bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescence energy transfer in the rapid-diffusion limit
Authors: Thomas David D. , Carlsen William F. , Stryer Lubert
Published: May 2006

Energy transfer is enhanced by translational diffusion of the donor and acceptor [Steinberg, I. Z. & Katchalski, E. (1968)
J. Chem. Phys.
48, 2404-2410]. The effect of diffusion on energy transfer depends on
D
τ
0
/
s
2
, in which
D
is the sum of the diffusion coefficients of the donor and acceptor, τ
0
is the lifetime of the donor in the absence of transfer, and
s
is the mean distance between donors and acceptors. In most previous studies,
D
τ
0
/
s
2
≪ 1, corresponding to the static limit. We report here steady-state and kinetic fluorescence experiments showing that
D
τ
0
/
s
2
≫ 1, the rapid-diffusion limit, can be attained by using Tb
3+
chelated to dipicolinate as a long-lived energy donor (τ
0
= 2.2 msec). The concentration of rhodamine B, the energy acceptor, resulting in 50% transfer was 0.67 μM, which is three orders of magnitude less than the concentration giving 50% transfer in the static limit. The dependence of the transfer efficiency on diffusion coefficients varying from 5 × 10
-11
to 1.5 × 10
-4
cm
2
/sec, spanning the range from the static limit to the rapid-diffusion limit, is in excellent agreement with theory. It is evident that energy donors with millisecond or longer excited state lifetimes can be used to probe translational motions in membranes and other assemblies. Energy transfer in the rapid diffusion limit is sensitive to the distance of closest approach (
a
) of the donor and acceptor. For a Tb·(DPA)
3
chelate trapped inside the aqueous space of a membrane vesicle containing eosin phosphatidylethanolamine,
a
= 10 Å. The transverse location of chromophores in model membranes and biological membranes can be determined by this technique.
Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein.
Authors: Emr S D , Schwartz M , Silhavy T J
Published: May 2006

Two mutant strains of Escherichia coli have been isolated in which the cellular location of an outer membrane protein, the phage lambda receptor (the lamB gene product), is altered. These mutations were initially selected in a strain containing a lamB-lacZ fusion. In the parent strain the protein coded for by the hybrid gene is located, at least in part, in the outer membrane. In the mutants it is located in the cytoplasm. The mutations responsible for the alteration of cellular location lie very early in the lamB gene, in a region corresponding to the NH2-terminus of the lambda receptor protein. One of these mutations is a small deletion internal to the lamB gene. When this mutation is present in an otherwise wild-type lamB gene, the protein produced is of lower molecular weight than normal receptor. The other mutation behaves as a point mutation; when it is present in an otherwise normal lamB gene, reversion can be demonstrated. The molecular weight of this mutant protein, which is located in the cytoplasm, is larger than that of the wild-type gene product by approximately 2000. It is suggested that these two mutations are in the portion of the lamB gene coding for a signal sequence and thereby block export of the protein.
Altered nucleosome spacing in newly replicated chromatin from Friend leukemia cells.
Authors: Murphy R F , Wallace R B , Bonner J
Published: May 2006

Chromatin from Friend leukemia cells labeled with [14C]thymidine for 24 hr followed by [3H]thymidine for 10 min is converted into nucleosomes by staphylococcal nuclease at only half the rate that total chromatin is converted. Polyacrylamide gel electrophoresis of nucleosomal DNA from cells labeled for 24 hr with [14C]thymidine followed by 10 min with [3H]thymidine demonstrates that the internucleosomal spacer of newly replicated chromatin is approximately 20 base pairs shorter than that of total chromatin. The implications of this difference for models of chromatin structure are discussed.
Translational control of transcription termination at the attenuator of the Escherichia coli tryptophan operon.
Authors: Zurawski G , Elseviers D , Stauffer G V , Yanofsky C
Published: May 2006

We have isolated two regulatory mutants altered in the leader region of the Escherichia coli tryptophan (trp) operon. In one mutant, trpL29, the AUG translation start codon for the trip leader peptide is replaced by AUA. The other mutant, trpL75, has a G leads to A change at residue 75, immediately after the UGA translation stop codon for the trp leader peptide. In vivo, trpL29 and trpL75 increase the efficiency of transcription termination at the trp attenuator 3- to 5-fold. trpL29 and trpL75 also fail to respond fully to tryptophan starvation and other conditions that normally relieve transcription termination at the trp attenuator. The trpL29 mutation, which presumably reduces synthesis of the trp leader peptide, is cis dominant. The effect of starvation for a number of the amino acids in the trp leader peptide was determined. Only starvation for tryptophan and arginine, amino acids that occur at residues 10, 11, and 12 of the 14-residue trp leader peptide, elicits relief of transcription termination. Our findings suggest that translation of trp leader RNA is involved in regulation of transcription termination at the attenuator. A model is discussed in which the location of the ribosome synthesizing the leader peptide is communicated to the RNA polymerase transcribing the leader region.
Binding of HLA antigen-containing liposomes to bacteria
Authors: Klareskog Lars , Banck Göran , Forsgren Arne , Peterson Per A.
Published: May 2006

