bluuebunny/crossref_metadata_2025 huggingface dataset:
Oscillations and control features in glycolysis: analysis of resonance effects.
Authors: Termonia Y , Ross J
Published: May 2006

We have presented [Termonia, Y. & Ross, J. (1981) Proc. Natl. Acad. Sci. USA 78, 2952-2956] an analysis of glycolysis based on experimental findings. Here, we give an interpretation based on the concepts of efficiency, resonance response, and control features available to highly nonlinear reaction kinetics. The comprehensive model of glycolysis, for which we presented numerical evaluation of concentration oscillations, entropy production, and average ATP/ADP ratios is separated into two subsystems: one for the phosphofructokinase (PFKase) reaction, the other for the pyruvate kinase (PKase) reaction. We analyze each subsystem separately and find that, for a range of parameter values around the best estimates obtained from experimental data, the PFKase reaction exhibits sustained oscillations. The PKase reaction, on the other hand, is in a stable stationary state and shows an oscillatory relaxation. The period of the PFKase reaction is within 25% of that obtained by computer simulation of the full model. Moreover, if, for a given set of kinetic parameters, the period of the oscillations in the PFKase reaction is of the same order of magnitude as the period of the oscillatory relaxation of the PKase reaction, then the onset of oscillatory behavior past marginal stability forces a tuning of the PFKase reaction period to that of the PKase reaction. Thus, the PKase reaction tunes the frequency of the primary oscillophor, the PFKase reaction, so that a resonance response results in the PKase reaction. We find this result for a substantial range of parameters. The sharp increase in the ATP/ADP ratio and the decrease in the dissipation (entropy production) are seen to be a result of the forced tuning to resonance. The origin of the remarkable control feature of the tuning of one part of the pathway by another that follows in the reaction mechanism is the coupling of the two parts and, in the case of glycolysis, this is realized by the intermediate fructose 1,6-diphosphate, which participates crucially in the PFKase and PKase reactions.
Ciliary membrane vesicles of paramecium contain the voltage-sensitive calcium channel.
Authors: Thiele J , Schultz J E
Published: May 2006

Calcium influx into ciliary membrane vesicles from paramecia was measured by using arsenazo III as metallochromic indicator and as intravesicular calcium trap. Influx was rapid and reversible. In ciliary membrane vesicles from paramecium pawn mutants which lack the voltage-sensitive calcium channel, calcium influx was significantly less than in wild-type paramecia. These data demonstrate that the voltage-dependent calcium channel of paramecia is functional in a cell-free system and available for biochemical studies.
Genetic divergence between rodent species assessed by using two-dimensional electrophoresis.
Authors: Aquadro C F , Avise J C
Published: May 2006

O'Farrell's technique of two-dimensional gel electrophoresis (2-DGE) has previously been applied to the study of intrapopulation genetic variation. This approach assays a larger, and in part nonoverlapping, cohort of protein encoding loci compared to conventional one-dimensional electrophoretic procedures (SGE) and has revealed substantially lower levels of mean heterozygosity. Here we extend this approach to analyze levels and patterns of genetic differentiation between species. We have used 2-DGE to compare an average of 189 polypeptides between six species of wild mice representing levels of evolutionary divergence ranging from different subspecies to different families. The magnitude of protein divergence estimated by 2-DGE was on the average only about one-half that predicted by SGE. This discrepancy may result from differences in sensitivities between the techniques or differences in the mean level of variation and divergence between the sets of loci assayed by the two methods. Nonetheless, the ranking of genetic distances by 2-DGE was identical to that by SGE. Thus, these results support the use of the simpler SGE techniques to estimate relative levels of genetic divergence.
Production of antigen-specific suppressive T cell factor by radiation leukemia virus-transformed suppressor T cells.
Authors: Ricciardi-Castagnoli P , Doria G , Adorini L
Published: May 2006

Hen egg-white lysozyme (HEL)-specific suppressor T cells induced in C57BL/6 mice have been selected by sequential passage over plates coated with goat anti-mouse Ig and HEL. These suppressor T cells, 80% I-J+, were infected in vitro with radiation leukemia virus (RadLV/Nu1) and injected intravenously into sublethally irradiated syngeneic recipients. After 4-6 months, 6 out of 20 injected mice developed thymic lymphomas, which were maintained by transplantation into histocompatible hosts and subsequently established as permanent cell lines. Cells of these six thymomas were screened for the presence of Thy 1.2, Lyt 1, Lyt 2, I-Jb, and Ig cell surface antigens by direct or indirect immunofluorescence. One tumor (thymoma L4) was found to express the expected phenotype of suppressor T cells (Thy 1.2+, Lyt 2+, I-J+). High-speed supernatants of extracts obtained from L4 cells were able to induce HEL-specific suppression in a T cell proliferative assay, demonstrating the presence of an antigen-specific suppressive T cell factor.
Functional deletion of different Ly-1 T-cell-inducer subset activities by Ly-2 suppressor T lymphocytes.
Authors: Green D R , Gershon R K , Eardley D D
Published: May 2006

