bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromatin higher-order structure studied by neutron scattering and scanning transmission electron microscopy.
Authors: Gerchman S E , Ramakrishnan V
Published: May 2006

Neutron scattering in solution and scanning transmission electron microscopy were simultaneously done on chicken erythrocyte chromatin at various salt and magnesium concentrations. We show that chromatin is organized into a higher-order structure even at low ionic strength and that the mass per unit length increases continuously as a function of salt concentration, reaching a limiting value of between six and seven nucleosomes per 11 nm. There is no evidence of a transition from a 10-nm to a 30-nm fiber. Fiber diameter is correlated with mass per unit length, showing that both increase during condensation. We also find that there is no essential difference between the mass per unit length measured by scanning transmission electron microscopy and neutron scattering in solution, showing that the ordered regions seen in micrographs are representative of chromatin in solution.
A DNA sequence of 15 base pairs is sufficient to mediate both glucocorticoid and progesterone induction of gene expression.
Authors: Strähle U , Klock G , Schütz G
Published: May 2006

To define the recognition sequence of the glucocorticoid receptor and its relationship with that of the progesterone receptor, oligonucleotides derived from the glucocorticoid response element of the tyrosine aminotransferase gene were tested upstream of a heterologous promoter for their capacity to mediate effects of these two steroids. We show that a 15-base-pair sequence with partial symmetry is sufficient to confer glucocorticoid inducibility on the promoter of the herpes simplex virus thymidine kinase gene. The same 15-base-pair sequence mediates induction by progesterone. Point mutations in the recognition sequence affect inducibility by glucocorticoids and progesterone similarly. Together with the strong conservation of the sequence of the DNA-binding domain of the two receptors, these data suggest that both proteins recognize a sequence that is similar, if not the same.
A small multigene family encodes Gi signal-transduction proteins.
Authors: Beals C R , Wilson C B , Perlmutter R M
Published: May 2006

The guanine nucleotide-binding regulatory proteins known as G proteins are receptor-associated signal-transduction molecules that are implicated in the control of a variety of metabolic processes. Recent evidence suggests that G proteins may mediate B-lymphocyte responses to bacterial lipopolysaccharide and may also transduce signals from the T-cell antigen receptor. Since these receptors are uniquely expressed on lymphoid cells, we used molecular cloning strategies to ask whether lymphocytes contain specialized G-protein alpha subunits to assist in signal transduction. Comparison of our two deduced human alpha i amino acid sequences with those previously determined for bovine and rodent G proteins permits the identification of three closely related but distinct types of alpha i molecules that comprise a small multigene family. Using gene-specific probes, we found that both of our alpha i genes are expressed in most cell types but in differing ratios. Our data support the view that a modest repertoire of extremely closely related G proteins mediates the transduction of signals derived from multiple different receptor molecules.
Pulsed-field electrophoresis: application of a computer model to the separation of large DNA molecules.
Authors: Lalande M , Noolandi J , Turmel C , Rousseau J , Slater G W
Published: May 2006

The biased reptation theory has been applied to the pulsed-field electrophoresis of DNA in agarose gels. A computer simulation of the theoretical model that calculates the mobility of large DNA molecules as a function of agarose pore size, DNA chain properties, and electric field conditions has been used to generate mobility curves for DNA molecules in the size range of the larger yeast chromosomes. Pulsed-field electrophoresis experiments resulting in the establishment of an electrophoretic karyotype for yeast, where the mobility of the DNA fragments is a monotonic function of molecular size for the entire size range that is resolved (200-2200 kilobase pairs), has been compared to the theoretical mobility curves generated by the computer model. The various physical mechanisms and experimental conditions responsible for band inversion and improved electrophoretic separation are identified and discussed in the framework of the model.
Genetic analysis of the interaction of the insertion sequence IS903 transposase with its terminal inverted repeats.
Authors: Derbyshire K M , Hwang L , Grindley N D
Published: May 2006

The insertion sequence IS903 has perfect, 18-base-pair terminal repeats that are the presumed binding sites of its transposase. We have isolated mutations throughout this inverted repeat and analyzed their effect on transposition. We show that every position in the inverted repeat (with the possible exception of position 4) is important for efficient transposition. Furthermore, various substitutions at a single position can have a wide range of effects. Analysis of these hierarchical effects suggests that transposase contacts the minor groove in the region from position 13 to position 16 but makes major groove (or more complex) interactions with the outer portion of the inverted repeat. Our data indicate that the transposase exhibits relaxed specificity for the "second" end of a transposed segment; the defect in transposition of virtually all mutant inverted repeats can be rescued by a wild-type end. However, this rescue exhibits a pronounced position effect; in most cases, it is efficient only when the wild-type end is close to the 3' end of the transposase gene. This confirms the cis-acting nature of the transposase protein and suggests the initial transposase-inverted repeat interaction is the rate-limiting step in transposition. From the behavior of transposons with one mutant and one wild-type end, we infer that the inverted repeat contains two functional domains--one for initial complex formation with transposase and the other for effective completion of transpositional recombination. To support this hypothesis we show that an end with a mutation in one domain can significantly rescue an end with a mutation in the other domain.
Selection by differential molecular survival: a possible mechanism of early chemical evolution.
Authors: de Duve C
Published: May 2006

