bluuebunny/crossref_metadata_2025 huggingface dataset:
Upper stratospheric photolysis of NaCl and KCl
Authors: Rowland F. Sherwood , Rogers Patricia J.
Published: May 2006

Sodium chloride has been postulated to be formed in the stratosphere by the reaction of NaOH with HCl, and an analogous reaction should occur also for KOH, with the formation of KCl. Photodissociation rates have been calculated for both NaCl and KCl using the published ultraviolet absorption cross sections below 300 nm. Both molecules absorb strongly and have photodissociation lifetimes of only a few minutes in the upper stratosphere. Neither molecule is an effective sink for chlorine above an altitude of about 35 km.
Lateral motion of beta receptors in membranes of cultured liver cells.
Authors: Henis Y I , Hekman M , Elson E L , Helmreich E J
Published: May 2006

We have studied the lateral mobility and distribution of beta receptors on Chang human liver cells by fluorescence photobleaching recovery and video intensification microscopy. The beta receptors were labeled with the fluorescent antagonist 7-(2-allylphenoxy)-2,2-dimethyl-6-hydroxy-1-(4-nitrobenzo-2-oxa-1,3-diazolyl)-1 ,4-diazaheptane (Alp-NBD). Sixty to 75% of the staining was specific (displaceable by unlabeled antagonists). Most of the antagonist-occupied beta receptors were immobile, because only 15-25% of their fluorescence recovered on the experimental time scale at 23 degrees C. This immobility correlates with the clustered distribution of Alp-NBD--beta-receptor complexes at 4 degrees C and 37 degrees C. The beta receptors appear to be aggregated prior to antagonist binding, because visible patches were observed immediately after labeling for 30 sec at 4 degrees C. Preincubation at 37 degrees C with (--)-isoproterenol, a beta agonist, prior to Alp-NBD labeling induced a time-dependent release of the beta receptors to a more homogeneous distribution and increased the mobile fraction to 70-80% (lateral diffusion coefficient = 1.4 X 10(-9) cm2/sec at 23 degrees C). This is not due to an effect on membrane fluidity, because the diffusion coefficient of a lipid probe was not altered. The time course of agonist-induced beta-receptor mobilization correlates with receptor loss and adenylate cyclase desensitization but is much slower than adenylate cyclase activation. This indicates that adenylate cyclase activation by beta receptors does not require macroscopic lateral mobility of the majority of the beta receptors.
Induction of prolactin-deficient variants of GH3 rat pituitary tumor cells by ethyl methanesulfonate: reversion by 5-azacytidine, a DNA methylation inhibitor.
Authors: Ivarie R D , Morris J A
Published: May 2006

GH3 cells are a rat pituitary tumor line expressing two pituitary peptide hormones, prolactin (rPRL) and growth hormone. Recently, it was found that the DNA alkylating agent ethyl methanesulfonate can induce the appearance of rPRL-deficient GH3 cell variants at a high frequency (ca. 20-30%). As shown here, such variants cannot be induced at high frequency by irradiation of wild-type GH3 cells with ultraviolet light, indicating that the effect may be specific to treatment with alkylating agents. Furthermore, the DNA methylation inhibitor 5-azacytidine reverted an ethyl methanesulfonate-induced rPRL-deficient variant into rPRL-expressing cells at high frequency (ca. 50%). The revertants were stable for at least 30-35 generations. These results support the hypothesis that the alkylating agent may promote the specific methylation of the rPRL gene or a gene regulating its activity, either one of which leads to inactivation of expression of the rPRL gene in GH3 cells.
Neutrophils are required for the DNA synthetic response of human lymphocytes to mevalonic acid: evidence suggesting that a nonsterol product of mevalonate is involved.
Authors: Larson R A , Chung J , Scanu A M , Yachnin S
Published: May 2006

Human peripheral blood lymphocytes, in the presence of human neutrophils, initiate DNA synthesis and cell cycling when exposed to mevalonic acid. The ability of lymphocytes to respond in this manner is a radiosensitive property of cells, whereas the help provided by neutrophils is maintained despite their exposure to x-irradiation. Other organic acid anions, including precursors of mevalonic acid biosynthesis and a variety of products of mevalonate metabolism, fail to initiate DNA synthesis when added to human lymphocytes. Because only the metabolically active R(--) enantiomer of mevalonic acid initiates lymphocyte DNA synthesis, we presume that physiological pathways of mevalonate metabolism are involved. The response to mevalonic acid of ML-236B (compactin)-inhibited lymphocytes is increased, and the threshold concentration of meyalonate at which lymphocyte DNA synthesis first appears is decreased, when the cells are cultured in lipoprotein-containing (as opposed to lipoprotein-depleted) medium. The response to mevalonic acid of lymphocytes cultured in lipoprotein-depleted medium can be enhanced by addition to the cultures of low density lipoprotein but not by addition of high density lipoprotein. Based upon the flux diversion hypothesis of mevalonate metabolism, these observations suggest that a nonsterol product of mevalonate metabolism may be responsible for the initiation of lymphocyte DNA synthesis by mevalonic acid.
Proton-hydroxide permeability of liposomes.
Authors: Deamer D W , Nichols J W
Published: May 2006

