bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of a yeast gene coding for arginine-specific carbamoyl-phosphate synthetase.
Authors: Lusty C J , Lu J
Published: May 2006

Several recombinant plasmids containing cpaII, the gene that encodes the large subunit of yeast arginine-specific carbamoyl-phosphate synthetase [carbamoyl-phosphate synthetase (glutamine-hydrolyzing), carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.3.5], have been isolated. The plasmids were selected by transformation of a yeast strain with a mutation in the structural gene of the large subunit of carbamoyl-phosphate synthetase. By using a recombinant pool with inserts of yeast nuclear DNA of 5-20 kilobase pairs, we obtained 13 transformants. Of five transformants studied, three have been found to have stable plasmid inserts. These plasmids could be amplified in Escherichia coli and transferred back into the yeast carbamoyl-phosphate synthetase-deficient strains with concomitant complementation of the nuclear mutation. Plasmids pJL2/T1 and pJL2/T5 contain identical nuclear DNA inserts of 5.9 kilobase pairs. Although the insert of plasmid pJL2/T3 is also 5.9 kilobase pairs long, the sequence overlap with pJL2/T1 and pJL2/T5 is only 4.5 kilobase pairs long. The T3 insert has an orientation in the vector opposite to that of the T1 and T5 inserts. The recombinant plasmids with the yeast cpaII gene fail to cross-hybridize with a cloned fragment of E. coli DNA containing the carA and carB genes for the bacterial carbamoyl-phosphate synthetase.
Epidermal growth factor carrier protein binds to cells via a complex with released carried protein nexin.
Authors: Knauer D J , Cunningham D D
Published: May 2006

Epidermal growth factor carrier protein (CP) is an arginine endopeptidase bound to epidermal growth factor (EGF) in vivo that processes pro-EGF to EGF and potentiates EGF action. Here, we provide a base for studying the biological functions of CP by showing that highly purified 125I-labeled CP, free of contaminating EGF, is specifically bound and internalized by normal human fibroblasts in serum-free medium. The characteristics of the binding reaction, however, were unusual and not consistent with direct interaction of CP with cell surface receptors. Subsequent experiments showed that cellular binding of 125I-labeled CP was mediated via a cell-secreted protein. We named the protein carrier protein nexin (CPN) because of its close functional similarity to protease nexin, which mediates cellular binding of thrombin or urokinase. Both CPN and protease nexin are secreted by cells, form covalent complexes with regulatory proteases in the extracellular environment, and mediate cellular binding of these proteases, apparently via a cell surface receptor for the nexin moiety of the complex. By several criteria, however, CPN and protease nexin are unique entities. This finding of a specific interaction of a growth factor carrier protein with cells suggests the possibility of additional physiological functions for these carriers in growth factor action or metabolism or both.
Developmental and hormonal regulation of protein H1 degrees in rodents.
Authors: Gjerset R , Gorka C , Hasthorpe S , Lawrence J J , Eisen H
Published: May 2006

The tissue and cellular distribution and regulation of the chromatin protein H1 degrees has been examined in developing and adult mouse and in rat. The protein appears in specific cell types of solid tissues only when the cells have terminated their maturation. This was found for brain, retina, striated and cardiac muscle, and liver. In tissues that depend on hormones for their function and maintenance, the expression of H1 degrees is dependent on the continued presence of the specific maintenance hormone. In regenerating rat liver the amount of H1 degrees decreases to one-third after the onset of DNA synthesis. The possible role of H1 degrees is discussed in light of these results.
Primary structure of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle.
Authors: Takio K , Smith S B , Krebs E G , Walsh K A , Titani K
Published: May 2006

The complete amino acid sequence of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle is presented. Primary fragments for the sequence determination were obtained by limited proteolysis with various proteases or by cleavage with cyanogen bromide. The sequence of the 400 amino acid residues has two homologous regions, strongly suggesting tandem gene duplication. The predicted secondary structure suggests the presence of 42% alpha-helix, 23% beta-strand, and 23 beta-turns. The molecular weight of the subunit, as derived from the sequence, is 45,084 including a phosphate group at residue 95. This is significantly less than earlier estimates based on NaDodSO4 gel electrophoresis and sedimentation experiments. The structure is discussed in terms of putative sites of interaction with cAMP and with the catalytic subunit.
Establishment of a cell line from lepidopteran wing imaginal discs: Induction of newly synthesized proteins by 20-hydroxyecdysone
Authors: Lynn D. E. , Miller S. G. , Oberlander H.
Published: May 2006

A cell line has been developed from the imaginal wing discs of the lepidopteran insect
Trichoplusia ni
(Hübner). The cells grow in suspension as fluid-filled, multicellular vesicles. The cell line has been designated IAL-TND1. Two-dimensional electrophoresis (isoelectric focusing and sodium dodecyl sulfate/polyacrylamide gels) of [
35
S]methionine-labeled proteins revealed similar patterns for IAL-TND1 cells and whole
T. ni
wing imaginal discs. Additionally, exposure to the insect steroid hormone 20-hydroxyecdysone stimulated the synthesis of the same new proteins in both whole wing discs and IAL-TND1 cell vesicles.
Frequency of UV-induced neoplastic transformation of diploid human fibroblasts is higher in xeroderma pigmentosum cells than in normal cells.
Authors: Maher V M , Rowan L A , Silinskas K C , Kateley S A , McCormick J J
Published: May 2006