Highly purified, detergent-solubilized HLA-A and -B antigens and HLA-D antigens were separately incorporated into liposomes. Detergent-solubilized transplantation antigens, but not papain-solubilized antigens lacking the membrane-integrated portions of the molecules, were bound to the liposomes. A considerable portion of the liposome-bound antigens displayed accessible antigenic sites, suggesting that they were oriented in the right-side-out direction. Liposomes containing the HLA-A and -B antigens or the HLA-D antigen interacted similarly with bacteria. The two types of liposomes bound efficiently to two strains of
Neisseria catarrhalis
(now classified as
Branhamella catarrhalis
) and to one strain of
Haemophilus influenzae
, weakly to one strain of
Escherichia coli
, and not at all to another strain of
E. coli
. The binding between the HLA antigen-containing liposomes and one strain of
N. catarrhalis
was abolished when Fab fragments directed against the heavy chains of HLA-A and -B antigens or against HLA-D antigens, respectively, were added. In contrast Fab fragments against β
2
-microglobulin did not measurably impede the bacteria-liposome interaction, suggesting that, with regard to the HLA-A and -B antigens, the heavy, but not the light, chains interacted with the bacteria. Additional experiments showed that
N. catarrhalis
preferentially interacted with transplantation antigens when mixed with detergent-solubilized lymphocyte membrane glycoproteins. These data suggest that HLA-A and -B and HLA-D antigens may have the function of interacting with foreign antigens such as bacteria.
Ligandin retains and albumin loses bilirubin binding capacity in liver cytosol.
Authors: Listowsky I , Gatmaitan Z , Arias I M
Published: May 2006

Circular dichroism methods were used to detect bilirubin-ligandin interactions in rat liver cytosol and fractions obtained at various stages during purification of ligandin. Ligandin retained its capacity to bind bilirubin in the presence of components of liver supernatant, but albumin, which binds bilirubin in serum, lost the capacity to bind bilirubin in liver supernatant. This was attributed to a greater binding specificity exhibited by ligandin. In their respective physiological milieus, albumin and ligandin are structurally adapted to bind ligands: albumin in serum, and ligandin in the cytosol of the liver cell. These studies are consistent with the hypothesis that the concentration of ligandin within the liver could regulate the net flux of certain organic anions from plasma into the liver.
Human placental cells transformed by tsA mutants of simian virus 40: a model system for the study of placental functions.
Authors: Chou J Y
Published: May 2006

Human placental cells were transformed with wild-type simian virus 40 (SV40) and temperature-sensitive SV40 mutants of the A and B classes. Four criteria for transformation were used: decreased generation time, increased saturation density, increased efficiency of growth on plastic, and ability to overgrow a nontransformed monolayer. Cell lines transformed by tsA mutants lost the transformed phenotype at the restrictive temperature (40 degrees); therefore, the A function of SV40 is required for the maintenance of the transformed phenotype activity, an inhibition of human chorionic gonadotropin synthesis, and an increase in thymidine kinase activity were seen when human placental cells transformed by wild-type or tsB mutants of SV40 were grown at 33 degrees or 40 degrees and when tsA transformants were grown at 33 degrees. When tsA transformants were grown at 40 degrees, alkaline phosphatase activity and human chorionic gonadotropin synthesis were greatly stimulated and thymidine kinase activity was greatly reduced, approximating their levels in the placenta.
Structure of psoralen-crosslinked ribosomal RNA from Drosophila melanogaster.
Authors: Wollenzien P L , Youvan D C , Hearst J E
Published: May 2006

Ribosomal RNA from Drosophila melanogaster photoreacted with hydroxymethyltrioxsalen has been examined by electron microscopy. Reproducible patterns of hairpins were found in both the 26S and 18S RNA. The frequency of these hairpins and the amount of incorporated drug were dependent upon the conditions under which the crosslinking was performed. A prominent central hairpin occurs in the 26S RNA and the break that interrupts the continuity of the RNA chain is located within it. In addition to several small hairpins, the crosslinked 18S RNA contains a large open loop.
Construction of a novel plasmid-phage hybrid: use of the hybrid to demonstrate ColE1 DNA replication in vivo in the absence of a ColE1-specified protein.
Authors: Kahn M , Helinski D R
Published: May 2006

A hybrid bacteriophage, P420, was constructed in vitro; it contains part of bacteriophage P4 and a 3.6-kilobase derivative of plasmid ColE1. In Escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. Replication of P420, directed by the ColE1 replicon, was found to occur after P420 phage infection of E. coli cells that had been incubated in the presence of chloramphenicol. Replication began without a lag period and resulted in the synthesis of covalently closed circles of P420 DNA. Like ColE1 DNA replication but unlike that of P4, replication was dependent on DNA polymerase I and was sensitive to rifampicin. The presence of a resident ColE1 plasmid in the infected cells resulted in an inhibition of the replication of the incoming P420 DNA. These results indicate that ColE1 does not require a plasmid-coded protein to replicate its DNA in vivo and demonstrate the utility of P4 bacteriophage for coupling bacteriophage properties to a plasmid replicon.
Use of anti-idiotypic antibodies as cell-surface receptor probes
Authors: Sege Karin , Peterson Per A.
Published: May 2006

Anti-idiotypic antibodies have been raised against antibodies to retinol-binding protein (RBP) and to insulin. After absorption the anti-idiotypic antibodies recognized the antigen-combining sites of the antibodies used as the immunogen but of no other antibodies. Some of the anti-idiotypic antibodies raised against antibodies to RBP bound specifically to rat intestine epithelial cells, which have a physiological cell-surface receptor for RBP. The RBP receptor mediates the uptake of retinol from RBP to the cells. This uptake was abolished in a concentration-dependent manner by the anti-idiotypic antibodies, which obviously competed with RBP for binding to the receptor.