Liposome permeability to net proton-hydroxide flux was compared under two conditions that have produced extreme values in previous studies. In the first [Nichols, J.W. & Deamer, D.W. (1980) Proc. Natl. Acad. Sci. USA 77, 2038-2042] small pH gradients were permitted to decay near pH 7 and permeability coefficients in the range of 10(-4) cm/sec were obtained. In the second [Nozaki, Y. & Tanford, C. (1981) Proc. Natl. Acad. Sci. USA 78, 4324-4328] the decay rate of large pH gradients (approximately equal to 3 pH units) was measured and apparent permeability coefficients in the range of 10(-9) cm/sec were reported. We have repeated the conditions of the latter study and have found that the decay of large pH gradients produces a diffusion potential that limits net proton-hydroxide flux. The permeability coefficients calculated from the flux are therefore underestimated and do not reflect intrinsic proton permeability. With the same liposome preparations under conditions in which small gradients decay so that diffusion potentials are negligible, the measured permeability coefficient is near 10(-4) cm/sec, as reported earlier.
Immunological evidence for the in vivo occurrence of a crosslinked complex of poly(ADP-ribosylated) histone H1.
Authors: Wong M , Kanai Y , Miwa M , Bustin M , Smulson M
Published: May 2006

The poly(ADP-ribosylation) of histones, which occurs within a limited and functionally specific domain of chromatin, is a novel post-translational modification. However, in the past it has been difficult to study this process in living cells because the substrate of the reaction (NAD) does not permeate the plasma membrane. In the current study, antibodies specific for histone H1 and poly(ADP-ribose) were used to study the occurrence of poly(ADP-ribose)+ species of H1 in vivo. Perchloric acid-extracted proteins from synchronously growing HeLa cells were fractionated by electrophoresis and transferred to nitrocellulose, and the transferred moieties were allowed to react with the specific antibodies and then with 125I-labeled protein A. The results conclusively demonstrate the natural occurrence of poly(ADP-ribose)-crosslinked complexes of histone H1 (i.e., H1 dimer), at the S/G2 phase transition of the cell cycle.
Cloning of a human complement component C4 gene.
Authors: Carroll M C , Porter R R
Published: May 2006

Six overlapping cosmid clones having an average insert size of 40 kilobase pairs were identified and isolated from a human genomic library by using a cDNA probe, Alu-7, specific for the amino acid sequence of C4d, a known region of the fourth component of human complement. Analysis of these genomic clones by restriction digestion and Southern blotting shows that all six probably contain the same complete C4 gene. Nucleotide sequence comparison of the genomic clone Cos-A and the cDNA clone Alu-7 shows an identical sequence except for the presence of a 1,500-base-pair intron in the genomic sequence. The amino acid sequence predicted from the nucleotide sequence agrees with the known C4d region amino acid sequence with one exception.
Phorbol diester receptor copurifies with protein kinase C.
Authors: Niedel J E , Kuhn L J , Vandenbark G R
Published: May 2006

The phorbol diester receptor present in the particulate fraction of rat brain was solubilized by divalent ion chelation in the absence of detergents. The soluble receptor was partially purified by (NH4)2SO4 precipitation, DEAE-cellulose, and gel filtration chromatography. The purified receptor required exogenous phospholipid for activity and displayed a Kd of 7 nM for [3H]phorbol 12, 13-dibutyrate. Biologically active phorbol analogs inhibited binding, whereas inactive analogs did not. The Ca2+-dependent, phospholipid-sensitive protein kinase C copurified with the phorbol receptor. Purified protein kinase C was activated directly by phorbol 12-myristate 13-acetate in the presence of phospholipid.
Isolation and spectral characterization of photochemical reaction centers from the thermophilic green bacterium
Chloroflexus aurantiacus
strain J-10-f1
Authors: Pierson Beverly K. , Thornber J. Philip
Published: May 2006

A rapid procedure has been devised to extract photochemically active reaction centers from the green bacterium
Chloroflexus aurantiacus
strain J-10-f1 and a mutant lacking colored carotenoids (73-3). The isolated material was completely free of antenna bacteriochlorophyll and cytochromes and nearly free of carotenoids and had near IR absorption maxima at 865, 815, and 756 nm. On oxidation, the peak at 865 nm was bleached and the remaining two peaks were shifted to 806 and 757 nm. Although these spectral characteristics show general similarities to those in reaction center preparations from purple bacteria, there are some distinct differences. Comparison of the spectra of reaction centers of
Chloroflexus
and
Rhodopseudomonas sphaeroides
, adjusted to the same absorbance at 865 nm, showed that the absorbance at 815 nm in
Chloroflexus
was much less and that at 757 nm was much greater than the equivalent absorbances in
R. sphaeroides
. Unlike reaction centers from
R. sphaeroides
and other photosynthetic bacteria that have two molecules of bacteriopheophytin and four molecules of bacteriochlorophyll per unit, reaction centers from
Chloroflexus
appear to have three molecules of each pigment per unit. A prominent shoulder at 793 nm disappears concomitantly with the bleaching at 865 nm on oxidation of
Chloroflexus
reaction centers; this spectral component may represent the higher energy transition in the near IR of the two bacteriochlorophylls forming P865. While
Chloroflexus
has a light-harvesting pigment system very similar to that of the Chlorobiaceae, its reaction center has optical absorption characteristics similar to those of the Rhodospirillaceae.
Identification of human neurophysins: complete amino acid sequences of MSEL- and VLDV-neurophysins.
Authors: Chauvet M T , Hurpet D , Chauvet J , Acher R
Published: May 2006

