bluuebunny/crossref_metadata_2025 huggingface dataset:
Positive selection of major histocompatibility complex-restricted suppressor T cells bearing the predominant idiotype in the immune response to lysozyme.
Authors: Araneo B A , Yowell R L , Metzger D W , Sercarz E E
Published: May 2006

The lysozyme system provides an excellent model for studying the role of multiple major histocompatibility complex (MHC) genes in the induction and regulation of Ir-gene controlled immune responses. Immunization of H-2b mice leads to concomitant activation of helper and suppressor activities by different epitopes on hen egg-white lysozyme (HEL) and thus phenotype unresponsiveness to native HEL. HEL-specific suppressor T cells in C57BL/10 nonresponder mice show MHC restriction, because their enrichment on antigen-pulsed macrophage monolayers requires syngeneic macrophages as well as HEL. The expression of the selected suppressor function requires interaction between the restricted suppressor precursor cell and an HEL-triggered, suppressor-inducer T cell. The MHC-restricted suppressor precursors bear the predominant idiotype found on anti-HEL antibodies, whereas MHC-restricted helpers do not.
Sequence homologies between A subunits of Escherichia coli and Vibrio cholerae enterotoxins.
Authors: Spicer E K , Kavanaugh W M , Dallas W S , Falkow S , Konigsberg W H , Schafer D E
Published: May 2006

The genes coding for the heat-labile enterotoxin LT produced by Escherichia coli have been cloned into the plasmid pBR313. Using DNA derived from the resulting chimeric plasmid, we determined the nucleotide sequence of two regions of the gene coding for the enzymatically active A subunit of LT. Translation of the nucleotide sequence gives the primary structure of the NH2-terminal and COOH-terminal regions of the LT A subunit. This permits direct comparison of the LT A subunit with the A subunit of cholera toxin. Our results show that the two toxins possess homologous sequences, of varying degrees, in both regions of their primary structure. The order of the component A1 and A2 polypeptides is A1-A2. The nucleotide sequence predicts the existence of a signal sequence of 18 amino acids at the NH2-terminus of the A subunit.
Tropospheric concentrations of methylchloroform, CH
3
CCl
3
, in January 1978 and estimates of the atmospheric residence times for hydrohalocarbons
Authors: Makide Yoshihiro , Rowland F. Sherwood
Published: May 2006

The ground level tropospheric concentrations of CH
3
CCl
3
were measured from 55°N to 53°S during the time period around Jan. 1, 1978. The northern temperate zone concentration of CH
3
CCl
3
averaged 94.6 ± 4.0 × 10
-12
by volume. The southern temperate zone concentration averaged 65.2 ± 1.3 × 10
-12
, for a worldwide average of 80 × 10
-12
by volume. The ratio of concentrations between the two zones is 1.45 ± 0.07. The observed CH
3
CCl
3
concentrations correspond to 0.52 ± 0.05 times the atmospheric release to that date, corresponding to an atmospheric residence time of 6.9 ± 1.2 yr. The atmospheric residence times for 22 other hydrohalocarbon molecules were estimated in comparison to that of CH
3
CCl
3
through the relative rates of reaction with OH radicals.
Yeast tRNA3Leu gene transcribed and spliced in a HeLa cell extract.
Authors: Standring D N , Venegas A , Rutter W J
Published: May 2006

A cloned yeast tRNA3Leu gene containing a 33-base intervening sequence (IVS) is selectively transcribed by a soluble extract from HeLa cells. The 130-nucleotide tRNA3Leu precursor RNA formed is colinear with the gene and contains approximately 4 leader nucleotides and up to 9 trailer nucleotides. The IVS is accurately and efficiently removed by an endogenous HeLa excision-ligase activity to yield the spliced tRNA, the free IVS, and the half-tRNA intermediates. The splicing reaction occurs without prior 5' and 3' maturation of the precursor but, with this exception, this pattern of synthesis and subsequent maturation of the tRNA3Leu precursor conforms to the scheme for tRNA biosynthesis deduced for the xenopus system. Indeed, the two systems utilize similar or identical tRNA3Leu precursors. Our results stress the extraordinary conservation of tRNA biosynthesis in eukaryotes and demonstrate that a HeLa extract provides a useful system for investigating this process.
Elongation of displacement-loop strands in human and mouse mitochondrial DNA is arrested near specific template sequences.
Authors: Doda J N , Wright C T , Clayton D A
Published: May 2006

Animal mitochondrial DNA (mtDNA) maintains a displacement loop (D loop) at the heavy strand origin of replication. These D loops represent sharply limited synthesis of heavy strands and provide a unique opportunity to examine the termination of DNA synthesis. Direct sizing at the nucleotide level indicates that the 3' ends of D-loop strands of human and mouse mtDNA are discrete and map within three to five nucleotides on the complementary template strand. In the case of human mtDNA, there is a single trinucleotide stop point 51-53 nucleotides downstream from a 15-nucleotide template sequence (3'T-A-A-C-C-C-A-A-A-A-A-T-A-C-A 5') which is repeated four times in the mouse mtDNA D-loop region 3'(T-A-A-Py-Py-A-A-A-T-T-A-C-A 5'). The stop points of the five major mouse D-loop strands are 24-63 nucleotides downstream from the four repeated template sequences. These results suggest that the arrest of D-loop strand elongation is an event determined by template sequence.
Sequence-specific contacts between the RNA polymerase of vesicular stomatitis virus and the leader RNA gene.
Authors: Keene J D , Thornton B J , Emerson S U
Published: May 2006