Anti-idiotypic antibodies against antibodies to insulin inhibited the binding of
125
I-labeled insulin to isolated rat epididymal fat cells, whereas anti-idiotypic antibodies raised against antibodies to RBP had no effect. Furthermore, on interacting with young rat thymocytes, anti-idiotypic antibodies against antibodies to insulin stimulated the uptake by the cells of α-aminoisobutyric acid, thereby mimicking the effect of insulin.

These results suggest that in some cases anti-idiotypic antibodies may be useful tools in elucidating structure-function relationships for cell-membrane receptors.
Peroxidized coelenterazine, the active group in the photoprotein aequorin.
Authors: Shimomura O , Johnson F H
Published: May 2006

The photoprotein aequorin emits light by an intramolecular reaction when Ca2+ is added under either aerobic or anaerobic conditions. Previously reported evidence has indicated two possibilities: (i) the functional group of aequorin is coelenterazine itself, a compond that plays key roles in the bioluminescence of various other types of organisms, or (ii) it is the enolized form of this compound. Present data rule out both of these possibilities, through elucidation of the structure of the yellow compound that is split off aequorin by treatment with NaHSO3. The yellow compound is now shown to be a tertiary alcohol of coelenterazine on the basis of chemical reactions, mass spectral data, and relationships to known derivatives of coelenterazine. From this structure and the method of forming the yellow compound from aequorin, aequorin evidently contains a peroxide of coelenterazine as the active group. The presence of such a peroxide is consistent with the fact that aequorin yields free coelenterazine upon treatment with Na2S2O4. Although there is no applicable technique at present to determine with assurance the specific state of the peroxide in the protein, a study with 18O tracer indicates that a linear peroxide structure is more likely than the alternative possibility of a dioxetane structure.
Stepwise mutation model and distribution of allelic frequencies in a finite population.
Authors: Kimura M , Ohta T
Published: May 2006

A mathematical theory is developed that enables us to derive a formula for the equilibrium distribution of allelic frequencies in a finite population when selectively neutral alleles are produced in stepwise fashion (stepwise mutation model). It is shown that the stepwise mutation model has a remarkable property that distinguishes it from the conventional infinite allele model (Kimura-Crow model): as the population size increases indefinitely while the product of the effective population size and the mutation rate is kept at a fixed value, the mean number of different alleles contained in the population rapidly reaches a plateau which is not much larger than the effective number of alleles (reciprocal of homozygosity).
Biochemical and immunochemical study of lysyl oxidase in experimental hepatic fibrosis in the rat.
Authors: Siegel R C , Chen K H , Greenspan J S , Aguiar J M
Published: May 2006

Lysyl oxidase catalyzes the crosslinking of collagen and elastin. Lysyl oxidase activity was measured and localized in rat liver during the evolution of hepatic fibrosis induced by CCl4. Enzyme activity measured with DL-[6-3H]-lysine-labeled collagen substrates in liver and plasma increased sharply after approximately 3 wk of injection, reached a maximum at 6 wk, and then decreased. The increase in activity correlated histologically with early connective tissue septa formation, and the magnitude of increase was significantly greater than that found for the intracellular collagen biosynthetic enzymes protocollagen prolyl hydroxylase and lysyl hydroxylase. Indirect immunofluorescence studies showed that lysyl oxidase was present in association with collagen in the extracellular space. However, it was not possible to correlate the distribution pattern with a particular liver cell type. These observations suggest that serial measurements of lysyl oxidase activity in liver or plasma may be useful for correlating changes in connective tissue formation with histologic connective tissue deposition.
Cycloheximide resistance can be mediated through either ribosomal subunit.
Authors: Sutton C A , Ares M , Hallberg R L
Published: May 2006

Two cycloheximide-resistant mutants of Tetrahymena thermophila were analyzed to determine the site of their cycloheximide resistance. The mutations in both strains had been previously shown to be genetically dominant and located at separate loci (denoted Chx-A and Chx-B). Strains carrying these mutations were readily distinguished by the extent to which they were resistant to the drug. The homozygous double mutant was more resistant than either single mutant. Cell-free extracts of wild type and of the three mutant strains, assayed for protein synthetic activity by both runoff of natural mRNA and poly(U)-dependent phenylalanine polymerization, demonstrated that in vitro the mutants were all more resistant than the wild type. Further fractionation of the cell-free systems into ribosomes and supernates localized cycloheximide resistance to the ribosome for both Chx-A and Chx-B homozygotes. Ribosome dissociation and pairwise subunit mixing in the in vitro system indicated that ribosome resistance was conferred by the 60S subunit from one strain whereas resistance in the other strain was mediated through the 40S subunit. This was further confirmed by reconstruction of all four cycloheximide-resistance "phenotypes" by mixing ribosomal subunits from appropriate strains. This finding suggests that the mechanisms by which these mutations confer resistance to cycloheximide are different.
Time of recombination in the Drosophila melanogaster oocyte: evidence from a temperature-sensitive recombination-deficient mutant.
Authors: Grell R F
Published: May 2006