A model is proposed to account for selective chemical evolution, progressing from a relatively simple initial set of abiotic synthetic phenomena up to the elaborately sophisticated processes that are almost certainly required to produce the complex molecules, such as replicatable RNA-like oligonucleotides, needed for a Darwinian form of selection to start operating. The model makes the following assumptions: (i) that a small number of micromolecular substances were present at high concentration; (ii) that a random assembly mechanism combined these molecules into a variety of multimeric compounds comprising a wide repertoire of rudimentary catalytic activities; and (iii) that a lytic system capable of breaking down the assembled products existed. The model assumes further that catalysts supplied with substrates were significantly protected against breakdown. It is shown that, by granting these assumptions, an increasingly complex network of metabolic pathways would progressively be established. At the same time, the catalysts concerned would accumulate selectively to become choice substrates for elongation and other modifications that could enhance their efficiency, as well as their survival. Chemical evolution would thus proceed by a dual process of metabolic extension and catalytic innovation. Such a process should be largely deterministic and predictable from initial conditions.
Isolation and characterization of cDNAs encoding the heavy chain of human inter-alpha-trypsin inhibitor (I alpha TI): unambiguous evidence for multipolypeptide chain structure of I alpha TI.
Authors: Salier J P , Diarra-Mehrpour M , Sesboue R , Bourguignon J , Benarous R , Ohkubo I , Kurachi S , Kurachi K , Martin J P
Published: May 2006

Human inter-alpha-trypsin inhibitor (I alpha TI) is a plasma glycoprotein of Mr 180,000, which has been described as a single polypeptide chain. Recently, however, we proposed that I alpha TI might be composed of a heavy (H) chain (Mr = 95,000) and a light (L) chain (Mr = 40,000) synthesized by two separate mRNAs. In the present study we have characterized cDNAs for the H chain of I alpha TI. These cDNAs collectively covered two sequences (977 and 1450 base pairs in length) with single open reading frames. The deduced amino acid sequences were highly homologous to each other and well matched with partial amino acid sequences obtained from purified serum I alpha TI. RNA blot analyses of liver RNAs with H- or L-chain cDNAs as probes clearly identified two distinct mRNAs of 3.3 and 1.3 kilobases, which corresponded to H or L chain, respectively. Poly(A)+ RNAs hybrid-selected with H-chain cDNAs coded for polypeptide chains of Mr 90,000-95,000. These results unambiguously establish that I alpha TI is made of multipolypeptides, possibly including one H and two L chains. The H chain contains potential calcium-binding sites and also regions homologous to the proposed reactive site for thiol-proteinase inhibitors. These data indicate that I alpha TI is a complex, multifunctional protein. mRNAs for both the H and L chains were found only in liver.
Protonophores induce plastoquinol oxidation and quench chloroplast fluorescence: Evidence for a cyclic, proton-conducting pathway in oxygenic photosynthesis
Authors: McCauley Steven W. , Melis Anastasios , Tang George M.-S. , Arnon Daniel I.
Published: May 2006

The photosynthetic apparatus converts light into chemical energy by a series of reactions that give rise to a coupled flow of electrons and protons that generate reducing power and ATP, respectively. A key intermediate in these reactions is plastoquinone (PQ), the most abundant electron and proton (hydrogen) carrier in photosynthetic membranes (thylakoids). PQ ultimately transfers electrons to a terminal electron acceptor by way of the Rieske Fe-S center of the cytochrome
bf
complex. In the absence of a terminal acceptor, electrons accumulate in the PQ pool, which is reduced to plastoquinol (PQH
2
), and also on a specialized PQ, Q
A
, which is reduced to an unprotonated semiquinone anion (Q
A
-
). The accumulation of Q
A
-
is measured by a rise in fluorescence yield and the accumulation of PQH
2
is measured by absorption difference spectrometry. We have found that in the absence of a terminal electron acceptor, two chemically diverse proton-conducting ionophores (protonophores), 2,6-di-
t
-butyl-4-(2′,2′-dicyanovinyl)phenol (SF 6847) and carbonylcyanide
p
-trifluoromethoxyphenylhydrazone (FCCP), induced oxidation of PQH
2
and quenching of chloroplast fluorescence, signifying oxidation of Q
A
-
. The two protonophores produced the same effects even when the only recognized pathway of PQH
2
oxidation by way of the cytochrome
bf
complex was inhibited by dibromothymoquinone. Two other uncouplers, gramicidin and nigericin, which are not protonophores but facilitate proton movement across membranes by other mechanisms, were ineffective. These findings are consistent with the operation in the oxygen-generating photosystem (photosystem II) of a cyclic, proton-conducting pathway.
Linoleic acid, but not cortisol, stimulates accumulation of casein by mouse mammary epithelial cells in serum-free collagen gel culture.
Authors: Levay-Young B K , Bandyopadhyay G K , Nandi S
Published: May 2006

