bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the heme-stabilized translational inhibitor of reticulocyte lysates by calcium ions and phospholipid.
Authors: De Haro C , De Herreros A G , Ochoa S
Published: May 2006

Hemin-supplemented reticulocyte lysates can be activated for translational inhibition by addition of Ca2+ or phospholipid. The fact that this inhibition is prevented or decreased in both cases either by the Ca2+ chelator EGTA or by polymyxin B, an inhibitor of the recently described Ca2+- and phospholipid-dependent protein kinases, suggests the involvement of both Ca2+ and phospholipid in this activation. The inhibition by Ca2+ or phospholipid is accompanied by phosphorylation of the 38-kilodalton subunit of the eukaryotic initiation factor 2 (eIF-2) and the 90-kilodalton band of the heme-controlled translational inhibitor (HCI) and can be reversed by high concentrations of eIF-2 or GTP. When incubation is conducted at 30 degrees C, the inhibition produced by Ca2+ is not reversed by EGTA after 15 min. However, at 20 degrees C, Ca2+ inhibition can be fully reversed as late as 90 min from the start of incubation and phosphorylation of the eIF-2 alpha-subunit is correspondingly decreased. These results are consistent with the idea that, like heme deprivation, the activation by Ca2+ and phospholipid promotes the first step of the reaction pro-inhibitor in equilibrium reversible inhibitor leads to irreversible inhibitor and suggest that, in the presence of hemin albeit by a different mechanism, this activation affects the same inhibitor that is activated in the absence of heme--namely, HCI. Whether this activation is direct or indirect--e.g., via a separate Ca2+- and phospholipid-dependent protein kinase--remains to be determined.
Biosynthesis of peroxisomal enzymes in the methylotrophic yeast
Hansenula polymorpha
Authors: Roa Michèle , Blobel Günter
Published: May 2006

The dramatic expansion of the peroxisomal compartment known to occur in the methanol-utilizing yeast
Hansenula polymorpha
on transfer from glucose- to methanol-containing media was shown to be accompanied by the synthesis of at least six major polypeptides that dominate the polypeptide pattern of total cell extracts analyzed by NaDodSO
4
/polyacrylamide gel electrophoresis. Two of these polypeptides have been identified by immunochemical methods as the monomers of the peroxisomal enzymes alcohol oxidase and catalase. We have studied the biosynthesis of these two peroxisomal enzymes, both by
in vitro
translation and by
in vivo
labeling experiments. By the criterion of mobility in NaDodSO
4
/polyacrylamide gel electrophoresis, the
in vitro
- and
in vivo
-synthesized monomers were indistinguishable from each other, both in the case of alcohol oxidase and in that of catalase. Thus, neither of these peroxisomal enzymes appear to be synthesized as larger precursors. However, further analysis of
in vitro
-synthesized versus mature peroxisomal alcohol oxidase showed that the
in vitro
-synthesized form sedimented as a 5S monomer and not, like the mature peroxisomal enzyme, as a 20S octamer. Moreover, the
in vitro
-synthesized form was highly susceptible to trypsin digestion whereas the mature 20S octamer appeared to be resistant.
Mitochondrial DNA heteroplasmy in Drosophila mauritiana.
Authors: Solignac M , Monnerot M , Mounolou J C
Published: May 2006

Mitochondrial DNA extracted from an isofemale strain of Drosophila mauritiana (subgroup melanogaster) appeared to be heterogeneous in size. A short genome [S; 18,500 base pairs (bp)] and a longer one (L; 19,000 bp) coexist in the preparation. The additional 500 bp have been located within the A+T-rich region. Hpa I digest patterns suggest that the S genome may carry a duplication of a 500-bp sequence including an Hpa I site and that the L genome may carry a triplication of the same sequence. At the 30th generation of the isofemale strain, 60 female genotypes were examined individually. Half of the files were pure either for the S or the L DNA. The remaining 50% exhibited various degrees of heteroplasmy for the two DNA types. Among metazoan animals, this D. mauritiana strain offers an exceptional situation with regard to the number of individuals heterogeneous for mtDNA and the relative stability of heteroplasmy through generations.
Structure-specific model of hemoglobin cooperativity.
Authors: Lee A W , Karplus M
Published: May 2006

A generalization of the Szabo-Karplus statistical mechanical model for hemoglobin cooperativity is formulated. The model fits the available thermodynamic and spectroscopic data with assumptions that are consistent with structural results and empirical energy function calculations. It provides a mechanism of hemoglobin cooperativity that is a generalization of the proposals of Monod, Wyman, and Changeux and of Perutz. The role of nonsalt-bridge related sources of constraints on ligand affinity and the mode of salt-bridge coupling to tertiary-quaternary structural changes are examined within the framework of the model. Analysis of proton release data for a range of pH values indicates that a pH-independent part of cooperativity must be present. The pH dependence of the first and last Adair constants point to partial linkage of salt bridges to ligation in the deoxy state and to a destabilized intra-beta-chain salt bridge in the unliganded oxy state.
Tunicamycin inhibits ganglioside biosynthesis in rat liver Golgi apparatus by blocking sugar nucleotide transport across the membrane vesicles.
Authors: Yusuf H K , Pohlentz G , Sandhoff K
Published: May 2006

