bluuebunny/crossref_metadata_2025 huggingface dataset:
Non-immunoglobulin-associated DNA rearrangements in mouse plasmacytomas.
Authors: Harris L J , Lang R B , Marcu K B
Published: May 2006

We have characterized a class of DNA rearrangements in plasmacytomas. These recombination events involve a DNA sequence whose origin is outside of the locus of the heavy chain constant region genes, CH. Therefore, we choose to refer to this sequence as non-immunoglobulin-associated rearranging DNA (NIARD). We have isolated two abortively rearranged C alpha genes, generated by NIARD events from the alpha-producing J558 myeloma. Restriction endonuclease maps of these sequences reveal two possible recombination sites in NIARD that are separated by approximately 6.5 kilobase pairs of DNA (defined as 5' and 3' sites). A NIARD rearrangement occurs in 15 out of 20 plasmacytomas tested, including gamma 3-, gamma 1-, gamma 2b-, gamma 2a-, and alpha-producers, but this event usually does not involve a CH switch (S) region. In fact, only S alpha appears to accept NIARD. However, NIARD did not undergo a rearrangement in eight IgA-producing hybridomas tested. One germ-line copy of NIARD (a 22-kilobase pair EcoRI fragment) is retained in all plasmacytomas. NIARD does not appear to possess repetitive DNA sequences homologous to S mu or S alpha. We discuss the possible role and implications of NIARD-like sequence rearrangements in allelic exclusion and chromosomal translocation events in plasmacytomas.
Purification of the cdc8 protein of Saccharomyces cerevisiae by complementation in an aphidicolin-sensitive in vitro DNA replication system.
Authors: Kuo C L , Campbell J L
Published: May 2006

DNA synthesis in vitro in Brij-treated Saccharomyces cerevisiae requires the product of the CDC8 gene (Hereford, L. M. & Hartwell, L. H. (1971) Nature (London) New Biol. 234, 171-172). Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8, a mutant containing a thermolabile cdc8 gene product. This constitutes a complementation assay by which the cdc8 gene product can be monitored during purification. A heat-stable protein responsible for this complementation has been partially purified from both wild-type A364a cells and from a cdc8 temperature-sensitive mutant. The complementation activity from the mutant is thermolabile when compared to the wild-type activity, indicating that CDC8 is the structural gene for the protein.
Purification of a specific reversible tyrosine-O-phosphate phosphatase.
Authors: Fukami Y , Lipmann F
Published: May 2006

A phosphatase specific for tyrosine-O-phosphate (Tyr-P) was separated from several nonspecific phosphatases present in the third instar larvae of Drosophila melanogaster. The enzyme hydrolyzed L-Tyr-P, with an apparent Km of 0.14 mM, but not D-Tyr-P after being freed from hydrolytic activity toward p-nitrophenyl phosphate, the common phosphatase substrate. Such purified preparations also catalyzed a reversible phosphate transfer reaction from unlabeled Tyr-P to [3H]tyrosine. The transfer activity was L4-14% of the hydrolytic activity, depending on the initial concentration of tyrosine (0.25-4.0 mM). The two activities coincided throughout purification. However, they differed in pH optimum, that of hydrolysis being 6.5-7 and that of phosphate transfer being 7.7.5. The two activities were also differentially inhibited by 1-p-bromotetramisole oxalate in the presence of EDTA and by Mn2+. Addition of Mg2+ did not affect either hydrolysis or phosphate transfer, but 5 mM Zn2+ was 65% inhibitory to both. Sodium fluoride strongly inhibited both reactions, and this inhibition was reversed by EDTA, while EDTA itself had no effect. Pi had no effect and no detectable incorporation of 32Pi into Tyr-P was observed, indicating that the phosphate transfer reaction is not a simple reversal of hydrolysis. No ATP-linked phosphorylation of tyrosine was found.
Partial purification and characterization of the mRNA for human thymidine kinase and hypoxanthine/guanine phosphoribosyltransferase.
Authors: Lin P F , Yamaizumi M , Murphy P D , Egg A , Ruddle F H
Published: May 2006

We used direct microinjection of poly(A)+RNA into individual hypoxanthine/guanine phosphoribosyltransferase-deficient or thymidine kinase-deficient cells and detected the specific in vivo translation products as an assay for human hypoxanthine/guanine phosphoribosyltransferase or thymidine kinase mRNAs. The incorporation of [3H]hypoxanthine or [3H]thymidine into cells in response to injected mRNA was assayed in situ by autoradiography. Methylmercuric hydroxide/agarose gel analysis showed that human hypoxanthine/guanine phosphoribosyltransferase mRNA contains approximately 1,530 nucleotides, which is twice the number required for its protein coding capacity. The mRNA for human cytoplasmic thymidine kinase is estimated to be approximately the same length; thus, the size of the cytosol thymidine kinase subunit can be predicted to be approximately 47,000 daltons, if the full coding capacity of its mRNA is utilized.
Two-fluid theory and thermodynamic properties of liquid mixtures: General theory
Authors: Brandani V. , Prausnitz J. M.
Published: May 2006