T-suppressor-cell activity was analyzed by use of an intermediate culture system that allows the study of T-cell interactions in the absence of concomitant inducer effects on B cells. Activated suppressor T cells were incubated with their potential target cells [immune Ly-1+,Ly-2- (Ly-1) cells] for 24-48 hr, and then the Ly-1 cells were reisolated by removing the suppressor cells with an appropriate antiserum (Ly-2). The functional consequences of the interactions that occurred during the incubation period were then assessed. Suppressor cells deleted the functional activity of two inducer-T-cell subsets; the helper T cell, which induces B-cell production of antibody, and the T cell that induces Ly2+ T suppressor cells. This latter activity is more sensitive to suppression than is the former. As suppressor T cells inactivate the cells that are responsible for their activity (i.e., their specialized Ly-1 inducer cells), a form of negative regulation of suppressor-T-cell activity, in which the down regulation of suppressor cells is effected by their removal of their own inducer cells, can be postulated. In addition, these findings show that clonal deletion and active suppression need not be mutually exclusive mechanisms of immune unresponsiveness. Suppressor cells can produce a functional deletion of immune activity that persists after they themselves are removed, for example, by antisera, or in physiological situations by the negative form of regulation postulated.
Phospholipid methylation and phospholipase A2 activation in cytotoxicity by human natural killer cells.
Authors: Hoffman T , Hirata F , Bougnoux P , Fraser B A , Goldfarb R H , Herberman R B , Axelrod J
Published: May 2006

The role of phospholipid methylation and phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) in natural killer (NK) function by human peripheral blood mononuclear cells was studied. Pretreatment of effector cells with a methyltransferase inhibitor, 3-deazaadenosine, in the presence of homocysteine thiolactone, reduced cytotoxicity in a dose-dependent fashion. This effect was closely associated with inhibition of methylation of lipids but not of nucleic acids or proteins. The suggestion for a role of phospholipid methylation was supported by the observation that the interaction between NK-susceptible tumor targets and peripheral blood mononuclear cells caused increased phospholipid methylation only when susceptible target cells were used. Phospholipase A2 was also implicated in human NK activity. Inhibitors of the enzyme such as tetracaine, mepacrine, Rosenthal's inhibitor, and corticosteroids impaired NK function. Rosenthal's inhibitor was also shown to exert an inhibitory effect on a purified NK-cell population obtained by the isolation of large granular lymphocytes on Percoll gradients. Peripheral blood mononuclear cells were also directly shown to display phospholipase A2-like activity, as measured by the decrease in radioactive arachidonate from prelabeled phospholipids, specifically phosphatidylcholine, in effector cells. These data suggest that enhanced phospholipid methylation occurs during the recognition function of NK cells. Consequent activation of phospholipase A2 might be involved in the mechanisms leading to lytic events within the target cell.
Functional clonal deletion in immunological tolerance to major histocompatibility complex antigens.
Authors: Nossal G J , Pike B L
Published: May 2006

CBA (H-2k) mice were rendered tolerant to H-2d antigens by injection of (CBA X BALB/c)F1 spleen cells at birth. At intervals of 2 days to 12 weeks, the frequencies of anti-H-2d cytotoxic T lymphocyte precursor cells (CTL-P) in thymus and spleen were determined by using a limiting-dilution microculture assay system for CTL-P. This assay, utilizing irradiated H-2d stimulator cells and concanavalin A-induced spleen cell conditioned medium, was shown to be linear over the range 30 to 100,000 responder cells and uninfluenced by IJ-positive cells. A profound and long-lasting deficit in activatable CTL-P, first demonstrable by day 5 of life in the thymus and day 8-10 in the spleen, developed in mice rendered tolerant, reaching a greater than 95% reduction by 6 weeks. Functional clonal deletion thus seems to be at least as important in the tolerant state as suppressor T cells. Repeated in vivo administration of anti-IJk serum partially inhibited clonal deletion, suggesting either that suppressor T cells are actively involved in producing clonal deletion or that IJk-bearing cells in the donor inoculum or the host represent an important factor.
Expression of cell surface markers after human B lymphocyte activation.
Authors: Stashenko P , Nadler L M , Hardy R , Schlossman S F
Published: May 2006

The fate of two recently described human B lymphocyte-specific antigens (B1 and B2) was studied after B-cell activation in vivo and in vitro. Whereas both B1 and B2 were present on virtually all B cells from normal lymph nodes, B2 was absent from approximately 50% of B cells from hyperplastic lymph nodes. When B cells from spleen, tonsil, or peripheral blood were stimulated in vitro with pokeweed mitogen, activated cells were found to lose B2 (days 4-5) and subsequently B1 (days 6-7). Temporally, B2 loss was accompanied by loss of surface IgD, expression of T10, and the development of intracytoplasmic IgM; B1 loss was correlated with the acquisition of surface IgG and the appearance of intracytoplasmic IgG. Peripheral blood B cells, on which B2 is normally only weakly expressed (B1++++B2+) in contrast to B cells from secondary lymphoid organs (B1++++B2++), exhibited a transitory increase in B2 expression to the B1++++B2++ phenotype prior to B2 disappearance during activation. Taken together with other findings, this observation suggests that peripheral blood may contain a relatively immature subpopulation of B cells.
Human T cell surface antigens bearing a structural relationship to HLA antigens.
Authors: Cotner T , Mashimo H , Kung P C , Goldstein G , Strominger J L
Published: May 2006

