bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacteriochlorophyll electronic transition moment directions in bacteriochlorophyll
a
-protein
Authors: Pearlstein Robert M. , Hemenger Richard P.
Published: May 2006

The low-temperature 800-nm band absorption and circular dichroism spectra of the bacteriochlorophyll (Bchl)
a
-protein from
Prosthecochloris aestuarii
strain 2K are analyzed theoretically. These spectra show considerable structure that is attributed primarily to resonance (exciton) interactions among the lowest singlet transitions of the Bchl
a
molecules contained in each protein. We calculate these spectra from the known arrangement of the Bchl molecules in the protein. With the conventional assignment of the lowest singlet transition of Bchl
a
as

Q
y

(
y
-polarized), agreement of calculated spectra with experiment is poor. All of our attempts, based on this conventional assignment, to improve the theoretical fits to absorption and circular dichroism spectra simultaneously are unsuccessful. However, by making the simple but unconventional assumption that the lowest singlet transition in each of the Bchl
a
molecules in each protein is
x
-polarized rather than
y
-polarized, we find good agreement between calculated and observed spectra. If these results are not fortuitous, they indicate that there is a systematic error in the protein structural model, that the conventional assignments of Bchl
a
transitions are incorrect, or that the protein environment provides a sufficiently strong perturbation to rotate the lowest singlet transition moment direction by ∼90°, presumably by changing the order of certain of the Bchl
a
orbitals.
Coordinate control of corticotropin, β-lipotropin, and β-endorphin release in mouse pituitary cell cultures
Authors: Allen Richard G. , Herbert Edward , Hinman Michael , Shibuya Haruo , Pert Candace B.
Published: May 2006

Hypothalamic extract stimulates the release of corticotropin (ACTH) and endorphins 2.5- to 30-fold in mouse pituitary tumor cell cultures (AtT-20/D
16v
line) and primary cell cultures from mouse anterior pituitary. ACTH and endorphin activities were measured by radioimmunoassay and immunoprecipitation. Pretreatment of tumor cell cultures with 1 μM dexamethasone reduced the stimulatory effect of the extract on release of ACTH and endorphins. Pretreatment of primary cell cultures with 10
-6
M dexamethasone reduced the stimulatory effect of both vasopressin and the extract on the release of ACTH and endorphins. Release of ACTH and endorphin was coupled in both kinds of cultures in the basal, stimulated, and inhibited states. The molecular weight forms of ACTH and endorphin in tumor cell culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Radioimmunoassay and immunoprecipitation show that the 13,000-dalton and 4500-dalton forms of ACTH were present in about equal amounts in medium from cultures incubated with or without hypothalamic extract for 15 min, 30 min, or 2 hr. Smaller amounts of the high molecular weight forms of ACTH (20,000- to 23,000-dalton and 31,000-dalton ACTH) were observed in the culture medium at these times. The predominant forms of endorphin released after 20 min or 3 hr of incubation had molecular weights of 31,000, 11,700 (β-lipotropic hormone-size material) and 3500 (β-endorphin-size material). No degradation of the forms of endorphin released into the culture medium was observed after incubating the culture medium for 1.5 hr in the absence of cells. The proportions of the different forms of endorphin and ACTH present in the culture medium resembles that seen in cell extracts.
Receptor-mediated uptake of low density lipoprotein reconstituted with 25-hydroxycholesteryl oleate suppresses 3-hydroxy-3-methylglutaryl-coenzyme A reductase and inhibits growth of human fibroblasts
Authors: Krieger Monty , Goldstein Joseph L. , Brown Michael S.
Published: May 2006

The free and esterified cholesterols of plasma low density lipoprotein (LDL) were extracted with heptane and replaced with 25-hydroxycholesteryl oleate. The resulting particle, designated r-[25-HC oleate]LDL, bound to LDL receptors on human fibroblasts, was taken up by adsorptive endocytosis and was hydrolyzed in lysosomes in a manner similar to that of native LDL. The r-[25-HC oleate]LDL suppressed 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate:NADP
+
oxidoreductase (CoA-acylating), EC 1.1.1.34], the enzyme catalyzing the rate-limiting step in cholesterol biosynthesis. This suppression did not occur when lysosomal hydrolysis of r-[25-HC oleate]LDL was inhibited by chloroquine. When fibroblasts were incubated with r-[25-HC oleate]LDL in the absence of a source of cholesterol, the cells developed an abnormal morphology, their growth was inhibited, and the cells died. The toxic effects of r-[25-HC oleate]LDL were prevented when the growth medium was supplemented with cholesterol in ethanol or with mevalonate, the product of the reductase reaction. These data suggest that the toxicity of r-[25-HC oleate]LDL was due to its suppression of reductase, which in turn caused cellular cholesterol deficiency. The r-[25-HC oleate]LDL did not suppress reductase activity nor did it alter the growth or morphology of mutant fibroblasts lacking LDL receptors, which were obtained from a patient with homozygous familial hypercholesterolemia. These experiments demonstrate the feasibility of using reconstituted LDL to selectively deliver hydrophobic compounds other than typical cholesteryl esters to cells possessing LDL receptors.
Does environmental variability limit niche overlap?
Authors: Turelli Michael
Published: May 2006