Methylation-protection studies with nucleocapsids from vesicular stomatitis virus indicate that the viral polymerase (L and NS proteins) contacts the genomic RNA template in the middle of the leader gene, 16-30 nucleotides from the 3' terminus. The data suggest that the NS protein binds to the sequence (formula, see text) and may function as an initiator protein for transcription.
Effect of vinblastine on distribution of murine leukemia virus-derived membrane-associated antigens.
Authors: Satake M , McMillan P N , Luftig R B
Published: May 2006

The effect of vinblastine on the distribution of murine leukemia virus-derived membrane-associated antigens was examined by using the indirect immunofluorescence of 3.7% formaldehyde-fixed MJD-54 (Moloney murine leukemia virus-infected) cells. On fixed, non-drug-treated cells, p30 antigen was distributed homogeneously and diffusely over the cell membrane. When cells were incubated with 10 microM vinblastine for 1 hr before fixation, the distribution of p30 antigen was greatly changed, fluorescence now being collected into poles (cap-like formation). In contrast to this distribution pattern for p30 antigen, gp70 antigen was distributed in a micropunctate pattern on the cell surface, with or without vinblastine pretreatment. These observations indicate that the distribution patterns of p30 and gp70 membrane antigens are completely different and that they are differently controlled by cytoplasmic microtubules. In addition, because the p30 membrane antigen visualized in these studies most likely represents viral Pr65gag precursor molecules which are localized directly under and associated with the plasma membrane, these results suggest that, under special conditions of fixation, it is possible to obtain a cap-like phenomenon for cytoplasmic (internal) membrane-oriented proteins.
Interferon increases the abundance of submembranous microfilaments in HeLa-S3 cells in suspension culture.
Authors: Wang E , Pfeffer L M , Tamm I
Published: May 2006

Human beta (fibroblast) interferon inhibits the proliferation of human HeLa-S3 carcinoma cells in suspension culture. Accompanying this effect, the lateral mobility of cell surface receptors for concanavalin A is decreased and the rigidity of the plasma membrane lipid bilayer is increased. The present findings show a marked increase in the number of polymerized actin-containing microfilaments 3 days after treatment of HeLa-S3 cells with beta-interferon (640 units/ml). The cortical region of the treated enlarged cells contains a thick and dense meshwork of 40-70 A microfilaments. The actin nature of the filaments was verified by their ability to bind heavy meromyosin. These results support the concept that beta-interferon induces a coordinated response in the plasma membrane and the underlying microfilaments in both tumor and normal cells.
Influence of H-2 antigen expression on killer T cell specificity, differentiation, and induction.
Authors: Fink P J , Bevan M J
Published: May 2006

Murine cytotoxic T lymphocytes (CTL) and helper cells are H-2 antigen restricted in their specificity: recognition of foreign antigen by these cells requires the concomitant recognition of self-H-2 molecules. Which H-2 antigens T cells treat as "self" is determined by the particular H-2 antigens expressed on radioresistant cells of the thymus in which these T cells mature. Using tetraparental [(P1 + P2) leads to F1] radiation chimeras with in situ F1 thymuses, we have found that the H-2 genotype of the stem cells does not influence their H-2 restriction specificity. This has allowed us to use tetraparental chimeras that have been thymectomized and grafted with parental (P1, P2, or both) thymus lobes to study the requirements for H-2-restricted T--T interactions during CTL ontogeny and induction. In animals that have received thymus grafts of both parental origins, CTL display no preference for maturation within a syngeneic thymus graft, a finding that is not compatible with a suggested requirement for intrathymic H-2-restricted T--T interactions in the maturation of precursor CTL. We have also grafted thymectomized tetraparental radiation chimeras with thymus grafts from only one parent to compare the induction of P1 and P2 CTL in environments in which peripheral (extrathymic) T cell interactions are restricted to one H-2 haplotype. Again, we find no evidence for preferential induction of CTL precursors syngeneic to the thymus graft, contrary to expectation if CTL induction requires that T helper cells restricted to thymic H-2 antigens interact directly with precursor CTL. In those animals with one parental thymus graft, there is variability in the ratios of P1 and P2 cells induced with several antigens, a finding that may be indicative of an H-2-restricted suppression mechanism operating in the periphery.
Episomal simian virus 40 genomes in human brain tumors.
Authors: Krieg P , Amtmann E , Jonas D , Fischer H , Zang K , Sauer G
Published: May 2006

Eight out of 35 human intracranial tumors were shown by restriction enzyme analysis to contain unintegrated simian virus 40 (SV40) DNA molecules. The relative amount of viral DNA was estimated to be the equivalent of one viral genome within every 10th to 20th cell. No infectious virus was detected in tissue cultures established from the tumors. From only one tumor was it possible to rescue, by cell fusion, infectious SV40 displaying wild-type properties. In those cases that permitted a more detailed analysis, the restriction enzyme cleavage patterns appeared to correspond to the wild-type patterns with one exception, in which the SV40 episomes displayed a deletion of approximately 70 base pairs close to the origin of DNA replication. From one tumor, the SV40 genomes were transferred into permissive CV-1 monkey cells by transfection with the total tumor DNA. Despite their persistence as episomes no infectious virus was produced. Furthermore, no viral antigens were detectable, although the SV40 messengers for the small and the large tumor antigens were present. These cells had, however, acquired the ability to form colonies in low concentrations of serum. Thus this report provides, by restriction enzyme analysis, direct evidence for the presence of SV40 DNA in human tumors.
Increased ascorbic acid content in chronic lymphocytic leukemia B lymphocytes.
Authors: Liebes L , Krigel R , Kuo S , Nevrla D , Pelle E , Silber R
Published: May 2006