Two human neurophysins have been purified from acetone-desiccated posterior pituitaries by acidic extraction, molecular sieving, and ion-exchange chromatography. The complete amino acid sequence of each protein has been determined by using a sequencer and characterizing two sets of overlapping enzymic peptides. The two neurophysins belong to two structural families previously defined as MSEL- and VLDV-neurophysins according to the nature of the residues in positions 2, 3, 6, and 7. (MSEL-neurophysins contain methionine-2, serine-3, glutamic acid-6, and leucine-7; VLDV-neurophysins contain valine-2, leucine-3, aspartic acid-6, and valine-7.) Human MSEL-neurophysin has only 93 residues instead of 95 usually found in MSEL-neurophysins from other mammalian species, probably because of a deletion of amino acids 91 and 92. Compared with bovine MSEL-neurophysin, nine variations (seven substitutions and two deletions) are observed. Human VLDV-neurophysin has 93 residues, as do the other mammalian VLDV-neurophysins. There are 11 substitutions when the comparison is made with bovine VLDV-neurophysin. Between the two human neurophysins, there are 26 variations. However, the central parts of the proteins (residues 10-70) are nearly identical. Furthermore, in this region identical substitutions are found in positions 29 and 60 of both neurophysins, suggesting either a single exon or some relationship between the two corresponding genes.
Evidence for a lipoxygenase mechanism in the biosynthesis of epoxide and dihydroxy leukotrienes from 15(S)-hydroperoxyicosatetraenoic acid by human platelets and porcine leukocytes.
Authors: Maas R L , Brash A R
Published: May 2006

Leukocyte preparations convert the hydroperoxy icosatetraenoic acids 5(S)-HPETE and 15(S)-HPETE to the unstable leukotriene epoxides LTA4 and 14,15-LTA4. In several ways, the conversion of 5- or 15-HPETE to leukotriene epoxide bears a formal mechanistic resemblance to the reaction catalyzed by the 12-lipoxygenase in the conversion of arachidonic acid to 12(S)-HPETE. Points of similarity include enzymatic removal of a hydrogen at carbon 10, double bond isomerization, and formation of a new carbon-to-oxygen bond. In the case of 15(S)-HPETE, two 8,15- and an erythro-14,15-dihydroxy acid (8,15- and 14,15-DiHETEs), which result from incorporation of molecular oxygen into each hydroxyl group, are coproducts in the formation of 14,15-LTA4. These facts prompted us to test the hypothesis that the biosynthesis of 14,15-LTA4 and of 8,15- and 14,15-DiHETEs from 15(S)-HPETE occurs by a mechanism similar to that observed in lipoxygenase reactions. Based on the results presented here, we conclude that the biosynthesis of 14,15-LTA4 and of 8,15- and 14,15-DiHETEs from 15(S)-HPETE occurs via a common intermediate and that, moreover, the formation of these metabolites from 15(S)-HPETE is catalyzed by an enzyme with many mechanistic features in common with the 12-lipoxygenase.
A computer model analysis of the active-site coupling mechanism in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.
Authors: Hackert M L , Oliver R M , Reed L J
Published: May 2006

A computer modeling system developed to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) accompanying release of lipoyl moieties by lipoamidase and by trypsin [Hackert, M.L., Oliver, R.M. & Reed, L.J. (1983) Proc. Natl. Acad. Sci. USA 80, 2226-2230] was used to analyze analogous data for the E. coli pyruvate dehydrogenase complex (PDC). The model studies indicate that the activity of PDC, as found for KGDC, is influenced by redundancies and random processes, which we describe as a multiple random coupling mechanism. In both complexes more than one lipoyl moiety services each pyruvate dehydrogenase (EC 1.2.4.1) or alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) (E1) subunit, and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data for PDC was obtained with a model that has four lipoyl domains with four or, more probably, eight lipoyl moieties servicing each E1 subunit. The lipoyl-lipoyl interaction network for PDC has lipoyl domain interactions similar to those found for KGDC plus the additional possibility of interaction of a lipoyl moiety and its paired mate on each dihydrolipoamide acetyltransferase (EC 2.3.1.12) (E2) subunit. The two lipoyl moieties on an E2 subunit in PDC appear to be functionally indistinguishable, each servicing the acetyltransferase site of that E2 subunit and a dihydrolipoamide dehydrogenase (EC 1.6.4.3) (E3) subunit if the latter is bound to that particular E2 subunit. The observed difference between inactivation of PDC by lipoamidase and by trypsin appears to be due to dead-end competitive inhibition by lipoyl domains that have been modified by excision of lipoyl moieties by lipoamidase.
Ordered and disordered phospholipid domains coexist in membranes containing the calcium pump protein of sarcoplasmic reticulum.
Authors: Lentz B R , Clubb K W , Barrow D A , Meissner G
Published: May 2006

Data are presented that lead to an alternative model for the organization and molecular dynamics of lipid molecules near the Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase; ATP phosphohydrolase, EC 3.6.1.3) of sarcoplasmic reticulum. Measurements of the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in progressively delipidated sarcoplasmic reticulum membranes have been quantitatively interpreted in terms of a layer of lipid of high anisotropy (the lipid annulus) coexisting with lipid layers of very low anisotropy. In addition, the Ca2+-ATPase has been reconstituted into pure 1,2-dipentadecanoyl 3-sn-phosphatidylcholine membranes over a range of lipid-to-protein ratios. High-sensitivity differential scanning calorimetry has demonstrated that roughly 30 lipid molecules per Ca2+-ATPase molecule (annular lipids) fail to undergo a calorimetrically detectable phase transition in the temperature range 4-44 degrees C. Roughly 100 lipid molecules beyond the annulus undergo a detectable phase transition at a temperature below the phase transition of pure lipid and with an enthalpy change [4.2 kcal/mol (1 kcal = 4.18 kJ)] about half that observed for pure lipid vesicles (7.7-7.8 kcal/mol). We propose that both the fluorometric and calorimetric data are consistent with a model in which a motionally inhibited lipid annulus is surrounded by a more extensive region of disrupted lipid packing order, which we have called the secondary lipid domain.
Selective gene expression induced by ecdysterone in cultured fat bodies of Drosophila.
Authors: Nakanishi Y , Garen A
Published: May 2006