Three cell surface antigens that are structurally related to the human major histocompatibility antigens (called HLA antigens) have been characterized from the leukemic T cell line MOLT-4. One antigen is a glycoprotein of Mr 49,000 recognized by two monoclonal antibodies. OKT6 and NA1/34, and is associated with a Mr 12,000 subunit that crossreacts serologically with beta 2-microglobulin but can be distinguished from it by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A second antigen, defined by the monoclonal antibody OKT10, is a Mr 46,000 protein associated with a small subunit distinct from beta 2-microglobulin. The OKT10 antigen is not restricted to T cells and is found on all T and B lymphoblastoid cell lines tested. The third protein is a beta 2-microglobulin-associated glycoprotein of Mr 43,000 that is serologically distinct from the OKT6 (NA1/34), OKT10, and HLA antigens. It is found on some, but not all, T cell lines but is absent from any other hematopoietic cell lines tested.
Cyclic GMP releases calcium from disc membranes of vertebrate photoreceptors.
Authors: Cavaggioni A , Sorbi R T
Published: May 2006

Physiological concentrations of cyclic guanosine 3',6'-monophosphate (cGMP) inhibit 45Ca uptake and increase 45Ca release from vertebrate photoreceptor rod outer segment disc membranes. These effects are specific for cGMP. Several facts, including the independence of these effects from added triphosphates, suggest that cGMP diminishes the Ca-binding capacity of the disc membranes. Preliminary data show that the apparent affinity constant of the cGMP-dependent Ca-binding sites of the disc membranes is of the same (or even higher) order of magnitude as that of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. As expected, the observed cGMP effects are not dependent on the light or dark conditions of the disc membranes.
Characterization of specific receptors for calcitonin in porcine lung.
Authors: Fouchereau-Peron M , Moukhtar M S , Benson A A , Milhaud G
Published: May 2006

The binding of salmon calcitonin was investigated in subcellular fractions obtained from normal porcine lung. Only the membrane fraction (density, 1.14 g/cm3) showed specific binding for calcitonin. Specific binding of 125I-labeled salmon calcitonin was competitively inhibited by concentrations of unlabeled homologous hormone in the range 0.01-1 nM. Half-maximal inhibition of binding was observed with 0.12 nM salmon calcitonin. Scatchard analysis of the data suggested the presence of one class of binding sites with a mean affinity constant of 0.9 X 10(10) M-1 and a mean receptor number of 40 X 10(8)/mg of protein. The binding of salmon calcitonin was highly specific; half-maximal inhibition of binding was observed with 63.8 nM bovine calcitonin, the hormone corticotropin having no effect in this system.
Metal-hydrogen bridge bonding of hydrocarbons on metal surfaces
Authors: Gavin Robert M. , Reutt Janice , Muetterties Earl L.
Published: May 2006

Molecular orbital studies implicate multicenter metal-hydrogen-carbon interactions as contributors to the bonding of chemisorbed hydrocarbons on clean metal surfaces. The most stable geometries appear to be those that achieve the maximum multicenter bonding to the coordinately unsaturated metal atoms in the vicinity of the anchoring metal-carbon interaction. Energy differences between possible surface sites are of the same magnitude as stabilization energies for three-center bonding of hydrogen atoms to the metal surface. Accordingly, secondary interactions of hydrogen with neighboring metal atoms may be significant determining factors in surface structures. The model predictions are compared with known structures and are used to propose a mechanism for hydrocarbon reactions on metal surfaces. These metal-hydrogen-carbon interactions are presumed to be intermediate points or states in C—H bond-breaking processes.
Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro.
Authors: Christie G E , Farnham P J , Platt T
Published: May 2006

Termination of transcription by Escherichia coli RNA polymerase in vitro appears to depend primarily on two structural features of the termination site--a G+C-rich region of dyad symmetry and a series of terminal uridine residues in the transcript. To determine whether these two features are sufficient to specify rho-independent termination in vitro, we have introduced new sequences within a tryptophan (trp) operon structural gene to create two sites with these characteristics. Transcription with wild-type RNA polymerase in vitro demonstrates that discrete termination occurs at one of these new sites, although at a low level. Use of the mutant RNA polymerase rpo203, which is more sensitive to certain weak terminators than is the wild-type enzyme, increases termination at both sites. We have compared the activity of our synthetic terminators with those of several termination sites in the E. coli trp operon. Under normal conditions of transcription in vitro, termination becomes more efficient with an increase in the length of the stem in the RNA hairpin or an increase in the number of consecutive uridine residues. Transcription with the rpo203 polymerase and with ribonucleotide analogs gives changes consistent with these general trends. These results support a model for termination involving separate but essential roles for the RNA hairpin and the stretch of uridines in the transcript.
Translational control of ribosomal protein L10 synthesis occurs prior to formation of first peptide bond
Authors: Robakis Nikolaos , Meza-Basso Luis , Brot Nathan , Weissbach Herbert
Published: May 2006

