bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetylcholine receptor channel ionic selectivity: ions experience an aqueous environment.
Authors: Lewis C A , Stevens C F
Published: May 2006

Alkali metal and alkaline earth cations pass readily through the acetylcholine receptor channel. Monovalent cations with larger crystal radii are more permeant than ones with smaller radii. For divalent ions, this selectivity sequence is reversed: smaller ions are more permeant than larger ones. This reversal in selectivity sequence with change of valence from 1 to 2 can be naturally accounted for by electrostatic interactions between the ion and its environment in the selectivity region of the channel. For monovalent ions, ion-dipole interactions dominate, and, for divalent ions, ion-induced dipole interactions are more important. The sign of these two types of effects is opposite and produces the reversal in the selectivity sequence. The magnitude of electrostatic interactions can be estimated from experimental data and suggests that the permeating ion's environment in the selectivity region of the channel is essentially like that in free water.
Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA
Authors: Akiyoshi D. E. , Morris R. O. , Hinz R. , Mischke Barbara Sue , Kosuge T. , Garfinkel D. J. , Gordon M. P. , Nester E. W.
Published: May 2006

Insertion of the transposon Tn
5
into the T-region of the octopine Ti plasmid of
Agrobacterium tumefaciens
gives rise to crown gall tumors having altered morphology. Three loci within the T-DNA that control tumor morphology have been detected [Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P. & Nester, E. W. (1981)
Cell
27, 143-153]. They influence tumor size (
tml
), production of roots (
tmr
), or production of shoots (
tms
). Cytokinin and auxin levels in such mutant tumors were examined by HPLC/radioimmunoassay and HPLC/fluorescence assay, respectively. Free indoleacetic acid levels (in pmol/g) were: uninfected tobacco stem tissues, 128; wild-type A348 tumors, 295;
tml
mutant tumors, 307;
tmr
mutant tumors, 129; and
tms
mutant tumors, 70. Average
trans
-ribosylzeatin levels were correspondingly: 0.97, 48, 40, 0.54, and 1,400 pmol/g.
trans
-Ribosylzeatin/indoleacetic acid ratios were as high as 24 in shoot-producing tumors and as low as 0.003 in root-producing tumors. The evidence strongly suggests that tumor phytohormone levels are determined by genes in the T-DNA.
Correct transcription of a cloned mouse immunoglobulin gene in vivo.
Authors: Picard D , Schaffner W
Published: May 2006

We have obtained correct transcripts from the mouse immunoglobulin lambda I light chain gene on transfection into human HeLa cells. Linkage to simian virus 40 (SV40) DNA containing a transcriptional "enhancer" element was required to raise lambda-chain gene transcription to a detectable level in our transient-expression assay. The transcripts had the same 5' end as authentic lambda I mRNA when the SV40 enhancer element was 150 base pairs upstream from the cap site. The situation was different when the lambda-chain gene promoter was separated from the SV40 sequences by more than 1 kilobase pair of spacer DNA; then, lambda-chain gene transcripts were not correctly initiated in human HeLa, monkey CV-1, and mouse 3T6 cells. In this respect, the lambda-chain gene behaves differently from the rabbit beta-globin gene, which can be activated by the SV40 enhancer over distances of several kilobases.
Isolation and characterization of the human apolipoprotein A-I gene.
Authors: Karathanasis S K , Zannis V I , Breslow J L
Published: May 2006

We have recently shown that an inherited polymorphism occurring in the human apolipoprotein A-I (apo A-I) gene is related to decreased high density lipoprotein and apo A-I levels in the plasma of two patients with severe premature atherosclerosis. Analysis of the molecular basis of this polymorphism and its possible effects on apo A-I gene expression requires direct comparison of both normal and polymorphic apo A-I alleles. Here we report the isolation and characterization of the normal human apo A-I gene and we show that the gene is interrupted by three intervening sequences, IVS-1, IVS-2, and IVS-3, occurring in the 5' noncoding region of apo A-I mRNA, the mRNA sequence coding for the signal peptide of apo A-I, and the sequence coding for the mature protein, respectively. In addition, the nucleotide sequence analysis of the apo A-I gene allowed determination of the complete amino acid sequence of the primary translation product of apo A-I mRNA. This amino acid sequence consists of 267 residues including a 24-residue-long amino-terminal extension (preprosegment). Finally, we show that the apo A-I gene contains six 66-base-pair-long tandemly repeated DNA segments, which suggests that the gene may have evolved by intragenic duplication events.
Structural similarities between viroids and transposable genetic elements.
Authors: Kiefer M C , Owens R A , Diener T O
Published: May 2006

The primary structures of the tomato planta macho and tomato apical stunt viroids have been determined, and probable secondary structures are proposed. Both viroids can assume the rodlike conformation with extensive base-pairing characteristic of all known viroids. Sequence homologies between the two viroids (75%) and with members of the potato spindle tuber viroid group (73-83%) indicate that they both belong to this group. Comparative sequence analysis of all members of the group reveals striking similarities with the ends of transposable genetic elements. These similarities, the presence of inverted repeats often ending with the dinucleotides U-G and C-A, and flanking imperfect direct repeats suggest that viroids may have originated from transposable elements or retroviral proviruses by deletion of interior portions of the viral (or element) DNA.
Chromophore structure of the physiologically active form (P
fr
) of phytochrome
Authors: Rüdiger W. , Thümmler F. , Cmiel E. , Schneider S.
Published: May 2006