The synthesis of ganglioside GM1 in intact rat liver Golgi-derived vesicles is stimulated by phosphatidylglycerol as much (about 20-fold) as by Triton X-100. The antibiotic tunicamycin inhibits strongly the synthesis, in the presence as well as in the absence of the phospholipid, but has no effect when Golgi membranes are solubilized with detergent. In Pronase-treated Golgi vesicles, which retain full enzyme activity, both phospholipid dependence and tunicamycin inhibition of the synthesis disappear completely. When freshly prepared Golgi vesicles are incubated with 125 microM UDP-[3H]Gal for 10 min at 30 degrees C, the nucleotide sugar is found to be transported into the vesicles at an approximate rate of about 85 pmol/mg of protein per min, 92% of which remains firmly bound to the membrane. Tunicamycin inhibits this transport in a concentration-dependent manner. These results indicate the existence of carrier proteins in rat liver Golgi vesicles, which mediate the transport of the sugar nucleotide UDP-Gal, and that the carriers face the cytoplasmic side of the vesicles. The results also show that, although the mechanism of phosphatidylglycerol-induced stimulation of the synthetic activity remains unclear, tunicamycin inhibits ganglioside biosynthesis by blocking the transport of the nucleotide sugar and not by inhibiting the transferase directly.
Transient expression of genes introduced into cultured cells of Drosophila.
Authors: Di Nocera P P , Dawid I B
Published: May 2006

Recombinant plasmids in which the sequence encoding the bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) has been placed under the control of Drosophila heat shock protein 70 (hsp 70) or copia promoters have been introduced into cultured cells of two Drosophila species (Schneider II line of Drosophila melanogaster and D. immigrans) as calcium-phosphate complexes. Within 1-2 days after transfection functional CAT enzyme was detected in cells exposed to either CAT recombinant. The expression of the bacterial information depends on the activity of the Drosophila promoters because plasmids in which the Drosophila DNA fragments were fused to the CAT coding sequence in inverted orientation did not support the synthesis of CAT enzyme activity. Low levels of CAT activity and of hybrid mRNA were detected in cells transformed with hsp-cat recombinants when the cells were maintained at room temperature, and both mRNA levels and CAT activity increased substantially after a brief exposure to 37 degrees C. hsp-cat mRNA has the same 5' terminus as authentic Drosophila hsp 70 messenger. These experiments document a practical system for the introduction and expression of isolated genes in cultured cells of Drosophila.
Structure and expression of the dnaQ mutator and the RNase H genes of Escherichia coli: overlap of the promoter regions.
Authors: Maki H , Horiuchi T , Sekiguchi M
Published: May 2006

A 1.6-kilobase-pair DNA fragment derived from the Escherichia coli chromosome was analyzed by Tn3 transposon insertion and deletion mapping to locate a mutator gene, dnaQ (mutD), and the rnh gene that codes for RNase H. When a strong promoter, PL of lambda phage, was placed at the right- and left-side of the cloned DNA fragment, the dnaQ protein and RNase H, respectively were overproduced. These results suggested that the two genes are transcribed in opposite directions and that their promoters are located in a narrow region between the genes. Nucleotide sequence analysis confirmed this and further revealed that transcriptional and translational initiation signals for the two genes overlap. From the sequence data it was deduced that the dnaQ protein and RNase H consist of 243 and 155 triplets and have molecular weights of 27,500 and 17,500, respectively. dnaQ81 amber mutant showed two codon alterations, CAG(glutamine-195) leads to TAG(amber) and ACA(threonine-193) leads to ATA(isoleucine). The dnaQ-lacZ and the rnh-lacZ fused genes were constructed and hybrid proteins with beta-galactosidase activity were produced. From beta-galactosidase levels it was estimated that the promoter for dnaQ is 5 times more active than that for rnh.
Intracellular calcium measurements by 19F NMR of fluorine-labeled chelators.
Authors: Smith G A , Hesketh R T , Metcalfe J C , Feeney J , Morris P G
Published: May 2006

Symmetrically substituted difluoro derivatives of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (nFBAPTA) show large 19F NMR chemical shifts on chelating divalent cations. The complexes of Ca2+ with 4FBAPTA and 5FBAPTA show fast and slow exchange behavior, respectively, and the chemical shift or the areas of the resonances from the free and complexed forms can be used to determine the free Ca2+ concentration. The measurement of the free Ca2+ concentration by either ligand is unaffected by free Mg2+ concentrations less than 10 mM, by pH 6-8, or by contaminating divalent ions of high affinity (Zn2+, Fe2+, Mn2+). The tetraacetoxymethyl ester derivative of 5FBAPTA was used to load mouse thymocytes with 5FBAPTA to intracellular concentrations of 1 mM, and the 19F spectrum indicated a free intracellular Ca2+ concentration [( Ca]i) of 250 nM. The [Ca]i was increased to 350 nM by addition of succinylated concanavalin A at mitogenic concentrations, and the addition of A23187 saturated the intracellular chelator with Ca2+ from the external medium. The method provides a measurement of [Ca]i and other divalent cation concentrations with direct identification of the ionic species chelated.
Construction and identification of mouse amelogenin cDNA clones.
Authors: Snead M L , Zeichner-David M , Chandra T , Robson K J , Woo S L , Slavkin H C
Published: May 2006