A stochastic theory of limiting similarity is presented that attempts to quantify the relationship between tolerable niche overlap among competing species and the degree of environmental fluctuation. The theory is based on a heuristic analytical approximation that provides conditions under which a rare invading species can increase in the presence of a community of established competitors. The major qualitative conclusion, derived from investigating two symmetric, discrete-time, stochastic analogs of the Lotka-Volterra competition equations, is that weak to moderate stochastic variation does not appear to limit significantly the similarity of competing species. This result is in sharp contrast to the conclusions of May and MacArthur's pioneering study of stochastic limiting similarity. A possible reason for this discrepancy is explored.
Nature of differential sympathetic discharges in chemoreceptor reflexes
Authors: Kollai Mark , Koizumi Kiyomi , McC. Brooks Chandler
Published: May 2006

In a study of autonomic reflexes it was found that some produce a generalized, bilaterally uniform response whereas others have an asymmetric or laterality of action. Recordings from vertebral nerve fibers (mainly vasoconstrictors to forelimb muscles), right and left cardiac sympathetics, and renal nerves show that baroreceptors evoke a bilaterally uniform inhibition but chemoreceptors of the carotid sinus and aortic arch initiate a differential discharge. In the chemoreceptor reflex the vagi are activated and bradycardia generally occurs. Vertebral and renal sympathetic fibers increase their activity bilaterally commensurate with the increase in arterial pressure. Sympathetic discharges to the heart, however, are not uniform; they show ipsilateral inhibition and a strong contralateral increase in activity. Stabilization of blood pressure or inactivation of baroreceptors abolishes the ipsilateral inhibition. In isolation, therefore, the chemoreceptor-induced cardiac sympathetic discharge is just quantitatively stronger contralaterally. In the absence of vagi, heart rate changes differ depending on which chemoreceptors are stimulated, because the pacemaker is on the right. Asymmetrical discharges do occur and, in the eventual response to stimulation of chemoreceptors, reflex interactions actually augment the laterality of effects. Peripheral interactions, in the sense that changes effected by one may induce another reflex, are responsible in part for the balances of autonomic activity ultimately seen as the body reacts to stimuli.
Cluster characterization in iron-sulfur proteins by magnetic circular dichroism.
Authors: Stephens P J , Thomson A J , Keiderling T A , Rawlings J , Rao K K , Hall D O
Published: May 2006

We report magnetic circular dichroism (MCD) spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothermophilus ferredoxin and Clostridium pasteurianum ferredoxin. The MCD is found to vary significantly with cluster oxidation state but is relatively insensitive to the nature of the protein. The spectra obtained are compared with the corresponding spectra of iron-sulfur proteins containing 2-Fe clusters. It is concluded that MCD is useful for the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins and is superior for this purpose to absorption and natural circular dichroism spectroscopy.
Contacts between Escherichia coli RNA polymerase and a lac operon promoter.
Authors: Johnsrud L
Published: May 2006

The chemical alkylating agent dimethyl sulfate can probe the interaction between Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and the purine bases of a promoter. This agent methylates the N7 position on guanine or the N3 position on adenine; the bound protein can either protect these positions or affect the reactivity to produce an enhanced methylation. The pattern of DNA residues in the lactose promoter protected from, or enhanced to, methylation by a specifically bound polymerase shows that the enzyme covers a region of at least 38 base pairs, stretching upstream from the origin of transcription. These protein-DNA contacts occur predominantly in the major groove of the DNA helix. Furthermore, this pattern of methylation shows that the polymerase unwinds the helix at the origin of transcription. The relationship between polymerase-DNA contacts defined by dimethyl sulfate and known features of promoter structure is discussed. To facilitate these experiments I have constructed a plasmid that permits a unique 5'-end labeling of each strand of a 95-base-pair fragment containing a lac operon promoter. This plasmid contains two copies of the lac promoter-operator region.
Rotational motion of the sarcoplasmic reticulum Ca2+-ATPase.
Authors: Thomas D D , Hidalgo C
Published: May 2006

Using saturation transfer electron paramagnetic resonance, we have detected the rotational motion of a spin label rigidly attached to the sarcoplasmic reticulum Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). At 4 degrees C, the spectrum indicates an effective rotational correlation time of 60 microsec, determined by comparison with reference spectra obtained from theoretical calculations and from experiments on model systems. This motion appears to correspond to rotation of the enzyme with respect to the membrane, because the motion persists when the membrane fragments are immobilized by sedimentation and the motion stops when the polypeptide chains, but not the membrane vesicles, are crosslinked by glutaraldehyde. The rotational mobility of the enzyme increases with increasing temperature, and this increase becomes more gradual when the temperature exceeds 20 degrees C; the same kind of temperature dependence has been observed previously for lipid fluidity and enzymatic activity.
Fibrinogen synthesis in serum-free hepatocyte cultures: Stimulation by glucocorticoids
Authors: Grieninger Gerd , Hertzberg Kathe M. , Pindyck Johanna
Published: May 2006