If neoplastic transformation of diploid human cells results from carcinogen-induced mutations, cells deficient in excision repair of UV-induced DNA damage should be significantly more sensitive to transformation by UV light than normal cells. We tested this hypothesis by irradiating fibroblasts from a xeroderma pigmentosum patient (XP7BE, complementation group D) with low doses of Uv light (254 nm) and cells from a normal person with much higher doses and comparing the frequency of transformation to anchorage independence. Both sets of cells exhibited a dose-dependent increase in transformation which corresponded to a dose-dependent decrease in survival. At doses that caused equal cell killing, the frequency of anchorage-independent cells was approximately equal. Colonies of XP7BE and normal cells isolated from agar, propagated, and injected into X-irradiated athymic mice produced fibrosarcomas in 100% of the animals. Normal cells irradiated shortly before the onset of DNA synthesis exhibited a high frequency of anchorage-independent cells; cells irradiated in early G1 showed no increase over background. These results agree with those we observed for UV induction of 6-thioguanine-resistant mutants in these cells and support the hypothesis that anchorage independence results from mutations induced by DNA replication on a damaged template.
Protease-catalyzed peptide bond formation: application to synthesis of the COOH-terminal octapeptide of cholecystokinin.
Authors: Kullmann W
Published: May 2006

This study of protease-catalyzed peptide synthesis reports the preparation of the COOH-terminal octapeptide amide of cholecystokinin. The octapeptide was assembled by chemical condensation of two tetrapeptide segments that had been synthesized through the concerted catalytic reactions of several proteases of different specificities. The resulting octapeptide derivative was subjected to catalytic transfer hydrogenation, followed by sulfation of its tyrosine residue and removal of the N alpha-protecting group. The homogeneous target peptide was obtained after purification via partition chromatography, gel filtration, and ion-exchange chromatography. The synthetic octapeptide stimulated amylase release from pancreatic acinar cells.
F-actin in conifer roots
Authors: Pesacreta Thomas C. , Carley William W. , Webb Watt W. , Parthasarathy Mandayam V.
Published: May 2006

The distribution of F-actin in the complex tissues of a higher plant organ has been visualized by fluorescence labeling the roots of the conifers
Chamaecyparis obtusa
and
Pseudotsuga menziesii
with F-actin-specific fluorescent dye-conjugated phallicidin. F-actin is present in the parenchymatous cells of the vascular tissue. Some vascular parenchyma cells possess larger numbers of F-actin-containing structures (microfilament bundles) than are known to exist in any other higher plant cell. Tissue type appears to be an important determinant of the presence or absence of F-actin in a cell. For example, in contrast to vascular cells, cortical cells show no indication of fluorescence labeling of F-actin after incubation with fluorescent phallicidin. Cytoplasmic streaming is seen only in vascular cells and in a pattern that reflects the intracellular distribution of F-actin.
Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12.
Authors: Pearson G D , Mekalanos J J
Published: May 2006

Hybridization probes derived from the A and B subunit genes of the heat-labile enterotoxin (LT) of Escherichia coli were used to analyze DNA from Vibrio cholera strain 569B for cholera toxin gene sequences. Southern blot analysis indicated that the cholera toxin A and B subunit genes were each duplicated in the strain. One of the two toxin subunit gene pairs was cloned as a 5.1-kilobase DNA insert in plasmid pBR322. E. coli cells carrying the recombinant plasmid pJM17 were shown to produce cholera toxin, which was found to be largely cell associated. Protein chemical analysis indicated that the toxin was in its unnicked form and required additional proteolytic processing by trypsin to exhibit full toxicity in tissue culture. The alteration in E. coli of the secretion and proteolytic processing of cholera toxin parallels that previously observed for LT. An in vitro generated insertion mutation in the A subunit gene on pJM17 was shown to abolish production of the A chain but still allow production of the B chain. These observations, together with restriction mapping data, have demonstrated that the cholera toxin and LT genes are very similar in their genetic organization.
Neutron-capture therapy of human cancer: in vitro results on the preparation of boron-labeled antibodies to carcinoembryonic antigen.
Authors: Mizusawa E , Dahlman H L , Bennett S J , Goldenberg D M , Hawthorne M F
Published: May 2006

Two samples of 2-phenyl-1,2-dicarba-closo-[1-3H]dodecaborane(12) were prepared by treating 1-lithio-2-phenyl-1,2-dicarba-closo-dodecaborane(12) with 3H2O (0.1 and 5.0 Ci/ml, respectively). These tritiated phenylcarborane samples were subsequently converted to corresponding samples of p-[1,2-dicarba-closo-[1-3H]dodecaboran(12)-2-yl]benzenediazonium ion ([3H]DBD) suitable for azo-coupling reactions. Reaction of the two tritiated diazonium ion samples with 2-napthol resulted in the formation of an azo dye (epsilon = 1.98 X 10(4) M-1 cm-1 at 485 nm). Experiments relating absorbance to 3H activity proved the two [3H]DBD sources to have 3.81 X 10(11) and 2.45 X 10(13) cpm of 3H per mol of tritiated carborane substituent. Purified antibodies to carcinoembryonic antigen were coupled to the [3H]DBD and, after extensive dialysis, the average number of carborane moieties per antibody molecule was determined by measuring the 3H activity associated with a known protein concentration. Further examination of these tritiated carborane-labeled antibodies by affinity chromatography proved that boron labeling did not destroy their immunoreactivity. Correlations of azo-coupling conditions (reactant ratios, pH) with immunoreactivity and antibody protein recovery are presented.
Correlation of regional brain metabolism with receptor localization during ketamine anesthesia: combined autoradiographic 2-[3H]deoxy-D-glucose receptor binding technique.
Authors: Hammer R P , Herkenham M , Pert C B , Quirion R
Published: May 2006