A two-step culture system has been developed to analyze the role of hormones in casein accumulation by mammary epithelial cells obtained from adrenalectomized and ovariectomized adult virgin mice. In the first step cells are grown inside collagen gel in medium containing insulin, epidermal growth factor (EGF), and linoleic acid for 9 days; these conditions stimulate very little casein accumulation. Following this growth phase the gels are released to float in medium containing insulin, prolactin, and linoleic acid. During this second phase the mammary cells will accumulate large amounts of casein, but only in the simultaneous presence of insulin, prolactin, and linoleic acid; in the absence of linoleic acid casein accumulation is greatly reduced. The casein accumulation is not dependent on the presence of the glucocorticoid cortisol and will occur in spite of the presence of the antiglucocorticoid agent RU 38 486. To determine if the response to cortisol observed in organ culture by other investigators might be mediated by stromal cells, epithelial cells were grown in collagen gel under fatty acid-free conditions and then cocultured with explants of mammary fat pads from adult virgin mice with or without mammary parenchyma. The cocultures were performed in fatty acid-free medium containing insulin and prolactin with or without cortisol. In the majority of experiments the mammary epithelial cells in the collagen gel accumulate more casein in the presence of cortisol than in its absence, irrespective of the presence of mammary parenchyma in the explant. Thus, mammary epithelial cells are directly dependent on insulin and prolactin for casein accumulation and indirectly dependent on cortisol by means of its effect on the stromal cells. This cortisol effect may be to cause release into the medium of linoleic acid or a metabolic product of linoleic acid from the stromal cells.
Correlations, descent measures: drift with migration and mutation.
Authors: Cockerham C C , Weir B S
Published: May 2006

The analysis of gene frequencies for a nested structure of genes within individuals, individuals within subpopulations, and subpopulations within populations is considered. Alternative parameterizations are provided by measures of correlation and of identity by descent, but the latter parameters provide more flexibility. The effects of population size, mating system, mutation, and migration can be incorporated into transition equations for identity measures and the structure of equilibrium populations can be determined; the procedures are illustrated for a finite island model. With parameters defined before estimation procedures are developed, problems of estimates depending on the numbers of sampled subpopulations are avoided, while the descent measures also avoid the approximations found in other treatments.
Substitution of tyrosine for either cysteine in beta-lactamase prevents release from the membrane during secretion.
Authors: Fitts R , Reuveny Z , van Amsterdam J , Mulholland J , Botstein D
Published: May 2006

Six independent secretion-defective mutations were found that result in failure to release protein from the membrane into the periplasmic space of Salmonella typhimurium after removal of the signal peptide. The mutant protein is found in a membrane-bound form accessible to trypsin added to intact spheroplasts. The phenotype of these mutations supports the existence in general of an intermediate in bacterial secretion. All six mutations changed one or the other of the two cysteine residues in the mature protein to tyrosine, suggesting that these residues are involved in the release of protein into the periplasmic space, most likely by affecting protein folding.
B-lymphocyte activation mediated by anti-immunoglobulin antibody in the absence of protein kinase C.
Authors: Mond J J , Feuerstein N , Finkelman F D , Huang F , Huang K P , Dennis G
Published: May 2006

B-cell activation induced by crosslinking of surface immunoglobulin is known to stimulate hydrolysis of phosphatidylinositol to diacylglycerol and inositol trisphosphate. We now provide evidence that alternative pathways of activation may also be recruited during such activation. We utilized depletion of protein kinase C activity to determine whether this enzyme is required under all conditions for anti-immunoglobulin-stimulated B-cell activation. Although anti-immunoglobulin does not induce B-cell proliferation in protein kinase C-depleted cells, it stimulates an earlier event in B-cell activation as reflected by its ability to enhance the expression of major histocompatibility complex-encoded class II molecules. Furthermore, the ribonucleoside 8-mercaptoguanosine restores the ability of anti-immunoglobulin to induce B-cell proliferation in protein kinase C-depleted cells. This restoration is also demonstrated by an enhancement of synthesis of a nuclear protein that we find is increased during B-cell mitogenesis. These results indicate that B-cell activation stimulated by anti-immunoglobulin may recruit pathways in addition to the one dependent on protein kinase C.
Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor.
Authors: Dewji N N , Wenger D A , O'Brien J S
Published: May 2006

Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a lambda gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of approximately 2 kilobases (lambda-S-1.2 and lambda-S-1.3) and both were both homologous with a previously isolated clone (lambda-S-1.1) for mature SAP-1. We report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is approximately 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide (23 amino acids) mature SAP-1 (78 amino acids) is generated by removing an additional 7 amino acids from the amino terminus and approximately 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which we designate as P-2. The molecular mass of glycosylated pro-SAP-1 is estimated at approximately 69 kDa, assuming glycosylation of all four sites. The value is close to the reported 70-kDa value for glycosylated pro-SAP-1. A computer search failed to reveal homology between P-2 and the sequence of any other protein; its function is uncertain. The 3' untranslated region is composed of 90 base pairs and is incomplete, since it does not contain a polyadenylylation site or a poly(A) tail.
O-acetylation of a cell-surface carbohydrate creates discrete molecular patterns during neural development.
Authors: Blum A S , Barnstable C J
Published: May 2006

The cell-surface antigen detected by the monoclonal antibody JONES is expressed in the retina and a number of other central nervous system regions of the rat during the latter part of embryonic development and the early postnatal period. In addition to the expression on certain neuroblast populations it is found on some but not all axons and is also expressed at high levels on the end feet of radial glia in regions through which axons actively grow. In the perinatal rat retina, almost all the antigenic activity was carried on a ganglioside migrating between GM1 and GM2. The epitope recognized by antibody JONES was base labile and treatment with 0.1 M sodium carbonate or ammonia vapor converted the antigen into GD3. Resistance to oxidation by sodium periodate and reformation of the epitope by chemical acetylation of base-treated gangliosides with N-acetylimidazole identify the antigen as 9-O-acetyl GD3. The acetylation of GD3 seems to be regulated independently from GD3 expression itself since acetylated and nonacetylated GD3 do not have identical immunocytochemical distributions in the developing central nervous system. In addition, five independent human melanoma cell lines varied substantially in their expression of 9-O-acetyl GD3, even though they all expressed high levels of GD3. Acetylation of ganglioside-linked sialic acid provides a mechanism for generating unique patterns of surface carbohydrates, which may influence cell interactions in development.
Abnormal gait sequence in locomotion after atropine treatment of catecholamine-deficient akinetic rats.
Authors: Pellis S M , Pellis V C , Chesire R M , Rowland N , Teitelbaum P
Published: May 2006

Excessive, abnormal locomotion occurs after a high dose (25-50 mg/kg) of atropine sulfate to rats already akinetic due to catecholamine deficiency from intraventricular administration of 6-hydroxydopamine. This abnormal locomotion involves an abnormal gait sequence [right (R) hindleg (H), left (L) foreleg (F), LH, RF] instead of the normal gait sequence (RH, RF, LH, LF). In such animals atropine progressively (i) decreases hindleg step size, (ii) decreases arching of the trunk, and (iii) increases foreleg step size. These factors combine to change the ratio of front/hind body support. If the body stretches too far and the hindleg step is too small, a given hindleg step supports insufficient weight to remove weight from the ipsilateral foreleg; consequently, the opposite foreleg must execute the next step, producing the abnormal gait sequence. Thus, atropine affects gait sequence indirectly; it acts on at least three variables that affect how body weight is distributed and shifted during locomotion. To maintain stability during such locomotion, gait sequence is appropriately altered.
Gene size differentially affects the binding of yeast transcription factor tau to two intragenic regions.
Authors: Baker R E , Camier S , Sentenac A , Hall B D
Published: May 2006

Yeast transcription factor tau (transcription factor IIIC) specifically interacts with tRNA genes, binding to both the A block and the B block elements of the internal promoter. To study the influence of A block-B block spacing, we analyzed the binding of purified tau protein to a series of internally deleted yeast tRNA(3Leu) genes with A and B blocks separated by 0 to 74 base pairs. Optimal binding occurred with genes having A block-B block distances of 30-60 base pairs; the relative helical orientation of the A and B blocks was unimportant. Results from DNase I "footprinting" and lambda exonuclease protection experiments were consistent with these findings and further revealed that changes in A block-B block distance primarily affect the ability of tau to interact with A block sequences; B block interactions are unaltered. When the A block-B block distance is 17 base pairs or less, tau interacts with a sequence located 15 base pairs upstream of the normal A block, and a new RNA initiation site is observed by in vitro transcription. We propose that the initial binding of tau to the B block activates transcription by enhancing its ability to bind at the A block, and that the A block interaction ultimately directs initiation by RNA polymerase III.
Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library.
Authors: Teitz T , Naiman T , Avissar S S , Bar S , Okayama H , Canaani D
Published: May 2006

In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion.
Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein).
Authors: Benovic J L , Kühn H , Weyand I , Codina J , Caron M G , Lefkowitz R J
Published: May 2006

