bluuebunny/crossref_metadata_2025 huggingface dataset:
Similarities and dissimilarities between calmodulin and a Chlamydomonas flagellar protein.
Authors: Van Eldik L J , Piperno G , Watterson D M
Published: May 2006

A protein that resembles vertebrate calmodulins and troponin C has been isolated from Chlamydomonas flagella by using a calmodulin purification protocol that included calcium-dependent affinity-based adsorption chromatography on phenothiazine-Sepharose conjugates. The flagellar protein resembled calmodulin in elution from reverse-phase columns, had a peptide map similar to that of calmodulin, and competed with vertebrate calmodulin in a radioimmunoassay using antisera against vertebrate calmodulin. However, this flagellar protein did not activate phosphodiesterase, lacked N epsilon-trimethyllysine, and had an isoelectric point approximately 0.3 pH unit higher than that of vertebrate calmodulin. When analyzed by polyacrylamide gel electrophoresis under various conditions, the Chlamydomonas protein migrated between vertebrate calmodulins and rabbit skeletal muscle troponin C and did not manifest a large calcium-dependent mobility shift. This calmodulin-like protein was identified as one of the approximately 200 35S-labeled components in Chlamydomonas flagella resolved by two-dimensional gel electrophoresis. These studies indicate that calmodulin and a structurally and functionally homologous protein are present in the same cell. These studies also demonstrate that caution is necessary: (i) in identifying a protein as a calmodulin, (ii) in using phenothiazines or antisera directed against vertebrate calmodulins as specific probes for calmodulin, and (iii) in the interpretation of experiments on biological systems in which calmodulin is substituted for the homologous calmodulin-like protein.
Fluorescence immunoassay based on long time correlations of number fluctuations.
Authors: Nicoli D F , Briggs J , Elings V B
Published: May 2006

We report the development of a fluorescence-based immunoassay technique relying on the physical phenomena of random number fluctuations and diffusion, which we review. By determining the autocorrelation of the fluctuations in the fluorescent intensity, this methid is able to measure the amount of labeled antigen or antibody that is bound to micrometer-sized carrier particles in solution. The principal advantage of this technique is its insensitivity to small, fast-diffusing sources. It also discriminates against weakly fluorescent contaminants of size comparable to the carrier particles. We demonstrate these attributes by using two model systems: a human IgG assay and an idealized system consisting of polystyrene fluorescent spheres and rhodamine dye.
Serotonin-induced hyperpolarization of an indentified Aplysia neuron is mediated by cyclic AMP.
Authors: Drummond A H , Benson J A , Levitan I B
Published: May 2006

Addition of serotonin to the medium bathing an Aplysia abdominal ganglion causes a change in the endogenous bursting activity of the identified neuron R15. At serotonin concentrations in the micromolar range, the predominant effect is an increase in depth and duration of the interburst hyperpolarization and consequent decrease in burst rate. At higher concentrations (10 microM) serototin can inhibit bursting completely. We have shown previously that these changes can be mimicked by bath application or intracellular injection of several cyclic AMP analogs substituted at the 8 position. Voltage clamp analysis indicates that serotonin and cyclic AMP analogs both cause an increase in membrane slope conductance in R15, with reversal potentials for the responses between -75 and -80 mV, close to the K+ equilibrium potential. When the K+ concentration in the bathing medium is changed, the reversal potentials change in a manner suggesting that serotonin and cyclic AMP analogs on K+ conductance are not additive. Furthermore, the effects of low concentrations of serotonin can be potentiated by the phosphodiesterase inhibitor Ro 20-1724. A pharmacological analysis indicates that the serotonin receptor that mediates hyperpolarization in R15 is similar to the serotonin receptor that we have shown to be coupled to adenylate cyclase. The present electrophysiological and pharmacological observations, together with our previous biochemical and pharmacological results, demonstrate that the serotonin-induced hyperpolarization of neuron R15 is mediated by cyclic AMP.
Construction of a composite tRNA gene by anticodon loop transplant.
Authors: Yarus M , McMillan C , Cline S , Bradley D , Snyder M
Published: May 2006

By using sites for the restriction nuclease Hpa II, the information for the anticodon stem and loop of an altered Su+2 amber suppressor tRNA (a mutant of tRNAGln) has been transplanted to a specially prepared tRNATrp gene, which lacks it homologous anticodon stem and loop sequence. The resulting tRNA gene was cloned under lac operator-promoter control. The result is a functional, hybrid, amber-suppressor tRNA that can exhibit a moderately high efficiency in translation. It appears less efficient, however, than Su+7 tRNA, the amber suppressor that results from a direct anticodon mutation in tRNATrp. As judged by its suppressor spectrum, which is almost identical to the spectra of Su+2, and Su+7, the recomposed tRNA inserts glutamine at amber sites. This experiment is the prototype of a series of construction that examine the role of the nucleotides in the anticodon region.
Chinese hamster lung cells synthesize and confine to the cellular domain a collagen composed solely of B chains.
Authors: Haralson M A , Mitchell W M , Rhodes R K , Kresina T F , Gay R , Miller E J
Published: May 2006