The determination of the biochemical phenotype of tooth epithelium requires specification by the dental mesenchyme. This is a general feature of epithelial-mesenchymal interaction in a number of different epidermal organ systems (e.g., salivary gland, mammary gland, feather, skin, and hair morphogenesis). To investigate these developmental processes, we have identified a cDNA clone representing the major group of gene products associated with enamel extracellular matrix formation. The mRNAs for mouse amelogenins, representing approximately equal to 90% of the total enamel proteins, have been isolated and partially characterized by specific immunoprecipitation. The poly(A)-containing RNAs were used for the synthesis and cloning of the mouse amelogenin cDNA. Recombinant plasmids containing amelogenin cDNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analyses. A cloned sequence was used to identify the expression of amelogenins during tooth development. The mouse cDNA sequence hybridized to genomic mouse and human DNAs. This amelogenin cDNA probe now enables molecular investigations of a number of classical problems in developmental biology.
Identification of a mutant human insulin predicted to contain a serine-for-phenylalanine substitution.
Authors: Shoelson S , Fickova M , Haneda M , Nahum A , Musso G , Kaiser E T , Rubenstein A H , Tager H
Published: May 2006

Using information gained from (i) the relative HPLC retention of an abnormal insulin present in the serum of a hyperinsulinemic diabetic patient and (ii) the loss of an Mbo II restriction site in one of the patient's insulin gene alleles, it was recently predicted that the mutant insulin contained a serine-for-phenylalanine substitution at position B24 or B25. We have now prepared human [SerB24]insulin and [SerB25]insulin by solid-phase peptide synthesis and semisynthesis using an improved approach whereby the protecting groups can be removed from the final product in a single step. During reversed-phase HPLC analysis, the two semisynthetic insulins were clearly separated from normal insulin and from each other. Analysis of the patient's immunoaffinity-purified serum insulin by HPLC and radioimmunoassay showed that the insulin eluted at the position of [SerB24]insulin and coeluted with the analog when the two were studied in admixture. Additional studies showed that [SerB24]insulin and [SerB25]insulin have about 16% and 0.5% of the activity of normal insulin, respectively, in stimulating glucose oxidation by isolated rat adipocytes. We conclude that the patient's abnormal insulin (insulin Los Angeles) is human [SerB24]insulin and that abnormal insulins with amino acid replacements at both positions B24 and B25 can be associated with human diabetes.
Thymosin alpha 11: a peptide related to thymosin alpha 1 isolated from calf thymosin fraction 5.
Authors: Caldarella J , Goodall G J , Felix A M , Heimer E P , Salvin S B , Horecker B L
Published: May 2006

Two peptides related to thymosin alpha 1 have been isolated from preparations of calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, is designated des-(25-28)-thymosin alpha 1. The other, named thymosin alpha 11, contains seven additional amino acid residues at the COOH terminus. The sequence of this peptide is: AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu- Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH. Thymosin alpha 11, in doses of less than 300 ng per mouse, protects susceptible inbred murine strains against opportunistic infections with Candida albicans. It is approximately equal to 30 times as potent as thymosin fraction 5 and approximately equal in potency to thymosin alpha 1.
cis and trans activation of globin gene transcription in transient assays.
Authors: Treisman R , Green M R , Maniatis T
Published: May 2006

We examined the effects of the simian virus 40 enhancer sequence on transcription of cloned human alpha- and beta-globin genes shortly after their introduction into cultured mammalian cells. We find that (i) detectable transcription of the beta-globin gene but not the alpha-globin gene requires linkage to the enhancer; (ii) the enhancer increases the amount of beta-globin RNA at least 100-fold but results in only a 5- to 10-fold increase in the amount of alpha-globin RNA; (iii) plasmid replication does not increase the level of beta-globin RNA, regardless of linkage to the enhancer, but does result in an approximately equal to 50-fold increase in the level of alpha-globin RNA; (iv) the enhancer is not required for and does not increase transcription of either gene in 293 cells, an adenovirus 5-transformed human kidney cell line. We also show that an enhancer sequence is not required for activity of the normally enhancer-dependent simian virus 40 early promoter in 293 cells, indicating that these cells contain a trans-acting factor(s) that circumvents the requirement for the enhancer sequence.
Fluorescence lifetimes in the bipartite model of the photosynthetic apparatus with α, β heterogeneity in photosystem II
Authors: Butler Warren L. , Magde Douglas , Berens Sylvia J.
Published: May 2006

Recent studies of the lifetime of fluorescence after picosecond pulse excitation of photosynthetic organisms revealed relatively complex decay kinetics that indicated a sum of three exponential components with lifetimes spanning the range from about 0.1-2.5 ns. These fluorescence lifetime data were examined in the context of a simple photochemical model for photosystem II that was used previously to account for fluorescence yield data obtained during continuous illumination. The model, which consists of a single fluorescing species of antenna chlorophyll and a reaction center, shows that, in general, the decay kinetics after pulse excitation should consist of the sum of two exponential decays. The model also shows that in going from open to closed reaction centers the lifetime of fluorescence may increase much more than the yield of fluorescence and surprisingly long fluorescence lifetimes can be obtained. However, conditions can be stated where fluorescence will decay essentially as a single component and with lifetime changes that are proportional to the yield changes. A heterogeneity was also introduced to distinguish photosystem II
α
units, which can transfer excitation energy among themselves but not the photosystem I, and photosystem II
β
units, which can transfer energy to photosystem I but not to other photosystem II units. It is proposed that the rather complex fluorescence lifetime data can be accounted for in large part by the simple photochemical model with the α, β heterogeneity in photosystem II.
Cloning of sequences expressed specifically in tumors of rat.
Authors: Yamamoto M , Maehara Y , Takahashi K , Endo H
Published: May 2006

