bluuebunny/crossref_metadata_2025 huggingface dataset:
ob17 cells transformed by the middle-T-only gene of polyoma virus differentiate in vitro and in vivo into adipose cells.
Authors: Grimaldi P , Czerucka D , Rassoulzadegan M , Cuzin F , Ailhaud G
Published: May 2006

Cell lines were derived from ob17 preadipocyte cells by focus formation after transfer of the complete early region of polyoma virus (ob17PY) or of a modified genome encoding only the middle T protein (ob17MT). Both ob17PY and ob17MT cell lines exhibited a high cloning efficiency in agarose medium containing 10% fetal bovine serum. Fully transformed ob17PY cells grew to high saturation densities and did not differentiate in vitro and in vivo. ob17MT cells and derived subclones did not grow in the absence of serum and were able to differentiate in vitro and to give rise in vivo to adipose tumors. Among these different clones an inverse relationship was observed in culture between their potentiality to overproliferate at low serum and their potentiality to convert into adipose cells. The expression of enzyme markers of adipose conversion was strictly dependent upon the presence of growth hormone. In addition, the hormonal requirements for differentiation were simpler than those of the original ob17 cells and the adipose conversion could take place in serum-free hormone-supplemented medium.
Dimethyl sulfoxide can initiate cell divisions of arrested callus protoplasts by promoting cortical microtubule assembly
Authors: Hahne Günther , Hoffmann Franz
Published: May 2006

A serious problem in the technology of plant cell culture is that isolated protoplasts from many species are reluctant to divide. We have succeeded in inducing consecutive divisions in a “naturally” arrested system—i.e., protoplasts from a hibiscus cell line, which do not divide under standard conditions—and in an artificially arrested system—i.e., colchicine-inhibited callus protoplasts of
Nicotiana glutinosa
, which do readily divide in the absence of colchicine. In both cases, the reinstallation of a net of cortical microtubules, which had been affected either by colchicine or by the protoplast isolation procedure, resulted in continuous divisions of the formerly arrested protoplasts. Several compounds known to support microtubule assembly
in vitro
were tested for their ability to promote microtubule assembly
in vivo
. Best results were obtained by addition of dimethyl sulfoxide to the culture medium. Unlimited amounts of callus could be produced with the dimethyl sulfoxide method from protoplasts which never developed a single callus in control experiments.
Failure to demonstrate mutations affecting protein structure or function in children with congenital defects or born prematurely.
Authors: Neel J V , Mohrenweiser H W
Published: May 2006

An effort has been made to confirm the report [Dubinin, N. P. & Altukhov, Y. P. (1979) Proc. Natl. Acad. Sci. USA 76, 5226-5229] that children born prematurely or exhibiting congenital defects can be shown to exhibit relatively high frequencies of rare (nonpolymorphic) electrophoretic variants of proteins and that a large proportion of these variants are due to mutation in either the father or the mother. In a series of 178 children who were comparable with those described in the earlier report, we failed to encounter a high frequency of these variants in some 5341 determinations involving 45 proteins, nor were any mutations observed. Data from 1583 determinations of enzyme activity on a subset of the panel of proteins were also unremarkable. We are thus unable to confirm the earlier report.
Translocation and rearrangement of c-myc into immunoglobulin alpha heavy chain locus in primary cells from acute lymphocytic leukemia.
Authors: Peschle C , Mavilio F , Sposi N M , Giampaolo A , Caré A , Bottero L , Bruno M , Mastroberardino G , Gastaldi R , Testa M G
Published: May 2006

We report the molecular analysis of an 8;14 reciprocal chromosome translocation in a case of acute lymphocytic leukemia (L3 type). DNA from primary leukemic cells was analyzed on the basis of restriction endonuclease mapping by hybridization with various human c-myc and Ig heavy chain probes. The breakpoint of the translocation is within an approximately equal to 200-base-pair region in the first intron of the c-myc gene. The first, untranslated exon thereby remains on chromosome 8q-, whereas the whole protein-coding region is rearranged in the C alpha 1 locus on chromosome 14q+. RNA transfer blot analysis showed high levels of at least two different c-myc transcripts originated from the translocated gene. Both differ in size from the normal 2.2- and 2.4-kilobase transcripts. Both c-myc structure and expression were apparently normalized in remission phase. These studies demonstrate rearrangement and abnormal expression of c-myc in primary cells from an acute leukemia patient, thus adding to the concept of a key role for c-onc in human oncogenesis.
Hydroxymethylglutaryl-coenzyme A reductase-containing hepatocytes are distributed periportally in normal and mevinolin-treated rat livers.
Authors: Singer I I , Kawka D W , Kazazis D M , Alberts A W , Chen J S , Huff J W , Ness G C
Published: May 2006