The acid-soluble collagen extracted from cultured Chinese hamster lung (CHL) cell layers has been isolated after limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis of this material under denaturing conditions showed the presence of collagen chains with an apparent molecular mass of 120,000 daltons both before and after reduction, indicating the absence of interchain disulfide bonds in the native molecule. When chromatographed on CM-cellulose under denaturing conditions, the majority (> 90%) of the CHL cell layer collagen chains eluted as relatively basic components slightly before the human alpha 2(I) chain and coincident with the human B chain. In addition, the CM-cellulose elution profiles of the cyanogen bromide peptides derived from the human B chain and from the CHL cell layer chain were essentially identical. Examination of CHL cells in culture by using affinity-purified antibody to human B chain revealed this collagen to be localized in an extracellular matrix surrounding the cells. Furthermore, analysis of the culture medium indicated the absence of any comparable collagen chain. These data provide additional evidence for the existence of a molecular form of collagen composed solely of B chains and suggest that this molecular form of collagen has an unusual affinity for the cell layer in this system.
Origin of DNA replication of bacteriophage f1 as the signal for termination.
Authors: Horiuchi K
Published: May 2006

Restriction fragments that contain the origin of DNA replication of bacteriophage f1 were inserted in vitro into circular f1 DNA molecules to form genomes that contain two origins. This DNA was used to transfect Escherichia coli. Analyses of the DNA of the progeny phage indicated that one origin and the DNA segment located between the two origins in the infecting DNA molecules had been eliminated. This result is interpreted to mean that the nucleotide sequence of the origin for plus (viral)-strand synthesis also serves as the signal for the termination of DNA synthesis.
Competitive intransitivity and size-frequency distributions of interacting populations
Authors: Buss L. W.
Published: May 2006

The competitive ranking of three marine sessile organisms was determined from overgrowth observations and found to be intrasitive. Overgrowth results were dependent upon relative vertical relief which, in turn, was related to colony area. Discriminant function analysis demonstrates that competitive rankings can be largely predicted on the basis of knowledge of the size-frequency distribution of interacting populations. Because size increases with age for these species, size-dependent competition implies age-dependent competition. The role of age structure in determining the results of inter-specific interactions has been curiously neglected, particularly as it relates to competitive ability, yet the age dependence of competitive ability suggests (
i
) the occurrence of intransitivity in purely exploitative systems, (
ii
) a mechanism for frequency-dependent behavior, (
iii
) the production of limit cycles, and (
iv
) the possibility of size refugia.
Genetic manipulation by means of microcell-mediated transfer of normal human chromosomes into recipient mouse cells.
Authors: McNeill C A , Brown R L
Published: May 2006

Microcell-mediated chromosome transfer is an innovative approach to the production of karyotypically simple hybrids. This method of gene transfer, employing micronuclei formed by prolonged Colcemid treatment, has been utilized for rodent systems. Expansion of this technology to include transfer of normal human genetic material has been hindered because large micronucleate populations from diploid human cells have been unobtainable. This report describes the production of micronuclei in 40-60% of normal human fibroblasts. These micronucleated cells have been enucleated by combining centrifugation and cytochalasin B treatment, and the resultant microcells have been purified and fused to recipient mouse (LMTK-) cells. Microcell hybrid clones containing a single human chromosome have been isolated from three separate fusion experiments. The time course for production of these hybrids, from fusion to karyotypic analysis, was 6 weeks. With a transfer frequency of about 2 x 10(-6), a single intact human chromosome has become a functioning element of the murine genome.
Rat dendritic cells function as accessory cells and control the production of a soluble factor required for mitogenic responses of T lymphocytes.
Authors: Klinkert W E , LaBadie J H , O'Brien J P , Beyer C F , Bowers W E
Published: May 2006

Transformation of T lymphocytes, induced by treatment with periodate or with neuraminidase plus galactose oxidase, requires the participation of accessory cells. Procedures were developed for the fractionation of rat lymph node cells, by which most of the lymphocytes can be recovered as a major population of cells that do not respond to mitogenic stimulation unless accessory cells from a separated minor population are added. Further purification led to a 1000-fold overall increase in accessory activity per cell, with a 50-70% yield. The purest preparations were virtually free of macrophages and contained more than 90% typical dendritic cells. Maximum responses occurred at a ratio of only one dendritic cell per 200 periodate-treated lymphocytes. This evidence thus indicates strongly that in rats, dendritic cells--not macrophages--function as accessory cells. Further, the number of dendritic cells in a preparation governed the magnitude of the mitogenic response and was limiting in the case of unfractionated lymph node cells. In addition, when oxidized with periodate or with neuraminidase plus galactose oxidase, the dendritic cell served as a very potent indirect stimulator of untreated responder lymphocytes. Both functions of the dendritic cell appeared to lack species specificity, since mouse dendritic cells were very active when tested with rat responder lymphocytes. A soluble factor (accessory cell-replacing factor), produced by cultures of lymph node or spleen cells subjected to oxidative mitogenesis, enabled otherwise unresponsive mitogen-treated lymphocytes to respond. Dendritic cells were required for the production of this factor but may not be solely responsible for its production.
Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity.
Authors: Olsson L , Kaplan H S
Published: May 2006