Fibrinogen synthesis was investigated in cultures of chicken embryo hepatocytes initiated and maintained in chemically defined, serum-free medium. 11-Hydroxy glucocorticoids caused a 3-fold stimulation of fibrinogen synthesis. Half-maximal stimulation was achieved with 1 nM corticosterone or hydrocortisone, as compared with only 0.1 nM dexamethasone. Increased fibrinogen production in the presence of these glucocorticoids was characterized by a 4-hr delay in onset, a sensitivity to actinomycin D, and a requirement for the continuous presence of the steroid. Crossed immunoelectrophoresis permitted analysis of the simultaneous effects of glucocorticoids on the synthesis of more than 20 plasma proteins secreted in culture. The absence of an effect on the synthesis of most of these proteins was in sharp contrast to the 3-fold increase in fibrinogen production. Sera from a variety of animals also stimulated an increase in fibrinogen synthesis that was similar in degree but less specific than that due to glucocorticoids and that partially masked the response of the cells to the steroid hormones. The presence of an anticoagulant in the medium was found to be necessary for detection of the fibrinogen secreted in culture. Although insulin was routinely included in the chemically defined medium, the cells synthesized fibrinogen and responded to glucocorticoids in the absence of hormonal supplementation of the medium. These findings are consistent with the thesis that variations in glucocorticoid levels contribute to the regulation of fibrinogen production in the intact animal.
Gene duplication in tetraploid fish: model for gene silencing at unlinked duplicated loci.
Authors: Bailey G S , Poulter R T , Stockwell P A
Published: May 2006

Several groups of fishes, including salmonids and catastomids, appear to have originated through genome duplication events. However, these two groups retain approximately 50% of the loci examined as functioning duplicates, despite the passage of 50 million years or more of mutation and selection. Although other effects are not excluded, this apparently slow rate of duplicate silencing can be explained in terms of the effects of selection against defective double homozygotes to unlinked duplicates. We have derived a computer simulation of genetic drift that affords direct evaluation of the effects of population size (N), mutation rate (micron), initial allele frequencies, back mutation, fitness, and time on the probability of fixation for null alleles at unlinked duplicate loci. The results show that this probability is approximately linearly related to population size for N greater than or equal to 10(3). Specifically, for naive populations, the time for 50% probability of gene silencing is approximately equal to 15N + micron-3/4 generations. The retention of 50% of the loci as functional duplicates may therefore result from the large effective size of salmonid and catastomid populations. The results also show that, under most conditions for populations of 2000--3000 or larger, unlinked duplicate loci will be sustained in the functional state longer than tandem (linked) duplicates and hence are available for evolution of new functions for a longer time.
Establishment of preadipocyte clonal line from epididymal fat pad of
ob/ob
mouse that responds to insulin and to lipolytic hormones
Authors: Négrel R. , Grimaldi P. , Ailhaud G.
Published: May 2006

A clonal cell line that responds to insulin and to lipolytic hormones has been established from the epididymal fat pad of the C57BL/6J
ob/ob
mouse. This line, designated ob 17, has a doubling time of 12.5 or 19 hr in 10% or 1% fetal calf serum, respectively. It presents a heterogeneous chromosome number with 40% of the cells containing 35-44 chromosomes and expresses the characteristic H2-LA antigen. After cessation of growth, ob 17 cells accumulate droplets of triglycerides; this accumulation occurs to a significant extent even in the
absence
of insulin normally added after confluence. Lipoprotein lipase activity is negligible in exponentially growing cells but appears at its maximal level just after confluence with or without insulin. Acid:CoA ligase and acylCoA:diglyceride acyltransferase develop later than lipoprotein lipase. The appearance of lipolytic and lipogenic enzymes, but not of triglycerides, seems to be independent of the presence of lipoproteins or of unesterified fatty acids in the culture medium. Therefore, the differentiation program becomes operative when growth is arrested, and differentiation occurs, providing a source of exogenous lipids. Differentiated ob 17 cells in which endogenous triglycerides have been prelabeled on the fatty acid moiety do respond to epinephrine and corticotropin by release of radioactive fatty acid. This lipolytic response is counteracted by prior addition of insulin. The ob 17 cell line appears to be a useful model for study of growth and differentiation of adipose cells as compared to preadipocyte cell lines from the nongenetically obese mouse.
Thy-1: a differentiation marker of potential mammary myoepithelial cells in vitro.
Authors: Lennon V A , Unger M , Dulbecco R
Published: May 2006

The rat mammary cell line Rama 25 [Bennett, D.C., Peachey, L.A., Durbin, H. & Rudland, P.S. (1978) Cell 15, 283--298] differentiates morphologically in vitro from a cuboidal form to a fusiform cell resembling myoepithelial cells. This differentiation occurs in all clonal isolates of the line. By using three different rabbit antisera specific for Thy-1, we have found that antigenic differentiation accompanies morphologic change to the fusiform state. Very few cuboidal cells had Thy-1 detectable on their surfaces in the living state; but after acetone fixation cytoplasmic Thy-1 was detected by immunofluorescence in all cells of the cuboidal type. Thy-1 specificity was established by the fact that immunofluorescence induced by rabbit anti-rat thymocyte serum was abolished by absorption with rat brain but not with erythrocytes, kidney, or liver; immunofluorescence induced by rabbit antiserum to purified Thy-1 glycoproteins from mouse lymphomas was absorbed by Thy-1 positive mouse and rat lymphomas. Surface Thy-1 provides a potentially valuable antigenic marker in the Rama 25 line for studying the differentiation of mammary myoepithelial cells.
Biochemistry of actomyosin-dependent cell motility (a review).
Authors: Korn E D
Published: May 2006