Expression of the LSP-2 and P1 genes was induced in cultured fat bodies of Drosophila third-instar larvae by supplementing the culture medium with ecdysterone. The fat bodies were isolated from ecdysterone-deficient larvae of the temperature-sensitive mutant ecd1, which were shifted from the permissive to the restrictive temperature either at the beginning of the third instar for the detection of LSP-2 induction or several hours later for the detection of P1 induction. During normal larval development, the LSP-2 gene is expressed before the P1 gene, and this order is also observed in the cultured fat bodies. Induction was demonstrated by increased amounts of LSP-2 and P1 transcripts in the ecdysterone-supplemented fat bodies. The amount of P1 transcript was determined by two methods: one involved measuring the hybridization of a labeled P1 DNA probe to total fat body RNA; the other involved labeling the newly synthesized RNA in nuclei isolated from cultured fat bodies and measuring the hybridization of the labeled RNA to P1 DNA. Only the second method was used for the LSP-2 transcript because the earlier expression of the LSP-2 gene results in a measurable accumulation of the transcript in the fat bodies before ecdysterone supplementation. The maximal level of P1 induction was reached within 2 hr after supplementation, and the induction was not affected by a concentration of cycloheximide that strongly inhibited total protein synthesis, suggesting that ecdysterone acts directly on the P1 gene rather than indirectly by inducing formation of proteins required for the subsequent induction of P1. Ecdysterone appears to function normally in the cultured fat body system because the LSP-2 and P1 genes are induced in culture in the same order as in vivo and a third gene, G12, that is not induced in fat bodies in vivo also is not induced in culture.
Purine-resistant Drosophila melanogaster result from mutations in the adenine phosphoribosyltransferase structural gene.
Authors: Johnson D H , Friedman T B
Published: May 2006

Mutants of Drosophila melanogaster selected for resistance to purine killing are deficient in adenine phosphoribosyltransferase (APRT; E.C. 2.4.2.7) activity. Genetic mapping and complementation analysis demonstrate that purine resistance, deficiency of APRT activity, and differences in the isoelectric point of APRT result from alterations at a single locus, Aprt (map position, 3:3.03). The level of APRT activity shows gene dose dependence in Aprt heterozygotes and in flies that are haploid for different Aprt alleles. Drosophila APRT is a dimer composed of apparently identical 23,000-dalton subunits. These results suggest that Aprt contains the structural gene for APRT.
Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus.
Authors: Jensen R , Sprague G F , Herskowitz I
Published: May 2006

The ultimate product of yeast mating-type interconversion is a stable a/alpha diploid cell. A haploid cell carrying the HO gene gives rise to a diploid cell in a two-step process: first, the cell switches mating type as a result of genetic rearrangement (cassette substitution) catalyzed by HO; then, cells of opposite type mate to form a/alpha diploids. Mating-type interconversion does not occur in a/alpha diploids despite the presence of the HO gene. We have identified a plasmid carrying the HO gene by screening a yeast clone bank (constructed in vector YEp13) for plasmids that allow mating-type switching by ho cells. The yeast segment responsible for mating-type interconversion integrates by homology at the ho locus, thus confirming that it carries HO. Using the HO gene as a probe, we find that strains with an active mating-type interconversion system produce HO RNA, whereas a/alpha HO/HO cells do not and that this inhibition requires products of both the MATa1 and MATa2 genes. Thus, mating-type interconversion does not occur in a/alpha HO/HO cells because the HO gene product is not synthesized. These results demonstrate the following: (i) The mating-type locus, proposed on genetic grounds to be a regulatory locus, controls expression of an unlinked gene (HO) at the level of RNA production. (ii) The HO gene is under negative feedback control: its expression is inhibited after successful completion of diploidization (formation of a/alpha diploids).
Initiation and replication of vesicular stomatitis virus genome RNA in a cell-free system.
Authors: Peluso R W , Moyer S A
Published: May 2006

A system for studying the in vitro replication of the RNA genomes of both wild-type vesicular stomatitis virus (VSV) and its defective interfering particle MS-T has been developed. After lysolecithin treatment of cells infected with VSV or VSV plus MS-T, a cell-free cytoplasmic extract is prepared which will support VSV mRNA synthesis and the synthesis of the 42S wild-type or 19S MS-T genome RNAs. The genome-length RNAs synthesized in vitro are assembled into RNase-resistant nucleocapsids. The level of 42S RNA synthesis in vitro (6-13% of total RNA synthesis) reflects the level of replication in vivo. Although the extracts of VSV-infected cells can also support the synthesis of VSV proteins, RNA replication is not dependent on de novo protein synthesis but utilizes the preformed soluble proteins present in the infected cell at the time the extract is prepared. The initiation of genomic RNA during in vitro replication can be demonstrated because detergent-disrupted, purified MS-T particles will replicate their RNA when added to either a total cytoplasmic extract from VSV-infected cells or the soluble protein fraction derived from such an extract.
Sequence specificity of actinomycin D and Netropsin binding to pBR322 DNA analyzed by protection from DNase I.
Authors: Lane M J , Dabrowiak J C , Vournakis J N
Published: May 2006

