bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a rare Epstein-Barr virus variant that enhances early antigen expression in Raji cells.
Authors: Rabson M , Heston L , Miller G
Published: May 2006

Early antigens (EAs) are made when the P3J-HR-1 strain of Epstein-Barr virus (EBV) infects cells that already harbor latent EB viral genomes. We wished to identify EBV genes that might participate in induction of EAs. We have recently isolated from the HR-1 line EB viral variants that are unable to induce EAs. We have now isolated a clone of HR-1 cells that releases virus with the capacity to induce EA. We compared the genome of the EB variant that possesses the capacity to induce EA with that of a variant that is unable to induce EA and with parental stock that was the source of both different biotypes of EBV. The variant that is able to induce EA contains, in molar or greater quantities, additional fragments of EBV DNA not found in the variant that lacks that capacity. These same DNA fragments are present in submolar quantities in the parental DNA, indicating that they represent a subpopulation in the parental viral DNA mixture. We thus provide evidence that EA induction is brought about by unusual forms of EBV DNA that are likely to act by regulating expression of the genome.
Monoclonal antibodies to human urokinase identify the single-chain pro-urokinase precursor.
Authors: Salerno G , Verde P , Nolli M L , Corti A , Szöts H , Meo T , Johnson J , Bullock S , Cassani G , Blasi F
Published: May 2006

Monoclonal antibodies have been obtained that recognize either the A or B chain of human urinary urokinase. These antibodies identify human urokinase-producing cells and the product of urokinase mRNA. Anti-urokinase monoclonal antibodies precipitate an approximately equal to 54,000-dalton protein synthesized in vitro in a rabbit reticulocyte cell-free system. This pro-urokinase appears to be the precursor of both A and B chains of human urinary urokinase. Urokinase mRNA in human kidney constitutes only 0.1% or less of total poly(A)+ RNA.
Perpendicular orientation and directional migration of amphibian neural crest cells in dc electrical fields.
Authors: Cooper M S , Keller R E
Published: May 2006

The behavior of cultured neural crest cells of Ambystoma mexicanum and Xenopus laevis in dc electrical fields was studied. In fields of 1-5 V/cm, isolated or confluent cells retract both their anode- and cathode-facing margins. Subsequently, the cells elongate, with protrusive activity confined to their narrow ends. In larger fields (greater than or equal to 5 V/cm), protrusions form on the cathode-facing sides of the perpendicularly oriented cells. The cells then begin migrating laterally, perpendicular to their long axes, towards the cathode. We suggest that the perpendicular alignment and cathode-directed migrations result from cytoskeletal changes mediated by modified ion fluxes through the anode-facing (hyperpolarized) and cathode-facing (depolarized) cell membranes. The breaking of cellular confluence in response to dc electric fields is also discussed.
A pattern of partially homologous recombination in mouse L cells.
Authors: Anderson R A , Kato S , Camerini-Otero R D
Published: May 2006

Herpes simplex virus thymidine kinase gene and pBR322 DNA (in large excess to the thymidine kinase gene) were introduced into mouse L cells by calcium phosphate DNA-mediated gene transfer. DNA fragments encompassing six junctions between the exogenous DNAs have been cloned and their nucleotide sequences determined. Analysis of these sequences has shown that stretches of partial homology involving from 20-50 base pairs are present near the points at which joining occurs between the donor molecules. The structure of the junction sequences suggests that the recombination event involves the alignment of the two donor DNA molecules at partially homologous regions followed by staggered cutting and joining. One donor molecule is always cut in the region of partial homology, while the second is cut at some distance that is a small multiple of 13.5 +/- 0.5 base pairs away (at 0, 14, 27, 39, 41, and 54 base pairs). In the three junctions where the second cut is far from the region of homology, a 17- to 19-base-pair segment of DNA separates the donor sequences. In all cases the origin of this "filler" DNA appears to be oligonucleotides derived from pBR322.
Mice completely suppressed for the expression of immunoglobulin kappa light chain.
Authors: Weiss S , Lehmann K , Raschke W C , Cohn M
Published: May 2006

Complete suppression of expression of immunoglobulin kappa light chain was achieved by injecting female mice from birth with a mixture of antisera against the mu heavy chain and kappa light chain (anti-mu and anti-kappa). Then their offspring were injected with anti-kappa from birth. This resulted in stable suppression as long as anti-kappa injections were continued. kappa light chain was not detectable either in serum or at the cellular level. The number of B cells in spleen and the concentration of immunoglobulin classes and subclasses in serum were normal. The normal levels were achieved by a compensating enhancement of lambda light chain expression. Analysis of the light chains of immunoglobulins secreted by spleen cells from suppressed mice after liposaccharide stimulation by two-dimensional gels showed lambda chain to have a limited heterogeneity. Primary responses to dinitrophenol, influenza strain A, and keyhole limpet hemocyanin were drastically affected, whereas secondary responses appeared to be quite normal, suggesting a surprisingly large potential repertoire.
Polypeptide hormone regulation of gene transcription: specific 5' genomic sequences are required for epidermal growth factor and phorbol ester regulation of prolactin gene expression.
Authors: Supowit S C , Potter E , Evans R M , Rosenfeld M G
Published: May 2006