Actins and myosins similar to the major proteins of muscle are the major molecular components of intricate mechanochemical systems that perform numerous vital motility and structural functions in all eukaryotic cells. In this article, after a brief summary of the morphological distribution and ultrastructure of actin, myosin, and interrelated proteins of nonmuscle cells, our present knowledge of their biochemistry is critically appraised from the perspective that understanding complex cellular processes depends ultimately on the identification, purification, and biochemical characterization of the proteins involved. Although few conclusions are reached, possible molecular mechanisms for cellular regulation of actin polymerization, filament association, actomyosin ATPase activity, and mechanochemical coupling are discussed and a number of potentially fruitful directions for further research are suggested. These include comparative biochemical investigations and the study of the interaction of heterologous proteins, but particular emphasis is given to the need for quantitative studies at the molecular level of motility proteins purified from a single cellular source.
Search for relationships among the hemolytic, phospholipolytic, and neurotoxic activities of snake venoms
Authors: Jeng T.-W. , Hendon R. A. , Fraenkel-Conrat H.
Published: May 2006

Several snake venom neurotoxins are larger and more complex than the well-studied group of postsynaptic toxins exemplified by α-bungarotoxin. Several of these, exemplified by β-bungarotoxin, show phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4) when tested in the presence of detergents. The high hemolytic activity of crotoxin, the neurotoxin of
Crotalus durissus terrificus
, in the presence of lecithin has been attributed to this activity. The phospholipase A2 activity of several snake venom proteins has now been compared under the physiological conditions of the hemolysis tests.

It appears that only the basic component of crotoxin, B, is enzymatically active, and that its activity is not inhibited by component A under these conditions, or in the presence of deoxycholate. Phosphatidylserine is found to be digested more readily than egg white phosphatidylcholine; and also causes hemolysis in conjunction with much lower levels of crotoxin. In neither case is calcium required or stimulating.

Phospholipase from
Crotalus adamanteus
, which is not neurotoxic, digests phosphatidylcholine more rapidly than does crotoxin, but phosphatidylserine more slowly; yet it is slightly less active than crotoxin in the hemolysis test with phosphatidylcholine, and much less with phosphatidylserine. The digestion of several phospholipids by either enzyme fails to release the expected protons in the absence of detergents at 37°.

β-Bungarotoxin, highly neurotoxic, has negligible phospholipase A2 activity in the absence of detergents, and is almost nonhemolytic in conjunction with all phospholipids tested.

Binding studies with
125
I-labeled compounds show that rabbit erythrocytes and ghosts have much greater affinity for crotoxin than for β-bungarotoxin and do not bind
Crotalus adamanteus
phospholipase. The crotoxin complex is split in the course of binding, with only component B, the hemolytic component, becoming bound. It appears that the role of component A may be to diminish the nonspecific binding tendency of component B.

Our data appear to be consistent with the concepts that affinity to membranes, particularly to specific sites on synaptic membranes, is the critical requirement for β type neurotoxicity, and that this property, at least in some instances, has evolved from phospholipase A2 enzymes, but does not necessarily require retention and expression of enzymatic activity.
Characterization of pro-opiocortin, a precursor to opioid peptides and corticotropin.
Authors: Rubinstein M , Stein S , Udenfriend S
Published: May 2006

The high molecular weight (approximately 30,000) precursor to opioid activity (pro-opiocortin) previously detected in extracts of rat pituitary was digested with trypsin and the resulting peptide mixture was resolved by high-performance reverse-phase chromatography. A peak of opioid activity was eluted at the position of the nonapeptide beta-LPH (61-69), which was also the same fragment obtained by trypsin digestion of betas-lipotropin or beta-endorphin. This identified the protein as a precursor to the endorphins and Met-enkephalin. No activity was detected in the position corresponding to the Leu5 analog of betas-LPH (61-69), thus ruling out the possibility of a beta-lipotropin-like precursor to Leu-enkephalin in pituitary extracts. Pro-opiocortin and beta-lipotropin are present in rat pituitary extracts in comparable amounts, approximately 10 pmol/mg of tissue.
Displacement-loop replication initiation sequence in animal mitochondrial DNA exists as a family of discrete lengths
Authors: Gillum Amanda M. , Clayton David A.
Published: May 2006

The single-stranded mitochondrial DNA (mtDNA) displacement-loop initiation sequence (7S mtDNA) is hydrogen-bonded at the origin of replication in animal cell mtDNA. Analysis of 7S mtDNA from several cell sources indicates that this initiation sequence exists as a family of fragments of relatively discrete lengths. mtDNA from both mouse L cells and mouse liver has four major sizes of 7S mtDNA fragments, ranging from 500 to 580 nucleotides in length. The 5′-end region of each of these species is the same; thus, the size heterogeneity is due primarily to differences in length at the 3′-end of these molecules. By contrast, 7S mtDNA from both human KB cells and human liver exists in three major forms, ranging from 555 to 615 nucleotides in length, due to differences at both terminal regions. The mtDNA initiation sequence from
Xenopus laevis
oocytes also exists in at least two forms, 1350 and 1510 nucleotides in length. Thus, the maintenance of multiple forms of mtDNA initiation sequence appears to be a general phenomenon of animal cells, although the precise mechanism of synthesis or processing of these forms is variable.

