bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA transcripts for I-J polypeptides are apparently not encoded between the I-A and I-E subregions of the murine major histocompatibility complex.
Authors: Kronenberg M , Steinmetz M , Kobori J , Kraig E , Kapp J A , Pierce C W , Sorensen C M , Suzuki G , Tada T , Hood L
Published: May 2006

The I-J subregion of the mouse major histocompatibility complex has been reported to encode antigenic determinants expressed by suppressor T cells. Previously, cosmid clones were obtained from mouse sperm DNA that contain all of the sequences between the I-A and I-E subregions, where I-J has been mapped genetically. However, hybridization of these sequences to RNA prepared from several I-J-positive suppressor T-cell hybridomas did not reveal the presence of a transcript. In addition, no rearrangements in this DNA were detected in the suppressor T cells that we have analyzed. Our results indicate that the I-J polypeptides are not encoded between the I-A and I-E subregions of the major histocompatibility complex. We discuss several hypotheses concerning the possible location and expression of I-J genes.
Crosslinking of the receptors for immunoglobulin E depolarizes the plasma membrane of rat basophilic leukemia cells.
Authors: Kanner B I , Metzger H
Published: May 2006

Aggregation of the receptor for IgE on mast cells, basophils, and a tumor analog, rat basophilic leukemia (RBL) cells, induces a calcium-dependent degranulation of the cells. We have measured the membrane potential (delta psi) of RBL cells during this reaction by using the tetraphenylphosphonium ion (Ph4P+) equilibration technique. We observed a 20-45% reduction in ionophore-sensitive Ph4P+ accumulation. The phenomenon persisted under conditions expected to collapse the mitochondrial membrane potential, consistent with the effect being due to a change in delta psi of the plasma membrane. We estimated that the change reflects a depolarization of 20 mV (from -90 to -70 mV, interior negative). Whereas degranulation fails to occur in the absence of external Ca2+, this was not true of the depolarization, indicating that the latter was not a consequence of secretion. When aggregation of the receptor is induced by reaction of the cell-bound IgE with a multivalent antigen, the secretory reaction can be halted by adding a univalent hapten. In this case, complete repolarization occurs. Equivalent depolarization was observed in the absence of Na+ but was diminished when both Ca2+ and Na+ were absent. Together, the data suggest that aggregation of the receptor opens ion channels and that the latter disappear promptly when the receptors are disaggregated. It is plausible that formation of these channels leads to the entry of Ca2+ and is an early and critical consequence of the aggregation of the receptors, thereby leading to degranulation.
Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon.
Authors: Bitoun R , Berman J , Zilberstein A , Holland D , Cohen J B , Givol D , Zamir A
Published: May 2006

The nifHDKY operon of Klebsiella pneumoniae encodes for structural polypeptides of nitrogenase and requires the nifA gene product for transcription. Mutations that allow transcription of the nifHDKY operon in absence of the nifA gene product were characterized in plasmids containing the regulatory region of nifHDKY and nifH fused in phase to lacZ. beta-Galactosidase activity served as a measure for nifH expression. Most mutations were located in the nif regulatory region and included insertion sequence 2 (IS2) insertions, a sequence duplication, and a base substitution. In Escherichia coli, beta-galactosidase activity expressed from the mutant plasmids in the absence of nifA was 6-30% of the nifA-activated, parental level. Expression from most mutant plasmids was further increased by nifA. In K. pneumoniae, IS2-containing plasmids expressed low levels of beta-galactosidase and responded poorly, if at all, to activation by nifA, whereas expression from other mutant types was similar to that observed in E. coli. Nucleotide sequence analysis of two mutants indicated that sequences within 41 base pairs upstream to the nifH coding sequence were involved in nif-specific regulation. The results suggest that an inverted repeat element in this region, which could theoretically form a cruciform structure in the DNA, is involved in the transcriptional control of the nifHDKY operon.
Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication.
Authors: Fuller R S , Kornberg A
Published: May 2006

Soluble protein fractions from Escherichia coli dnaA+ cells but not dnaA temperature-sensitive cells replicate plasmids containing the E. coli chromosomal origin of replication (oriC). Complementation of these mutant fractions provided an assay for dnaA protein activity in initiation of replication at oriC. From a strain (constructed in vitro) that overproduces the dnaA protein more than 200-fold, the 52,000-dalton polypeptide was purified to near homogeneity. Although the protein tends to aggregate, monomer-sized protein purified by high-performance liquid chromatography is fully active for replication. It binds specifically and tightly to oriC in a supercoiled plasmid as judged by a Millipore filter-binding assay and by protection of the unique HindIII site within the oriC sequence. In the oriC replication reaction, dnaA protein acts at an early step preceding DNA synthesis.
Ontophyletics of the nervous system: development of the corpus callosum and evolution of axon tracts.
Authors: Katz M J , Lasek R J , Silver J
Published: May 2006

The evolution of nervous systems has included significant changes in the axon tracts of the central nervous system. These evolutionary changes required changes in axonal growth in embryos. During development, many axons reach their targets by following guidance cues that are organized as pathways in the embryonic substrate, and the overall pattern of the major axon tracts in the adult can be traced back to the fundamental pattern of such substrate pathways. Embryological and comparative anatomical studies suggest that most axon tracts, such as the anterior commissure, have evolved by the modified use of preexisting substrate pathways. On the other hand, recent developmental studies suggest that a few entirely new substrate pathways have arisen during evolution; these apparently provided opportunities for the formation of completely new axon tracts. The corpus callosum, which is found only in placental mammals, may be such a truly new axon tract. We propose that the evolution of the corpus callosum is founded on the emergence of a new preaxonal substrate pathway, the "glial sling," which bridges the two halves of the embryonic forebrain only in placental mammals.
High-frequency structural gene deletion as the basis for functional hemizygosity of the adenine phosphoribosyltransferase locus in Chinese hamster ovary cells.
Authors: Adair G M , Stallings R L , Nairn R S , Siciliano M J
Published: May 2006