A temperature-sensitive recombination-deficient mutant, rec-126, has been isolated that permits high frequencies of recombination at the permissive temperature (25 degrees) but greatly decreases recombination at the restrictive temperature (31 degrees). The sensitive period for response of female germ cells carrying this mutant to the restrictive temperature has been defined. Sensitivity begins very close to the time the oocyte enters premeiotic interphase and initiates DNA synthesis; it continues for the duration of premeiotic-S; and it terminates with the completion of S. This time span precisely coincides with the sensitive period for enhancement of recombination by heat in the normal genome and is further characterized by the presence of the synaptonemal complex. These results provide compelling evidence for identifying premeiotic-S as the time of meiotic recombination.
Transport of long-chain fatty acids by Escherichia coli: mapping and characterization of mutants in the fadL gene.
Authors: Nunn W D , Simons R W
Published: May 2006

A new locus (fadL) that is required for the utilization of long-chain fatty acids has been mapped and partially characterized in an Escherichia coli mutant. The fadL locus has been mapped at 50 min on the chromosome. A mutant bearing a defect in this locus cannot utilize long-chain fatty acids as a sole carbon source. Derivatives of this mutant that can grow on decanoate (termed fadR) are capable of growth on medium-chain but not long-chain fatty acids. It is believed that the fadL mutants is defective in the transport of long-chain fatty acids into the cell for the following reasons: (i) fadR fadL strains can oxidize in vivo decanoate but not oleate; (ii) neither fadL nor fadR fadL strains can incorporate oleate into their membrane lipids; (iii) the activity of the acyl-CoA synthetase (EC 6.2.1.3) in fadR fadL strains is comparable to the acyl-CoA synthetase activity in fadR fadL+ strains; and (iv) in vitro extracts from fadR fadL+ strains. If the above hypothesis is correct, the uptake of long-chain fatty acids by E. coli requires at least two gene products.
Developmental change in human intestinal alkaline phosphatase.
Authors: Mulivor R A , Hannig V L , Harris H
Published: May 2006

Starch gel electrophoresis and inhibition studies with L-phenylalanine, L-homoarginine, L-leucine, L-leucylglycylglycine, and L-phenylalanylglycylglycine were carried out on a series of human alkaline phosphatases [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] derived from fetal and adult liver, kidney, bone, and intestine. No differences between adult and fetal liver, kidney, or bone alkaline phosphatases were observed by either electrophoretic or inhibition studies. However, the fetal intestinal enzyme could be clearly distinguished from the adult intestinal enzyme by its greater anodal electrophoretic mobility and its retardation after treatment with neuraminidase. Even after extensive neuraminidase treatment, its anodal mobility was still slightly greater than that of adult intestinal alkaline phosphatase. Fetal and adult intestinal enzymes showed the same inhibition profiles with the series of inhibitors both before and after treatment with neuraminidase. A survey of intestinal samples from fetuses and premature infants of various gestational ages indicated that the changeover from the synthesis of fetal to adult intestinal enzyme begins at about 28-32 weeks of gestation. The difference between the fetal and adult forms of intestinal alkaline phosphatase may represent the expression of different gene loci or a difference in post-translational modification.
Monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants.
Authors: Wiktor T J , Koprowski H
Published: May 2006

Somatic cell hybrids (hybridomas) between mouse myeloma cells and spleen cells derived from BALB/c mice immunized with inactivated rabies vaccine were found to produce antibodies to rabies virus. Monoclonal antibodies with different specificities were obtained either from the mass culture directly after fusion or from clones derived from a single-cell cloning procedure. Several strains of fixed or street rabies virus were analyzed by virus neutralization procedures which demonstrated differences in their antigenic composition. Hybridoma antibodies were able to protect experimental animals from lethal rabies virus infection.
Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor.
Authors: Read M S , Shermer R W , Brinkhous K M
Published: May 2006

A platelet-aggregating activity was found in many snake venoms, predominantly those of the genus Bothrops, that is apparent only in the presence of the platelet-aggregating von Willebrand factor of plasma. It is designated "venom coagglutinin." The coagglutinin can be largely separated from the thrombin-like enzyme of the venoms by ion-exchange chromatography. The venom factor acts on formaldehyde-fixed platelets and is effective with decalcified, heparinized, and afibrinogenemic plasmas but not with severe von Willebrand disease plasmas or with normal plasmas in which the von Willebrand factor has been neutralized by specific antibodies. Use of this coagglutinin permits the assay of von Willebrand factor without the many disadvantages of the ristocetin test. The coagglutinin is active with human, dog, pig, and bovine plasmas and with platelets of any one of these species. This broad-spectrum activity without regard to species contrasts with the ristocetin-resistance of many combinations of plasma and platelets from various species. The assay provides a procedure for studying human, porcine, and canine von Willebrand disease. The lack of species specificity of the coagglutinin suggests that it may be a universal activator of the von Willebrand factor-platelet reaction.
Rapid inactivation of cyclooxygenase activity after stimulation of intact platelets
Authors: Lapetina E. G. , Cuatrecasas P.
Published: May 2006