LKB film autoradiography of 2-]3H]deoxy-D-glucose uptake shows that ketamine, administered in anesthetic doses, alters the pattern of metabolic activity in rat hippocampus. The labeled metabolic marker can be washed out of the slide-mounted tissue sections by preincubation to permit in vitro autoradiography of drug and neurotransmitter receptors in the same animal. In this way, opiate and phencyclidine receptor distributions may be correlated with patterns of glucose utilization in adjacent sections. If the observed relative enhancement of 2-deoxy-D-glucose uptake in the stratum moleculare of hippocampus reflects elevated metabolism in nerve terminals there, then the binding of ketamine to phencyclidine receptors on neurons in distant afferent sites, such as entorhinal cortex, may initiate the physiologic and metabolic effects.
"Onion skin" replication of integrated polyoma virus DNA and flanking sequences in polyoma-transformed rat cells: termination within a specific cellular DNA segment.
Authors: Baran N , Neer A , Manor H
Published: May 2006

Replication of integrated polyoma virus DNA and flanking cellular sequences was studied in an inducible line of polyoma-transformed rat cells, designated the LPT line, that contains a single viral integration site. Chromosomal DNAs were purified from LPT cells treated with the virus-inducing agent mitomycin C and from untreated cells and were digested with restriction enzymes. The digests were analyzed by the Southern blotting technique. The virus DNA and a recombinant plasmid containing flanking cell DNA were used as hybridization probes. The analysis showed that mitomycin C treatment caused a more than 10-fold amplification of restriction fragments extending up to about 2.0 kilobase pairs into the cellular DNA flanking one end of the viral insertions, defined as the left joint. Fragments extending beyond this region were not amplified. These results showed that (i) integrated polyoma virus DNA undergoes multiple rounds of replication in mitomycin C-treated LPT cells and (ii) the replication extends into the flanking sequences and is arrested within a 0.40-kilobase-pair cellular DNA segment located about 2.0 kilobase pairs beyond the left joint. This segment may include a terminator of a normal cellular replicon.
Anionic lipid headgroups as a proton-conducting pathway along the surface of membranes: a hypothesis.
Authors: Haines T H
Published: May 2006

Evidence has been gathering from several laboratories that protons in proton-pumping membranes move along or within the bilayer rather than exchange with the bulk phase. These experiments are typically conducted on the natural membrane in vivo or in vitro or on fragments of natural membrane. Anionic lipids are present in all proton-pumping membranes. Model studies on the protonation state of the fatty acids of liposomes containing entrapped water show that the bilayers always contain mixtures of protonated and deprotonated carboxylates. Protonated fatty acids form stable acid-anion pairs with deprotonated fatty acids through unusually strong hydrogen bonds. Such acid-anion dimers have a single negative charge, which is shared by the four negative oxygens of both headgroups. The two pK values of the resulting dimer will be significantly different from the pK of the monomeric species, so that the dimer will be stable over a wide pH range. It is proposed that anionic lipid headgroups in biological membranes share protons as acid-anion dimers and that anionic lipids thus trap and conduct protons along the headgroup domain of bilayers that contain such anionic lipids. Protons pumped from the other side of the membrane may enter and move within the headgroup sheet because the protonation rate of negatively charged proton acceptors is 5 orders of magnitude faster than that of water. Protons trapped in the acidic headgroup sheet need not leave this region in order to be utilized by a responsive proton-translocating pore (a transport protein using the proton gradient). Experiments suggest the proton concentration in the headgroup domain may vary widely and the anionic lipid headgroup sheet may therefore function as a proton buffer. Due to the Gouy-Chapman-Stern layer at polyanionic surfaces, anionic lipids will also sequester protons from the bulk solution at low and moderate ionic strengths. At high ionic strength metal cations may replace protons sequestered near the headgroups, but these cations cannot substitute for protons in the "proton-conducting pathway," which is based on hydrogen bonding.
Femtosecond photolysis of CO-ligated protoheme and hemoproteins: appearance of deoxy species with a 350-fsec time constant.
Authors: Martin J L , Migus A , Poyart C , Lecarpentier Y , Astier R , Antonetti A
Published: May 2006

Photolysis of HbCO, MbCO, and CO-protoheme has been investigated by measuring transient differential spectra and kinetics of induced absorption after excitation with a 250-fsec laser pulse at 307 nm. Probing was performed by a part of a continuum pulse between 395 and 445 nm. Photodissociation of the three liganded species occurred within the pulse duration. By contrast, the formation of deoxy species appeared with a mean (+/- SD) response time of 350 +/- 50 fsec. This time constant was identical for the three species and independent of the presence or absence of the protein structure. Our results suggest the formation of a transient high-spin in plane iron (II) species which relaxes in 350 fsec to a high-spin stable state with concerted kinetics of CO departure and iron displacement. The spin transition is suspected to occur via liganded excited states which relax in part to non-reactive states with a 3.2-psec time constant.
HLA-A2 and HLA-B7 antigens are phosphorylated in vitro by rous sarcoma virus kinase (pp60v-src) at a tyrosine residue encoded in a highly conserved exon of the intracellular domain.
Authors: Guild B C , Erikson R L , Strominger J L
Published: May 2006