Mevinolin is a potent inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34), an enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis. We have been studying the hepatic distribution of reductase with immunofluorescence microscopy and liver ultrastructure with electron microscopy in normal and drug-treated rats. In control animals, only about 20% of the hepatocytes were reductase positive. These cells were localized in the periportal lobular zones. The numbers of positive hepatocytes in animals given mevinolin or cholestyramine (or both) were directly proportional to the activities of the HMG-CoA reductase determined biochemically. This induction of HMG-CoA reductase immunofluorescence was centered periportally. Rats given 0.075% mevinolin alone had a homogeneous distribution of reductase staining in their hepatocyte cytoplasm, whereas a combination of 0.25% mevinolin and 3% cholestyramine caused a 150-fold increase in enzyme activity and induced prominent juxtanuclear immunofluorescent globules of HMG-CoA reductase in all hepatocytes. With electron microscopy, these bodies were composed of tightly packed stacks of smooth endoplasmic reticulum cysternae and aggregates of branched smooth endoplasmic reticulum tubules. Our data suggest that a subpopulation of periportal rat hepatocytes may be uniquely specialized for cholesterol synthesis.
Heterogeneity of microtubule-associated protein 2 during rat brain development.
Authors: Binder L I , Frankfurter A , Kim H , Caceres A , Payne M R , Rebhun L I
Published: May 2006

The electrophoretic pattern of the large microtubule-associated protein, MAP2, changes during rat brain development. Immunoblots of NaDodSO4 extracts obtained from the cerebral cortex, cerebellum, and thalamus at 10-15 days after birth reveal only a single electrophoretic species when probed with any of three MAP2 monoclonal antibodies. By contrast, adult MAP2 contains two immunoreactive species, MAP2a and MAP2b. The single band of MAP2 from immature brain electrophoretically comigrates with adult MAP2b. Between postnatal days 17 and 18, immature MAP2 simultaneously resolves into two species in both the cerebellum and cerebral cortex. Immunoblots of NaDodSO4 extracts from spinal cord demonstrate the adult complement of MAP2 by day 10, indicating that MAP2 does not change coordinately throughout the entire central nervous system. In vitro cAMP-dependent phosphorylation of immature MAP2 causes a band split reminiscent of that seen during brain development in vivo. The possibility that the developmentally regulated changes observed in MAP2 during brain maturation are due to timed phosphorylation events is discussed.
Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.
Authors: John M E , John M C , Ashley P , MacDonald R J , Simpson E R , Waterman M R
Published: May 2006

Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts approximately equal to 425 and approximately equal to 950 base pairs long, respectively, which are specific for bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450scc with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P-450scc is encoded by mRNA species approximately equal to 2000 bases long, a majority of which are polyadenylylated. P-450scc mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450scc mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450scc may be encoded by a single gene.
Thymine glycol and thymidine glycol in human and rat urine: a possible assay for oxidative DNA damage.
Authors: Cathcart R , Schwiers E , Saul R L , Ames B N
Published: May 2006

Thymine glycol is a DNA damage product of ionizing radiation and other oxidative mutagens. In an attempt to find a noninvasive assay for oxidative DNA damage in individuals, we have developed an HPLC assay for free thymine glycol and thymidine glycol in urine. Our results indicate that humans excrete about 32 nmol of the two glycols per day. Rats, which have a higher specific metabolic rate and a shorter life span, excrete about 15 times more thymine glycol plus thymidine glycol per kg of body weight than do humans. We present evidence that thymine glycol and thymidine glycol are likely to be derived from repair of oxidized DNA, rather than from alternative sources such as the diet or bacterial flora. This noninvasive assay of DNA oxidation products may allow the direct testing of current theories which relate oxidative metabolism to the processes of aging and cancer in man.
Effect of single amino acid replacements on the thermal stability of the NH2-terminal domain of phage lambda repressor.
Authors: Hecht M H , Sturtevant J M , Sauer R T
Published: May 2006

The thermal stabilities of mutant phage lambda repressors that have single amino acid replacements in the NH2-terminal domain have been studied by means of circular dichroism and differential scanning calorimetry. The variations in stability determined by these physical methods correlate with the resistance to proteolysis at various temperatures and can be compared with the temperature-sensitive activity of the mutants in vivo. In general, mutant proteins bearing solvent-exposed substitutions have thermal stabilities identical to wild type, whereas buried substitutions reduce stability. In one case, a single amino acid replacement increases the thermal stability of the repressor.
Diacylglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells: role of Ca2+-sensitive phospholipid-dependent protein kinase.
Authors: Rozengurt E , Rodriguez-Pena A , Coombs M , Sinnett-Smith J
Published: May 2006

The synthetic diacylglycerol 1-oleoyl-2-acetylglycerol competes directly with [3H]phorbol 12,13-dibutyrate for common binding sites in monolayer cultures of Swiss 3T3 cells and rapidly stimulates the phosphorylation of a Mr 80,000 cellular protein that has recently been shown to reflect the activation of protein kinase C in intact cells. Thus, this diacylglycerol provided a useful tool to determine whether exogenously added diacylglycerols can mimic the potent tumor promoter phorbol ester in eliciting DNA synthesis and cell division in quiescent cells. We found that OAG acts synergistically with insulin and other growth factors to stimulate reinitiation of cell proliferation, and several lines of evidence indicate that OAG shares with phorbol esters a common pathway of mitogenic action via stimulation of protein kinase C activity in intact 3T3 cells. The findings support the hypothesis that diacylglycerols represent endogenous analogs of phorbol esters and raise the possibility that diacylglycerols generated in the plasma membrane could act as a mitogenic signal for quiescent cells.
Biological activities of v-myc and rearranged c-myc oncogenes in rat fibroblast cells in culture.
Authors: Mougneau E , Lemieux L , Rassoulzadegan M , Cuzin F
Published: May 2006