The beta-adrenergic receptor kinase is an enzyme, possibly analogous to rhodopsin kinase, that multiply phosphorylates the beta-adrenergic receptor only when it is occupied by stimulatory agonists. Since this kinase may play an important role in mediating the process of homologous, or agonist-specific, desensitization, we investigated the functional consequences of receptor phosphorylation by the kinase and possible analogies with the mechanism of action of rhodopsin kinase. Pure hamster lung beta 2-adrenergic receptor, reconstituted in phospholipid vesicles, was assessed for its ability to mediate agonist-promoted stimulation of the GTPase activity of coreconstituted stimulatory guanine nucleotide-binding regulatory protein. When the receptor was phosphorylated by partially (approximately 350-fold) purified preparations of beta-adrenergic receptor kinase, as much as 80% inactivation of its functional activity was observed. However, the use of more highly purified enzyme preparations led to a dramatic decrease in the ability of phosphorylation to inactivate the receptor such that pure enzyme preparations (approximately 20,000-fold purified) caused only minimal (approximately 1off/- 7%) inactivation. Addition of pure retinal arrestin (48-kDa protein or S antigen), which is involved in enhancing the inactivating effect of rhodopsin phosphorylation by rhodopsin kinase, led to partial restoration of the functional effect of beta-adrenergic receptor kinase-promoted phosphorylation (41 +/- 3% inactivation). These results suggest the possibility that a protein analogous to retinal arrestin may exist in other tissues and function in concert with beta-adrenergic receptor kinase to regulate the activity of adenylate cyclase-coupled receptors.
Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor.
Authors: Debouck C , Gorniak J G , Strickler J E , Meek T D , Metcalf B W , Rosenberg M
Published: May 2006

The mature gag and pol proteins of human immunodeficiency virus (HIV) and all retroviruses derive from large gag and gag-pol polyprotein precursors by posttranslational cleavage. A highly specific, virally encoded protease is required for this essential proteolytic processing. In this study, the HIV protease gene product was expressed in Escherichia coli and shown to autocatalyze its maturation from a larger precursor. In addition, this bacterially produced HIV protease specifically processed an HIV p55 gag polyprotein precursor when coexpressed in E. coli. This system will allow detailed structure-function analysis of the HIV protease and provides a simple assay for the development of potential therapeutic agents directed against this critical viral enzyme.
Two separate domains within vesicular stomatitis virus phosphoprotein support transcription when added in trans.
Authors: Chattopadhyay D , Banerjee A K
Published: May 2006

The structural phosphoprotein NS of vesicular stomatitis virus, in association with the virion-associated RNA polymerase L protein, transcribes the genome ribonucleoprotein template in vitro. It contains an acidic N-terminal domain and two distinct domains at the C-terminal end that are involved in binding to the polymerase protein and the template RNA enwrapped with the nucleocapsid protein. In the present study, the portions of the NS gene that encode the N- and C-terminal domains of the protein were cloned in pGEM vectors and expressed by in vitro transcription and translation. It was shown that two polypeptides obtained by translation of the encoded mRNAs support RNA synthesis in vitro in a reconstitution reaction when they are added together in trans. Moreover, the N-terminal domain can be functionally substituted by structurally similar polypeptides.
Lipophosphoglycan of Leishmania major that vaccinates against cutaneous leishmaniasis contains an alkylglycerophosphoinositol lipid anchor.
Authors: McConville M J , Bacic A , Mitchell G F , Handman E
Published: May 2006

The major cell surface glycoconjugate of Leishmania major, a putative parasite receptor for macrophages, is a lipophosphoglycan containing 81.6% (wt/wt) carbohydrate, 17.0% (wt/wt) phosphate, and 1.4% (wt/wt) lipid. It has been purified to homogeneity by hydrophobic chromatography and consists of a polydisperse family of molecules with Mr 5000-40,000. It contains galactose, mannose, glucose, arabinose, glucosamine, and inositol in the molar ratio of 51:21:5:6:1:1. The lipophosphoglycan has a complex structure, consisting mainly of tri- and tetrasaccharide units linked by phosphodiester bonds, which are cleaved by HF hydrolysis. The phosphate groups are located on the 6-hydroxyl of both galactose and mannose residues. The lipophosphoglycan is anchored to the parasite surface by a 1-O-alkyl-sn-glycero-3-phosphoinositol moiety. This conclusion is supported by analysis of the products of nitrous acid deamination, HF hydrolysis, and Staphylococcus aureus phosphatidylinositol specific-phospholipase C treatment. The 24:0 and 26:0 alkyl chains accounted for 93% of the ether-linked fatty acids in the lipid anchor. The results are also consistent with a glycosidic linkage between the inositol and a non-N-acetylated glucosamine residue. The lipophosphoglycan membrane anchor shares limited structural homology with the glycosylphosphatidylinositol anchors of several eukaryotic proteins, indicating that this type of membrane anchor is not limited to proteins. Vaccination of mice with the purified L. major lipophosphoglycan in liposomes induced resistance against cutaneous leishmaniasis.
Differential turnover of tyrosinated and detyrosinated microtubules.
Authors: Webster D R , Gundersen G G , Bulinski J C , Borisy G G
Published: May 2006