A simplified DNA-directed
in vitro
system has been developed to study the regulation of the synthesis of ribosomal protein L10 by measuring the formation of the first dipeptide, fMet-Ala. The results show that the inhibition of L10 synthesis by L10 (autoregulation) occurs at or prior to the formation of the first peptide bond.
A Rous sarcoma virus provirus is flanked by short direct repeats of a cellular DNA sequence present in only one copy prior to integration.
Authors: Hughes S H , Mutschler A , Bishop J M , Varmus H E
Published: May 2006

The Rous sarcoma virus (RSV)-transformed rat cell line RSV-NRK-2 contains a single complete RSV provirus. We have obtained recombinant lambda clones that contain both ends of the RSV provirus and the flanking rat sequences. The provirus is integrated in unique DNA and is present in only one of the two homologous chromosomes. The rat sequences into which the RSV provirus integrated were also cloned from the RSV-NRK-2 cell line. The sequences of the regions involved in the recombination event have been determined and compared. Our data suggest that, compared with the sequence of viral DNA in the large circular form of unintegrated viral DNA, the provirus lacks two base pairs at each end and that the provirus is flanked by a six-base-pair direct repeat of cellular DNA. This six-base-pair repeat was apparently created during the integration event because this sequence was present only once at the integration site before the provirus was inserted. A survey of eight other independent RSV transformed rat cell lines demonstrates that, in agreement with earlier results, the RSV proviruses have entered different segments of rat cell DNA. We have also determined the sequence of a second virus DNA-host cell DNA junction from a second RSV-transformed rat cell line (RSV-NRK-4) and find that there are no obvious similarities between the two integration sites or between the integration sites and the termini of viral DNA.
Folding units in globular proteins.
Authors: Lesk A M , Rose G D
Published: May 2006

We present a method to identify all compact, contiguous-chain, structural units in a globular protein from x-ray coordinates. These units are then used to describe a complete set of hierarchic folding pathways for the molecule. Our analysis shows that the larger units are combinations of smaller units, giving rise to a structural hierarchy ranging from the whole protein monomer through supersecondary structures down to individual helices and strands. It turns out that there is more than one way to assemble the protein by self-association of its compact units. However, the number of possible pathways is small--small enough to be exhaustively explored by a computer program. The hierarchic organization of compact units in protein molecules is consistent with a model for folding by hierarchic condensation. In this model, neighboring hydrophobic chain sites interact to form folding clusters, with further stepwise cluster association giving rise to a population of folding intermediates.
Retroregulation of the int gene of bacteriophage lambda: control of translation completion.
Authors: Schindler D , Echols H
Published: May 2006

Bacteriophage lambda regulates the integration--excision reaction as a crucial aspect of the choice of pathway during lysogenic or lytic viral development. This control involves differential expression of the tightly linked, partially overlapping int and xis genes from two promoter sites: pI, positively regulated by cII/cIII proteins, and pL, positively regulated by N protein. After lambda infection, Int is synthesized from the pI transcript under cII regulation; however, very little Int is produced from the pL RNA because of the existence of a cis-acting regulatory element, sib, on the opposite side of the int gene from the pL promoter. Presumably sib serves to prevent unwanted synthesis of Int protein during the lytic response; the Int protein necessary for excisive recombination from a prophage can be supplied by pL transcription because sib is separated from int by prophage insertion. We have studied the effect of sib on nearby lambda genes by means of gel electrophoresis of labeled proteins from infected cells. Deletion of the sib region greatly enhances production of Int protein without substantial effect on Xis production; thus, sib regulation normally is highly specific for Int. When the sib region is moved past int and xis by deletion, regulation of the adjacent gene for the protein Ea22 occurs, suggesting that sib regulation can work on other genes. Although synthesis of wild-type Int is severely inhibited by sib, shorter Int protein fragments generated by nonsense mutations escape sib regulation, indicating that the regulation is translational and occurs near the completion stage of protein synthesis. Regulation by sib thus exhibits novel regulatory features: distal location, recombinational control, and regulation of the completion of protein synthesis. Because Int and Ea22 control is lost in a RNase III- host, we suggest that sib regulation might involve RNase III cleavage of a RNA duplex region that includes sib and the regulatory target (normally the int gene). We note such a potential site within int.
Membrane attack complex of complement: distribution of subunits between the hydrocarbon phase of target membranes and water.
Authors: Podack E R , Stoffel W , Esser A F , Müller-Eberhard H J
Published: May 2006

Membrane destruction by complement is effected by the membrane attack complex (MAC) which is the dimer of a fusion product of the complement proteins C5b, C6, C7, C8, and C9. Phospholipid bilayer vesicles were used as target membranes for the MAC and its intermediate complexes. The subunits of these membrane-bound complexes were explored as to their relative exposure to the hydrocarbon phase of the lipid bilayer and to water surrounding the lipid vesicles. Protein exposed to the aqueous phase was labeled with 125I; protein exposed to the hydrocarbon phase was labeled by using tritiated azido phospholipids and irradiation. Analysis of the membrane-bound MAC showed that subunits C5b, C8 beta, and C9 were exposed to the aqueous phase. The subunits C8 alpha-gamma and C9 were primarily in contact with the hydrocarbon phase. C6 and C7 were little exposed to either phase, suggesting that these proteins are inaccessible within the MAC. Analysis of the intermediate complexes showed that C5b was the subunit most exposed to water in membrane-bound C5b-7, and C5b and C8 beta were the water-exposed subunits in C5b-8. Subunit exposure to the hydrocarbon phase of the lipid bilayer changed during MAC assembly. Whereas all three subunits of C5b-7 carried the phospholipid photolabel; most of the label was bound to the C8 subunit in C5b-8 and to C9 in the MAC. It is proposed that contact with the hydrocarbon core of membranes is established by C5b-7 through each of its subunits, by C5b-8 through C8, and by the MAC through C8 and, particularly, C9.
Heterogeneous platinum-catalyzed hydrogenation of dialkyl(diolefin)platinum(II) complexes: A new route to platinum surface alkyls
Authors: McCarthy Thomas J. , Shih Yen-Shiang , Whitesides George M.
Published: May 2006