We report the establishment of human-human hybridomas producing monoclonal antibody of predefined antigenic specificity. The U-266 human myeloma cell line was incubated in the presence of 8-azaguanine, and a rapidly growing, 8-azaguanine-resistant, hypoxanthine/amethopterin/thymidine (HAT) medium-sensitive mutant line, U-266AR1, was selected. These cells were fused with lymphoid cells from uninvolved spleens removed at staging laparotomy from patients with untreated Hodgkin's disease who had been previously sensitized to the chemical allergen 2,4-dinitrochlorobenzne. Hybrid cell cultures growing in HAT medium were screened for IgG production. Positive cultures were selected and their supernatants were tested in a solid-phase radioimmunoassay for reactivity with dinitrophenyl hapten coupled to bovine serum albumin. Cultures producing specific antibody were subcloned and expanded, and their antibody products were shown to be monoclonal by biosynthetic labeling and sodium dodecyl sulfate/polyacrylamide gel electrophoresis.
Brain clathrin: immunofluorescent patterns in cultured cells and tissues.
Authors: Bloom W S , Fields K L , Yen S H , Haver K , Schook W , Puszkin S
Published: May 2006

High-purity bovine brain clathrin elicited antibodies, which were separated by affinity chromatography on clathrin conjugated with CNBr-activated Sepharose 4B. Indirect immunofluorescent labeling on cultured rat fibroblasts showed the characteristic dotted fluorescence throughout the entire cytoplasm and concentrated patches of fluorescence in areas where the Golgi apparatus and GERL (Golgi apparatus-endoplasmic reticulum-lysosome complex) exist next to the nucleus. Cerebellar tissue sections exhibited profuse immunofluorescence in the granular layer; dissociated cultures of neonatal rat cerebellar cells showed intense fluorescence in the cytoplasm and processes of granule cell neurons. The data suggest that clathrin is an abundant protein distinctly localized in the cytoplasm of cells and clearly correlated in its distribution and arrangement with actin stress fibers.
Light-induced calcium release by intact retinal rods.
Authors: Gold G H , Korenbrot J I
Published: May 2006

Brief illumination of the isolated toad retina produces a transient increase in the extracellular free Ca concentration in the photoreceptor layer. This change in concentration arises from the release of Ca2+ by the rod outer segments. The release begins within 200 msec after the stimulus flash. The peak amplitude of the concentration change increases linearly with light intensity up to approximately 100 absorbed photons per rod and saturates at about 10(7) absorbed photons per rod. In the linear range, the amount of Ca released corresponds to about 2 x 10(4) Ca2+ per absorbed photon per rod. The high stoichiometry of this release, in relation to the low free intracellular Ca concentration, suggests that the release reflects a light-dependent increase in the intracellular free Ca concentration. This light-activated Ca release occurs in the absence of a detectable receptor potential (in low-Na Ringer's solution) and, therefore, must reflect one of the initial stages in the transduction process.
Organization of transcriptional signals in plasmids pBR322 and pACYC184.
Authors: Stüber D , Bujard H
Published: May 2006

Electron microscopic analysis of in vitro transcriptional complexes of pBR322 and pACYC184 revealed five and six major transcriptional units, respectively, in these two plasmid vectors. These units are transcribed with various efficiencies, depending upon the individual promoter strengths, which differ in pBR322 up to 10-fold. A most interesting signal arrangement was found at the beginning of the tetracycline resistance region, where two partially overlapping promoters (P1 and P2) initiate transcription crosswise in opposite directions. Whereas P2 is known to promote tetracycline resistance and to be inactivated by HindIII cleavage, P1 is able to transcribe DNA integrated at that site and probably contributes to the expression of the beta-lactamase gene in pBR322. In pACYC184, besides P1, P2, and the cat (chloramphenicol resistance) promoter (P5), two initiation sites (P3 and P4) were mapped in a region that appears to be part of insertion sequence 1. The maps of transcription signals permit a more predictable utilization of these cloning vehicles and also allow the reinterpretation of earlier cloning results.
Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7.
Authors: Tabor S , Richardson C C
Published: May 2006

The gene 4 protein of bacteriophage T7 recognizes specific sequences on single-stranded DNA and then catalyzes the synthesis of tetraribonucleotide primers complementary to the template. With phi X174 DNA as a template, the gene 4 protein enables T7 DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) to initiate DNA synthesis at 13 major sites. DNA sequence analysis of the 5' termini of the newly synthesized DNA shows the predominant recognition sequences for the gene 4 protein to be 3'-C-T-G-G-G-5' or 3'-C-T-G-G-T-5'; the products of synthesis at these sites are RNA primers having the sequences pppA-C-C-C or pppA-C-C-A. The gene 4 protein can also synthesize primers at the sequences 3'-C-T-G-G-AC-5' and 3'-C-T-G-T-N-5', although these sites are used less than 10% as frequently as the predominant sites. Comparison of the utilization of primer sites suggests that the gene 4 protein binds randomly to single-stranded DNA and then translocates along the DNA in a unidirectional 5'-to-3' direction with regard to the DNA strand in search of recognition sequences. Models are presented for the role of the gene 4 protein in the initiation of lagging-strand synthesis and in the initiation of DNA replication at the origin.
Regulation of glutamine synthetase activity by adenylylation in the Gram-positive bacterium Streptomyces cattleya.
Authors: Streicher S L , Tyler B
Published: May 2006

The enzymatic activity of glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from the Gram-positive bacterium Streptomyces cattleya is regulated by covalent modification. In whole cells containing high levels of GS the addition of ammonium chloride leads to a rapid decline in GS activity. Crude extracts prepared from such ammonia-shocked cells had very low levels of GS activity as measured by biosynthetic and gamma-glutamyltransferase assays. Incubation of the crude extracts with snake venom phosphodiesterase restored GS activity. In cell extracts, GS was also inactivated by an ATP- and glutamine-dependent reaction. Radioactive labeling studies demonstrated the incorporation of an AmP moiety into GS protein upon modification. Our results suggest a covalent modification of GS in a Gram-positive bacterium. This modification appears to be adenylylation of the GS subunit similar to that found in the Gram-negative bacteria.
Water structure-dependent charge transport in proteins.
Authors: Gascoyne P R , Pethig R , Szent-Györgyi A
Published: May 2006

