interactions of the different forms of cytochrome P-450 in the drug-metabolizing enzyme systems.
Structural and metabolic relationship between the molybdenum cofactor and urothione.
Authors: Johnson J L , Rajagopalan K V
Published: May 2006

The molybdenum cofactor isolated from sulfite oxidase (sulfite: ferricytochrome c oxidoreductase, EC 1.8.2.1) and xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.2.1.37) in the presence of iodine and KI (form A) has been shown to contain a pterin nucleus with an unidentified substituent in the 6 position [Johnson, J. L., Hainline, B. E. & Rajagopalan, K. V. (1980) J. Biol. Chem. 255, 1783-1786]. A second inactive form of the cofactor was isolated aerobically but in the absence of iodine and KI. The latter cofactor derivative (form B) is highly fluorescent, has a visible absorption band at 395 nm and, like form A, contains a phosphate group. Cleavage of the phosphate ester bond with alkaline phosphatase exposes a glycol function that is sensitive to periodate. Oxidation of form B with alkaline permanganate yields a highly polar compound with properties of a sulfonic acid, suggesting that the active molybdenum cofactor might contain sulfur. The sulfur-containing pterin urothione characterized by Goto et al. [Goto, M., Sakurai, A., Ohta, K. & Yamakami, H. (1969) J. Biochem. 65, 611-620] had been isolated from human urine. The permanganate oxidation product of urothione, characterized by Goto et al. as pterin-6-carboxylic-7-sulfonic acid, is identical to that obtained from form B. Because urothione also contains a periodate-sensitive glycol substituent, a structural relationship is suggested. The finding that urine samples from patients deficient in the molybdenum cofactor are devoid of urothione demonstrates a metabolic link between the two molecules.
Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus.
Authors: González J M , Brown B J , Carlton B C
Published: May 2006

The recently discovered high-frequency transfer of plasmids between strains of Bacillus thuringiensis was used to study the genetic relationship between plasmids and production of the insecticidal delta-endotoxin crystal. Three strains of B. thuringiensis transmitted the Cry+ (crystal-producing) phenotype to Cry- (acrystalliferous) B. thuringiensis recipients. Agarose gel electrophoresis showed that one specific plasmid from each donor strain was always present in Cry+ "transcipients." The size of the transmissible crystal-coding plasmid varied with the donor strain, being 75 MDal (megadaltons) in size in HD-2, 50 MDal in HD-73, and 44 MDal in HD-263. Immunological analysis showed the Cry+ transcipients to be hybrid strains, having flagella of the recipient serotype and crystals of the donor serotype. These results demonstrate that the structural genes for the delta-endotoxin are plasmid borne. Crystal-coding plasmids also transferred into two strains of the related species Bacillus cereus and yielded transcipients that produced crystals of the same antigenicity as the donor strain.
A T15-idiotype-positive T suppressor hybridoma does not use the T15 VH gene segment.
Authors: Nakanishi K , Sugimura K , Yaoita Y , Maeda K , Kashiwamura S , Honjo T , Kishimoto T
Published: May 2006

The T suppressive factor (TsF) released from a T15-idiotype-positive phosphocholine (PCho)-specific T hybridoma, F18-3-4, which was formed by fusion between BALB/c T cells and BW5147 thymoma, was immunochemically characterized. TsF inhibited the in vitro induction of both IgE and IgG1 antibody responses of 2,4-dinitrophenyl keyhole limpet hemocyanin (DNP-KLH)-primed spleen cells in the presence of PCho-KLH-DNP. TsF had the ability to bind to PCho determinants and possessed T15 idiotype determinants as well as Iad products. However, we were unable to detect either the rearrangement of the T15 VH gene or the presence of T15 VH gene transcripts in hybridomas by DNA and RNA blot hybridization analyses with the T15 VH DNA probe.
Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA.
Authors: Montoya J , Christianson T , Levens D , Rabinowitz M , Attardi G
Published: May 2006

The initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mitochondrial DNA have been investigated by mapping experiments utilizing in vitro "capped" mitochondrial RNA molecules or nascent RNA chains. Mitochondrial poly(A)-containing RNA molecules were labeled at their 5' ends with [alpha-32P]GTP and guanylyltransferase ("capping" enzyme) and mapped on the mitochondrial genome by DNA transfer hybridization and S1 nuclease protection experiments. A mapping site for the capped 5' ends was found on the H strand very near to the 5' terminus of the 12S rRNA gene, and another site was found on the L strand very near to the 5' terminus of the 7S RNA coding sequence. In parallel experiments, the 5' ends of the nascent chains isolated from mitochondrial DNA transcription complexes were similarly mapped very near to the 5' termini of the 12S rRNA gene and of the 7S RNA coding sequence. The in vitro capped RNA molecules and the nascent chains thus presumably identify the same transcriptional initiation sites on the H strand and the L strand. The occurrence of a second possible initiation site for H-strand transcription 90-110 nucleotides upstream of that described above--i.e., 20-40 nucleotides upstream of the tRNAPhe gene--had been previously indicated by a mapping analysis of the nascent RNA chains and has been confirmed in the present work. The presence of two initiation sites for H-strand transcription can be correlated with other types of evidence that point to two different transcription events leading to the synthesis of a polycistronic molecule corresponding to the almost entire H strand and to the synthesis of the rRNA species.
Functional reconstitution of beta-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase.
Authors: Pedersen S E , Ross E M
Published: May 2006

A procedure for the functional reconstitution of beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. beta-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of beta-adrenergic agonists. This stimulation of the activation of G/F was specific for beta-adrenergic agonists and could be specifically blocked by beta-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active beta-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F by receptor was reconstituted.
Deletion mutants defining the Escherichia coli replication factor Y effector site sequences in pBR322 DNA.
Authors: Soeller W C , Marians K J
Published: May 2006

The Escherichia coli DNA replication factor Y, along with other genetically undefined replication proteins, is involved in a dnaB-, dnaC-, and dnaG-dependent pathway of primer formation on phi X174 single-stranded circular DNA. In addition, replication factor Y has a site-specific, single-stranded DNA-dependent ATPase activity. We have previously demonstrated the presence of two factor Y effector sites on pBR322 DNA. When inserted into the filamentous phage f1R229, these sites can function as rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent origins of DNA replication. We report here the construction of deletion mutants of the two pBR322 factor Y effector sites. These deleted sites no longer function as effectors for factor Y ATPase activity nor as templates for rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent DNA synthesis. We conclude that the DNA sequences required for factor Y ATPase activity and origin function are likely to be identical.
Glucose-induced insulin release depends on functional cooperation between islet cells.
Authors: Pipeleers D , in't Veld P I , Maes E , Van De Winkel M
Published: May 2006

Similar to other endocrine glands, the endocrine pancreas displays a characteristic topography of its constituent cells. The functional significance of this structural organization was examined by measuring the secretory activity of the B cell in rat islet cell preparations of different composition. Glucose released 30-fold more insulin from B cells lodged within intact islets as from purified single B cells; structurally coupled B cells and single B cells isolated with A cells or incubated with glucagon responded 4- and 2-fold, respectively, more effectively to glucose than single B cells alone. Glucose homeostasis is thus dependent not only on the number and integrity of the insulin-containing B cells but also on their interactions with neighboring B and non-B cells. This study provides direct support for the concept that the microanatomy of the islet creates the anatomical basis for functional cooperation between islet cells and hence for an appropriate glucose-induced insulin release.
Genomic clones coding for some of the initial genes expressed during Drosophila development.
Authors: Sina B J , Pellegrini M
Published: May 2006

Preblastoderm Drosophila embryos were made permeable and labeled in vivo with [32P]phosphate-containing medium. Cytoplasmic polyadenylylated RNA was extracted from these embryos and used to screen a library of Drosophila genomic DNA sequences cloned in phage lambda. Ten cloned sequences were selected for further study. These sequences were not complementary to mitochondrial DNA, nor did they contain the repeated nuclear genes coding for rRNA or histones. The cloned sequences each encode one or more unique genes expressed in preblastoderm embryos. RNA blot analysis indicated that some of these genes are also expressed at other times during embryogenesis. These results show that, in spite of the rapid nuclear divisions taking place during the preblastoderm stage, Drosophila nuclear genes are transcribed and that a subset of these genes show variable, stage-specific levels of expression during early embryogenesis.
Differential synthesis of spore coat proteins in prespore and prestalk cells of Dictyostelium.
Authors: Devine K M , Morrissey J H , Loomis W F
Published: May 2006