Chromopeptides were prepared by proteolytic digestion of phytochrome (far-red absorbing form, P
fr
) and of phycocyanin. The phycocyanobilin peptide, the chromophore of which is
Z,Z,Z
-configurated, was modified to the
Z,Z,E
isomeric chromophore. It has been demonstrated earlier that the P
fr
chromopeptide and the
Z,Z,E
-configurated phycocyanin chromopeptide behave similarly with regard to spectral and chromatographic properties and reactivity. We present evidence here, obtained by high-resolution
1
H NMR spectroscopy, that both the modified phycocyanobilin chromophore and the phytochrome chromophore obtained directly from P
fr
are 15
E
-configurated.
Rapid recovery of chloroplast mutations affecting ribulosebisphosphate carboxylase/oxygenase in
Chlamydomonas reinhardtii
Authors: Spreitzer Robert J. , Ogren William L.
Published: May 2006

Based on the unique ability of chloroplast genes to recombine in
Chlamydomonas reinhardtii
, a collection of acetate-requiring mutants was screened for recombination with a mutation affecting ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu-1,5-
P
2
carboxylase/oxygenase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39]. This chloroplast mutation,
rcl-u
-1-10-6C, causes the absence of Rbu-1,5-
P
2
carboxylase/oxygenase activities and alters the isoelectric point of the larger subunit. Several mutants that displayed little or no recombination with 10-6C were recovered, and two lacked carboxylase activity. These new chloroplast mutants lack both large and small Rbu-1,5-
P
2
carboxylase/oxygenase subunits. The approach demonstrated here permits the routine recovery of chloroplast mutations affecting this enzyme. Multiple mutations in the Rbu-1,5-
P
2
carboxylase/oxygenase large-subunit gene can be used to investigate the function and regulation of this enzyme and the regulation of chloroplast genes in general.
Inhibition of muscle differentiation by trypanosoma cruzi.
Authors: Rowin K S , Tanowitz H B , Wittner M , Nguyen H T , Nadal-Ginard B
Published: May 2006

L6E9 rat myoblasts were infected in tissue culture with the myotropic Brazil strain of Trypanosoma cruzi. The effect of parasite infection on the ability of myoblasts to differentiate into myotubes was studied. Both morphological and biochemical differentiation were found to be profoundly affected by parasitic infection in a dose-related fashion. Evidence is presented to suggest that infected myoblasts can no longer differentiate. Differentiation, once underway, seemed unaffected by the parasitic infection; biochemical markers of differentiation remained intact.
Genomic cloning and preliminary characterization of the human thymidine kinase gene.
Authors: Lin P F , Zhao S Y , Ruddle F H
Published: May 2006

In this report, we describe the cloning of the human cytoplasmic thymidine kinase (tk-C; EC 2.7.1.21) gene and its preliminary characterization. The tk-C sequences were isolated from a phage genomic library made from DNA of a transfected mouse cell carrying the human tk-C gene. The human transforming sequences were identified by homology with human Alu sequences. Six recombinant phages were isolated and five were competent to transfer human TK-C activity to TK-deficient mouse cells when transferred in pairs. Conclusively, sequences homologous to these clones are present in all human TK+ transformants examined. We estimate the maximal size of the tk-C gene to be 14 kilobase pairs and its minimal size to be between 4 and 5 kilobase pairs. The gene contains many noncoding inserts and numerous Alu sequences.
Kpn I family of long-dispersed repeated DNA sequences of man: evidence for entry into genomic DNA of DNA copies of poly(A)-terminated Kpn I RNAs.
Authors: DiGiovanni L , Haynes S R , Misra R , Jelinek W R
Published: May 2006

We have isolated eight cDNA clones complementary to the human Kpn I repeat and determined the base sequence of three. We have also determined a portion of the base sequences of three human Kpn I family members. The three cDNA sequences are extensively homologous with the 3' ends of the three genomic Kpn I family members and with a simian Kpn I family member recently described [Thayer, R. E. & Singer, M. F. (1983) Mol. Cell. Biol. 6, 967-973]. The genomic repeats terminate in regions of sequence rich in dAMP residues close to sequences at the 3' ends of the cDNA clones; a precise 3'-terminal nucleotide cannot be distinguished. These structural features are consistent with the dispersal of at least some Kpn I family members by entry into genomic DNA of copies of Kpn I RNA transcripts. Each cDNA contains a long poly(dAMP) homopolymer at its 3' end and either one or two A-A-T-A-A-A polyadenylylation signal sequences upstream from it, suggesting that Kpn I family members may be transcribed by RNA polymerase II.
Identification of a transposon Tn3 sequence required for transposition immunity.
Authors: Lee C H , Bhagwat A , Heffron F
Published: May 2006

A plasmid containing transposon Tn3 is immune to further insertions of Tn3. This phenomenon works in cis and is referred to as transposition immunity. We have used the ability of Tn3 to form cointegrates between two plasmids to develop a quantitative assay to detect transposition immunity. Presence of Tn3 on both the plasmids reduces the cointegration frequency to less than 1/100 of parental. Using this assay, we have determined that (i) tnpR is not required for immunity, (ii) only the terminal 38 base pairs of Tn3 need be present to confer immunity, and (iii) other parts of Tn3 appear not to confer immunity.
ATP-dependent protein kinase-catalyzed phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the phosphotransferase system in Streptococcus pyogenes.
Authors: Deutscher J , Saier M H
Published: May 2006