Human lymphocyte extracts analyzed by high-performance liquid chromatography reveal a major UV-absorbing peak that was shown to be ascorbic acid by spectral, chemical, and enzymatic criteria. Because this peak appeared very prominent in the elution profile of chronic lymphocytic leukemia (CLL) lymphocyte extracts, we measured the ascorbic acid content in lymphocytes from the blood of normal subjects and untreated patients with chronic lymphocytic leukemia. A significantly higher concentration of 111 +/- 15.3 nmol per 10(8) cells (mean +/- SEM) was found in CLL lymphocytes than in normal blood lymphocytes, which contained 42.2 +/- 3.3 nmol per 10(8) cells. Selective enrichment with B and T cells showed that this difference was limited to the chronic lymphocytic leukemia B cell, which had a 5- to 15-fold higher content of ascorbic acid than normal B cells had. In contrast, the ascorbic acid level was similar in normal and CLL T cells. The very high ascorbic acid content provides the chronic lymphocytic leukemia B cell with a reducing substance that could react with oxidants or free radicals.
Amygdalin (Laetrile) and prunasin beta-glucosidases: distribution in germ-free rat and in human tumor tissue.
Authors: Newmark J , Brady R O , Grimley P M , Gal A E , Waller S G , Thistlethwaite J R
Published: May 2006

Amygdalin, the gentiobioside derivative of mandelonitrile commonly referred to as Laetrile, is presently under intensive investigation as a potential cancer chemotherapeutic agent. Because of this interest, we investigated the activity of beta-glucosidases that cleave glucose from amygdalin and from prunasin (mandelonitrile monoglucoside) in tissues from germ-free rats and in normal and neoplastic human tissues. Rat and human small intestinal mucosa contain high levels of activity of glucosidases that act on both of these cyanogenic glucosides. Release of glucose from these compounds was not detected in any of the human neoplastic tissues examined in the present study. These observations are consistent with reports of cyanide toxicity through the oral use of amygdalin or prunasin and pose serious questions concerning the alleged tumoricidal effect of amygdalin.
Dopaminergic modulation of adenylate cyclase stimulation by vasoactive intestinal peptide in anterior pituitary.
Authors: Onali P , Schwartz J P , Costa E
Published: May 2006

The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by vasoactive intestinal peptide (VIP) was used as a model to investigate the molecular mechanisms triggered by the occupancy of dopamine recognition sites in rat anterior pituitary. Dopamine failed to change the basal enzyme activity, but it inhibited the stimulation of adenylate cyclase elicited by VIP. Apomorphine, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, and 2-bromo-alpha-ergocryptine mimicked the effect of dopamine, whereas (-)-sulpiride and and classical neuroleptics antagonized it. Dopamine failed to modulate the activation of pituitary adenylate cyclase by prostaglandin E1, which does not increase prolactin secretion. From these results we infer that stimulation of D-2 (dopamine) receptors may affect pituitary secretion by inhibiting the activation of anterior pituitary adenylate cyclase by VIP or other secretagogues.
Further perspectives on the charge transfer transitions of blue copper proteins and the ligand moieties in stellacyanin
Authors: McMillin David R. , Morris Margaret C.
Published: May 2006

The charge transfer spectra of plastocyanin and azurin are analyzed in detail, taking into account the structural information that is available. The number and relative energies of the low-lying charge transfer bands attributable to cysteine and methionine sulfur are considered with reference to the energy levels associated with the donor centers and to experimental work on relevant complexes. Two cysteine → Cu(II) charge transfer transitions are assigned to bands that occur near 620 nm and near 770 nm. Energy considerations suggest that the methionine → Cu(II) transition occurs in the vicinity of 550 nm for both plastocyanin and azurin. The existence of a disulfide linkage in stellacyanin is established by thiol titrations, and the possibility exists that the fourth ligand in stellacyanin may involve disulfide sulfur. If so, the cystine → Cu(II) transition is also expected to appear around 550 nm.
Molecular basis of auxin-regulated extension growth and role of dextranase
Authors: Heyn Anton N. J.
Published: May 2006

The first step in the extension growth of the plant cell is a process in which the cell wall becomes ductile or plastic, after which the actual enlargement takes place passively under the influence of turgor. The nature of this process has not been explained, although much research has been carried out concerning it. In the present report, it is shown that a specific enzyme, which is identical or nearly so with dextranase (α-1,6-D-glucan 6-glucanohydrolase, EC 3.2.1.11) and is associated with the cell walls of growing coleoptiles, plays a prominent role in this process. The action of this enzyme is dependent on the level of growth hormone, auxin, in the tissue. Under its action, certain cell wall components are broken down to yield arabinose and glucose. These sugars are also released during autolysis of cell wall material. The molecular linkages broken in the process are probably the arabinogalactan crosslinks of the hemicellulose matrix, which are the main constituents of the wall containing arabinose. This is substantiated by the finding that dextranase can break down arabinan and compounds containing arabinose chains with the release of arabinose, just as in the action of the enzyme on the wall. The breaking of these crosslinks will impart the necessary plasticity to the wall for cell extension to occur.
Comparative inhibition of mitochondrial and cytosolic phosphoenolpyruvate carboxykinases by stereospecific substrate analogues.
Authors: Duffy T H , Markovitz P J , Chuang D T , Utter M F , Nowak T
Published: May 2006