The major spore coat proteins (SP60, SP70, and SP96) of Dictyostelium discoideum have been analyzed by using two-dimensional nonequilibrium pH gradient electrophoresis. These proteins have been characterized with respect to electrophoretic behavior, metachromatic staining with silver, and posttranslational modifications; these techniques allow unambiguous identification of these proteins in silver-stained gels and autofluorograms of total cell extracts. They are synthesized by cells in the prespore region of migrating slugs, ultimately becoming major components of mature spores, but are not detectable either in cells in the prestalk region of slugs or in mature stalk cells. The ease with which these relatively abundant proteins can be recognized on two-dimensional gels makes them very useful markers for spatial and temporal differentiation in this organism.
Vaccinia virus: a selectable eukaryotic cloning and expression vector.
Authors: Mackett M , Smith G L , Moss B
Published: May 2006

Foreign DNA was inserted into two nonessential regions of the vaccinia virus genome by homologous recombination in cells infected with virus and transfected with plasmids containing the foreign DNA elements flanked by vaccinia virus DNA. Thymidine kinase-negative (TK-) recombinants were selected after inserting foreign DNA into the coding region of the TK gene of wild-type vaccinia virus; TK+ recombinants were selected after inserting the herpesvirus TK gene into TK- mutants of vaccinia virus. For TK+ expression, it was necessary to insert a 275-base-pair DNA fragment containing the initiation site and sequences upstream of an early vaccinia virus transcript next to the coding sequences of the herpesvirus gene. The unique ability of the herpesvirus TK to phosphorylate 125I-labeled deoxycytidine provided independent confirmation of gene expression. These studies demonstrate the use of vaccinia virus as a selectable cloning and expression vector, confirm the map location of the vaccinia virus TK gene, and provide initial information regarding the location of vaccinia virus transcriptional regulatory sequences.
Control of transposon Tn5 transposition in Escherichia coli.
Authors: Sasakawa C , Lowe J B , McDivitt L , Berg D E
Published: May 2006

Tn5 is a composite transposable element in which the insertion sequences IS50R and IS50L bracket a central region encoding kanamycin resistance (kanr). IS50R encodes a functional transposase, whereas IS50L contains the promoter of the kanr gene. To determine the relative activities of IS50R and IS50L in transposition we examined the structures of chimeric DNA molecules generated by insertion of segments of pBR322::Tn5 dimeric plasmids into red- lambda phage in recA- Escherichia coli. Restriction endonuclease analyses showed that the inserted sequences contained direct terminal repeats of pairs of IS50R or of IS50L elements and that the frequencies of usage of IS50R vs. IS50L depended on the position and orientation of Tn5 in the plasmid vector: IS50R was used preferentially when Tn5 was in transcriptionally quiescent regions of the vector (in either orientation) or when IS50L was immediately downstream from a strong promoter in the vector. In contrast, IS50L was used preferentially when IS50R was downstream from a strong promoter. We conclude IS50R tends to be used preferentially but that when transcription impinges on the end of an IS50 element the participation of that element in transposition is inhibited.
Evidence for a major cluster of lymphocyte differentiation antigens on murine chromosome 2.
Authors: Meruelo D , Offer M , Rossomando A
Published: May 2006

The region of chromosome 2 between H-13 and H-3 has been shown to contain loci coding for a variety of other alloantigens, including Ly-4 and the locus coding for beta 2-microglobulin. Herein we show that Ly-6 and Ly-11 are coded for by genes in a segment of chromosome 2 adjacent to the H-3-H-13 region and that this segment of chromosome also contains the tightly linked loci coding for antigens Ala-1, DAG, H9/25, H-30, Ly-8, and ThB. In addition, at least one locus (and probably more) affecting susceptibility to leukemia induction is found within this gene cluster.
Prostaglandin biosynthesis in the human fetal adrenal gland: Regulation by glucocorticosteroids
Authors: Mitchell Murray D. , Carr Bruce R. , Mason J. Ian , Simpson Evan R.
Published: May 2006