The two-fluid theory of binary mixtures postulates that the extensive thermodynamic properties of a binary mixture may be expressed by the contributions of two hypothetical fluids that mix ideally. This postulate, coupled with an expression for the partition function of the hypothetical fluid, permits evaluation of the properties of binary liquid mixtures by using only two adjustable binary parameters. Particular attention is given to the problem of nonrandomness in mixtures. A quantitative description of nonrandomness is achieved by combining the two-fluid concept with a hypothesis for ensemble averaging of a distribution of nearest-neighbor pairs.
Random walk properties from lattice bond enumeration: Steady-state diffusion on two- and three-dimensional lattices with traps
Authors: Shuler Kurt E. , Mohanty Udayan
Published: May 2006

We have applied the lattice bond enumeration method to the calculation of the steady-state diffusion in a lattice with fixed traps. We show that, to first order in density of traps, our random walk calculations for the effective diffusion constant in lattices with periodically arrayed traps are in exact agreement with calculations carried out previously for randomly arrayed traps embedded in a three-dimensional continuum medium (fluid). Our lattice random walk results are independent of dimension for
d
> 1, and we conjecture that this is also true for the continuum diffusion model.
A remark on minimal hypercones
Authors: Schoen Richard
Published: May 2006

Let
M
be a minimal hypersurface in the sphere. It is shown that if the cone over
M
is a stable critical point for the area functional, then
M
admits a conformally equivalent metric with positive scalar curvature. This gives both topological and geometric restrictions on such hypersurfaces
M
.
Conversion of proinsulin to insulin: involvement of a 31,500 molecular weight thiol protease.
Authors: Docherty K , Carroll R J , Steiner D F
Published: May 2006

A lysed crude secretory granule fraction from rat islets of Langerhans was shown to process endogenous proinsulin to insulin with a pH optimum of 5.0--6.0. The converting activity in the lysed fraction was not inhibited by serine protease inhibitors (diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin) or metalloprotease inhibitors (EDTA and o-phenanthroline) but was inhibited by some thiol protease reagents (p-chloromercuribenzenesulfonic acid, antipain, and leupeptin) but not by others (N-ethylmaleimide and iodoacetamide). N alpha-p-Tosyl-L-lysyl chloromethyl ketone only mildly inhibited at higher concentrations, whereas L-alanyl-L-lysyl-L-arginyl chloromethyl ketone was a powerful inhibitor. L-Alanyl-L-lysyl-L-arginyl chloromethyl ketone was [125I]iodotyrosylated and used as an affinity labeling agent for the converting activity. Because the crude granule preparation contained contaminating lysosomes the affinity labeling of the granule preparation proteins was compared with that in liver lysosomes purified from rats injected with Triton WR1339. In the crude granule fraction the affinity label bound in a cysteine-enhanced manner to a single 31,500 molecular weight protein, but in purified liver lysosomes the major affinity-labeled protein had a molecular weight of 25,000 and minor 31,500 and 35,000 molecular weight proteins were also labeled. Evidence suggests that these proteins are thiol proteases and that in islets the 31,500 molecular weight thiol protease is involved in the conversion of proinsulin to insulin.
IgG2a monoclonal antibodies inhibit human tumor growth through interaction with effector cells.
Authors: Herlyn D , Koprowski H
Published: May 2006

Monoclonal antibodies of IgG2a isotype specifically inhibited growth of human tumors in nude mice. Twenty-three monoclonal antibodies of other isotypes showed no tumoricidal reactivity. Complement depletion of nude mice had no effect on tumor suppression by monoclonal antibody. The role of T and killer cells as mediators of the monoclonal antibody effect in nude mice was virtually excluded. On the other hand, macrophages were strongly incriminated as effector cells because silica treatment of nude mice abolished the tumoricidal effect of monoclonal antibody. IgG2a monoclonal antibody-dependent macrophage-mediated cytotoxicity assays with human tumor cells in culture resulted in specific lysis of tumor cells.
RNA processing errors in patients with beta-thalassemia.
Authors: Ley T J , Anagnou N P , Pepe G , Nienhuis A W
Published: May 2006

We have developed a method that permits rapid identification of the consequences of mutations that alter beta-globin RNA processing in erythroid cells. S1 nuclease mapping techniques were used to analyze total bone marrow RNA obtained fron 15 patients who are clinically homozygous for beta-thalassemia and from 5 patients with erythroid hyperplasia from other causes. This analysis was facilitated by the use of single-stranded uniformly labeled DNA probes of high specific activity that were prepared by using recombinant phage M13-beta-globin DNA templates. Two abnormalities of RNA processing were found to occur with high frequency in these patients. Nine thalassemic patients were found to have increased levels of an RNA species that retains all sequences transcribed from intervening sequence 1, implying the presence of mutations that decrease the correct splicing of this intron. Seven of 15 thalassemic patients were found to have an abnormally processed RNA species that retains 19 nucleotides transcribed from the 3' end of intron 1; this abnormality is caused by the G leads to A substitution in intron 1 that is known to create an alternative splice acceptor site.
Conformational substates in a protein: structure and dynamics of metmyoglobin at 80 K.
Authors: Hartmann H , Parak F , Steigemann W , Petsko G A , Ponzi D R , Frauenfelder H
Published: May 2006