Trypsin, thrombin, and ionophore A23187 activate phospholipid breakdown of platelets that have been labeled with [
14
C]arachidonate, releasing their cyclooxygenase and lipoxygenase products. Intact platelets can also very effectively directly degrade low concentrations of exogenous, free [
14
C]arachidonate. Pretreatment of platelets with trypsin, thrombin, or ionophore A23187 for a minimum time of 30 sec leads to complete inactivation of cyclooxygenase activity, as demonstrated by subsequent exposure to [
14
C]arachidonate. Lipoxygenase activity is lost after 5 min. The thrombin-induced inactivation of cyclooxygenase and lipoxygenase is prevented by cyclic AMP (which inhibits the stimulated activity of phospholipase A
2
), although cyclic AMP does not affect the degradation of exogenous [
14
C]arachidonate. Exposure of platelets labeled with [
14
C]arachidonate to unlabeled arachidonate under conditions that lead to use of the latter also results in a similarly rapid inhibition of cyclooxygenase activity, as determined by subsequent challenge with thrombin. Under these conditions lipoxygenase activity is much less markedly inactivated. The arachidonate-induced inhibition of cyclooxygenase activity is not prevented by cyclic AMP. Trypsin does not induce platelet aggregation, and platelets whose cyclooxygenase activity has been inactivated are intact insofar as they are still able to undergo aggregation. These studies demonstrate that operation in intact platelets of the cyclooxygenase pathway, through use of endogenous or exogenous substrate, leads to a very rapid, irreversible inactivation of this enzyme. The lipoxygenase pathway is also progressively impaired, but much less rapidly than the cyclooxygenase enzyme and much less markedly on use of exogenous compared to endogenous substrate. The possible consequences of these physiological processes of spontaneous inactivation are considered.
Photobilirubin: an early bilirubin photoproduct detected by absorbance difference spectroscopy.
Authors: Lightner D A , Wooldridge T A , McDonagh A F
Published: May 2006

Absorption of light converts bilirubin-IXalpha in solution to a mixture of what are probably cis-trans geometric isomers. This reaction is much faster than other photochemical reactions of bilirubin and reaches photoequilibrium before losses due to photooxidation are significant. At room temperature in the dark in the presence of trifluoroacetic acid or iodine or simply on standing, the photoproducts revert to the natural isomer. They also revert under visible light. Their formation and reversion can be followed by chromatography on polyamide and by absorbance difference spectroscopy.
High affinity binding of chylomicron remnants to rat liver plasma membranes.
Authors: Carrella M , Cooper A D
Published: May 2006

The binding of chylomicron remnants rat liver plasma membranes was studied. Liver membranes bound up to 8 times more remnants than they bound chylomicrons. The remnant particle appeared to bind to the membrane as a unit. Remnant binding was greatest to liver plasma membrane; only one third as much binding was observed with whole liver homogenate, and virtually no binding occurred to erythrocyte membranes or glass. Binding was saturable and had kinetics compatible with the existence of a high affinity site. Half-maximal binding occurred at 27 micron. Competitive binding studies revealed no competition with albumin, a triglyceride dispersion, cholesterol/lecithin vesicles, very low density lipoprotein, or low density lipoprotein. Some displacement of remnant binding was observed with chylomicrons and high density lipoprotein. Binding was decreased by treatment of the membranes with trypsin or the presence of heparin in the incubation medium. These studies suggest that there is a high affinity receptor for the chylomicron remnant on the surface of the hepatocyte.
Recognition of differentiation antigens of spermatogenesis in the mouse by using antibodies from spleen cell-myeloma hybrids after syngeneic immunization.
Authors: Bechtol K B , Brown S C , Kennett R H
Published: May 2006

Two differentiation antigens of spermatogenesis in the mouse are defined by monoclonal antibodies from hybridomas produced between the myeloma P3-X63Ag8 and spleen lymphocytes immunized with syngeneic testis cells. These antigens are on testis cells, and not liver, kidney, brain, spleen, or whole ovary. They are species but not strain specific and trypsin sensitive but collagenase insensitive. On the basis of their appearance during the onset of spermatogenesis they appear to be differentiation antigens expressed during different but overlapping time windows during spermatogenesis.
Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region.
Authors: Oxender D L , Zurawski G , Yanofsky C
Published: May 2006

The secondary structure of the terminated trp leader transcript from Escherichia coli was analyzed by RNase T1 partial digestion. Base-paired regions were recovered by nondenaturing gel electrophoresis and identified by denaturing gel electrophoresis and fingerprinting. The tandem tryptophan codons in the leader peptide coding region were found to be base paired with a more distal region of the transcript. This and other secondary structures that the trp leader RNA can form help explain the physiological response of the operon as well as the behavior of regulatory mutants.
Spliced adenovirus-associated virus RNA.
Authors: Laughlin C A , Westphal H , Carter B J
Published: May 2006