Turnover of tyrosinated and detyrosinated microtubules ([Tyr]MTs and [Glu]MTs, respectively) was analyzed by the combined use of hapten-mediated immunocytochemistry and peptide-specific antibodies. Cells were microinjected with hapten-labeled tubulin and then processed for triple-label immunofluorescence to determine the pattern of incorporation of the injected subunits into [Tyr]- and [Glu]-MTs. Within 2 min of microinjection, hapten-labeled domains were present at the ends of virtually all [Tyr]MTs but were absent from most [Glu]MTs, demonstrating that [Tyr]MTs grew, whereas most [Glu]MTs did not. After 1 hr of incubation, all [Tyr]MTs analyzed were copolymers of endogenous and hapten-labeled subunits, indicating complete and rapid turnover of these MTs. However, the majority of [Glu]MTs were not hapten-labeled, indicating that they had not turned over. Even 16 hr after injection, cells that had not divided retained a small proportion of [Glu]MTs lacking hapten, implying that some had persisted for most of a cell generation. At mitosis, all MTs were hapten-labeled, indicating that the stable interphase [Glu]MTs had depolymerized. The results establish that the MT network is heterogeneous in its turnover rate, being composed of at least two populations: [Tyr]MTs that turn over rapidly and [Glu]MTs that turn over slowly.
Frequencies of independent and simultaneous selection of Chinese hamster cells for methotrexate and doxorubicin (adriamycin) resistance.
Authors: Rice G C , Ling V , Schimke R T
Published: May 2006

We have determined the frequency with which Chinese hamster cells become resistant to either methotrexate or doxorubicin (former generic name, adriamycin) alone or to the two drugs simultaneously. We find that the frequency of acquisition of simultaneous resistance is 10-100 times higher than that predicted from the frequency of each resistance selected independently. In approximately 50% of cloned resistant variants, resistance is the result of amplification of the dihydrofolate reductase gene (methotrexate) and/or of the multiple-drug-resistance P-glycoprotein gene (doxorubicin). Prior exposure of cells to hypoxia markedly enhances these resistance frequencies. Our results indicate that the simultaneous emergence of resistance to these two cancer chemotherapeutic agents are not independent events, and we interpret them to constitute two consequences of the same basic process occurring at a high frequency.
Ecdysteroid biosynthesis and embryonic development are disturbed in insects (
Locusta migratoria
) reared on plant diet (
Triticum sativum
) with a selectively modified sterol profile
Authors: Costet Marie F. , El Achouri Mostafa , Charlet Maurice , Lanot Rene , Benveniste Pierre , Hoffmann Jules A.
Published: May 2006

Wheat seedlings germinating in the presence of the systemic fungicide fenpropimorph accumulate 9β,19-cyclopropylsterols (95% of total sterols) in place of Δ
5
-sterols, which are normally produced in these plants. Adult females of the phytophagous insect
Locusta migratoria
show a dramatic decrease in their cholesterol content when reared on fenpropimorph-treated wheat. These females lay eggs with the ecdysteroid concentration reduced by up to 80% as compared to controls. Injection of fenpropimorph to the insects or feeding them on wheat coated with the fungicide (normal sterol composition) does not affect their sterol or ecdysteroid profiles; addition of cholesterol to fenpropimorph-treated wheat prior to feeding restores normal ecdysteroid titers in the insects. The severe reduction of the ecdysteroid content in eggs laid by females reared on fenpropimorph-treated wheat is associated with a series of developmental arrests and/or abnormalities. The results show that the dietary 9β,19-cyclopropylsterols cannot be used by
Locusta
in place of Δ
5
-sterols for ecdysteroid biosynthesis. They suggest that the selective inhibition of specific enzymes in the sterol biosynthetic pathway of the plants can be used as a strategy to control insect development.
Isolation, identification, and synthesis of 2-carboxyarabinitol 1-phosphate, a diurnal regulator of ribulose-bisphosphate carboxylase activity
Authors: Berry Joseph A. , Lorimer George H. , Pierce John , Seemann Jeffrey R. , Meek James , Freas Suzan
Published: May 2006