The sequence of 42 nucleotides at the 5′-end of 7S mtDNA from mouse L cells has been determined and found to be rich in dGuo and dThd residues, with no apparent palindromes or potential secondary structures. We thus present sequence information on the replication origin of mtDNA, as defined by the naturally occurring 7S mtDNA.
Regulation of transcription of the late genes of bacteriophage T7.
Authors: McAllister W T , Wu H L
Published: May 2006

The transcription program of bacteriophage T7 in vivo was analyzed by hybridizing T7 mRNAs, labeled at intervals after infection, to Hpa I restriction fragments of T7 DNA. Transcripcion of the late genes is temporally regulated: class II genes are transcribed between 4 and 16 min after infection; most class III genes are transcribed from 8 min after infection until lysis. Genes 8--10 are transcribed as both class II and class III genes. The rate of T7 RNA synthesis decreases sharply at 10 min after infection. The rapid decrease in the rate of T7 RNA synthesis and the shutoff of class II RNA synthesis were not observed in cells infected with phage defective in gene 3.5 (lysozyme). Although the decrease in the rate of T7 RNA synthesis is independent of DNA replication, the failure to shut off class II RNA synthesis normally in 3.5-- -infected cells may reflect the role of T7 lysozyme in DNA replication. In vitro, the regions of T7 DNA transcribed by the phage RNA polymerase were found to be dependent upon ionic conditions.
Nucleotide sequences of the separate origins of synthesis of bacteriophage G4 viral and complementary DNA strands.
Authors: Fiddes J C , Barrell B G , Godson G N
Published: May 2006

Bacteriophage G4 has physically separated origins of synthesis of its viral and complementary DNA strands. Chain termination and "plus and minus" DNA sequencing methods have been used to obtain the nucleotide sequence of these two origins. The unique origin at which the complementary DNA strand is initiated has located in the untranslated region between genes F and G. This sequence, which has considerable secondary structure, contains a stretch which is complementary to the RNA primer that is observed during synthesis in vitro of the G4 complementary DNA strand [Bouché, J.P., Rowen, L. & Kornberg, A. (1978) J. Biol. Chem., in press]. This G4 origin shows extensive sequence homology with the bacteriophage lambda origin of DNA replication [Denniston-Thompson, K., Moore, D. D., Kruger, D. E., Furth, M. E. & Blattner, F. R. (1977) Science 198, 1051-1056]. The sequence around the site in gene A at which G4 viral DNA strand synthesis is initiated by the nicking action of the cistron A protein is very similar to that of bacteriophage phiX174. An (A + T)-rich stretch flanked by (G + C)-rich sequences may be involved in the interaction between the DNA and protein.
Flash photolysis of human serum albumin: characterization of the indole triplet absorption spectrum and decay at ambient temperature.
Authors: Hicks B , White M , Ghiron C A , Kuntz R R , Volkert W A
Published: May 2006

The method of flash photolysis was used to identify the transient absorption spectrum and to characterize the decay kinetics of the indole triplet of human serum albumin. This protein was studied because it contains a single indole side chain which is deeply buried in an expandable oily region and because the phosphorescence of the homologous indole in bovine serum albumin could not be detected at ambient temperatures. The transient was identified on the following basis: (i) its triplet-triplet absorption spectrum was similar to those previously reported for indole and tryptophan; (ii) it was quenched by small quantities of oxygen; and (iii) it was photobleached by 370- to 700-nm light. In a nitrogen-saturated solution at room temperature, the indole triplet decays exponentially for more than a factor of 10 with a lifetime of 0.5 msec. These observations suggest that, because of its exponential decay and relatively long lifetime, the triplet will be more valuable than the indole singlet as an intrinsic reporter group for the study of the structure and dynamics of proteins in solution.
Tetrakis(acetoxymercuri)methane: a polymetallic reagent for labeling sulfur in nucleic acids.
Authors: Strothkamp K G , Lehmann J , Lippard S J
Published: May 2006

Tetrakis(acetoxymercuri)methane binds to the sulfur atom of 6-thioguanosine and also to the 4-thiouridine residue of Escherichia coli tRNAVal. A 4:1 complex is formed between 6-thioguanosine and tetrakis(acetyoxymercuri)methane. Addition of 3 equivalents of N,N-dimethyl-2-amino-ethanethiol hydrochloride to tetrakis(acetoxymercuri)methane effectively blocks three of the four mercury atoms, rendering the compound monofunctional toward 6-thioguanosine. Under appropriate conditions, tetrakis(acetyoxymercuri)methane, in the presence or absence of N,N-dimethyl-2-amino-ethanethiol hydrochloride, binds to the 4-thiouridine residue in E. coli tRNAVal without forming intermolecular crosslinks. These results suggest that tetrakis(acetoxymercuri)methane will be a useful polymetallic reagent for labeling sulfur sites in polynucleotides. It may also prove to be a valuable reagent for preparing heavy metal derivatives of proteins for x-ray crystallographic study.
Identification of pyramidal cells as the critical elements in hippocampal neuronal plasticity during learning.
Authors: Berger T W , Thompson R F
Published: May 2006