A direct approach to determining the sequence specificities of equilibrium binding drugs by using the DNase protection technique is described. The method utilizes singly end-labeled restriction fragments and partial digestion of the drug fragment complex with DNase I. Microdensitometry of autoradiograms produced after electrophoretic separation of digestion products allows determination of sequences that are affected by drug binding. The feasibility of the technique for locating small ligands bound to DNA and its eventual use as a quantitative thermodynamic approach to studying ligand binding to heterogeneous DNA as a function of sequence is illustrated by using actinomycin D and Netropsin.
Clusters of point mutations are found exclusively around rearranged antibody variable genes.
Authors: Gearhart P J , Bogenhagen D F
Published: May 2006

We have examined the nucleotide sequences of a series of murine antibody genes derived from one kappa light chain gene in order to gain insight into the mechanism that specifically mutates variable genes. Six rearranged VK167 genes from hybridoma and myeloma cells were cloned from bacteriophage lambda libraries. The sequences were compared to the germ-line sequence of the VK167 gene, the JK genes, and the CK gene to identify sites of mutation. Four of six rearranged genes had extensive mutation which occurred exclusively in a 1-kilobase region of DNA centered around the V-J gene. No mutations were found at more distant sites in the intervening sequence or in the constant gene. The frequency of mutation was approximately 0.5% (32 mutations per 6,749 base pairs). Mutations were mostly due to nucleotide substitutions with no preference for transitions or transversions. The location of mutations around each gene indicates that they occur in clusters at random sites. The observation of mutations in the intervening sequence downstream from the JK5 gene rules out models for the mechanism of mutagenesis that rely solely on gene conversion or recombination. The distribution and high frequency of mutations are most easily explained by a mechanism of error-prone repair that occurs during several cycles of cell division.
Regulation of low density lipoprotein receptors by plasma lipoproteins from patients with abetalipoproteinemia.
Authors: Illingworth D R , Alam N A , Sundberg E E , Hagemenas F C , Layman D L
Published: May 2006

Despite an absence of low density lipoproteins (LDLs) and chylomicron remnants from plasma, the rates of cholesterol synthesis or the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased. These observations suggest that other lipoprotein particles present in the plasma of patients with abetalipoproteinemia may regulate LDL receptor activity and the rates of cellular cholesterol synthesis in this disorder. In the present report we have studied the effects of lipoprotein fractions from the plasma of normal subjects, patients with abetalipoproteinemia, and a patient with dysbetalipoproteinemia on the binding, internalization, and degradation of 125I-labeled LDL (125I-LDL) by cultured human fibroblasts. LDL from normal subjects or the high density lipoprotein fraction HDL2 from the plasma of patients with abetalipoproteinemia effectively down-regulated LDL receptor activity (greater than 50% inhibition at 20 micrograms of protein per ml). HDL2 from the plasma of patients with abetalipoproteinemia also effectively reduced the binding, internalization, and degradation of 125I-LDL by cultured human fibroblasts. 125I-HDL2 from the plasma of patients with abetalipoproteinemia was bound, internalized, and degraded by cultured human fibroblasts; this process was competitively inhibited by unlabeled normal LDL or HDL2 from abetalipoproteinemic plasma and was 1/6th to 1/8th times as high when 125I-HDL2 was incubated with fibroblasts from a patient with receptor-negative homozygous familial hypercholesterolemia. We conclude that lipoproteins present in the HDL2 fraction of plasma from patients with abetalipoproteinemia (which are relatively rich in apoprotein E) are effective regulators of LDL receptor activity in normal human fibroblasts. These in vitro findings may explain why the in vivo rates of cholesterol synthesis and the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased.
ATP stimulates proteolysis in reticulocyte extracts by repressing an endogenous protease inhibitor.
Authors: Speiser S , Etlinger J D
Published: May 2006

An endogenous inhibitor of the reticulocyte ATP-dependent proteolytic system has been purified partially by ammonium sulfate precipitation from rabbit reticulocyte and erythrocyte extracts. Inhibitor-free protease rapidly degrades 21-40% of the substrate [14C]methyl-alpha-casein per hour, resembling ATP-dependent activity in reticulocyte extracts. This proteolytic activity is not stimulated by ATP and does not respond to ubiquitin. Adding back the inhibitory fraction to reticulocyte inhibitor-free protease results in a significant decrease (65-75%) in proteolysis, both in the presence and absence of ATP. In contrast, inhibition is repressed when both ATP and the ubiquitin-containing fraction are present, resulting in an 80-350% stimulation of proteolysis by these components. These results suggest that ATP, in the presence of ubiquitin, may act in releasing the protease(s) from its endogenous inhibitor. Erythrocyte extracts, unlike reticulocyte extracts, exhibit low levels of ATP-dependent proteolytic activity. However, ion-exchange chromatography reveals that erythrocytes contain levels of proteolytic activity that are comparable to the reticulocyte's inhibitor-free protease. Addition of ubiquitin and inhibitor to erythrocyte protease results in a highly ATP-dependent activity that resembles levels of ATP-dependence (3- to 4-fold) seen in reticulocyte extracts. Thus, the proteolytic and inhibitory components of the ATP-dependent proteolytic system appear to be retained with reticulocyte maturation. However, some other component(s) of the system are lost or modified with maturation so that the protease remains inactive.
Purification and initial characterization of a type beta transforming growth factor from human placenta.
Authors: Frolik C A , Dart L L , Meyers C A , Smith D M , Sporn M B
Published: May 2006