The CHO-AT3-2 Chinese hamster ovary cell line is functionally hemizygous for the adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) locus. Class 1 APRT +/- heterozygotes, such as CHO-AT3-2, can be isolated at high spontaneous frequencies from wild-type CHO cell populations. Simon et al. [Simon, A. E., Taylor, M. W., Bradley, W. E. C. & Thompson, L. (1982) Mol. Cell. Biol. 2, 1126-1133] have proposed that a high-frequency event that inactivates one APRT allele might be responsible for both the spontaneous generation of class 1 APRT +/- heterozygotes and the high-frequency occurrence of APRT- mutants in class 2 APRT +/- heterozygote populations. This event appears to occur at only one of the two APRT alleles. To investigate the nature of this high-frequency event, and to determine the genetic basis for functional hemizygosity of the APRT locus in CHO-AT3-2 cells, we have mapped the APRT locus by using CHO-AT3-2-mouse somatic cell hybrids. Our data confirm that CHO-AT3-2 cells have a single functional APRT allele, which is located on the Z7 chromosome. Karyotypic analysis of CHO-AT3-2 revealed an interstitial deletion on the long arm of the Z4 chromosome, in the very region where the other APRT allele should be located. To determine whether the Z4q interstitial deletion had resulted in physical loss of the APRT gene, DNA from CHO-AT3-2-mouse cell hybrids that had either lost or retained the Z4q- chromosome was analyzed for the presence of CHO APRT coding sequences. Our data suggest that allele-specific high-frequency structural gene deletion events involving the long arm of chromosome Z4 are responsible for the spontaneous generation of functional hemizygosity at the APRT locus in CHO cells.
Changes in surface antigens of immature thymocytes under the influence of T-cell growth factor and thymic factors.
Authors: Chen S S , Tung J S , Gillis S , Good R A , Hadden J W
Published: May 2006

Peanut agglutinin-positive thymocytes, peanut agglutinin-negative thymocytes, cortisone-resistant thymocytes, and unfractionated thymocytes were prepared from congeneic C57BL/6 Tla mice. By using surface iodination and immunoprecipitation of solubilized antigen with specific antisera (e.g., anti-H-2D, anti-TL, anti-Qa2/3, and anti-gp70), the released specific antigens were electrophoresed on polyacrylamide gels, and their radioactivity was measured. The relative percentages of surface antigens H-2D, TL, Qa2/3, and gp70 were 3.2%, 47.5%, 2.5%, and 46.8%, respectively, for peanut agglutinin-positive thymocytes; 31.8%, 4.4%, 32.7%, and 31.1%, respectively, for cortisone-resistant thymocytes; 13.2%, 28.7%, 12.3%, and 45.8%, respectively, for peanut agglutinin-negative thymocytes; and 7.7%, 27.1%, 4.3%, and 60.9%, respectively, for unfractionated thymocytes. After incubation with thymosin fraction V or T-cell growth factor (interleukin II) for 20 hr, the changes in surface antigens of peanut agglutinin-positive thymocytes closely correlated with their normal maturation (i.e., H-2D increases and TL decreases). Thymic factors (e.g., thymosin alpha 1, thymopoietin pentapeptide, facteur thymic serique) had only small or no effects on surface antigens of peanut agglutinin-positive thymocytes. The results suggest that peptides yet to be identified in thymosin fraction V may play an important role in intrathymic evolution and that T-cell growth factor is possibly a peripheral signal derived from activated T cells that modulates T-cell receptors and may be a critical regulator of intrathymic cellular development.
T cells discriminate between Ia antigens expressed on allogeneic accessory cells and B cells: a potential function for carbohydrate side chains on Ia molecules.
Authors: Cowing C , Chapdelaine J M
Published: May 2006

Previous studies have shown that the peptides obtained from accessory cell and B-cell Ia molecules are identical but that the alpha chains of B-cell Ia molecules are more extensively sialylated than those of accessory cells. The present studies were designed to determine whether this glycosylation difference can account for the functional difference in the capacity of the two cell types to activate alloreactive T cells. The experimental data show that normal resting B cells lack the capacity to induce DNA synthesis or differentiation in alloreactive T cells. T cells do recognize polymorphisms in B-cell Ia molecules, however, because they can be specifically primed for a subsequent proliferative stimulus of the same haplotype. The mitogenic signal for T cells is delivered by either allogeneic accessory cells or neuraminidase-treated B cells. Therefore, the T-cell receptor(s) may contain a site specific for the nonpolymorphic asialocarbohydrate moiety on the alpha chains of accessory cell Ia molecules.
Juvenile and adult metachromatic leukodystrophy: partial restoration of arylsulfatase A (cerebroside sulfatase) activity by inhibitors of thiol proteinases.
Authors: von Figura K , Steckel F , Hasilik A
Published: May 2006