Dielectric and conductivity measurements are reported for bovine serum albumin as a function of hydration. Strong evidence is found for the existence of mobile charges whose short- and long-range hopping motion strongly depends on the physical state of the protein-bound water. These charges are considered to be protons. Insights into the nature of the electrical properties of protein-methylglyoxal complexes are provided, and the possibilities for correlated proton-electron motions are outlined.
Emergence of beta-adrenergic sensitivity in the developing chicken heart.
Authors: Lipshultz S , Shanfeld J , Chacko S
Published: May 2006

Muscle cells dissociated from 5-day embryonic chicken hearts showed dose-dependent increases in both chronotropic rates of contraction and cyclic AMP (cAMP) levels in response to epinephrine (EPI), an effect that could be blocked by beta-adrenergic antagonists. However, 2- to 2.5-day embryonic chicken myocardial cells, although similar to 5-day heart cells with respect to the organization of myofibrils, failed to respond to EPI either by increased rates of contraction or by elevated levels of intracellular cAMP. Development of beta-adrenergic sensitivity in 2- to 2.5-day cells did not occur even after several days of growth in culture. However, addition of an extract prepared from 11-day chicken embryos to 2- to 2.5-day muscle cell cultures at any point during in vitro growth resulted in the development of sensitivity to EPI as measured by increases in both the beating frequency and cAMP levels after a 48-hr incubation in embryo extract (EE). The basal level of cAMP in cells unresponsive to EPI is 5 times that in EPI-sensitive muscle cells from older hearts (5 days). The high basal level of cAMP in these cells is reduced to a level characteristic of cells from older hearts when treated with the EE. Once sensitivity was acquired, it was retained as a stable trait of the muscle cells in culture. Furthermore, EE-treated 2- to 2.5-day cells showed less reduction of positive chronotropy in response to multiple doses of EPI than did cells prepared from 5-day hearts. Fractionation of EE on Sephadex G-200 showed that the activity was present in the large-molecule fractions. It is concluded that the development of beta-adrenergic sensitivity can be induced in unresponsive heart cells cultured from 2- to 2.5-day embryos by a factor(s) in the EE that is not adsorbed on Sephadex G-200.
In vitro synthesis of rat fibrinogen: identification of preA alpha, preB beta, and pre gamma polypeptides.
Authors: Nickerson J M , Fuller G M
Published: May 2006

Vertebrate fibrinogen consists of two sets of three nonidentical polypeptides that are synthesized in the liver. The subunits of fibrinogen have been synthesized in a cell-free, membrane-free translation system and compared with (alpha), polypeptides of fibrinogen purified from rat plasma and (b) subunits synthesized and secreted by hepatoma cells grown in culture. Rat hepatoma monolayers were grown with or without tunicamycin to prevent or allow glycosylation of the B beta and gamma subunits, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis indicated that each of the polypeptides translated in vitro from mRNA is larger than its corresponding nonglycosylated fibrinogen chain. The primary translation A alpha, B beta, and gamma chains are larger than their authentic nonglycosylated counterparts by 600, 1100, and 3000 daltons, respectively. Furthermore, the preA alpha and preB beta translation products are thrombin sensitive. These results strongly imply that signal peptides exist on each of the primary translation products of fibrinogen.
Hoechst 33342 dye uptake as a probe of membrane permeability changes in mammalian cells.
Authors: Lalande M E , Ling V , Miller R G
Published: May 2006

Flow cytometric analysis of the uptake of the DNA-specific and fluorescent probe Hoechst 33342 (HO342) offers a simple and rapid method for measuring membrane transport rates in mammalian cells and identifying cellular subpopulations that differ in their membrane transport rates. In a Chinese hamster ovary wild-type cell line and in three colchicine-resistant lines derived from it, the rate of uptake of HO342 dye decreases as the stepwise resistance to colchicine increases. A colchicine-sensitive revertant cell line shows an increased rate of dye uptake. Drug resistance in these lines has previously been shown to be related to changes in transport rate. In a manner similar to colchicine, the rate of uptake of HO342 dye shows nonsaturation kinetics. The effects of KCN, a metabolic inhibitor, on HO342 dye uptake, both in the presence and in the absence of glucose, is similar to that previously observed for colchicine uptake. When murine spleen cells are stained with HO342 under appropriate conditions, one sees two populations of lymphocytes differing in HO342 fluorescence intensity, a difference not related to DNA content. The two subpopulations show the same relative difference in both colchicine and HO342 uptake. HO342 dye appears, therefore, to enter mammalian cells by the same mechanism as colchicine--i.e., by unmediated diffusion--and can be used as a probe of cytoplasmic membrane permeability. Potential applications of the dye in studies of drug resistance, detection of activated T cells, and recognition of lymphocyte subpopulations are discussed.
Immigration and extinction probabilities for individual species: relation to incidence functions and species colonization curves.
Authors: Gilpin M E , Diamond J M
Published: May 2006