The sequences specifically transcribed in tumor cells are believed to be closely related to transformed phenotypes. For the isolation of such sequences, a cDNA clone library was constructed by using poly(A)+ RNAs from azo-dye-induced rat ascites hepatomas. Thirty-one tumor RNA-responsive clones were isolated by screening 4,000 clones of this library with conventional techniques, differential colony hybridization, and RNA blot hybridization. These clones were categorized into two groups with respect to their size distribution of mRNAs from which clones were derived. The first group was complementary to a single distinct species, either about 1.5 or 0.6 kilobases in length, of poly(A)+ RNA, and the second showed no distinct bands but a smear on a RNA blot. Semiquantitative RNA dot blot assays revealed that the sequences of these clones were expressed very little, if at all, in normal and regenerating livers, while generally high in ascites hepatomas. This specificity was also true for other solid lines of tumors, such as Morris hepatoma 5123D of Buffalo rat and Walker 256 carcinosarcoma of Wistar rat. The smear class sequences were transcribed from middle-repetitive sequences of DNA, indicating that a class of middle-repetitive sequences is specifically transcribed in tumor cells.
Human fetal to adult hemoglobin switching: changes in chromatin structure of the beta-globin gene locus.
Authors: Groudine M , Kohwi-Shigematsu T , Gelinas R , Stamatoyannopoulos G , Papayannopoulou T
Published: May 2006

We have investigated the chromatin structure of the chromosomal DNA regions containing the human G gamma-, A gamma-, delta-, and beta-globin structural genes in both fetal and adult erythropoietic tissues and in two human erythroleukemia cells lines before and after induction. Our results indicate that DNase I introduces specific cuts into the beta-globin gene cluster in erythroid cells but not in leukocytes. The predominant sites are located at the 5' sides of the G gamma-, A gamma-, delta-, and beta-globin genes, within 200 base pairs of the respective cap sites. Examination of fetal liver cells has revealed the presence of hypersensitive sites at the 5' side of all four genes, whereas analysis of adult bone marrow has revealed the characteristic sites near the delta- and beta-globin genes but no hypersensitive sites at the 5' termini of the G gamma- or A gamma-globin genes. The presence of delta and beta hypersensitive sites in fetal cells suggests that the increment in expression of the delta and beta genes during development most likely involves the modulation of another pathway to gene expression. Using isolated nuclei from HEL and K562 cells, we have found that the G gamma, A gamma, delta, and beta genes are preferentially sensitive [relative to the pro-alpha2(I) collagen gene] to mild digestion with DNase I, whereas these genes are as resistant as collagen genes in cells that do not express globin. These findings are discussed within the context of chromatin structural correlates of hemoglobin switching.
Expression of the chloramphenicol acetyltransferase gene in mammalian cells under the control of adenovirus type 12 promoters: effect of promoter methylation on gene expression.
Authors: Kruczek I , Doerfler W
Published: May 2006

The effect of DNA methylation at specific promoter sites on gene expression was tested by using a sensitive and quantitative assay system. The plasmid pSVO CAT contains the prokaryotic gene chloramphenicol acetyltransferase (CAT) and a HindIII site in front of it for experimental promoter insertion. Upon insertion into pSVO CAT, the E1a and protein IX gene promoters from adenovirus type 12 (Ad12) DNA were capable of mediating CAT expression upon transfection in mouse cells. In many viral and nonviral eukaryotic genes, DNA methylation at highly specific sites in the promoter region can attain a regulatory function in gene expression. One of the important sites is the 5' C-C-G-G 3' sequence. The CAT-promoting activity of the early simian virus 40 promoter in plasmid pSV2 CAT is refractory to methylation by the Hpa II or Hha I DNA methyltransferase at 5' C-C-G-G 3' or 5' G-C-G-C 3' sequences, respectively, because this promoter lacks such sites. The CAT coding sequence of this plasmid carries four Hpa II and no Hha I sites. Methylation of the Hpa II sites in the coding region does not affect expression. The E1a promoter of Ad12 DNA comprising the leftmost 525 base pairs of the viral genome carries two 5' C-C-G-G 3' and three 5' G-C-G-C 3' sites upstream from the leftmost "TATA" signal. Methylation of the Hpa II or Hha I sites incapacitates this promoter. The promoter of protein IX gene of Ad12 DNA contains one 5' C-C-G-G 3' and one 5' G-C-G-C 3' site downstream and two 5' G-C-G-C 3' sites greater than 300 base pairs upstream from the TATA motif and probably outside the promoter. The protein IX promoter is not inactivated by methylation of these sites. These data demonstrate that critical 5' methylations in the promoter region decrease or eliminate transcription; methylations of sites too far upstream or probably any sites downstream from the TATA site do not affect expression.
Isolation and characterization of a cDNA clone for the murine I-E beta polypeptide chain.
Authors: Mengle-Gaw L , McDevitt H O
Published: May 2006