A fusion gene containing 5' rat prolactin genomic sequences ligated to the structural portion of the rat growth hormone gene ( grl ) was introduced by DNA-mediated gene transfer into mammalian cells by using a chimeric plasmid vector. Clonal transfected cell lines produced a mRNA that used the authentic 5' initiation site and that was processed to the predicted size. The intracellular levels of this RNA product were increased 2.5- to 5-fold by exposure of the cells to epidermal growth factor (EGF) and 2- to 3-fold by exposure of the cells to a potent phorbol ester, phorbol 12-myristate 13-acetate, apparently due to regulation at the level of gene transcription. Substitution of the 5' prolactin DNA sequences by 5' growth hormone DNA sequences resulted in the loss of EGF inducibility. A genomic sequence in or near the 5' flanking portion of the prolactin gene therefore appears to confer polypeptide hormone transcriptional regulation upon the gene.
Nucleotide sequence of avian carcinoma virus MH2: two potential onc genes, one related to avian virus MC29 and the other related to murine sarcoma virus 3611.
Authors: Kan N C , Flordellis C S , Mark G E , Duesberg P H , Papas T S
Published: May 2006

The 5.2-kilobase (kb) RNA genome of avian carcinoma virus MH2 has the genetic structure 5'-delta gag (0.2 kb)- mht (1.2 kb)-myc (1.4 kb)-c (0.4 kb)-poly(A) (0.2 kb)-3'. delta gag is a partial retroviral core protein gene, mht and myc are cell-derived MH2-specific sequences, and c is the 3'-terminal retroviral vector sequence. Here we have determined the nucleotide sequence of 3.5 kb from the 3' end of delta gag to the 3' end of molecularly cloned proviral MH2 DNA, in order to elucidate the genetic structure of the virus and to compare it with other mht - and myc-containing oncogenic viruses as well as with the chicken proto-myc gene. The following results were obtained: (i) delta gag- mht forms a hybrid gene with a contiguous reading frame of 2682 nucleotides that terminates with a stop codon near the 3' end of mht . The 3' 969 nucleotides of mht up to the stop codon are 80% sequence related to the onc-specific raf sequence of murine sarcoma virus 3611 (94% homologous at the deduced amino acid level). (ii) The myc sequence is preceded by an RNA splice acceptor site shared with the cellular proto-myc gene, beyond which it is colinear up to a 3'-termination codon and 40 noncoding nucleotides with the myc sequences of avian retrovirus MC29 and chicken proto-myc. Thus, myc forms, together with a 5' retroviral exon, a second MH2-specific gene. (iii) myc is followed by the 3'-terminal c region of about 400 nucleotides, which is colinear with that of Rous sarcoma virus except for a substitution near the 5' end of the long terminal repeat. It is concluded that MH2 contains two genes with oncogenic potential, the delta gag- mht gene, which is closely related to the delta gag-raf transforming gene of MSV 3611, and the myc gene, which is related to the transforming gene of MC29. Furthermore, it may be concluded that the cellular proto-onc genes, which on sequence transduction become viral onc genes, are a small group because among the 19 known onc sequences, 5 are shared by different taxonomic groups of viruses of which the mht /raf homology is the closest determined so far.
Synthesis and characterization of cDNA encoding a cartilage-specific short collagen.
Authors: Ninomiya Y , Olsen B R
Published: May 2006

Hyaline cartilage contains a unique set of collagenous proteins. Type II collagen is the most abundant, constituting about 85% of the total cartilage collagen. In addition, several minor collagenous components have been described. To study the structure and developmental regulation of chondrocyte-specific collagens, we have constructed a cDNA library from embryonic chicken sternal cartilage mRNA. We report here on the isolation and characterization of a 3200 base-pair-long cDNA that codes for a collagenous polypeptide of unusual structure in that the total length of the molecule is only about half of pro alpha 1(II) collagen chains. The mRNA for this polypeptide is considerably smaller than mRNA encoding the pro alpha chains of interstitial collagens. In addition, the peptide encoded by the cDNA appears to contain at least three domains with triple-helical potential separated by short, noncollagenous peptides. Between the three collagenous domains are several cysteinyl residues.
Fc-mediated binding of IgG to vimentin-type intermediate filaments in vascular endothelial cells.
Authors: Hansson G K , Starkebaum G A , Benditt E P , Schwartz S M
Published: May 2006

Prior studies have shown that vascular endothelial cells bind circulating IgG intracellularly during cell death. We now demonstrate that all endothelial cells have intracellular binding sites for IgG and that these binding sites are exposed to circulating IgG only if the plasma membrane is damaged. The binding sites are located on the cytoskeletal intermediate filaments and can be detected also in other cells containing vimentin-type intermediate filaments. Monoclonal human IgG1 exhibited saturable, high-affinity binding to vimentin-enriched cytoskeletons. Binding was inhibited by Fc fragments but not by Fab, F(ab')2, or pFc' fragments, suggesting that the binding site on IgG is located in the C gamma 2 domain of the Fc fragment. Binding of IgG to intermediate filaments may be important for the destruction and removal of damaged cells.
Shared antigenic determinant between the Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons.
Authors: Jacob M H , Berg D K , Lindstrom J M
Published: May 2006