HLA-A2 and -B7 antigens are phosphorylated by Rous sarcoma kinase (pp60v-src) in vitro. The phosphate group is attached to the heavy chains as determined by NaDodSO4/polyacrylamide gel electrophoresis. The site of phosphorylation was localized to the COOH-terminal intracellular domain by its susceptibility to limited trypsin proteolysis. Furthermore, the 32P-labeled amino acid is a single tyrosine residue located in the COOH terminus of the heavy chain. The protein sequences of known class I human and murine intracellular domains contain a highly conserved sequence -K-G-G-X-Y- located NH2-terminally to the single tyrosine residue of this domain. The DNA sequences that encode class I antigen intracellular domains were compared by computer with a homology matrix program. Exon 6 which encodes the conserved tyrosine-containing protein sequence in both human and mouse is 75% homologous across species and 90-100% homologous within species. The significance of the high degree of conservation within exon 6 is discussed.
Critique of a proposed stability criterion for chemical systems far from equilibrium
Authors: Escher Claus , Ross John
Published: May 2006

Noyes [Noyes, R. M. (1981)
Proc. Natl. Acad. Sci. USA
78, 7248-7249] has obtained a result for the Gibbs free energy per unit mass in a continuous flow stirred tank reactor, which states that the steady-state free energy increases monotonically with decreasing residence time for any stable stationary point. We show that his derivation is based on an erroneous stability criterion. We further investigate the dependence of the Gibbs free energy on residence time for the case of mass-action kinetics and ideal mixtures. We then apply this development to Schlögl's trimolecular reaction mechanism [Schlögl, F. (1971)
Z. Phys
. 248, 446-458] and show that Noyes' conjecture is violated for this simple example.
Isolation and partial characterization of a new human collagen with an extended triple-helical structural domain.
Authors: Bentz H , Morris N P , Murray L W , Sakai L Y , Hollister D W , Burgeson R E
Published: May 2006

The collagens are a family of major connective tissue proteins that are known to be the products of at least 11 distinct genes. Primary structural differences between the individual collagen types are thought to reflect functional diversity. We have isolated a previously unknown collagen gene product, termed "long-chain" (LC) collagen, from human chorioamniotic membranes by limited pepsin digestion. Comparison of the isolated alpha-chain subunit to the alpha chains of other collagen types by amino acid composition, peptide mapping with either cyanogen bromide fragmentation or staphylococcal V8 protease digestion, chromatographic elution position, and relative molecular weight indicates that this protein is a product of a previously unrecognized gene. We report structural studies indicating that this molecule contains three identical alpha-chain subunits that are each approximately molecular weight 170,000. The amino acid composition of LC alpha chains suggests that they are about 90% triple helical. Comparisons of the length of segment-long-spacing (SLS) crystallites made from LC molecules with those from types I and V collagens indicate that the LC molecule is substantially longer than these collagens and somewhat longer than the reported length of type IV collagen. This finding suggests that LC collagen represents an additional class of collagen molecules. We suggest that these molecules be referred to as type VII collagen.
Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.
Authors: Cenatiempo Y , Twardowski T , Redfield B , Reid B R , Dauerman H , Weissbach H , Brot N
Published: May 2006

An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val. The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNAi, and rabbit liver or Escherichia coli Val-tRNA1Val. Picomole quantities of fMet-Val are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure. In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMet-Val-His are synthesized, corresponding to the NH2-terminal sequence of alpha- and beta-globin, respectively. Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the alpha- and beta-globin mRNA species.
A 64-kilodalton membrane protein of Bacillus subtilis covered by secreting ribosomes.
Authors: Horiuchi S , Tai P C , Davis B D
Published: May 2006

The complexed (ribosome-bearing) membrane fraction of Bacillus subtilis contains several proteins (CM-proteins) that are virtually absent from the ribosome-free fraction and hence might be components of the apparatus of protein secretion. We have determined, by trypsin digestion and by labeling with a nonpenetrating reagent (diazoiodosulfanilic acid), the accessibility of four of these proteins on the two surfaces of the membrane, as exposed either in protoplasts or in inverted membrane vesicles. The 68-kilodalton protein is a transmembrane protein and the 45-kilodalton protein faces only the external surface, whereas the 31-kilodalton protein is inaccessible from either side. Of particular interest is the 64-kilodalton protein: it can be digested by trypsin, and can bind antibody, on the cytoplasmic surface, but only after the ribosomes have been released. This protein is thus evidently a component of the apparatus of protein secretion, closely covered by secreting ribosomes. Whether the other CM-proteins are also involved in protein secretion is uncertain.
Uncoupling of oxidative phosphorylation in rat liver mitochondria by general anesthetics.
Authors: Rottenberg H
Published: May 2006