Phosphoenolpyruvate carboxykinase [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] was purified from the liver of several species; the mitochondrial enzyme and the cytosolic enzyme were separated from each other. The species included guinea pig, monkey, rat, and chicken. Each enzyme was assayed for inhibition by the substrate analogues E-2-phosphoenolbutyrate and Z-2-phosphoenolbutyrate. Each enzyme tested displayed the same stereospecificity: the E diastereoisomer was the more potent inhibitor than the Z. These results suggest an active site homology for all carboxykinases. The absolute values for Ki measured show that in almost every case the mitochondrial enzyme is more susceptible to inhibition by these analogues than is the cytosolic enzyme. The Ki values are one-fifth those for the mitochondrial enzymes. These results imply subtle differences in ligand interactions at the active sites of these enzymes.
SOS induction and autoregulation of the himA gene for site-specific recombination in Escherichia coli.
Authors: Miller H I , Kirk M , Echols H
Published: May 2006

The himA gene of EScherichia coli controls the lysogenization of bacteriophage lambda at the level of catalysis of site-specific recombination and expression of the lambda int and cI genes required for lysogenic development. We have analyzed the regulation of himA by two methods: (i) beta-galactosidase synthesis from a lacZ gene inserted into the himA gene and (ii) detection of radioactive HimA protein after fractionation by two-dimensional gel electrophoresis. We find that himA- mutations produce enhanced expression of the himA gene, indicating that HimA protein controls its own synthesis. The himA gene is also induced by treatment of cells with UV or mitomycin C, suggesting control by the inducible DNA repair (SOS) system regulated by the LexA and RecA proteins. Regulation of himA follows the pattern expected for a typical SOS gene: constitutive high expression in mutants that have inactive LexA or the altered RecA conferred by the recA441 (tif1) mutation and low noninducible expression in a mutant that has a deleted recA gene. We conclude that the himA gene is a component of the inducible SoS response, repressed by LexA and induced by the capacity of activated RecA to cleave LexA. We suggest that HimA may be subject to SOS induction because it functions as an "acquisitionase" for new genetic material and thus is of special utility under conditions of impaired capacity for growth of the bacterial population.
DNA sequences homologous to vertebrate oncogenes are conserved in Drosophila melanogaster.
Authors: Shilo B Z , Weinberg R A
Published: May 2006

Sequences homologous to the oncogene sequences of acute RNA tumor viruses have been shown to be highly conserved within vertebrates. In the present work, eight different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of organisms of other phyla. Five of these probes hybridized to the DNA of Drosophila melanogaster. Abelson leukemia virus probe detected a single homologous DNA fragment in Drosophila DNA. In contrast, probes prepared from the genomes of Harvey, avian, and feline sarcoma viruses and avian myelocytomatosis virus hybridized with multiple homologous sequences in Drosophila DNA. The identification of sequences homologous to vertebrate oncogenes in invertebrates demonstrates both a high degree of conservation of these genes and a wide distribution among divergent species. It seems likely that sequences homologous to vertebrate oncogenes play a crucial role in metazoan metabolism.
Solution structural studies of the A and Z forms of DNA.
Authors: Wu H M , Dattagupta N , Crothers D M
Published: May 2006

We report transient electric dichroism studies of short double-helical DNA and poly[d(G-C)] fragments in alcohol/water mixtures. The limiting reduced dichroism and the rotational correlation time changed abruptly in the alcohol concentration range expected for the DNA B-to-A and poly[d(G-C)] B-to-Z transitions. The Z form of poly[d(G-C)] was also induced by mitomycin C crosslinking in aqueous solution. The rotational correlation times observed for A- and Z-DNA were approximately consistent with dimensions determined by crystallographic and fiber diffraction analysis: the estimated rise per base pair was 2.8 A for A-DNA and 3.7 A for Z-DNA in solution. In addition, the observed limiting reduced dichroism values for A- and Z-DNA were close to the theoretical limit of -1.5, requiring a structure in which the base transition moments are effectively perpendicular to the double-helix axis. This is the result expected for any DNA double helix having dyad symmetry in which the base pairs are flat and the base transition moments lie predominantly in the short axis of the base pair and therefore close to a helix dyad axis. Only B-DNA deviates from this rule, strongly reinforcing our earlier conclusion that the base pairs in B-DNA are not flat but are propeller twisted, either statically or as a dynamic average.
Solubilization and localization of triolein in phosphatidylcholine bilayers: a 13C NMR study.
Authors: Hamilton J A , Small D M
Published: May 2006

Cosonicated mixtures of egg phosphatidylcholine and small amounts (less than 5% wt/wt) of triolein have been studied by 13C NMR spectroscopy. The 50.3-MHz 13C NMR spectrum of vesicles preparations containing 90% isotopically substituted [1-13C]triolein showed two carbonyl resonances at chemical shift values that indicate hydrogen bonding of H2O molecules with the carbonyl groups. The extent of hydration, estimated from the chemical shift values that indicate hydrogen bonding of H2O molecules with the chemical shift values (173.07 ppm and 172.39 is approximately 50%. The data suggest that the triolein is located in the bilayer with the three carbonyl groups at the aqueous interface. The acyl chains are extended toward the bilayer interior, with a conformation of the glyceryl region such that the primary (alpha) carbonyls are closer to the aqueous medium than is the secondary (beta) carbonyl. Thus, triolein is present in the bilayer in an orientation appropriate for enzymatic hydrolysis, with the second substrate (H2O) in close proximity to the hydrolytic site, and with a conformation that could explain, in part, enzymatic specificity for hydrolysis at the alpha position. Spectra of vesicles containing greater than or equal to 3% triolein showed two additional carbonyl peaks characteristic of pure (neat) triolein. This allowed a determination of the maximum solubility (approximately 2.8%) of surface-oriented triolein in the bilayer phase. Beyond this limit all excess triolein partitions into a separate oil phase.
Chi activity during transduction-associated recombination.
Authors: Dower N A , Stahl F W
Published: May 2006