Monoclonal antibodies raised against purified acetylcholine receptor from muscle and electric organ were tested for cross-reaction with surface components on chicken ciliary ganglion neurons. Indirect immunofluorescence indicated that antibodies to a determinant in the "main immunogenic region" of the receptor bind to the neurons in culture. Ultrastructural studies on 16-day embryonic ganglia, using horseradish peroxidase-conjugated monoclonal antibody, revealed that most of the conjugate labeling was associated with synaptic membrane on the neurons. A lesser amount of labeling was associated with the short processes extending from the neuronal somata in the region of preganglionic innervation. The labeling was blocked by coincubation with unlabeled antibodies of appropriate specificity and not by nonimmune serum. The pattern of labeling was clearly different from that previously found for a horseradish peroxidase conjugate of alpha-bungarotoxin: the toxin conjugate bound extensively to the short processes but not to synaptic membrane on the neurons. The synaptic antigen identified here by the cross-reacting antibodies is a candidate for the synaptic acetylcholine receptor on chicken ciliary ganglion neurons.
Goblin (ankyrin) in striated muscle: identification of the potential membrane receptor for erythroid spectrin in muscle cells.
Authors: Nelson W J , Lazarides E
Published: May 2006

Goblin , a high molecular weight (Mr, 260,000) polypeptide of avian erythrocyte plasma membranes characterized by hormone-dependent phosphorylation, is shown by a variety of criteria to be the avian equivalent of ankyrin, the membrane attachment protein for spectrin; a polyclonal monospecific goblin antiserum reacts specifically with ankyrin from mammalian erythrocyte ghosts; goblin and ankyrin have highly homologous, although distinct, two-dimensional peptide maps; and, in reconstitution experiments, goblin binds to spectrin and band 3 in approximately the same molar ratio as ankyrin. Immunoautoradiography and immunofluorescence with goblin antiserum reveal that a serologically related polypeptide (Mr, 235,000) is present in highly purified membrane fractions of mammalian myocardium and in whole extracts of adult chicken cardiac and skeletal muscle-nonerythroid tissues which express predominantly the erythroid (alpha beta-) spectrin phenotype. Erythroid spectrin and goblin (ankyrin) are codistributed in skeletal muscle at the sarcolemma as discrete foci adjacent to the Z lines and, in pectoral muscle, also at the periphery of the Z discs. These spatial relationships indicate that goblin and spectrin in muscle cells form a structural framework that serves as the attachment site for the myofiber at the level of the Z line on the sarcolemma.
Inducer-mediated commitment of murine erythroleukemia cells to terminal cell division: the expression of commitment.
Authors: Murate T , Kaneda T , Rifkind R A , Marks P A
Published: May 2006

Murine erythroleukemia cells (MELC) are transformed cells that can be induced to differentiate by a variety of agents, such as hexamethylenebisacetamide (HMBA) and dimethyl sulfoxide. Dexamethasone suppresses HMBA-mediated MELC differentiation, but MELC retain a memory for their exposure to HMBA since, on transfer from culture with HMBA and dexamethasone to medium without additions, a portion of the cells express characteristics of terminal differentiation. This study characterizes the steroid suppressed steps in the multi-step process of inducer-mediated MELC terminal differentiation. MELC in culture with HMBA and dexamethasone show low levels of commitment to terminal cell division; upon transfer to culture with inducer alone there is a rapid increase in the proportion of committed cells. The magnitude of this rapid or "step-up" expression of commitment increased with the length of prior culture with inducer and steroid. This step-up expression is not inhibited by actinomycin D or cordycepin but is blocked by cycloheximide. HMBA is required for step-up expression of commitment. In the absence of inducer, there is a rapid decay in the capacity for step-up expression. Thus, HMBA initiates a series of changes leading to the accumulation of factors--which may be mRNAs--whose expression is blocked by dexamethasone. Hemin, which induces MELC accumulation of globin mRNA but not commitment to terminal cell division, cannot, as does HMBA or dimethyl sulfoxide, cause step-up expression of commitment.
Six HLA-D region alpha-chain genes on human chromosome 6: polymorphisms and associations of DC alpha-related sequences with DR types.
Authors: Spielman R S , Lee J , Bodmer W F , Bodmer J G , Trowsdale J
Published: May 2006

Analysis of cosmid clones containing genes related to the HLA-DR alpha chain calls for at least six HLA-D region alpha-chain coding sequences in man; namely, DR alpha, DC alpha, DX alpha (very closely related to DC alpha), SB alpha 1, SB alpha 2 (two closely linked genes on the same cosmid clones), and DZ alpha. The first four genes have been described previously. SB alpha 2 and DZ alpha are recently identified genes, characterized by their unique and, from a limited study, nonpolymorphic bands when used as probes for human DNA on Southern blots. All of the genes are present in somatic cell hybrids containing a human X/6 translocation chromosome, and so they are all presumably in the HLA region. The polymorphisms in the region of the DC alpha and related DX alpha genes were studied with Southern blots of DNA from a set of mostly homozygous HLA-D-typing cell lines. With EcoRI, the band patterns for the DC alpha gene corresponded to the major cross-reactive HLA-DR serotypes associated with DC (namely MT1, -2, and -3) while the DX alpha band was invariant. Both genes were polymorphic with the enzyme Taq I. Within some DR types additional polymorphic variation was detected at the DNA level, implying the existence of subtypes. The pattern of polymorphisms for DC alpha, and to a lesser extent for DX alpha, suggests that these genes may play an important role in certain HLA-D associations with disease.
Schistosoma mansoni egg granuloma size reduction in challenged baboons after vaccination with irradiated cryopreserved schistosomula.
Authors: Damian R T , Roberts M L , Powell M R , Clark J D , Lewis F A , Stirewalt M A
Published: May 2006