The Ia antigens, encoded within the I region of the major histocompatibility complex, are a group of cell surface glycoproteins that are involved in the control of immune responsiveness. We isolated and determined the sequence of a 1,045-base-pair cDNA clone for one of the murine immune response genes, Ek beta. Comparison of the predicted amino acid sequence of the product of Ek beta with that of Ed beta shows that most of the amino acid differences are clustered in short stretches of peptide sequence in the first external protein domain. This clustering of allelic variation suggests that observed haplotype-specific immune responsiveness to certain antigens may be controlled, at least in part, by differences in configuration defined by these regions of allelic variability in the NH2-terminal domain of the E beta chain.
Monoclonal antibody cross-reactions between Drosophila and human brain.
Authors: Miller C A , Benzer S
Published: May 2006

A panel of 146 monoclonal antibodies (MAbs), obtained with Drosophila melanogaster tissue as primary immunogen, was tested for cross-reactivity with the human central nervous system. Sites examined included spinal cord, cerebellum, hippocampus, and optic nerve. Nonnervous tissues tested were liver and lymph node. Approximately half of the antibodies reacted with one or more sites in the human central nervous system, identifying regional, cell class, and subcellular antigens. Some recognized neuronal, glial, or axonal subsets. Immunoblot analysis revealed that some antibodies reacted with similar antigen patterns in both species.
Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P-450 in rat adrenal gland.
Authors: Privalle C T , Crivello J F , Jefcoate C R
Published: May 2006

Rat adrenal mitochondria accumulated cholesterol during ether stress in vivo when side-chain cleavage was inhibited by aminoglutethimide (control = 14.6 vs. aminoglutethimide = 26.5 micrograms of cholesterol per mg of protein). This accumulation was insensitive to simultaneous administration of cycloheximide (24.2 micrograms/mg), but side chain cleavage in the mitochondria was greatly decreased. Outer and inner mitochondrial membrane fractions were separated by discontinuous Ficoll gradient centrifugation. Quantitation of marker enzymes for inner, outer, and microsomal enzymes indicated that outer membranes contained less than 5% inner membranes. The inner membrane fraction contained less than 7% outer membrane and included 90% of mitochondrial cytochrome P-450. Electron microscopy revealed outer membranes as circular intact ghosts, whereas inner membranes were largely intact and retained vesicular structure typical of intact adrenal cortex mitochondria. Administration of aminoglutethimide effected a 2-fold increase in inner membrane cholesterol (9.4 vs. 20.1 micrograms/mg) but simultaneous administration of cycloheximide completely blocked this increase (10.9 micrograms/mg). We conclude that: (i) in the presence of aminoglutethimide, stress stimulates accumulation of cholesterol in the inner membrane of adrenal mitochondria; and (ii) transfer of cholesterol from outer to inner membranes requires a cycloheximide-sensitive agent.
Localization, synthesis, and activity of an antigenic site on influenza virus hemagglutinin.
Authors: Atassi M Z , Webster R G
Published: May 2006

This paper reports the antigenicity of the fusion region of the influenza virus hemagglutinin (HA). Two peptides, comprising the fusion region (residues 1-11 of the HA2 part of HA) of strain A and strain B influenza virus, were synthesized and their abilities to bind rabbit, goat, and human anti-influenza antibodies were determined. In addition, 30 anti-HA monoclonal antibodies were examined for their ability to bind the synthetic peptides. In quantitative immunoadsorbent titrations, the two peptides bound considerable amounts of antibodies in rabbit and goat antisera against virus or HA of the A or B strain as well as in several human sera from patients recovering from influenza A. Of the 30 anti-HA monoclonal antibodies, 5 bound completely and 4 bound partially to the peptides. Antibodies were raised in rabbits against the peptides by immunizing with peptide-bovine serum albumin conjugates or with the free peptides. Anti-peptide antibodies were bound by HA and by the intact virus of the respective strain. However, these antisera failed to exhibit significant virus neutralizing activity. In contrast, the monoclonal antibodies that reacted with these peptides inhibited viral infectivity. The results clearly show that residues 1-11 of HA2 represent an important antigenic site on influenza virus.
Light-dependent assembly of ribulose-1,5-bisphosphate carboxylase
Authors: Bloom Mark V. , Milos Patrice , Roy Harry
Published: May 2006

Ribulose-1,5-bisphosphate carboxylase [Ru
P
2
Case; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is composed of eight small subunits (
M
r
, 14,000) and eight large subunits (
M
r
, 55,000). Newly synthesized large subunits are associated with two complexes having sedimentation coefficients of 7 and 29 S. Assembly of Ru
P
2
Case occurs in isolated intact chloroplasts in the light but not in the dark. When extracts of chloroplasts are treated with ATP or GTP, Ru
P
2
Case assembly is accelerated while the 29S large subunit complex is maintained. In the presence of Mg
2+
, ATP brings about almost complete dissociation of the 29S complex, whereas GTP and a nonhydrolyzable analog of ATP are without effect. These results indicate the existence of a complex set of reactions involving nucleotides, Mg
2+
, and several putative intermediates in Ru
P
2
Case assembly. It is postulated that these reactions at least partly account for the light dependence of Ru
P
2
Case assembly. In particular, ATP and GTP promote the assembly of large subunits into Ru
P
2
Case.
A lectin-like receptor is involved in invasion of erythrocytes by Plasmodium falciparum.
Authors: Jungery M , Pasvol G , Newbold C I , Weatherall D J
Published: May 2006

Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.
Increased expression of myc-related oncogene mRNA characterizes most BALB/c plasmacytomas induced by pristane or Abelson murine leukemia virus.
Authors: Mushinski J F , Bauer S R , Potter M , Reddy E P
Published: May 2006

RNA blots of poly(A)-containing RNA from normal livers and spleens and from a number of transplantable hematopoietic and lymphoid BALB/c tumors, including early and late generation plasmacytomas, were hybridized with probes for four onc genes. abl RNA was abundant only in those tumors producing Abelson virus, bas RNA was found in approximately equal amounts in normal tissues and plasmacytomas, and myb RNA was absent in normal liver and plasmacytomas. Normal liver and spleen RNA showed faint traces of myc hybridization, but myc RNA was increased in most plasmacytomas. In one plasmacytoma, TEPC 1165, a particularly abundant amount of myc RNA was found, principally as a 3.5-kilobase band. In the other plasmacytomas, bands of 2.4- or 1.8-kilobase myc RNA were found. Southern blots of DNA from tumors that contained 2.4-kilobase or larger myc RNA showed myc hybridization to an EcoRI fragment of about 21 kilobase pairs, similar to the myc band in normal DNA. EcoRI digests of DNA from two tumors that expressed myc RNA of 1.8 kilobases showed an additional smaller myc band, suggesting that the myc gene is rearranged in these plasmacytomas. The basis for increased myc gene transcription in plasmacytomas is not understood, but the evidence suggests that different mechanisms may be operating in different plasmacytomas. Apparently, neither myc gene amplification nor myc gene rearrangement is required for increased myc transcription.
Crystallization of cytochrome bc1 complex.
Authors: Ozawa T , Tanaka M , Shimomura Y
Published: May 2006

Complex III (cytochrome bc1 particle; ubiquinol:ferricytochrome c oxidoreductase, EC 1.10.2.2) was purified from beef heart mitochondria by a combination of hydrophobic interaction and affinity chromatography. By washing the complex with detergent on the hydrophobic interaction column, phospholipids were effectively depleted; 7 mol of cardiolipin per mol of cytochrome c1 was retained in the final sample. NaDodSO4 gel electrophoresis showed nine polypeptide subunits in the sample. The molecular weight of the complex was estimated to be approximately equal to 225,000 from the specific heme c1 content and the subunit composition. The purified complex was crystallized by slow removal of the detergent in which the complex was dispersed. Electron micrographs and electron diffraction patterns showed that the crystal is hexagonal with unit cell dimensions a = b = 113 A, c = 132 A, and with angles alpha = beta = 90 degrees, gamma = 120 degrees. The role of bound cardiolipin in the structural integrity of the complex was discussed.
Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence.
Authors: Zyskind J W , Cleary J M , Brusilow W S , Harding N E , Smith D W
Published: May 2006

The chromosomal replication origin (oriC) of Vibrio harveyi has been isolated on a plasmid and shown to function as an origin in Escherichia coli. The nucleotide sequence of the V. harveyi oriC was determined. From a comparison of this sequence with oriC sequences of five enteric bacteria, we derived a consensus sequence of bacterial origins that function in E. coli. This consensus sequence identifies 122 positions within oriC where nucleotide substitutions can occur without loss of origin function. These positions are clustered rather than scattered. Four interrelated nine-base-pair repeats and eight of the dam methylation G-A-T-C sites are conserved in the consensus sequence. Very few relative insertion-deletion changes occur, and these are localized to one region of oriC. The genes for three polypeptides linked to the V. harveyi oriC were identified by using in vitro protein synthesis directed by deletion derivative plasmid templates. One of these genes, coding for a 58,000 Mr polypeptide and located 3.0 kilobase pairs from the V. harveyi oriC region, is lethal to E. coli when many copies (approximately 40 per cell) are present (high copy lethal or HCL gene). In addition, nucleotide sequence analysis showed that a different gene, the gid gene to the left of oriC, is highly conserved between E. coli and V. harveyi, whereas the coding region to the right of oriC is much less conserved.
Inverse relationship between blood levels of high density lipoprotein subfraction 2 and magnitude of postprandial lipemia.
Authors: Patsch J R , Karlin J B , Scott L W , Smith L C , Gotto A M
Published: May 2006

Triglyceridemic response to a standard oral fat meal was determined in 28 healthy subjects and related to the levels of several lipids, lipoproteins, and apolipoproteins in the post-absorptive plasma. A fatty test meal was administered orally, and postprandial plasma triglyceride levels were determined. In the fasting blood samples, concentrations of apolipoproteins (apo) A-I, A-II, and B were determined by radioimmunoassay, and those of high density lipoprotein (HDL) subfractions HDL2 and HDL3, by zonal ultracentrifugation. The magnitude of triglyceridemic response showed a negative correlation with the plasma levels of HDL2 (r = -0.860, P less than 0.001), HDL-associated cholesterol (r = -0.605, P less than 0.001), and apoA-I (r = -0.459, P less than 0.02). No correlation was found between the triglyceridemic response and the levels of total cholesterol, HDL3, and apoA-II. Triglyceridemic response was correlated positively with fasting triglyceride concentrations (r = 0.450, P less than 0.02) and apoB levels (r = 0.396, P less than 0.03). In two subjects followed for 3 yr, when HDL2 levels rose or fell, the triglyceridemic response decreased or increased, respectively (r = -0.944; r = -0.863). Our data indicate that normolipidemic individuals with high HDL2 levels in the plasma are able to clear alimentary fat at a faster rate than normolipidemic subjects with low HDL2 levels. The pronounced difference in severity and duration of postprandial lipemia among subjects with varying HDL2 levels may help to explain the negative correlation between the risk of atherosclerosis and HDL cholesterol levels.
Botrocetin (venom coagglutinin): reaction with a broad spectrum of multimeric forms of factor VIII macromolecular complex.
Authors: Brinkhous K M , Read M S , Fricke W A , Wagner R H
Published: May 2006