Chi is a genetic element that stimulates phage lambda recombination by the Escherichia coli recBC pathway during lytic infection [Stahl, F. W. (1979) Annu. Rev. Genet. 13, 7--24]. Herein we show that chi in lambda prophage influences exchange distribution in P1 phage-mediated transduction and in conjugation. This demonstration encourages the view that chi may influence genetic exchange in E. coli in the total absence of lambda.
In vivo effects of antibodies to immune response gene products: prevention of experimental allergic encephalitis.
Authors: Steinman L , Rosenbaum J T , Sriram S , McDevitt H O
Published: May 2006

Prevention of experimental allergic encephalitis in SJL/J [H-2s] mice was achieved with in vivo administration of antibody reactive with I-As gene products prior to immunization with spinal cord antigen. No protection was evident in animals that received antisera specific for I-Js gene products. Administration of antibody to I-As beginning 5 days after immunization with spinal cord antigen delayed, but did not prevent, the onset of experimental allergic encephalitis.
A plausible two-state model for cytochrome c oxidase.
Authors: Wilson M T , Peterson J , Antonini E , Brunori M , Colosimo A , Wyman J
Published: May 2006

The catalytic properties of pulsed and resting cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1), expressed in terms of a minimal kinetic scheme and simulated by numerical computations, were successfully described. A two-state model, in which the relative amounts of the enzyme present in each conformation are regulated by the rates of electron flux and O2 binding on one side and the interconversion rates on the other, accounts for the activation of cytochrome c oxidase during turnover.
Restrictions on permissible paths for changing efficiencies of operation of flow reactors
Authors: Noyes Richard M.
Published: May 2006

Sometimes a continuous flow stirred tank reactor can exhibit two or more different states of chemical reaction for the same flow rate. If such a situation is possible, a state of low reactive efficiency can be converted to one of higher efficiency by first decreasing the flow rate and then increasing it to the original value. This transition can never be accomplished by first increasing and then decreasing the flow rate. Similarly, a state of high reactive efficiency can be converted to one of lower efficiency only by first increasing and then decreasing the flow rate. These restrictions on permissible order of operations seem to differ from restrictions on efficiencies of cyclic heat engines and on directions of chemical change in closed systems as derived previously from thermodynamics.
First approximation of a stereochemical rationale for the genetic code based on the topography and physicochemical properties of "cavities" constructed from models of DNA.
Authors: Hendry L B , Bransome E D , Hutson M S , Campbell L K
Published: May 2006

To examine the question of whether or not the genetic code has a stereochemical basis, we used artificial constructs of the topography and physicochemical features of unique "cavities" formed by removal of the second codon base in B-DNA. The effects of base changes on the stereochemistry of the cavities are consistent with the pattern of the genetic code. Fits into the cavities of the side chains of the 20 L amino acids involved in protein synthesis can be demonstrated by using conventional physicochemical principles of hydrogen bonding and steric constraints. The specificity of the fits is remarkably consistent with the genetic code.
Metabolic labeling of lutropin with [35S]sulfate.
Authors: Hortin G , Natowicz M , Pierce J , Baenziger J , Parsons T , Boime I
Published: May 2006

Chemical analyses have previously detected sulfate linked to the oligosaccharides of lutropin isolated from bovine and human pituitaries. To determine whether lutropin could be metabolically labeled with sulfate, isolated bovine and rat pituitaries were incubated with [35S]sulfate. In both species, two major labeled products were immunoprecipitated with antisera specific to lutropin subunits. Incorporation into the subunits occurred posttranslationally since it was not blocked by cycloheximide, which did, however, block the incorporation of radiolabeled methionine. Metabolic labeling with [35S]sulfate provides a valuable approach for examining the biosynthetic processing of lutropin and the physiological role of sulfate in this hormone.
Synthesis of adenosine triphosphate in respiration-inhibited submitochondrial particles induced by microsecond electric pulses
Authors: Teissie Justin , Knox Barry E. , Tsong Tian Yow , Wehrle Janna
Published: May 2006