Young male baboons born in captivity were immunized with an attenuated, cryopreserved schistosomular vaccine derived from gamma-irradiated (50 krad) cercariae of the Puerto Rican strain of Schistosoma mansoni. Protection against a heterologous Kenyan strain of S. mansoni, after percutaneous infection, was assessed. Partial protection (33-53% reduction in worm burden) was obtained in three of six vaccinated-challenged baboons, an unremarkable result. Of greater interest was the fact that all six unvaccinated-challenged control baboons, but only one of the six vaccinated-challenged baboons, had macroscopic egg granulomas on their liver surfaces. This difference in granuloma size was substantiated by measuring hepatic and colonic granulomas. The mean (+/- SEM) hepatic and colonic granuloma diameters for the six unvaccinated baboons were 406 +/- 38 micron and 313 +/- 27 micron, respectively, and for the five "typical" vaccinated-challenged baboons the mean diameters were 283 +/- 27 micron and 202 +/- 23 micron, respectively. Both hepatic and colonic granulomas were significantly smaller in the five typical vaccinated-challenged baboons. Not only did the exceptional vaccinated-challenged baboon have very large hepatic and colonic granulomas, but also it was the only one of its group whose mesenteric lymph node cells were not suppressed in their in vitro proliferative response to a schistosome antigen. These results strongly suggest that granuloma size reduction in the majority of the vaccinated baboons was the result of immunoregulation--i.e., the small postvaccination granulomas were "modulated." Despite their small size, hepatic granulomas in the typical vaccinated baboons were apparently as effective in sequestering egg toxins and preventing hepatocyte damage as the larger granulomas of the control baboons. Smaller, less obstructive granulomas are thought to be more beneficial to the host than large, vigorous granulomas, with respect to lessening chronic disease. The present results give encouragement that a vaccine to ameliorate disease in human schistosomiasis is possible. This effect should add to the attractiveness of partial protection against challenge infections conferred by attenuated larval vaccines, as reported by others, to yield a dually beneficial vaccine for human use.
Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus.
Authors: Lai M M , Baric R S , Brayton P R , Stohlman S A
Published: May 2006

Mouse hepatitis virus, which replicates in cytoplasm, contains leader RNA sequences at the 5' end of the virus-specific mRNAs. We have sequenced this leader RNA by synthesizing cDNA from a synthetic oligodeoxyribonucleotide primer (15-mer) that is complementary to the sequences at the junction site between the leader and body sequences of the mRNAs. The leader sequences on each mRNA have exactly the same size, which span approximately equal to 70 nucleotides. Leader cDNA fragments obtained from several mRNA species were sequenced and found to be identical. Computer analysis of the leader RNA sequences shows that they share extensive sequence homology with the long-terminal-repeat region of several mammalian sarcoma viruses, suggesting possible common functions. This is a novel case of spliced leader sequences in the mRNAs of a cytoplasmic virus. An identical leader sequence is also present at the 5' end of the virion genomic RNA. The leader RNA is thus probably encoded by the virion genomic RNA template and is fused to the different body sequences of the various mRNAs. Since conventional RNA splicing is not involved, a novel mechanism for fusing two noncontiguous RNA segments in the cytoplasm must be utilized during viral transcription. Several minor cDNA bands longer than the leader were also synthesized, suggesting the possible presence of partially homologous sequences in other parts of the genome RNA.
Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70.
Authors: Nunberg J H , Rodgers G , Gilbert J H , Snead R M
Published: May 2006

A method is presented whereby antigenic determinants recognized by specific monoclonal antibodies can be mapped to specific sites on a protein sequence with high resolution. Short DNase I-generated DNA fragments encoding portions of the protein of interest are molecularly cloned into the EcoRI site of the beta-galactosidase gene of phage lambda Charon 16 so as to obtain expression of random protein fragments as fusion proteins. The monoclonal antibody is used to screen the phage library to isolate phage expressing the specific antigenic determinant. DNA of immunoreactive phage can be analyzed rapidly and subcloned to allow DNA sequence determination. The method is generally applicable and permits antigenic determinants of functionally interesting monoclonal antibodies to be mapped and related to specific protein sequences. We have used this procedure to determine the region of the feline leukemia virus envelope protein gp70 recognized by a virus-neutralizing monoclonal antibody, cl.25. Antibody binding was mapped to a 14-amino acid region in the amino-terminal half of gp70. This region may be directly involved in an essential function of the gp70 protein, perhaps in gp70-mediated host recognition functions. Synthetic peptides derived from this region may provide useful vaccine antigens for the prevention of feline leukemia virus-associated disease in cats.
NMR study of chloride ion interactions with thylakoid membranes
Authors: Baianu I. C. , Critchley C. , Govindjee  , Gutowsky H. S.
Published: May 2006