Platinum metal catalyzes the reduction of dialkyl(diolefin)platinum(II) complexes by dihydrogen to alkanes and platinum(0). The reaction involves adsorption of the platinum(II) complex on the platinum(0) catalyst surface with conversion of the alkyl moieties to platinum surface alkyls; these appear as alkane products. The platinum atom originally present in the soluble organoplatinum species becomes part of the platinum(0) surface.
Rubel's universal differential equation
Authors: Duffin R. J.
Published: May 2006

Fourth-order differential equations such as 16

y′
m
y

′
2
- 32

y
m
y
n
y

′ + 17
y

0
3

= 0 are developed. It is shown that the equation is “universal” in the sense that any continuous function can be approximated with arbitrary accuracy over the whole
x
axis by a solution
y
(
x
) of the equation. This solution is a piecewise polynomial of degree 9 and of class
C
4
.
Selecting the highest probability in binomial or multinomial trials
Authors: Levin Bruce , Robbins Herbert
Published: May 2006

Some sequential procedures are considered for selecting the binomial population with largest success probability or for selecting the multinomial outcome with highest cell probability. Procedures with and without sequential elimination of inferior populations are evaluated with respect to the expected probability of the population selected.
Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates.
Authors: Lamothe P , Baril B , Chi A , Lee L , Baril E
Published: May 2006

Three forms of DNA polymerase alpha [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] were partially purified from the combined nuclear extract and postmicrosomal supernatant solution of synchronized HeLa cells. These enzymes, designated DNA polymerases alpha 1, alpha 2, and alpha 3, on the basis of their order of elution from DEAE-Bio-Gel, differ in their abilities to utilize single-strand DNA templates. DNA polymerase alpha 2 has equal catalytic activities with activated and single-strand DNAs as template-primers. DNA polymerase alpha 1 has only partial catalytic activity with single-strand DNA templates, and DNA polymerase alpha 3 is essentially inactive with this template. Successive steps of hydrophobic affinity chromatography and phosphocellulose chromatography of DNA polymerase alpha 2 resolved the polymerase alpha activity and two protein factors (C1 and C2) that are required for its catalytic activity with a DNA template-primer that contains extended single-strand regions. In the absence of the factors, DNA polymerase alpha activity is measurable with activated but not single-strand DNA templates. In the presence of the C1 and C2 factors DNA polymerase alpha activity with single-strand DNA templates is restored to about 75% of the catalytic activity of DNA polymerase alpha 2 with this template.
Histone-dependent and histone-independent forms of an ADP-ribosyltransferase from human and turkey erythrocytes.
Authors: Moss J , Stanley S J
Published: May 2006

An ADP-ribosyltransferase from turkey erythrocytes, which catalyzes the mono(ADP-ribosylation) of guanidino compounds such as arginine and of many purified and crude cellular proteins, appears to exist both in high-activity, histone-independent and low-activity, histone-dependent forms. At low salt concentrations, the activity of the transferase with agmatine as acceptor was less than 10% that observed in the presence of 200 mM NaCl. In the absence of salts, ADP-ribosylation of agmatine was stimulated greater than 10-fold by histones, and activity approached that observed with high salt concentration; under these conditions, the histones did not serve as ADP-ribose acceptors themselves. Histone also activated the highly purified ADP-ribosyltransferase from human erythrocytes. Enzyme activity was increased in the presence of salt and was then relatively independent of histones. DNA was not required for the stimulation of ADP-ribosylation by histone; incubation of the transferase and histone with DNase did not significantly decrease enzymatic activity. Additional DNA in the assay decreased the effect of histone. The erythrocyte ADP-ribosyltransferase from diverse species thus appears to exist in two forms: one is dependent on histones for activity and one which, in the presence of salt, has high intrinsic activity and is independent of histone. The fact that the active forms of the transferase generated in the presence of salt or histone have similar catalytic activity suggests that these forms of transferase may be identical. It would appear that the enzymatic activity of transferase from different species may be controlled by histones.
31P NMR studies of intact anchorage-dependent mouse embryo fibroblasts.
Authors: Ugurbil K , Guernsey D L , Brown T R , Glynn P , Tobkes N , Edelman I S
Published: May 2006