We describe the structure of cytoplasmic RNA species transcribed from the DNA of adenovirus-associated virus, a defective parvovirus. The RNA was hybridized with minus strand template DNA and visualized in the electron microscope. Alternatively, the DNA.RNA duplex molecules were digested with nuclease S1 or Escherichia coli exonuclease VII and analyzed by agarose gel electrophoresis. A set of RNA species was observed with 5' terminal at map positions 5, 13, 19, or 39 and a 3' terminus and poly(A) tail at position 96 (one map unit is equivalent to 1% of genome length). Most of these RNAs are spliced and lack sequences approximately between positions 40 and 49. Some RNA preparations also contained unspliced molecules with 5' and 3' terminal at positions similar to those in the spliced RNA.
Chromophore organization in photosynthetic reaction centers: High-resolution magnetophotoselection
Authors: Boxer Steven G. , Roelofs Mark G.
Published: May 2006

The electron spin resonance spectrum of the triplet excited state of
Rhodopseudomonas spheroides
R-26 reaction centers has been studied after excitation with the polarized narrow-bandwidth output of a tunable dye laser from 520 to 670 nm. A theory is developed relating experimental observables to the angles between the electronic transition dipole moment of the excited chromophore and the principle magnetic axis system of the triplet state of the dimeric trap. Data is presented which demonstrates that the treatment is correct and useful, and angles are obtained for the

Q
x

transitions of bacteriopheophytin. High-resolution magnetophotoselection data in the region of the bacteriochlorophyll

Q
x

transitions can be combined with polarized photobleaching experiments to provide direct information on the structure of dimeric trap.
A practicable immunological approach to block spermatogenesis without loss of androgens.
Authors: Talwar G P , Naz R K , Das C , Das R P
Published: May 2006

The intrinsic capacity of the immune system to elicit immune response selectively against late developing sperm proteins has been mobilized to intercept spermatogenesis. Bacillus Calmette-Guérin given in appropriate doses intratesticularly is effective in bringing about this effect. In dogs and rhesus monkeys, the sperm count in the semen declined precipitously, and almost complete azospermia was attained in 4-6 weeks after immunization. The few sperm cells that were present were immotile. Examination of serial sections of testes in immunized rats showed about 98% of the tubules to be devoid of sperm. The tubules were partially or fully atrophied. The basement membrane was, however, intact and the pertubular cell layer was normal. Sertoli cell nuclei were apparently normal but the cytoplasm was vacuolated and, in most cells, partially disintegrated. The lumen of the tubules was exhausted of formed elements and at times filled with eosinophilic debris. Leydig cells were present and hyperplasia of interstitial cells was seen, with massive infiltration of leukocytes. Blood testosterone levels were in the normal range and Leydig cells were responsive to gonadotropins. Libido was intact. The method was applicable to a variety of mammalian species. The implications of the results are discussed.
Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in ColE1 DNA by means of specific antibodies.
Authors: de Murcia G , Lang M C , Freund A M , Fuchs R P , Duane M P , Sage E , Leng M
Published: May 2006

ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[14C]acetylaminofluorene in the range of 0-15 N-2-[14C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and native DNA that had reacted with N-acetoxy-N-2-acetylaminofluorene were shown by electron microscopy to recognize specifically the acetylaminofluorene-modified ColE1 DNA. The antibodies bound to DNA were visualized either per se or after reaction with goat anti-rabbit immunoglobulins coupled with ferritin. There was a linear relationship between the average number of antibodies bound per DNA molecule and the number of N-2-(deoxyguanosine-8yl)-acetylaminofluorene residues per DNA molecule. The slope of this straight line was equal to 0.4. Due to the bivalence of the immunoglobulins one would expect a value of 0.5; we actually observed an important fraction of the bound antibodies crosslinking two parts of the same (or of another) DNA molecule.
In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells.
Authors: Zackroff R V , Goldman R D
Published: May 2006

Intermediate filaments (IF) from baby hamster kidney (BHK-21) cells can be disassembled at low ionic strength and reassembled upon addition of salt. Turbidimetric analyses show that reassembled IF exhibit the light scattering properties of long rods under physiological conditions (5 mM Na+/K+ phosphate, pH 7.2/170 mM NaCl at 21 degrees C). IF weight concentration, determined by centrifugation, is directly proportional to the optical density at 3000 nm. Thus, turbidity can be used as a quantitative assay for IF assembly. Turbidimetric and centrifugation analyses both indicate that IF assembly exhibits a critical protein concentration of 0.05-0.15 mg/ml. Above the critical concentration, IF weight concentration at steady-state is a linear function of the total protein concentration. Negative stain observations at early stages of the assembly process suggest lateral association of protofilaments to form short IF. This lateral association is accompanied by a rapid turbidity increase which is then followed by IF elongation and a slower turbidity increase to plateau. Further purification of IF by low/high-NaCl-induced cycles of disassembly/reassembly results in retention of 54- and 55-kilodalton (decamin) polypeptides. These results constitute a quantitative description of in vitro reassembly of IF from homogeneous cultures of nonkeratinizing cells and establish conditions for further studies on the regulation of IF assembly.
Structural order of lipids and proteins in membranes: evaluation of fluorescence anisotropy data.
Authors: Jähnig F
Published: May 2006