The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5-
P
2
) carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] of leaves of
Phaseolus vulgaris
is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5-
P
2
carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5-
P
2
carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5-
P
2
carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5-
P
2
carboxylase. The compound was analyzed by GC/MS,
13
C NMR, and
1
H NMR and shown to be 2-carboxyarabinitol 1-phosphate [(2-
C
-phosphohydroxymethyl)-D-ribonic acid]. Verification of structure was obtained by comparison of the inhibitory activity of the isolated compound with that of 2-carboxy-D-arabinitol 1-phosphate synthesized
in vitro
. This compound (but not 2-carboxy-D-arabinitol 5-phosphate) inhibited Rbu-1,5-
P
2
carboxylase in a way that was kinetically identical to that of the isolated, naturally occurring compound. The structure of the isolated compound differs from the Rbu-1,5-
P
2
carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate (
K
d
= 32 nM) compared with the bisphosphate (
K
d
< 10 pM). The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5-
P
2
carboxylase activity
in vivo
.
Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro.
Authors: Yang L , Wold M S , Li J J , Kelly T J , Liu L F
Published: May 2006

We examined the roles of DNA topoisomerases in the replication of simian virus 40 (SV40) DNA in a cell-free system composed of an extract from HeLa cells supplemented with purified SV40 tumor antigen. When the activities of both topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) in the extract were blocked with specific inhibitors or antibodies, DNA synthesis was decreased by a factor of 15-20. Addition of purified HeLa DNA topoisomerase II to extracts immunologically depleted of both topoisomerases completely restored replication, and the replication products consisted largely of monomeric daughter molecules. Addition of purified HeLa DNA topoisomerase I to depleted extracts restored DNA synthesis, but the primary products were multiply intertwined, catenated daughter molecules. We conclude that DNA topoisomerases have at least two roles in the replication of SV40 DNA. Either topoisomerase I or topoisomerase II is sufficient to provide the unlinking activity necessary for fork propagation during SV40 DNA replication. However, topoisomerase II is uniquely required for the segregation of newly synthesized daughter molecules.
Clastogenic action of hydroperoxy-5,8,11,13-icosatetraenoic acids on the mouse embryo fibroblasts C3H/10T1/2.
Authors: Ochi T , Cerutti P A
Published: May 2006

Phorbol 12-myristate 13-acetate induces the release of a low molecular weight clastogenic factor from monocytes. Hydroperoxy-5,8,11,13-icosatetraenoic acids represent major components of clastogenic factor. We report that several isomeric hydroperoxy-5,8,11,13-icosatetraenoic acids efficiently induce DNA strand breakage and/or alkali-labile sites in the mouse embryo fibroblasts C3H/10T1/2. Fe chelation by desferrioxamine suppresses breakage by approximately equal to 42% indicating the participation of Fe-catalyzed radical reactions. An additional 37% inhibition is observed upon addition of the Ca2+ chelators EGTA and quin-2. This result suggests that hydroxyperoxy-5,8,11,13-icosatetraenoic acid may activate a Ca2+-dependent nuclease. The addition of the antioxidant enzymes CuZn-superoxide dismutase and catalase had no effect, while glutathione peroxidase suppressed strand breakage by 90%. To our knowledge, our results yield a first insight into the mechanism of action of monocyte clastogenic factor and the role of inflammation in tumor promotion.
Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans.
Authors: Recsei P A , Gruss A D , Novick R P
Published: May 2006

A 1.5-kilobase-pair fragment of DNA that contains the lysostaphin gene from Staphylococcus simulans and its flanking sequences has been cloned and completely sequenced. The gene encodes a preproenzyme of Mr 42,000. The NH2-terminal sequence of the preproenzyme is composed of a signal peptide followed by seven tandem repeats of a 13-amino acid sequence. Conversion of prolysostaphin to the mature enzyme occurs extracellularly in cultures of S. simulans and involves removal of the NH2-terminal portion of the proenzyme that contains the tandem repeats. The high degree of homology of the repeats suggests that they have arisen by duplication of a 39-base-pair sequence of DNA. In S. simulans, the lysostaphin gene is present on a large beta-lactamase plasmid.
Isolation of enzyme cDNA clones by enzyme immunodetection assay: isolation of a peptide acetyltransferase.
Authors: Eberwine J H , Barchas J D , Hewlett W A , Evans C J
Published: May 2006

The biological activity of many proteins and peptides can be profoundly affected by enzyme-catalyzed covalent modifications such as acetylation, sulfation, glycosylation, or amidation. This article describes the cloning of such an enzyme, a peptide acetyltransferase from rat brain that catalyzes the amino-terminal acetylation of endorphins and perhaps other substrates in vivo. Blot-hybridization analysis suggests that the mRNA encoding the acetyltransferase is approximately 2.0 kilobases, is present in whole rat brain and rat hypothalamus, and is slightly larger in mouse AtT20 tumor cells. The acetyltransferase was cloned by using a strategy whereby a cDNA expression library was screened with a solid-phase enzyme-activity assay; this technique combines the use of the substrate coupled to a solid support and subsequent recognition of the product by using a specific antiserum. We have called this method the enzyme immunodetection assay (EIDA). The EIDA should prove useful in the isolation of other clones for proteins that possess enzymatic activity upon expression in bacterial hosts.
Synthesis of sodium channels in the cell bodies of squid giant axons.
Authors: Brismar T , Gilly W F
Published: May 2006