31P NMR spectra of "normal" and x-ray-transformed anchorage-dependent mouse embryo fibroblasts (C3H/10T 1/2) at 145.7 MHz and 37 degrees C were obtained by using a solenoid coil probe with a sensitivity approximately 2.6 times higher than that of the standard Helmholtz coil probe. The cells were grown as monolayers on the surface of microcarrier beads. Results show that these cells have low levels of NMR-detectable ADP, are highly impermeable to protons, are capable of maintaining high ATP levels in the absence of respiration, and, when ATP generation is blocked, ultimately hydrolyze their existing ATP to a nonphosphorylated product and Pi. Only small differences were observed between the spectra of "normal" and transformed cells. However, the latter accumulated 2-deoxyglucose 6-phosphate at a faster rate.
Rat alpha-lactalbumin has a 17-residue-long COOH-terminal hydrophobic extension as judged by sequence analysis of the cDNA clones.
Authors: Dandekar A M , Qasba P K
Published: May 2006

cDNA for rat alpha-lactalbumin has been cloned in bacterial plasmid, and its sequence has been analyzed. The DNA sequence analysis shows that rat alpha-lactalbumin has 17 extra residues beyond the COOH terminus of the alpha-lactalbumin isolated and sequenced to date from other species. The predicted COOH-terminal sequence is hydrophobic and proline rich and bears some resemblance to beta-casein sequences.
Initiation factor-independent translation of mRNAs from Gram-positive bacteria.
Authors: McLaughlin J R , Murray C L , Rabinowitz J C
Published: May 2006

Initiation factor-independent translation of mRNA derived from bacillus phage phi29 DNA occurs with translation systems derived from Bacillus subtilis or Escherichia coli. This is in sharp contrast to the strict dependence on ribosome salt wash fraction of E. coli ribosomes for the translation of T7 and other mRNAs derived from Gram-negative organisms.
The human beta-globin gene and a functional viral thymidine kinase gene in developing mice.
Authors: Wagner E F , Stewart T A , Mintz B
Published: May 2006

Two foreign cloned genes--one encoding a tissue-specific protein and one encoding a constitutive enzyme--were introduced into mouse eggs by microinjection into a pronucleus shortly after fertilization. They were the adult human genomic beta-globin gene and the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene of herpes simplex virus (HSV), ligated in the pBR322 plasmid. Thirty-three developing mice were autopsied in late fetal life; all appeared normal. Blot hybridization tests revealed that the DNA of as many as five (15%) of the fetuses (from separate litters), and of their corresponding placentas, contained copies of the human beta-globin gene and of the HSV tk gene that had been retained and replicated without significant loss or rearrangement. The estimated total numbers of copies per cell were 3-50 for the donor globin gene and 3-20 for the donor tk. In some of the fetuses, these totals included some copies of molecular weight higher than that of the intact sequence; the additional segments may have arisen through changes such as deletions or duplications. The foreign genes in the five positive fetuses appear to be present in high molecular weight DNA. Assays capable of distinguishing between foreign and native TK indicated that at least one of the fetuses with the HSV tk gene had some TK enzyme of the HSV type and, therefore, at least one gene copy that was being accurately transcribed and translated to produce a functional protein, despite the absence of selective pressure. Thus, pure recombinant genes introduced into mice at the onset of their development can remain intact and be stably incorporated and even expressed. These experiments provide a practical basis for novel investigations of the developmental control of normal gene expression in vivo of the causes and possible cures of genetic diseases.
Cloning arg3, the gene for ornithine carbamoyltransferase from Saccharomyces cerevisiae: expression in Escherichia coli requires secondary mutations; production of plasmid beta-lactamase in yeast.
Authors: Crabeel M , Messenguy F , Lacroute F , Glansdorff N
Published: May 2006

The yeast arg3 gene, coding for ornithine carbamoyltransferase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), has been cloned on a hybrid pBR322-2-micrometers plasmid. The cloned gene gives a normal regulatory response in yeast. It is not expressed at 35 degrees C when a mutation preventing mRNA export from the nucleus at this temperature is included in the genetic make-up of the carrier strain. In Escherichia coli, no functional expression can be observed from the native yeast arg3 gene. The study of a mutant plasmid (M1) producing low levels of yeast carbamoyltransferase in E. coli has permitted the localization and orientation of arg3 on the plasmid. The mutation involved is a deletion that alters the regulatory response of arg3 in yeast. The plasmid bla gene produces detectable amounts of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) in yeast: the data provide an estimate of the beta-lactamase activity associated with one exemplar of the plasmid expressing arg3 (0.6 units).
A new hypothalamic substance, and not luteinizing hormone-releasing hormone, is detected immunocytochemically by antibody to luteinizing hormone-releasing hormone.
Authors: Sternberger L A , Greenwald J L , Hock D , Elger K , Forssmann W G
Published: May 2006