The role of Cl
-
in photosynthetic O
2
evolution has been investigated by observing the
35
Cl NMR linewidth under a variety of conditions in aqueous suspensions of chloroplasts, primarily for the halophytes
Avicennia germinans, Avicennia marina
, and
Aster tripolium
but also for spinach. The line broadening shows there is weak, ionic binding of Cl
-
to thylakoids, the bound Cl
-
exchanging rapidly (>>10
4
sec
-1
) with free Cl
-
in solution. The binding is necessary for O
2
evolution to occur. Michaelis-Menten constants obtained from the Cl
-
dependence of the O
2
evolution rate are ≈15-70 mM for the halophytes compared with 0.6 mM for spinach (0.5 mM with Br
-
). There appear to be two types of Cl
-
binding sites in halophytes, of which the stronger is the activator, at lower [Cl
-
], of O
2
evolution. The
35
Cl line broadening includes a nonspecific interaction, which becomes apparent at high Cl
-
concentrations (≥0.5 M).
Evolution of multigene families under interchromosomal gene conversion.
Authors: Nagylaki T
Published: May 2006

A model for the evolution of the probabilities of genetic identity within and between loci of a multigene family in a finite population is formulated and investigated. Unbiased interchromosomal gene conversion, equal crossing-over between tandemly repeated genes, random genetic drift, and mutation to new alleles are incorporated. Generations are discrete and nonoverlapping; the diploid, monoecious population mates at random. Formulae for the equilibrium values of the probabilities of identity and for the rate of convergence are deduced. At equilibrium, the amount of intralocus homology, f, always exceeds the amount of interlocus homology, ĝ. The equilibrium homologies f and ĝ and the characteristic convergence time T are independent of the crossover rate. As the population size and the number of repeats increase, f and ĝ decrease and T increases; as the rate of gene conversion increases, f and T decrease whereas ĝ increases. The time T can be sufficiently short to imply that interchromosomal gene conversion may be an important mechanism for maintaining sequence homogeneity among repeated genes.
Direct demonstration of increased intracellular concentration of free calcium as measured by quin-2 in stimulated rat peritoneal mast cell.
Authors: White J R , Ishizaka T , Ishizaka K , Sha'afi R
Published: May 2006

Rat mast cells, passively sensitized with monoclonal mouse IgE antibody, were stimulated with multi-valent antigen, and an increase in cytoplasmic Ca2+ was determined by using the fluorescent probe quin-2. The increase in quin-2 fluorescence reached maximum within 20 sec after the antigen challenge and then gradually declined. A substantial increase in quin-2 fluorescence was observed in the presence of EGTA, indicating that bridging of cell-bound IgE antibody molecules by antigen induced not only Ca2+ influx but also mobilization of intracellular Ca2+. Phosphatidylserine added to the medium enhanced both the antigen-induced histamine release and the increase in quin-2 fluorescence and slowed the rate at which the quin-2 signal returned to basal levels. Both the antigen-induced increase in quin-2 fluorescence and histamine release were inhibited by pretreatment of mast cells with inhibitors of methyltransferases, theophylline, or cromoglycate. It was also found that methyltransferase inhibitors and theophylline inhibited not only stimulus-dependent calcium influx but also release of bound calcium from intracellular stores. Other secretagogues, compound 48/80 (1 microgram/ml) and Ca ionophore A23187 (0.1 microM), induced a rapid increase in cytoplasmic Ca2+ in rat mast cells and subsequent histamine release. In contrast, the cocarcinogenic compound phorbol 12-myristate 13-acetate caused histamine release without increasing the quin-2 fluorescence.
Heterogeneity of vertebrate brain tubulins.
Authors: Field D J , Collins R A , Lee J C
Published: May 2006

We have examined the extent of brain tubulin heterogeneity in six vertebrate species commonly used in tubulin research (rat, calf, pig, chicken, human, and lamb) using isoelectric focusing, two-dimensional electrophoresis, and peptide mapping procedures that provide higher resolution than previously available. The extent of heterogeneity is extremely similar in all of these organisms, as judged by number, range of isoelectric points, and distribution of the isotubulins. A minimum of 6 alpha and 12 beta tubulins was resolved from all sources. Even the pattern of spots on two-dimensional peptide maps is remarkably similar. These similarities suggest that the populations of tubulin in all of these brains should have similar overall physical properties. It is particularly interesting that chicken, which has only four or five beta-tubulin genes, contains approximately 12 beta tubulins. Thus, post-translational modification must generate at least some of the tubulin heterogeneity. Mammalian species, which contain 15-20 tubulin DNA sequences, do not show any more tubulin protein heterogeneity than does chicken. This suggests that expression of only a small number of the mammalian genes may be required to generate the observed tubulin heterogeneity.
Isolation and characterization of a gene for a major light-harvesting polypeptide from
Cyanophora paradoxa
Authors: Lemaux Peggy G. , Grossman Arthur
Published: May 2006

Antibodies raised against mixtures of phycobilisome polypeptides from the eukaryotic alga
Cyanidium caldarium
were used in an immunological screen to detect expression of phycobiliprotein genes in an
Escherichia coli
library containing segments of plastid (chloroplast, cyanelle) DNA from another eukaryotic alga,
Cyanophora paradoxa
. The four candidate clones obtained were mapped by restriction analysis and found to be overlapping. The clone with the smallest insert (1.4 kilobases) was partially sequenced and a coding region similar to the carboxyl terminus of the phycobiliprotein subunit β-phycocyanin was found. The coding region for the β-phycocyanin gene in
C. paradoxa
has been mapped to the small single copy region on the cyanelle genome, and its orientation has been determined. A short probe unique to a conserved chromophore binding site shared by at least two phycobiliprotein subunits has now been generated from the carboxyl terminus of the β-phycocyanin gene. This probe may be useful in identifying specific phycobiliprotein subunit genes, β-phycocyanin, β-phycoerythrocyanin, and possibly β-phycoerythrin, in other eukaryotic algae and in prokaryotic cyanobacteria.
Structure of the cholesteryl ester core of human plasma low density lipoproteins: selective deuteration and neutron small-angle scattering.
Authors: Laggner P , Kostner G M , Degovics G , Worcester D L
Published: May 2006