Phosphorylation of ADP to ATP was induced in nonrespiring submitochondrial particles (SMP) from rat liver by the application of electric pulses with field strengths of 10-35 kV/cm and a decay time of 60 μs. In all cases respiration was inhibited completely by using cyanide or rotenone. Newly formed ATP was measured by two independent methods, (
i
) the luciferase/luciferin bioluminescence assay and (
ii
) synthesis of [
32
P]ATP from ADP and
32
P
i
. Both methods gave consistent and essentially identical results. Above 10 kV/cm the amount of ATP synthesized increased with increasing field strength, and at 30 kV/cm, approximately 40 pmol of ATP was synthesized per mg of SMP protein per pulse. ATP synthesis was shown to be related to the field-induced transmembrane potential, not to Joule heating of the suspension. Synthesis was abolished by the uncouplers carbonyl cyanide
p
-trifluoromethoxyphenylhydrazone, carbonyl cyanide
m
-chlorophenylhydrazone, and 2,4-dinitrophenol. The ionophores valinomycin and A23187 reduced the level of synthesis by 75% and 50%, respectively. ATP synthesis was also blocked by inhibitors of the F
0
F
1
ATPase complex, oligomycin,
N,N
′-dicyclohexyl carbodiimide, venturicidin, and aurovertin. The activities of the adenine nucleotide translocator and adenylate kinase, as well as release of bound nucleotides, could be excluded as sources of the new ATP. The data indicate that the minimal applied field at which ATP synthesis could be detected is approximately 8 kV/cm, corresponding to a maximal induced membrane potential of 60 mV in SMP. The maximal synthesis occurred at around 30 kV/cm, or an induced transmembrane potential of 200 mV. The duration of the applied pulse was also found to be critical, with 8 μ
s
being the minimal triggering time for the synthesis. The induction of ATP synthesis in nonrespiring SMP by an externally applied electrical field is a direct demonstration of the transformation, by the mitochondrial inner membrane, of electrical energy into the chemical bond energy of ATP.
Poly(L-lysine) has different membrane transport and drug-carrier properties when complexed with heparin.
Authors: Shen W C , Ryser H J
Published: May 2006

Methotrexate (MTX) conjugated to a Mr 3000 poly(L-Lys) markedly inhibits the growth of Pro-3 MtxRII5-3 Chinese hamster ovarian cells, a mutant line known to be drug resistant because of defective MTX transport. In these cells, membrane transport of [3H]MTX-poly(Lys) is sharply decreased by addition of 0.5- to 2.5-fold heparin but remains at 15-20% of control in 2.5- to 50-fold heparin excess. Heparin addition at first markedly inhibits but, at high concentration, restores the growth inhibitory effect of MTX-poly(Lys). In excess heparin, MTX-poly(Lys) is transported as a heparin complex. Because reduced transport (15-20%) is sufficient to cause a 90% inhibition of cell growth, MTX-poly(Lys) apparently gains pharmacologic potency when compared to heparin. This gain can be related to a greater inhibitory effect on dihydrofolate reductase and to a different mode of transport. The inhibitory effect of MTX-poly(Lys) on dihydrofolate reductase in vitro is increased nearly 100-fold in the presence of excess heparin but remains less than that of free MTX. Unlike that of MTX-poly(Lys), the transport of MTX-poly(Lys)-heparin has the characteristics and efficiency of a receptor-mediated process. It proceeds by endocytosis but is not, as in the case of uncomplexed conjugate, followed by the intracellular generation of pharmacologically active breakdown products that would account for cytotoxicity. These observations raise the possibility that at least part of the MTX-poly(Lys)-heparin reaches cellular dihydrofolate reductase in the form of macromolecular complexes that escape from entrapment in endocytotic structures. Our data illustrate a way to overcome drug resistance by taking advantage of the specific uptake of a macromolecular drug carrier. They offer a method of drug delivery in which heparin improves selectivity and decreases the unwanted toxicity inherent to polycationic carriers.
Phenylketonuric Tetrahymena: phenylalanine hydroxylase mutants and other tyrosine auxotrophs.
Authors: Sanford Y M , Orias E
Published: May 2006

Nineteen tyrosine auxotrophs of the ciliated protozoan Tetrahymena thermophila have been isolated and biochemically examined. These mutants are defective in the conversion of phenylalanine to tyrosine; this is analogous to the defect that causes phenylketonuria in humans. After nitrosoguanidine mutagenesis and self-fertilization, progeny clones were screened for tyrosine auxotrophy and positively identified by using growth tests and in vivo radiometric assays for phenylalanine-to-tyrosine conversion. Mutants in one complementation group (locus) lacked phenylalanine hydroxylase activity; mutants in three other loci appeared to be deficient in the unconjugated pteridine cofactor that is necessary for the function of the hydroxylase. Another mutant lacked the dihydropteridine reductase activity required to regenerate the reduced form of the pteridine cofactor. Because hydroxylation of tyrosine to dopa and of tryptophan to 5-hydroxytryptophan may require the same cofactor and pterin reductase as phenylalanine hydroxylase, these mutants may also prove useful for the study of the role of catecholamines and serotonin, substances known to be present in Tetrahymena.
T-Cell lymphoma model for the analysis of interleukin 1-mediated T-cell activation.
Authors: Gillis S , Mizel S B
Published: May 2006