The general anesthetics chloroform and halothane inhibit ATP synthesis in rat liver mitochondria, in the millimolar concentration range (1-12 mM), in parallel with a reduction of respiratory control and the ratio of ATP produced to oxygen consumed. In these effects, halothane and chloroform are similar to classical, protonophoric, uncouplers. The rate of ADP-stimulated respiration or the rate of uncoupler-stimulated respiration is not affected. Like classical uncouplers, halothane and chloroform also stimulate mitochondrial ATPase activity. However, the extent of stimulation by these agents is larger than by protonophoric uncouplers and, more significantly, ATPase activity stimulated by carbonylcyanide m-chlorophenylhydrazone is further stimulated by these agents. In the presence of the Ca2+ chelator EGTA, halothane and chloroform have no measurable effect on the magnitude of the proton electrochemical potential, delta mu H. In the absence of EGTA these anesthetics have a small effect on delta mu H, apparently due to stimulation of Ca2+ cycling. Under these conditions the membrane potential is decreased while delta pH is increased, but the total value of delta mu H is only slightly decreased. The uncoupling activity of the anesthetics is the same in the presence of absence of EGTA. Thus, in contrast to protonophoric uncouplers, the uncoupling effect of general anesthetics does not depend on the collapse of delta mu H. In the same concentration range in which anesthetics uncouple oxidative phosphorylation both halothane and chloroform increase membrane fluidity, as measured by the partitioning of the hydrophobic spin probe 5-doxyldecane. These findings suggest a role for intramembrane processes in energy conversion that is not dependent on the bulk delta mu H.
Spider leg autotomy induced by prey venom injection: An adaptive response to “pain”?
Authors: Eisner Thomas , Camazine Scott
Published: May 2006

Field observations showed orb-weaving spiders (
Argiope
spp.) to undergo leg autotomy if they are stung in a leg by venomous insect prey (
Phymata fasciata
). The response occurs within seconds, before the venom can take lethal action by spread to the body of the spiders. Autotomy is induced also by honeybee venom and wasp venom, as well as by several venom components (serotonin, histamine, phospholipase A
2
, melittin) known to be responsible for the pain characteristically elicited by venom injection in humans. The sensing mechanism by which spiders detect injected harmful chemicals such as venoms therefore may be fundamentally similar to the one in humans that is coupled with the perception of pain.
Manifold amplification of in vivo immunity in normal and immunodeficient mice by ribonucleosides derivatized at C8 of guanine.
Authors: Goodman M G , Weigle W O
Published: May 2006

Antigen-specific lymphocyte activation is generally thought to be initiated by the binding of antigen to specific membrane receptors, followed by transduction of a signal across the cell membrane. In addition, at least two supplementary signals apparently are required for induction of specific antibody synthesis. Various exogenous agents have been shown to augment the magnitude of specific responses to antigen. Certain of these adjuvants are polyclonal activators as well. We recently described a new class of activator, the C8-substituted-guanine ribonucleosides, that traverses the cell membrane and bypasses classical triggering mechanisms to activate the lymphocyte at an intracellular site. The current studies demonstrate that this new class of activator exerts powerful in vivo adjuvant activity when administered to mice after antigen. This effect is highly dose- and time-dependent. When nucleoside administration is delayed until 3-4 days after immunization, enhancement of the response persists but the optimal dose of nucleoside decreases by 2 orders of magnitude, indicating that these compounds are highly effective modulators of the immune response well after the initiating events have occurred. Amplification of the antibody response occurs over a wide range of antigen concentrations but is maximal at optimal antigenic concentrations. Enhancement of antibody responses in vivo is absolutely dependent upon derivatization of guanosine at C8; native guanosine is ineffective. When administered after antigen (sheep erythrocytes) to male (CBA/N X CBA/CaJ)F1 mice (which bear the xid B-cell defect) in an effort to bolster their impaired response to this T cell-dependent antigen, the brominated nucleoside induced a degree of responsiveness equivalent to that generated in the normal (female) counterparts injected with antigen and 8-bromoguanosine.
Visualization of human C4b-binding protein and its complexes with vitamin K-dependent protein S and complement protein C4b.
Authors: Dahlbäck B , Smith C A , Müller-Eberhard H J
Published: May 2006

C4b-binding protein (C4bp) participates in the regulation of the C3 convertase of the classical pathway of complement. By binding to C4b, which is one of the structural subunits of this enzyme, C4bp accelerates the decay-dissociation of the enzyme and renders C4b susceptible to degradation by factor I (C3b inactivator). C4bp is a high molecular weight plasma protein (Mr = 570,000) composed of apparently identical subunits (Mr = 70,000) linked by disulfide bonds. In plasma and in purified form C4bp also forms a bimolecular complex (Kd = 0.9 X 10(-7) M) with protein S, a recently identified vitamin K-dependent plasma protein. The binding sites on C4bp for protein S and C4b are distinct and noncompetitive and protein S does not influence the function of C4bp as a regulator of the C3 convertase. C4bp, C4b, and protein S were visualized by electron microscopy by negative staining. C4bp was found to have an unusual spider-like structure. It is composed of seven thin (30 A), elongated (330 A), and flexible subunits that are linked to a small central body. Protein S exhibited two globular domains of equal size with a center-to-center distance of approximately equal to 50 A. Protein S was found to bind to the C4bp through only one of its domains by attaching to a short subunit that is distinct from the other seven subunits. C4b imaged as an irregular, relatively compact molecule. It was found to interact with the peripheral ends of the elongated subunits, suggesting seven C4b-binding sites per molecule of C4bp.
Inhibition of growth of a prolactin-secreting pituitary tumor in rats by analogs of luteinizing hormone-releasing hormone and somatostatin.
Authors: de Quijada M G , Redding T W , Coy D H , Torres-Aleman I , Schally A V
Published: May 2006