HPr, a phosphate carrier protein of the streptococcal phosphotransferase system, is phosphorylated at the N-1 position of a single histidyl residue in a reaction requiring phosphoenolpyruvate (P-ePrv), Mg2+, and enzyme I (P-ePrv-HPr phosphotransferase, EC2.7.3.9). We demonstrate that in addition to this reaction, a seryl residue within HPr can be phosphorylated in an ATP-dependent process. This reaction is catalyzed by a protein kinase with an approximate Mr of 20,000. In whole cells the kinase activity is stimulated by glucose, whereas in crude extracts the activity is stimulated by glycolytic intermediates such as glucose 6-phosphate, fructose 1,6-diphosphate, and 2-phosphoglycerate. P-(Ser)-HPr cannot transfer its phosphate group via enzyme II to a sugar as does the P-(His)-HPr. Instead, a phosphatase (Mr = 70,000) was found to hydrolyze the phosphate group of P-(Ser)-HPr. The phosphatase reaction is strongly inhibited by the addition of P-ePrv and enzyme I. Protein kinase-catalyzed phosphorylation of the enzyme constituents of the phosphotransferase system in Escherichia coli has also been demonstrated. These observations lead us to suggest that phosphorylation of a seryl residue in HPr is involved in the regulation of sugar transport in the bacteria cell.
Structural similarities between the development-specific protein S from a gram-negative bacterium, Myxococcus xanthus, and calmodulin.
Authors: Inouye S , Franceschini T , Inouye M
Published: May 2006

During differentiation of Myxococcus xanthus, a large amount of protein S is produced and assembled on the surface of the myxospore by a process that specifically requires Ca2+. The gene for protein S has been cloned, and two tandemly repeated homologous genes have been found to be within a short distance of each other in the M. xanthus chromosome. We determined the DNA sequence of 3,692 bp encompassing both genes and deduced the amino acid sequences of the two gene products. The gene 1 (upstream) product and the gene 2 (downstream) product show extensive amino acid sequence homology (88%). However, from their structures, protein S was found to be produced from gene 2, indicating that gene 2 is specifically turned on during differentiation. The structure of protein S shows striking similarities with calmodulin: protein S is composed of four internally homologous domains. In particular, the first and the third domains, consisting of 38 residues each, show a high level of homology (79%), and the second and the fourth domains, consisting of 40 residues each, show homology of 65%. In the first and the third domains, there is a common sequence of nine residues, Glu (or Asp)-Asn-Asn-Thr-Ile-Ser-Ser-Val-Lys, which is highly homologous to one of the proposed Ca2+-binding sequences in bovine brain calmodulin, Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys.
Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.
Authors: Germino J , Gray J G , Charbonneau H , Vanaman T , Bastia D
Published: May 2006

The initiation of DNA replication of plasmid R6K is triggered by a 35-kilodalton initiator protein. The initiator protein had been elusive because of its lability and the lack of a convenient assay procedure to aid its purification. Using recombinant DNA techniques, we have fused the cistron of the initiator near its COOH-terminal end, in the correct reading frame, to the lacZ cistron of Escherichia coli at the ninth codon from the NH2 terminus. The fused cistron yielded a protein that was not only stable in vivo but also had dual activities: initiation of DNA replication in vivo and in vitro and hydrolysis of beta-galactoside. Using an affinity column that is specific for beta-galactosidase, we have demonstrated the rapid purification of the hybrid protein to near homogeneity. Exploiting the polymeric structure of the initiator, we have also isolated the nonfused form of the initiator protein, associated through subunit interaction with the beta-galactosidase-fused protein, which permits its purification by affinity chromatography. NH2-terminal amino acid sequence analysis of the heteropolymer has not only shown that the fused and nonfused initiators have the same sequence but also confirmed the protein sequence of the initiator as predicted from its nucleotide sequence. The techniques described here should be generally useful for the isolation of other proteins that are difficult to purify by conventional procedures.
Partial cDNA sequence to a hamster gene corrects defect in Escherichia coli pyrB mutant.
Authors: Davidson J N , Niswander L A
Published: May 2006

The first three enzymes of pyrimidine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyl-transferase, and dihydro-orotase) are carried on a multifunctional protein in mammalian cells and are on separate proteins in bacteria. A plasmid containing a cDNA sequence corresponding to 80% of a hamster mRNA for this protein was transformed into Escherichia coli mutants lacking aspartate carbamoyltransferase (pyrB) or dihydro-orotase (pyrC). Only pyrB transformants were able to grow in the absence of uracil. Plasmid recovered from primary transformants was similar in size to the original plasmid and could yield prototrophs after secondary transformation of E. coli pyrB mutants. When cell extracts were prepared from pyrB transformants, high levels of aspartate carbamoyltransferase activity were found, and the enzyme had properties identical to the mammalian enzyme, including lack of allosteric regulation, precipitation by antiserum specific to the hamster multifunctional protein, and presence of a strong aggregation center. These results demonstrate that (i) a partial hamster protein can complement E. coli defective in pyrimidine biosynthesis, (ii) the order of the enzyme domains of the multifunctional protein is likely to be NH2-dihydro-orotase-carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-COOH, and (iii) the enzyme domains appear to be self-contained at the DNA and protein levels. The protocol described here may be a general means for studying the domains of multifunctional proteins and for isolating other mammalian genes for which bacterial mutants have been prepared. It also permits study of the structure and function of the same gene in both prokaryotic and eukaryotic cells and may provide new insight into the evolution of complex genes.
Use of a monoclonal antibody as a substrate for mature neurons in vitro.
Authors: MacLeish P R , Barnstable C J , Townes-Anderson E
Published: May 2006