Arylsulfatase A polypeptides were examined in cultured fibroblasts from a patient with juvenile metachromatic leukodystrophy and three patients with the adult form of the disease, with the aid of metabolic labeling and immunoprecipitation. The mutant cells were severely deficient in the arylsulfatase polypeptides. The apparent rate of synthesis, however, as estimated from the secretion of polypeptides or activity by cells incubated in the presence of 10 mM NH4Cl was 20-50% of control. In the absence of NH4Cl, the mutant enzyme was rapidly degraded upon transport into lysosomes. In the presence of inhibitors of thiol proteinases arylsulfatase A polypeptides were partially protected from degradation, and the catalytic activity of arylsulfatase A was increased. In addition, the treatment partially corrected the capacity of the cells to degrade cerebroside sulfates. Inhibitors of thiol proteinases may be of therapeutic value in variants of metachromatic leukodystrophy, in which an unstable arylsulfatase A is synthesized.
Consequences of UMP synthase deficiency in cattle.
Authors: Robinson J L , Drabik M R , Dombrowski D B , Clark J H
Published: May 2006

Several dairy cows have been identified as partially deficient in UMP synthase. Although erythrocytes of normal cows contained 2.54 units of enzyme per ml, four cows were discovered with only 1.08 units per ml. Cows deficient in UMP synthase secreted milk with abnormally high levels of orotate, 300-1,000 micrograms of orotate per ml compared to 80 micrograms/ml for normal cows. The deficiency also was accompanied by a lactation-induced orotic aciduria. Although bovine urinary orotate was generally less than 10 micrograms/ml, the urine of the deficient cows, when lactating, contained 20-200 micrograms/ml. Their plasma orotate also was elevated. Genetic transmission of the condition was suggested by a common bull in the pedigrees of all deficient animals. Indeed, these cows, with half the normal level of UMP synthase, are probably heterozygotes with a 50% chance of passing the deficient allele to their progeny. For these putative heterozygotes, the condition is apparently benign because longevity and production were unaffected. However, the existence of a gene for UMP synthase deficiency in the dairy cow population poses a hazard with respect to the conception of homozygotic, deficient animals. These, in analogy with a comparable human condition, would be expected to exhibit high perinatal morbidity and mortality.
Linewidth of single laser-cooled
24
Mg
+
ion in radiofrequency trap
Authors: Nagourney W. , Janik G. , Dehmelt H.
Published: May 2006

A single Mg
+
has been successfully trapped and cooled in a small radiofrequency trap. The ion was cooled by using the radiation from a single-frequency ring dye laser whose output was doubled in frequency with an NH
4
2
H
2
PO
4
temperature phasematched crystal; a power of about 22 μW or less was sufficient for all of the experiments. The ion temperature was estimated by a computer fit of the experimental resonance line profile; the resulting linewidth was compared to previously published Mg
+
3
2
P
3/2
Hanle-effect linewidths. The result is a temperature of 5
-5
+15
mK, which is lower than that attained previously.
Characterization of monoclonal antibodies against mitochondrial F1-ATPase.
Authors: Moradi-Ameli M , Godinot C
Published: May 2006

Four stable murine hybridoma clones producing homogeneous antibodies against pig heart mitochondrial F1-ATPase have been established. All antibodies exhibit specific binding to F1-ATPase. Three of them interact with the beta subunit and one binds strongly to the alpha and barely to the beta subunit. All of them exhibit linear Scatchard plots and high binding affinities, with dissociation constants between 4.7 X 10(-8) M and 6.5 X 10(-10) M. The minimal number of moles of IgG bound at saturation per mole of immobilized F1-ATPase is 2.2 for the anti-alpha-subunit antibody and 2.5 for one anti-beta-subunit antibody. This suggests that more than two copies of the alpha and beta subunits are present in the enzyme. Two antibodies seem to recognize F1-ATPase equally before or after denaturation with sodium dodecyl sulfate. The two other antibodies exhibited a much higher affinity for the nondissociated enzyme, indicating that they are very sensitive to a specific conformation of the enzyme.
Pig brain contains cholecystokinin octapeptide and several cholecystokinin desoctapeptides.
Authors: Eng J , Shiina Y , Pan Y C , Blacher R , Chang M , Stein S , Yalow R S
Published: May 2006

A sequential method employing methanol extraction of the COOH-terminal fragment of cholecystokinin (CCK) from pig brain followed by HCl extraction of the more basic CCK peptides was used as the first step in purification of these peptides. Recovery was monitored with two different assays, one directed to the COOH terminus of CCK and the other to the NH2 terminus. The amino acid content and sequence were determined for each of five peptides after purification. The only peptide containing COOH-terminal immunoreactivity was CCK-octapeptide (CCK8). The other four peptides did not contain CCK8 and had lost one or two additional amino acids, perhaps as a consequence of the action of carboxypeptidases. These peptides were shown to be CCK33-desnonapeptide, CCK39-desnonapeptide and -desdecapeptide, and a large molecular weight precursor, CCK58-desnonapeptide, containing 19 amino acids (Ala-Val-Gln-Lys-Val-Asp-Gly-Glu-Ser-Arg-Ala-His-Leu-Gly-Ala-Leu-Leu-Ala-Arg) NH2-terminal to CCK39. The three NH2-terminal fragments of CCK58, CCK39, and CCK33 were about equally prominent. The brain, unlike the gut, appears to cleave CCK8 rapidly from a precursor peptide but to process the NH2-terminal portions of the molecule more slowly and incompletely.
Promotion of lipolysis in rat adipocytes by pertussis toxin: reversal of endogenous inhibition.
Authors: Olansky L , Myers G A , Pohl S L , Hewlett E L
Published: May 2006