A polypeptide transforming growth factor (TGF) that induces anchorage-dependent rat kidney fibroblasts to grow in soft agar has been isolated from human placenta and purified to homogeneity. This polypeptide is classified as a type beta TGF because it does not compete with epidermal growth factor (EGF) for membrane receptor sites but does require EGF for induction of anchorage-independent growth of indicator cells. Purification of this peptide was achieved by acid/ethanol extraction of the placenta, followed by gel filtration, cation exchange, and HPLC of the acid-soluble proteins. Homogeneity of the TGF-beta from the final column was shown by its constant specific activity and amino acid composition across the peak of soft agar colony-forming activity and by its migration as a single band at Mr 23,000-25,000 on NaDodSO4/polyacrylamide gel electrophoresis. Under reducing conditions, the protein migrated on a gel as a single band at Mr 13,000. The purified placental TGF-beta caused half-maximal growth stimulation of indicator cells in soft agar at 64-72 pg/ml (3 pM) in the presence of EGF at 2 ng/ml (0.34 nM).
Ferredoxin/flavoprotein-linked pathway for the reduction of thioredoxin.
Authors: Hammel K E , Cornwell K L , Buchanan B B
Published: May 2006

Thioredoxins are small redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of important biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Reduced ferredoxin is known to serve as the source of reducing power for the reduction of thioredoxins only in photosynthetic cells that evolve oxygen. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction is considered to be NADPH. We now report evidence that Clostridium pasteurianum, an anaerobic bacterium normally living in the soil unexposed to light, resembles photosynthetic cells in that it uses reduced ferredoxin as the reductant for thioredoxin. Moreover, the transfer of electrons from reduced ferredoxin to thioredoxin is catalyzed by a flavoprotein enzyme that has not been detected in other organisms. Our results reveal the existence of a pathway for the reduction of thioredoxin in which ferredoxin, reduced fermentatively either by molecular hydrogen or by a carbon substrate, provides the reducing power for the flavoprotein enzyme ferredoxin-thioredoxin reductase, which in turn reduces thioredoxin.
Thermodynamics and kinetics of protein incorporation into membranes.
Authors: Jähnig F
Published: May 2006

The free energy and enthalpy of protein incorporation into membranes are calculated with special emphasis on the hitherto neglected effects of immobilization of protein and perturbation of lipid order in the membrane. The free energy change is found to be determined by the hydrophobic effect as the driving force for incorporation and the protein immobilization effect which leads to a considerable reduction of the free energy gained from the hydrophobic effect. For incorporation of a hydrophobic, bilayer-spanning alpha-helix, the free energy change obtained is of the order of -15 kcal/mol (1 cal = 4.184 J) in agreement with experimental results. The lipid perturbation effect yields only a small contribution to the free energy change due to an energy/entropy compensation inherent in lipid order. This effect dominates the enthalpy change, giving rise to values on the order of 100 kcal/mol with a pronounced temperature dependence around the lipid phase transition as observed experimentally. The kinetics of protein incorporation are even more strongly affected by the lipid perturbation effect, leading to an abrupt decrease of the rate of incorporation below the lipid phase transition.
Increase of proenkephalin mRNA and enkephalin content of rat striatum after daily injection of haloperidol for 2 to 3 weeks.
Authors: Tang F , Costa E , Schwartz J P
Published: May 2006

Proenkephalin mRNA has been detected in striatum, hypothalamus, cortex, cerebellum, hippocampus, midbrain, and brain stem of rat by RNA ("Northern") blot analysis using a 918-base-pair DNA hybridization probe complementary to proenkephalin mRNA [Comb, M., Seeburg, P. H., Adelman, J., Eiden, L. & Herbert, E. (1982) Nature (London) 295, 663-666]. The size of the mRNA species in all brain regions is approximately 1,400 bases, and it was found to be comparable with that of bovine adrenal medulla and human pheochromocytoma. The data were quantified by densitometric scanning of the autoradiograms: the area under the peak is proportional to the amount of standard proenkephalin mRNA from bovine adrenal medulla. The relative content of proenkephalin mRNA in the various brain regions correlates generally with the content of [Met5]enkephalin-like immunoactivity of these regions. Rats receiving daily intraperitoneal injection of haloperidol (1 or 2 mg/kg) show a fourfold increase of proenkephalin mRNA content in striatum but not other brain regions. In agreement with previous reports, [Met5]enkephalin-like immunoactivity increased twofold in striatum.
Sequence dependence of base-pair stacking in right-handed DNA in solution: proton nuclear Overhauser effect NMR measurements.
Authors: Patel D J , Kozlowski S A , Bhatt R
Published: May 2006