Adjacent paraffin sections of rat hypothalami fixed in Bouin's fluid were treated either with buffer or with luteinizing hormone-releasing hormone (LHRH) before immunocytochemical staining with anti-LHRH. Upon buffer pretreatment, pituitary gonadotrophs were unstained and hypothalamic fibers were stained. Upon LHRH pretreatment, pituitary gonadotrophs were stained (receptor reaction) and hypothalamic fibers were unstained. Extension of washes and use of series of neutralizing antisera between LHRH application and immunocytochemical staining, as well as the absence of inhibiting concentrations of LHRH in the later washes and neutralizing antisera removed from the sections, excluded the possibility that the disappearance of visualization of hypothalamic fibers was due to blockage of anti-LHRH in immunocytochemical staining. The results suggested that LHRH removed from the sections an immunocytochemically stainable but as yet unknown analog of LHRH and replaced it with LHRH, which in turn became lost during subsequent immunocytochemical processing. This idea was confirmed by the isolation by high-pressure liquid chromatography of a peak, distinct from LHRH, upon treatment of hypothalami with LHRH. It is suggested that the new substance may be carrier-held and that this substance, rather than LHRH, is normally detected by immunocytochemistry with anti-LHRH. Added LHRH binds not only to high-affinity pituitary receptors but also to low-affinity hypothalamic carriers.
Changes in the structure of nuclei between the magic neutron numbers 50 and 82 as indicated by a rotating-cluster analysis of the energy values of the first 2
+
excited states of isotopes of cadmium, tin, and tellurium
Authors: Pauling Linus
Published: May 2006

Values of
R
, the radius of rotation of the rotating cluster, are calculated from the observed values of the energy of the lowest 2
+
states of the even isotopes of
48
Cd,
50
Sn, and
52
Te with the assumption that the cluster is α,
p
2
, and α, respectively.
R
shows a maximum at ≈
N
= 58, a minimum at ≈
N
= 62, and a second maximum at ≈
N
= 70. The increase to the first maximum is interpreted as resulting from the overcrowding of spherons (alphas and tritons) in the mantle (outer layer) of the nuclei, causing the cluster to change from rotating in the mantle to skimming over its surface; the decrease to the minimum results from the addition of three dineutrons to the core, expanding the mantle and permitting the rotating cluster to begin to drop back into it; and the increase to the second maximum results from the overcrowding of the larger mantle surrounding the core containing the semi-magic number 14 of neutrons rather than the magic number 8 for
N
= 50. The decrease after the second maximum results from the further increase in the number of core neutrons to 20, corresponding to the magic number 82. Some additional evidence for the change to an intermediate structure between
N
= 50 and
N
= 82 is also discussed.
Structure of a chromosomal gene for human interferon β
Authors: Ohno Shigeo , Taniguchi Tadatsugu
Published: May 2006

We have cloned and analyzed a chromosomal DNA segment containing the human interferon β
1
gene from a human gene library. The nucleotide sequence of the protein-coding and the noncoding regions of the chromosomal gene was identical to the cDNA sequence reported previously. In the region upstream from the putative transcription initiation site, significant nucleotide sequence homology was observed between interferon β
1
and α
1
genes. This region thus may play a role in expression of the interferon genes. From the sequence data and the result of nuclease S1 mapping experiments, we conclude that, like the interferon α
1
gene, the interferon β
1
gene is devoid of intervening sequences.
Molecular cloning of mRNA sequences encoding rat lens crystallins.
Authors: Dodemont H J , Andreoli P M , Moormann R J , Ramaekers F C , Schoenmakers J G , Bloemendal H
Published: May 2006

To provide access to crystallin-specific DNA sequences, we have constructed plasmid clones bearing duplex DNA sequences complementary to crystallin mRNAs isolated from rat lens. Optimization of the cDNA reaction conditions enabled us to fractionate three double-stranded (ds) cDNA groups. Molecular cloning of dC-tailed ds cDNAs into the Pst I site of dG-tailed pBR322 yielded crystallin-specific clones of each group. By means of positive hybridization selection and translation, recombinant plasmids containing cDNA sequences coding for rat lens polypeptides from alpha-, beta-, and gamma-crystallins could be identified. The established cDNA clones have been used for a blot-hybridization analysis to map the crystallin mRNAs from which they originated. Both procedures revealed a high degree of homology between the gamma-crystallin sequences. From the beta-crystallin class, the beta H-specific cDNA coding for the beta B1a polypeptide was obtained. The alpha A-chain clone did not show any cross-hybridization to the alpha B-chain mRNA despite the existence of 60% homology between the corresponding gene products. As this clone hybridized to both alpha A2 and alpha AIns mRNAs, sequence analysis was applied for further characterization. The results showed that the cloned cDNA corresponds to the alpha A2 sequence exclusively.
Biosynthesis of porphyrins and heme from gamma, delta-dioxovalerate by intact hepatocytes.
Authors: Morton K A , Kushner J P , Burnham B F , Horton W J
Published: May 2006