Pertussis toxin (PT), a protein produced by Bordetella pertussis, was studied for its effect on lipolysis in isolated rat epididymal adipocytes. Exposure of adipocytes to pertussis toxin resulted in a significant increase in cyclic AMP levels and lipolysis after a lag of 1-2 hr. Both the maximal rate of lipolysis and the time lag (beyond 1 hr) were PT concentration-dependent. Heat treatment (95 degrees C, 30 min) or incubation with specific antibody directed against PT eliminated the ability of toxin to increase lipolysis. Cell-free culture medium from B. pertussis, but not from nontoxigenic Bordetella species, had the same effect on lipolysis as purified toxin. Comparison of the PT effect with the known lipolytic effect of cholera toxin (CT) revealed that the two toxins elicited responses that were indistinguishable in time course and magnitude. In contrast, the adenylate cyclase (EC 4.6.1.1) activities in membranes prepared from PT- or CT-treated adipocytes were different. Adenylate cyclase activity in membranes from control (untreated) adipocytes was inhibited 35-64% by the adenosine analogue N6-(L-2-phenylisopropyl)-adenosine. As expected from previous studies, membranes from CT-treated adipocytes demonstrated an increased basal activity but showed the same proportional inhibition by N6-(L-2-phenylisopropyl)-adenosine as controls. On the other hand, membranes from adipocytes exposed to PT (400 ng/ml for 4 hr) showed no increase in basal adenylate cyclase activity but had reduced sensitivity to N6-(L-2-phenylisopropyl)-adenosine inhibition, with the maximal effect ranging from 11 to 30% at 10(-6) M N6-(L-2-phenylisopropyl)-adenosine. These data support the hypothesis that PT promotes cyclic AMP-dependent lipolysis in a manner quantitatively equivalent to CT, but by a different mechanism involving increased cyclic AMP levels resulting from loss of responsiveness to endogenous inhibitors such as adenosine.
Harmonic dynamics of proteins: normal modes and fluctuations in bovine pancreatic trypsin inhibitor.
Authors: Brooks B , Karplus M
Published: May 2006

A normal mode analysis making use of an empirical potential function including local and nonlocal (nonbonded) interactions is performed for the bovine pancreatic trypsin inhibitor in the full conformational space of the molecule (1,740 degrees of freedom); that is, all bond lengths and angles, as well as dihedral angles, are included for the 580-atom system consisting of all heavy atoms and polar hydrogens. The heavy-atom frequency spectrum shows a dense distribution between 3 and 1,800 cm-1, with 350 modes below 216 cm-1. Most of the low-frequency modes, of which many have significant anharmonic character, are found to be delocalized over the protein. The root-mean-square amplitudes of the atomic fluctuations are calculated at 300 K from the normal modes and compared with those obtained from a solution molecular dynamics simulation based on the same potential function; very good agreement is obtained for the variation in the main-chain fluctuations as a function of residue number, though larger differences occur for the side chains. The fluctuations are generally, though not always, dominated by frequencies below 30 cm-1, in accord with the results of the dynamics simulation. The vibrational contributions to the thermodynamic properties of the protein are calculated as a function of temperature; the effects of perturbations on the spectrum, suggested for ligand or substrate binding, are examined. The analysis demonstrates that, in spite of the anharmonic contributions to the potential, a normal mode description can provide useful results concerning the internal motions of proteins.
Consequences of cell membrane attack by complement: release of arachidonate and formation of inflammatory derivatives.
Authors: Imagawa D K , Osifchin N E , Paznekas W A , Shin M L , Mayer M M
Published: May 2006

Treatment of [3H]arachidonic acid [( 3H]C20:4)-labeled and antibody-sensitized Ehrlich ascites tumor cells with guinea pig or rabbit serum complement (C) released up to about 20 or 25% of the incorporated [3H]C20:4 into the aqueous phase as a consequence of C-induced hydrolysis of cellular phospholipid. The dose-response curve of release of [3H]C20:4 from Ehrlich ascites tumor cells, with respect to C, was approximately in the same range as the cytolytic response. In the case of [3H]C20:4-labeled and antibody-sensitized peritoneal mouse macrophages, treatment with C induced release of about 11% of the incorporated 3H as C20:4 and about 6% as prostaglandins, thromboxane B2, and hydroxyicosatetraenoic acids. C6- and C8-deficient rabbit and human sera, respectively, induced release of small amounts of [3H]C20:4 from Ehrlich ascites tumor cells and macrophages; these deficient sera also released traces of oxygenated derivatives from macrophages. Addition of purified C6 or C8 effectively restored release from both cell types, indicating that the terminal C proteins, up to and including C8, are required for the major part of the release. Our results do not rule out a possible requirement for C9.
Differential agglutination by soybean agglutinin of human leukemia and neuroblastoma cell lines: potential application to autologous bone marrow transplantation.
Authors: Reisner Y
Published: May 2006

Normal human bone marrow cells were mixed with radioactively labeled tumor cells from different leukemia and neuroblastoma cell lines, and the cell mixtures were separated by differential agglutination with soybean agglutinin. It is shown that the cell fraction unagglutinated by soybean agglutinin, which was previously found to be capable of reconstituting the hematopoietic system of lethally irradiated recipients, can be purged of tumor cells with varying efficiency depending on the tumor cell expression of soybean agglutinin receptors as detected by flow cytofluorimetry with fluoresceinated soybean agglutinin.
Naloxone-inaccessible sigma receptor in rat central nervous system.
Authors: Tam S W
Published: May 2006

