bluuebunny/crossref_metadata_2025 huggingface dataset:
Transit of pp60v-src to the plasma membrane.
Authors: Courtneidge S A , Bishop J M
Published: May 2006

The protein kinase (pp60v-src) encoded by the transforming gene (v-src) of Rous sarcoma virus is synthesized on free polyribosomes and then translocated to the plasma membrane of infected cells. Neither the mechanism of the translocation nor the physiological significance of the membrane localization has been elucidated. We have explored these problems by pursuing previous observations of a complex between pp60v-src and two cellular proteins with molecular weights of 50,000 and 89,000. We found the complex located entirely in the cytoplasm, where it appears to form immediately after the synthesis of pp60v-src. While in the complex, pp60v-src has little detectable kinase activity and is phosphorylated predominantly on serine. After transfer from the complex to the plasma membrane, pp60v-src becomes phosphorylated on tyrosine as well as serine and acquires kinase activity. Under restrictive conditions, temperature-sensitive pp60v-src is produced in normal quantities, but translocation to the plasma membrane is diminished. As an apparent consequence, the cytoplasmic complex accumulates to abnormal abundance. Alternatively, temperature-sensitive pp60v-src that has been synthesized and translocated to the plasma membrane under permissive conditions appears to be released from the membrane and returns to the cytoplasmic complex when the infected cells are shifted to the restrictive temperature. We conclude that the cytoplasmic complex may be the vehicle by which pp60v-src reaches the plasma membrane. It is possible that other proteins may follow a similar route to the membrane. Binding to plasma membrane appears to be a discrete step in the biogenesis of pp60v-src and may be essential to the function of the protein.
Electron paramagnetic resonance crystallography of bacterial catalase: g-Contour mapping method of analysis.
Authors: Dickinson L C , Chien J C , Marie A L , Parak F
Published: May 2006

Single crystals of bacterial catalase from Micrococcus luteus have been examined by EPR at 77 K. X-ray perfect crystals gave a set of four prominent resonances in all three orthogonal planes which yielded eight heme direction cosine matrices to an accuracy of +/- 2 degrees as expected for the P4(2)2(1)2 space group and unit cell parameters previously determined. These matrices are related by D4 symmetry operation of the space group. There were additional weaker resonances only resolved in two or even one plane. A method of g-contour mapping was devised to solve for the orientations of hemes that give rise to these weaker resonances. Three additional sets of heme orientations, also following D4 symmetry, were determined. All of the above sites have the same principal g values, 2.0, 5.4, and 6.6. The EPR crystallographic results imply that several conformational substates may be trapped at 77 K.
Cell types and morphogenesis in the mammary gland.
Authors: Dulbecco R , Henahan M , Armstrong B
Published: May 2006

Cell types present in the mammary gland and their evolution were studied by labeling female rats with radioactive thymidine at various phases of the estrus cycle. The results suggest that the stem cells for mammary development are present in the terminal end buds and that they generate a lineage for lumenal cells and possibly a distinct one for myoepithelial cells. Growth and differentiation are controlled by both hormones and local factors.
Development and characterization of a tissue culture cell line with essential fatty acid deficiency.
Authors: Laposata M , Prescott S M , Bross T E , Majerus P W
Published: May 2006

We have developed an essential fatty acid-deficient cell line from a parental cell line, HSDM1C1, which metabolizes arachidonic acid to prostaglandin E2 (PGE2). This cell line, designated EFD-1, is depleted of arachidonate, is unable to synthesize PGE2 in response to bradykinin, and has changes in fatty acid composition characteristic of tissues from animals with essential fatty acid deficiency. Within 15 min of repletion by arachidonate, the ability to synthesize PGE2 is restored. Linoleate also is able to restore PGE2 synthesis, indicating that deficient cells contain both the rate-limiting delta 6 desaturase enzyme and the delta 5 desaturase enzyme, which are required to form arachidonate. When parental cells are incubated in lipid-free medium, there is rapid induction of the ability to convert linoleate to arachidonate. Arachidonate prevents this induction, suggesting that icosanoid precursor availability controls the rate of arachidonate formation.
Two human c-onc genes are located on the long arm of chromosome 8.
Authors: Neel B G , Jhanwar S C , Chaganti R S , Hayward W S
Published: May 2006

We have used in situ chromosome hybridization techniques to map the human cellular counterparts (c-onc genes) of the transforming genes of two RNA tumor viruses on human meiotic pachytene and somatic metaphase chromosomes. We find that the human c-mos gene is located on chromosome 8 at a position corresponding to band 8q22 on the somatic map. The human c-myc gene is found on chromosome 8 at position 8q24. These regions on the long arm of chromosome 8 have been previously reported to be involved in specific translocations found in the M-2 subset of acute nonlymphoblastic leukemias. Burkitt lymphoma, and other forms of non-Hodgkin lymphoma, and a familial abnormality that predisposes to renal cell carcinoma. These results suggest that translocations of the human c-mos or c-myc genes may be causally related to neoplastic transformation.
Anti-idiotypes against anti-H-2 monoclonal antibodies: structural analysis of the molecules induced by in vivo anti-idiotype treatment.
Authors: Bluestone J A , Krutzsch H C , Auchincloss H , Cazenave P A , Kindt T J , Sachs D H
Published: May 2006

Previous studies have shown that treatment of mice in vivo with xenogeneic anti-idiotype produced against a monoclonal anti-H-2Kk antibody, 11-4.1, leads to the induction of molecules (Id') that inhibit the binding of anti-idiotype to idiotype. To investigate the nature of these Id' molecules, spleens from such anti-idiotype-treated mice were fused with the SP2/0 myeloma to produce monoclonal Id' antibodies. All four monoclonal Id' antibodies were found to react with goat and rabbit anti-11-4.1 in addition to the pig anti-idiotype used for their induction and one of the four, J1-8-1, reacted with syngeneic BALB/c anti-11-4.1. Partial amino acid sequences were determined for the heavy and light chains of these monoclonal antibodies. J1-8-1 heavy chain had an NH2-terminal amino acid sequence identical to that of 11-4.1 for the 39 NH2-terminal residues assigned, whereas its light chain and the heavy and light chains of the other Id' molecules differed markedly from those of the 11-4.1 antibody. Isolated heavy chains and light chains of J1-8-1 and 11-4.1 were reassociated in homologous and heterologous pairs. When J1-8-1 heavy chains and 11-4.1 light chains were mixed in equimolar concentrations, anti-H-2Kk reactivity was found at a level approximately 10% of that observed for reassociated 11-4.1 homologous heavy and light chains. The finding that in vivo anti-idiotype treatment can trigger Id' molecules structurally similar to the original idiotype has implications regarding the mechanism of induction of Id' molecules and the regulation of repertoire expression by idiotypic networks.
On a generalization of a theorem of Erich Hecke
Authors: Lee Ronnie , Weintraub Steven H.
Published: May 2006

E. Hecke initiated the application of representation theory to the study of cusp forms. He showed that, for
p
a prime congruent to 3 mod 4, the difference of multiplicities of certain conjugate representations of
SL
(

F
p

) on cusp forms of degree 1, level
p
, and weight ≥2 is given by the class number
h
(-
p
) of the field
Q
(√-
p
). We apply the holomorphic Lefschetz theorem to actions on the Igusa compactification of the Siegel moduli space of degree 2 to compute the values of characters of the representations of
Sp
4
(

F
p

) on certain spaces of cusp forms of degree 2 and level
p
at parabolic elements of this group. Our results imply that here too, the difference in multiplicities of conjugate representations of
Sp
4
(

F
p

) is a multiple of
h
(-
p
).
Lipoprotein lipase suppression in 3T3-L1 cells by an endotoxin-induced mediator from exudate cells.
Authors: Kawakami M , Pekala P H , Lane M D , Cerami A
Published: May 2006

Conditioned medium from cultures of mouse peritoneal exudate cells incubated wih endotoxin contains a mediator that markedly suppresses (greater than 90%) lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) activity in differentiating 3T3-L1 mouse preadipocytes. The effect is dependent upon the amount of mediator and is evident as early as 30 min after the addition of the mediator-containing medium to 3T3-L1 cell cultures. Neither endotoxin nor conditioned medium from cultures of exudate cells not exposed to endotoxin shows the presence of the mediator. Lysates of the exudate cells are also unable to suppress the lipase activity. Increasing the amount of insulin does not reverse this suppression, even at 1000 times the concentration used for standard experiments. The lipoprotein lipase suppression mediator present in the conditioned medium of endotoxin-treated exudate cells is heat labile and has an apparent molecular weight of at least 12,000. The mediator does not inhibit lipoprotein lipase activity directly nor does it affect the half-life of enzyme activity released in the medium. The present study demonstrates that endotoxin promotes the release of a mediator from exudate cells that suppresses the activity of lipoprotein lipase in 3T3-L1 preadipocytes.
Inferences from the equations used for describing the chemistry of contraction.
Authors: Morales M F
Published: May 2006

Schemes describing the chemical kinetic behavior of actin/myosin/ATP (i.e., contractile) systems are often describable as circles of isomerizations. In this paper explicit formulas for the steady-state concentrations of intermediate complexes and of the net reaction rate are given, and then the power spectrum of the concentration fluctuations is derived explicitly for certain cases, in a manner applicable to experiments.
Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.
Authors: Weiher H , Schaller H
Published: May 2006

A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands of a DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C leads to T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence.
Hydrogen exchange in RNase A: neutron diffraction study.
Authors: Wlodawer A , Sjölin L
Published: May 2006

Hydrogen exchange has been studied in a single crystal of RNase A [ribonuclease (pancreatic), EC 3.1.27.5] in the course of a neutron structure investigation. Refinement of the occupancies of amide hydrogens provided information about the kind of isotope present in each site and also provided estimates of the errors associated with the measurement. Twenty-eight of the 120 peptide amide hydrogens were found to be at least partially protected from exchange during approximately 1 year required for crystal preparation and data collection. Most of the protected hydrogens were involved in hydrogen bonds with main-chain carbonyl groups. A contiguous region of the beta-sheet containing residues 75, 106--109, 116, and 118 had a large number of protected hydrogens, indicating its low flexibility and the lack of accessibility to solvent. Residues 11--13 from the alpha-helix near the amino terminus were protected, in good agreement with a model of cooperative unwinding of this helix, starting from the free (amino) end.
Differences in temporal aspects of mutagenesis and cytotoxicity in Chinese hamster cells treated with methylating agents and thymidine.
Authors: Peterson A R , Peterson H
Published: May 2006

Equitoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MeMes) produced different frequencies of 8-azaguanine-resistant mutants and different amounts of N7-methylguanine, O6-methylguanine (m6G), and N3-methyladenine in the DNA of V79 Chinese hamster cells. Thus, neither the cytotoxicities nor the mutagenicities of these methylating agents could be attributed solely to nitrogen or to oxygen methylations in the DNA. However, MNNG produced 12-fold more m6G and 5-fold more mutants than did MeMes, indicating that a substantial part of the MNNG-induced mutations resulted from m6G--thymine mispairing during DNA replication. The expression as mutants of mutagenic oxygen methylations in the DNA of cells treated with MNNG was enhanced by thymidine (dThd) and deoxycytidine (dCyd), but these nucleosides did not significantly enhance MeMes-induced mutagenesis. The cytotoxicities of MNNG and MeMes were also increased by 10 microM dThd in proportion to the amount of m6G in the DNA. These increases in cytotoxicity were abolished by dCyd, which did not greatly reduce the dThd-induced enhancements of mutagenesis. Moreover, when dThd was present only during the 2-hr treatment with MNNG, maximal cytotoxicity occurred, but MNNG-induced mutagenesis was not increased. Maximal mutagenesis occurred when the dThd was present throughout the first doubling time of the MNNG-treated cells. Thus, the expression of the cytotoxicity and the mutagenicity associated with m6G in the DNA of V79 cells occurred by quite different mechanisms.
Abortive termination of bioBFCD RNA synthesized in vitro from the bioABFCD operon of Escherichia coli K-12.
Authors: Nath S K , Guha A
Published: May 2006

The nature of divergent transcription from the bioABFCD gene cluster of Escherichia coli K-12 has been studied in vitro by using DNA restriction fragments as templates. The in vitro RNA transcript initiated at the promoter pa was found to be similar to the in vivo bioA transcript, whereas the transcription initiated at pB produced a small piece of RNA less than 200 nucleotides long. Substituting ITP for GTP or using an S100 cell-free extract as a source of antiterminator permitted transcription of the bioBFCD gene cluster initiating at the promoter pB to be read through. The possible site for in vitro transcription termination in the bioB region is discussed.
Ochotona princeps (pika) myoglobin: an appraisal of lagomorph phylogeny.
Authors: Dene H , Goodman M , McKenna M C , Romero-Herrera A E
Published: May 2006

Myoglobin was purified from skeletal muscle of the pika (Ochotona princeps) and its primary structure was determined. This sequence was added to the set of 64 already known vertebrate myoglobin sequences and used to evaluate the phylogenetic position of the pika among other mammals, using a computerized search procedure based on maximum parsimony criteria. The pika is clearly related to the rabbit (Oryctolagus cuniculus), with which it is traditionally associated as a member of the order Lagomorpha. A monophyletic group composed of Lagomorpha, Scandentia, and Carnivora is a consistent feature of the dendrograms produced. An association of Carnivora and Lagomorpha casts doubt on the results of those investigations using rabbit antisera in systematic studies and depending on carnivores as an outside reference group for cladogram construction.
DNA transfer of focus- and tumor-forming ability into nontumorigenic CHEF cells.
Authors: Smith B L , Anisowicz A , Chodosh L A , Sager R
Published: May 2006

CHEF/18 fibroblastic cells derived from a Chinese hamster embryo are diploid and nontumorigenic and require multiple steps of chemical treatment and selection to produce tumorigenic derivatives. In this report, CHEF/18 cells and a mutant capable of growing in medium with a low concentration of serum, LS1-1, were recipients in DNA transfer experiments using the calcium phosphate coprecipitation method. Focus formation with donor DNAs from tumor-derived CHEF cells and from human bladder carcinoma cell line EJ gave yields of 0.02-0.59 focus per microgram of DNA per 10(6) recipients. In one experiment in which CHEF/18 cells were transfected with EJ DNA, the presence of human DNA was detected in five of seven foci by using a cloned Alu sequence. Cells from one of these foci gave rise to tumors in nude mice, and the DNA produced secondary CHEF/18 transfectants. Because normal human cells as well as CHEF/18 cells require multiple stages to become tumorigenic, these findings suggest that EJ cells contain tumor-inducing DNA as the result of prior changes that occurred during the development of this carcinoma.
Isolation and characterization of retrovirus from cell lines of human adult T-cell leukemia and its implication in the disease.
Authors: Yoshida M , Miyoshi I , Hinuma Y
Published: May 2006

A retrovirus (ATLV) was unequivocally demonstrated in human adult T-cell leukemia (ATL) cell lines by density (1.152-1.155 g/cm3) in a sucrose gradient, reverse transcriptase activity insensitive to actinomycin D, RNA labeled with [3H]uridine, and specific proteins with molecular weights of 11,000, 14,000, 17,000, 24,000, and 45,000. Furthermore, cDNA prepared by endogenous reaction with detergent-treated virions hybridized to 35S RNA containing poly(A), which was inducible by IdUrd treatment of a T-cell line derived from leukemic cells of the ATL, and the integrated form of ATLV proviral DNA was detected in T-cell lines derived from ATL. The ATLV proviral DNA was also detected in fresh peripheral lymphocytes from all five patients with ATL tested so far but not in those from healthy adults. On the other hand, ATLV protein of Mr 42,000 was found to be at least one of the ATL-associated antigen(s) that were previously detected in ATL-leukemic cells by all sera from patients with ATL. These findings on the close association of ATLV protein and proviral DNA with ATL are direct evidence for the possible involvement of the retrovirus ATLV in leukemogenesis of human ATL.
Limited proteolysis of liver aldolase and fructose 1,6-bisphosphatase by lysosomal proteinases: effect on complex formation.
Authors: Pontremoli S , Melloni E , Michetti M , Salamino F , Sparatore B , Horecker B L
Published: May 2006

Cathepsin M, which catalyzes inactivation of both rabbit liver fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) and rabbit liver fructose 1,6-bisphosphatase (Fru-P2ase; EC 3.1.3.11), has been characterized as a peptidyl peptidase. Modification of the COOH terminus of aldolase by cathepsin M or by Fru-P2ase converting enzyme 2 abolishes its ability to bind to phosphocellulose P11 and to form the complex with Fru-P2ase. On the other hand, modification of the COOH terminus of Fru-P2ase does not affect its interaction with aldolase. This property is lost, however, when Fru-P2ase is modified in the NH2-terminal region by the converting enzyme or by subtilisin. The results suggest that interaction of aldolase and Fru-P2ase may involve the exposed COOH-terminal region of the former and an exposed proteinase-sensitive region located between residues 57 and 67 of the latter.
Beta subunits of human choriogonadotropin and ovine lutropin are biologically active.
Authors: Moudgal N R , Li C H
Published: May 2006

Highly purified beta subunits of human choriogonadotropin and ovine lutropin were found to possess measurable steroidogenic activity in the in vitro rat Leydig cell assay. In addition, beta subunits of these two gonadotropins were able to inhibit the biological activity of human choriogonadotropin in the rat Leydig cell assay.
Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion.
Authors: Tatemoto K
Published: May 2006

A new candidate hormone--designated peptide YY (PYY)--has been isolated from extracts of porcine upper intestinal tissue by using a novel chemical assay method. This peptide has been detected in the extracts by the presence of its unusual COOH-terminal tyrosine amide structure. The peptide consists of a linear chain of 36 amino acids and its complete amino acid sequence is: Tyr-Pro-Ala-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Ser-Arg-Tyr -Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2. This peptide has a distinct sequence homology to the pancreatic polypeptide. It is therefore proposed that PYY and the pancreatic polypeptide together form a new peptide family. PYY has been found to strongly inhibit both secretin- and cholecystokinin-stimulated pancreatic secretion in the anesthetized cat.
Reconstitution of rods from tobacco mosaic virus protein and RNA modified with bulky carcinogens.
Authors: Fraenkel-Conrat H , Singer B , Takanami Y , Santella R M , Grunberger D
Published: May 2006

Tobacco mosaic virus (TMV) RNA was treated with radioactive N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF) and (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BaP diol epoxide) to obtain 3-25 adducts per molecule. Modified full length 30S RNAs and unmodified RNA were reconstituted for various time periods with TMV protein. The particulate products were separated by ultracentrifugation, and the amounts of virus-like material were quantitated by UV spectrophotometry. The length distribution and general appearance of the virus-like rods were studied by electron microscopy. Neither type of carcinogen prevented typical rod formation, but the rate of formation and the maximal yield of reconstituted particles diminished with increasing modification by both agents. The rod length distribution also showed progressively lesser numbers of full-length virus rods. The particulate material contained approximately the same number of adducts as the modified RNA. Thus, it appears that these carcinogen modifications of guanine residues at the N-2 or C-8 atoms did not prevent orderly protein assembly on the RNA but instead slowed up this process and frequently stopped it, possibly at sites where adducts happen to be clustered.
Inverted repeats of Tn5 are transposable elements.
Authors: Berg D E , Johnsrud L , McDivitt L , Ramabhadran R , Hirschel B J
Published: May 2006

Experiments presented here show that each of the 1.5-kilobase inverted repeats of the kanamycin-resistance transposon Tn5 is transposable; we designate them IS50-L (left) and IS50-R (right). By DNA sequence analyses, IS50 is 1533 base pairs (bp) long and generates 9-bp direct repeats of target sequences. The ends of IS50 comprise a hyphenated 8-of-9-bp inverted repeat and are not used with equal efficiency; the outside ends are more active than the inside ends, suggesting that a strong transposase recognition site at the outside ends, suggesting that a strong transposase recognition site at the outside end extends beyond the 8 bp common to both ends.
Light-dependent effects of a hydrolysis-resistant analog of GTP on rod photoresponses in the toad retina.
Authors: Clack J W , Oakley B , Pepperberg D R
Published: May 2006

Responses to 100-ms flashes were recorded intracellularly from dark- and light-adapted rod photoreceptors in the isolated retina of the toad, Bufo marinus. Properties of photoresponses were analyzed under each condition of adaptation when retinas were superfused with 1.0 mM guanosine 5'-[beta, gamma-methylene]triphosphate (p[CH2]ppG), a hydrolysis-resistant analog od GTP. When applied to retinas that previously had been subjected to intense light (approximately 30% bleach), p[CH2]ppG increased both the amplitude and duration of photoresponses. By contrast, treatment of dark-adapted retinas with p[CH2]ppG did not alter these response parameters. When similarly applied to either dark- or light-adapted retinas, GTP had no effects on amplitude or duration of photoresponses. These results are discussed in terms of GTP-dependent mechanisms for rod adaptation.
Preparation of a monoclonal antibody to rat liver glucose-6-phosphate dehydrogenase and the study of its immunoreactivity with native and inactivated enzyme.
Authors: Dao M L , Johnson B C , Hartman P E
Published: May 2006

A monoclonal antibody of the IgG class was prepared against rat liver glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) by the hybridoma technique. This antibody does not affect the catalytic activity of the enzyme and shows crossreactivity with the palmitoyl CoA-inactivated G6PD. By solid phase radioimmunoassay, the presence of crossreacting materials in comparable amounts was determined in liver homogenate supernatants from rats that had been starved and refed a high-sucrose diet (containing a high level of G6PD activity) and from rats that had been starved and refed a high-fat diet (containing a low level of G6PD activity). These findings indicate that G6PD is present in an inactive form in rats fed a high-fat diet. The monoclonal antibody will facilitate isolation and characterization of the inactive variant G6PD.
Mevalonic acid in human plasma: relationship of concentration and circadian rhythm to cholesterol synthesis rates in man.
Authors: Parker T S , McNamara D J , Brown C , Garrigan O , Kolb R , Batwin H , Ahrens E H
Published: May 2006

We tested the hypothesis that the rate of cholesterol synthesis in tissues determines the concentrations of mevalonic acid (MVA) in plasma. We found that plasma MVA concentrations were correlated (i) with increased rates of whole-body cholesterol synthesis (measured by sterol-balance methods) in patients treated with cholestryamine resin (ii) with decreased rates of whole-body sterol synthesis (indicated by conversion of labeled acetate to sterol in freshly isolated mononuclear leukocytes) in out-patients after 4 weeks on a cholesterol-rich diet. In addition, a diurnal rhythm of plasma MVA concentrations was observed in patients whose activities were strictly controlled on a metabolic ward. At the peak of the rhythm (between midnight and 3 a.m.) MVA concentrations were 3-5 times greater than at the nadir (between 9 a.m. and noon). Furthermore, a relationship between the diurnal rhythm of plasma MVA and endogenous cholesterol synthesis is suggested by our finding that the plasma MVA rhythm was suppressed by cholesterol feeding (1,200 mg/day) and abolished by a 12-day fast. The presence in human plasma of MVA, an obligate precursor of cholesterol, in amounts apparent related to the rate of cholesterol synthesis offers a noninvasive, nonisotopic method for studying cholesterol synthesis in man.
Indirect induction of erythroid differentiation in mouse Friend cells: evidence for two intracellular reactions involved in the differentiation.
Authors: Nomura S , Oishi M
Published: May 2006

The mechanism of in vitro erythroid differentiation in mouse Friend cells was studied by employing cell fusion between two genetically marked Friend cells and other nonerythroid cells, including BHK (baby hamster kidney) and FM3A (mouse mammary gland) cells. We were able to induce erythroid differentiation indirectly by fusing Friend cells that had been exposed briefly to dimethyl sulfoxide prior to fusion with nonerythroid cells that had been treated with ultraviolet light (or other DNA-damaging agents). The results suggest that two distinct reactions are involved in erythroid differentiation in Friend cells in vitro. One reaction, originating from the damaged DNA (or inhibition of DNA replication as a consequence), exhibits an inducible nature, is nonspecific to Friend cells, and is trans-acting. The other reaction is specific to Friend cells and most likely is cis-acting. We also present evidence from the cell fusion experiments that a typical tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, inhibits erythroid differentiation by affecting the latter reaction. The biological significance of these findings is discussed.
Presence of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, a precursor of a mutagenic nitroso compound, in soy sauce.
Authors: Wakabayashi K , Ochiai M , Saitô H , Tsuda M , Suwa Y , Nagao M , Sugimura T
Published: May 2006

After treatment with nitrite, Japanese soy sauce was strongly mutagenic to Salmonella typhimurium TA100 without S9 mixture. Two precursors of the mutagen were isolated from Japanese soy sauce, and these were identified as (-)-(1S,3S)-1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid [(-)-(1S,3S)-MTCA] and its stereoisomer (-)-(1R,3S)-MTCA. After treatment with nitrite, 1-mg samples of these compounds induced 17,400 and 13,000 revertants of TA100, respectively, without S9 mixture. Quantitative analysis of various kinds of soy sauces produced in Japan showed the presence of 82-678 micrograms of MTCA per ml. The mutagenicities of these compounds with nitrite accounted for 16-61% of the total mutagenicity of soy sauce with nitrite. Most soy sauces produced in the United States were less mutagenic than those produced in Japan and little, if any, of these two precursors of the mutagen was found in them. A major reaction product of (-)-(1S,3S)-MTCA and nitrite was a compound having a nitroso substitution at position N-2, but this compound was not mutagenic. Thus, the mutagen(s) formed from (-)-(1S,3S)-MTCA and nitrite was a minor product(s), and its specific mutagenic activity must be very high.
Isolation of genes expressed preferentially during sporulation in the yeast Saccharomyces cerevisiae.
Authors: Clancy M J , Buten-Magee B , Straight D J , Kennedy A L , Partridge R M , Magee P T
Published: May 2006

A library of Saccharomyces cerevisiae DNA in the vector lambda Charon 28 was probed for sequences complementary to cDNA made from poly(A)+ RNA isolated from the well-sporulating yeast strain AP1 a/alpha. The RNA was isolated from cells that had been incubated 7, 9, 11, and 13 hr in sporulation medium. DNA complementary to poly(A)+ RNA from alpha/alpha(nonsporulating) AP1 was used as a control, and 46 bacteriophage that gave a stronger response with a/alpha cDNA than with alpha/alpha cDNA were obtained in a screening of three yeast genomes worth of DNA. Two of the bacteriophage appeared to contain a/alpha-specific genes, in that they hybridized to cDNA from vegetative a/alpha RNA. The rest appeared to correspond to a/alpha genes expressed preferentially during sporulation. Restriction endonuclease analysis of four of the cloned sequences revealed a single major region of transcription in each; these regions ranged in size from 2.5 to 4.0 kilobases. RNA blot analysis showed that, in three of the four cases, transcripts of two different sizes were homologous to the cloned sequence. In all four cases, the homologous transcripts appeared at about 7 hr and were decreasing in amount by 13 hr. These results provide evidence for transcriptional control of genes expressed during sporulation and for at least one group of genes that is turned on at about the time of meiosis I in sporulation.
Monoclonal antibodies to renal brush border membrane maltase: age-associated antigenic alterations.
Authors: Reiss U , Sacktor B
Published: May 2006

Monoclonal antibodies to maltase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from young adult and aged rats were prepared by the hybridoma technique. Four cell lines producing antibodies of the IgG1 subclass to maltase were established. Two, designated 1F12E1 and 8B1G6, produced monoclonal antibodies specific for the catalytically active form of the enzyme found predominantly in enzyme preparations from young animals. The other two clones designated 7G10H3 and 2E1C10 produced monoclonal antibodies that reacted exclusively with an enzymatically inactive form of maltase found mostly in enzyme preparations from aged rats. The increased prevalence of an inactive form of the enzyme in the old rat accounts for the decreased maltase-specific activity previously reported in the senescent rat. The active and inactive maltase species were separated by immunoaffinity chromatography by using the monoclonal antibodies as ligands. The separated forms of the enzyme were not distinguished by NaDodSO4/polyacrylamide gel electrophoresis, peptide mapping of the CNBr-cleaved proteins, and the NH2-terminal residues of these peptides. This study demonstrates the presence of an altered, antigenically distinct enzyme in senescent animals. Critical issues on the mechanism of the aging process may be addressed by application of these findings.
Characterization of an interferon receptor on human lymphoblastoid cells.
Authors: Faltynek C R , Branca A A , McCandless S , Baglioni C
Published: May 2006

A cell-free assay was developed to measure the binding of iodinated human interferon-alpha 2 to membranes prepared from lymphoblastoid Daudi cells. The kinetics of binding were similar at 0 degrees C and 30 degrees C, with 1.3-fold more interferon bound at the higher temperature. Membrane preparations treated with Triton X-100 proved to be a convenient source of solubilized receptor. An assay was developed to measure the binding of 125I-labeled interferon (125I-interferon) to solubilized receptors, based on the precipitation of interferon-receptor complexes with polyethylene glycol. Optimal binding with this assay was obtained at 0 degrees C. The solubilized receptor was analyzed by zonal sedimentation centrifugation and gel filtration. Sedimentation analysis in H2O and 2H2O gradients provided the sedimentation coefficient and the partial specific volume of the receptor-Triton X-100 complex. Gel filtration chromatography provided the Stokes radius of this complex. From these data we calculated several physical parameters, including Mr = 95,000 for the protein portion of the complex. The receptor is a highly asymmetric and hydrophobic membrane protein. 125I-Interferon could be crosslinked to receptors of intact Daudi cells or of isolated membranes by use of disuccinimidyl suberate. The covalently linked 125I-interferon-receptor complexes were analyzed by gel electrophoresis. A single band with Mr = 140,000 was detected in gel autoradiographs. If one molecule of interferon is present in this complex, the Mr of the receptor is close to 120,000. Possible reasons for the different Mr values obtained with the two analytical procedures used are discussed.
Preferential utilization of exogenously supplied leucine for protein synthesis in estradiol-induced and uninduced cockerel liver explants.
Authors: Gehrke L , Ilan J
Published: May 2006

A cockerel liver explant system has been used to study protein synthesis and ribosome transit times. After a 2-hr preincubation of explant tissue in the presence of a large concentration of nonradioactive leucine, a small quantity of [3H]leucine was added and the kinetics of uptake of [3H]leucine into the intracellular acid-soluble leucine pool was compared to the incorporation of [3H]leucine into protein. Incorporation of [3H]leucine into protein reaches a linear rate almost immediately after addition of label, whereas the acid-soluble pool does not reach constant specific activity until much later. The length of time needed to reach a linear rate of incorporation of [3H]leucine into protein is approximately equal to the length of time needed to equilibrate nascent polypeptide chains with labeled precursor--that is, one average ribosome transit time. Therefore, it seems that the immediate precursor pool for protein synthesis reaches constant specific activity almost instantly after addition of [3H]leucine. The results indicate that at least part of the supply of leucine for protein synthesis is derived directly from the exogenous incubation medium and not from the intracellular acid-soluble amino acid pool.
Whole-body irradiation of deuterated mice by the 10B(n, alpha)7Li reaction.
Authors: Slatkin D N , Stoner R D , Gremme A M , Fairchild R G , Laissue J A
Published: May 2006

Specific pathogen-free mice, 8-12 wk of age, were supplied with either acidified tap water or acidified 30 atom % 2H2O in tap water. Thirteen days later, when body water deuterium was about 20 atom %, mice were irradiated either by neutrons or by x-rays after intraperitoneal injection of boric acid. Mortality from whole-body neutron-boron radiation, unlike mortality from whole-body x-radiation, was not lowered by such deuteration. Time intervals to death of neutron-irradiated mice were compatible with the gastrointestinal syndrome. Neither species nor numbers of colonic bacteria were measurably altered by deuteration alone. Because the toxic, nonlethal range of deuterium substitution for aqueous hydrogen in mammals is approximately 1/5th to 1/3rd, these results indicate that partial deuteration of human tissues would improve neutron capture therapy of deep tumors. Neutron penetration would be enhanced and damage to normal tissues from photons would be decreased. The number of deuterium recoils due to neutron capture by hydrogen would also be decreased.
Defects in functional expression of an influenza virus hemagglutinin lacking the signal peptide sequences.
Authors: Sekikawa K , Lai C J
Published: May 2006

We have investigated the requirement of the signal sequence for expression of influenza virus hemagglutinin (HA). For this purpose we used a recombinant prepared from a late-region deletion mutant of simian virus 40 (SV40) and cloned influenza HA DNA; the influenza DNA was inserted into the late region of SV40 previously occupied by the deleted sequences coding for SV40 capsid proteins. A simple in-phase deletion was made in the HA DNA, resulting in loss of 11 internal amino acids from the 16 amino acid signal peptide. This deletion HA recombinant was then used to infect African green monkey kidney cells. Mutant HA was not detected on the cell surface but stably accumulated in the cytoplasm at a level similar to that of wild-type HA. NaDodSO4/polyacrylamide gel analysis of lysates from infected cells showed that mutant HA was not glycosylated. Significantly, the amount of mutant HA synthesized was not affected by tunicamycin. In contrast, wild-type HA was decreased more than 90% by tunicamycin. These findings suggest that mutant polypeptide is synthesized on free polyribosomes rather than on membrane-bound polyribosomes. The mutant HA failed to agglutinate erythrocytes, probably due to a defect directly or indirectly associated with the lack of carbohydrate side chains.
Translocation pathway in the catalysis of active transport.
Authors: Tanford C
Published: May 2006

Possible pathways for translocation across the membrane in active transport processes are examined theoretically. Thermodynamic and kinetic requirements are readily satisfied by an alternating-access mechanism of the kind that has been proposed in the past by several investigators. The essential features of this mechanism (for transport of a single species) are shown to be defined by four explicit conditions. (i) The transport protein must have at least two distinct conformational states, each accessible from only one side of the membrane. (ii) Binding affinity for the transported species is high in the state accessible from the uptake side of the membrane and much lower in the state accessible from the discharge side. (iii) The change from one conformation to the other involves movement of the binding site itself (with the transported species remaining attached) or rearrangement within the site that is topologically equivalent to such movement. (iv) Return to the original conformation occurs with unoccupied binding sites. The analysis demonstrates that a passage through the membrane that is simultaneously accessible from both sides cannot be used for active transport regardless of what the energetics of opening or closing of the passage may be. Even movement from one fixed site to another within the protein, without access to the outside, is virtually excluded as a possible element of the central mechanism. A ligand conduction mechanism for ATP-linked ion transport is in principle conceivable but is subject to restrictions that make it improbable.
On an operator arising in the Calderón-Zygmund method of rotations and the Bramble-Hilbert lemma
Authors: Fefferman R.
Published: May 2006

In this article sharp estimates are obtained for an operator that is a quadratic average with respect to
v
of one-dimensional Hardy-Littlewood maximal functions in the directions
v
. This operator arises naturally in Fourier analysis and numerical analysis.
Bactobilin: blue bile pigment isolated from Clostridium tetanomorphum.
Authors: Brumm P J , Fried J , Friedmann H C
Published: May 2006

A blue bile pigment, possessing four acetic and four propionic acid side chains has been isolated from extracts of the anaerobic microorganism Clostridium tetanomorphum and in smaller amounts from Propionibacterium shermanii. The compound could be prepared in larger amounts by incubation of C. tetanomorphum enzyme extracts with added delta-aminolevulinic acid. The ultraviolet-visible, infrared, and proton magnetic resonance spectra of the pigment indicate a chromophore of the biliverdin type. Field-desorption mass spectrometry of the purified methyl ester showed a strong molecular ion at m/e = 962. This corresponds to the molecular weight expected for the octamethyl ester of a bilatriene type of bile pigment structurally derived from uroporphyrin III or I. Of the five possible structures, two could be eliminated by proton magnetic resonance spectroscopy. The name bactobilin is proposed for this previously unreported bile pigment.
Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors.
Authors: Davis A R , Bos T J , Nayak D P
Published: May 2006

We have replaced the late genes of simian virus 40 (SV40) with a cloned cDNA copy of the neuraminidase (NA; EC 3.2.1.18) gene of the WSN (H1N1) strain of human influenza virus. When the SV40-NA recombinant virus was complemented in a lytic infection of monkey cells with a helper virus containing an early region deletion mutant, influenza NA was expressed and readily detected by immunofluorescence as well as by immunoprecipitation of in vivo labeled proteins with monoclonal antibodies against NA. In addition, the expressed NA exhibited enzymatic activity by cleaving the sialic acid residue from alpha-2,3-sialyllactitol. The expressed protein was glycosylated and transported to the cell surface, and it possessed the same molecular weight as the NA of WSN virus grown in monkey cells. Because the structure of NA is quite different from that of other integral membrane proteins and includes an anchoring region at the NH2 terminus consisting of hydrophobic amino acids, we also constructed deletion mutants of NA in this region. Replacement of DNA coding for the first 10 NH2-terminal amino acids with SV40 and linker sequences had no apparent effect on NA expression, glycosylation, transport to the cell surface, or enzymatic activity. However, further deletion of NA DNA encoding the first 26 amino acids abolished NA expression. These data suggest that the hydrophobic NH2-terminal region is multifunctional and is important in biosynthesis and translocation of NA across the membrane as well as in anchoring the protein.
Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.
Authors: Saber M A , Zern M A , Shafritz D A
Published: May 2006

We present a simple and improved method for in situ localization of albumin and collagen mRNAs in isolated mouse hepatocytes. The cells were isolated by collagenase perfusion, mincing, and differential centrifugation. Nick-translated 3H-labeled mouse albumin cDNA (pmalb-2) and chicken pro-alpha 2(I) collagen cDNA (pCg45) probes were then hybridized with the cells in silane-treated microcentrifuge tubes. The cells were transferred and fixed to a microscope slide and hybridization was evaluated semiquantitatively by counting exposure of grains in autoradiographic emulsion placed over the cells. With this method of in situ hybridization, all hepatocytes appear to have significant, but highly variable, amounts of albumin mRNA. In addition, type I procollagen mRNA appears to be present at low abundance in hepatocytes. These results indicate that in situ hybridization can effectively demonstrate the presence of specific low- or high-abundance mRNAs in isolated well-differentiated eukaryotic cells.
Complete sequence of a chicken lambda light chain immunoglobulin derived from the nucleotide sequence of its mRNA.
Authors: Reynaud C A , Dahan A , Weill J C
Published: May 2006

Recombinant cDNA plasmids have been constructed from chicken spleen poly(A)-containing RNA. Two clones have been selected and provide the sequence determination of a chicken lambda immunoglobulin light chain: they include the complete variable, constant, and 3' untranslated regions of the chicken lambda light chain mRNA and part of the leader sequence. Comparison of the chicken light chain constant region with both human and mouse lambda constant sequences indicates 61% homology at the amino acid level. Unexpectedly, the chicken variable sequence is 53-63% homologous to human variable sequences when it is compared to the various lambda subgroups and only 42% homologous to the mouse V lambda 1 sequence. The degree of homology between the variable regions of these three species does not easily correlate with their phylogenetic relationship.
Frequency-dependent activation of glucose utilization in the superior cervical ganglion by electrical stimulation of cervical sympathetic trunk.
Authors: Yarowsky P , Kadekaro M , Sokoloff L
Published: May 2006

Electrical stimulation of the distal stump of the transected cervical sympathetic trunk produces a frequency-dependent activation of glucose utilization, measured by the deoxy[14C]glucose method, in the superior cervical ganglion of the urethane-anesthetized rat. The frequency dependence falls between 0-15 Hz; at 20 Hz the activation of glucose utilization is no greater than at 15 Hz. Deafferentation of the superior cervical ganglion is transection of the cervical sympathetic trunk does not diminish the rate of glucose utilization in the ganglion in the urethane-anesthetized rat. These results indicate that the rate of energy metabolism in an innervated neural structure is, at least in part, regulated by the impulse frequency of the electrical input to the structure, and this regulation may be an essential component of the mechanism of the coupling of metabolic activity to functional activity in the nervous system.
Genomic environment of variant surface antigen genes of Trypanosoma equiperdum.
Authors: Raibaud A , Gaillard C , Longacre S , Hibner U , Buck G , Bernardi G , Eisen H
Published: May 2006

Expression of variant antigen genes in Trypanosoma equiperdum is accompanied by the duplication of a silent basic copy gene and the transposition of the copy to an expression site elsewhere in the genome. We have analyzed the genomic locations of both the basic and expression-linked copies of the T. equiperdum gene for variable surface glycoprotein VSG-1. Both copies are situated proximal to termini in both extracted DNA and in chromatin. The regions between the VSG-1 genes and the termini have a very high buoyant density in CsCl and contain an unidentified nucleoside that replaces deoxycytidine.