Two distinct forms of the myc oncogene were assayed for their ability to induce, in cultured rat fibroblast cells, the alterations of cellular growth controls observed upon transfer of the gene of polyoma virus encoding only the large T protein (plt). Both of these rearranged myc genes and the plt gene had been previously shown to cooperate with ras oncogenes for transformation of rat embryo fibroblasts (REF) and were thought to induce the same early step ("immortalization") of the tumoral transformation pathway. We now report that these two different oncogenes elicite the same response in the following biological assays: (i) reduction of the requirements in serum factors for growth in culture of cells of the established FR3T3 line; (ii) expression of transformed properties in low serum medium after transfer into FR3T3 cells expressing only the middle T protein of polyoma virus (MTT lines); (iii) conferring on REF cells the ability to grow as clonal colonies after seeding at low cell density; (iv) conferring on REF cells the ability to grow continuously in cell culture. These congruent phenotypes suggest that the activities of the large T and myc proteins result in the induction of the same molecular events. These results also provide simple biological assays and selective systems for oncogenes of the myc class.
Long-acting delivery systems for peptides: inhibition of rat prostate tumors by controlled release of [D-Trp6]luteinizing hormone-releasing hormone from injectable microcapsules.
Authors: Redding T W , Schally A V , Tice T R , Meyers W E
Published: May 2006

Intramuscular injection of [6-D-tryptophan]-luteinizing hormone-releasing hormone [( D-Trp6]LH-RH) in microcapsules of poly(DL-lactide-co-glycolide), designed to release a controlled dose of the peptide over a 30-day period, decreased the weights of androgen-dependent Dunning prostate tumors in rats and suppressed serum testosterone levels more effectively than daily subcutaneous administration of equivalent or double doses of unencapsulated [D-Trp6]LH-RH. The microcapsules or daily injections of [D-Trp6]LH-RH also significantly decreased tumor volumes. Microcapsules of [D-Trp6]LH-RH or related analogs that can be injected once a month should make the treatment of patients with prostate carcinoma and other neoplasms or disorders more convenient and efficacious.
Liver uptake of chylomicron remnants with high and low apoprotein E:C ratios.
Authors: Borensztajn J , Kotlar T J
Published: May 2006

The ability of the isolated perfused rat liver to differentiate between chylomicrons and remnants with either high or low apoprotein E:C ratios was investigated. Remnants were prepared in hepatectomized rats injected with chylomicrons double-labeled with [3H]cholesterol and 14C-labeled fatty acids. By densitometric scanning of polyacrylamide gels, the apoprotein E:C ratio of the chylomicrons was 0.8 and that of the remnants was 1.5. When livers were perfused with these lipoproteins in a recirculatory system for 4 min, uptake of remnants was about 3-fold greater than that of chylomicrons. Preparation of remnants as well as chylomicrons with a low apoprotein E:C ratio was achieved by (i) removal of all apoproteins from the surface of the lipoproteins by trypsin digestion, followed by (ii) transfer of soluble apoproteins from serum lipoproteins to the apoprotein-free particles. The apoprotein E:C ratio of the reconstituted lipoproteins was decreased from 1.5 to 0.3 for remnants and from 0.8 to 0.2 for chylomicrons. In spite of these changes in apoprotein E:C ratios, the hepatic uptake of the reconstituted lipoproteins with low apoprotein E:C ratios was similar to their unmodified controls. These results indicate that the hepatic discrimination between chylomicrons and remnants is not determined by the relative amounts of apoproteins E and C on the surface of the lipoproteins.
Modulation of cholecystokinin concentrations in the rat hippocampus by chelation of heavy metals.
Authors: Stengaard-Pedersen K , Larsson L I , Fredens K , Rehfeld J F
Published: May 2006

Previously, we have reported that enkephalins, cholecystokinin, and heavy metals show roughly parallel distributional patterns in the hippocampus. A substantial body of evidence indicates that cholecystokinin-octapeptide (CCK-8) and enkephalins act as neurotransmitters. A CCK-8 degrading enzyme was recently detected in brain synaptosomes. Its activity depended on free thiol groups and the presence of a heavy metal. Since the heavy metal-containing neuropil is closely related to CCK-immunoreactive nerve terminals, we have investigated the effect of metal chelation on CCK components in the rat hippocampus. In vivo treatment of rats with a single dose of the chelating agent diethyldithiocarbamate caused a reversible chelation of heavy metals in the hippocampus. This effect was paralleled by a 3-fold increase in hippocampal content of CCK-8 and a smaller increase in the intermediate forms of CCK (CCK-58, CCK-39, CCK-33). Diethyldithiocarbamate also decreased the spontaneous motility and aggressiveness of the rats. These data show reversible changes of neuronal CCK processing by a drug, and hence they provide additional evidence that CCK is involved in the regulation of neuronal activities.
Expression of Epstein-Barr virus (EBV) DNA and cloned DNA fragments in human lymphocytes following Sendai virus envelope-mediated gene transfer.
Authors: Volsky D J , Gross T , Sinangil F , Kuszynski C , Bartzatt R , Dambaugh T , Kieff E
Published: May 2006

Purified EBV DNA and cloned DNA fragments were trapped in Sendai virus (SV) envelopes during envelope reconstitution. The DNA-loaded reconstituted envelopes (RSVE/DNA) served as gene-transfer vehicles using the capability of RSVE to fuse with normal and tumor cells. The efficiency of RSVE-mediated EBV DNA transfer into lymphoid tumor cells and fresh human lymphocytes was 5-10% of the enveloped 3H-labeled EcoRI fragment B of EBV DNA. Purified intracellular EBV (B95-8 strain) DNA induced EBV nuclear antigen (EBNA) in 0.2-1% of human lymphocytes, transiently stimulated cellular DNA synthesis, but did not fully transform cells. Cloned Sal I F1 fragment [approximately equal to 9 kilobase pairs (kbp)] and a smaller BamHI K (5.2 kbp) fragment from the same region of B95-8 EBV DNA induced EBNA in 2-4% of human lymphocytes but did not stimulate DNA synthesis nor transform cells. Cloned BamHI D1 fragment (approximately equal to 9 kbp) from AG-876 virus DNA, or a combination of cloned BamHI X and H fragments (approximately equal to 2 and 7 kbp, respectively) from the similar region of B95-8 virus DNA, significantly stimulated lymphocyte DNA synthesis, but EBNA could not be detected and transformation was not achieved. Early antigen and viral capsid antigen were not observed with any of the fragments tested. Our results suggest that the induction of EBNA and stimulation of lymphocyte proliferation are not controlled by the same region of EBV DNA.
Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro.
Authors: Maderious A , Chen-Kiang S
Published: May 2006

The major late transcriptional unit of adenovirus type 2 has served as a model for studying transcription in eukaryotes. We report that pausing and premature termination are intrinsic to the transcription of this transcriptional unit by RNA polymerase II. In vivo and in isolated nuclei, transcription pauses at discrete sites proximal to the initiation site and can prematurely terminate at nucleotide 175 and possibly also at nucleotide 120. The prematurely terminated RNAs are not associated with the transcription complexes and accumulate in the cell nucleus in vivo, whereas paused RNAs remain associated with the transcription complexes and elongate into full-length transcripts. Pausing is also reproduced in the transcription complexes in a soluble system. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole enhances pausing but not premature termination, and its action is reversible. The proposed premature termination site at nucleotide 175 in adenovirus type 2 bears sequence homology to the tR1 site in coliphage lambda.
Electrical polarity in embryos of wild carrot precedes cotyledon differentiation
Authors: Brawley Susan H. , Wetherell Donald F. , Robinson Kenneth R.
Published: May 2006

Endogenous electrical currents traverse embryos of a higher plant, the wild carrot
Daucus carota
L. Current enters the apical pole and leaves the region near the presumptive radicle in the radially symmetric globular embryo. Current also enters the exposed surfaces of incipient globular embryos. This electrical polarity precedes differentiation of vascular tissue and cotyledon development. Localized current is observed at both growing ends of the embryos in subsequent stages of embryogenesis. Inward current is found at the cotyledons; outward current is found at the radicle/root. Exogenous indole-3-acetic acid (3 μM) reversibly inhibits these currents. Little current traverses the surface of intermediate regions of the embryo. The ionic gradients generated by these currents may be important in accumulation of metabolites and in other developmental processes within the embryo.
Leukotrienes in the rat central nervous system.
Authors: Lindgren J A , Hökfelt T , Dahlén S E , Patrono C , Samuelsson B
Published: May 2006

Leukotrienes C4, D4, and E4 were isolated after incubation of rat brain tissue in vitro with the ionophore A23187 and arachidonic acid. Identification of the compounds was carried out using high-performance liquid chromatography, radioimmunoassay, and bioassay. Average production of leukotrienes C4, D4, and E4 during 10 min of incubation was estimated to 25, 8, and 0.7 pmol per g of brain tissue (wet weight), respectively. Radioimmunoassay determinations indicated in vitro biosynthesis of leukotriene C4 in most regions of the brain, with the highest levels obtained in the hypothalamus and the median eminence. In slices from the caudate nucleus, ionophore A23187 caused a dose-dependent stimulation of leukotriene C4 formation with maximal effect at 5 microM. Leukotriene C4 synthesis of rat brain tissue was inhibited by 30 microM nordihydroguaiaretic acid. Finally, using the indirect immunofluorescence technique, nerve endings in the median eminence and cell bodies in the preoptic area reacting with antibodies raised against leukotriene C4 were observed.
Different channel properties of Torpedo acetylcholine receptor monomers and dimers reconstituted in planar membranes.
Authors: Schindler H , Spillecke F , Neumann E
Published: May 2006

It is demonstrated that the monomeric and dimeric structures of the nicotinic acetylcholine receptor of Torpedo californica electric tissue, reconstituted in planar lipid bilayers, are functionally different. The native dimer D of Mr 500,000 (heavy-form) exhibits a "single" channel conductance about twice as large as that of the monomer M of Mr 250,000 (light form). Under conditions where monomers aggregate, the conductance changes from the level of the monomer M to that of dimers M2. The dimer conductances (D and M2) seem to result from synchronous opening and closing of the two channels in the dimer, giving the impression of "single channel" activity. This channel cooperativity is apparently mediated by noncovalent interactions between the two monomers, since it requires no disulfide linkage between monomers. Both the monomers M and the dimers D and M2 show at least one substate of lower conductivity. The relative population of the two conductance levels depends on the ion type (Na+ and K+), indicating ion-specific channel states. Since the channel conductance of isolated dimers resembles those obtained from unextracted microsacs, the dimer with two synchronized channels appears to be the in vivo predominant gating unit. In the linear association of dimers, observed in the native membrane, channel synchronization may extend to more than two channels as suggested by oligomeric channel cooperativity in associations of monomers and dimers.
The survival of brain transplants is enhanced by extracts from injured brain.
Authors: Nieto-Sampedro M , Whittemore S R , Needels D L , Larson J , Cotman C W
Published: May 2006

Fragments of rat embryo corpus striatum implanted into wound cavities in the cortex of neonatal rat hosts showed very poor survival when transplantation was performed immediately after the cavity was made. Consistent survival of the transplants was made possible by supplying the implant with extracts from the injured brain of neonatal rats. These extracts also supported in vitro survival of embryonic striatal neurons, suggesting that transplanted cells benefit from injury-induced neuronotrophic factors.
Single catalytic site model for the oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase.
Authors: Speck S H , Dye D , Margoliash E
Published: May 2006

A single catalytic site model is proposed to account for the multiphasic kinetics of oxidation of ferrocytochrome c by cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). This model involves nonproductive binding of substrate to sites near the catalytic site on cytochrome c oxidase for cytochrome c, decreasing the binding constant for cytochrome c at the catalytic site. This substrate inhibition results in an increase in the first-order rate constant for the dissociation of the ferricytochrome c-cytochrome c oxidase complex, the rate-limiting step in the steady-state turnover of electrons between cytochrome c and cytochrome c oxidase in the spectrophotometric assay, yielding increases in the initial rate as well as the Michaelis constant--namely, multiple kinetic phases.
Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis.
Authors: Yansura D G , Henner D J
Published: May 2006

The Escherichia coli lac operator has been placed on the 3' side of the promoter for the penicillinase gene of Bacillus licheniformis, creating a hybrid promoter controllable by the E. coli lac repressor. The E. coli lac repressor gene has been placed under the control of a promoter and ribosome-binding site that allows expression in Bacillus subtilis. When the penicillinase gene that contains the lac operator is expressed in B. subtilis on a plasmid that also produces the lac repressor, the expression of the penicillinase gene can be modulated by isopropyl beta-D-thiogalactoside (IPTG), an inducer of the lac operon in E. coli. A similar system was constructed from a promoter of the B. subtilis phage SPO-1 and the leukocyte interferon A gene, which allowed the controlled expression of interferon in B. subtilis. These two examples show that a functional control system can be introduced into B. subtilis from E. coli.
Asymmetric acetylcholinesterase is assembled in the Golgi apparatus.
Authors: Rotundo R L
Published: May 2006

The synthesis, assembly, and processing of the multiple molecular forms of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) in quail muscle cultures was studied by using lectins to distinguish enzyme molecules residing in different subcellular compartments. Special emphasis was given to the assembly of asymmetric AcChoEase molecules because these appear to be the predominant, if not unique, forms of AcChoEase at the vertebrate neuromuscular junction. All cell surface and secreted AcChoEase forms bind to immobilized wheat germ agglutinin, ricin, and concanavalin A, indicating that they have complex oligosaccharides. After treatment of muscle cells with a membrane-permeable irreversible AcChoEase inhibitor, there is a rapid reappearance of the globular monomeric, dimeric, and tetrameric AcChoEase forms. However, the collagen-tailed asymmetric form does not appear until about 90 min after treatment. Analysis of the AcChoEase oligosaccharides with lectins indicates maturation to complex forms over a 90-min period. A large fraction of the intracellular globular AcChoEase molecules bind only to concanavalin A, indicating that they are assembled in the rough endoplasmic reticulum. In contrast, all intracellular asymmetric AcChoEase binds to wheat germ agglutinin, and a significant fraction binds to ricin, indicating that this unique AcChoEase form is assembled from subunits that have previously acquired complex sugars. I conclude that assembly of asymmetric AcChoEase, hence acquisition of information specifying basal lamina localization, occurs in the Golgi apparatus.
In vivo formation of gene fusions encoding hybrid beta-galactosidase proteins in one step with a transposable Mu-lac transducing phage.
Authors: Casadaban M J , Chou J
Published: May 2006

A Mu-lac bacteriophage transposon, MudII301 (Ap, lac), was constructed to form hybrid protein gene fusions. When it integrates into structural genes in the appropriate direction and reading phase, transcription and translation from outside gene controlling regions can proceed across 116 nucleotides from the right end of Mu into lacZ codons to form hybrid proteins that are enzymatically active for beta-galactosidase. Integration can be obtained either by infection to form lysogens or by transposition during growth of a lysogen. The size of the hybrid protein product either corresponds to or, in the cases of translation restart or protein degradation, is a minimal estimate of the distance of the Mu insertion from the translation initiation site of the gene. Hybrid proteins formed by insertions in randomly selected genes and in the araB and A genes were examined by polyacrylamide gel electrophoresis.
nusB: a protein factor necessary for transcription antitermination in vitro by phage lambda N gene product.
Authors: Ghosh B , Das A
Published: May 2006

We demonstrate that the protein product of the Escherichia coli nusB gene is essential for transcription antitermination in vitro by phage lambda N gene product. We recently have described a convenient biochemical assay for N protein activity in the S30-coupled transcription translation system and demonstrated that N action requires the 69-kDa L factor (nusA), the product of E. coli nusA gene. Using a complementation assay for the restoration of N activity specifically in the nusB mutant extract, we have purified the nusB complementing activity. This activity is due to a 15-kDa polypeptide that is overproduced in E. coli containing multiple copies of the nusB gene. We find that nusA and nusB are required for N activity to suppress a rho-dependent as well as a rho-independent terminator. The requirement for nusB protein in antitermination could not be overcome by an excess of nusA or N protein, nor could an excess of nusB overcome the requirements for nusA in antitermination. Our results suggest that the formation of an antitermination apparatus by N requires nusA and nusB proteins in equimolar amounts.
Triazine herbicide resistance in the photosynthetic bacterium
Rhodopseudomonas sphaeroides
Authors: Brown Alfred E. , Gilbert Carl W. , Guy Rachel , Arntzen Charles J.
Published: May 2006

The photoaffinity herbicide azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-
s
-triazine) selectively labels the L subunit of the reaction center of the photosynthetic bacterium
Rhodopseudomonas sphaeroides
. Herbicide-resistant mutants retain the L subunit and have altered binding properties for methylthio- and chloro-substituted triazines as well as altered equilibrium constants for electron transfer between primary and secondary electron acceptors. We suggest that a subtle alteration in the L subunit is responsible for herbicide resistance and that the L subunit is the functional analog of the 32-kDa Q
B
protein of chloroplast membranes.
Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.
Authors: Zarucki-Schulz T , Kulomaa M S , Headon D R , Weigel N L , Baez M , Edwards D P , McGuire W L , Schrader W T , O'Malley B W
Published: May 2006

A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 108,000 protein when translated in vitro and which was immunoprecipitable by the alpha PR-B antibody. This cDNA represents a 470-base-pair portion of the PR-B nucleotide sequence. Additional clones have been subsequently isolated from the recombinant library using the insert from pPRB-1 as a specific probe. A mRNA size of approximately 3000 nucleotides was determined for the chicken progesterone receptor B subunit by formaldehyde/agarose gel electrophoresis and blot hybridization using pPRB-1 as a probe. Preliminary studies show that withdrawal of hormone from chickens treated chronically with estrogen leads to a dramatic decrease in the cellular RNA concentration of receptor B, indicating that target tissue levels of receptor B RNA are under hormonal control.
Radial distributions of density within macromolecular complexes determined from dark-field electron micrographs.
Authors: Steven A C , Hainfeld J F , Trus B L , Steinert P M , Wall J S
Published: May 2006

A procedure has been developed for direct determination of radial distributions of density in filamentous and spheroidal particles by analyzing dark-field scanning transmission electron micrographs of unstained freeze-dried specimens. Unlike electron microscopic methods based on staining or shadowing with heavy atoms, this approach can be used to probe the internal structure of macromolecular complexes. As an experimental proving ground, we have applied the procedure to tobacco mosaic virus (TMV) and to RNA-free helical polymers of TMV coat protein. Both structures are found to project outermost diameters of 17.6 +/- 0.4 nm, to have empty axial holes approximately equal to 3.5 nm in diameter, and to have density peaks at radii of 2.5 +/- 0.5 and 6.7 +/- 0.3 nm. Thus visualized, the only significant difference between them is the presence in the virion of an additional density peak at 4.1 +/- 0.5 nm contributed by its internalized RNA molecule. We have also used the procedure to monitor the structural expression of radiation damage in the low electron dose regime prior to the onset of significant mass loss. Changes in the radial density profiles are detected at average doses as low as approximately equal to 400 electrons per nm2: the trend is for the internal structure of these particles to fuse toward a state of uniform density, although the values of their outermost diameters remain unaffected.
Loss of reciprocal modulations of growth and liver function of hepatoma cells in culture by contact with cells or cell membranes.
Authors: Nakamura T , Nakayama Y , Teramoto H , Nawa K , Ichihara A
Published: May 2006

In mature rat hepatocytes in primary culture, many metabolic functions and cell growth are controlled reciprocally by cell density, and this reciprocal regulation is mediated by a cell-surface modulator through cell-cell contact. Cultured RY-121B cells from Reuber hepatoma and MH1C1 cells from Morris hepatoma, which retain some liver-specific functions, did not show any cell-density dependency of either cell growth or hepatocyte-specific functions, such as induction of tyrosine aminotransferase by dexamethasone. However, when RY-121B cells were cocultured with a low density of rat hepatocytes as monolayers in direct contact, they exerted contact-dependent control of DNA synthesis and of differentiated function of the hepatocytes. Furthermore, plasma membranes from various tumor cells including these hepatoma cells had strong modulator activity on primary cultures of normal rat hepatocytes, and their activity mimicked the reciprocal effects of cell density on DNA synthesis and induction of tyrosine aminotransferase. On the contrary, addition of plasma membranes from normal adult rat liver to sparse cultures of RY-121B or MH1C1 cells did not cause any inhibition of active DNA synthesis or enhancement of induction of tyrosine aminotransferase in these cells. These results suggest that hepatoma cells have lost cell density-dependent regulations of many cellular activities and cell growth because they have lost the ability to respond to the cell surface modulator, although they retain modulator activity on their plasma membranes.
Human genomic library screened with 17-base oligonucleotide probes yields a novel interferon gene.
Authors: Torczynski R M , Fuke M , Bollon A P
Published: May 2006

A method is presented that has permitted a human genomic library to be screened for low-copy genes using 17-base synthetic oligonucleotides as probes. Parallel screening with two different 17-base probes permitted the unambiguous identification of clones containing interferon-alpha (IFN-alpha) genes. The isolated human IFN-alpha genes were sequenced, and one appears to be IFN-alpha L; the other is one not previously described, which we have designated IFN-alpha WA. The IFN-alpha WA sequence differs from those of IFN-alpha genes A-L at approximately equal to 10% of the positions and is most similar to IFN-alpha C, -alpha F, and -alpha H. IFN-alpha WA has been found to encode amino acids that differ from those conserved at each of five positions in all previously reported IFN-alpha species. The IFN-alpha WA gene codes for an active interferon, which has been expressed in Escherichia coli using an M13-lacZ fusion as an expression vector. About 5 X 10(6) units of IFN-alpha WA were obtained per liter of bacterial culture. The described screening procedure using short probes should permit the isolation of genes for which sequence information is available from animal or plant genomic libraries.
Enkephalin convertase localization by [3H]guanidinoethylmercaptosuccinic acid autoradiography: selective association with enkephalin-containing neurons.
Authors: Lynch D R , Strittmatter S M , Snyder S H
Published: May 2006

Enkephalin convertase, an enkephalin-forming carboxypeptidase, is potently inhibited by guanidinoethylmercaptosuccinic acid (GEMSA). We have localized enkephalin convertase in rat brain by in vitro autoradiography with [3H]GEMSA. [3H]GEMSA-associated silver grains are highly concentrated in the median eminence, bed nucleus of the stria terminalis, lateral septum, dentate gyrus, hippocampus, central nucleus of the amygdala, preoptic hypothalamus, magnocellular nuclei of the hypothalamus, interpeduncular nucleus, dorsal parabrachial nucleus, locus coeruleus, nucleus of the solitary tract, and the substantia gelatinosa of the spinal trigeminal tract. This distribution corresponds closely with immunocytochemical localizations of enkephalin-containing cells and axons, indicating that enkephalin convertase is selectively involved in enkephalin biosynthesis.
Identification of the tyrosine protein kinase from LSTRA cells by use of site-specific antibodies.
Authors: Casnellie J E , Gentry L E , Rohrschneider L R , Krebs E G
Published: May 2006

The lymphoma cell line LSTRA contains an elevated level of tyrosine protein kinase activity. It has been suggested that this elevated level of activity is due to the presence of a phosphoprotein with a molecular weight of 56,000 (pp56, formerly referred to as a 58,000-dalton protein). This paper describes the preparation of antibodies against pp56 through the use of a synthetic peptide that contains the sequence around the site of tyrosine phosphorylation in pp56, which is identical to the phosphorylation site in pp60src. These antipeptide antibodies specifically immunoprecipitated 32P-labeled pp56 from detergent extracts of LSTRA cells. In immunoblotting experiments, pp56 was the major antigen detected in the particulate fraction from LSTRA cells by the antipeptide antibodies. The antibodies were also used to show that the level of pp56 is greatly elevated in LSTRA cells. Incubation of the detergent extract of the particulate fraction from LSTRA cells with the antipeptide antibodies resulted in inhibition of most of the LSTRA cell tyrosine protein kinase activity. These results indicate that pp56 is the tyrosine protein kinase whose activity is elevated in LSTRA cells. This enzyme may be a member of the large family of protein kinases involved in the regulation of cell growth.
Cyclic GMP may serve as a second messenger in peptide-induced muscle degeneration in an insect.
Authors: Schwartz L M , Truman J W
Published: May 2006

At the end of metamorphosis, the intersegmental muscles of the moth Antheraea polyphemus undergo rapid degeneration in response to the peptide eclosion hormone (EH). Muscle death was preceded by a 22-fold increase in muscle guanosine-3',5'-cyclic monophosphate (cGMP) titers, which peaked 60 min after peptide exposure; adenosine-3'5'-cyclic monophosphate (cAMP) titers remained unchanged. EH induced a dose-dependent increase in muscle cGMP content with a threshold dose similar to that needed to induce cell death. Exogenous cGMP, but not cAMP, mimicked the action of EH. Sodium nitroprusside, a potent stimulator of guanylate cyclase, and methylated xanthines, a class of 3',5'-cyclic-nucleotide phosphodiesterase inhibitors, also induced the selective death of these muscles. It is concluded that an elevation of cGMP level is involved in EH-induced muscle degeneration. The intersegmental muscles become sensitive to EH at the end of adult development in response to the declining titers of the steroid molting hormones, the ecdysteroids. At earlier times, treatment with EH, exogenous cGMP, sodium nitroprusside, or methylated xanthines was ineffective in causing cell death. Nevertheless, treatment with EH at this time resulted in a marked increase in intersegmental-muscle cGMP. Thus, the onset of physiological responsiveness to the peptide hormone presumably results from biochemical changes distal to the EH receptors and guanylate cyclase.
Isolation of cDNA clones specifying the fourth component of mouse complement and its isotype, sex-limited protein.
Authors: Nonaka M , Takahashi M , Natsuume-Sakai S , Nonaka M , Tanaka S , Shimizu A , Honjo T
Published: May 2006

cDNA clones specific for the fourth component of mouse complement (C4) and its hormonally regulated isotype, sex-linked protein (Slp), were isolated using as a probe a 20-mer synthetic oligonucleotide corresponding to a known sequence of human C4 cDNA. Two types of clones, one specific for C4 (pFC4/10, with a 3.7 kilobase insert) and one specific for Slp (pFSlp/1, with a 4.7 kilobase insert), were isolated from liver cDNA libraries constructed from the Slp-producing FM mouse strain. The cDNA inserts of these clones shared 70% of the restriction sites determined. Only one type of clone was isolated from the Slp-negative DBA/1 strain; this type showed restriction maps indistinguishable from that of pFC4/10. pFC4/10 and pFSlp/1 displayed extensive homology: 94% nucleotide homology and 89% derived amino acid homology in the C4a region and 92% nucleotide homology and 89% derived amino acid homology in the thiol-ester region. An Arg-Gln-Lys-Arg sequence in the beta-alpha junction and a Cys-Ala-Glu-Gln sequence in the thiol-ester site were identified for both proteins. A remarkable divergency between C4 and Slp sequences was recognized in the region immediately following the C4a sequence.
Antigen conformation determines processing requirements for T-cell activation.
Authors: Streicher H Z , Berkower I J , Busch M , Gurd F R , Berzofsky J A
Published: May 2006

We studied the difference in requirements for processing and presentation to a single T-cell clone of four different forms of the same epitope of sperm whale myoglobin--namely, on the native protein, on two conformationally altered forms of the protein, or as a 22-residue antigenic peptide fragment. The T-cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys-140. As inhibitors of macrophage processing of antigen, we used several agents that inhibit lysosomal function: the weak bases chloroquine and NH4Cl, the cationic ionophore monensin, and the competitive protease inhibitor leupeptin. When these agents were used to inhibit processing of antigen by presenting cells and then washed out before T cells were added to culture, they inhibited the presentation of native antigen but not of fragment 132-153. To our surprise, the intact but denatured form, S-methylmyoglobin, behaved like the fragment not like the native protein. Apomyoglobin was intermediate in susceptibility to inhibition. Thus, native myoglobin requires a processing step that appears to involve lysosomal proteolysis, which is not required by fragment 132-153 or the denatured unfolded forms. For an antigen the size of myoglobin (Mr 17,800), it appears that unfolding of the native conformation, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater accessibility in the latter of sites, such as hydrophobic residues, buried in the native protein, we propose that processing may be necessary to expose these sites, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell.
De novo cellular synthesis of sulfated proteoglycans of the developing renal glomerulus in vivo.
Authors: Kanwar Y S , Jakubowski M L , Rosenzweig L J , Gibbons J T
Published: May 2006

The site of cellular synthesis of glomerular proteoglycans was investigated in developing glomeruli of 4- to 5-day-old rats. [35S]Sulfate was administered intravenously and animals were sacrificed 15 min to 12 hr later. The outermost layers of the kidney cortices were utilized for characterization of proteoglycans and electron microscopic autoradiography. Sepharose CL-6B chromatography and cellulose acetate electrophoresis revealed that most (approximately equal to 96%) of the radioactivity was associated with heparan sulfate-proteoglycan synthesized during maturation of glomerular capillaries. Tissue autoradiography revealed the following: (i) during the S-shaped body stage, there is rapid incorporation of [35S]sulfate by mesenchymal cells into the cleft region (site for development of future glomerular extracellular matrices); (ii) during the precapillary stage, mesenchyme-derived cells showed higher incorporation of radioisotope than did epithelial cells; and (iii) during the mature capillary stage, all glomerular cell types (mesangial, endothelial, and epithelial) incorporated [35S]sulfate, incorporation by mesangial cells being the greatest. Radiolabeling was also higher in the mesangial matrix than in the glomerular basement membrane of peripheral capillary loops. Synthesis of a single major species of sulfated glycosaminoglycan by cells of different embryologic origin may be unique to glomerular capillaries.
Expression of cellular oncogenes during embryonic and fetal development of the mouse.
Authors: Slamon D J , Cline M J
Published: May 2006

Cellular oncogenes are conserved with great fidelity across a broad span of evolution. This avid conservation suggests possible roles in critical physiologic functions. Little, however, is known about their activity in normal cellular processes. In this study, we examined the expression pattern of eight cellular oncogenes during embryonic and fetal development of the mouse. Five of these genes (c-myc, c-erb, c-Ha-ras, c-src, and c-sis) were expressed at appreciable levels, and four were modulated in a consistent manner during the course of prenatal development.