Human fetal adrenal (HFA) tissue was maintained in organ culture to evaluate the biosynthesis of prostaglandins and hormonal regulation of prostaglandin formation by this tissue. The HFA tissue secreted substantial amounts of prostaglandin E
2
, prostaglandin F
2α
, 13,14-dihydro-15-ketoprostaglandin F
2α
, 6-ketoprostaglandin F
1α
, and thromboxane B
2
; secretion of prostaglandin D
2
could not be demonstrated. Prostaglandin biosynthesis in HFA tissue was inhibited in a time-dependent manner by corticotropin (ACTH; 0.4 μM); by the fourth day of culture, the extent of inhibition of biosynthesis of each prostaglandin was 60-90%. Progesterone (1 μM), cortisol (1 μM), and dexamethasone (1 μM) inhibited prostaglandin biosynthesis whereas estradiol (1 μM) did not. Of the compounds tested for inhibitory activity, dexamethasone was the most potent. An inhibitor of 11β-hydroxylase activity (metyrapone; 0.1 mM) effectively eliminated the inhibition of prostaglandin biosynthesis caused by corticotropin and progesterone. Metyrapone treatment alone caused a 3-fold increase in prostaglandin biosynthesis by fetal adrenal tissues. Similar stimulatory effects resulted from treatment with inhibitors of (
i
) 3β-hydroxysteroid dehydrogenase (cyanoketone; 15 μM), (
ii
) steroid 17α-hydroxylase (SU 10603; 19 μM), and (
iii
) cholesterol side-chain cleavage (aminoglutethimide; 1 mM). Inhibition of prostaglandin biosynthesis by dexamethasone in the presence or absence of metyrapone was concentration dependent and 50% inhibition could be demonstrated at 1 nM. A competitive inhibitor of the binding of glucocorticosteroids to cytoplasmic receptors (cortisol 21-mesylate; 1 μM) significantly reduced the inhibition of prostaglandin biosynthesis effected by dexamethasone (10 nM). These findings suggest that prostaglandin biosynthesis in the HFA gland is regulated by endogenously synthesized glucocorticosteroids, the actions of which are mediated by a glucocorticosteroid receptor. Such glucocorticosteroids induce the synthesis of a substance that inhibits prostaglandin biosynthesis.
High actin concentrations in brain dendritic spines and postsynaptic densities.
Authors: Matus A , Ackermann M , Pehling G , Byers H R , Fujiwara K
Published: May 2006

Antibodies against actin were used to corroborate the presence of actin as a major component protein of isolated brain postsynaptic densities. The same antibodies also were used as an immunohistochemical stain to study the distribution of actin in sections of intact brain tissue. This showed two major sites where actin is concentrated: smooth muscle cells around blood vessels and postsynaptic sites. In the postsynaptic area the highest concentration of actin occurs in postsynaptic densities and there also is intense staining in the surrounding cytoplasm, especially within dendritic spines. Antiactin staining was much weaker in other parts of neurons and in glial cells. The high concentration of actin in dendritic spines may be related to shape changes that these structures have been found to undergo in response to prolonged afferent stimulation.
Isolation of Drosophila proteins that bind selectively to left-handed Z-DNA.
Authors: Nordheim A , Tesser P , Azorin F , Kwon Y H , Möller A , Rich A
Published: May 2006

An affinity column for isolating Z-DNA binding proteins was made by attaching brominated poly(dG-dC) to Sephadex. Proteins from Drosophila nuclei were prepared and those that could bind to Escherichia coli B-DNA were removed from the solution. The remaining proteins were passed over the Z-DNA affinity column and then eluted with NaCl. Using both direct and competitive filter binding assays, we found that the eluted proteins bind to brominated poly(dG-dC) (Z-DNA) and poly(dG-m5dC) but not to poly(dG-dC) (B-DNA), native or denatured E. coli or calf thymus DNA, or brominated oligonucleotides. The proteins also bind to negatively supercoiled plasmids carrying Z-DNA sequences but not to relaxed or linearized plasmids in which the Z-DNA conformation is no longer present. Gel analysis reveals a mixture of several large proteins up to approximately 150,000 daltons.