The crystal structure of sperm whale metmyoglobin has been determined at 80 K to a resolution of 2A. The overall structure at 80 K is similar to that at 300 K except that the volume is smaller. Refinement of the structure by the method of restrained least squares (current R = 0.175) permits the assignment of isotropic atomic mean-square displacements to all nonhydrogen atoms. Comparison with the values obtained earlier at 250-300 K indicates that the protein at 80 K is more rigid. The average experimentally determined Debye-Waller factor, B, for the protein is 14A2 at 300 K and 5A2 at 80 K. Plots of backbone mean-square displacement vs. temperature show a discontinuity of slope for at least one-third of all residues. This behavior is in good agreement with the temperature dependence of the mean-square displacement of the heme iron as measured by Mössbauer absorption. The magnitudes of the smallest mean-square displacements observed at 80 K indicate that intramolecular motions can be frozen out to a surprisingly large degree. Even at 80 K, however, some atoms in myoglobin still have mean-square displacements greater than 0.1A2, thus providing evidence for conformational substates.
Fragments of the simian virus 40 transforming gene facilitate transformation of rat embryo cells.
Authors: Colby W W , Shenk T
Published: May 2006

Segments of the simian virus 40 (SV40) genome that encode only fragments of large tumor antigen can facilitate immortalization of secondary rat embryo cells. The phenotypes of the immortalized cells range from nearly "normal" to fully transformed. All of the cell lines contain SV40 sequences and express unstable NH2-terminal fragments of large tumor antigen. SV40 small tumor antigen does not appear to be essential for either immortalization or transformation.
Argininosuccinate synthetase of bovine liver: chemical and physical properties.
Authors: Ratner S
Published: May 2006

Homogeneous crystalline argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] prepared from bovine liver according to Rochovansky et al. [Rochovansky, O., Kodowaki, H. & Ratner, S. (1977)J. Biol. Chem. 252, 5287-5294] has been characterized with respect to amino acid composition and other chemical and physical properties. The total residue molecular weights derived from the amino acid analysis are in agreement with values previously obtained by physical means for the catalytically active tetramer, 185,000, and the monomer, 46,500. The enzyme is focused sharply at pH 7.6 as a single protein. Additional properties reported include 2.74 X 10(5) M-1 cm-1 for the molar absorption coefficient, based on the absolute value for protein, and 0.747 ml/g for the chemically based partial specific volume.
Genetic instability in Drosophila melanogaster: deletion induction by insertion sequences.
Authors: Green M M
Published: May 2006

Females of Drosophila melanogaster heteroallelic or homoallelic for X chromosome giant (gt) mutants generate deletions involving the wild-type alleles at two X chromosome gene loci: yellow body color (y) and white eye color (w). The deletions, bidirectional in the case of y and with fixed endpoint in the case of w, are associated with particular X chromosomes. Distinctive insertion sequences, located proximal to the target loci, are presumed to generate the deletions.
Modulation of the epidermal growth factor receptor by platelet-derived growth factor and choleragen: Effects on mitogenesis
Authors: Wharton Walker , Leof Edward , Pledger W. J. , O'Keefe E. J.
Published: May 2006

The addition of fresh medium supplemented with partially purified platelet-derived growth factor (PDGF) to quiescent density-arrested cultures of BALB/c-3T3 cells decreases the subsequent binding of radiolabeled epidermal growth factor (EGF). The decrease in EGF binding can be observed 1 hr after the addition of PDGF. This effect is maximal in 2-3 hr, and binding remains diminished for at least 6 hr. These effects can be accounted for by a decrease in the number of EGF receptors with no change in receptor affinity. The action of PDGF is concentration dependent, but even at very high concentrations of PDGF the reduction in EGF binding is never more than 50%. Similar decreases in EGF binding are produced by other treatments that render BALB/c-3T3 cells competent, such as the addition of fibroblast growth factor or medium previously exposed to the macrophage-like cell line P388D
1
. Cholera toxin (choleragen), which alone had no effect on EGF binding, dramatically potentiated the ability of PDGF to down regulate EGF receptors. Two to three hours after the addition of PDGF and choleragen, EGF binding was reduced by 80-90% compared with control values. The ability of PDGF and choleragen together to decrease EGF binding was substantially inhibited by cycloheximide. Autoradiography of [
3
H]thymidine-labeled cells shows that choleragen potentiates the action of PDGF; lower concentrations of PDGF are required to make cells competent after choleragen treatment. Furthermore, cells treated with PDGF and choleragen no longer require EGF for traverse of G
1
phase and initiation of DNA synthesis in defined medium. The reduction in receptor number produced by choleragen and PDGF, which may be due to internalization of the EGF receptor, may mimic the action of EGF and thereby remove the EGF requirement for DNA synthesis.
Dissection of a replication origin of Xenopus DNA.
Authors: Chambers J C , Watanabe S , Taylor J H
Published: May 2006

A previously cloned 503-base pair (bp) EcoRI segment of genomic DNA from Xenopus laevis selected for enhancement of replication of its vector plasmid was moved to the EcoRI site of pBR322. This plasmid designated pJCC31 and five other clones, which were made by cleaving the 503-bp segment in relation to a dispersed repeated sequence and subcloning, were compared with pBR322 for replication by microinjection into Xenopus eggs. The replication measured by incorporation of a 32P-labeled nucleotide as well as semiconservative segregation and dilution of N6-methyladenine at the EcoRI sites showed pJCC31 to be about 15 times as efficient as pBR322. The next most efficient subclone, pJCC31-2, contains an insert with a complete 320-bp dispersed repeated sequence bracketed by an 8-bp direct repeat. This observation, along with our previous report that repeated sequences of the Alu family in the human genome enhanced replication of the vector plasmid nearly as much as that of the presumptive Xenopus origin, leads to the hypothesis that members of a subset of the short dispersed repeated sequences in vertebrates function as origins for chromosomal replication. Preliminary studies also show that the presumptive Xenopus origin contains a RNA polymerase promoter that increases the transcription of the plasmid when it is microinjected into Xenopus oocytes.
Geometry and analysis of Shimizu
L
-functions
Authors: Atiyah M. F. , Donnelly H. , Singer I. M.
Published: May 2006