It has been postulated that the psychotomimetic effects of opiates of the benzomorphan series are due to their activity at the sigma receptor. Therefore, the binding of (+/-)-[3H]ethylketocyclazocine ( [3H]EKC), a benzomorphan, to synaptosomal membranes of rat central nervous tissue was studied. Surprisingly, high concentrations of naloxone, a mu, delta, and kappa receptor antagonist, only inhibited about 80% of the specifically bound [3H]EKC in the spinal cord. This suggested that the remaining 20% of the binding sites were not mu, delta, or kappa. The Scatchard plot of the binding of [3H]EKC was nonlinear but became linear in the presence of naloxone (1 microM), suggesting a single class of naloxone-inaccessible receptor sites. This biochemically readily distinguishable receptor type bound the dextrorotatory isomer of EKC stereoselectively. The sigma agonist N-allylnormetazocine [(+)-SKF 10,047] stereoselectively competed with the binding of [3H]EKC to this naloxone-inaccessible binding site. A number of opiates that have psychotomimetic activity also competed for binding to this binding site. This binding site is designated as sigma binding site according to the nomenclature originally suggested by Martin et al. [Martin, W. R., Eades, C. G., Thompson, J. A., Huppler, R. E. & Gilbert, P. E. (1976) J. Pharmacol. Exp. Ther. 197, 517-532]. The drug selectivity and regional distribution of this sigma binding site in the rat central nervous system are different from that of the mu and delta opioid receptors and phencyclidine receptors. The concentration of the sigma binding site is highest in the spinal cord, pons and medulla, and cerebellum.
Correlation between rate constant for reduction and redox potential as a basis for systematic investigation of reaction mechanisms of electron transfer proteins.
Authors: Meyer T E , Przysiecki C T , Watkins J A , Bhattacharyya A , Simondsen R P , Cusanovich M A , Tollin G
Published: May 2006

Rate constants for the reduction of approximately 40 electron transfer proteins by photoreduced flavins have been determined by laser flash photolysis techniques. The data for a series of 12 homologous cytochromes and 10 homologous high redox potential ferredoxins (HiPIPs) are in excellent agreement with semi-empirical equations relating rate constant and thermodynamic redox potential that have proven applicable to nonbiological electron transfer systems. These correlations allow the establishment of relative reactivities within structurally homologous classes of biological oxidation-reduction proteins, including cytochromes and HiPIPs, and a variety of nonhomologous heme-, iron-sulfur-, copper-, and flavin-containing proteins. A qualitative correspondence is shown to exist between such relative reactivity and the extent of solvent exposure of the redox centers in a particular structural class. The implications of these results are considered, and it is concluded that free energy relationships provide a sound basis for systematic analysis of reaction mechanisms of electron transfer proteins.
Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta.
Authors: Greenfield L , Bjorn M J , Horn G , Fong D , Buck G A , Collier R J , Kaplan D A
Published: May 2006

A 1,942-base-pair DNA segment encoding the structural gene for diphtheria toxin was sequenced, and the primary structure of the toxin was deduced. Restriction enzyme fragments corresponding to nontoxic or hypotoxic peptides of the toxin were isolated from corynebacteriophage beta and cloned into Escherichia coli on plasmid pBR322, and the sequence was determined. The mature toxin molecule deduced from the sequence has 535 amino acid residues and a molecular weight of 58,342. The deduced sequence for the fragment A moiety was the same as that determined at the protein level, except for a single serine residue, which had been mispositioned in the earlier study. Several differences were noted with respect to the partial sequence data available on the fragment B moiety, some or all of which may reflect genetic variations among populations of corynephages carrying the toxin gene. The DNA sequence predicts a 25-residue leader peptide preceding the mature protein, which is presumably involved in secretion of the toxin from lysogenized Corynebacterium diphtheriae. We infer that initiation of translation probably occurs at a GTG codon (codon -25). Cloned restriction fragments containing sequences for the amino-terminal region of toxin, together with 5' flanking regions, were expressed in E. coli. Toxin-related peptides were synthesized and secreted into the periplasmic space. These results provide a basis for applying recombinant DNA methods to the study of diphtheria toxin and for producing novel, genetically altered forms of the toxin suited to the construction of new classes of immunotoxins.
Surface properties of an amphiphilic peptide hormone and of its analog: corticotropin-releasing factor and sauvagine.
Authors: Lau S H , Rivier J , Vale W , Kaiser E T , Kézdy F J
Published: May 2006