The structural arrangement of cholesteryl esters in human plasma low density lipoproteins (LDL) has been studied by selective deuteration and neutron small-angle scattering. LDL were labeled by in vitro exchange with two different kinds of deuterated cholesteryl esters, one labeled in the fatty acyl chain (cholesteryl myristate-d27) and the other in the branched side chain of cholesterol (cholesteryl-25,26,27-d7 oleate). Neutron scattering data from deuterated and protonated LDL were compared to identify the locations of the fatty acyl and cholesterol side chain moieties. Below the thermotropic transition, radii of gyration of 60 A and 70 A were obtained for these two domains, respectively, indicating that the cholesteryl nuclei are situated more distantly from the center than the fatty acyl chains. At 37 degrees C, above the thermotropic transition of the cholesteryl esters in LDL, both parts have similar radii of gyration of approximately 56 A. This information is used in a discussion of possible structural models for the apolar lipid core of LDL.
Protein 4.1: its association with the human erythrocyte membrane.
Authors: Shiffer K A , Goodman S R
Published: May 2006

125I-labeled protein 4.1a and 4.1b have equal ability to reassociate with inside-out erythrocyte vesicles that were depleted of protein 4.1 in addition to other peripheral membrane proteins. The reassociation of 125I-labeled protein 4.1 to protein 4.1-depleted vesicles at 4 degrees C is salt dependent, pH dependent, and saturable with a Kd of 42-50 nM and an extrapolated maximal binding capacity of 120-140 micrograms of protein 4.1 bound per mg of vesicle protein or 60-70 micrograms of protein 4.1 bound per mg of ghost protein, correlating with the protein 4.1 content in the erythrocyte membrane (6-7% of the total membrane protein). Selective proteolytic cleavage of these vesicles with papain (5 micrograms/ml at 4 degrees C) eliminates greater than 60% of the high-affinity binding sites; therefore, we conclude that the interaction of protein 4.1 with the cytoplasmic membrane surface is through a specific high-affinity protein-protein association.
Differential regulation of the duplicated isocytochrome c genes in yeast.
Authors: Laz T M , Pietras D F , Sherman F
Published: May 2006

The two unlinked genes CYC1 and CYC7 encode iso-1-cytochrome c and iso-2-cytochrome c, respectively, in the yeast Saccharomyces cerevisiae. An examination of the steady-state level of CYC1 and CYC7 mRNAs in normal and mutant strains grown under different conditions, along with previous results of apoprotein levels, demonstrate that CYC1 and CYC7 have similar and different modes of regulation. Both CYC1 and CYC7 mRNAs are diminished after anaerobic growth. In contrast, CYC1 mRNA but not CYC7 mRNA is decreased by heme deficiency in hem1 mutants. Although both CYC1 and CYC7 mRNAs are substantially lowered after growth in glucose medium, there is a difference in the kinetics of glucose derepression. CYC1 mRNA levels rise in the early logarithmic phase of growth before complete exhaustion of glucose, whereas CYC7 mRNA levels rise in the late logarithmic phase when the level of CYC1 mRNA has plateaued. For a brief period before cessation of growth, the level of CYC7 mRNA attains a level corresponding to the high derepressed level of CYC1 mRNA. The high amount of CYC7 mRNA is surprising because iso-2-cytochrome c constitutes only 5% of the total cytochrome c complement in derepressed cells. We suggest that iso-2-cytochrome c has the potential to comprise a major proportion of cytochrome c under certain physiologic conditions that have not been experimentally defined. The cyc3 mutant, which lacks the ability to attach heme groups to apocytochromes c, contains both CYC1 and CYC7 mRNAs in normal amounts. Yet, cyc3 mutants contain only apoiso-2-cytochrome c and not apoiso-1-cytochrome c. The lack of accumulation of apoiso-1-cytochrome c in cyc3 mutants, which contain CYC1 mRNA, suggests that apoiso-1-cytochrome c is extensively regulated by a post-transcriptional process.
Function of the thalamic reticular complex: the searchlight hypothesis.
Authors: Crick F
Published: May 2006

It is suggested that in the brain the internal attentional searchlight, proposed by Treisman and others, is controlled by the reticular complex of the thalamus (including the closely related perigeniculate nucleus) and that the expression of the searchlight is the production of rapid bursts of firing in a subset of thalamic neurons. It is also suggested that the conjunctions produced by the attentional searchlight are mediated by rapidly modifiable synapses--here called Malsburg synapses--and especially by rapid bursts acting on them. The activation of Malsburg synapses is envisaged as producing transient cell assemblies, including "vertical" ones that temporarily unite neurons at different levels in the neural hierarchy.
Statistical thermodynamics of amphiphile chains in micelles
Authors: Ben-Shaul A. , Szleifer I. , Gelbart W. M.
Published: May 2006