The values of 0 of Shimizu
L
-functions are realized as the signature defects of framed manifolds. This settles a conjecture of Hirzebruch [Hirzebruch, F. (1973)
Enseign. Math.
19, 183-281] affirmatively. The proof employs the spectral theory of elliptic operators.
Molecular structure and evolutionary origin of human cardiac muscle actin gene.
Authors: Hamada H , Petrino M G , Kakunaga T
Published: May 2006

Two recombinant phages that contain cardiac muscle actin gene were isolated from a human DNA library and their structures were determined. Restriction analysis indicates that both clones carry the same EcoRI 13-kilobase fragment where the coding sequence is mapped. The cloned DNA hybridized with polyadenylylated RNA from human fibroblasts, which directs the synthesis of cytoplasmic beta- and gamma-actin in vitro. However, sequence determination of the cloned DNA showed that the entire coding sequence perfectly matched the amino acid sequence of cardiac muscle actin. The initiation codon is followed by a cysteine codon that is not found at the amino-terminal site of any actin isoform, suggesting the necessity of post-translational processing for in vivo actin synthesis. There are five introns interrupting exons at codons 41/42, 150, 204, 267, and 327/328. Surprisingly, these intron locations are exactly the same as those of the rat skeletal muscle actin gene but different from those of nonmuscle beta-actin gene. Nucleotide sequences of all exon/intron boundaries agree with the G-T/A-G rule (G-T at the 5' and A-G at the 3' termini of each intron). The 3'-untranslated sequence has no homology to that of nonmuscle beta- or gamma-actin gene, but Southern blot hybridization has shown that this region has considerable homology to that of one of the other actin genes. These results indicate that the recombinant phages, which we have isolated, contain cardiac muscle actin gene and that cardiac muscle actin gene and skeletal muscle actin genes are derived from their ancestor gene at a relatively recent time in evolutionary development.
Internalized insulin receptors are recycled to the cell surface in rat hepatocytes.
Authors: Fehlmann M , Carpentier J L , Van Obberghen E , Freychet P , Thamm P , Saunders D , Brandenburg D , Orci L
Published: May 2006

We have followed the fate of cell surface insulin receptors in isolated rat hepatocytes by both a biochemical and a morphological approach. Hepatocytes were labeled with the photoreactive and biologically active 125I-labeled insulin analogue, [2-nitro-4-azidophenylacetylB2]des-PheB1-insulin, under conditions that allow for minimal internalization (2 hr at 15 degrees C). Analysis of the cell-associated radioactivity by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed the specific labeling of a major insulin receptor subunit with Mr 130,000 and a minor degradation product with Mr 125,000. When the cells were exposed at 15 degrees C to trypsin at the end of the association period, these two bands were no longer observed, indicating that the labeled receptors were at the cell surface. This trypsin sensitivity of the receptor disappeared within 30-60 min of incubation of the cells at 37 degrees C, reflecting the internalization of the hormone-receptor complexes. Over the subsequent 4 hr of incubation, this was followed by a progressive reappearance of the receptor complexes at the cell surface, as indicated by the recovery of trypsin sensitivity of the labeled insulin receptors. An identical (both chronologically and quantitatively) journey of the insulin receptors was observed when the labeled material was studied by quantitative electron microscopic autoradiography. Thus, when the cells were incubated at 37 degrees C there was a rapid decrease (30-60 min) in the percentage of autoradiographic grains associated with the plasma membrane, followed by a progressive increase in this percentage over the subsequent 4 hr of incubation. In conclusion, using a biochemical and morphological approach to trace the photoaffinity-labeled insulin receptor, we have shown that the internalized hormone-receptor complex is recycled back to the cell surface.
On the nature of crossreactions observed with antibodies directed to defined epitopes.
Authors: Nigg E A , Walter G , Singer S J
Published: May 2006

Antibodies directed against a synthetic peptide (src-c) containing the six carboxyl-terminal amino acids of p60src, the transforming protein of Rous sarcoma virus, recognize p60src. However, when used at sufficiently high concentrations they also react with a number of constituents of untransformed cells. These reactions can be completely inhibited by src-c peptide. Crossreactivities are to different components in cells from different species and cannot be attributed to p60c-src, the ubiquitous cellular homologue of p60src. By indirect immunofluorescence microscopy and immunochemical techniques we have identified three cytoskeletal proteins, myosin, tubulin, and vimentin, as well as an unknown intranuclear antigen, as major targets of anti-src-c antibodies in different untransformed cells. These crossreactivities probably reflect identities or similarities in the amino acid sequence of the immunogenic peptide and segments of the otherwise unrelated crossreactive proteins. These findings are discussed with respect to the interpretation of crossreactivities that are occasionally observed with anti-peptide sera and with monoclonal antibodies.
Immunological similarity between a cyanobacterial enzyme and a nuclear DNA-encoded plastid-specific isozyme from spinach
Authors: Weeden Norman F. , Higgins Ratchford C. , Gottlieb L. D.
Published: May 2006