A mouse monoclonal antibody was produced against salamander retinal membranes. It binds to the retina as well as to a wide variety of other salamander tissues and is called Sal-1. Because retinal neurons dissociated from the mature salamander retina adhere poorly to standard substrates, cells were plated onto coverslips previously treated with Sal-1. This previous treatment resulted in a dramatic increase in cell-substrate adhesion. At low concentrations, the antibody had no detectable effect on the light response and fine structure of freshly dissociated rod cells. After several weeks in culture, retinal neurons continued to be attached to Sal-1-treated coverslips and appeared healthy. Furthermore, many cells had extended elaborate cell processes and achieved morphologies characteristic of mature neurons. These results show that antibodies can be used as substrates for the culture of mature neurons. This technique may also prove useful for in vitro studies of a variety of cell systems.
Identification of the epsilon-subunit of Escherichia coli DNA polymerase III holoenzyme as the dnaQ gene product: a fidelity subunit for DNA replication.
Authors: Scheuermann R , Tam S , Burgers P M , Lu C , Echols H
Published: May 2006

Based on extensive genetic and biochemical studies, the multisubunit DNA polymerase III holoenzyme is considered responsible for the chain-elongation stage in replication of the genome of Escherichia coli and is thus expected to be the major determinant of fidelity as well. Previous experiments have shown that two mutations conferring a very high mutation rate on E. coli, mutD5 and dnaQ49, decrease severely the 3' leads to 5' exonucleolytic editing activity of the polymerase III holoenzyme. To identify more precisely the nature of these mutations, we have carried out genetic mapping and complementation experiments. From these studies and experiments by others, we conclude that the most potent general mutator mutations in E. coli occur in a single gene, dnaQ. To define further the role of the dnaQ gene, we have used two-dimensional gel electrophoresis to compare the labeled dnaQ gene product with purified polymerase III holoenzyme. The dnaQ product comigrates with the epsilon-subunit, a 25-kilodalton protein of the polymerase III "core" enzyme. We conclude that the epsilon-subunit of polymerase III holoenzyme has a special role in defining the accuracy of DNA replication, probably through control of the 3' leads to 5' exonuclease activity.
De novo purine synthesis in cultured rat embryos undergoing organogenesis.
Authors: Rowe P B , McEwen S E
Published: May 2006

The cultured rat embryo undergoing organogenesis (9.5-11.5 days of gestation) together with its associated yolk sac synthesize purine nucleotides via the de novo synthetic pathway. Although both the embryo and its yolk sac contain significant levels of the purine base salvage enzymes adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the culture medium that consists largely of rat serum contains no measurable quantities of salvageable purine bases or nucleosides but high activity levels of purine catabolic enzymes. Short-term pulse-chase experiments with adenine and guanine, carried out under virtually serum-free conditions, confirmed that purine base salvage mechanisms were active and that there was no significant net transfer of purines between the embryo and its yolk sac. A comparison between the specific radioactivities of the [14C]glycine added to the culture medium for the studies of the de novo synthetic pathway and the purine bases in both the cellular nucleotides and the nucleic acids indicated the existence of a large glycine pool, which almost certainly was derived from the degradation of medium serum proteins by the yolk sac. Although there are no clear-cut data available on the in vivo plasma levels of purines that could be potentially utilized to meet the demands of the embryo, it is evident that the de novo pathway is adequately developed to meet these needs.
Monoclonal AKR/J thymic leukemias contain multiple JH immunoglobulin gene rearrangements.
Authors: Herr W , Perlmutter A P , Gilbert W
Published: May 2006

We have examined the patterns of heavy-chain immunoglobulin gene rearrangement and provirus integration in seven murine leukemia virus-induced thymic leukemias from AKR/J mice. In five of the seven tumors examined, there were between two and five rearrangements of the heavy-chain immunoglobulin J (JH) segment. Except for one case, the rearranged JH segments are present in less than one copy per cell, indicating that these tumors contain subpopulations of thymocytes with differing JH rearrangements. Nevertheless, each tumor is probably of monoclonal origin because the cells in a given tumor contain a common set of randomly integrated murine leukemia proviruses. Our results indicate that the JH segments rearranged within a few cell divisions after tumor cell proliferation began and may, therefore, identify a specific stage in T-cell differentiation when tumorigenesis occurs.
DNA sequences necessary for packaging of bacteriophage lambda DNA.
Authors: Hohn B
Published: May 2006