The probability distribution of amphiphile chain conformations in micelles of different geometries is derived through maximization of their packing entropy. A lattice model, first suggested by Dill and Flory, is used to represent the possible chain conformations in the micellar core. The polar heads of the chains are assumed to be anchored to the micellar surface, with the other chain segments occupying all lattice sites in the interior of the micelle. This “volume-filling” requirement, the connectivity of the chains, and the geometry of the micelle define constraints on the possible probability distributions of chain conformations. The actual distribution is derived by maximizing the chain's entropy subject to these constraints; “reversals” of the chains back towards the micellar surface are explicitly included. Results are presented for amphiphiles organized in planar bilayers and in cylindrical and spherical micelles of different sizes. It is found that, for all three geometries, the bond order parameters decrease as a function of the bond distance from the polar head, in accordance with recent experimental data. The entropy differences associated with geometrical changes are shown to be significant, suggesting thereby the need to include curvature (environmental)-dependent “tail” contributions in statistical thermodynamic treatments of micellization.
Geodesic flow on SO(4) and the intersection of quadrics
Authors: Adler Mark , van Moerbeke Pierre
Published: May 2006

This note studies the relationship between the complete integrability of geodesic flow on SO(4) with a left-invariant metric and the geometry of the intersection of four quadrics in P
6
.
Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis.
Authors: Germino J , Bastia D
Published: May 2006

We have developed a rapid and general technique for purification of a protein encoded by a cistron contained in a recombinant DNA clone. The technique consists of fusing the target cistron DNA in the correct reading frame to a marker cistron via a piece of DNA that codes for a linker peptide. The target cistron in the example presented here is the replication initiator cistron of the plasmid R6K. The linker is a DNA fragment encoding 60 amino acids from the triple helical region of chicken pro alpha-2 collagen, and the marker cistron encodes the beta-galactosidase protein of Escherichia coli. The tripartite hybrid protein was rapidly purified by selective binding to and elution from a beta-galactosidase specific-affinity column. The hybrid protein was then digested with a purified microbial collagenase to cleave the linker, and high-pressure liquid chromatography allowed the rapid isolation of the target protein from the marker protein. Using this technique, we have purified the highly labile R6K replication initiator to homogeneity, and we have resolved the protein into NH2-terminal and COOH-terminal segments. We have further shown, by in vitro binding, that the COOH-terminal segment has at least one DNA-binding domain. The domain binds to the same restriction fragments of the R6K chromosome as the intact or beta-galactosidase-tagged initiator protein.
In vitro-synthesized adenovirus 2 messenger RNA precursors are accurately spliced by nuclear extracts.
Authors: Goldenberg C J
Published: May 2006

Precursor mRNAs were synthesized in vitro from a plasmid in which the early region 2 gene of adenovirus 2 is fused to an efficient bacteriophage promoter (Salmonella phage 6). The RNAs were purified and used as substrates for in vitro splicing in the presence of nuclear extracts prepared from MOPC-315 mouse myeloma cells. The in vitro splicing was accurate at the nucleotide level. The reaction occurs rapidly and without any detectable lag. The concentration of the pre-mRNA precursor during incubation appears to be an important factor for high efficiency (60%-80%) of in vitro RNA splicing. Fractionation of the splicing components as well as modifications of the DNA template to study the nucleotide-sequence requirement for in vitro splicing can now be accomplished with this system.
Identification of 17 beta-estradiol as the estrogenic substance in Saccharomyces cerevisiae.
Authors: Feldman D , Tökés L G , Stathis P A , Miller S C , Kurz W , Harvey D
Published: May 2006

Saccharomyces cerevisiae possesses a high-affinity estrogen binding protein and an endogenous ligand that displaces [3H]estradiol from both the yeast binding protein and mammalian estrogen receptors. Semipurified preparations of this ligand have been shown to exhibit estrogenic activity in mammalian systems. We now describe the purification procedure and ultimate identification of the estrogenic substance in extracts of S. cerevisiae as 17 beta-estradiol. Organic solvent extracts of commercially obtained dried yeast were sequentially chromatographed on silica gel columns and then subjected to a series of reversed phase HPLC fractionations. Active ligand was monitored by [3H]estradiol displacement in a rat uterine cytosol assay. After seven chromatography steps, the purified and highly active ligand exhibited a single peak with retention times identical to those of 17 beta-estradiol on both HPLC and GC. The yeast material was identified as 17 beta-estradiol by its UV absorbance and mass spectrometric fragmentation pattern. In addition, radioimmunoassay confirmed the presence of approximately the same mass of 17 beta-estradiol (approximately equal to 800 ng/1.5 kg of yeast) as estimated both by a competitive binding assay with estrogen receptor and by mass spectrometry. Extraneous contamination by estradiol was excluded by repeat experiments with different batches of starting material and demonstration of estradiol by RIA in conditioned medium and cell pellets of laboratory-grown S. cerevisiae whereas non-conditioned medium did not possess the steroid. We conclude that 17 beta-estradiol is a yeast product.
Structure of deoxyhemoglobin Cowtown [His HC3(146) beta----Leu]: origin of the alkaline Bohr effect and electrostatic interactions in hemoglobin.
Authors: Perutz M F , Fermi G , Shih T B
Published: May 2006