We develop a dynamic model of island biogeography based on immigration and extinction probabilities of individual species rather than on the usual biogeographic parameters of the number of species immigrating or going extinct per unit time. In the real world, these probabilities vary enormously among species, and three field studies suggest lognormal distributions. Based on such distributions of individual probabilities, our model proceeds by calculating the conditional probability of species occurrence--the likelihood that a given species will occur on a given island--as a function of total species number on the island. The model succeeds in predicting two observed features of species communities; (i) staggered, sigmoidally-shaped incidence functions ("differing area requirements of different species") and (ii) concave immigration and extinction curves.
Detection of a common feature in several human tumor cell lines--a 53,000-dalton protein.
Authors: Crawford L V , Pim D C , Gurney E G , Goodfellow P , Taylor-Papadimitriou J
Published: May 2006

Human cell lines, whether derived from spontaneous tumors or transformed in vitro with simian virus 40, were found to contain a 53,000-dalton phosphoprotein (pp53) in contrast to normal human cells in which this protein was not detected. Isoelectric focusing showed that pp53 comprised several species in both simian virus 40-transformed and tumor cells. Comparison of the pp53 species from the various cell lines by partial proteolysis showed that they were similar but not identical. Among the 13 tumor cell lines examined, only 1 line, HeLa, did not contain detectable pp53. All the other tumor cell lines contained pp53, and it is suggested that this protein may be associated with their transformed state.
Fc (IgG) receptor distributions in homogeneous and heterogeneous cell populations by flow microfluorometry.
Authors: Titus J A , Sharrow S O , Connolly J M , Segal D M
Published: May 2006

A flow microfluorometric method has been developed for quantitating the numbers of Fc receptors on individual cells. The cells were equilibrated at 0 degrees C with radiolabeled, affinity-crosslinked rabbit IgG dimers, washed, and treated with fluorescent antibodies against rabbit IgG. The stained cells were analyzed for fluorescence emission by using a fluorescence-activated cell sorter and for bound dimer molecules by using a gamma counter. Standard curves relating fluorescence emission to numbers of dimer molecules bound to cells were used to determine Fc receptor distributions on P388D1 cells, human peripheral blood lymphocytes, and normal mouse spleen cells. Essentially all of the P388D1 cells bore Fc receptors, distributed in a skewed Gaussian profile having a peak at 2 X 10(5) receptors per cell. Human peripheral blood lymphocytes and mouse spleen cells contained positive and negative subpopulations. The percentage of positive cells in human lymphocytes from different donors ranged from 50 to 25; the receptor distributions of these cells were symmetrical and similar in all donors in shape and average receptor density (4.2 X 10(4) receptors per cell). Mouse spleen cells contained 55% positive cells with nonsymmetrical heterogeneous distributions of receptor densities. These cells peaked at 1 to 2 X 10(4) receptors per cell, but significant numbers of cells had receptor densities 10- to 20-fold greater.
Thymus-dependent membrane antigens in man: Inhibition of cell-mediated lympholysis by monoclonal antibodies to T

H
2

antigen
Authors: Evans Robert L. , Wall Donna W. , Platsoucas Chris D. , Siegal Frederick P. , Fikrig Senih M. , Testa Carolyn M. , Good Robert A.
Published: May 2006

In prior studies a heteroantiserum to a surface membrane component termed T

H
2

was used to define two subsets of human T cells (T

H
2

+
and T

H
2

-
), which were found to express distinct sets of activities
in vitro
. In the present studies we prepared monoclonal antibodies to surface determinants that are restricted to T cells belonging to each of these two subsets. Two antibodies, termed αLeu-2a and αLeu-2b, which seem to define the same surface antigen identified by the original T

H
2

antiserum, reacted with 57-84% of thymocytes and 22-46% of the erythrocyte-rosette-forming cells (ERF-C) in peripheral blood. Two other monoclonal antibodies, termed αLeu-3a and αLeu-3b, reacted with the same subpopulation of thymocytes (78-89%) and peripheral blood ERF-C (47-78%) but, unlike αLeu-2a and αLeu-2b, did not exhibit cross-blocking; i.e., labeling cells with αLeu-3a did not inhibit the subsequent binding of αLeu-3b. T cells reactive with αLeu-2a were shown to be unreactive with αLeu-3a, indicating that two separate subpopulations of T cells, Leu-2 (formerly T

H
2

+
) and Leu-3 (T

H
2

-
) T cells, were thereby defined. These two T cell subsets make up the subpopulation of ERF-C (80-95%) previously defined by a monoclonal antibody to a T cell membrane antigen (Leu-1) that has a thymus-dependent distribution on normal lymphocytes but is expressed by some surface-immunoglobulin-positive (sIg
+
) leukemic lymphocytes. None of the Leu antibodies reported here reacted with sIg
+
, Leu-1
+
leukemic cells, nor did they react with normal hematopoietic cells or lymphoid cells that had surface markers characteristic of B cells. Studies of the blocking effects of Leu antibodies on killing in cell-mediated lympholysis by effector T cells were carried out in the absence of complement. These experiments established the following points: (
i
) αLeu-2a abolished the killing by cytotoxic T cells of allogeneic phytohemagglutinin-stimulated blasts, (
ii
) inhibition of killing by αLeu-2b was markedly less than inhibition by αLeu-2a, and (
iii
) other antibodies, including αLeu-1, αLeu-3a, and αLeu-3b, had little or no effect on killing in cell-mediated lympholysis. The relevance of these findings to prior studies done in the mouse and in man are discussed.
Isolation and partial nucleotide sequence of a cDNA clone for human histocompatibility antigen HLA-B by use of an oligodeoxynucleotide primer.
Authors: Sood A K , Pereira D , Weissman S M
Published: May 2006