We investigated the effects of [D-Trp6]LH-RH [agonistic analog of luteinizing hormone-releasing hormone (LH-RH)], N-Ac-[D-p-Cl-Phe1,2,D-Trp3,D-Phe6,D-Ala10]LH-RH (antagonistic analog), and [D-5-methoxy-Trp8]somatostatin (somatostatin analog) on the growth of the prolactin and corticotropin-secreting pituitary tumor 7315a in female Buffalo rats. Chronic administration of [D-Trp6]LH-RH in a dose of 25 micrograms/day, starting 18 days after inoculation with the tumor, inhibited the growth of the pituitary tumor. Tumor weight and volume also were reduced when this agonist was administered 3 days after inoculation. The antagonistic LH-RH analog, injected in a dose of 50-100 micrograms for 14-24 days, also significantly inhibited the growth of pituitary tumor. Chronic administration of the somatostatin analog in a dose of 25 micrograms twice a day likewise decreased tumor weights in comparison with controls. The inhibition of pituitary tumor growth by LH-RH agonist, LH-RH antagonist, and somatostatin analog was accompanied by a decrease in serum prolactin levels. It was concluded that LH-RH agonist, LH-RH antagonist, and somatostatin analog can inhibit the growth of estrogen-dependent prolactin/corticotropin-secreting pituitary tumor in rats.
Nucleotide sequence of Abelson murine leukemia virus genome: structural similarity of its transforming gene product to other onc gene products with tyrosine-specific kinase activity.
Authors: Reddy E P , Smith M J , Srinivasan A
Published: May 2006

The nucleotide sequence of the proviral genome of Abelson murine leukemia virus (A-MuLV), an acute transforming virus of murine origin, has been determined. Like other transforming viruses, A-MuLV contains sequences derived from its helper virus, Moloney murine leukemia virus (M-MuLV), and a cell-derived protooncogene (abl) insertion sequence. By comparison of the A-MuLV sequence with that of M-MuLV, it was possible to precisely localize and define sequences contributed by the host cellular DNA. From the nucleotide sequence, we have predicted the amino acid sequence of p120gag-abl, the product of the A-MuLV gag-abl hybrid gene. The amino acid sequence of the putative abl gene, when compared with the sequences of other tyrosine-specific protein kinases (src, fes, fps, and yes), revealed significant homologies, indicating that all these functionally related transforming genes are derived from divergent members of the same protooncogene family. In addition to the gag-abl sequence, the proviral genome was found to contain an additional open reading frame that could code for an 18,000-dalton protein, whose role is at present undetermined.
Isolation of novel human genomic DNA clones related to human interferon-beta 1 cDNA.
Authors: Sehgal P B , May L T , Sagar A D , LaForge K S , Inouye M
Published: May 2006

Southern blot-hybridization analyses of human DNA (from Namalwa lymphoblastoid cells) digested with the restriction endonuclease EcoRI were carried out under optimal conditions with two human fibroblast interferon (IFN-beta 1) cDNA probes, pD19 and pD24, which contain IFN-beta 1 inserts 0.8 and 0.7 kilobase (kb) long, respectively. The analyses revealed the presence of several hybridizable DNA fragments, including two of lengths 6.8 and 5.5 kb, in addition to the classical IFN-beta 1 genomic DNA fragment of length approximately equal to 2.0 kb. We have screened a human DNA library in lambda bacteriophage Charon 4A by using a 32P-labeled IFN-beta 1 insert cDNA (pD24) and thereby isolated six strongly positive human genomic DNA clones. One of these (lambda B37) represents the classical human IFN-beta 1 gene; another (lambda B37) contains a 6.8-kb EcoRI DNA fragment(s) which cross-hybridizes with the IFN-beta 1 cDNA insert probes pD19 and pD24; and the remaining four (which are identical to each other and are exemplified by lambda B4) contain two EcoRI DNA fragments approximately 5.5 and 9 kb long which also cross-hybridize the IFN-beta 1 cDNA probes. A mRNA 0.9 kb long derived from the classical IFN-beta1 gene is expressed in poly(I) . poly(C)-induced human diploid fibroblasts (FS-4 strain). Induced FS-4 cells also contain polyadenylylated RNA 1.8, 3, 5, and approximately equal to 8 kb long derived from the lambda B3 gene, all of which appear to code for biologically active human IFN-beta as tested by using the Xenopus laevis oocyte translation assay. These data strongly indicate that lambda B3 represents a novel functional IFN-beta gene. A 12-kb polyadenylylated RNA, derived from lambda B4, is expressed constitutively at a low level in FS-4 cells, but the amount of this RNA increases 5-7 hr after exposure of the cells to poly(I) . poly(C).
Homology of amino acid sequences of rat liver cathepsins B and H with that of papain.
Authors: Takio K , Towatari T , Katunuma N , Teller D C , Titani K
Published: May 2006