Synthetic corticotropin (adrenocorticotropic hormone)-releasing factor [CRF; for the sequence, see Vale, W., Spiess, J., Rivier, C. & Rivier, J. (1981) Science 213, 1394-1397] in aqueous solution exists predominantly as a random coil. At concentrations greater than 1 microM, the peptide shows a tendency to self-aggregate with a concurrent slight increase in the apparent alpha-helical content as measured by the CD spectrum. The alpha-helix formed by this molecule is highly amphiphilic--i.e., the hydrophilic and hydrophobic regions are segregated on opposite faces of the helix. As predicted from the potential amphiphilic structure, CRF binds avidly to the surface of single bilayer egg phosphatidylcholine vesicles. This binding appears to obey a simple Langmuir isotherm with the following parameters: Kd = 1.3 +/- 0.6 X 10(-7) M and capacity at saturation (N) = 11.0 +/- 1.0 mmol of peptide per mol of phospholipid. CRF also readily forms an insoluble monolayer at the air-water interface. The monolayer is composed of monomers of the hormone with molecular areas, A'0 = 22 A2 per amino acid, suggesting a compact secondary structure. Judged from the collapse pressure (19.0 +/- 0.1 dyne/cm; 1 dyne = 10 microN) of the monolayer, the amphiphilicity of CRF approximates that of plasma apolipoproteins, a class of proteins of the most pronounced amphiphilic character. These results suggest that the binding of CRF to the cell membrane is accompanied by the induction of an alpha-helical secondary structure and it is this predominantly helical form that is the biologically active form of the peptide.
Yeast plasmid 2-micron circle promotes recombination within bacterial transposon Tn5.
Authors: Jayaram M , Broach J R
Published: May 2006

The site-specific recombination system (FLP) encoded by the yeast plasmid 2-micron circle can also act in yeast on the inverted repeats of the bacterial transposon Tn5. The efficiency of this recombination is dependent on the location of Tn5 within the 2-micron circle genome but can be as high as that observed for 2-micron circle itself. Comparison of the DNA sequences between the Tn5 repeat and the 2-micron circle recombination region reveals certain strikingly similar structural features that might be important in the recombination reaction.
Construction and testing of mouse--human heteromyelomas for human monoclonal antibody production.
Authors: Teng N N , Lam K S , Calvo Riera F , Kaplan H S
Published: May 2006

FU-266, a mutant human myeloma cell line sensitive to hypoxanthine/aminopterin/thymidine (HAT), was transfected by protoplast fusion with DNA of the recombinant plasmid vector pSV2-neoR, thus acquiring a dominant marker conferring resistance to the antibiotic G-418. One of the resultant neoR clones, E-1, was fused to irradiated (500 rads) or unirradiated cells of the HAT-sensitive, G-418-sensitive, nonproducer mouse myeloma line X63-Ag8.653. Hybrid clones were selected in G-418 plus ouabain, thus preserving their HAT sensitivity. Small numbers of human chromosomes were retained in all such hybrids, but most of them ceased secreting human myeloma (IgE(lambda). Selected hybrid clones were then tested as malignant fusion partners in a series of fusions with polyclonally activated human B lymphocytes and with antigen-primed human B lymphocytes, in some instances after transformation of the latter with Epstein-Barr virus. The yield of viable chimeric hybridomas has been consistently high, as has the proportion of hybridomas secreting human immunoglobulin molecules unpermuted with mouse or human myeloma heavy or light chains. Secretion by many subcloned hybridomas has been stable for over 6 months, and several antigen-specific human monoclonal antibodies have been generated. Thus these heteromyeloma cell lines appear to have significant advantages for human monoclonal antibody production.
Cloning and sequence analysis of cDNA for human renin precursor.
Authors: Imai T , Miyazaki H , Hirose S , Hori H , Hayashi T , Kageyama R , Ohkubo H , Nakanishi S , Murakami K
Published: May 2006

The primary structure of human renin precursor has been deduced from its cDNA sequence. A library of cDNA clones was constructed from human kidney poly(A)+ RNA by applying the vector/primer method of Okayama and Berg. The library was screened for human renin sequences by hybridization with the previously cloned mouse renin cDNA. Of the 240,000 colonies screened, 35 colonies that were positive for hybridization were isolated. Two recombinant plasmids containing long inserts of about 1,300 and 1,600 base pairs were selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the human renin precursor consists of 406 amino acids with a pre and a pro segment carrying 20 and 46 amino acids, respectively. A high degree of sequence homology was found upon comparison of the mouse and human renins. Close similarities were also observed in the primary structures of renin and aspartyl proteinases that have known three-dimensional structures, suggesting a similar tertiary structure for renin.
Use of Monte Carlo calculations in the study of microtubule subunit kinetics.
Authors: Chen Y , Hill T L
Published: May 2006

GTP-tubulin forms a cap on microtubule ends during aggregation. The bulk of the microtubule is GDP-tubulin. This complicates the usual simple kinetic theory of subunit exchange at microtubule ends to such an extent that Monte Carlo calculations are needed to handle the complications, except in special cases. The Monte Carlo method is introduced here, for this problem, and illustrated with steady-state and transient examples. Monte Carlo transients are needed to simulate dilution experiments. Preliminary results (with M. F. Carlier) have been obtained applying these theoretical procedures to experimental data.
Effects of hypolipidemic therapy on cholesterol homeostasis in freshly isolated mononuclear cells from patients with heterozygous familial hypercholesterolemia.
Authors: Sundberg E E , Illingworth D R
Published: May 2006