The activity of single neurons recorded from rabbit hippocampus during classical conditioning of the nictitating membrane reflex was studied. All cells were first categorized according to their responses after fornix stimulation--i.i., antidromic activation, orthodromic activation, or no activation. The majority of cells that were antidromically activated--pyramidal cells--showed a highly positive correlation between the pattern of unit discharge and the topography of the nicititating membrane response within trial periods. Units that were orthodromically driven by fornix stimulation tended to inhibit during the presentation of trial stimuli, whereas most non-activated cells maintained low spontaneous levels of activity at all times. Thus, the major output neurons of the hippocampus appear to be the neuroanatomical substrate for the large and rapidly developing neuronal plasticity induced by this classical conditioning paradigm.
Characterization of the histone core complex.
Authors: Chung S Y , Hill W E , Doty P
Published: May 2006

A stable histone core complex containing equimolar ratios of H2A, H2B, H3, and H4 has been isolated from chicken erythrocyte chromatin in high salt. This complex has been characterized by sedimentation and chemical cross-linking studies. In velocity ultracentrifugation, only one single sharp sedimentation boundary has been observed with sedimentation coefficient S020,w = 3.8 +/- 0.1. The molecular weight of the histone core complex has been investigated by low-speed sedimentation equilibrium studies over a wide range of protein concentration. Analysis of the apparent weight-average molecular weight as a function of concentration indicates that the histone core complex in 2 M NaCl, pH 9.0, is in equilibrium between a tetramer (H2A)(H2B)(H3)(H4) and an octamer [(H2A)(H2B)(H3)(H4)]2 species with a tetramer molecular weight of 55,000. The equilibrium constant K is approximately 1.2 X 10(-5) liter per mol at 10 degrees. Evidence of such a tetramer-octamer equilibrium in solution is also supported by the results of our chemical crosslinking experiments on the histone core complex.
Ontogenetic development of kainate neurotoxicity: correlates with glutamatergic innervation.
Authors: Campochiaro P , Coyle J T
Published: May 2006

Stereotaxic injection of kainic acid into the striatum of adult rats causes degeneration of neurons intrinsic to the striatum but spares axons of passage and of termination of extrinsic neurons. Neurochemical and histologic studies demonstrate that striatal neurons are almost insensitive to kainate at 7 days after birth and that their vulnerability increases with age; by 3 weeks after birth, striatal injection of kainate produces a lesion comparable to that of the adult. The intensity and duration of the acute behavioral response to kainate also increases with age. The maturational increase in striatal neuronal sensitivity to kainate correlates with the development of glutamatergic innervation to the striatum, as measured by [3H]glutamate uptake by synaptosomes, and with the development of a postsynaptic, high-affinity receptor site for kainate. These ontogenetic studies provide additional evidence that kainate's neurotoxicity is a receptor-mediated event related to glutamatergic innervation of vulnerable neurons.
Kinetics of hemoglobin and transition state theory.
Authors: Szabo A
Published: May 2006

Experimental evidence indicates that the way cooperativity in hemoglobin is manifested kinetically depends on the nature of the ligand. It is shown that the qualitative differences in the kinetics of NO, O2, CO, and bulky isocyanides can be understood in structural terms in a framework based on transition state theory.
Potent inhibition of dynein adenosinetriphosphatase and of the motility of cilia and sperm flagella by vanadate.
Authors: Gibbons I R , Cosson M P , Evans J A , Gibbons B H , Houck B , Martinson K H , Sale W S , Tang W J
Published: May 2006

The motility of demembranated sea urchin sperm flagella and that of embryo cilia reactivated with 0.1 mM ATP are completely inhibited by 4 micron and 0.5 micron vanadium(V) [V(V), in vanadate], respectively. The Mg2+-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3)of the latent form of dynein 1 is inhibited 50% by 0.5-1 micron V(V), while the Ca2+-activated ATPase activity is much less sensitive. The inhibition of flagellar beat frequency and of dynein 1 ATPase activity by V(V) appears not to be competitive with ATP. In agreement with other reports, the inhibition of (Na,K)-ATPase by V(V) shows a slow onset in the presence of ATP and is relatively rapid in its absence. With dynein, however, the inhibition occurs at a rapid rate whether or not ATP is present. Catechol at a concentration of 1 mM reverses the V(V) inhibition of reactivated sperm motility, dynein ATPase, and (Na, K)-ATPase. Myosin and actomyosin ATPases show no inhibition by concentrations of V(V) up to 500 micron. The inhibition by V(V) provides a possible technique for distinguishing between the actions of dynein and myosin in different forms of cell motility.
Novel enzyme from rat liver that cleaves an ADP-ribosyl histone linkage.
Authors: Okayama H , Honda M , Hayaishi O
Published: May 2006