Several laboratories have recently demonstrated that the requirement for macrophages in mitogen-induced production of murine T-cell interleukin 2 (IL-2; formerly referred to as "T-cell growth factor") could be circumvented by using the macrophage-derived peptide interleukin 1 (IL-1; formerly referred to as "lymphocyte-activating factor"). Using two cloned T-cell lymphomas, we investigated the mechanism through which IL-1 exerted its effect on IL-2 production. One of the cell lines used (LBRM-33 5A4) produces large concentrations of IL-2 upon mitogen stimulation, whereas the second (LBRM-33 1A5) is incapable of producing IL-2 in response to mitogen. It was observed that addition of purified IL-1 to nonproducer 1A5 cells converted them to a state in which subsequent mitogen stimulation triggered production of IL-2. The concentration of IL-2 produced by IL-1 treated 1A5 cells was equivalent in magnitude to that generated by mitogen-stimulated 5A4 cells (500-1000 units/ml, or approximately 1000 times the concentration of IL-2 contained in conventional preparations of murine mitogen-conditioned medium). The observations that (i) brief exposure to IL-1 was sufficient for 1A5 cell conversion to IL-2 production and (ii) IL-1 could actively be absorbed from culture medium by live or fixed 1A5 cells led us to propose the existence of IL-1 receptors on responsive 1A5 cells. On the basis of these experiments, we have postulated that IL-1 mediates its effect on immune reactivity (enhancement of thymocyte mitogenesis and induction of antibody and cytotoxic T cell responses) by maturation of a subset of immature T cells to the point where they are capable of IL-2 production. Subsequent release of IL-2 after ligand activation allows for clonal expansion of activated T cells which mediate particular effector functions.
Genetic and serological analysis of the expression of crossreactive idiotypic determinants on anti-p-azobenzenarsonate antibodies and p-azobenzenarsonate-specific suppressor T cell factors.
Authors: Sy M S , Brown A , Bach B A , Benacerraf B , Gottlieb P D , Nisonoff A , Greene M I
Published: May 2006

Data are reported on the genetic control and structure of antigen-specific suppressive molecules obtained from azobenzenearsonate (ABA)-specific suppressor T cells (Ts). Ts-derived suppressor factors (TsFs) were previously shown to bear determinants encoded by genes of the I-J subregion of the H-3 major histocompatibility complex and structures encoded by VH (variable region of heavy chains) genes linked to the Igh locus. To further characterize TsF we have made use of a K light (L) chain variable region (VK) genetic marker linked to the locus governing expression of a crossreactive idiotype on anti-ABA antibodies. The experiments evaluated the possible contribution to TsF of structures under control of the VK locus. TsF was prepared in strains of mice with known Igh and VK genes. Mice carrying Igh-1e genes produced TsF that was retained by an immunoadsorbent containing bound anti-idiotype antibodies; the genetic constitution at the VK locus was not relevant. By using a panel of anti-idiotypic antibodies prepared against the antigen-binding site of antibody carrying the idiotype, against determinants outside the site or against VH structures, we determined that idiotypic structures on the TsF are associated with V-region elements associated with the H chain and not with the binding site formed by a VH-BL combination.
Effects of Lyt antibodies on T-cell functions: augmentation by anti-Lyt-1 as opposed to inhibition by anti-Lyt-2.
Authors: Hollander N , Pillemer E , Weissman I L
Published: May 2006

Monoclonal anti-Lyt-1 (alpha Lyt-1) and alpha Lyt-2 manifest inverse effects on allogeneic mixed lymphocyte reactions when added to the reaction mixtures without complement. alpha Lyt-1 augments cell proliferation and generation of cytotoxic cells but has no effect on cell-mediated cytolysis, whereas alpha Lyt-2 blocks cell proliferation, generation of killer cells, and cytolytic activity of killer cells. The augmenting effect of alpha Lyt-1 cannot be attributed to a direct mitogenic effect on T cells. Both inhibition by alpha Lyt-2 and potentiated by alpha Lyt-1 require interaction of responder cells with the antibodies during the first 24 hr of the mixed lymphocyte reaction, indicating that early stages of the reaction are sensitive to Lyt antibodies. The enhancing effect of alpha Lyt-1 on alloantigen-induced T-cell proliferation is associated with augmented production of T-cell growth factors. When alpha Lyt-1 is present in mixed lymphocyte cultures, the supernatant media collected after 24 or 48 hr of culture induce higher proliferation of activated T cells compared to media of mixed lymphocyte cultures incubated in the absence of antibodies or in the presence of alpha Lyt-2 which has no effect on secretion of growth factors. The differences in the effects of alpha Lyt-1 and alpha Lyt-2 could not be attributed to differences in heavy chain constant region functions because both were of the same lambda 2A immunoglobulin class and were used at the same concentration. The data suggest a possible role for Lyt-1 molecules in early activation and mitogenesis processes such as production of growth factors.
Translation of tyrosine hydroxylase from poly(A)-mRNA in pheochromocytoma cells is enhanced by dexamethasone.
Authors: Baetge E E , Kaplan B B , Reis D J , Joh T H
Published: May 2006

Polysomal poly(A)-mRNA was purified from a clonal cell line of rat pheochromocytoma (PC 12) and translated in a reticulocyte cell-free protein-synthesizing system. Tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahyropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] was isolated from other protein by immunoprecipitation and NaDodSO4/polyacrylamide gel electrophoresis. The molecular weight and relative proportion of tyrosine hydroxylase to other proteins synthesized in vitro were identical to those of the enzyme synthesized in vivo in cultured cells. Incubation of PC 12 cells with 10 microM dexamethasone increased the activity and amount of tyrosine hydroxylase 2.5-fold. The ratio of tyrosine hydroxylase to total protein translated from poly(A)-mRNA isolated from cells treated with dexamethasone was 2.5 times higher than the ratio of enzyme to total protein translated from an equal amount of poly(A)-mRNA from untreated cells. The dexamethasone-elicited induction of tyrosine hydroxylase in PC 12 cells therefore is a result of an increased "relative" amount or activity of tyrosine hydroxylase mRNA.
Lysosomotropic amines inhibit mitogenesis induced by growth factors.
Authors: King A C , Hernaez-Davis L , Cuatrecasas P
Published: May 2006