The purpose of this study was to assess the ability of hepatocytes to synthesize porphyrins and heme from delta-aminolevulinic acid derived from gamma, delta-dioxovalerate and alanine. An alternate pathway for delta-aminolevulinic acid synthesis, in contrast to the condensation of succinate and glycine by delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] has been suggested. This has been supported by the isolation of gamma, delta-dioxovalerate transaminase from liver mitochondria (Varticovski, L., Kushner, J. P. & Burnham, B. F. (1980) J. Biol. Chem. 255, 3742-3747). Gamma, delta-dioxovalerate transaminase catalyzes the formation of delta-aminolevulinic acid by a transamination reaction involving gamma, delta-dioxovalerate and alanine. To assess the significance of this alternate route, we prepared suspensions of respiring rat hepatocytes, which were incubated with optimal concentrations of various additives and then analyzed spectrophotometrically for synthesized porphyrins. No porphyrin synthesis was detected in cell suspensions incubated with succinate(1 mM) and glycine (20 mM). Cell suspensions incubated with gamma, delta-dioxovalerate (0.5-1.0 mM) and alanine (20 mM) synthesized 0.19 nmol of porphyrin per 10(7) cells per 2 hr (SD, 0.057). Cell suspensions incubated with delta-aminolevulinic acid (0.1 mM) synthesized 1.26 nmol of porphyrin per 10(7) cells per 2 hr (range, 1.18-1.32). Incubations with chemically synthesized gamma, delta-dioxo[5-14C]valerate were followed by extraction and purification of porphyrin esters and heme. Liquid scintillation counting revealed radiolabel incorporation into both porphyrins and heme. These studies demonstrate significant tetrapyrrole synthesis by the gamma, delta-dioxovalerate transaminase reaction. That gamma, delta-dioxovalerate is an important precursor of heme in vivo must be considered.
Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli.
Authors: Davis A R , Nayak D P , Ueda M , Hiti A L , Dowbenko D , Kleid D G
Published: May 2006

Antigenic determinants of influenza virus hemagglutinin were expressed in Escherichia coli. DNA coding for presequences of hemagglutinin were removed and an ATG codon was placed before DNA coding for mature hemagglutinin. A number of expression plasmids were constructed in which various segments of this reconstructed hemagglutinin DNA were fused to DNA coding for bacterial beta-galactosidase. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.
Biosynthesis and periplasmic segregation of human proinsulin in Escherichia coli.
Authors: Chan S J , Weiss J , Konrad M , White T , Bahl C , Yu S D , Marks D , Steiner D F
Published: May 2006

A plasmid containing human preproinsulin cDNA inserted into the endonuclease Pst I site of the ampicillinase gene of plasmid pBR322 was modified by excision of large portions of the ampicillinase-coding region to produce a variety of gene fusion combinations, many of which generated proteins detectable with antisera to insulin or human C peptide. In one case a perfect hybrid of the NH2-terminal half of the leader sequence of ampicillinase (residues -23 to -12) with the human preproinsulin prepeptide beginning at residue -13 was formed; the result was the synthesis and secretion of human proinsulin into the periplasmic space. We have characterized this protein immunologically and also by labeling it biosynthetically or by iodination followed by immunoprecipitation and automated amino acid sequence analysis. It contains the A and B chain regions of insulin as well as specific human C peptide immunodeterminants and is convertible to an insulin-like component by tryptic digestion. These results demonstrate that human proinsulin can be produced by bacteria and that this biosynthetic approach should prove feasible for the production of adequate amounts of human proinsulin for a variety of clinical studies and human insulin for therapeutic purposes.
Cloning of bacteriophage fd gene 2 and construction of a plasmid dependent on fd gene 2 protein.
Authors: Meyer T F , Geider K
Published: May 2006

Bacteriophage fd gene 2 was cloned in plasmid pBR325. Cells carrying the hybrid plasmid produce about 200 times more enzymatically active fd gene 2 protein than did cells infected with phage fd wild type, as measured by replication of phage fd replicative form I in vitro. Cloned gene 2 supports replication of an artificial phage fd miniplasmid consisting of the origin of bacteriophage fd replication and a gene coding for kanamycin resistance. This plasmid occurs in high copy numbers and is viable only in cells carrying the cloned fd gene 2 or in cells infected with phage fd. Because the miniplasmid is not propagated in natural hosts, it can be considered a safe cloning vector. Its fusion with the gene 2 hybrid plasmid provides an autonomous replicon independent of the polA function of the host cell. fd gene 2 is the only phage-encoded trans-acting function required for replication of double-stranded fd DNA in vivo.
Both alpha and beta subunits of human choriogonadotropin photoaffinity label the hormone receptor.
Authors: Ji I , Ji T H
Published: May 2006

It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor.
Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.
Authors: Ohtsuka E , Tanaka S , Tanaka T , Miyake T , Markham A F , Nakagawa E , Wakabayashi T , Taniyama Y , Nishikawa S , Fukumoto R , Uemura H , Doi T , Tokunaga T , Ikehara M
Published: May 2006

A RNA molecule has been synthesized that is identical in sequence to Escherichia coli tRNAfMet except that it lacks the base modifications present in the E. coli tRNA. This was achieved by enzymatic joining of chemically synthesized oligonucleotides with chain lengths of 3-10 which were synthesized by the phosphodiester or phosphotriester method. First, quarter molecules of tRNA were constructed by joining of chemically synthesized fragments with RNA ligase. The 5'-quarter molecule (bases 1-20) served as an acceptor in joining reactions with the 3',5'-bisphosphorylated donor molecule (bases 21-34). The 5'-half molecule thus obtained was treated with phosphatase and joined to the 3'-half molecule which was prepared by ligation of the other quarter molecules (bases 35-60, acceptor; bases 61-77, donor) followed by 5'-phosphorylation with polynucleotide kinase. The synthetic tRNA was characterized by oligonucleotide pattern and was partially active in aminoacylation with E. coli methionyl-tRNA synthetase.