A novel enzyme that splits a bond between ADP-ribose and histone was discovered and partially purified from rat liver cytosol. The 105,000 X g supernatant of rat liver homogenate was precipitated by 45% saturated ammonium sulfate and then chromatographed on a DEAE-cellulose column. The enzyme activity was eluted in a single peak at about 0.2 M NaCl and clearly separated from poly(ADP-ribose) glycohydrolase which came out at 0.13 M NaCl. In contrast to the latter enzyme, this new enzyme catalyzed the spliting of a linkage between ADP-ribose and a protein portion in mono ADP-ribosylated histone H2B but little, if any, of the glycosidic ribosyl(1"-2') ribose bonds within poly(ADP-ribose). Analysis of the reaction product by paper chromatography and Dowex 1 column chromatography indicated that the split product contained the ADP-ribose moiety but was not exactly identical with ADP-ribose. Available evidence suggested that it was either an altered ADP-ribose molecule produced by a structural rearrangement or ADP-ribose itself linked to an unidentified compound. The enzyme had a pH optimum of about 6.0 and was inhibited by 80-90% in the presence of 5 mM ADP-ribose.
Tumorigenicity and its suppression in cybrids of mouse and Chinese hamster cell lines.
Authors: Howell A N , Sager R
Published: May 2006

The effect of cytoplasm upon the expression of tumorigenicity was examined with a pair of mouse and a pair of Chinese hamster cell lines, in intraspecies cybrids formed by reciprocal fusions between either tumorigenic or nontumorigenic cells and cytoplasms derived from them. With the mouse cells, 3T3 and the simian virus 40-transformed line SVT2, the cybrid clones were tumorigenic when SVT2 cells were fused with 3T3 cytoplasts, but not in the reciprocal fusion. With the hamster cells, CHEF/18 and the spontaneous transformant CHEF/16, however, tumorigenicity was partially suppressed in cybrid clones formed by fusion of tumorigenic CHEF/16 cells with CHEF/18 cytoplasts; cybrids were nontumorigenic in the reciprocal fusion. Thus, cybrid analysis has shown that tumorigenicity is not cytoplasmically transmitted in these two cell pairs, but suppression of tumor-forming ability may be cytoplasmically transmitted in the hamster cybrids.
Identification of the
Mtv
-2 gene responsible for the early appearance of mammary tumors in the GR mouse by nucleic acid hybridization
Authors: Michalides R. , Deemter L. Van , Nusse R. , Nie R. Van
Published: May 2006

In the mouse strain GR, the
Mtv
-2 gene controls the expression of large amounts of mammary tumor virus (MTV) antigens in the milk at first lactation. It also controls the early appearance of mammary tumors. We have investigated the number of MTV proviral sequences associated with this
Mtv
-2 gene by nucleic acid hybridization between MTV [
3
H]cDNA and DNA from GR, B10, and GR-
Mtv
-2
-
mice. B10 and GR-
Mtv
-2
-
mice lack
Mtv
-2 gene expression. The molecular hybridizations revealed that the DNA of GR mice contains 12 copies of MTV proviral sequences, whereas only 4 copies are present in the DNA of B10 and GR-
Mtv
-2
-
mice. We therefore conclude that the
Mtv
-2 gene in the GR mouse strain is associated with eight additional MTV proviral sequences. The four Mtv proviral sequences in the GR-
Mtv
-2
-
DNA might represent another
Mtv
gene in the GR mouse. Different amounts of MTV RNA are detected in mammary glands at first lactation of B10 and GR-
Mtv
-2
-
mice, even though both contain four copies of MTV proviral sequences. This indicates a difference between these two mouse strains either in the regulation of expression of these MTV proviral sequences or in the location of these sequences in the murine genome.
Complement C3 convertase: Cell surface restriction of β1H control and generation of restriction on neuraminidase-treated cells
Authors: Pangburn Michael K. , Müller-Eberhard Hans J.
Published: May 2006

The alternative or properdin pathway of complement is primarily controlled by the endopeptidase C3b inactivator (C3bINA) and the nonproteolytic glycoprotein β1H. The molecular mechanisms of control were investigated by performing binding studies of radiolabeled complement proteins to C3b bearing sheep erythrocytes (E
S
C3b). C3b was found to have distinct binding sites for β1H, C3bINA, Factor B, and properdin. β1H binding increased C3bINA binding 30-fold, while Factor B binding prevented C3bINA action on C3b and was competitive with β1H binding. Properdin binding, which facilitates Factor B interaction with C3b, had no effect on the β1H and C3bINA sites. Activators such as rabbit erythrocytes (E
R
) have previously been shown to interfere with the effectiveness of the control by C3bINA and β1H, thereby allowing unrestricted formation of C3 convertase. Such restriction of control does not occur on the surface of E
S
, a nonactivator of the alternative pathway. On the basis of comparative binding studies, restriction of control is explained entirely by reduced binding of β1H to E
R
C3b relative to E
S
C3b. Access of properdin, Factor B, C3bINA, and the Fab fragment of anti-C3 to the two cell types was unrestricted. Restriction of β1H control could be generated on the surface of E
S
by removal of cell-surface sialic acid with neuraminidase (acylneuraminyl hydrolase; EC 3.2.1.18). This enzymatic treatment converted E
S
from a nonactivator to an activator of the alternative pathway.
Insulin binding to solubilized material from fat cell membranes: Evidence for two binding species
Authors: Krupp Michael N. , Livingston James N.
Published: May 2006