Single-crystal x-ray studies of d(C-G-C-G-A-A-T-T-C-G-C-G) exhibit base-pair propeller twisting [Dickerson, R. E. & Drew, H. R. (1981) J. Mol. Biol. 149, 761-786] that results in close contacts between adjacent purines in the minor groove in pyrimidine (3'-5')-purine steps and in the major groove in purine (3'-5')-pyrimidine steps [Calladine, C. R. (1982) J. Mol. Biol. 161, 343-362]. These observations require an approximately 3.4 A separation between the minor groove edges of adenosines on adjacent base pairs for the dA-dA step but predict a smaller separation for the dT-dA step and a larger separation for the dA-dT step in a D(A-T-T-A).d(T-A-A-T) fragment. We have confirmed these predictions from steady-state nuclear Overhauser effect measurements between assigned minor groove adenosine H-2 protons on adjacent base pairs in the proton NMR spectrum of the d(C1-G2-A3-T4-T5-A6-T6-A5-A4-T3-C2-G1) self-complementary dodecanucleotide duplex (henceforth called the Pribnow 12-mer) in solution. The measured cross-relaxation rates (product of steady-state nuclear Overhauser effect and selective spin- lattice relaxation rates) translate to interproton separations between adjacent adenosine H-2 protons of 4.22 A in the (dA3-dT4).(dA4-dT3) step, of 3.56 A in the (dT4-dT5).dA5-dA4) step, and of 3.17 A in the (dT5-dA6).(dT6-dA5) step for the Pribnow 12-mer duplex with an isotropic rotational correlation time of 9 ns at 5 degrees C. These proton NMR results show that the sequence-dependent base-pair stacking resulting from base-pair propeller twisting of defined handedness for right-handed DNA in the solid state is maintained in aqueous solution.
Subfractionation of rat liver Golgi apparatus: separation of enzyme activities involved in the biosynthesis of the phosphomannosyl recognition marker in lysosomal enzymes.
Authors: Deutscher S L , Creek K E , Merion M , Hirschberg C B
Published: May 2006

A highly purified Golgi apparatus preparation from rat liver was subfractionated on a Percoll gradient into two major protein peaks of similar size that migrated at densities of 1.028 and 1.051 g/ml. The lighter protein peak contained 70--80% of the total activities of the oligosaccharide-processing enzymes alpha-1,2-mannosidase and mannosidase II and of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyl-1-phosphotransferase (alpha-N-acetylglucosaminylphosphotransferase), an enzyme involved in the biosynthesis of the mannose 6-phosphate recognition marker of lysosomal enzymes. These enzyme activities were enriched 2-fold in specific activity over that of the heavy protein peak. In contrast, 80% of the alpha-N-acetylglucosaminylphosphodiesterase, an enzyme that exposes 6-phosphomonoesters of mannose on the oligosaccharide chains of lysosomal enzymes, migrated in a region of slightly higher density than did the protein peak of density 1.051 g/ml. Sialyltransferase (SiaTase) and galactosyltransferase (Gal-Tase) activities distributed almost equally among the two protein peaks. Controls rule out that the two protein peaks were the result of aggregation/deaggregation and that enzyme activities were altered by Percoll per se. Lysosomal enzyme activities migrated in a region essentially devoid of Golgi apparatus-associated enzyme activities. These results suggest a physical separation within the Golgi apparatus of some of the enzymes involved in the biosynthesis and processing of the oligosaccharides on glycoproteins, including those responsible for the formation of the mannose 6-phosphate recognition marker on lysosomal enzymes.
Monoclonal antibodies specific for Hantaan virus.
Authors: Franko M C , Gibbs C J , Lee P W , Gajdusek D C
Published: May 2006

Six hybridoma cell line producing monoclonal antibodies to Hantaan virus were established by fusion of NS-1 mouse myeloma cells with spleen cells of mice immunized with Hantaan virus strain 76-118. The specificity of these monoclonal antibodies was established by immunoblotting analysis and immunofluorescence. Five of the clones reacted with antigens on the cell surface and in the cytoplasm, and one clone reacted with a determinant expressed only in the cytoplasm of the infected cells. Two of the clones produced antibodies that reacted with a Mr 50,000 polypeptide in virus-infected cellular extracts and purified virus preparations. The monoclonal antibodies were used to examine the antigenic relationship among Hantaan virus strains and between Hantaan virus and Prospect Hill virus and the virus of nephropathia epidemica. Three antibodies were capable of distinguishing between the Lee strain and the 760-118 strain of Hantaan virus and three additional antibodies reacted with determinants shared by both virus strains. None of the six reacted with Prospect Hill virus or the virus of nephropathia epidemica.
Topography of transcription: path of the leading end of nascent RNA through the Escherichia coli transcription complex.
Authors: Hanna M M , Meares C F
Published: May 2006

A cleavable dinucleotide photoaffinity reagent was prepared and used to map the path of the leading end of the RNA transcript across the surface of Escherichia coli RNA polymerase/T7 DNA transcription complexes. By using 5'-(4-azidophenacylthio)phosphoryladenylyl(3'-5')uridine, transcription was specifically initiated at the A1 promoter of bacteriophage T7 D111 or D123 DNA. Transcription complexes containing radiolabeled RNA chains of various lengths (4-116 nucleotides) were prepared, and the 5' end of the RNA transcript was then covalently attached to the nearby polymerase subunits or DNA by irradiation with UV light. The photoaffinity-labeled enzyme subunits and DNA were separated, the radiolabeled RNAs were cleaved from each, and the lengths and sequences of RNA attached to each component were determined. The leading end of RNA chains up to 12 bases long was found to label the DNA and the beta and beta' subunits of RNA polymerase, with more than 90% of the label going to the DNA. When the RNA transcript reached 12 bases in length, the 5' end diverged from the DNA and only the beta and beta' enzyme subunits were labeled thereafter. These two subunits were heavily labeled by RNA chains 12 to as many as 94 bases long. No significant labeling of the alpha subunit occurred. The sigma subunit was not labeled by RNAs longer than the trinucleotide.
Molecular cloning of the cDNA coding for rat ornithine transcarbamoylase.
Authors: Horwich A L , Kraus J P , Williams K , Kalousek F , Konigsberg W , Rosenberg L E
Published: May 2006