Botrocetin, originally called venom coagglutinin, is a Bothrops factor that causes aggregation of blood platelets in the presence of the von Willebrand component of the factor VIII macromolecular complex. The complex consists of a series of multimers with a molecular weight of about 1-20 x 10(6). Ristocetin, another agent that causes platelet aggregation dependent on von Willebrand factor, reacts with only the higher molecular weight multimers. We report on the reactivity of botrocetin in relation to the multimeric structure of the factor VIII complex. Several plasmas or plasma fractions with abnormal distribution of the multimeric sizes were examined, including variant von Willebrand disease type IIA with lack of the higher molecular weight forms, commercial antihemophilic factor concentrates with a preponderance of lower molecular weight forms, cryoprecipitate-free plasma containing mainly the smaller multimers, and a chromatographic fraction of plasma containing only the highest molecular weight polymers. Factor VIII-related antigen content was adjusted to 25-100%. All of the preparations lacking the high molecular weight forms caused prompt platelet aggregation with botrocetin, but none of them caused aggregation in the ristocetin test made isochronal with the botrocetin test. The very high molecular weight polymers were equally effective with botrocetin and ristocetin. These findings indicate that the Bothrops factor is reactive with a broad spectrum of high to low molecular weight forms of the factor VIII complex, suggesting that bioassays of von Willebrand factor with botrocetin should correlate better with immunoassays for factor VIII-related antigen and could reflect better the full platelet-aggregating function of the complex than do ristocetin determinations.
Site-to-site directed immobilization of enzymes with bis-NAD analogues.
Authors: Månsson M O , Siegbahn N , Mosbach K
Published: May 2006

Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) have been crosslinked with glutaraldehyde on agarose beads. The crosslinking was performed while the two enzymes were spatially arranged with their active sites facing one another with the aid of a bis-NAD analogue. Subsequently the bis-NAD analogue was allowed to diffuse out. By using a third enzyme, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3), which was also coupled to the same beads and which competes with lactate dehydrogenase for the NADH produced by alcohol dehydrogenase, the effect of site-to-site directed immobilization was studied. It was found that much more NADH than was theoretically expected (50% instead of 19% of produced NADH) was oxidized by lactate dehydrogenase, which indicates that the NADH was preferentially channeled to lactate dehydrogenase due to the juxtapositioned active sites of the two enzymes.
Reverse transcription of trypanosome variable antigen mRNAs initiated by a specific oligonucleotide primer.
Authors: Merritt S C , Tschudi C , Konigsberg W H , Richards F F
Published: May 2006

African trypanosomes change their antigenicity by successively expressing different members of a group of related but highly diverse proteins, the variant surface glycoproteins (VSGs). We describe a synthetic oligonucleotide that can prime specifically reverse transcription of VSG mRNA out of total trypanosome poly(A)+ RNA. The specificity of this priming was verified by cDNA sequence analysis of the transcription products and by the demonstration of variant-specific hybridization of the individual cDNAs to cellular RNA. The oligonucleotide primer also was used as a probe for the conserved sequence found on these VSG mRNAs in trypanosome genomic DNA libraries. A large number of primer-positive clones were detected in a Trypanosoma gambiense genomic library, but very few positive signals were found in a library of Trypanosoma congolense genomic DNA.
Purification and characterization of sheep brain cold-stable microtubules.
Authors: Pirollet F , Job D , Fischer E H , Margolis R L
Published: May 2006

The isolation of cold-stable microtubules in high yields, described previously only from rodents, was extended to the brain of higher animals. Under optimal conditions, yields of 30 mg of cold-stable microtubles per 100 g of sheep brain could be obtained routinely. Material purified by two polymerization cycles displayed the same stability to cold temperature or to millimolar concentrations of calcium and the same lability to calmodulin and to ATP as did the purified material obtained from the rat [Job, D., Rauch, C.T., Fischer, E.H. & Margolis, R.L. (1982) Biochemistry 21, 509]. Furthermore, DE-52 chromatography of this material yielded a fraction that restored cold stability when added to cold-labile microtubules. Known to bind to calmodulin and to enhance microtubule assembly, tau proteins had no cold-stabilizing activity. Protein profiles of the cold-stabilizing fraction from sheep and rat brain were similar to one another but showed no protein bands corresponding to the tau proteins.
Sendai virus-mediated lysis of liposomes requires cholesterol.
Authors: Kundrot C E , Spangler E A , Kendall D A , MacDonald R C , MacDonald R I
Published: May 2006