Giant axons in squid are formed by fusion of axons from many small cell bodies in the giant fiber lobe (GFL) of the stellate ganglion. Somata of GFL cells in vivo are inexcitable and do not have measurable sodium current (INa) when studied with microelectrode or patch-electrode voltage-clamp techniques. If GFL cells are separated from the giant axons and maintained in primary culture, axon-like INa can be recorded from the somata after several days. Incorporation of Na channels into GFL cell bodies requires protein synthesis, intracellular microtubule-based transport, and the lack of a morphologically defined axon to serve as a sink for channels synthesized in culture.
Intramembrane translocation and posttranslational palmitoylation of the chloroplast 32-kDa herbicide-binding protein.
Authors: Mattoo A K , Edelman M
Published: May 2006

The 32-kDa herbicide-binding protein, a component of photosystem II, is synthesized as a membrane-associated 33.5-kDa precursor within the chloroplast. We show that membrane attachment of the precursor and processing to the 32-kDa form occur in the unstacked stromal lamellae. Once processed, the 32-kDa protein translocates, within the thylakoids, to the topologically distinct stacked granal lamellae. Posttranslational palmitoylation of the processed 32-kDa protein is also shown to occur. This modification takes place in a membrane-protected domain and is mainly confined to the protein assembled in the granal lamellae, where functional photosystem II centers are concentrated.
Structure of A-DNA in solution.
Authors: Charney E , Chen H H
Published: May 2006

The structure of DNA in aqueous trifluoroethanol solutions containing more than 70% (vol/vol) trifluoroethanol, where it has been proposed to have the A-conformation, has been studied by the technique of electric linear dichroism. The data demonstrate that both linear phi X174 DNA [5386 base pairs (bp) long] and a 172-bp fragment of sea urchin 5S RNA gene are in the double-helical A-form in the alcoholic solution and that two of the important structural parameters, the base tilt and the base-pair translation along the helical axis, are the same as in the A-form structure of crystalline double-helical d(GGTATACC). Despite its molecular length, the limiting reduced dichroism (-1.41) of the phi X174 DNA in low-salt aqueous solution is near the theoretical limit (-1.50) for DNA with base planes perpendicular to the helical axis. Transformation of this DNA and also of the 172-bp fragment to the A-structure in alcohol solutions reduces the experimental value of the dichroism to about -1.04, which agrees almost exactly with the value of -1.03 calculated for the A-form crystal. The base-pair translation along the helical axis was determined from relaxation of the DNA dichroism when the electric field is turned off. The 172-bp fragment is found to have a base-pair translation of only 2.6 A in the A-structure and is thus the same as in A-DNA fibers and in the A-form oligomers crystals. We conclude from the coincidence of the base-pair translation and the apparent values of the base tilt that the A structures are identical or nearly so in the solution and solid states. Comparison of the phi X174 data with data on smaller DNA fragments and with the data on the 172-bp sea urchin gene fragment unambiguously confirms that, in aqueous solution, the experimental reduced electric dichroism of B-DNA is highly dependent on molecular length, while the dichroism of the A-DNA form in alcoholic solutions is independent of molecular length at least in the range from 172 to 5386 bp.
Molecular cloning of a gene that is necessary for G1 progression in mammalian cells.
Authors: Greco A , Ittmann M , Basilico C
Published: May 2006

We have cloned a human cDNA that complements the mutation of ts11, a temperature-sensitive (ts) mutant of the BHK hamster cell line that at the nonpermissive temperature is blocked in progression through the G1 phase of the cell growth cycle. After transfecting human chromosomal DNA into ts11 cells and selecting for cells that had acquired a non-ts phenotype, we screened a genomic library constructed in the EMBL3 lambda vector from a secondary non-ts transformant and isolated a recombinant phage containing human DNA sequences that were uniformly present in primary and secondary non-ts transformants. Genomic probes that recognized an mRNA of about 2 kilobases in human cells were used to isolate from a cDNA expression library two cDNA plasmids that could efficiently transform ts11 cells to a non-ts phenotype. Sequencing of one of these cDNAs revealed a single open reading frame, which could encode a 540 amino acid protein. The ts11 gene has at least two other homologs in human DNA and thus it appears to be part of a small gene/pseudogene family. Experiments with serum-synchronized cells indicate that the expression of the ts11 gene, which is necessary for G1 progression, is itself cell-cycle regulated, being induced in approximately mid-G1.