The stimulation of DNA synthesis by epidermal growth factor, insulin, and serum is inhibited by a variety of alkylamines when present for the duration of the stimulatory preincubation (20-24 hr). These results contradict an earlier report [Maxfield, F. R., Davies, P. J. A., Klempner, L., Willingham, M. C. & Pastan, I. (1979) Proc. Natl. Acad. Sci. USA 76, 5731-5735] and can be explained by differences in incubation conditions. The most straightforward interpretation of our results is that the mitogenic activities of growth factors are blocked by agents that inhibit the intracellular processing of hormone-receptor complexes. Therefore, the continued internalization and degradation of growth factors or their receptors within cells may play an important role in inducing mitogenesis in cultured human fibroblasts and may explain the prolonged requirement for epidermal growth factor in the culture medium (8 hr) to elicit a mitogenic response. We also found that bacitracin, a potent inhibitor of the enzyme transglutaminase, neither prevents receptor internalization or degradation in human fibroblasts nor inhibits the mitogenic activity of epidermal growth factor. These results suggest that transglutaminase activity may not be relevant to the mechanisms of growth-factor-induced receptor internalization or mitogenesis.
Mechanism of the actomyosin ATPase: effect of actin on the ATP hydrolysis step.
Authors: Stein L A , Chock P B , Eisenberg E
Published: May 2006

The Lymn-Taylor model for the actomyosin ATPase suggests that during each cycle of ATP hydrolysis the complex of myosin subfragment 1 (S-1) with actin must dissociate into S-1.ATP plus actin before ATP hydrolysis can occur. In the present study we tested whether such a mandatory detachment step occurs by measuring the effect of actin on the rate and magnitude of the ATP hydrolysis step (initial Pi burst) and on the steady-state ATPase rate. We find that the rate of the initial Pi burst markedly increases at high actin concentration although the Lymn-Taylor model predicts the rate should remain nearly constant or decrease. In addition, at high actin concentration, the magnitude of the initial Pi burst is much larger than is predicted by the Lymn-Taylor model. Finally, at 360 microM actin, at which more than 90% of the S-1.ATP is bound to actin, there is no inhibition of the steady-state ATPase activity although the Lymn-Taylor model predicts that 70% inhibition should occur. We conclude that the acto-S-1 complex is not dissociated by ATP during each cycle of ATP hydrolysis; in fact, the rate of the initial Pi burst appears to be even faster when S-1.ATP is bound to actin than when it is dissociated.
Nitroaniline diamine.poly(dA-dT) complexes: 1H and 19F NMR parameters for full intercalation of aromatic rings into DNA.
Authors: Patel D J , Gabbay E J
Published: May 2006

High-resolution proton, fluorine, and phosphorus NMR studies have been undertaken on complexes of methyl- and trifluoromethyl-substituted nitroaniline diamines with the synthetic DNA poly(dA-dT) in 10 mM buffer solution. We demonstrate full intercalation of the nitroaniline group of these reporter molecules between base pairs, based on large upfield proton shifts (1.3-1.7 ppm) at all four aromatic proton markers on complex formation. The temperature and pH dependences of the thymidine H-3 Watson-Crick proton chemical shift and line width require the formation of intact and stable base pairs in this intercalative complex in solution. The 19F chemical shift of the trifluoromethyl-labeled nitroaniline diamine shifts downfield by approximately 2 ppm on formation of the synthetic DNA complex and most likely reflects the nonpolar environment of the aromatic ring when sandwiched between base pairs. A sequence specificity in the binding of the nitroaniline dication to poly(dA-dT) is implied by the observation of two partially resolved 31P resonances with the phosphodiester at the intercalation site shifting downfield by approximately 0.4 ppm on complex formation.
Preferential replication of repeated DNA sequences in nuclei isolated from soybean cells grown in suspension culture.
Authors: Caboche M , Lark K G
Published: May 2006

Nuclei isolated from suspension cultures of soybean cells are able to replicate DNA in vitro for short periods of time. The reaction is not blocked by dideoxythymidine triphosphate, suggesting that DNA polymerase alpha is involved. Reassociation kinetics, restriction analysis, and isopycnic centrifugation of the DNA synthesized in vitro show that repeated sequences are preferentially replicated in isolated nuclei. However, some repeated sequences are not replicated in vitro, including those coding for ribosomal RNA. The addition of cytoplasmic or yeast extracts clearly stimulates DNA replication in vitro but does not improve the ability of nuclei to replicate single copy DNA. Incubation of cells with cytokinins prior to the isolation of nuclei modifies the pattern of replication of repeated sequences in vitro. These experiments suggest that the replication of single copy and repeated nuclear sequences have different requirements and may be controlled in a different way in plant cells.
Enzyme-linked sandwich immunoassay for insulin using laser fluorimetric detection.
Authors: Lidofsky S D , Hinsberg W D , Zare R N
Published: May 2006

Human serum samples are assayed for insulin by an enzyme-linked sandwich immunoassay. Horseradish peroxidase is used as an enzyme label for antibody, and enzyme activity is measured by means of the fluorogenic substrate, p-hydroxyphenylacetic acid. The product is detected by excitation of fluorescence with the 325-nm line of a continuous-wave helium/cadmium ion laser on line with reverse-phase high-pressure liquid chromatography. The incubation period is 90 min and the limit of detection of insulin is 30 pM, corresponding to 5 microunits/ml. This method correlates highly with radioimmunoassay, with coefficient of correlation r = 0.95.