We have isolated a cDNA clone for one of the HLA-B locus alloantigens by hybridization with a 30-nucleotide-long DNA probe. The probe was isolated from a reverse transcriptase (RNA-dependent DNA nucleotidyltransferase)-catalyzed cDNA synthesis reaction on poly(A)-mRNA in which an oligonucleotide (5'-32P)dC-T-T-C-T-C-C-A-C-A-TOH served as a primer and in which dideoxynucleoside triphosphates were used to reduce the size and heterogeneity of the cDNA products. The desired cDNA clone was isolated from a library of recombinant cDNA clones in the plasmid pBR322. The partial nucleotide sequence of the cDNA clone corresponds to the amino acid sequence of HLA-B7 antigen. The approach described in this paper is extremely sensitive and may be useful in cloning other genes for which the corresponding mRNA is present at low levels. This cDNA clone is nearly full length and can be used to isolate and to study the genes within the HLA region and to obtain expression of HLA-B peptides in cells.
Unique primed start of phage phi X174 DNA replication and mobility of the primosome in a direction opposite chain synthesis.
Authors: Arai K , Kornberg A
Published: May 2006

A specific fragment of the phi X174 viral circle sustains the primed start of complementary DNA strand synthesis in vitro, even though the intact circle permits primed starts at many sites. The 300-nucleotide fragment from restriction nuclease digestion contains the recognition site for protein n', a DNA-dependent ATPase essential for priming phi X174 DNA replication. This n' recognition site contains within it a 44-nucleotide sequence with a potential hairpin structure and may be regarded as the starting signal for replication [Shlomai, J. & Kornberg, A. (1980) Proc. Natl. Acad. Sci. USA 77, 799-803]. After initiation on the 3' side of this sequence, the priming system (primosome) repeatedly generates primers by moving processively on the DNA template in a direction opposite to chain elongation. This primosome mobility is an attractive model for the discontinuous phase of Escherichia coli chromosome replication, in which processive primosome movement with the replicating fork is proposed for repeated initiations of nascent replication fragments.
Photosensitized electron transfer processes in SiO
2
colloids and sodium lauryl sulfate micellar systems: Correlation of quantum yields with interfacial surface potentials
Authors: Laane Colja , Willner Itamar , Otvos John W. , Calvin Melvin
Published: May 2006

The effectiveness of negatively charged colloidal SiO
2
particles in controlling photosensitized electron transfer reactions has been studied and compared with that of the negatively charged sodium lauryl sulfate (NaLauSO
4
) micellar system. In particular, the photosensitized reduction of the zwitterionic electron acceptor propylviologen sulfonate (PVS
0
) with tris(2,2′-bipyridinium)ruthenium(II) [Ru(bipy)
3
2+
] as the sensitizer and triethanolamine as the electron donor is found to have a quantum yield of 0.033 for formation of the radical anion (PVS
[unk]
) in the SiO
2
colloid compared with 0.005 in the homogeneous system and 0.0086 in a NaLauSO
4
micellar solution. The higher quantum yields obtained with the SiO
2
colloidal system are attributed to substantial stabilization against back reaction of the intermediate photoproducts—i.e., Ru(bipy)
3
3+
and PVS
[unk]
—by electrostatic repulsion of the reduced electron acceptor from the negatively charged particle surface. The binding properties of the SiO
2
particles and NaLauSO
4
micelles were investigated by flow dialysis. The results show that the sensitizer binds to both interfaces and that the SiO
2
interface is characterized by a much higher surface potential than the micellar interface (≈-170 mV vs. -85 mV). The effect of ionic strength on the surface potential was estimated from the Gouy-Chapman theory, and the measured quantum yields of photosensitized electron transfer were correlated with surface potential at different ionic strengths. This correlation shows that the quantum yield is not affected by surface potentials smaller than ≈-40 mV. At larger potentials, the quantum yield increases rapidly. The quantum yield obtained in the micellar system at different strengths fits nicely on the correlation curve for the colloid SiO
2
system. These results indicate that the surface potential is the dominant factor in the quantum yield improvement for PVS
0
reduction.
Fluorometric assay for adenosine 3',5'-cyclic monophosphate-dependent protein kinase and phosphoprotein phosphatase activities.
Authors: Wright D E , Noiman E S , Chock P B , Chau V
Published: May 2006

A novel peptide substrate for adenosine 3',5'-cyclic monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), Leu-Arg-Arg-Trp-Ser-Leu-Gly, was synthesized. Phosphorylation of the peptide causes a 20% increase in the peptide fluorescence intensity at 358 nm. Values of Km and kcat for the phosphorylation reaction at pH 7.0 (25 degrees C), were determined to be 2.7 +/- 0.5 microM and 5.5 +/- 0.4 sec-1, respectively. The phosphorylated peptide was shown to be an effective substrate for phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) with a Km of 113 +/- 10 microM and a kcat of 2.4 +/- 0.2 sec-1 in the presence of 2.5 mM MnCl2. Changes in the peptide fluorescence intensity as a function of its phosphorylation state provide a highly sensitive assay of cyclic AMP-dependent protein kinase and phosphoprotein phosphatase activities.
The
M
r
80,000 common forms of neurophysin and vasopressin from bovine neurohypophysis have corticotropin- and β-endorphin-like sequences and liberate by proteolysis biologically active corticotropin
Authors: Lauber Marc , Nicolas Pierre , Boussetta Hamadi , Fahy Christine , Béguin Pierre , Camier Maryse , Vaudry Hubert , Cohen Paul
Published: May 2006

