bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletions in the 3'-terminal tRNA-like structure of brome mosaic virus RNA differentially affect aminoacylation and replication in vitro.
Authors: Bujarski J J , Dreher T W , Hall T C
Published: May 2006

Deletions in cDNA clones covering the 3' 201 nucleotides of brome mosaic virus RNA 3 were produced by S1 nuclease treatment of cloned DNA linearized at several different restriction sites. Transcription of these clones yielded RNAs containing structural alterations in the 3'-terminal tRNA-like structure that is involved in aminoacylation and replication. Replicase template activity, but not aminoacylation activity, was especially sensitive to deletions in arm C, which contains a tyrosyl anticodon. Deletions in arm B were detrimental to aminoacylation, but the proportion of replicase template activity lost depended on the site of the deletion. Removal of arm D had little effect on aminoacylation and, in some instances, resulted in a 2-fold stimulation of replicase template activity.
Ionic inhibition of formation of RecA nucleoprotein networks blocks homologous pairing.
Authors: Chow S A , Radding C M
Published: May 2006

Conditions that favor the complete coating of single-stranded DNA by RecA protein promote the association of these presynaptic filaments with naked double-stranded DNA to form large nucleoprotein networks before homologous pairing occurs. These RecA nucleoprotein networks sequester virtually all of the DNA in the reaction mixture. Conditions that are suboptimal for the formation of the RecA presynaptic filament rendered both the formation of RecA-DNA networks and the subsequent formation of joint molecules sensitive to inhibition by excess ATP or by pyrophosphate when these were added during synapsis. The rate of homologous pairing was directly related to the degree of inhibition of network formation. Various multivalent cations added during synapsis restored both the formation of networks and the pairing of homologous molecules. These observations support the view that the nucleoprotein network is a synaptic intermediate by means of which RecA protein facilitates the conjunction of DNA molecules and the subsequent processive search for homology. Inhibition by multivalent anions and restoration by multivalent cations suggests in addition, that negative charge repulsion inhibits the binding of naked duplex DNA to presynaptic filaments.
Quantitative and qualitative changes in histone gene expression during early mouse embryo development.
Authors: Graves R A , Marzluff W F , Giebelhaus D H , Schultz G A
Published: May 2006

There are large amounts of histone mRNA present in mouse eggs. These RNAs are rapidly degraded, as are other mRNAs, after fertilization and prior to the second cleavage. During cleavage, the histone mRNA accumulates as the embryo divides. The same sets of histone genes are expressed in eggs and embryos, although there are large qualitative differences in the amounts of particular histone mRNAs. The function of the egg histone mRNA is unknown. The amount of histone mRNA in cleaving and blastocyst embryos is probably sufficient to code for the blastocyst histone proteins.
Light regulation of the synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase in peas: Evidence for translational control
Authors: Inamine Gordon , Nash Barbara , Weissbach Herbert , Brot Nathan
Published: May 2006

The specific activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) increases 30- to 50-fold when dark-grown pea seedlings are shifted into the light. The large subunit (LS) of this multimeric protein is known to be synthesized in the chloroplast, but plastids from dark-grown cells contain relatively low levels of LS. However, despite the low level of LS synthesis in the plastids of dark-grown plants, these organelles contain significant levels of LS mRNA. Hybridization studies showed that the amount of LS mRNA increased about 3-fold, relative to total plant RNA, when dark-grown plants were illuminated. This increase in LS mRNA can be accounted for by a similar increase in chloroplast genome copy number. It was found that the amount of translatable LS mRNA per μg of plastid RNA is similar when isolated from either dark-grown plants or dark-grown plants subjected to light. These results suggest that although light can increase the level of LS mRNA by increasing the copy number of this gene, the primary regulation of LS synthesis by light in pea chloroplasts is at the level of translation.
On the correlation between HII phase and the contact-induced destabilization of phosphatidylethanolamine-containing membranes.
Authors: Bentz J , Ellens H , Lai M Z , Szoka F C
Published: May 2006

The abundance of phosphatidylethanolamine (PtdEtn) in biological membranes and the capacity of this lipid to sustain nonbilayer structures have been promoted as evidence for a role of PtdEtn in biological fusion processes. To date there has been no direct evidence of a connection between the kinetics of bilayer destabilization and the polymorphism accessible to PtdEtn. We have developed a model system to examine this point directly using the proton-induced destabilization of PtdEtn/cholesterylhemisuccinate unilamellar liposomes. We find that the initial rate of bilayer mixing rapidly increases with temperature and reaches a maximal level just below the HII-phase transition temperature. The leakage from these liposomes rapidly increases, both in rate and extent, within the HII-phase transition temperature range. Of an even greater significance is that at no temperature is there any mixing of aqueous contents within the liposomes. Thus, these lipids can begin to undergo the lamellar- to HII-phase transition at the stage of two apposed liposomes. However, the nonbilayer structures formed do not cause fusion--i.e., the concomitant mixing of aqueous contents.
Nerve growth factor rapidly induces ornithine decarboxylase mRNA in PC12 rat pheochromocytoma cells.
Authors: Feinstein S C , Dana S L , McConlogue L , Shooter E M , Coffino P
Published: May 2006

The mechanism by which nerve growth factor (NGF) stimulates ornithine decarboxylase (OrnDCase; EC 4.1.1.17) activity in the rat pheochromocytoma cell line PC12 was investigated. As demonstrated previously, NGF rapidly induces OrnDCase activity in a dose-dependent manner, with maximal enzymatic activity at 4-6 hr after exposure to NGF. Activity subsequently returns to near basal levels. A cloned OrnDCase cDNA was used to analyze the levels of OrnDCase RNA. In response to NGF administration, OrnDCase RNA levels were induced. The time course of the OrnDCase RNA induction paralleled that of the enzyme activity induction, and the magnitude of both inductions was quantitatively the same. Increased concentration of OrnDCase RNA was clearly detected at the earliest time point examined, 2 hr. No change was observed in the size of OrnDCase RNA. The dose-response curves for both RNA and enzyme activity inductions were also similar. Thus, increased OrnDCase RNA levels fully account for, and are responsible for, the induction of activity. Further, one-third of the OrnDCase RNA induction was unaffected by cycloheximide treatment but was fully blocked by actinomycin D treatment, suggesting that NGF acts through at least two mechanisms to mediate the OrnDCase induction. The first mechanism is cycloheximide insensitive and the second is mediated through an event requiring ongoing protein synthesis. Both mechanisms require ongoing transcription, as evidenced by the complete sensitivity of the induction process to actinomycin D.
mRNA stabilization controls the expression of a class of developmentally regulated genes in
Dictyostelium discoideum
Authors: Mangiarotti Giorgio , Giorda Roberto , Ceccarelli Adriano , Perlo Carla
Published: May 2006

During the development of
Dictyostelium discoideum
, several thousand new mRNA species appear in the cytoplasm after the cells have formed stable aggregates. Here we show that six of these late mRNAs, corresponding to six clones randomly chosen from a genomic library, are synthesized from the very beginning of development at a rate comparable to that observed late in development but that transcripts do not accumulate until after aggregation. The early- and late-synthesized mRNAs are identical in size and compete with each other for hybridization to the genomic clones. The early-synthesized mRNAs do not accumulate in the cytoplasm in the preaggregation stage because they are very unstable. Their stability, estimated from the kinetics of incorporation during continuous labeling with
32
P, increases by perhaps an order of magnitude in the postaggregation stage. We conclude that mRNA stabilization is the major controlling factor of the expression of these genes.
Structure and expression of genes encoding murine Qa-2 class I antigens.
Authors: Mellor A L , Antoniou J , Robinson P J
Published: May 2006

DNA structural analysis of the Qa region in two BALB/c mouse substrains with different Qa-2 phenotypes reveals that a deletion of DNA has occurred in BALB/cBy (Qa-2-) mice relative to BALB/c (Qa-2+) mice. We propose that this deletion arises from unequal crossing-over and recombination between adjacent BALB/c class I genes and results in the generation of a hybrid class I gene in BALB/cBy mice. Furthermore, we suggest that this is a direct cause of the change in Qa-2 phenotype. Further support for this model was obtained from transfection experiments in which cloned genes from the equivalent part of the Qa region in C57BL/10 mice were introduced into L cells. Four C57BL/10 genes, arranged in two almost identical pairs, encode polypeptides that are precipitated from lysates of transfectants with anti-Qa-2/3 antiserum. Although loss of one pair of these genes in BALB/c mice has no qualitative effect on Qa-2 phenotype, the loss of both pairs of genes via gene fusion leads to the loss of the Qa-2+ phenotype in BALB/cBy mice.
Auditory representation of autogenous song in the song system of white-crowned sparrows
Authors: Margoliash Daniel , Konishi Masakazu
Published: May 2006

The HVc (hyperstriatum ventrale, pars caudale) is a forebrain nucleus in the motor pathway for the control of song. Neurons in the HVc also exhibit auditory responses. A subset of these auditory neurons in the white-crowned sparrow (
Zonotrichia leucophrys
) have been shown to be highly selective for the individual bird's own (autogenous) song. By using multiunit recording techniques to sample from a large population, we demonstrate that the entire population of auditory neurons in the HVc is selective for autogenous song. The selectivity of these neurons must reflect the song-learning process, for the acoustic parameters of a sparrow's song are acquired by learning. By testing with laboratory-reared birds, we show that HVc auditory neurons prefer autogenous song over the tutor model to which the birds were exposed early in life. Thus, these neurons must be specified at or after the time song crystallizes. Since song is learned by reference to auditory feedback, HVc auditory neurons may guide the development of the motor program for song. The maintenance of a precise auditory representation of autogenous song into adulthood can contribute to the ability to distinguish the fine differences among conspecific songs.
Protein RepC is involved in copy number control of the broad host range plasmid RSF1010.
Authors: Haring V , Scholz P , Scherzinger E , Frey J , Derbyshire K , Hatfull G , Willetts N S , Bagdasarian M
Published: May 2006

Essential replication (rep) genes of the broad host range plasmid RSF1010 have been cloned onto controlled expression vectors and their protein products have been visualized, after induction, by NaDodSO4/polyacrylamide gel electrophoresis of whole cell lysates. During this induction the replication of a coresident RSF1010 replicon, pKT210, was analyzed by quantitative DNA X DNA hybridization. The initiation of pKT210 replication was stimulated 6-fold by a simultaneous overproduction of the RepA and RepC proteins compared to cells in which only the RepA protein was overproduced. An enhanced synthesis of the RepB protein resulted in a 1.6-fold stimulation of pKT210 replication, whereas an overproduction of the RepA protein alone had no effect. Purified RepC protein has been shown to bind preferentially to DNA carrying the replication origin of RSF1010. Within this segment it was bound specifically to those DNA fragments that contained the 20-base-pair direct repeats of the origin region. These results suggest that RepC protein acts as a positive replication regulator, that its concentration is rate-limiting, and that the replication rate of RSF1010 is controlled, at least in part, at the level of RepC synthesis.
Isolation and characterization of the product of the methionine-regulatory gene metJ of Escherichia coli K-12.
Authors: Smith A A , Greene R C , Kirby T W , Hindenach B R
Published: May 2006

We have modified a previously isolated metJ plasmid by removing a segment of DNA including the rop gene. Bacterial strains carrying this plasmid produce elevated levels of the metJ gene product, presumably because of the high number of gene copies in the cell. We have isolated the metJ gene product in nearly homogeneous form from such a strain. The subunit size and the amino acid composition are the same as those predicted from the DNA sequence of the metJ gene. Sedimentation equilibrium measurements show that the native metJ gene product is a dimer. The purified dimer protects a short segment of DNA in the regulatory region of the metB and metJ genes from hydrolysis by DNase I.
Efficient insertion of genes into the mouse germ line via retroviral vectors.
Authors: van der Putten H , Botteri F M , Miller A D , Rosenfeld M G , Fan H , Evans R M , Verma I M
Published: May 2006

We present a general strategy for the efficient insertion of recombinant retroviral vector DNA into the mouse germ line via infection of preimplantation mouse embryos. Transgenic mice were generated that harbor a replication-competent recombinant retrovirus (delta Mo + Py M-MuLV) that lacks the Moloney murine leukemia virus (M-MuLV)-type enhancer sequence in the long terminal repeat (LTR). Instead, the LTR contains an enhancer element that permits polyoma virus F101 to grow in undifferentiated F9 embryonal carcinoma cells. Expression studies in different tissues of animals transgenic for delta Mo + Py M-MuLV indicate possibilities to target and modulate expression of retroviral recombinants in mice via their LTR enhancer sequences. In addition, 16 transgenic mice were generated that harbor proviral DNA of a defective recombinant retrovirus carrying a mutant dihydrofolate reductase gene.
Bird remains from an archaeological site on Henderson Island, South Pacific: Man-caused extinctions on an “uninhabited” island
Authors: Steadman David W. , Olson Storrs L.
Published: May 2006

Long thought never to have been inhabited and to be in a pristine ecological state, Henderson Island (southeast Pacific) is now known to have been colonized and then abandoned by Polynesians. Bones from an archaeological site on the island associated with
14
C dates of ≈800 and ≈500 years B.P. include specimens of 12 species of birds, of which 3, a storm-petrel and two pigeons (
Nesofregetta fuliginosa, Ducula
cf.
aurorae
or
D. pacifica
, and
Ducula
cf.
galeata
), no longer occur on Henderson, and two others (
Puffinus nativitatis
and
Sula sula
) still visit but are not known to breed. The vanished species were presumably exterminated by Polynesians and the biota of Henderson Island can thus no longer be regarded as being in an unaltered state. The prehistoric abandonment of various small, unarable islands by Polynesians may have been due to the depletion of seabirds and pigeons, the only readily available food source. The species of pigeons identified from Henderson are known historically only from distant archipelagos and have never before been found sympatrically. Distributional patterns resulting from man-caused extinctions may give rise to erroneous interpretations of the relationships and evolutionary history of insular organisms. Certain endangered species, such as
Ducula galeata
, might effectively be preserved by reintroduction to abandoned islands that they occupied before human intervention.
Recognition of interspecies hybrid class I histocompatibility antigens by antigen-specific cytolytic T lymphocytes.
Authors: Maziarz R , Allen H , Strominger J L , Flavell R , Biro P A , Burakoff S J
Published: May 2006

Two reciprocal interspecies hybrid class I histocompatibility genes have been constructed between genomic clones of human HLA-A2 and murine H-2Kb. The proteins encoded by these genes have been designated A21+2/Kb, where the polymorphic domains, alpha 1 and alpha 2, of HLA-A2 are linked to the carboxyl-terminal domains (alpha 3, transmembrane, and intracytoplasmic domains) of H-2Kb, and Kb1+2/A2, where the alpha 1 and alpha 2 domains of the H-2Kb antigen are linked to the carboxyl-terminal domains of HLA-A2. These genes have been transfected and expressed in recipient mouse L cells and human RD (rhabdomyosarcoma) cells. Both hybrid antigens were found to be serologically intact when tested with a panel of antigen-specific monoclonal antibodies. The monoclonal antibody W6/32, which recognizes a monomorphic determinant on all HLA-A, -B, and -C antigens, recognizes the alpha 1 and/or the alpha 2 domain, rather than the more conserved alpha 3 domain. Human cytolytic T lymphocytes (CTL) specific for the HLA-A2 antigen recognized the A2 and A21+2/Kb proteins only when expressed in human cells and not when expressed in mouse cells, even when surface antigen levels were 10-fold greater on the mouse cells than on the human cells. In contrast, a long-term, murine anti-H-2b CTL line not only lysed mouse L-cell lines that expressed the parental Kb and hybrid Kb1+2/A2 antigens but also lysed the Kb and Kb1+2/A2 human cell RD transformants as well. In both cases, the level of CTL recognition and lysis of the transformants that expressed the native antigen Kb was greater than of those transformants that expressed the hybrid antigen Kb1+2/A2. These data suggest that the carboxyl-terminal domains play some role in CTL allorecognition. The lack of human CTL recognition of HLA molecules expressed in mouse L cells, however, cannot be explained by the presence of a xenogeneic carboxyl terminus. Since murine CTL can recognize their target antigen when expressed on the surface of human cells, the possibility remains either that a ligand necessary for other molecular interactions of human CTL may be absent on mouse target cells or that murine and human CTL differ in affinity of binding to target antigens in the absence of accessory-molecule interactions.
Brood care by male bumble bees
Authors: Cameron Sydney A.
Published: May 2006

Male
Bombus griseocollis
(Hymenoptera: Apidae) share in the brood care of nestmates by incubating pupae (usually during the first few days after they emerge as adults). Male posture during incubation of a pupa is identical to that observed for females. Pupae incubated by males were 4°C-6°C above the temperature of unincubated pupae. Although this increase was not as great as that caused by workers or queens, it was an important factor in warming pupae. Incubating males may benefit nestmates incidentally without lowering their own individual fitness.
Structure of canine pulmonary surfactant apoprotein: cDNA and complete amino acid sequence.
Authors: Benson B , Hawgood S , Schilling J , Clements J , Damm D , Cordell B , White R T
Published: May 2006

The apoproteins of pulmonary surfactant (PSAP) are thought to be critical for normal surfactant function. They bind to surfactant phospholipids and enhance their ability to form surface films in vitro. These acidic glycoproteins have monomeric molecular weights of 36,000, 32,000, and 28,000 (PSAP-36, -32, and -28). Each member of this family of proteins has a similar amino acid composition and their differences in electrophoretic mobility are due in part to glycosylation. We have derived the full amino acid sequence of PSAP-32 from the nucleotide sequence of PSAP cDNA. A cDNA library was prepared from canine lung poly(A)+ RNA and screened with oligonucleotide probes that were based on the NH2-terminal amino acids of PSAP-32 determined by Edman degradation. This protein has the striking feature of collagen-like and non-collagen-like sequences in the same polypeptide chain. There are 24 Gly-Xaa-Yaa triplets, where Yaa is often hydroxyproline. These repeats comprise one-third of PSAP near the NH2 terminus. The remaining two-thirds of PSAP is resistant to bacterial collagenase digestion and contains a possible N-glycosylation site near the carboxyl terminus. The NH2-terminal one-third of PSAP-32 probably contains the cysteine involved in interchain disulfide bonds.
Inhibition of chymotrypsin by heparin cofactor II.
Authors: Church F C , Noyes C M , Griffith M J
Published: May 2006

Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. The rate of thrombin inhibition by heparin cofactor II is accelerated (greater than or equal to 1000-fold) in the presence of the glycosaminoglycans, heparin and dermatan sulfate. We have found that chymotrypsin A alpha is also inhibited by heparin cofactor II with a second-order rate constant value of 1.8 X 10(6) M-1 X min-1 at pH 8.0 and 25 degrees C. However, there was no measurable effect of heparin or dermatan sulfate on the rate of chymotrypsin inhibition. Arginine-modified heparin cofactor II showed a comparable percentage loss of both antichymotrypsin and antithrombin activities. Heparin cofactor II and chymotrypsin formed a stable complex with a Mr value near 90,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis; this suggests a 1:1 reaction stoichiometry. The chymotrypsin cleavage site in heparin cofactor II was the same as that for thrombin, and primary structure analysis of the inhibitor showed a P'1-P'8 sequence of Ser-Thr-Gln-Val-Arg-Phe-Thr-Val ... . The results indicate that, in contrast to alpha 1-antichymotrypsin, which does not inhibit trypsin-like enzymes, including thrombin, heparin cofactor II can effectively inhibit both thrombin and chymotrypsin.
Cleavage of cruciform DNA structures by an activity from Saccharomyces cerevisiae.
Authors: West S C , Körner A
Published: May 2006

Protein extracts from Saccharomyces cerevisiae have been fractionated to reveal a nuclease activity that cleaves cruciform structures in DNA. Negatively supercoiled plasmids that contain inverted repeats that are extruded into cruciform structures have been used as DNA substrates. The sites of cleavage of pColIR215 DNA are located within the extruded cruciform stems and are symmetrically opposed to each other across the cruciform junction. Neither relaxed duplex DNA nor single-stranded DNA serve as substrates. The native molecular weight of the activity was estimated to be approximately equal to 200,000 by gel filtration.
Growing Xenopus oocytes have spare translational capacity.
Authors: Taylor M A , Johnson A D , Smith L D
Published: May 2006

Previous studies have shown that exogenous mRNAs injected into full-grown (stage 6) Xenopus oocytes are translated only at the expense of endogenous messages; translational capacity is limited. In this report, we demonstrate that injection of globin mRNA into small, stage 4 oocytes results in an increase in total protein synthesis without a concomitant decrease in the translation of endogenous mRNAs. The absence of competition with endogenous messages in stage 4 oocytes, injected with globin mRNA, compared with stage 6 oocytes, was not due to differential turnover of the injected mRNA. Hybridization of RNA from mRNA-injected oocytes at both stages revealed that similar amounts of globin mRNA were present. These results are interpreted to mean that protein synthesis in growing oocytes is limited by the availability of mRNA and not components of the translational machinery. This conclusion, however, does not apply to all mRNA classes. The capacity of stage 4 oocytes to translate a mRNA (zein) on membrane-bound polysomes is as restricted as reported previously for stage 6 oocytes. This result suggests that putative membrane binding sites or rough endoplasmic reticulum content are limited in oocytes at both stages. The possible role of association of injected mRNA with endogenous proteins that prevent translation is discussed.
Purification and characterization of a human tumor necrosis factor from the LuKII cell line.
Authors: Rubin B Y , Anderson S L , Sullivan S A , Williamson B D , Carswell E A , Old L J
Published: May 2006

A factor with tumor necrosis factor (TNF) activity produced by the LuKII human lymphoblastoid cell line [designated TNF(LuKII)] was purified sequentially by using controlled-pore glass, lentil lectin-Sepharose, and procion red agarose chromatography, yielding TNF with a specific activity of 1.5 X 10(7) units per mg of protein and an isoelectric point of approximately equal to 6.7. Purified TNF(LuKII) fractionated by NaDodSO4/PAGE under reducing as well as nonreducing conditions was found to contain seven protein bands of Mr 80,000, 70,000, 43,000, 25,000, 23,000, 21,000, and 19,000. The proteins of Mr 80,000 and 70,000 could not be dissociated into lower molecular weight components. Peptide mapping analysis and immunoblotting analysis revealed that the seven protein bands in the purified TNF(LuKII) preparations are related. After fractionation of TNF(LuKII) by NaDodSO4/PAGE under reducing conditions, TNF activity was recovered from the regions of Mr 70,000 and 19,000-25,000. Purified human TNF(LuKII) (i) produces hemorrhagic necrosis of Meth A mouse sarcoma in the standard in vivo mouse TNF assay; (ii) has the same pattern of reactivity as mouse TNF (cytotoxic/cytostatic/no effect) on a panel of human cancer cell lines; and (iii) has its anticellular effect potentiated by interferon, also a feature of mouse TNF.
Phorbol ester induces a differential effect on the effector function of human allospecific cytotoxic T lymphocyte and natural killer clones.
Authors: Bensussan A , Tourvieille B , Chen L K , Dausset J , Sasportes M
Published: May 2006

Six-day allosensitized human peripheral blood lymphocytes treated for 12-18 hr with phorbol 12-myristate 13-acetate (PMA) were found to lose the capacity to kill the specific target in a standard cell-mediated lympholysis (CML) assay, but they were still effective in their ability to kill the tumor cell line K562. We investigated which antigens on the cell membrane involved in alloimmune recognition might be modified by PMA, since it was found that in a lectin-dependent CML assay, the lytic mechanism was not impaired. For this purpose, T3+, T4+ allospecific cytotoxic T lymphocyte (CTL) clones and either T3+ or T3- natural killer (NK) clones were generated from 6-day allostimulated lymphocytes. The results indicated that PMA inhibited the cytolytic function of both alloimmune CTL and NK T3+ clones. In contrast, PMA did not modify the effector cell function of T3- NK clones. Phenotypic analysis of T-cell surface antigens from T3+ clones showed that T3 molecule expression on the cell membrane was reduced by 80-90% after PMA treatment, whereas expression of both accessory T4 molecules, involved in antigen recognition, and receptor for interleukin 2 was increased. Moreover, the loss of function was transitory and could be restored 4 days after PMA treatment when the T3 molecules were fully reexpressed at the cell surface. Taken together, these data strongly suggest that (i) PMA prevents cell-mediated cytotoxicity by modulating the disulfide-linked heterodimer associated to T3 and described as the receptor for antigen on the cell surface of major histocompatibility complex (MHC) and non-MHC specific CTL clones, without affecting the lytic mechanism per se, and (ii) the expression of the receptor for the antigen present on the tumor cell line K562 is not decreased on T3- NK clones after PMA treatment and must be different from that on T3+ T-cell clones.
Calcium binding in pigmented and albino eyes.
Authors: Dräger U C
Published: May 2006

The localization of calcium binding sites in eyes was determined autoradiographically after extracting endogenous Ca from tissue sections and replacing it with 45Ca. The strongest labeling was associated with pigmented tissues due to the high concentration of melanin, which was shown to bind Ca effectively and in a pH-dependent fashion. The second strongest binding was over the tapetum lucidum of the cat eye, and moderate labeling was associated with eye muscles and epithelium and endothelium of the cornea. The neural retina was generally more lightly labeled than the surrounding tissue of the eye; here the plexiform layers stood out in comparison to the nuclear layers, as did a band located internal to the photoreceptor outer segments. The possibility that the Ca buffering capacity of melanin may represent the common denominator for the various neurological defects found in hypopigmentation mutants is discussed.
On the mechanism of renaturation of complementary DNA strands by the recA protein of Escherichia coli.
Authors: Bryant F R , Lehman I R
Published: May 2006

The renaturation of complementary DNA strands by the recA protein of Escherichia coli has been found to exhibit the following features. (i) Optimal renaturation occurs at recA protein levels below that required to saturate the DNA strands; saturating amounts of recA protein significantly reduce the rate of reaction. (ii) The reaction proceeds in the absence of a nucleotide cofactor but is markedly stimulated by ATP in the presence of 10 mM Mg2+. A similar stimulation occurs in the absence of ATP when the Mg2+ concentration is increased from 10 mM to 30-40 mM. (iii) Both the ATP-stimulated and the Mg2+-stimulated reactions follow apparent first-order kinetics. These results, taken together with the known effects of ATP and Mg2+ on the state of aggregation of recA protein, suggest that the association of recA monomers may play an important role in recA protein-promoted DNA renaturation.
Purification of the Rous sarcoma virus src kinase by casein-agarose and tyrosine-agarose affinity chromatography.
Authors: Fukami Y , Lipmann F
Published: May 2006

A simple and effective purification method for the src kinase, the transforming gene product of Rous sarcoma virus, has been developed by using affinity chromatography on casein-agarose and tyrosine-agarose columns. NaDodSO4/polyacrylamide gel electrophoresis and silver staining analysis showed that the purified kinase preparation was composed of a predominant polypeptide of 60,000-Da. In most of the preparations, however, three minor proteins (54,000, 52,000, and 15,000 Da) were also detected, and they were partially characterized. As one of the exogenous substrates, calmodulin was found to be phosphorylated on tyrosine by the purified src kinase.
Detection of larger polypeptides structurally and functionally related to type I transforming growth factor.
Authors: Linsley P S , Hargreaves W R , Twardzik D R , Todaro G J
Published: May 2006

Peptides corresponding to various regions of type I rat transforming growth factor (rTGF) have been chemically synthesized, conjugated to carrier protein, and used to immunized rabbits. An antiserum raised against one of these peptides, corresponding to the carboxyl-terminal 17 amino acids of rTGF, has been used to develop a competitive RIA for the immunizing peptide. In this assay, the antiserum reacts only with a restricted region of the immunizing peptide corresponding to the 11 carboxyl-terminal amino acids of the rTGF molecule. Intact rTGF competes as well as the immunizing peptide on a molar basis, indicating that the cognate sequence in rTGF is recognized by this antiserum. Mouse epidermal growth factor (mEGF) was ineffective as a competitor, even at a 10,000-fold greater concentration, showing that the RIA was capable of distinguishing TGF from functionally related proteins. Several immunoreactive species were detected by gel filtration of conditioned medium from cultured retrovirus-transformed rat cells; both a low molecular weight species that corresponds to rTGF, and a higher molecular weight specie(s) copurify with biologic activity in the EGF radioreceptor assay. Immunoblotting analysis of the higher molecular weight peak fractions revealed three polypeptides (MrS, 24,000, 40,000, and 42,000) that reacted specifically with the antiserum. These findings suggest the existence of a family of larger polypeptides containing both structural and functional determinants of rTGF.
Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity.
Authors: Manne V , Bekesi E , Kung H F
Published: May 2006

Several ras genes have been expressed at high levels in Escherichia coli and the resultant ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. We were able to detect a weak GTPase activity associated with the purified proteins. The normal cellular ras protein (p21N) exhibits approximately equal to 10 times higher GTPase activity than the "activated" proteins. Even though the turnover rate of the reaction is very low (0.02 mol of GTP hydrolyzed per mol of p21N protein per minute), the reaction appears to be catalytic; one molecule of p21N hydrolyzes more than one molecule of GTP. The GTPase and the GDP binding activities both have been recovered from a Mr 23,000 protein eluted following NaDodSO4/polyacrylamide gel electrophoresis, suggesting that these two activities are associated with the same protein. Mg2+ ions and dithiothreitol are required for GTPase activity and the optimal pH is between 7 and 8. Guanidine X HCl, which is required for solubilizing bacterially expressed ras protein, is strongly inhibitory to GTPase activity at concentrations higher than 0.5 M.
Adolescence in Tetrahymena thermophila.
Authors: Rogers M B , Karrer K M
Published: May 2006

The development of sexual maturity in Tetrahymena thermophila has been shown to include an intermediate stage, adolescence, during which cells are capable of mating with mature cells but not other adolescent cells. When the progeny of successfully mated cells are grown logarithmically and tested frequently for the ability to mate, they are unable to form mating pairs for about 65 generations. This period is known as immaturity. During the next stage, the progeny pair with mature cells but not with other adolescent cells despite the presence of complementary mating types. Adolescence persists for 20-25 generations before the cells attain maturity, which is defined as the ability to mate with any cell of different mating type. Once paired with mature cells, adolescents successfully complete conjugation. Cytological preparations show that both members of the pair undergo meiosis and form macronuclear anlagen. The proteins synthesized during a mating between adolescents and mature cells are similar to those synthesized during a mating between mature cells as determined by two-dimensional gel analysis. Both the adolescent cell and the mature partner contribute genetic markers to the progeny.
Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein.
Authors: Bouillaud F , Ricquier D , Thibault J , Weissenbach J
Published: May 2006

The uncoupling protein (UCP) of mammalian brown fat is a specialized and unique component responsible for energy dissipation as heat. Translation and immunoprecipitation from sucrose-fractionated mRNA indicated that the mRNA of UCP sedimented at 14-16 S. A recombinant cDNA library prepared from mRNA of thermoactive brown fat enriched for UCP mRNA has been constructed and cloned in Escherichia coli. Recombinant plasmids were screened by differential colony hybridization to a cDNA probe complementary to poly(A)+ RNA isolated from thermogenic or from weakly thermogenic brown fat. Several differentially hybridizing plasmids were shown to contain UCP cDNA sequences by their ability to select a mRNA coding for an in vitro translation product that was immunoprecipitable with antibodies against UCP. Blot hybridization of brown fat mRNA to a 32P-labeled UCP cDNA probe revealed two major species of mRNA (15S and 18S). As compared to non-thermogenic tissue, a strikingly increased hybridization to the probe was observed with brown fat mRNA from thermoactive tissue. Moreover, hybridization was observed with RNA of brown adipose tissue from rat, hamster, or mouse but not with RNA from rat or mouse liver.
Acquired immunological tolerance of foreign cells is impaired by recombinant interleukin 2 or vitamin A acetate.
Authors: Malkovský M , Medawar P B , Thatcher D R , Toy J , Hunt R , Rayfield L S , Doré C
Published: May 2006

The susceptibility of newborn mice to the inception of tolerance after exposure to antigen is associated with their deficiency in the production of endogenous interleukin 2 (IL-2). As further evidence of the complicity of IL-2 in the inception and maintenance of tolerance, it is shown here that a solid and long-lasting state of tolerance induced by the intravenous injection into newborn CBA mice of lymphoid cells from (CBA X C57BL/10ScSn)F1 hybrids can be brought to an end by the administration of exogenous IL-2 or by supplementing an otherwise normal diet with vitamin A acetate, the effect of which is to increase the proportion of the moiety of the T-cell population that produces IL-2. These results indicate that certain nonspecific stimuli can influence whether immunological tolerance is maintained.
Murine monoclonal antibodies to the myelin-associated glycoprotein react with large granular lymphocytes of human blood.
Authors: Dobersen M J , Gascon P , Trost S , Hammer J A , Goodman S , Noronha A B , O'Shannessy D J , Brady R O , Quarles R H
Published: May 2006

Monoclonal antibodies prepared to human myelin-associated glycoprotein were shown to react with a population of human peripheral blood mononuclear cells. The population is similar to the large granular lymphocytes or natural killer cells defined by antibody Leu 7 (also called HNK-1). The population also includes cells exhibiting the Leu 2 marker for suppressor/cytotoxic T cells. The results indicate a shared antigenicity between the nervous system and the immune system and may be relevant to the pathogenesis of demyelinating diseases.
Different effects of N-ethylmaleimide on M1 and M2 muscarine receptors in rat brain.
Authors: Flynn D D , Potter L T
Published: May 2006

N-Ethylmaleimide (MalNEt) disclosed three differences between M2 muscarine receptors in membranes from the rat brainstem and M1 receptors in the hippocampus. At 0.1 mM, MalNEt completely interconverted the higher affinity state of M2 receptors for carbachol to a lower affinity state, while having no effect on the two affinity states of M1. This "uncoupling" effect is similar to that produced by guanine nucleotides and appears to be due to separation of an agonist-receptor complex from a guanine nucleotide-binding protein. Higher MalNEt concentrations (1-5 mM) increased the affinity of uncoupled M2 receptors, again without effect on M1 states. Finally, in MalNEt, the affinity of M2 receptors for carbachol was different from values for M1 receptors. Thus, MalNEt is an excellent agent for distinguishing M1 and M2 receptors and the two states of M2 receptors. MalNEt had no effect on the affinity or M1-selectivity of the antagonist pirenzepine.
A unique subspecies of histone H4 mRNA from rat myoblasts contains poly(A).
Authors: Bird R C , Jacobs F A , Stein G , Stein J , Sells B H
Published: May 2006

Fractionation of rat L6 myoblast histone H4 mRNA into its three component subspecies revealed that one of the major subspecies (H4-1) contained poly(A). The unique poly(A)+ H4 mRNA makes up about 8% of the total polysomal H4 mRNA population detected. Unlike the poly(A)- histone mRNAs, whose levels are reduced by greater than 95% when myoblasts differentiate into myotubes, the poly(A)+ subspecies is reduced by only 70%. The poly(A)+ H4 mRNA from myotubes incubated with actinomycin D decays with a half-life of 37-42 min, which is similar to that obtained for the poly(A)- H4 mRNAs in myoblasts. Both the poly(A)+ and poly(A)- subspecies decay at an increased rate after inhibition of DNA synthesis. In myoblasts the poly(A)+ H4 mRNA exists almost exclusively in the polysomal compartment (greater than 95%) with little (less than 5%) in the free ribonucleoprotein (mRNA-protein or mRNP) complex compartment of the cell. Poly(A)- histone H4 mRNA subspecies, on the other hand, are distributed with approximately 80% in the polysomal compartment and 20% in the free mRNP complex compartment. The unique poly(A)+ H4 mRNA is unusual, not only in that it contains poly(A) but also in its behavior compared to poly(A)- H4 mRNAs during terminal differentiation.
The cellular defect in alpha 1-proteinase inhibitor (alpha 1-PI) deficiency is expressed in human monocytes and in Xenopus oocytes injected with human liver mRNA.
Authors: Perlmutter D H , Kay R M , Cole F S , Rossing T H , Van Thiel D , Colten H R
Published: May 2006

To determine the basis for low serum concentrations of alpha 1-proteinase inhibitor (alpha 1PI) in individuals with homozygous alpha 1PI deficiency (hereafter referred to as PiZZ), biosynthesis and secretion of alpha 1PI were studied in Xenopus oocytes microinjected with hepatic mRNA and in blood monocytes (an extrahepatic site of alpha 1PI gene expression). Although both the usual alpha 1PI (hereafter referred to as PiMM) and PiZZ alpha 1PI were secreted in functionally active form, the rate of secretion of alpha 1PI was significantly and selectively decreased in Xenopus oocytes injected with PiZZ liver mRNA and in monocytes from PiZZ individuals. The apparent size of alpha 1PI in the intracellular compartment of Xenopus oocytes injected with PiZZ liver mRNA was different from the corresponding intracellular PiMM alpha 1PI in oocytes injected with PiMM liver mRNA. There were also differences in the relative ratio of native and complexed alpha 1PI secreted by monocytes from individuals with PiMM and PiZZ phenotypes.
Nucleotide sequence, evolution, and expression of the fetal globin gene of the spider monkey Ateles geoffroyi.
Authors: Giebel L B , van Santen V L , Slightom J L , Spritz R A
Published: May 2006

The single gamma-globin gene of the New World spider monkey Ateles geoffroyi is similar to other primate genes of the beta-globin gene cluster ("beta-like" globin genes). The number of nonsynonymous nucleotide substitutions between the coding regions of Ateles and other primate gamma-globin genes suggests that the Platyrrhine and Catarrhine evolutionary lines diverged approximately equal to 40 million years ago, an estimate reasonably consistent with the fossil record. However, the number of synonymous coding region and noncoding base differences is much smaller than predicted by various molecular "clocks." This suggests that the rate of synonymous coding and noncoding base substitution has not been constant per absolute time in primate lineages. Expression of the cloned Ateles gamma-globin gene in cultured monkey cells showed that the sequence AAUAAA near the mRNA 3' terminus is not sufficient to define the site of transcript polyadenylylation.
Sex- and cell-specific regulation of yolk polypeptide genes introduced into Drosophila by P-element-mediated gene transfer.
Authors: Tamura T , Kunert C , Postlethwait J
Published: May 2006

To find whether cis-acting regulatory sequences necessary for sex- and cell-specific expression of two yolk polypeptide genes (Yps) reside near the structural genes themselves, we introduced a 5.0-kilobase genomic DNA segment containing a 3' truncated Yp1 and a complete Yp2 into five different autosomal locations by P-element-mediated gene transfer. Transcripts from the introduced Yp1 were not found in male flies but appeared on a normal developmental schedule in adult females, accumulating in their body walls and ovarian follicles but not in guts or malpighian tubules. Protein from the introduced Yp2 allele was present in female hemolymph and vitellogenic ovaries but was lacking from male hemolymph. We conclude that sequences necessary for the correct stage-, cell-, and sex-specific expression of the Yp1 and Yp2 genes are included in this genomic fragment. These results combined with published work suggest that two tissue-specific, cis-acting, bidirectional, positive regulatory elements placed on either side of a centrally located HindIII site govern expression of both Yp genes--one element specific for fat body and the other specific for ovarian follicle cells.
Regulated expression of ras gene constructs in Dictyostelium transformants.
Authors: Reymond C D , Nellen W , Firtel R A
Published: May 2006

Constructs were made in which approximately equal to 500 base pairs of the 5' flanking region of the ras gene of Dictyostelium discoideum and variable amounts of the coding region were linked to a ras cDNA in a transformation vector. These constructs then were used to transform Dictyostelium cells and their regulation was examined. In Dictyostelium transformants, transcripts from the ras gene constructs were found at high levels in cells in fast-shaking cultures containing cAMP, whereas transcripts were either not detectable or present at very low levels in cultures lacking exogenous cAMP. In slow-shaking culture, a significantly lower level of ras RNA was detected. When normal developing aggregates were dissociated, RNA from the ras constructs decreased rapidly but then reaccumulated in the presence of cAMP. These results show that the sequences necessary for the response to external cAMP are present within an approximately equal to 650-base-pair region upstream from the ATG start codon and/or within portions of the protein-coding region. Moreover, the proper regulation of ras gene expression in high-copy-number transformants suggests that trans-acting factors which may control transcription are not limiting. Vector constructs were also examined in which the cDNA was present in the opposite orientation compared to the gene fragment (antisense orientation). When these were transfected into cells, no transformants were obtained, suggesting that expression of the ras gene is essential for vegetative growth.
Blood group A cross-reacting epitope defined by monoclonal antibodies NCC-LU-35 and -81 expressed in cancer of blood group O or B individuals: its identification as Tn antigen.
Authors: Hirohashi S , Clausen H , Yamada T , Shimosato Y , Hakomori S
Published: May 2006

Two monoclonal antibodies, NCC-LU-35 and NCC-LU-81, have been established after immunization of mice with membrane preparations of human lung cancer Lu65 tumor xenograft cells grown in vivo and intact cells cultured in vitro, respectively. These two antibodies react specifically with a majority of human adenocarcinomas, irrespective of the host's blood group ABO status, as well as with normal tissues and erythrocytes of blood group A individuals. The antigenicity is associated with a high molecular weight mucin-like glycoprotein separated by gel filtration of Lu65 tumor extracts. The epitope of the mucin-like glycoprotein has been identified as alpha-N-acetylgalactosaminyl residue directly linked O-glycosidically to serine or threonine residues of polypeptides. This epitope was serologically detected several years ago and given the name Tn. Our identification of the epitope is based on the following results: The antigen is sensitive to alpha-N-acetylgalactosaminidase, but not to sialidase or alpha-fucosidase. Various mono- and difucosyl A determinants, either type 1 or type 2 chain, cross-react with both antibodies. The reactivity with both antibodies can be created by treatment of glycophorin A of normal erythrocytes with sialidase followed by beta-galactosidase. N-[3H]acetylgalactosamine can be released by galactose oxidase/NaB3H4 treatment from the Lu65 mucin-like glycoprotein but not from the mucin-like glycoprotein of normal colonic mucosa upon reductive beta-elimination (alkaline borohydride treatment). The antigen may be one of the tumor-associated A cross-reacting antigens occurring in a wide variety of human adenocarcinomas of hosts belonging to all ABO blood groups.