The extent of DNA flanking the "cohered cohesive end" site of bacteriophage lambda DNA, which is required for packaging, was determined by using defined DNA fragments and a cosmid in vivo packaging assay. From the right end of lambda DNA a 20- to 36-base-pair stretch extending from the center of the cohered cohesive ends is shown to be required, whereas the packaging efficiency of cosmids extending to 70 base pairs into the left lambda arm is reduced to 10% (compared to a fragment extending until about 80 base pairs). A 60-base-pair stretch of the left arm leaves an efficiency of only 1%. The segment thus delineated, by the nature of the assay, is both necessary and sufficient for the binding of packaging proteins to the DNA, the packaging of DNA itself, the DNA cleavage, and successful injection of the DNA into a bacterial host. By contrast, in vitro packaging of restriction fragments of mature lambda DNA directly demonstrated the selectivity of the packaging proteins for the fragment originating from the left end of the DNA. The results of the two complementary experiments are discussed in terms of the various steps before, during, and after packaging for which different sequences flanking and including the cohered cohesive ends might be required.
Role of basic residues in the phosphorylation of synthetic peptides by myosin light chain kinase.
Authors: Kemp B E , Pearson R B , House C
Published: May 2006

The substrate specificity of the chicken gizzard myosin light chain kinase has been studied by using a series of synthetic peptide analogs of the NH2-terminal sequence of the chicken gizzard myosin light chain (Mr = 20,000). An 18-residue synthetic peptide, (sequence in text) corresponding to the sequence reported by Maita et al. [Maita, T., Chen, J. I. & Matsuda, G. (1981) Eur. J. Biochem. 117, 417-424], was phosphorylated with a 22-fold higher Km and a Vmax that was decreased to 1% of the native protein substrate. This peptide was also an inferior substrate when compared with an 18-residue synthetic peptide with an alternative sequence, (sequence: see text) which was phosphorylated with an apparent Km of 6.9 microM, comparable to the native protein substrate of 8.6 microM, and a Vmax of 3.9 mumol X min-1 X mg-1, 11% of that for the protein substrate. The kinetic of phosphorylation of shortened peptides corresponding to both sequences, together with peptides with appropriate substitutions, indicated that basic residues were the primary determinants of specificity for the smooth muscle myosin light chain kinase. In the latter peptide sequence, lysine residues 11 and 12 and the arginine at position 13 had a major influence on the kinetics of peptide phosphorylation.
Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.
Authors: Kosower N S , Glaser T , Kosower E M
Published: May 2006

Rat, but not human, erythrocytes undergo fusion promoted by the membrane-mobility agent 2-(2-methoxyethoxy)-ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C). The difference in behavior is correlated with rat erythrocyte membrane protein degradation caused by Ca2+-activated proteases. The human erythrocyte is deficient in such protease activity. Membrane protein degradation is a necessary, but not sufficient, requirement for membrane fusion. Membrane protein degradation probably releases membrane components from certain constraints. In addition, the motion of membrane components precedes fusion and must be promoted by reagents such as A2C, leading to the creation of fusion-potent lipid areas. This sequence of chemical and physical events occurs in other fusion processes.
Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of leukotriene C4.
Authors: Weller P F , Lee C W , Foster D W , Corey E J , Austen K F , Lewis R A
Published: May 2006

5-Lipoxygenase pathway-derived products of arachidonic acid released by human eosinophils activated in vitro have been measured by using radioimmunoassays specific for leukotriene B4 (LTB4) and for sulfidopeptide leukotrienes including leukotriene C4 (LTC4). Eosinophil-enriched leukocytes (mean, 85% eosinophils) from five hypereosinophilic donors activated with 5.0 microM ionophore A23187 for 15 min at 37 degrees C in the presence of 50 mM L-serine released 69 +/- 28 and 1.5 +/- 0.8 (mean +/- SEM) ng of LTC4 and LTB4, respectively, per 10(6) cells; ratios of LTC4 to LTB4 ranged from 16 to 149. Eosinophils stimulated with ionophore (2.5 microM) or phorbol myristate acetate (1 microgram per ml) metabolized exogenously added LTC4 to products that coeluted on reverse-phase high-performance liquid chromatography with synthetic S-diastereoisomeric LTC4 sulfoxides and 6-trans-LTB4 diastereoisomers, and this metabolic inactivation was inhibited by L-serine or catalase. Ionophore-activated eosinophils purified from three normal donors also preferentially generated LTC4 (38 +/- 3 ng per 10(6) cells) relative to LTB4 (6.0 +/- 3.1 ng per 10(6) cells), whereas neutrophils from the same donors released LTB4 (48 +/- 21 ng per 10(6) cells) in a greater than 7-fold excess to LTC4. The predominant production by human eosinophils of LTC4 with its potent smooth muscle spasmogenic and vasoactive properties may contribute to the pathobiology of allergic and other diseases associated with eosinophilia.
Nutrition-endocrine interactions: induction of reciprocal changes in the delta 4-5 alpha-reduction of testosterone and the cytochrome P-450-dependent oxidation of estradiol by dietary macronutrients in man.
Authors: Kappas A , Anderson K E , Conney A H , Pantuck E J , Fishman J , Bradlow H L
Published: May 2006