The amino acid sequences of rat liver lysosomal thiol endopeptidases, cathepsins B and H, are presented and compared with that of the plant thiol protease papain. The 252-residue sequence of cathepsin B and the 220-residue sequence of cathepsin H were determined largely by automated Edman degradation of their intact polypeptide chains and of the two chains of each enzyme generated by limited proteolysis. Subfragments of the chains were produced by enzymatic digestion and by chemical cleavage of methionyl and tryptophanyl bonds. Comparison of the amino acid sequences of cathepsins B and H with each other and with that of papain demonstrates a striking homology among their primary structures. Sequence identity is extremely high in regions which, according to the three-dimensional structure of papain, constitute the catalytic site. The results not only reveal the first structural features of mammalian thiol endopeptidases but also provide insight into the evolutionary relationships among plant and mammalian thiol proteases.
Neutron scattering studies of the isolated C1r2C1s2 subunit of first component of human complement in solution.
Authors: Boyd J , Burton D R , Perkins S J , Villiers C L , Dwek R A , Arlaud G J
Published: May 2006

The subunit complex C1r2C1s2 of the first component of complement was investigated by small-angle neutron scattering in both the activated and unactivated forms. From these experiments, a molecular weight of 390,000 for C1r2C1s2 was found. The matchpoint was determined to be 43% 2H2O. Both results are consistent with composition data. The partial specific volume is 0.751 ml/mg. The radius of gyration at infinite contrast was found to be 17 nm for C1r2C1s2 and 1.1 nm for the cross section. Models for C1r2C1s2 were computed by the method of hard spheres, in which C1r2C1s2 was represented by spheres 0.87 nm diameter arranged in a straight rod of length 59 nm and a circular cross section of 3.2 nm. This rod can be bent at one or two places by up to 60 degrees without significant effect on the calculated radii of gyration. The model is in agreement with published ultracentrifugation and electron microscopy data.
Ultrasonic absorption evidence for structural fluctuations in frog virus 3 and its subparticles.
Authors: Robach Y , Michels B , Cerf R , Braunwald J , Tripier-Darcy F
Published: May 2006

The structural fluctuations specific to self-assemblies of biological molecules have been investigated further with ultrasonic techniques by using frog virus 3 (FV3). We compared the ultrasonic properties of complete FV3 virions and of several subparticles that may be obtained from this DNA virus: (i) the central nucleoprotein core versus its component DNA and proteins in a dissociated state; (ii) the core versus the capsidless subparticle, consisting of the core surrounded by the lipid membrane; and (iii) the complete virus versus the capsidless subparticle. The ultrasonic absorption by the core particle was quite large compared with the absorption by other nucleoprotein assemblies, suggesting that the core contains some organized structure. Both the core and the complete virus absorbed ultrasound more than did the capsidless subparticle. The difference spectrum for the virion relative to the capsidless subparticle may represent a single relaxation and is analyzed, by using a recent model, in terms of volume fluctuations due to radial movements in the virion. These fluctuations are much smaller than can be detected in virus crystals with present-day x-ray techniques.
Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase
Authors: Shinozaki Kazuo , Yamada Chieko , Takahata Naoyuki , Sugiura Masahiro
Published: May 2006

Ribulose-1,5-bisphosphate carboxylase/oxygenase consists of large subunits (LS) and small subunits. In plants, the LS is encoded in chloroplast DNA and the small subunit, in nuclear DNA. In cyanobacteria, both subunits are thought to be encoded in chromosomal DNA because of prokaryotes. The gene for the LS of ribulose-1,5-bisphosphate carboxylase/oxygenase from a cyanobacterium,
Anacystis nidulans
6301, has been cloned in pBR322 and subjected to sequence analysis. The coding region contains 1,416 base pairs (472 codons). The deduced amino acid sequence of
A. nidulans
LS protein shows 80% homology with sequences of maize, spinach, and tobacco LS proteins; the nucleotide sequence of
A. nidulans
LS gene shows 70% homology with sequences of the plant genes. Between
A. nidulans
LS and the plant LS proteins there is exact sequence homology around the lysine residue to which the activator CO
2
binds and around the two lysine residues to which ribulose 1,5-bisphosphate binds. The amino acid sequence where the LS binds to the small subunit is also highly conserved. From comparison of the LS proteins of
A. nidulans
and the three plants, the rate of amino acid substitution is estimated to be 0.25-0.5 × 10
-9
per year per site, which is far below the median value of various types of proteins (1.2 × 10
-9
for hemoglobin α). The LS protein is thus a conserved protein.
Isolation and physical mapping of the gene encoding the major sigma factor of Bacillus subtilis RNA polymerase.
Authors: Price C W , Gitt M A , Doi R H
Published: May 2006

At least four sigma factors separately bind the Bacillus subtilis RNA polymerase core (beta beta' alpha 2), each conferring a different promoter specificity on the holoenzyme in vitro. Using the Broome-Gilbert immunological screening, we isolated recombinant lambda phages that carry rpoD, the gene for the most abundant sigma factor, sigma 55. These phages encode a 55,000-dalton protein whose size, immunological properties, and peptide map identify it as sigma 55. All the phages have in common two adjacent 3.5-kilobase EcoRI fragments from the B. subtilis chromosome; most carry additional genomic DNA. Deletion analysis localized rpoD to a 1.6-kilobase region, suggested the direction of its transcription, and found two additional genes near rpoD, which code for proteins of 62,000 and 17,000 daltons.
Molecular area effects in
Cepaea
Authors: Ochman Howard , Jones J. S. , Selander Robert K.
Published: May 2006