Ornithine transcarbamoylase is a mitochondrial matrix enzyme composed of three identical subunits encoded on the X chromosome. The subunit is synthesized on cytoplasmic polysomes as a precursor that is cleaved during transport into mitochondria. We report here the isolation and characterization of cDNA clones containing sequences corresponding to the mRNA encoding the ornithine transcarbamoylase subunit. cDNA was synthesized using rat liver mRNA enriched by polysome immunoadsorption for the low-abundance messenger species encoding the enzyme subunit. After insertion of cDNA into plasmid pBR322 and cloning in Escherichia coli, identification of the desired plasmids was accomplished by (i) differential colony hybridization using cDNA probes synthesized from mRNA of various tissues; (ii) differential blot hybridization using cDNA probes synthesized from mRNA enriched for or depleted of the ornithine transcarbamoylase message; (iii) hybrid-selected translation assays; and (iv) most definitively, structural analysis, which matched 25 consecutive amino acid residues determined by sequential Edman analysis of the carboxyl-terminal portion of the purified enzyme subunit with coding sequence present in the insert of one of the plasmids.
Evidence for the general base mechanism in carboxypeptidase A-catalyzed reactions: partitioning studies on nucleophiles and H2(18)O kinetic isotope effects.
Authors: Breslow R , Chin J , Hilvert D , Trainor G
Published: May 2006

Methanol does not detectably compete with water in carboxypeptidase-catalyzed cleavage of any substrate, although it is preferentially reactive in a model for the proposed nucleophilic mechanism for the enzyme that involves an anhydride intermediate. To test for such a common intermediate in the cleavage of related peptide and ester substrates, a method has been developed to examine H2(16)O-H2(18)O kinetic isotope-partitioning effects. The finding that benzoylglycylphenylalanine has an isotope effect of 1.019 +/- 0.002 while benzoylglycyl-beta-L-phenyl-lactate shows a small inverse isotope effect excludes most versions of a nucleophilic mechanism having a common anhydride intermediate. The bulk of the available evidence strongly favors the previously proposed general base mechanism.
Self-adjointness of the Fourier expansion of quantized interaction field Lagrangians
Authors: Paneitz S. M. , Segal I. E.
Published: May 2006

Regularity properties significantly stronger than were previously known are developed for four-dimensional non-linear conformally invariant quantized fields. The Fourier coefficients of the interaction Lagrangian in the interaction representation—i.e., evaluated after substitution of the associated quantized free field—is a densely defined operator on the associated free field Hilbert space K. These Fourier coefficients are with respect to a natural basis in the universal cosmos ˜M, to which such fields canonically and maximally extend from Minkowski space-time M
0
, which is covariantly a submanifold of ˜M. However, conformally invariant free fields over M
0
and ˜M are canonically identifiable. The
k
th Fourier coefficient of the interaction Lagrangian has domain inclusive of all vectors in K to which arbitrary powers of the free hamiltonian in ˜M are applicable. Its adjoint in the rigorous Hilbert space sense is

a
-k

in the case of a hermitian Lagrangian. In particular (
k
= 0) the leading term in the perturbative expansion of the
S
-matrix for a conformally invariant quantized field in M
0
is a self-adjoint operator. Thus, e.g., if ϕ(
x
) denotes the free massless neutral scalar field in M
0
, then ∫M
0
:ϕ(
x
)
4
:
d
4
x
is a self-adjoint operator. No coupling constant renormalization is involved here.
Alpha-pyridine nucleotides as substrates for a plasmid-specified dihydrofolate reductase.
Authors: Smith S L , Burchall J J
Published: May 2006

The alpha epimers of pyridine nucleotides are almost totally inactive as reductants in dehydrogenase reactions. In contrast, the R plasmid R67-specified dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) isolated from trimethoprim-resistant Escherichia coli utilized alpha-NADPH and alpha-NADH in addition to the "normal" beta-epimers. The enzymes from bacterial and mammalian sources used only beta-NADPH and beta-NADH. THe Km value for alpha-NADPH (16 microM) was 4-fold greater than that for beta-NADPH (4 microM), while the maximal velocity of the alpha-NADPH-catalyzed reaction was 70% of that seen with the beta-NADPH. beta-NADP+ and alpha-NADP+ were competitive inhibitors of the R67 enzyme. Pyridine nucleotide analogues such as deamino- and acetyl-NADPH were used readily by bacterial, plasmid, and mammalian enzymes, whereas thio-NADPH was used only by the plasmid enzyme. These data suggest that the enzyme from R plasmid R67 possesses a pyridine nucleotide binding site different from that of other dihydrofolate reductases and dehydrogenases.
Tubulin genes of Trypanosoma brucei: a tightly clustered family of alternating genes.
Authors: Seebeck T , Whittaker P A , Imboden M A , Hardman N , Braun R
Published: May 2006

African trypanosomes are the causative agents of many medically and economically important diseases of man and domestic animals. The cell body of these blood-dwelling protozoa is enveloped with a dense layer of pellicular microtubules, which confer both motility and mechanical stability on these cells; microtubules are also important components of the flagellum. The major structural components of the microtubuli are two related proteins, alpha- and beta-tubulin. We have analyzed the genomic organization of alpha- and beta-tubulin genes in Trypanosoma brucei. In this organism, the majority of these genes are arranged in a single, tightly packed cluster of alternating alpha- and beta-tubulin genes with a basic repeat length together of 3.6 kilobase pairs. A genomic library of T. brucei was constructed by using the phage vector A 1059, and recombinant phages carrying tubulin genes were isolated by screening the library with heterologous chicken tubulin cDNA probes. The results of restriction endonuclease and hybridization analysis of DNA isolated from recombinant phages, and subcloned fragments thereof, were compatible with the restriction maps derived from digestion and Southern blot hybridization of genomic DNA.