The in vivo biotransformations of drugs known to be metabolized by enzymes localized in the endoplasmic reticulum of liver can be greatly altered by diet in humans, as we have shown previously. Steroid hormones also are metabolized extensively by hepatic microsomal enzymes; therefore, we examined the possibility that testosterone and estradiol biotransformations, as assessed with radiolabeled tracer methods, could be influenced by dietary macronutrients. Normal males were fed a high-protein diet for 2 weeks, followed by a high-carbohydrate diet for an additional 2 weeks. The delta 4-5 alpha-reduction of testosterone was considerably diminished, while the cytochrome P-450-dependent hydroxylation of estradiol at the C2 position was substantially enhanced during ingestion of the high-protein diet as compared with the high-carbohydrate diet. These results indicate that dietary macronutrients can significantly alter major metabolic pathways for testosterone and estradiol in man. The mechanism by which reciprocal changes in the delta 4-5 alpha-reduction of testosterone and the cytochrome P-450-mediated oxidation of estradiol are produced by diets is not known. Similar changes in steroid delta 4-5 alpha-reduction and cytochrome P-450-dependent chemical oxidations have been observed in circumstances in which the mixed-function oxidase system in liver is induced by agents such as phenobarbital, hexachlorobenzene, dioxin, and polyhalogenated biphenyls. Thus, the alterations in steroid hormone metabolism produced by dietary macronutrients in man mimic those that can be produced by drugs and environmental chemicals.
Systematic variation of metal—metal bond order in metalloporphyrin dimers
Authors: Collman James P. , Barnes Craig E. , Woo L. Keith
Published: May 2006

A general method for synthesizing a novel class of metalloporphyrin dimers containing unbridged metal—metal bonds is presented. By choosing the appropriate metal, the bond order in these dimers can be varied systematically. The synthesis and characterization of osmium(II) and molybdenum(II) porphyrin dimers containing the respective metal—metal double and quadruple bonds are described. A simple molecular orbital treatment that accounts for the metal—metal bonding, spin state, and geometry in these complexes is discussed.
Characterization and partial nucleotide sequence of endogenous type C retrovirus segments in human chromosomal DNA.
Authors: Repaske R , O'Neill R R , Steele P E , Martin M A
Published: May 2006

Twenty-six different murine leukemia virus (MuLV)-related clones have been isolated from a human DNA library and characterized by restriction enzyme mapping and reciprocal nucleic acid hybridization reactions. The sequence of approximately 2,600 nucleotides, spanning more than 4.0 kilobases, of one of the MuLV-related cloned human DNAs was also determined. The deduced amino acid sequence permitted the alignment of this prototype cloned human DNA segment with the p12 gag, p30 gag, p10 gag, and pol regions of Moloney MuLV. A majority of the endogenous type C retrovirus-related segments present in human DNA are approximately 6.0 kilobases in size and appear to contain a deletion of env sequences.
Transport and direct utilization of gamma-glutamylcyst(e)ine for glutathione synthesis.
Authors: Anderson M E , Meister A
Published: May 2006

Administration of gamma-glutamylcystine or of gamma-glutamylcysteine disulfide to mice leads to significantly increased levels of glutathione in the kidney as compared to controls given glutamate plus cysteine (or cystinylbisglycine). Studies with gamma-glutamylcystine selectively labeled with 35S in either the internal or external S atom indicate preferential utilization of the gamma-glutamylcysteine moiety of this compound for glutathione synthesis. Mice depleted of glutathione by treatment with buthionine sulfoximine do not significantly use the disulfides gamma-glutamylcystine or gamma-glutamylcysteine disulfide but do use gamma-glutamylcysteine for glutathione synthesis. These findings suggest a pathway in which gamma-glutamylcystine, formed by transpeptidation between glutathione and cystine, is transported and reduced by transhydrogenation with glutathione to cysteine and gamma-glutamylcysteine; the latter is used directly for glutathione synthesis. The findings show transport of gamma-glutamyl amino acids, indicate an alternative pathway of glutathione synthesis, and demonstrate a means of increasing kidney glutathione levels.
Phencyclidine ("angel dust") analogs and sigma opiate benzomorphans cause cerebral arterial spasm.
Authors: Altura B T , Quirion R , Pert C B , Altura B M
Published: May 2006

Several psychotomimetic phencyclidine (PCP) analogs--N-ethyl-l-phenylcyclohexylamine (PCE), N-[1-(2-thienyl)cyclohexyl]piperidine (TCP), N-[1-(thienyl)cyclohexyl ))pyrrolidine (THP), ketamine, and N,N-dimethyl-l-phenyl-cyclohexylamine (PCDEA)--were tested on basilar and middle cerebral arteries of the dog in vitro and found to induce contraction in these blood vessels with a maximal contractile activity (i.e., intrinsic activity) similar to that of PCP. The concentration-effect curves of these compounds were found to be parallel to the curve of PCP (P less than 0.01). The relative potency was PCE greater than TCP greater than PCP greater than THP greater than PCDEA greater than ketamine. A PCP analog with no psychotomimetic activity, 1-piperidinocyclohexanecarbonitrile (PCC), did not induce the blood vessels to contract, nor did the opiate morphine. Three psychotomimetic benzomorphans--pentazocine, cyclazocine, and N-allylnorcyclazocine--were found to: (i) also produce contraction; and (ii) have concentration--effect curves parallel to the curve of PCP, but with reduced intrinsic activities (i.e., maximal tensions were lowered) compared to PCP. A kappa opiate, ethylketocyclazocine, relaxed the blood vessels in a dose-dependent manner. This study provides direct evidence for a distinct PCP receptor on cerebral blood vessels and suggests that certain benzomorphans may produce cerebral vasospasm via PCP-receptor interactions.
Detection of autonomous replicating sequences (ars) in the genome of Epstein-Barr virus.
Authors: Henry B E , Raab-Traub N J , Pagano J S
Published: May 2006