Enzyme polymorphisms in the land snail
Cepaea nemoralis
in the central Pyrenees show concordant geographic patterns of strong differentiation that are not correlated with the distributions of characters of shell color and banding or with the major pattern of variation in climate and vegetation type. Three regions of relative genetic uniformity separated by steep clines in allele frequencies are designated as “molecular area effects.” A model of allopatric differentiation of populations in temporary geographic isolation during the last period of Pleistocene glaciation, followed by invasion of the Pyrenees and hybridization in secondary contact, is proposed to account for the present-day pattern of genetic differentiation. The genetic structure of the Pyrenean populations of
C. nemoralis
is not interpretable in terms of stasipatric or parapatric models of speciation.
Regulation of carbohydrate metabolism by 2,5-anhydro-D-mannitol.
Authors: Riquelme P T , Wernette-Hammond M E , Kneer N M , Lardy H A
Published: May 2006

In hepatocytes isolated from fasted rats, 2,5-anhydromannitol inhibits gluconeogenesis from lactate plus pyruvate and from substrates that enter the gluconeogenic pathway as triose phosphate. This fructose analog has no effect, however, on gluconeogenesis from xylitol, a substrate that enters the pathway primarily as fructose 6-phosphate. The sensitivity of gluconeogenesis to 2,5-anhydromannitol depends on the substrate metabolized; concentrations of 2,5-anhydromannitol required for 50% inhibition increase in the order lactate plus pyruvate less than dihydroxyacetone less than glycerol less than sorbitol less than fructose. The inhibition by 2,5-anhydromannitol of gluconeogenesis from dihydroxyacetone is accompanied by an increase in lactate formation and by two distinct crossovers in gluconeogenic-glycolytic metabolite patterns-i.e., increases in pyruvate concentrations with decreases in phosphoenolpyruvate and increases in fructose-1,6-bisphosphate concentrations with little change in fructose 6-phosphate. In addition, 2,5-anhydromannitol blocks the ability of glucagon to stimulate gluconeogenesis and inhibit lactate production from dihydroxyacetone. 2,5-Anhydromannitol decreases cellular fructose 2,6-bisphosphate content in hepatocytes; therefore the effects of the fructose analog are not mediated by fructose 2,6-bisphosphate, a naturally occurring allosteric regulator. 2,5-Anhydromannitol also inhibits gluconeogenesis in hepatocytes isolated from fasted diabetic rats, but higher concentrations of the analog are required.
Computer simulation of the dynamics of hydrated protein crystals and its comparison with x-ray data.
Authors: van Gunsteren W F , Berendsen H J , Hermans J , Hol W G , Postma J P
Published: May 2006

The structure and dynamics of the full unit cell of a protein (bovine pancreatic trypsin inhibitor) containing 4 protein molecules and 560 water molecules have been simulated by using the molecular dynamics method. The obtained structure, atom positional fluctuations, and structure factors are compared with x-ray values. A way of calculating the motional contributions to structure factors is proposed.
NMR study of slowly exchanging imino protons in yeast tRNAasp.
Authors: Figueroa N , Keith G , Leroy J L , Plateau P , Roy S , Gueron M
Published: May 2006

We have monitored the exchange of imino and amino protons by NMR after quick transfer of yeast tRNAAsp in 2H2O solvent. When the concentration of exchange-catalyzing buffer is not too high, one imino proton exchanges considerably more slowly than any other (e.g., 100 hr versus 4 hr for the second-slowest imino proton at 18 degrees C in 15 mM Mg). This provides excellent conditions for identification, by the nuclear Overhauser effect, of the slowest exchanging proton, which we show to be the imino proton of the U-8 . A-14 reverse Hoogsteen tertiary-structure base pair; other slowly exchanging protons are identified as imino protons from A . U-11 and G . psi-13. In preliminary experiments, we find that the exchange of these protons is catalyzed by cacodylate or Tris buffer. The lifetimes of two other imino protons, ca. 10 min at 28 degrees C, are buffer independent. Slowly exchanging amino protons have also been observed. Correlation with the exchange of the uracil-8 imino proton suggests that they may be from adenine-14.
Immunocytochemical localization of alpha-D-mannosidase II in the Golgi apparatus of rat liver.
Authors: Novikoff P M , Tulsiani D R , Touster O , Yam A , Novikoff A B
Published: May 2006

Mannosidase II is involved in the trimming of alpha-1,6-mannosyl residues during the biosynthesis of glycoproteins containing N-linked oligosaccharides of the complex type. A highly specific polyclonal antibody (IgG) was isolated from rabbits immunized with a homogeneous preparation of mannosidase II prepared from rat liver. With this antibody, light and electron microscopic immunocytochemical studies on rat liver reveal that essentially all mannosidase II in hepatocytes is localized in the Golgi apparatus, the only other site with reaction product being the endoplasmic reticulum. The indirect immunocytochemical method used in this study involved three major steps: exposure of aldehyde-fixed tissue to immune and nonimmune IgG, treatment with staphylococcal protein A labeled with horseradish peroxidase, and incubation in diaminobenzidine to reveal sites of peroxidase activity. The procedures described overcome major problems in immunocytochemistry, allowing preservation of antigenic sites and maintaining adequate ultrastructural integrity. The in situ localization of other carbohydrate-processing enzymes, involved in either trimming or attachment of sugar residues, should be possible with this procedure. Because biosynthetic precursors of the processing enzymes may be revealed by an immunocytochemical approach, it is potentially significant that mannosidase II reaction product is present in areas of the endoplasmic reticulum as well as in the Golgi apparatus.