Hemoglobin Cowtown [His HC3(146)-beta----Leu] exhibits high oxygen affinity and a halved alkaline Bohr effect. X-ray analysis shows the COOH-terminal leucine to be in equilibrium between two positions: one with the salt bridge between the terminal carboxyl and Lys C5(40)alpha intact and the leucyl side chain leaning against main chain atoms of helices F and FG and the other with the terminal salt bridge broken and the leucyl side chain touching Pro C2(37)alpha. Structural changes are confined to the immediate neighborhood of the COOH terminus, showing the halving of the alkaline Bohr effect to be due directly to the loss of the histidine, without significant contributions from changes in pK values of other ionizable groups due to structural changes elsewhere.
Macroscopic models for studies of electrostatic interactions in proteins: limitations and applicability.
Authors: Warshel A , Russell S T , Churg A K
Published: May 2006

The validity of macroscopic models for calculations of electrostatic energies in proteins is examined. The Tanford-Kirkwood (TK) model is extended to include the self energy of the ionized groups. It is shown that ionized groups cannot exist inside nonpolar regions of proteins and argued that the experimental finding of ions inside proteins proves that the corresponding local environment is polar. The modified TK model (MTK model), which adjusts charge-charge interactions by the corresponding solvent accessibilities, is found to be inconsistent with the TK model, on which it is based. The MTK model corresponds to a polar interior whereas the TK model assumes a nonpolar interior. It is shown that models that assume a high dielectric constant for proteins give reasonable results for interactions between charged groups at equilibrium. It is then explained why, in contradiction to common belief, protein interiors are polar around charged groups. It is argued that in focusing on charge-charge interactions one overlooks the key contribution of the protein dipoles in determining the self energy of charges in the interior of proteins.
Adenosine-dependent activation of tyrosine hydroxylase is defective in adenosine kinase-deficient PC12 cells.
Authors: Erny R , Wagner J A
Published: May 2006

(R)-N6-Phenylisopropyladenosine (PIA) stimulates dopa production 3- to 5-fold in PC12 cells, with a half-maximal effective concentration (EC50) of 50 nM. This increase can be explained by a stable activation of tyrosine hydroxylase [TyrOHase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] when it is phosphorylated by a cAMP-dependent protein kinase. The activation of TyrOHase is mediated by the adenosine-dependent activation of adenylate cyclase (EC50 = 600 nM). PIA (10 microM) is as effective as cholera toxin or dibutyryl cAMP in activating TyrOHase in wild-type cells. Adenosine kinase-deficient mutants of PC12 were found to be resistant to PIA-dependent activation of TyrOHase (EC50 = 100-1000 nM). This phenomenon was explored in detail in one adenosine kinase-deficient mutant and was shown to occur because the mutant was resistant to the adenosine-dependent activation of adenylate cyclase. In this mutant, TyrOHase was activated 14-fold by cholera toxin, suggesting that activated TyrOHase is about 14 times as active as unactivated TyrOHase. These studies with kinase-deficient PC12 cells provide genetic evidence that adenosine-dependent activation of TyrOHase is mediated by acute increases in cAMP. When the adenosine receptor found on PC12 cells is expressed in vivo, it might function as either a presynaptic (i.e., localized on the nerve terminal) or a postsynaptic (i.e., localized on the cell body or dendrite) receptor that regulates rates of transmitter synthesis in response to cell activity.
Psychotomimetic opiate receptors labeled and visualized with (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine.
Authors: Largent B L , Gundlach A L , Snyder S H
Published: May 2006

3-(3-Hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP) has been proposed as a selective dopamine autoreceptor agonist in the central nervous system. This report describes the pharmacology and localization of specific high-affinity binding sites for (+)-[3H]3-PPP in brain. The drug specificity of (+)-[3H]3-PPP binding is identical to that of sigma receptors, which may mediate psychotomimetic effects of some opiates. Haloperidol and the opioid derivatives, pentazocine, cyclazocine, and SKF 10,047 are potent inhibitors of (+)-[3H]3-PPP binding. Stereoselectivity is exhibited for the (+) isomers of cyclazocine and SKF 10,047 at the sigma site, opposite to the stereoselectivity seen at mu, delta, and kappa opiate receptors. (+)-[3H]3-PPP does not label dopamine receptors, as potent dopamine agonists and antagonists are weak inhibitors of binding and the localization of specific (+)-[3H]3-PPP binding sites does not parallel that of dopamine neurons. Discrete localizations of (+)-[3H]3-PPP binding sites in many brain areas including limbic, midbrain, brainstem, and cerebellar regions may explain psychotomimetic actions of opiates and behavioral effects of 3-PPP.
The role of a signaling protein in bacterial sensing: behavioral effects of increased gene expression.
Authors: Clegg D O , Koshland D E
Published: May 2006

A recombinant DNA approach has been used to study intracellular signaling in the bacterial sensing system. The Escherichia coli cheY gene, whose function is unknown, has been subcloned behind the synthetic inducible tac promoter. The resulting plasmid directs the synthesis of the Y protein in response to isopropyl beta-D-thiogalactoside, independent of its usual operon control. When this construct was introduced into wild-type and mutant cells, the Y protein caused a clockwise rotational bias in the flagellar motors. This effect was observed even in heavily biased counterclockwise strains lacking most of the central chemotaxis processing genes. The results show that the Y protein has a direct influence on flagellar rotation not requiring other processing genes of the sensing system. The Y protein appears to bind directly to a part of the flagellar motor, probably the flaA gene product, and it is probably the key element in biasing the motor toward the clockwise rotational direction.