Epstein-Barr virus (EBV) DNA was analyzed for the presence of autonomous replicating sequences (designated ars) in a eukaryotic system consisting of a uracil auxotroph of Saccharomyces cerevisiae, YNN27, and a pBR322 hybrid plasmid, YIp5, containing the yeast uracil gene but apparently lacking a eukaryotic origin of replication. Cloned EBV DNA EcoRI restriction fragments, A, B, and DIJhet, were judged to function in this capacity by their ability to convert YNN27 cells to the uracil phenotype after transformation with each EBV-specific fragment ligated into YIp5. Additional analyses to confirm and to specify further the location of the ars were performed by cleavage of EcoRI fragments A and B into smaller BamHI fragments, which were subsequently cloned in YIp5 and tested for their ability to function as ars. BamHI fragment X, obtained from EcoRI fragment A, and BamHI fragment R, obtained from EcoRI fragment B, showed ars behavior. The successful recovery of the appropriate virus DNA segments in plasmid form from transformed yeast cells and the ability of these yeast cells to be propagated further substantiated the ars capability of the three EBV fragments.
Crystallization of a common cold virus, human rhinovirus 14: “Isomorphism” with poliovirus crystals
Authors: Erickson John W. , Frankenberger Elizabeth A. , Rossmann Michael G. , Fout G. Shay , Medappa K. C. , Rueckert Roland R.
Published: May 2006

Crystals of rhinovirus 14 have been grown reproducibly. They diffract x-rays to a resolution of at least 3.5 Å. The orthorhombic crystal unit cell contains two virions, each situated on a crystallographic twofold axis. At less than 30-Å resolution, the space group approximates to
1
222 with the particles possessing 222 pseudo crystallographic symmetry. The crystals are “isomorphous” with type I polio crystals [Finch, J. T. & Klug, A. (1959)
Nature
(
London
) 183, 1709-1714; Hogle, J. M. (1982)
J. Mol. Biol.
160, 663-668], suggesting some similarities of structure between enteroviruses and rhinoviruses.
Association of pp36, a phosphorylated form of the presumed target protein for the src protein of Rous sarcoma virus, with the membrane of chicken cells transformed by Rous sarcoma virus.
Authors: Amini S , Kaji A
Published: May 2006

A cellular protein with a molecular mass of approximately 36 kilodaltons is the presumed target protein of the src protein [the transforming protein encoded by Rous sarcoma virus (RSV)]. The cellular location of the phosphorylated 36-kilo-dalton protein (pp36) in chicken embryo fibroblasts transformed by the Schmidt-Ruppin strain of RSV has been investigated. In these studies, two-dimensional electrophoresis was used for detection of the phosphoproteins in total cell extracts and also in fractionated subcellular components. We conclude that pp36 is localized in the plasma membrane, on the basis of the following observations. (i) Fractionation of 32P-labeled cell extracts showed that pp36 is almost exclusively localized in the crude membrane fraction and no appreciable amount was found in nuclear or cytoplasmic fractions. (ii) On further fractionation of the crude membrane fraction, pp36 was localized mostly in the plasma membrane rather than in other membranous fractions. (iii) Isolated plasma membrane by itself phosphorylated the 36-kilodalton protein on incubation with [gamma-32P]ATP.
Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.
Authors: Purrello F , Burnham D B , Goldfine I D
Published: May 2006

The direct addition of insulin to highly purified nuclear envelopes prepared from the livers of diabetic rats resulted in a decrease in the incorporation of 32P into trichloroacetic acid-precipitable proteins. Autoradiography of 32P-labeled envelopes, solubilized in sodium dodecyl sulfate and subjected to electrophoresis, revealed that insulin decreased the phosphorylation of all major protein bands. Insulin produced detectable effects at concentrations between 0.1 and 1 pM, maximal effects at 10 pM, and progressively diminished effects at higher concentrations. Two insulin analogs, desdipeptide proinsulin and desoctapeptide insulin, had approximately 10% and 1%, respectively, the activity of native insulin. When nuclear envelopes were first phosphorylated with [gamma-32P]ATP and insulin was then added with an excess of unlabeled ATP, dephosphorylation was enhanced, suggesting that insulin was regulating nuclear envelope phosphatase activity. The direct addition of insulin to isolated rat liver nuclei in the presence of ATP stimulated the release of previously 14C-labeled trichloroacetic acid-precipitable mRNA-like material, and the direct addition of insulin to nuclear envelopes stimulated the activity of nucleoside triphosphatase, the enzyme that participates in mRNA nucleocytoplasmic transport. Moreover, the dose-response curves for these functions mirrored insulin's inhibition of nuclear envelope phosphorylation. These data suggest, therefore, a mechanism whereby insulin directly inhibits the phosphorylation of the nuclear envelope, leading in turn to the regulation of mRNA metabolism.
Efficient isolation of genes by using antibody probes.
Authors: Young R A , Davis R W
Published: May 2006

A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method uses an expression vector, lambda gt11 (lac5 nin5 cI857 S100), that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins. Efficient screening of antigen-producing clones in lambda gt11 recombinant cDNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities. The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences. Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification. Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells.
Monensin blocks the maturation of receptors for insulin and somatomedin C: Identification of receptor precursors
Authors: Jacobs Steven , Kull Frederick C. , Cuatrecasas Pedro
Published: May 2006

Cultured human lymphoid (IM-9) cells were labeled with [
35
S]methionine in the presence and absence of monensin, a carboxylic ionophore that inhibits post-translational protein maturation. Labeled receptors for insulin and somatomedin C were immunoprecipitated with antibodies specific for each receptor. Monensin inhibits the biosynthesis of mature α and β subunits of both receptors and leads to the accumulation of immunoreactive polypeptides with molecular weights of 180,000. These 180,000 molecular weight polypeptides exist as disulfide-linked dimers and may be biosynthetic precursors of both α and β subunits. In the presence of monensin, small amounts of immunoreactive polypeptides with molecular weights 115,000 and 89,000 also are produced. These may be abnormally processed forms of the α and β subunits lacking residues normally added during terminal glycosylation. In cells treated with monensin, the polypeptides of molecular weights 180,000 and 115,000 can be affinity-labeled with
125
I-labeled insulin. These labeled polypeptides are immunoprecipitated by antibodies specific for insulin receptors but not by antibodies specific for somatomedin-C receptors. This indicates that the putative precursors for insulin and somatomedin-C receptors are distinct polypeptides, although they have similar molecular weights and similar modes of processing. A possible structural relationship between the precursors for these receptors and the type II insulin-like growth factor receptor is discussed.
Rates of enzyme-catalyzed exchange determined by two-dimensional NMR: a study of glucose 6-phosphate anomerization and isomerization.
Authors: Balaban R S , Ferretti J A
Published: May 2006

The application of two-dimensional (2D) Fourier-transform NMR to the determination of rate constants of complex enzyme-catalyzed reactions in the steady state is described. The yeast phosphoglucose isomerase (EC 5.3.1.9)-catalyzed anomerization of glucose 6-phosphate (Glc-6-P) as well as its isomerization to fructose 6-phosphate (Fru-6-P) was chosen as an example. The 2D technique permitted the simultaneous monitoring of the time course of the anomerization and isomerization steps, from which the various reaction rates were determined. The results obtained in the steady state demonstrate the usefulness of the 2D technique by confirming that the anomerization of Glc-6-P is enzyme catalyzed and that the isomerization of the alpha anomer of Glc-6-P to Fru-6-P is at least 10 times faster than the isomerization of the beta anomer of Glc-6-P. These results are compared with reaction rates obtained by rapid-quench methods and the mechanistic implications are discussed. Extrapolation of these results suggests that the 2D Fourier-transform NMR method should be applicable in intact biological tissues.
Cold-sensitive expression of transformation by a host range mutant of type 5 adenovirus.
Authors: Babiss L E , Ginsberg H S , Fisher P B
Published: May 2006

hr1, an E1a (0-4.5 map units) host range mutant of type 5 adenovirus (Ad5), transformed a cloned rat embryo fibroblast (CREF) cell line at about a 5-fold higher frequency than wild-type (wt) Ad5 when cells were cultured at 37 degrees C. However, if the cells were infected with hr1 and maintained at 32 degrees C morphological transformation did not occur. When infected cells were shifted from 32 degrees C to 37 degrees C 2 wk postinfection, the frequency of transformation by 6 wk was identical to that of cells grown continuously at 37 degrees C, whereas cultures shifted from 37 degrees C to 32 degrees C 2 wk postinfection displayed a greater than 96% reduction in morphological transformation. hr1-transformed cells had a fibroblastic morphology as contrasted with the typical epithelioid morphology of wt Ad5-transformed cells, but hr1- and wt Ad5-transformed cells had similar saturation densities, growth rates, and agar cloning efficiencies when assayed at 37 degrees C. However, when cells transformed by hr1 at 37 degrees C were grown at 32 degrees C, they had a saturation density close to that of normal CREF cells and grew at a lower efficiency in agar than wt-transformed cells. DNA transfer/hybridization analysis of two hr1-transformed cloned cell lines, A2 and B3, indicated that A2 cells contained a complete integrated copy of the Ad5 genome, whereas in B3 cells only part of the Ad5 genome was integrated. RNA transfer and RNA/DNA filter hybridization analyses indicated that the types of viral messenger RNAs and the relative amounts of RNA transcribed were similar in the A2 and B3 cell lines when they were grown at 32 degrees C and 37 degrees C. Indirect immunofluorescence, with antisera from hamsters bearing Ad-induced tumors, indicated a temperature dependence in staining--i.e., cells grown at 37 degrees C or shifted from 32 degrees C to 37 degrees C contained intense, particulate staining in the nuclear region, whereas the staining was decreased significantly in cells cultured at 32 degrees C and in cells shifted from 37 degrees C to 32 degrees C. These findings indicate that the gene product affected by the hr1 mutation is cold sensitive and is essential for the expression of the characteristics that identify the transformed cell.