Freshly isolated mononuclear leukocytes have been reported to show changes in cholesterol synthesis and high-affinity degradation of low-density lipoproteins (LDL) that parallel those that occur in the liver. To examine whether hypolipidemic therapy in patients with heterozygous familial hypercholesterolemia influences cholesterol homeostasis in their mononuclear cells we assessed the effects of colestipol and nicotinic acid (alone and in combination) on the rates of high-affinity 125I-labeled LDL degradation and on the rates of cholesterol and phosphatidylcholine biosynthesis by freshly isolated cells. Rates of 125I-labeled LDL degradation were lower in mononuclear cells from patients with heterozygous familial hypercholesterolemia on no medication (3.1 ng per 4 X 10(6) cells per 5 hr) than in cells from normal control subjects (6.1 ng per 4 X 10(6) cells per 5 hr) and, in the former patients, the values were not significantly affected by therapy with nicotinic acid. In contrast, freshly isolated mononuclear cells from patients receiving colestipol degraded 125I-labeled LDL at near-normal rates (5.0 ng per 4 X 10(6) cells per 5 hr). The rates of cholesterol synthesis were also higher in mononuclear cells isolated from patients treated with colestipol than in cells from untreated patients or from those receiving nicotinic acid; in contrast the rate of synthesis of phosphatidylcholine did not show any consistent changes. Similar results were obtained in a smaller number of patients studied longitudinally, in which colestipol therapy significantly increased rates of cholesterol synthesis and high-affinity degradation of 125I-labeled LDL by freshly isolated mononuclear cells. We conclude that previously observed changes in cholesterol homeostasis in the liver of patients treated with bile acid sequestrants are paralleled by similar changes in freshly isolated mononuclear cells and that these cells offer an accessible model for further studies on how diet and pharmacologic agents influence cellular cholesterol homeostasis in humans.
Singular integrals related to the Radon transform and boundary value problems
Authors: Phong D. H. , Stein E. M.
Published: May 2006

Two classes of integral operators are introduced that combine features of Calderón—Zygmund singular integrals, the Hilbert integral, and the Radon transform. Examples and applications arising from integral geometry and several complex variables are discussed.
Coordinate regulation of unsaturated phospholipid, RNA, and protein synthesis in Mycoplasma capricolum by cholesterol.
Authors: Dahl J S , Dahl C E
Published: May 2006

The effect of cholesterol, epicoprostanol, and phosphatidylcholine on phospholipid, RNA, and protein synthesis was investigated in the sterol auxotroph Mycoplasma capricolum. Cells growing poorly on lanosterol were stimulated to grow more rapidly by supplementing the medium with either 2 micrograms of cholesterol or 2.2 micrograms of egg phosphatidylcholine per ml. In such cells cholesterol caused a sequential stimulation of phospholipid, RNA, and protein synthesis. Enhanced oleate incorporation into phospholipid occurred early; the rates of RNA and protein synthesis increased later. In cells supplemented with phosphatidylcholine only RNA and protein syntheses were enhanced. The addition of 2 micrograms of epicoprostanol per ml to cells growing on lanosterol promptly inhibited the rate of unsaturated phospholipid synthesis and subsequently the rate of growth. Inhibition of both processes was relieved by supplying 2 micrograms of cholesterol or 2.2 micrograms of phosphatidylcholine per ml along with the inhibitory sterol. The results suggest that cholesterol in small amounts exerts a positive regulatory effect and epicoprostanol exerts a negative one on unsaturated phospholipid synthesis and, in turn, that RNA and protein synthesis are coordinately controlled with phospholipid synthesis. The previously reported phenomenon of sterol synergism and the postulated novel role of sterols in membranes.
Keratin gene expression in mouse epidermis and cultured epidermal cells.
Authors: Roop D R , Hawley-Nelson P , Cheng C K , Yuspa S H
Published: May 2006

The major differentiation products of mouse epidermis are keratins of 40-70 kilodaltons (kDal). We have prepared a library of cDNA clones from total poly(A)+ RNA from newborn mouse epidermis. Clones corresponding to the major in vivo keratins of 55, 59, and 67 kDal have been isolated and characterized. By RNA blot analysis of poly(A)+ RNA from newborn mouse epidermis, we have identified RNA species that are approximately 1,600, 2,000, and 2,400 nucleotides in length and are complementary to the cDNAs for the 55-, 59-, and 67-kDal keratins, respectively. Analysis of RNA from primary cultures of newborn mouse epidermis by this same technique shows greatly reduced levels of these RNAs. Transcripts complementary to all three cloned cDNAs are abundant in 14- to 16-day embryonic and adult mouse skin. Thus, altered expression in culture does not appear to be due to induction of a developmentally programmed switch by placing the cells in culture but instead is due to factors modulating expression within the culture system. Because the 55-, 59-, and 67-kDal keratins are the major proteins in epidermis they probably represent keratin associated with terminal differentiation. The expression data suggest that cultured cells are blocked in expression of differentiation keratins but instead synthesize other keratin family members probably related to cytoskeletal functions.
Immunoglobulin gene expression in transformed lymphoid cells.
Authors: Oi V T , Morrison S L , Herzenberg L A , Berg P
Published: May 2006

Myeloma, hybridoma, and thymoma cell lines have been successfully transfected for the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt) by using the plasmid vector pSV2-gpt. The transformed cells synthesize the bacterial enzyme 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase (XGPRT; EC 2.4.2.22) and have been maintained in selective medium for over 4 months. Lymphoid cell lines expressing a K immunoglobulin light chain were obtained by transfecting cells with pSV2-gpt containing a rearranged K light chain genomic segment from the S107 myeloma cell line. The S107 light chain is synthesized in gpt-transformed J558L myeloma cells and is identical to the light chain synthesized by the S107 myeloma cell line, as judged by immunoprecipitation and two-dimensional gel electrophoresis. Furthermore, this light chain is synthesized and secreted as part of an intact antibody molecule by transformed hybridoma cells that normally secrete an IgGl (gamma, K) antibody molecule. No light chain synthesis was detected in a similarly transformed rat myeloma or a mouse thymoma line.
A unitary mechanism of calcium antagonist drug action.
Authors: Murphy K M , Gould R J , Largent B L , Snyder S H
Published: May 2006