The immunochemical properties of the plastid and cytosolic isozymes of phosphoglucose isomerase (glucosephosphate isomerase; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) in spinach (
Spinacia oleracea
) and the single phosphoglucose isomerase enzyme from the cyanobacterium
Synechococcus
sp. were compared by an application of the enzyme-linked immunosorbent assay. Utilizing antibodies made in rabbits against subunits of purified plastid and cytosolic phosphoglucose isomerase isozymes from spinach, we demonstrate that the plastid isozyme is immunochemically more similar to the cyanobacterial enzyme than to the spinach cytosolic counterpart. The antiserum to plastid phosphoglucose isomerase crossreacted strongly with plastid phosphoglucose isomerases from other flowering plants. The antiserum to cytosolic phosphoglucose isomerase crossreacted with other plant cytosolic phosphoglucose isomerase isozymes. The results are consistent with the hypothesis [Weeden, N. F. (1981)
J. Mol. Evol.
17, 133-139] that the nuclear gene specifying plastid phosphoglucose isomerase was derived from a prokaryote after the incorporation of a plastid-like symbiont into the ancestral plant cell.
Inhibition of in vitro natural killer activity by the third component of complement: role for the C3a fragment.
Authors: Charriaut C , Senik A , Kolb J P , Barel M , Frade R
Published: May 2006

Purified human native third component of complement, C3, was found to inhibit in vitro natural killer (NK) cell cytotoxicity in both mouse and human systems. The effect was dose and time dependent, a 50% inhibition being reached with 190 nM C3 (35 micrograms/ml) added during the NK assay or after a 30-min preincubation of the effector cells with this C3 concentration. C3 was shown to act at the effector-cell population level because pretreatment of the target cells did not modify the NK lysis. The inhibition was not due to general cytotoxicity nor to cell agglutination. Moreover, another in vitro cytotoxicity system (represented by alloreactive cytotoxic lymphocytes) was not affected by purified C3. Structural analysis of the active part of the C3 molecule shows that the C3-induced inhibition is supported by the C3a fragment. Release of carboxyl-terminal arginine residue by carboxypeptidase B, converting C3a into des-Arg77-C3a, did not alter the inhibitory effect displayed by this fragment. These results suggest that C3a may play an important role in the regulation of NK activity.
Restriction point control of cell growth by a labile protein: evidence for increased stability in transformed cells.
Authors: Campisi J , Medrano E E , Morreo G , Pardee A B
Published: May 2006

It has been proposed that animal cells must accumulate a labile protein(s) before they can pass the restriction (R) point in the G1 phase of the cell cycle [Rossow, P. W., Riddle, V. G. H. & Pardee, A. B. (1979) Proc. Natl. Acad. Sci. USA 76, 4446--4450]. Here, we present evidence that this R protein acquires increased stability in transformed 3T3 cells, thereby allowing these cells to continue growth under conditions that arrest untransformed cells. Low doses of cycloheximide or histidinol drastically reduced the rate at which normal 3T3 (A31) fibroblasts in early G1 could enter DNA synthesis. These drugs had less effect on entry of two tumorigenic A31 derivatives, BPA31 and SVA31, in S, although measurement of [3H]leucine incorporation showed that the inhibitors were equally effective in the three cell lines. The hypothesis is that the transformed lines are less sensitive because moderate inhibition of their R protein synthesis is compensated by lower rates of protein degradation. To test this idea, we completely inhibited cytoplasmic protein synthesis for several hours shortly before A31 and BPA31 cells had reached the R point. After removal of inhibitor, A31 cells showed delays in the onset of S that were in excess of the inhibitor pulse, consistent with decay of labile protein during the pulse. BPA31 cells showed no excess delays, suggesting a much more stable R protein. The half-life of the R protein was estimated as 2.5 hr in A31 cells, indicating that, in these cells, R protein synthesis starts at the beginning of G1. In the BPA31 cells the R protein showed no signs of decay for at least 8 hr.
Central GABAergic innervation of neurointermediate pituitary lobe: biochemical and immunocytochemical study in the rat.
Authors: Oertel W H , Mugnaini E , Tappaz M L , Weise V K , Dahl A L , Schmechel D E , Kopin I J
Published: May 2006

Activity of glutamic acid decarboxylase GluDCase, the biosynthetic enzyme of gamma-aminobutyric acid (GABA) was measured in low-speed homogenate supernatant of the neural and intermediate (neurointermediate) lobe (28--30 pmol of CO2 per microgram of protein per hr) and of the anterior lobe (2--4 pmol of CO2 per microgram of protein per hr). In the neurointermediate lobe, stalk transection reduced the GluDCase activity by more than 95%. By using an antiserum to rat brain GluDCase and the unlabeled antibody--peroxidase method of Sternberger, GluDCase immunoreactivity was localized in many terminals within the neurointermediate lobe of the hypophysis. In pars intermedia, immunoreactive terminals occurred in apposition to secretory cells and to glial cells and were near nonimmunoreactive axonal profiles; in pars neuralis they were apposed to pituicytes and to unlabeled axons including the neurosecretory terminals and were along fenestrated portal capillaries. GluDCase immunoreactive axons terminals exhibited diverse morphological features and would not have been identified as a distinct population without the GluDCase antiserum. No GluDCase-immunoreactivity was found in the anterior pituitary lobe. Stalk transection abolished GluDCase immunoreactivity in the neurointermediate lobe. These data provide biochemical and morphological evidence for a central GABAergic innervation of neural and intermediate lobes of the hypophysis.
Symplectic geometry and positivity of pseudo-differential operators
Authors: Fefferman C. , Phong D. H.
Published: May 2006

In this paper we establish positivity for pseudo-differential operators under a condition that is essentially also necessary. The proof is based on a microlocalization procedure and a geometric lemma.
Yeast invertase polymorphism is correlated with variable states of oligosaccharide chain phosphorylation.
Authors: Frevert J , Ballou C E
Published: May 2006

Saccharomyces cerevisiae invertase (EC 3.2.1.26) isolated from wild-type strain X2180 can be resolved by isoelectric focusing into at least seven bands revealed by an activity stain. Most of this polymorphism is eliminated in mutants that are defective in phosphorylation of the mannoprotein carbohydrate chains (mnn4 and mnn6). In contrast to strain X2180, invertase from the mnn9 mutant, which makes mannoprotein lacking the outer portion of the polymannose chains, shows only two major bands on isoelectric focusing. Although mnn2 mannoprotein is though not to have any branches in its outer chain, the invertase of this mutant shows at least six bands on isoelectric focusing, and digestion of this invertase with an endo-alph aI leads to 6-mannanase that removes the unbranched outer chain produces an invertase with two bands that are similar to those from the mnn9 mutant. The invertase from mnn2 cells, grown with [32P]orthophosphate and precipitated with specific antiserum, gives at least five radioactive bands on isoelectric focusing, and after digestion with the endomannanase the radioactivity no longer migrates with the residual invertase. Mutants with shortened and unbranched outer chains (mnn2 mnn7, mnn2 mnn8, and mnn2 mnn10) give invertase patterns similar to mnn2. The results suggest that multiple states of outer chain phosphorylation lead to isoelectric polymorphism of S. cerevisiae external invertase and, because invertase has nine carbohydrate chains, no more than one phosphate group per chain would be required to account for this property.
Metabolism and mechanism of action of 5-fluorodeoxycytidine.
Authors: Newman E M , Santi D V
Published: May 2006

5-Fluoro-2'-deoxycytidine (FdCyd) is a potent inhibitor of growth of tissue culture cells. The major cytotoxic event appears to be inhibition of thymidylate synthetase as evidenced by reversal of the cytotoxicity with thymidine but not deoxycytidine and by the effect of FdCyd on nucleotide pools, which is characteristic of specific inhibition of this enzyme. The metabolism of FdCyd was established by using a method in which its cytotoxicity was compared in several S-49 mutant cell lines having defined single or double deficiencies of enzymes involved in nucleoside and nucleotide metabolism. Our results indicate that FdCyd is metabolized to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of thymidylate synthetase by two pathways: (i) sequential reactions catalyzed by deoxycytidine kinase and deoxycytidylate deaminase and (ii) sequential reactions catalyzed by cytidine deaminase and thymidine kinase. We have shown that metabolism of FdCyd can be directed through the former pathway by inhibition of cytidine deaminase with tetrahydrouridine. Since cytidine deaminase appears to be responsible for catabolism of FdCyd in animals, our results suggest that the antineoplastic effects of FdCyd should be examined in combination with inhibitors of cytidine deaminase.
Micrococcal nuclease cleavage of nucleotide linked to glutamine synthetase yields phosphotyrosine at the ligation site.
Authors: Martensen T M , Stadtman E R
Published: May 2006

The activity of micrococcal nuclease was studied on a novel substrate, denatured adenylylated glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2], which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP. The products of the digestion were adenosine and O-phosphotyrosylglutamine synthetase. The Km of the macromolecular substrate with the nuclease was 1/40 that of the synthetic substrate, nitrophenyl-pdT, which is commonly used for assay of the enzyme. Native adenylylated glutamine synthetase was not deadenosylated by micrococcal nuclease under the conditions that permit rapid deadenosylation of denatured glutamine synthetase. Failure to attack native glutamine synthetase is probably not due to steric factors because the native enzyme is deadenylylated by snake venom phosphodiesterase under identical conditions. The inability of micrococcal nuclease to deadenosylate native glutamine synthetase may be due to the formation of an inactive complex because the native protein inhibited the nuclease activity on the denatured protein.
A novel repeated element with Z-DNA-forming potential is widely found in evolutionarily diverse eukaryotic genomes.
Authors: Hamada H , Petrino M G , Kakunaga T
Published: May 2006

By Southern blotting and hybridization analysis using 32P-labeled poly(dT-dG) . poly(dC-dA) as a probe, we have found, in eukaryotic genomes, a huge number of stretches of dT-dG alternating sequence, a sequence that has been shown to adopt the Z-DNA conformation under some conditions. This sequence was found in all eukaryotic genomes examined from yeast to human, indicating extraordinary evolutionary conservation. The number of the sequence ranged from about 100 in yeast to tens of thousands in higher eukaryotes. Comparison of nucleotide sequences of dT-dG alternating regions and its flanking regions in several cloned genes showed that the repeated element [the Z(T-G) element]] consists only of dT-dG alternating sequence with variable length. The presence of another purine-pyrimidine alternating sequence was also surveyed in eukaryotic genomes by Southern blot hybridization using 32P-labeled poly(dG-dC) . poly(dG-dC) as the probe. The stretches of dC-dG alternating sequence [the Z(C-G) element] were found to be moderately repetitive in human, mouse, and salmon genomes. However, a few and no copies of the Z(C-G) element were found in yeast and calf genomes, respectively. These results provide evidence for the abundance of potential Z-DNA-forming sequences in nature.
Evidence for another cell-adhesion molecule in Dictyostelium discoideum.
Authors: Springer W R , Barondes S H
Published: May 2006

We raised a rabbit antiserum that completely blocked cell-cell adhesion of aggregating Dictyostelium discoideum cells in an in vitro assay. All adhesion-blocking activity of this antiserum was adsorbed with a D. discoideum fraction containing molecules having molecular weights as high as about 10(6), even after the material had been extensively digested with Pronase. The properties of this fraction indicate that the antigenic determinants in this macromolecule are saccharide residues. Antigen-rich material is found on vegetative cells but accumulates on or around differentiating D. discoideum cells as they aggregate. The cell surface of an aggregating cell contains about 5 X 10(5) antigenic sites. Antigen is also abundant in the medium of D. discoideum cells starved in suspension, which proved the most convenient starting material for its purification. Like several other macromolecules already discovered in D. discoideum by using this immunological approach, the material identified here may play a direct or indirect role in cell-cell adhesion and merits tentative consideration as a cell-adhesion molecule.
Partial purification and characterization of a renotropic fraction from ovine pituitaries.
Authors: Nomura K , Puett D , Nicholson W E , Liddle G W
Published: May 2006

It has been previously established that hypophysectomy leads to renal atrophy in rats and that a crude pituitary-derived fraction is effective in restoring kidney weight to the level expected for intact animals of the same body weight. This paper reports that considerable purification of the crude renotropic fraction from ovine pituitaries has been achieved and that the purified fraction is capable of restoring kidney weights of hypophysectomized castrated rats to normal values. For example, after five daily subcutaneous injections (135 micrograms/day) there were significant increases in dry kidney weight and total renal protein and DNA. The pituitary-derived fraction was devoid of somatotropin, contained only trace amounts of corticotropin, gamma-lipotropin, vasopressin, and prolactin, and had only low levels of thyrotropin and follitropin. Daily injections of prolactin, thyrotropin, and follitropin in doses of 20 micrograms each failed to stimulate renal growth in hypophysectomized rats. Thus, it seems highly unlikely that these factors are responsible for the observed renal hyperplasia after treatment with the pituitary fraction. The purified renotropic fraction had an isoelectric pH between 8 and 9. On polyacrylamide gel electrophoresis in the presence of detergent and a reducing agent, the renotropic fraction exhibited two major bands and one minor band with mobilities that corresponded to those of a standard lutropin preparation. The renotropic fraction exhibited considerable crossreactivity with an antiserum directed against the lutropin alpha subunit, suggesting the presence of the common glycoprotein hormone subunit. Moreover, the purified fraction stimulated steroid production by Leydig tumor cells in vitro. It is noteworthy, however, that standard ovine lutropin at 135 micrograms/day failed to exhibit renotropic activity in hypophysectomized castrated rats, although effects were noted at twice that dose. It appears that the renotropic activity represents a pituitary substance that can be separated from lutropin only with difficulty.
Opioid polypeptides in guinea pig pancreas.
Authors: Stern A S , Wurzburger R J , Barkey R , Spector S
Published: May 2006

Extracts from guinea pig pancreas were found to contain high molecular weight enkephalin-containing polypeptides as well as free [Met]enkephalin and [Leu]enkephalin and the hexa-, hepta-, and octapeptides derived from proenkephalin. Within the limits of sensitivity of our assay (10 fmol/g of tissue), beta-endorphin and beta-lipotropin were undetectable.
Structure of columns in monkey striate cortex induced by luminant-contrast and color-contrast stimulation.
Authors: Crawford M L , Meharg L S , Johnston D A
Published: May 2006

Color and black and white line stimulation produce qualitatively and quantitatively different 2-deoxy[14C]glucose column patterns in the striate cortex of macaque monkeys. Microdensitometry of these stimulus-dependent columns reveals interlaminar density differences that serve as a signature for the stimulus conditions which produced them. Such columns are individually distinct and form a three-dimensional mosaic throughout striate cortex.
Partial characterization of a phosphorylated intermediate associated with the plasma membrane ATPase of corn roots
Authors: Briskin Donald P. , Leonard Robert T.
Published: May 2006

The phosphorylated protein associated with a deoxycholate-extracted plasma membrane fraction from corn (
Zea mays
L. var WF9 × Mol7) roots was characterized in order to correlate its properties with those of plasma membrane ATPase. Its phosphorylation, like that of plasma membrane ATPase, was dependent on Mg
2+
, substrate specific for ATP, insensitive to azide, oligomycin, or molybdate, and sensitive to
N,N
′-dicyclohexylcarbodiimide, diethylstilbestrol, or vanadate. Monovalent cations affected the phosphorylation of the protein in a manner consistent with their stimulatory effects on ATPase. For K
+
, this was shown to occur through an increase in the turnover of the phosphoenzyme. Analysis of the phosphorylated protein by NaDodSO
4
/polyacrylamide gel electrophoresis revealed the presence of a single labeled polypeptide with a molecular weight of about 100,000. Phosphorylation of this polypeptide was dependent on Mg
2+
, sensitive to K
+
, and inhibited by vanadate. It is concluded that this polypeptide represents the catalytic subunit of the plasma membrane ATPase. These results are discussed in terms of a model for the coupling of metabolic energy to H
+
and K
+
transport in higher plants.
Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon.
Authors: Johnston S A , Hopper J E
Published: May 2006

GAL4 is a classically defined positive regulatory gene controlling the five inducible structural genes of galactose/melibiose utilization in yeast. The positive regulatory function of the GAL4 gene product in turn is controlled by the product of another gene, the negative regulator GAL80. We have cloned a 3.1-kilobase fragment containing GAL4 by homologous complementation using the multicopy chimeric vector YEp24 and demonstrated that multiple copies of GAL4 in yeast have pronounced dosage effects on the expression of the structural genes. Yeast transformed with GAL4-bearing plasmid become constitutive for expression of the galactose/melibiose genes, even in normally repressing (glucose) medium. Multiple copies of the GAL4 plasmid also increase expression of the structural genes in inducing (galactose) medium and can partially overcome the effects of a dominant super-repressor mutant, GAL80S. Using an internal deletion in GAL4, we have demonstrated that these dosage effects are due to overproduction of GAL4 positive regulatory product rather than an effect of the flanking sequences titrating out a negative regulator. These results point to the importance of competitive interplay between the positive and negative regulatory proteins in the control of this system. We have also used the dosage effect of GAL4 plasmid in combination with different GAL4 and GAL80 alleles to create new phenotypes. We interpret these phenotypes as indicating that (i) the repressing effects of glucose, at least in part, are mediated by the product of the negative regulatory gene, GAL80, and (ii) the GAL80 protein may have specific interactions with the control regions of the structural genes.
Inhibition of cell growth by a hypothalamic peptide.
Authors: Redding T W , Schally A V
Published: May 2006

A fraction purified from acetic acid extracts of porcine hypothalami was found to contain significant antimitogenic activity when tested in normal and neoplastic cell lines. Addition of this hypothalamic material (1-100 micrograms/ml) to culture media significantly inhibited [3H]thymidine incorporation into cellular DNA in several cell lines. Amino acid incorporation into pituitary proteins and uridine incorporation into RNA were also significantly reduced by this factor(s). Addition to the culture media of this hypothalamic material at 5 micrograms/ml and 50 micrograms/ml per day decreased by 17% and 36%, respectively, cell numbers of 3T6 fibroblast cell cultures. Time-response curves showed that the inhibition of [3H]thymidine incorporation into DNA in 3T6 fibroblast cells begins within 2 hr after adding this fraction to the culture medium. The inhibitory action cannot be explained by a direct cytotoxic effect since 3T6 cells labeled with 51Cr and incubated for 6 hr in the presence of this hypothalamic fraction fail to show an increase in the release of 51Cr into the medium as compared with controls. Incubation with trypsin and chymotrypsin completely abolished the antimitogenic activity of this material and pepsin decreased it. This strongly suggests that the antimitogenic activity exhibited by this fraction is due to a polypeptide(s). These observations provide evidence for the presence in the mammalian hypothalamus of an antimitogenic peptide(s) that may be involved in the regulation of cell proliferation.
Fluorine-19 chemical shifts as structural probes of metal-sulfur clusters and the cofactor of nitrogenase.
Authors: Mascharak P K , Smith M C , Armstrong W H , Burgess B K , Holm R H
Published: May 2006

Several properties of the FeMo-cofactor (co) of nitrogenase in N-methylformamide solution at ambient temperature have been investigated by means of 19F NMR spectroscopy. With C6H5CF3 reference signals the magnetic moment per Mo atom was found to be approximately equal to 3.9 BM, consistent with S = 3/2 ground state identified by other spectroscopic methods at low temperature. Reaction of FeMo-co with 1.0 eq of RFS- (RF = p-C6H4CF3, p-C6H4F) afforded isotropically shifted signals indicative of binding to a paramagnetic cluster. By comparison with the spectra of Fe-S and Fe-Mo-S species derivatized with RFS-, including the cubane-type MoFe3S4 clusters with S = 3/2 ground states, it was concluded that the essential FeMo-co cluster structure remains intact and a Fe atom is the probable thiolate binding site. An interaction of FeMo-co with C6H5S- had been detected earlier by low temperature EPR spectroscopy. The binding site assignment is based on large observed isotropic shifts (ca. -12ppm) compared to the much smaller values found for Mo-SRF ligands in MoFe3S4 clusters and anticipated in FeMo-co on the basis of recent spectroscopic results. Isotropic 19F shifts have proven extremely sensitive to electronic and structural features of Fe-S and Fe-Mo-S clusters. The inclusion of a 19F NMR label in FeMo-co should prove of utility in further investigation of cofactor properties and reactions.
Rules governing the composition of revolving clusters in quasiband and prolate-deformation states of atomic nuclei
Authors: Pauling Linus
Published: May 2006

A set of rules, involving the magic and semimagic values of neutron and proton numbers and the proton/neutron ratio, is formulated for the composition of the revolving clusters producing the values of the moment of inertia given by the differences in energy of the adjacent levels in quasibands and bands of nuclei. The cluster compositions assigned with use of these rules to isotopes of Kr, Sr, Zr, Mo, and the actinon nuclei and to successive levels of the ground-state band of
158
Er lead to reasonable values of the radius of revolution (the distance from the center of the nonrevolving sphere to the center of the cluster). These values correspond to a spheron diameter of about 3.20 fm.