We have tested the hypothesis that the high
M
r
forms common to both neurophysin and vasopressin detected in bovine neurohypophysis extracts (Nicolas, P., Camier, M., Lauber, M., Masse, M.-J. O., Möhring, J. & Cohen, P. (1980)
Proc. Natl. Acad. Sci. USA
77, 2587-2591) might also contain the sequences of other known neuropeptides. The following evidence indicates that corticotropin- and β-endorphin-like sequences are associated with similar high
M
r
forms and are included in these
M
r
80,000 molecules. During the fractionation steps of high
M
r
material, both corticotropin and β-endorphin immunoreactive species were found to coelute with the neurophysin and vasopressin ones, either under
M
r
140,000 (in 0.1 M formic acid) or
M
r
70,000-80,000 (in 6 M guanidine) elution volumes. Corticotropin immunoreactivity was found to cofocus at pIs 6.05 and 5.8 with the
M
r
80,000 neurophysin-containing species. This material was submitted to affinity chromatography on purified anti-neurophysin antibodies covalently attached to Sepharose 4B. Both the corticotropin and β-endorphin immunoreactivities, together with the neurophysin and vasopressin immunoreactivities, were retained on the immunoadsorbent and codesorbed by either a drastic pH change or by selective displacement with an excess of neurophysin. Comparison of the tryptic-digest maps of either the
M
r
68,000 fragment immunoprecipitated by anti-corticotropin antibodies or the
M
r
68,000 fragment released after precipitation of the
M
r
80,000 species by anti-neurophysin antibodies indicated large sequence homologies. Exposure of either the
M
r
80,000 or 68,000 components to mild proteolytic activities resulted in the formation of lower-size fragments. The resulting corticotropin-like immunoreactive material, recovered under the elution volume of standard
125
I-labeled corticotropin-(1-24), was tested for its ability to activate glucocorticoid biogenesis by the amphibian interrenal tissue (adrenal) in perifusion. It was found to exhibit a noticeable activity qualitatively undistinguishable from the one of the reference human corticotropin-(1-39). The name neurohypophyseal “coenophorin” (from the Greek word for common) is proposed for this class of
M
r
80,000 polypeptides that might represent the common precursor store-house for a set of neuropeptides produced in the hypothalamo-neurohypophyseal tract.
Conformational transition in the myosin hinge upon activation of muscle.
Authors: Ueno H , Harrington W F
Published: May 2006

We have determined the rates of chymotryptic proteolysis of the myosin hinge region in glycerinated rabbit psoas fibers and myofibrils in in rigor-inducing, activating, and relaxing buffers. The time course of formation of light meromyosin (LMM) provides a specific probe for cleavage within the hinge domain. In rigor-inducing and relaxing buffers proteolysis within the hinge is depressed, but on activation LMM is formed at a markedly increased rate, which is dependent on the concentration of MgATP. Peptide bond cleavage occurs at four widely separated sites spanning the length of the hinge domain. Only a trivial amount of proteolysis occurs at the head--rod swivel or within the heavy chain of the head itself (S-1 subunit) in rigor-inducing and relaxing solvents, and we find no significant change on activation. The rate of formation of LMM in rigor-inducing buffer is unchanged by addition of MgADP, Pi, or magnesium adenosine 5'-[beta, gamma-imido]triphosphate or in activating solvent at zero overlap between thick and thin filaments. These results provide evidence for a conformational (helix--coil) transition within the myosin hinge upon activation of skeletal muscle.
Preparation of tritium- or deuterium-labeled vitamin D analogs by a convenient general method
Authors: Paaren Herbert E. , Fivizzani Mary A. , Schnoes Heinrich K. , DeLuca Hector F.
Published: May 2006

The three-step conversion of vitamin D analogs to 6-oxo-3,5-cyclovitamin D derivatives followed by reduction with a tritide or deuteride reagent and subsequent cycloreversion gives 6-tritio(deutero)vitamin D derivatives and corresponding 5,6-
trans
-analogs. The method is general and affords the 6-labeled-vitamin D analogs in ≈20% overall yield.
Effect of surface modification on aggregation of phospholipid vesicles.
Authors: Wu P S , Tin G W , Baldeschwieler J D , Shen T Y , Ponpipom M M
Published: May 2006

Phospholipid vesicles have been extensively investigated because of their usefulness as models for biological membranes and their potential application as carriers for drug delivery. However, preparations of small sonicated vesicles tend to aggregate and fuse (on storage at room temperature and at 4 degrees C), resulting in significant changes in turbidity, rate of uptake by macrophage, and proton NMR linewidths. By modification of the surface of phospholipid vesicles with charged groups such as beta-aminogalactose that extend significantly from the vesicle surface, it is possible to obtain preparations that are stable for greater than 7 days.
Single channel currents induced by complement in antibody-coated cell membranes.
Authors: Jackson M B , Stephens C L , Lecar H
Published: May 2006

An extracellular patch electrode was used to record ionic currents from individual complement-induced channels in the membranes of antibody-coated skeletal muscle. The amplitude of the single-channel currents leads to an estimate of 90 pS for the unit conductance. The kinetics of channel opening and closing show marked variability and complexity. Channels flicker open and closed repeatedly, indicating that once these lesions form, they undergo rapid structural transitions between discrete conducting and nonconducting states.
Random walk properties from lattice bond enumeration: Anisotropic diffusion in lattices with periodic and randomly distributed scatterers
Authors: Shuler Kurt E. , Mohanty Udayan
Published: May 2006