[3H]Nitrendipine binding to drug receptor sites associated with calcium channels is allosterically regulated by a diverse group of calcium channel antagonists. Verapamil, D-600 (methoxyverapamil), tiapamil, lidoflazine, flunarizine, cinnarizine, and prenylamine all reduce [3H]nitrendipine binding affinity. By contrast, diltiazem, a benzothiazepine calcium channel antagonist, enhances [3H]nitrendipine binding. All these drug effects involve a single site allosterically linked to the [3H]nitrendipine binding site. Inhibition of [3H]nitrendipine binding by prenylamine, lidoflazine, or tiapamil is reversed by D-600 and diltiazem, which alone respectively slightly reduce or enhance [3H]nitrendipine binding. Diltiazem reverses the inhibition of [3H]nitrendipine binding by D-600. Our prediction that drugs allosterically regulating [3H]nitrendipine binding should be calcium antagonists is confirmed by calcium antagonism in guinea pig ileum observed with the antihistamine dimethindene, the neuroleptics thioridazine and mesoridazine, and the anticholinergic biperiden.
Phosphorylation induces a decrease in the biological activity of the protein inhibitor (GABA-modulin) of gamma-aminobutyric acid binding sites.
Authors: Wise B C , Guidotti A , Costa E
Published: May 2006

gamma-Aminobutyric acid (GABA)-modulin is a brain protein of Mr 16,500 that down-regulates the high-affinity binding site for GABA which is located in crude synaptic membranes. This protein can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase and by a partially purified preparation of calmodulin-sensitive Ca2+-dependent protein kinase. The GABA-modulin sites that are phosphorylated by the two enzymes are different, as revealed by HPLC analysis of tryptic digests. The capacity of GABA-modulin to decrease the number of sites that bind [3H]muscimol was completely abolished by phosphorylation of this protein with the cAMP-dependent protein kinase but not with the Ca2+-dependent enzyme. GABA-modulin present in crude synaptic membranes prepared from rat cortex also was shown to be phosphorylated by endogenous protein kinases activated by cAMP, Ca2+ and calmodulin, and Ca2+ and phosphatidylserine. These results suggest a potentially important role for protein kinase and GABA-modulin in the regulation of the number of GABA recognition sites.
A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements ("costameres") mark sites of attachment between myofibrils and sarcolemma.
Authors: Pardo J V , Siliciano J D , Craig S W
Published: May 2006

We have found that vinculin is localized at the sarcolemma of skeletal muscle cells in a two-dimensional orthogonal lattice. Perpendicular to the longitudinal axis of the cell, bands of vinculin encircle the muscle cell and repeat along its length with a periodicity corresponding to the subjacent sarcomeres. Because of their appearance and probable function, we call the transverse elements of the lattice "costameres" (Latin costa, rib; Greek meros, part). Costameres have a substructure consisting of densely clustered patches of vinculin; the patches are segregated into two rows which flank the Z line and overlie the I band of the underlying sarcomere. It is likely that the costameres are physically coupled to the underlying myofibrils because: (i) the costameres broaden and narrow in concert with the underlying I band in stretched and contracted muscle, and (ii) adjacent but misaligned myofibrils are mirrored by corresponding discontinuities in the overlying costameres. We hypothesize that the sarcolemmal lattice, detected because vinculin is one of its molecular components, integrates the contractile apparatus with the sarcolemma during lengthening and shortening of the muscle cells.
Enzyme relationships in a sorbitol pathway that bypasses glycolysis and pentose phosphates in glucose metabolism.
Authors: Jeffery J , Jörnvall H
Published: May 2006

A pathway from glucose via sorbitol bypasses the control points of hexokinase and phosphofructokinase in glucose metabolism. It also may produce glycerol, linking the bypass to lipid synthesis. Utilization of this bypass is favored by a plentiful supply of glucose--hence, conditions under which glycolysis also is active. The bypass further involves oxidation of NADPH, so the pentose phosphate pathway and the bypass are mutually facilitative. Possible consequences in different organs under normal and pathological, especially diabetic, conditions are detailed. Enzymes with related structures (for example, sorbitol dehydrogenase and alcohol dehydrogenase, and possibly, aldehyde reductase and aldose reductase, respectively) are linked functionally by this scheme. Some enzymes of the bypass also feature in glycolysis (aldolase and alcohol dehydrogenase), and these enzymes, with the reductases involved, are proteins known to occur in different classes or multiple isozyme forms. Two of the enzymes (aldolase and alcohol dehydrogenase) both involve classes with and without a catalytic metal (zinc). The existence of parallel pathways and the occurrence of similar enzymic steps in one pathway may help to explain the abundance and multiplicity of enzymes such as reductases, aldolases, and alcohol dehydrogenases.
A physical interpretation for the natural photosynthetic process
Authors: Hill Robert , Rich Peter R.
Published: May 2006

The efficiency of the process of photosynthesis is shown to depend on the molecular conversion of power. This requires establishment of a discipline that is now implicit in current thought and that offers a definition of relationship between equilibrium state and power. The quantum aspect for the microscopic process is different from the macroscopic system idealized as the heat engine and is required for the interpretation of molecular machinery. By using three postulates the ideal maximal efficiency for the molecular energy conversion is calculated from the data, which are assembled in the form of the “Z scheme” for photosynthesis. The observed and the calculated efficiencies for a green plant are substantially in agreement.