The components of fat cell membranes responsible for the binding of insulin were solubilized by treatment with the nonionic detergent Triton X-100. By using a polyethylene glycol precipitation method to assay specific insulin binding, the soluble preparation was shown to have insulin-binding characteristics similar to those of intact fat cells. Further studies of this preparation by polyacrylamide gel electrophoresis in the presence of
125
I-labeled insulin demonstrated two distinct insulin binding activities, designated species I and II. The two species were separated by electrophoresis in the absence of iodo-labeled hormone and eluted from the gel. Scatchard analysis of the insulin binding data for species I showed a curvilinear plot with the initial portion having a
K
d
of 1.3 × 10
-10
M. The Scatchard plot for species II was linear with a
K
d
of 6.0 × 10
-9
M. Desoctapeptide insulin and glucagon failed to compete for the insulin-binding sites in both species whereas desalanine insulin was an effective competitor. High concentrations of proinsulin competed with the iodo-labeled hormone for binding to species I but not to species II. In the presence of a low concentration of
125
I-labeled insulin (0.3 nM) some species I activity appeared to be converted to species II activity; there was no evidence of interconversion between the two species in the absence of insulin. Neither species degraded insulin as measured by trichloroacetic acid precipitation or rebinding to intact fat cells. These findings indicate the existence in the adipocyte plasma membrane of two insulin-binding species that have distinct physicochemical properties.
Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts
Authors: Smith Charles Allen , Hanawalt Philip C.
Published: May 2006

The repair mode of DNA replication has been demonstrated in isolated nuclei from UV-irradiated human cells. Nuclei are incubated in a mixture containing [
3
H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The
3
H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is UV dependent and proceeds at approximately the
in vivo
rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg
2+
-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote UV-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.
Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from Electrophorus electricus electroplax membranes.
Authors: Agnew W S , Levinson S R , Brabson J S , Raftery M A
Published: May 2006

The tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from electroplax membranes of Electrophorus electricus has been purified. The toxin-binding site could be efficiently solubilized with Lubrol-PX, resulting in an extract of high initial specific activity. Purification was facilitated by the development of a rapid, quantitative binding assay. The binding component was stabilized during purification by the use of mixed lipid/detergent micelles of defined composition, and by the saturation of the site with tetrodotoxin. The purification was achieved by means of a highly selective adsorption of the toxin-binding component to DEASE-Sephadex A-25, followed by desorption at high ionic strength and chromatography over Sepharose 6B. Final peak specific activities were at least 50% of the specific activity expected for a pure, undenatured toxin-binding componenet of 230,000 molecular weight. The purified material exhibited a sedimentation coefficient of approximately 8 S and an unusual Stokes radius of 95 A. Purified material showed a relatively simple pattern on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, being comprised of only three polypeptides.
Pre-proparathyroid hormone; amino acid sequence, chemical synthesis, and some biological studies of the precursor region.
Authors: Habener J F , Rosenblatt M , Kemper B , Kronenberg H M , Rich A , Potts J T
Published: May 2006

The precursor of bovine proparathyroid hormone was synthesized by translation of parathyroid mRNA in a wheat-germ cell-free system. The amino acid sequence of the NH2-terminal extension (the pre sequence) was determined by repetitive Edman degradation of the polypeptide labeled with radioactive amino acids (radiosequencing). The pre sequence of pre-proparathyroid hormone is (formula: see text) which is followed by the sequence of proparathyroid hormone. It is significant that 20 of the 25 amino acids in the sequence are hydrophobic. This high hydrophobicity is consistent with the proposed role of the pre sequence as a membrane-penetrating peptide. The precursor-specific sequence of 31 amino acids was snythesized chemically by the solid-phase technique. This synthetic peptide was shown to bind to the microsomal fraction of homogenates prepared from extracts of parathyroid glands, a finding consistent with the proposed role of the precursor peptide in the attachment of the nascent chain--mRNA--ribosome complex to the endoplasmic reticulum.
Transformation-dependent secretion of a low molecular weight protein by murine fibroblasts.
Authors: Gottesman M M
Published: May 2006

A protocol has been devised to radiolabel proteins secreted by murine fibroblasts in vitro. A radiolabeled polypeptide of molecular weight 35,000 is released into medium in relatively large amounts by transformed cells and in much smaller amounts by nontransformed fibroblasts. This major excreted polypeptide (MEP) is found in the medium of spontaneously transformed mouse cells and in the medium of mouse cells transformed by a DNA tumor virus, RNA tumor viruses, or methylcholanthrene. The appearance of MEP appears to be well correlated with anchorage independence in these transformed cells. MEP can be localized within the cytoplasm of transformed but not untransformed cells by indirect immunofluorescence. The presence of MEP within murine fibroblasts or in their culture medium serves as a novel biochemical marker of transformation. A biological role for this protein has not been assigned.