Vesicles were constituted with glycophorin, the Sendai virus receptor of human erythrocytes, and loaded with calcein, a polar derivative of fluorescein, at self-quenching concentrations. On exposure to Sendai virus and mild hypo-osmotic stress, vesicles of the appropriate composition released a significant portion of their internal contents, as indicated by an increase in calcein fluorescence. Susceptible liposomes were not induced to leak by heat-inactivated virus or by trypsin-treated virus. The response of the vesicles to virus attachment is thus analogous to virus-induced hemolysis and presumably involves fusion of the vesicle and virus membranes. In addition to glycophorin and phosphatidylcholine, cholesterol was absolutely required for the lytic response to the virus. The need for cholesterol was not attributable to inactivation of the virus by liposomes without cholesterol. The presence of gangliosides increased the encapsulated volume of the liposomes, but gangliosides did not effectively substitute for glycophorin. Thin-layer chromatography of lipid extracted from incubated virus and liposomes containing a small amount of a fluorescent phosphatidylcholine indicated that phosphatidylcholine in the vesicle is not chemically altered by functional interaction with the virus.
Mechanism of estrogen action: Indirect effect of estradiol-17β on proliferation of quail oviduct cells
Authors: Laugier Christian , Pageaux Jean-Francois , Soto Ana M. , Sonnenschein Carlos
Published: May 2006

Experimental data were collected to test whether the effect of estrogens required a direct action of the steroid on their target cells for induction of (
i
) cell multiplication and (
ii
) cell-type-specific protein synthesis. Ovariectomized quails were perfused for 24 hr with several doses of estradiol-17β (E
2
) (0.05-6.8 ng/min) through either the jugular vein or the portal vein. E
2
plasma concentrations increased progressively when the perfusion rate through the jugular vein was 0.5 ng/min and higher. With the portal vein, by contrast, E
2
plasma concentrations increased over the concentration in unperfused ovariectomized animals only when the perfusion rate was above 2 ng/min. An increase in DNA concentration per oviduct was observed regardless of the route of administration and the rate of perfusion, starting at 0.5 ng/min. Nuclear estrophilins increased when E
2
was perfused through the jugular vein at rates of 0.5 ng/min or greater. This same parameter was not increased in oviducts of quail perfused through the portal vein even at a perfusion rate of 2.0 ng/min. Progestophilins were induced in the oviducts of quail perfused through the jugular vein at rates of 2 ng/min and above; on the other hand, progestophilins were induced in birds perfused through the portal vein at rates above 2 ng/min. Ovalbumin was not induced in quail oviducts at any rate and route of perfusion. The induction of the synthesis of cell-type-specific protein (progestophilins, in this case) seems to require, however, the direct action of E
2
. The E
2
concentrations effecting the induction of progestophilins were higher than those necessary to effect the proliferation of oviduct cells. These results suggest that the E
2
effect on cell proliferation is indirect, it involves an intermediary step at the liver, and it does not require increased concentration of nuclear estrophilins.
Inhibition of Micrococcus luteus DNA topoisomerase I by UV photoproducts.
Authors: Pedrini A M , Ciarrocchi G
Published: May 2006

The activity of Micrococcus luteus DNA topoisomerase I on UV-irradiated supercoiled DNA was studied under either processive or distributive reaction conditions. Changes in DNA structure caused by UV irradiation reduce the rate of DNA relaxation at very low concentration of photoproducts. Under processive conditions the inhibition of the topoisomerase I by photoproducts can be quantitated by measuring the amount of substrate left in the replicative form I band. The mode of action of DNA topoisomerase I was affected by the presence of photoproducts in the DNA substrate, although the ability of the enzyme to form a covalent complex with UV-irradiated supercoiled DNA was not changed. The inhibition of topoisomerase I by UV photoproducts has been compared to the effects of single-stranded DNA and UV-irradiated duplex linear DNA on the enzyme, and the results suggest that the inhibition by photoproducts is caused by changes in the conformation of the supercoil. Our findings indicate the possibility that DNA topoisomerase I plays a role in repair.
Isolation and sequence characterization of a cDNA clone of human antithrombin III.
Authors: Chandra T , Stackhouse R , Kidd V J , Woo S L
Published: May 2006

A human liver cDNA library was constructed by using poly(A)-containing RNA isolated from a human liver biopsy specimen. This library is comprised of 40,000 independent transformants with an average inserted DNA length of 1,200 base pairs. By using the previously cloned baboon antithrombin III cDNA as a specific hybridization probe, greater than 30 human antithrombin III cDNA clones were identified from this library. The clone with the longest DNA insert was selected for sequence analysis. This antithrombin III cDNA clone contains 1,479 base pairs of inserted human DNA and was designated phATIII 113. It contains DNA sequences that code for a signal peptide and the entire mature antithrombin III protein which is comprised of 432 amino acid residues.
Protons block the dark current of isolated retinal rods.
Authors: Mueller P , Pugh E N
Published: May 2006

Membrane currents of isolated frog rods were recorded with the suction pipette technique and tested by perfusion techniques for their sensitivity to H+. The following facts have been established. (i) Increased [H+] suppresses the Na+ conductance of the outer segment rapidly and reversibly. (ii) H+ acts in the rod interior. (iii) The [H+] necessary to cause a 50% decrement in Na+ conductance is inversely related to the [Ca2+] over 5 orders of magnitude. (iv) The sensitivity to H+ and the sensitivity to light, as a function of [Ca2+], have the same slope. Thus, H+ act like light in effecting membrane current suppression but behave as if their effect is mediated through Ca2+. Based on these results and properties of rod disk membrane phosphodiesterase, we propose that protons produced in the light-activated hydrolysis of cGMP liberate Ca2+ from the disks by ion exchange.