A lattice bond enumeration method recently devised for the study of random walk problems is used to rederive, with relative ease, the classic Maxwell-Rayleigh formulas for the effective de conductivity of a heterogeneous solid composed of periodically (or randomly) arrayed spheres in a medium of a different material.
Boundary behavior of functions holomorphic in domains of finite type
Authors: Nagel Alexander , Stein Elias M. , Wainger Stephen
Published: May 2006

We study several notions of distance and ball on the boundary of domains of finite type in C

n

. We use these notions to develop a theory of maximal functions and area integrals for functions holomorphic in such domains.
Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli.
Authors: Rose J K , Shafferman A
Published: May 2006

Bacterial plasmids that directed expression of the vesicular stomatitis virus glycoprotein (G-protein) gene under control of the tryptophan operon regulatory region were constructed. A plasmid directing the synthesis of a G-protein-like protein (containing the NH2-terminal segment of seven amino acids encoded by the trpE gene fused to the complete G-protein sequence lacking only its NH2-terminal methionine) could be transformed into trpR+ (repressed) but not into trpR- (derepressed) cells. This result suggested initially that derepressed synthesis of the G-protein-like protein encoded by this plasmid was lethal in Escherichia coli. Deletion of the sequence encoding the large hydrophobic segment near the COOH terminus of G-protein did not overcome this lethality. Lethality of derepressed synthesis was overcome by deletion of the G-protein gene region encoding 10 amino acids in the hydrophobic NH2-terminal domain (signal peptide). Tryptic peptide mapping demonstrated that the G-protein-like protein and some truncated proteins encoded by the plasmid contained G-protein protein sequences. Antisera to vesicular stomatitis virus precipitated the G-protein-like protein, showing that it shares antigenic determinants with the authentic G-protein protein.
Calcium-dependent interaction of S100b, troponin C, and calmodulin with an immobilized phenothiazine.
Authors: Marshak D R , Watterson D M , Van Eldik L J
Published: May 2006

We have purified the brain-specific protein S100b by affinity-based adsorption chromatography on phenothiazine-Sepharose conjugates and studied the interaction of this and other calcium-modulated proteins with the immobilized antipsychotic drug. Bovine brain calmodulin, rabbit skeletal muscle troponin C, and bovine brain S100b bind to phenothiazine-Sepharose in a calcium-dependent manner. These three proteins competitively inhibit the calcium-dependent binding of 125I-labeled chicken gizzard calmodulin to the immobilized drug. However, carp parvalbumin and chicken intestinal vitamin D-dependent calcium binding protein do not inhibit the phenothiazine--calmodulin interaction. These results suggest that the known amino acid sequence homology among calmodulin, troponin C, and S100b may be reflected in a similar functional domain present in these proteins but absent in parvalbumin and vitamin D-dependent protein.
Electron nuclear double resonance spectroscopy of a hen egg-white lysozyme--Cu2+ complex in tetragonal single crystals.
Authors: Hutchison C A , Singel D J
Published: May 2006

We have used the electron paramagnetic resonance of Cu2+ bound in a tetragonal single crystal of hen egg-white lysozyme to obtain the electron nuclear double resonance spectra of protons in the vicinity of the Cu2+ at the site designated as B by Teichberg et al. [Teichberg, V. I., Sharon, N., Moult, J., Smilansky, A. & Yonath, A. (1974) J. Mol. Biol. 87, 357-368]. The values of the hyperfine interaction parameters and the coordinates of eight protons are reported. The configuration of the H2O molecules coordinated to the Cu2+ and their relationships to the protein molecule structure are discussed.
Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors.
Authors: Zidovetzki R , Yarden Y , Schlessinger J , Jovin T M
Published: May 2006

The rotational diffusion of epidermal growth factor (EGF)--receptor complexes on living human epidermoid carcinoma cells (A-431) has been measured by phosphorescence emission and anisotropy in the mu s time domain. A biologically active phosphorescent conjugate of EGF, erythrosin-EGF, was applied to living cells. The hormone--receptor complexes were mobile with rotational correlation times in the range 25--90 mu s when labeled and measured at 4 degrees C. Prolonged incubation and exposure to higher temperatures (23 and 37 degrees C) resulted in longer times up to 350 mu s, indicative of the progressive formation of microclusters, estimated to contain 10-50 receptors. Upon internalization of the hormone--receptor complexes, visible patches were observed by fluorescence microscopy, and the rotational correlation times were shorter, indicating a decrease in size of the dynamic unit. The sign of the rotational relaxation also varied with localization and processing of the hormones. The rate of lateral diffusion of EGF--receptor complexes, measured under similar conditions by fluorescence photobleaching recovery, increased with temperature in contrast to the rotational motion.
Isolation and amino acid sequence of a morphogenetic peptide from hydra
Authors: Schaller H. Chica , Bodenmüller Heinz
Published: May 2006

From
Anthopleura elegantissima
and from
Hydra attenuata
, a morphogenetic peptide—the head activator—was isolated in pure form. The sequence of the head activator was established by enzymatic and by chemical degradation of the whole peptide or fragments thereof and subsequent analysis of the amino acids by micromethods. The head activator from both sources was identical and has the sequence:

<pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe.