bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-free synthesis and segregation of beta 2-microglobulin.
Authors: Lingappa V R , Cunningham B A , Jazwinski S M , Hopp T P , Blobel G , Edelman G M
Published: May 2006

beta2-Microglobulin has been synthesized in vitro by using a rabbit reticulocyte lysate system and mRNA from the mouse tumor cell line EL4. The molecule is synthesized as a precursor with an NH2-terminal extension of 19 amino acids: Ser-X-Ser-Val-X-Leu-Val-Phe-Leu-Val-Leu-Val-Ser-Leu-X-Gly-Leu-Tyr-X. The processing and segregation of this peripheral membrane protein are directly comparable to those of secretory proteins and integral membrane proteins: addition of dog pancreas microsomal membranes during translation caused conversion to the processed chain, but addition of membranes after synthesis did not; only the processed chain sedimented with the membrane vesicles and was protected from proteolysis by the vesicles; and processing of nascent beta 2-microglobulin was blocked by competitive inhibitors that prevent processing and segregation of secretory and integral membrane proteins. These results suggest that the signal sequences of secretory proteins, integral membrane proteins, and peripheral membrane proteins have a common function and a common receptor on the cytoplasmic face of dog pancreas microsomal membranes. This system also provides a means for studying in vitro the expression and function of the major histocompatibility antigens that are associated with beta 2-microglobulin on cell surfaces.
Protein kinase activity associated with the D2 hybrid protein related to simian virus 40 T antigen: Some characteristics of the reaction products
Authors: Baumann Ernst A. , Hand Roger
Published: May 2006

Protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) has been found associated with the D2 hybrid protein, a highly purified protein of 107,000 daltons specified by the adenovirus-simian virus 40 (SV40) hybrid Ad2
+
D2, which has many properties associated with authentic SV40 T antigen [Tjian, R. & Robbins, A. (1979)
Proc. Natl. Acad. Sci. USA
76, 610-614]. We have now examined some of the biochemical characteristics of the reaction products. Acceptors for the terminal phosphoryl group of [γ-
32
P]ATP are the purified protein itself and at least four proteins extracted from nuclei of uninfected cells. Purified histones do not serve as substrate for the enzyme. Phosphorylation is markedly reduced by heating the D2 hybrid protein to 50°C for 30 min. The products of phosphorylation are stable to treatment with ethanol/ether, DNase, and RNase, but completely degraded by digestion with Pronase, demonstrating their protein nature. The phosphate bonds are liable to hot alkali and sensitive to digestion with alkaline phosphatase but stable to treatment with hot acid or hydroxylamine. These results provide evidence that
32
P is incorporated into
O
-phosphoserine or
O
-phosphothreonine residues of acceptor proteins, indicating that the enzymatic activity is characteristic for protein kinase, and that cell-specified nuclear proteins other than histones may serve as substrates for the enzyme.
T4 DNA-delay proteins, required for specific DNA replication, form a complex that has ATP-dependent DNA topoisomerase activity.
Authors: Stetler G L , King G J , Huang W M
Published: May 2006

Under some conditions, T4 DNA replication requires the products of the DNA-delay genes, genes 39, 52, 58, and 60. By using an in vitro complementation assay that stimulates DNA replication in T4 39(-)-infected cell extracts, T4 gene 39 protein has been purified. The purified fraction also contains complementing activities for T4 genes 52 and 60. On sodium dodecyl sulfate/polyacrylamide gel analysis the purified preparation exhibits three protein components: a 51,000-dalton protein corresponding to the product of gene 52, a 64,000-dalton protein corresponding to the product of gene 39, and a 110,000-dalton protein. This purified fraction shows a DNA topoisomerase activity that untwists superhelical DNA in an ATP- and Mg2+-dependent reaction. The analogs adenylyl imidodiphosphate and adenyl [beta, gamma-methylene]diphosphonate cannot be used to replace ATP. The topoisomerase activity is not sensitive to the antibiotics oxolinic acid and novobiocin, known antagonists of Escherichia coli DNA gyrase. The possible relationship among the three polypeptides and their biological activities is discussed.
Effect of interferon on concentrations of cyclic nucleotides in cultured cells.
Authors: Tovey M G , Rochette-Egly C , Castagna M
Published: May 2006

Constant intracellular concentrations of both adenosine 3',5'-cyclic-monophosphate (cyclic AMP) and guanosine 3',5'-cyclic-monophosphate (cyclic GMP) were obtained when leukemia L1210 cells were cultivated under steady-state conditions in the chemostat. In this sensitive and controlled system addition of mouse interferon resulted in a rapid (5-10 min) increase in the intracellular concentration of cyclic GMP, which preceded by several hours an increase in the intracellular concentration of cyclic AMP. In contrast to the effect of interferon, addition of prostaglandin E1 induced a rapid increase in the intracellular concentration of cyclic AMP without markedly affecting the intracellular concentration of cyclic GMP. It is suggested that the rapid effect of interferon on cyclic GMP plays a role in mediating some of the effects of interferon on cells.
Morphological revertants of an avian sarcoma virus-transformed mammalian cell line exhibit tumorigenicity and contain pp60src.
Authors: Lau A F , Krzyzek R A , Brugge J S , Erikson R L , Schollmeyer J , Faras A J
Published: May 2006

The biological and biochemical properties of Rous sarcoma virus-transformed and revertant field vole cells were investigated. Revertant vole cells appear morphologically similar to normal, uninfected cells, yet, like transformed vole cells, they are fully capable of growing in agar suspension and producing tumors in athymic nude mice. These highly tumorigenic, yet morphologically normal appearing, vole cells express viral-specific antigens such as the gag gene product (Pr76) but lack the env gene protein (gp85). Moreover, they contain the src gene protein, pp60src. These results support the concept of the pleiotropic nature of the src gene product and in addition suggest that pp60src may have multiply mechanisms of action. With this revertant cell system it may be feasible to distinguish between those biochemical functions of the src gene product that are important for tumorigenicity in vivo and those that are related to in vitro morphological transformation.
Major intracellular cations and growth control: Correspondence among magnesium content, protein synthesis, and the onset of DNA synthesis in BALB/c3T3 cells
Authors: Rubin A. H. , Terasaki M. , Sanui H.
Published: May 2006

Omission of Ca
2+
from the medium of confluent BALB/c3T3 cells for a period of 17 hr causes a large decrease in the number of cells synthesizing DNA. This effect is reversed by raising the Mg
2+
concentration of the medium to 20 mM. However, if the [Mg
2+
] is greater than 20 mM (“ultra-high” Mg
2+
), there is again a decrease in the number of cells synthesizing DNA. The synthesis of protein has a similar dependence on Mg
2+
concentration in Ca
2+
-deficient medium, but it responds within 45 min of the shift in cation concentrations rather than the 10 hr that is required for the change in DNA synthesis to become apparent. Cells in the ultrahigh Mg
2+
concentrations that are at first inhibitory to protein synthesis later return to maximal protein synthesis. This delayed increase in protein synthesis is reflected in a delayed increase in DNA synthesis. Intracellular concentrations of Mg
2+
in Ca
2+
-deficient media increase in proportion to extracellular Mg
2+
concentrations. Cells in medium with 30 mM Mg
2+
have a high intracellular content of Mg
2+
at 3 hr but have decreased their intracellular content by 17 hr, a time at which protein synthesis has been restored to normal. Intracellular Na
+
and K
+
concentrations also change in Ca
2+
-deficient medium, but independent variation of these ions shows that protein synthesis is relatively insensitive to their concentration. Intracellular Ca
2+
remains fairly constant under all these conditions. The rate of protein synthesis of intact cells changes as a function of intracellular Mg
2+
content in a manner very similar to that which has been reported for cell-free systems. We conclude that protein synthesis is very sensitive to small changes in intracellular [Mg
2+
] within physiological range and that the onset of DNA synthesis is dependent on the rate of protein synthesis. Regulation of the availability of Mg
2+
within the cell therefore presents a plausible mechanism for growth control.
Loss of epidermal growth factor requirement and malignant transformation
Authors: Cherington Paul V. , Smith Barbara L. , Pardee Arthur B.
Published: May 2006

Serum provides growth factors that regulate and limit the growth of normal cells in tissue culture. Animal cells that are malignantly transformed usually exhibit diminished serum requirements for growth in culture. We have used a defined, serum-free medium to determine which of these growth factors becomes dispensable for the growth of transformed Syrian and Chinese hamster fibroblast cells. The medium's four growth factors—epidermal growth factor (EGF), insulin, fibroblast growth factor, and transferrin—were added or omitted as desired. A decreased requirement for EGF was most closely related to tumorigenicity of chemically (ethyl methanesulfonate) transformed cells in
nude
mice. All lines examined retained their requirement for transferrin, which is needed throughout the growth cycle, in contrast to the other factors, which are needed primarily in G
1
phase. Lines that had lost their EGF requirement but had retained their insulin requirement were arrested in G
1
by insulin deficiency, indicating that their growth control system remained. Mutagenesis with ethyl methanesulfonate can also create requirements of the transformed cells for unknown factors in serum. We conclude that an initial step that reduces the serum requirement in culture, and in tumorigenesis, is relaxation of the growth-regulatory function of EGF.
Non-H-2 antigens on fibroblasts and an embryocarcinoma cell line react with xenoantisera against H-2 antigens.
Authors: Kvist S , Ostberg L , Peterson P A
Published: May 2006

The murine embryocarcinoma cell line F9 lacks the classical transplantation antigens. However, rabbit anti-H-2 antigen sera, recognizing "backbone structures" of H-2 K and D antigens, react with three types of molecules manufactured by F9 cells. A 49,000- and a 25,000-dalton chain are glycoproteins located on the cell surface. The third component with an apparent molecular weight of 15,000, and the 25,000-dalton chain seem to be unrelated to the 49,000-dalton glycoprotein. The 25,000- and 15,000-dalton components are not manufactured by splenocytes, thymocytes, hepatocytes, or various lymphoma, mastocytoma, and plasmacytoma cell lines. However, fibroblasts derived from both embryos and adult animals synthesize 25,000- and 15,000-dalton molecules reactive with the rabbit anti-H-2 antigen sera. The 49,000-, 25,000-, and 15,000-dalton molecules are not recognized by a syngeneic anti-F9 cell serum.
13-Azaprostanoic acid: a specific antagonist of the human blood platelet thromboxane/endoperoxide receptor.
Authors: Le Breton G C , Venton D L , Enke S E , Halushka P V
Published: May 2006

A newly synthesized 13-aza derivative of prostanoic acid (13-APA) specifically inhibited human platelet aggregation induced by arachidonic acid, prostaglandin H2, or the stable endoperoxide analog (15S)-hydroxy-9 alpha,11 alpha-)epoxymethano)-prosta-5Z,13E-dienoic acid. 13-APA also inhibited [14C]serotonin release in response to arachidonic acid, ADP, or thrombin, but did not inhibit primary aggregation induced by ADP or thrombin. 13-APA completely blocked prostaglandin H2-induced aggregation in indomethacin-treated resuspended platelets but did not inhibit thromboxane synthesis. We therefore conclude that 13-APA acts as a direct antagonist of the platelet thromboxane/endoperoxide receptor.
Cloning immunoglobulin gamma 2b chain gene of mouse: characterization and partial sequence determination.
Authors: Kataoka T , Yamawaki-Kataoka Y , Yamagishi H , Honjo T
Published: May 2006

DNA from newborn mice was digested with restriction endonuclease EcoRI, and a 6.6-kilobase fragment encoding immunoglobulin gamma 2b chain mRNA derived from MPC 11 myeloma was enriched about 100-fold by RPC-5 column chromatography and agarose gell electrophoresis. The 6.6-kilobase fragment was cloned with lambda gt WES.lambda B as EK2 vector. The cloned phage (lambda WES.IgH22) contained the constant region gene of the gamma 2b chain but not the variable region gene of MPC 11 mRNA. The constant region genes of the other gamma chains (i.e., gamma 1, gamma 2a, and gamma 3) were not present in lambda gt WES.IgH22 DNA. R-loop mapping indicates that the gamma 2b chain structural gene is divided into two parts (330 +/- 60 SD base pairs and 930 +/- 110 SD base pairs) by an intervening sequence (360 +/- 100 SD base pairs). The nucleotide sequence around the junction of the hinge region and CH2 domain was determined and shown to match the amino acid sequence of the initial part of the CH2 domain of the gamma 2b chain. The base sequence upstream from the junction, however, is unrelated to the amino acid sequence of the CH1 domain and the hinge region of all the gamma chains whose sequences have been determined. These results indicate that the gamma 2b chain gene is interrupted at the junction of the hinge region and CH2 domain by an intervening sequence. The existence of two more intervening sequences, one between the CH1 domain and the hinge region and the other between the CH2 and CH3 domains, is discussed.
Differential activities of glycolipid glycosyltransferases in Tay-Sachs disease: studies in cultured cells from cerebrum.
Authors: Basu M , Presper K A , Basu S , Hoffman L M , Brooks S E
Published: May 2006

Four different glycolipid:glycosyltransferase activities involved in the biosynthesis in vitro of gangliosides and blood group-related glycosphingolipids have been tested in a simian virus 40-transformed glial cell culture derived from the cerebrum of a fetus with Tay-Sachs disease (TSD). The TSD cultured brain cells contained little activity of either UDP-Gal:GM2(beta 1-3)galactosyltransferase (GalT-3; EC 2.4.1.62), which catalyzes the formation of GM1a from GM2 (tay-Sachs) ganglioside, or GDP-Fuc:nLcOse4Cer (alpha 1-2)fucosyltransferase (FucT-2; EC 2.4.1.89), which catalyzes the formation of H1 glycolipid from nLcOse4Cer. These cells contained a potent inhibitor of the second reaction (catalyzed by a Golgi-rich membrane fraction from bovine spleen), whereas no inhibition of the first reaction (catalyzed by a membrane fraction from 14-day-old embryonic chicken brain) was observed. The activity of UDP-Gal:LcOse3Cer(beta 1-4)galactosyltransferase (GalT-4; EC 2.4.1.86) was 30- to 80-fold higher than the activity of GalT-3. The presence of CMP-AcNeu:nLcOse4Cer sialyltransferase activity and the absence of either GalT-3 or FucT-2 suggested a probable pathway for the synthesis of sialylneolactotetraosylceramide [GM1b(GlcNAc)] in addition to a specific blockage of GM1a ganglioside synthesis from GM2 in these TSD transformed cells.
Promoter mutations in the transfer RNA gene tyrT of Escherichia coli.
Authors: Berman M L , Landy A
Published: May 2006

The DNA sequences for nine independent promoter mutants of a tyrosine tRNA gene, tyrT of Escherichia coli, are reported. The nine mutations involve six transitions, two transversions, and one deletion. They are located at four different sites in the first 30 base pairs preceding the start point of transcription. The changes found are: a T.A to A.T transversion at position -8 (two mutants); a T.A to C.G transition at position -8 (three mutants); a T.A to C.G transition at position -13 (two mutants); a T.A to C.G transition at position -16 (one mutant); and a deletion of a G.C base pair at position -26/27 (one mutant). Four of the five different mutant tyrT promoters have alterations at positions that might have been expected from DNA sequence studies with other prokaryote promoters. One of these mutants (a G.C deletion at position -26/27) occurs in a stretch of eight consecutive G.C base pairs which may be characteristic of stable RNA promoters.
Race-specific molecules that protect soybeans from
Phytophthora megasperma
var.
sojae
Authors: Wade Mark , Albersheim Peter
Published: May 2006

Phytophthora megasperma
var.
sojae
(A. A. Hildebrand) is a fungal stem and root rot-causing pathogen of soybeans. Glycoproteins secreted into the medium of the aseptically cultured fungus have been partially purified by (NH
4
)
2
SO
4
precipitation and by column chromatography on norleucine-substituted Sepharose 4B and on DEAE-cellulose. Glycoprotein preparations from
P. megasperma
var.
sojae
races 1, 2, and 3 have been tested on four cultivars of soybeans. The partially purified glycoproteins from incompatible races of the pathogen (races that cannot successfully infect the plant), but not those from compatible races (races that can kill the plant), protect soybean seedlings from attack by compatible races. The seedlings are protected by introducing the glycoproteins into hypocotyl wounds of seedlings either 90 min prior to or at the time of inoculation of the wounds with mycelia of one of the pathogens. The glycoprotein preparations are poor nonspecific elicitors of phytoalexin accumulation; the glycoproteins have less than 1.0% of the elicitor activity of the glucans present in the mycelial walls of the pathogen.
Cytoplasmic inheritance of erythromycin resistance in human cells.
Authors: Doersen C J , Stanbridge E J
Published: May 2006

An erythromycin-resistant mutant, ERY2301, was isolated from ethidium bromide-treated HeLa cells in the presence of erythromycin at 300 micrograms/ml. ERY2301 cells were enucleated and the anucleate cytoplasts were fused with D98/AH-2, a hypoxanthine phosphoribosyltransferase-deficient variant of HeLa cells. The resultant cybrids were isolated in a double selective medium containing erythromycin and 6-thioguanine. Cybrid formation occurred at a frequency of 10(-3) to 10(-4). In vitro protein synthesis by intact and Triton X-100 treated mitochondria isolated from ERY2301 was resistant to the macrolide antibiotics erythromycin and carbomycin, but was sensitive to chloramphenicol. These results suggest that the site of erythromycin resistance in ERY2301 may be at the level of mitochondrial protein synthesis and indicate that this trait is cytoplasmically inherited and, therefore, presumably encoded in the mitochondrial genome.
Nitrogen control in
Salmonella
: Regulation by the
glnR
and
glnF
gene products
Authors: Kustu Sydney , Burton Doris , Garcia Emilio , McCarter Linda , McFarland Nancy
Published: May 2006

The product of the
glnR
gene is required for nitrogen regulation of the synthesis of glutamine synthesis (Gln synthetase) [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] and two periplasmic transport proteins that are subject to nitrogen control in
Salmonella
. Strains with mutations to loss of function of the
glnR
product [e.g., a strain with a Tn10 insertion or one with an ICR-induced (frameshift) mutation in
glnR
] have about 3% as much Gln synthetase as a fully derepressed wild-type strain and are unable to increase synthesis of this enzyme or periplasmic transport proteins in response to nitrogen limitation. The structural gene for Gln synthetase,
glnA
, and those for the periplasmic transport proteins are unlinked on the chromosome; thus,
glnR
appears to encode a diffusible positive regulatory element. Consistent with this, the mutant
glnR
allele is recessive to the wild-type allele with regard to expression of
glnA
(synthesis of Gln synthetase). Although
glnR
is closely linked to
glnA
, strains with mutations to complete loss of function of the
glnR
product can be distinguished from
glnA
strains by their ability to produce detectable Gln synthetase and to grow in the absence of glutamine. To demonstrate unequivocally that
glnR
is distinct from
glnA
, we have purified and characterized Gln synthetase from a strain with a Tn10 insertion in
glnR
. Because the properties of Gln synthetase from the insertion mutant, most importantly the carboxyl-terminal sequence of amino acids, are the same as those of synthetase from wild type, the Tn10 insertion cannot be in
glnA
(if it were, the carboxyl terminus of Gln synthetase would have to be altered); therefore we conclude that the Tn10 insertion is in a regulatory gene,
glnR
, which is distinct from
glnA
. A model for the function of the
glnR
product together with the previously defined
glnF
product in mediating nitrogen control is discussed.
Selective and unidirectional membrane redistribution of an H-2 antigen with an antibody-clustered viral antigen: relationship to mechanisms of cytotoxic T-cell interactions.
Authors: Geiger B , Rosenthal K L , Klein J , Zinkernagel R M , Singer S J
Published: May 2006

We have studied the co-redistribution of vesicular stomatitis virus (VSV) antigen and of individual H-2 antigens on the surfaces of mouse cells, and in parallel we have also used these VSV-infected cells as targets in cytotoxic T-cell killing experiments. Antibody-induced patching and capping of the VSV antigen caused an extensive co-patching and co-capping of the H-2Kb antigen but not of the H-2Db antigen. In reciprocal experiments, the antibody-induced patching of the H-2Kb or H-2Db antigen did not result in a co-patching of the VSV antigen. Radioimmunoassays showed that the relative numbers of H-2Kb, H-2Db, and VSV antigens on the surfaces of the cells exhibiting such nonreciprocal co-redistributions were closely similar. Furthermore, the H-2 restricted cytotoxic T-cell lysis of these target cells showed a marked preference for H-2Kb compared to H-2Db compatibility. We propose that the VSV and H-2 antigens are molecularly independent entities in the unpreturbed target cell membrane but that the antibody-induced clustering of the VSV antigen causes a selective and unidirectional co-redistribution (which we designate as syn-capping) of H-2Kb with the VSV antigen clusters. It is suggested that such a T-cell-induced syn-capping process involving an antigen and an H-2 molecule on the target cell may play a critical role in the mechanism of cytotoxic T-cell killing.
Bacterial lipopolysaccharide activates suppressor B lymphocytes.
Authors: Koenig S , Hoffmann M K
Published: May 2006

Lipopolysaccharide (LPS) extracted from the outer cell wall of Gram-negative bacteria modulates the immune response in vivo and in vitro. Depending on the experimental conditions, it may enhance or inhibit the production of humoral antibody. The pathway by which LPS suppresses antibody production is examined in this study. C57BL/6 spleen cells incubated with LPS (greater than 10 micrograms/ml) not only fail to produce antibody to sheep erythrocytes in vitro but also, when transferred 24 hr after stimulation with LPS, inhibit antibody production in spleen cells that were not treated with LPS. This observation suggested that LPS activates suppressor cells. We have identified a suppressor B cell as mediator of LPS-induced immune suppression and determined its cell surface antigen phenotype as Ig+, Ia+, CR+, Ly-B-2+,PC1-.LPS does not induce suppressor macrophages or suppressor T cells, nor are macrophages or T cells required for the generation of suppressor B cells by LPS.
Some sequence similarities among cloned mouse DNA segments that code for lambda and kappa light chains of immunoglobulins.
Authors: Wu T T , Kabat E A , Bilofsky H
Published: May 2006

A comparison between the cloned mouse DNA segments that were found to code for the lambda and kappa light chains of immunoglobulins established that there were seven short nucleotide sequences, two of which matched 6 out of 7, two 7 out of 8, two 8 out of 9, and one 9 out of 10 bases; these sequences were located either at homologous amino acid positions or at positions displaced by four amino acids or less. They all occurred in the framework regions (FRs), five next to the complementarity-determining regions (CDRs). Three of these were unique and did not occur elsewhere in the immunoglobulin nucleotides sequenced thus far or in DNA's of phage phi X174, phage G4, or simian virus 40. Five could serve as sites of joining by recombination or insertion of CDR to FR segments, and the invariant tryptophan that is the first residue of the second FR might serve as a sixth. These sites are consistent with the mini-gene or insertional hypotheses for the generation of antibody diversity but could also serve as points of recognition for a mutator enzyme or could serve to limit somatic mutation to the CDRs.
Cloning DNA sequences from influenza viral RNA segments.
Authors: Lai C J , Markoff L J , Zimmerman S , Cohen B , Berndt J A , Chanock R M
Published: May 2006

DNA sequences corresponding to gene segments that code for the nonstructural protein, the matrix protein, and the hemagglutinin of influenza A virus [strain A/Udorn/72 (H3N2)] were cloned in Escherichia coli pBR 322. Initially, positive and negative cDNA strands were prepared separately by reverse transcription. The positive strands of cDNA were transcribed from genomic RNA segments by using a specific dodecamer DNA sequence as a primer; the negative strands of cDNA were transcribed from cytoplasmic viral mRNA segments by using an oligo(dT) primer. DNA duplexes corresponding in size to the virus RNA segments were then purified, inserted into the plasmid DNA, and used for transformation of E. coli. The influenza virus-specific DNA sequences isolated from recombinant plasmid molecules were characterized by mapping restriction enzyme cleavage sites. In addition, the orientation of cloned DNA was determined with reference to the 3' terminus of viral RNA.
Structure of potato inhibitor complex of carboxypeptidase A at 5.5-A resolution.
Authors: Rees D C , Lipscomb W N
Published: May 2006

The complex of the 39-amino inhibitor (potato) of bovine carboxypeptidase A (carboxypeptidase; peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) was crystallized in space group P32. There are two protein-inhibitor complexes in the asymmetric unit. These crystals exhibited pseudo-P3221 symmetry due to twinning about the a3 axis. Heavy atom difference Patterson maps and rotation functions indicated, however, that the noncrystallographic twofold axis that relates these two complexes is nearly coincident with the a3 axis. Consequently, to a good approximation at low resolution, the space group of the complex is P3221 and the effects of twinning may be ignored. The structure was solved by using multiple isomorphous replacement and molecular replacement techniques. At 5.5-A resolution, the multiple isomorphous replacement map was readily interpretable in terms of the known native carboxypeptidase A structure plus extra density around the active site. The position of this extra density is consistent with the binding mode for extended substrate proposed from earlier model building studies with the native enzyme (Lipscomb, W.N., Hartsuck, J.A., Reeke, G.N., Quiocho, F.A. Bethge, P.H., Ludwig, M.L., Steitz, T.A., Muirhead, H. & Coppola, J.C. (1968) Brookhaven Symp. Biol. 21, 24-90).
Selective protection of stereospecific enkephalin and opiate binding against inactivation by N-ethylmaleimide: evidence for two classes of opiate receptors.
Authors: Smith J R , Simon E J
Published: May 2006

Stereospecific binding of 3H-labeled [D-Ala2,D-Leu5]enkephalin is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide. This inactivation, like that of opiate binding, exhibits pseudo-first-order kinetics with a half-inactivation time of 10-12 min. The presence of opiates or enkephalins during incubation with N-ethylmaleimide provides good protection. Ouantitative studies of protection demonstrate that naltrexone and morphine are 20 and 8 times, respectively, more effective in protecting the binding of [3H]naltrexone than that of [3H]enkephalin. [D-Ala2,Leu]Enkephalin and [D-Ala2,Met]enkephalin, however, are more effective (7 and 30 times, respectively) for the protection of 3H-labeled [D-Ala2,D-Leu5]enkephalin binding. These results provide strong evidence for the existence of two classes of opiate receptor in rat brain.
Organ chemiluminescence: noninvasive assay for oxidative radical reactions.
Authors: Boveris A , Cadenas E , Reiter R , Filipkowski M , Nakase Y , Chance B
Published: May 2006

In situ and perfused rat livers showed a spontaneous chemiluminescence of 7-12 counts/sec . cm2 (corresponding to 7-12 x 10(3) photons/sec . cm2); chemiluminescence was increased up to 30 times by infusion of exogenous hydroperoxides. The chemiluminescence of the perfused liver was oxygen dependent. Ethyl, t-butyl, and cumene hydroperoxides were almost equally effective in inducing light emission in the perfused liver. Glutathione release and chemiluminescence showed a parallel increase upon hydroperoxide supply to the perfused liver. A partial spectral analysis of the chemiluminescence of the perfused liver showed a predominance of red-light-emitting species, presumably arising from the singlet oxygen dimol-emission peaks. Many side reactions derived from the complex free radical sequence of lipid peroxidation could afford the chemistry leading to light emission, which represents only about 10(-14) of the utilization of peroxide.
Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells.
Authors: Poste G , Nicolson G L
Published: May 2006

B16 mouse melanoma sublines in culture spontaneously shed intact plasma membrane vesicles. These vesicles can be fused with the plasma membrane of cells from homologous and heterologous B16 sublines by using polyethylene glycol and phytohemagglutinin-P. Fusion of vesicles from a highly metastatic subline (F10) that localizes exclusively in the lung with cells from a poorly metastatic subline (F1) significantly increased the ability of F1 cells to become arrested in the lung and form metastases in this organ. In contrast, fusion of F1 vesicles with F10 cells did not alter the ability of vesicle-modified cells to localize in the lung or form lung metastases. F10 vesicle-modified F1 cells reverted to their original arrest behavior and metastatic capacity after removal of F10 vesicle components from the plasma membrane. The changes in the arrest and metastatic behavior of F10 vesicle-modified F1 cells were highly highly specific. Vesicles from other B16 sublines that are poorly metastatic and show limited localization in the lung (F1, FLLr, and F10Lr) did not modify the arrest behavior and metastatic capacity of FU cells. These results suggest that the differences in the abilities of the F1 and F10 sublines to localize in the lung are determined by differences in cell surface properties.
Protein bodies of mung bean cotyledons as autophagic organelles
Authors: Van der Wilden Willem , Herman Eliot M. , Chrispeels Maarten J.
Published: May 2006

We present evidence that protein bodies constitute the principal lytic compartment in storage parenchyma cells of mung bean cotyledons and propose that they play a role in cellular autophagy. We developed a method to isolate protein bodies by incubating tissue slices with cell wall-degrading enzymes and fractionating the cellular organelles on a Ficoll gradient. About 75-80% of the protein bodies present in the protoplasts were recovered intact in a band at the 5/25% Ficoll interface. This band contained a similar proportion of the cellular α-mannosidase,
N
-acetyl-β-glucosaminidase, ribonuclease, acid phosphatase, phosphodiesterase, and phospholipase D. β-Amylase was present in the cells but not in the protein bodies. Ultrastructural observations showed that on the 3rd day of seedling growth protein bodies contain small vesicles (0.3-1.0 μm) with a cytoplasmic content (ribosomes, membrane vesicles, mitochondria). Later in seedling growth these vesicles appeared empty. We believe that these are autophagic vesicles resulting from invaginations of the protein body membrane and that their cytoplasmic contents are digested by the acid hydrolases present in the protein bodies.
Model for capping derived from inhibition of surface receptor capping by free fatty acids.
Authors: Klausner R D , Bhalla D K , Dragsten P , Hoover R L , Karnovsky M J
Published: May 2006

When low concentrations (2-5 mole %) of cis unsaturated free fatty acids (group A) are intercalated into lymphocyte plasma membrane, capping is inhibited. No effect is seen with trans unsaturated or saturated fatty acids (group B). The capping inhibition is reversible with increasing doses of extracellular calcium. Fluorescence photobleaching recovery has shown that the group A free fatty acids do not inhibit the receptor immobilization associated with patch formation, but inhibit the final energy-dependent movement of the patched receptors into a cap. We have also shown that the group A free fatty acids cause a shift in membrane-bound calcium to the lipid phase from probable protein-associated sites. We have incorporated these findings into a model for capping and membrane-cytoskeletal interactions.
Differential lectin binding to cellular membranes in the epidermis of the newborn rat.
Authors: Brabec R K , Peters B P , Bernstein I A , Gray R H , Goldstein I J
Published: May 2006

Three fluorescein-labeled lectins have been shown to exhibit specificity for the surface of cells in different layers of the epidermis in the newborn rat. An isolectin from seeds of Bandeiraea simplicifolia with specificity for alpha-D-galactosyl end groups labeled the basal and lower spinous cells; a lectin from Ulex europaeus exhibiting specificity for alpha-L-fucosyl units outlines the surface of spinous cells, and a second lectin from B. simplicifolia, with specificity for N-acetyl-D-glucosamine, labels the cornified cells. Appropriate blocking experiments have confirmed the specific nature of the binding.
Colchicine inhibits epidermal growth factor degradation in 3T3 cells.
Authors: Brown K D , Friedkin M , Rozengurt E
Published: May 2006

Colchicine (2 microM) did not affect the initial rate of association of 125I-labeled epidermal growth factor (125I-EGF) to Swiss 3T3 cells but continued incubation (up to 24 hr) led to an increase in cell-associated radioactivity. The effect is also produced by Colcemid, vinblastine, and podophyllotoxin but not by lumicolchicine. Disruption of microtubules with colchicine does not alter the rate of "down regulation" of EGF receptors, suggesting the binding and internalization of the factor proceed unchanged. However, colchicine markedly decreases the rate of appearance of acid-soluble radioactivity from cells either incubated continuously with 125I-EGF for 24 hr or exposed to the radioactive peptide for only 1 or 3 hr. The results indicate that colchicine decreases the rate of degradation of internalized 125I-EGF. Because antitubulin agents enhance the mitogenic effect of EGF our results suggest that peptide degradation can be dissociated from the long-term biological effect.
Induction of a basement membrane glycoprotein in embryonic kidney: possible role of laminin in morphogenesis.
Authors: Ekblom P , Alitalo K , Vaheri A , Timpl R , Saxén L
Published: May 2006

The glycoprotein laminin is found exclusively in the basement membranes of adult tissues, not in the mesenchymal stroma. We studied the appearance and distribution of laminin during the early formation of kidney tubules in mouse embryos and in an in vitro transfilter model system. In immunofluorescence using affinity-purified antibodies, the distribution of laminin showed a clear correlation, both spatially and temporally, to the early stages of tubule formation. In vivo, laminin was first detected in a punctate pattern in areas where the pretubular aggregates form; later, it became confined to the basement membranes of the tubules. In experiments in vitro, the nephrogenic mesenchyme was found to form tubules after 12-24 hr of transfilter contact with the inductor. The first laminin spots were found after 12 hr of culture, 24 hr before overt morphogenesis. As the mesenchymal cells began to aggregate and elongate (at 36 hr), laminin was detected in those cells destined to become epithelial, and at 48 hr it was not found in cells remaining in the stroma. In more mature tubules (at 72 hr), laminin was seen as a sharp band in the basement membranes. It is suggested that laminin is involved in the increased cell adhesiveness during the early aggregation of the nephrogenic mesenchyme.
Immunoselection of mutants deficient in cell surface glycoproteins encoded by murine erythroleukemia viruses.
Authors: Kabat D , Ruta M , Murray M J , Polonoff E
Published: May 2006

We have described a heterogeneously processed glycoprotein with an apparent molecular weight of 55,000 (gp55) that is encoded by the Friend spleen focus-forming virus, an acute erythroleukemia virus [Dresler, S., Ruta, M., Murray, M.J. & Kabat, D. (1976) J. Virol. 30, 564-573]. Several lines of evidence suggest that a small proportion of the gp55 in infected cells is located on the surface membranes. First, different nonproducer cell lines infected with cloned Friend spleen focus-forming virus were efficiently killed in the presence of complement by cytotoxic antisera that react with gp55. Furthermore, a clone of cells selected for resistance to the cytotoxic antibody synthesized an altered intracellular form of gp55. This immunoselection procedure appears to also be useful for isolating glycoprotein mutants of other RNA tumor viruses. Analysis of cell surface proteins labeled with [125I]iodine supported the idea that gp55 occurs on the plasma membranes. However, the cell surface gp55 had more highly processed oligosaccharides than the majority of the gp55, which occurs within the infected cells. In addition, we have found that leukemia cells from mice infected with the erythroleukemic Rauscher virus complex contain a membrane glycoprotein that appears similar to the gp55 encoded by Friend spleen focus-forming virus. The encoding of similar glycoproteins by independently isolated acute erythroleukemia viruses suggests that these glycoproteins may be important in leukemogenesis.
Oxidative mechanisms of monocyte-mediated cytotoxicity.
Authors: Weiss S J , LoBuglio A F , Kessler H B
Published: May 2006

Human monocytes stimulated with phorbol myristate acetate were able to rapidly destroy autologous erythrocyte targets. Monocyte-mediated cytotoxicity was related to phorbol myristate acetate concentration and monocyte number. Purified preparations of lymphocytes were incapable of mediating erythrocyte lysis in this system. The ability of phorbol myristate acetate-stimulated monocytes to lyse erythrocyte targets was markedly impaired by catalase or superoxide dismutase but not by heat-inactivated enzymes or albumin. Despite a simultaneous requirement for superoxide anion and hydrogen peroxide in the cytotoxic event, a variety of hydroxyl radical and singlet oxygen scavengers did not effect cytolysis. However, tryptophan significantly inhibited cytotoxicity. The myeloperoxidase inhibitor cyanide enhanced erythrocyte destruction, whereas azide reduced it modestly. The inability of cyanide to reduce cytotoxicity coupled with the protective effect of superoxide dismutase suggests that cytotoxicity is independent of the classic myeloperoxidase system. We conclude that monocytes, stimulated with phorbol myristate acetate, generate superoxide anion and hydrogen peroxide, which together play an integral role in this cytotoxic mechanism.
Cholecystokinin-converting enzymes in brain.
Authors: Malesci A , Straus E , Yalow R S
Published: May 2006

Crude extracts of porcine cerebral cortical tissue convert cholecystokinin (CCK) to its COOH-terminal fragments, the dodecapeptide (CCK-12) and the octapeptide (CCK-8). The Sephadex G-75 void volume eluate of the crude extract cleaves the arginine-isoleucine bond and effects conversion only to CCK-12; the Sephadex G-50 void volume eluate of the same extract cleaves the arginine-aspartate bond as well, so that both CCK-12 and CCK-8 are end products. Thus, there are at least two enzymes; the one involved in the conversion to CCK-12 is of larger molecular radius than the other. The Km for the cleavage of CCK at the arginine-isoleucine bond by the Sephadex G-75 void volume eluate enzyme is 1.1 X 10(-6) M; the Km for trypsin cleavage of the same bond is 4.7 x 10(-6) M. The lower Vmax for the brain enzyme (1.5 x 10(-11) mol/min per g of extract) compared with trypsin (66 x 10(-11) mol/min per g of trypsin) simply reflects the lesser degree of purify of the brain extract than of the highly purified trypsin.
Method for preparing cultures of central neurons: cytochemical and immunochemical studies.
Authors: Sotelo J , Gibbs C J , Gajdusek D C , Toh B H , Wurth M
Published: May 2006

We report a simplified method for culturing fetal central nervous system cells predominantly inducing neurons that grow, differentiate, and live in vitro for as long as 10 weeks. These central nervous system cells form a confluent cell culture in which about 80% of the cells are fully differentiated neurons producing interconnecting axons and dendrite processes and live upon a sparse underlying population of fibrillary and protoplasmic astrocytes, oligodendrocytes, and fibroblasts. Morphological and cytochemical characteristics of these cell types, based on immunofluorescent cell specific markers and silver staining of neurons, are presented.
Immunocytochemical localization of the sex steroid-binding protein of plasma in tissues of the adult monkey Macaca nemestrina.
Authors: Bordin S , Petra P H
Published: May 2006

The sex steroid-binding protein (SBP) present in the serum of the monkey Macaca nemestrina is shown to exist in cells of tissue involved in reproduction. The localization was demonstrated by immunofluorescence with monospecific antibodies raised against homogeneous human SBP. These antibodies were previously shown to crossreact with monkey SBP. The protein appears to be located in the cytoplasm of epithelial cells lining the prostate alveoli, the ducti of the epididymis, and the seminiferous tubula of the testes of the monkey. The protein is also present in the cytoplasm of parenchymal cells of the liver, where SBP is believed to be synthesized, and in cells of the adrenal cortex, where steroids are known to be synthesized. Controls appear dark and illustrate specificity of the immunofluorescence, ruling out both tissue autofluorescence and other nonspecific interactions. In all cases, the relative intensity of fluorescence appears minimal in the nuclei of cells. Experiments performed with cultured MCF-7 cells indicate that SBP can across the plasma membrane and enter into the cytoplasm but not into the nucleus. Additional studies indicate that the monospecific antibodies do not crossreact with the monkey prostate androgen receptor, as shown by ultracentrifugation in sucrose densty gradients. The physiological significance of these observations is not known; however, the existence of this protein in cells of target tissues for sex steroids introduces a new dimension in our thinking about the role of this protein in androgen action.
Nucleotide sequence of a cloned structural gene coding for a precursor of pancreatic somatostatin.
Authors: Goodman R H , Jacobs J W , Chin W W , Lund P K , Dee P C , Habener J F
Published: May 2006

We have constructed and cloned, in bacteria, recombinant plasmids containing DNA complementary to mRNA coding for a pancreatic pre-prosomatostatin, a product of the cell-free translation of pancreatic islet mRNAs shown previously by immunoprecipitation to be a precursor of somatostatin. A clone containing a nearly full-length cDNA insert of 550 base pairs was identified and appeared to contain the entire coding sequence for the somatostatin precursor in addition to portions of the 5' and 3' untranslated regions. mRNA coding for the pre-prosomatostatin is 600-630 bases long as determined by agarose gel electrophoresis and hybridization with labeled cDNA. Analyses of the nucleotide sequence of the cDNA revealed a protein of 119 amino acid beginning with methionine followed by a typical leader sequence containing 18 hydrophobic amino acids. The tetradecapeptide somatostatin, identical in sequence to mammalian hypothalamic somatostatin, is located at the carboxy terminus followed immediately by a stop codon. An ARg-Lys sequence immediately preceding the sequence of somatostatin is typical of a prohormone cleavage site. A sequence Ala-Pro-Arg-Glu preceding the Arg-Lys cleavage site is identical to that found in porcine prosomatostatin. The evolutionary conservation of the identical amino acid sequence of the somatostatin tetradecapeptide from fish to mammals is remarkable. In addition, similar conservation, in fish and mammals, of the sequence Ala-Pro-Arg-Glu-Arg-Lys preceding the coding region for somatostatin suggets that this particular sequence may have biologic importance in cellular processing of the somatostatin precursor.
Calcium-dependent hormonal regulation of amino acid transport and cyclic AMP accumulation in rat hepatocyte monolayer cultures.
Authors: Kelley D S , Evanson T , Potter V R
Published: May 2006

The effect of glucagon, epinephrine, norepinephrine, dexamethasone, insulin, and dexamethasone plus glucagon on the transport of 2-aminoisobutyric acid (AIB) and that of glucagon on the production of cyclic AMP were examined in rat hepatocyte monolayer cultures under three different culture conditions involving calcium. The hepatocytes were studied in calcium-contaning medium after treatment with or without 0.033% dimethyl sulfoxide, the solvent for the calcium ionophore A23187 (calcium controls); calcium-free medium after treatment with A23187 (calcium-depleted); and calcium-containing medium after treatment with ionophore (calcium-restored). The basal and hormonally regulated rates of AIB transport for hepatocytes in calcium control and calcium-depleted cultures were comparable. The restoration of calcium in calcium-restored cultures increased the basal and the hormonally stimulated transport of AIB when compared to the other conditions. Calcium markedly enhanced the stimulation of AIB transport in cultures treated with glucagon, catecholamines, and dexamethasone plus glucagon. The level of cyclic AMP production in response to glucagon in calcium control and calcium-depleted cultures was the same and it was conspicuously higher than the level in calcium-restored cultures. Varying the concentration of calcium in the medium used to maintain the hepatocytes in calcium control cultures did not affect the stimulation of AIB transport or cyclic AMP production by glucagon. However, in calcium-restored cultures, increasing the calcium concentration of the medium resulted in increased stimulation of AIB transport and decreased production of cyclic AMP by glucagon. In the calcium-restored cultures, calcium in the absence of glucagon enhanced AIB transport but had no effect on cyclic AMP production. Cultures maintained for 6 hr in calcium-free medium after the depletion of calcium showed a 6- to 7-fold increase in the production of cyclic AMP in response to glucagon, but no stimulation of AIB transport. We suggest that mobilization of cellular calcium by glucagon either directly or through cyclic AMP mediates its stimulation of amino acid transport.
Regulation of Tn5 transposition in Salmonella typhimurium.
Authors: Biek D , Roth J R
Published: May 2006

The drug-resistance element Tn5 transposes with high frequency immediately after entry into a cell. Establishment of Tn5 within a cell results in a decrease in this transposition frequency. This phenomenon resembles "zygotic induction" of repressible operons and prophages. Evidence is presented that Tn5 transposition is under negative control by a factor encoded within the element itself. Established Tn5 elements (that contain point mutations inactivating the resistance gene) are able to inhibit transposition of an incoming Tn5 element by a factor of 12- to 70-fold. Several deletion derivatives of Tn5 lack the ability to inhibit transposition.