The limiting long-time value of fluorescence anisotropy in membranes is correlated with the orientational order parameter, which characterizes the structural anisotropy of membranes. Existing experimental results for diphenylhexatriene in lipid bilayers are evaluated for the order parameter of lipid order. Steady-state measurements of fluorescence anisotropy can provide the order parameter in good approximation. Proteins in a fluid lipid phase increase the lipid order parameter so determined. Upon comparison with the order parameter from deuterium magnetic resonance, it is concluded that proteins increase the order of the surrounding lipids in off-normal directions. Order parameters of protein order obtained from the limiting value of protein fluorescence anisotropy are discussed with respect to the influence of lipid order on protein order.
Cytoskeleton-disrupting drugs enhance effect of growth factors and hormones on initiation of DNA synthesis.
Authors: Otto A M , Zumbé A , Gibson L , Kubler A M , Jimenez de Asua L
Published: May 2006

Addition of growth factors, such as prostaglandin F2 alpha or fibroblastic growth factor, to quiescent Swiss mouse 3T3 cells resulted in an abrupt increase in the rate of initiation of DNA synthesis after a lag phase of 13-15 hr. This increase could be quantified by a rate constant k. Addition of colchicine, Colcemid, or vinblastine had a synergistic effect on the initiation of DNA synthesis triggered by PGF2 alpha or FGF by increasing the value of k. These drugs alone had no effect. Colchicine had a synergistic effect only if added within 8 hr of the PGF2 alpha or FGF addition. Also, colchicine exerted its full effect when it was present only for the first 5 hr with either growth factor. These results suggest that an intact cytoskeleton is not required for the initiation of DNA synthesis. Furthermore, cytoskeleton-disrupting drugs enhance the stimulatory effect of the growth factors.
A human thymus-leukemia antigen defined by hybridoma monoclonal antibodies.
Authors: Levy R , Dilley J , Fox R I , Warnke R
Published: May 2006

A series of mouse hybridomas producing monoclonal antibodies against human acute lymphocytic leukemia (ALL) cells was generated and screened for tumor specificity. Among 1200 primary cultures, 60 produced an antibody that could distinguish between the immunizing leukemia cells and an isologous B lymphoblastoid cell line. Of these, two produced an antibody that detects an antigen expressed preferentially on ALL cells and on a subpopulation of normal cells found in the cortex of the thymus. Other normal human lymphoid cells from lymph nodes, spleen, bone marrow, and peripheral blood express only low levels of this antigen. High levels of this "thymus-leukemia" antigen were found on T-ALL cells, T-ALL-derived cell lines, and some "null" ALL cells. By contrast, B-cell leukemias, B lymphoblastoid cell lines, and normal and malignant myeloid cells contain either low or undetectable amounts of this antigen. The thymus-leukemia antigen has been isolated from the membranes of leukemia cells by detergent solubilization and subsequent immunoprecipitation with the monoclonal antibody. Preliminary biochemical characterization shows the antigen to be associated with a polypeptide of Mr approximately 28,000.
Classical conditioning in Aplysia californica.
Authors: Walters E T , Carew T J , Kandel E R
Published: May 2006

A form of aversive classical conditioning is described in which a chemosensory conditioned stimulus rapidly acquires the ability to modulate a defensive response (escape locomotion). Because Aplysia show both sensitization and classical conditioning, it is now possible to begin to examine the relationship between nonassociative and associative learning on behavioral and cellular levels.
Conformation of nucleosides: circular dichroism study on the syn-anti conformational equilibrium of 2-substituted benzimidazole nucleosides.
Authors: Miles D W , Townsend L B , Miles D L , Eyring H
Published: May 2006

The solution conformations of 2-substituted derivatives of 1-(beta-D-ribofuranosyl)benzimidazole have been determined by circular dichroism spectroscopy in aqueous solutions. It is shown that analogs with methyl, amino, or methylamino substituents at position 2 of the benzimidazole ring (position 8 of the purine ring) have predominantly anti conformations, whereas analogs with chloro, aza, methoxy, or methylmercapto substituents have predominantly syn conformations. The preferred solution conformations of the benzimidazole nucleosides and analogous purine nucleosides are compared. The results demonstrate that the replacement of nitrogen by carbon at position 3 of the purine ring of purine (beta) nucleosides leads to important conformational consequences, which are strengthened or neutralized by substituents at position 8 of the purine ring.
Thymidylate synthetase purified to homogeneity from human leukemic cells.
Authors: Lockshin A , Moran R G , Danenberg P V
Published: May 2006

Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) from a human leukemic cell line has been purified to homogeneity with one-step affinity column chromatography. The purified enzyme has a specific activity of 3.8 micron/min per mg of protein, which corresponds to a turnover number of 250. These are the highest values reported for a thymidylate synthetase from neoplastic tissue. A ratio of 1.7 mol of 5-fluoro-2'-deoxyuridylate binds per mol of enzyme in the presence of 5,10-methylenetetrahydrofolate. The ternary complex so formed migrates intact on denaturing gels and can be precipitated with trichloroacetic acid; however, urea dissociates the ternary complex. The human thymidylate synthetase is composed of two subunits of 33,000 daltons each. It contains more residues of cysteine, glycine, and arginine and fewer of histidine than the well-studied thymidylate synthetase from Lactobacillus casei.
Heterogeneous nuclear RNA double-stranded regions probed in living HeLa cells by crosslinking with the psoralen derivative aminomethyltrioxsalen.
Authors: Calvet J P , Pederson T
Published: May 2006

The psoralen derivative aminomethyltrioxsalen (AMT, 4'-aminomethyl-4,5',8-trimethylpsoralen) has been employed as a probe for heterogeneous nuclear RNA (hnRNA) double-stranded regions in experiments with living HeLa cells. hnRNA ribonucleoprotein (hnRNP) particles were purified from untreated or AMT-treated cells after irradiation with 365-nm light, and double-stranded hnRNA regions (dsRNA) were isolated by RNase A + T1 digestion of hnRNP, followed by preparative Cs2SO4 isopycnic centrifugation. The purified, hnRNP-derived dsRNA was then assayed for interstrand crosslinks by measurement of its "snapback" to RNase-resistant form after thermal denaturation. By this procedure, the amount of crosslinked dsRNA was found to be increased 3- to 7-fold in cells exposed to AMT in vivo. The levels of crosslinking in vivo compared favorably with those observed in model experiments with pure dsRNA in vitro. These results establish that double-stranded hnRNA regions exist in the living cell, and they further demonstrate that these base-paired regions are organized as rather accessible sites within the nucleus.
Heme biosynthesis in Friend erythroleukemia cells: control by ferrochelatase.
Authors: Rutherford T , Thompson G G , Moore M R
Published: May 2006

The activities of the enzymes of heme biosynthesis (except protoporphyrin oxidase) have been followed during the induction of Friend cells in culture. All the enzyme activities increased after induction with dimethyl sulfoxide. The activities of the intermediate enzymes were much higher than those of delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37], the initial enzyme, or ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the final enzyme of the pathway. Ferrochelatase activity was not detectable in the uninduced cell. delta-Aminolevulinate synthase activity increased during the first 24 hr of induction; porphobilinogen deaminase activity began to increase after 48 hr and ferrochelatase activity, after 72 hr. However, the induction of heme synthesis followed the same time course as that of ferrochelatase activity, not that of delta-aminolevulinate synthase activity. The cellular growth medium was found to contain traces of protoporphyrins. Thus, ferrochelatase is shown to be rate limiting for heme synthesis during early stages of Friend cell induction. A Friend cell variant (Fw), which is not inducible except in the presence of exogenous hemin, was also studied. All the enzymes of heme synthesis except ferrochelatase were inducible by butyric acid. Ferrochelatase was not inducible by butyric acid or hemin plus butyric acid. These cells also excrete protoporphyrin, The failure to induce ferrochelatase activity is believed to be the cause of, not a consequence of, the noninducibility of this cell line.
Induction of amplified synthesis and secretion of IgM by fusion of murine 'b lymphoma with myeloma cells.
Authors: Laskov R , Kim K J , Asofsky R
Published: May 2006

Murine B lymphoma cultured cell lines bearing membrane IgM and lacking the ability to secrete measurable amounts of IgM were fused with drug-resistant cell lines derived from the IgG2b-producing MPC-11 myeloma. Many of the hybrid clones synthesized and secreted large amounts of IgM, as judged by radial and double immunodiffusion in agar of culture supernatants and by polyacrylamide gel electrophoresis of biosynthetically labeled IgM. When fused with an IgG2b-producing myeloma, many of the hybrids also produced IgG2b, indicating that there was no suppression of the parental IgG synthesis in the hybrids. The amount of IgM or IgG2b secreted by the hybrids was higher than that secreted by myeloma cell lines. The synthesis of the gamma2b heavy chain of the MPC-11 myeloma was not reexpressed by fusion of a gamma2b nonproducer myeloma cell variant with B lymphoma. No other classes of Ig heavy chains, besides the gamma2b and the mu chains, were found to be secreted by any of more than 100 B lymphoma--myeloma hybrids examined. The results of the present work suggest that myeloma cells may fuse not only with normal plasma cells but also with less-mature normal B lymphocytes and that this fusion "induces" the maturation of B lymphocytes into IgM-secreting cells.
Regulation of protein synthesis in rabbit reticulocyte lysates: Additional initiation factor required for formation of ternary complex (eIF-2·GTP·Met-tRNA
f
) and demonstration of inhibitory effect of heme-regulated protein kinase
Authors: Ranu Rajinder Singh , London Irving M.
Published: May 2006

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA
f
and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA
f
complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978)
Proc. Natl. Acad. Sci. USA
75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg
2+
, ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA
f
. In the absence of Mg
2+
, however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity—hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.