bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin activates a tyrosine-specific protein kinase in extracts of 3T3-L1 adipocytes and human placenta.
Authors: Petruzzelli L M , Ganguly S , Smith C J , Cobb M H , Rubin C S , Rosen O M
Published: May 2006

Insulin activates a tyrosine-specific cAMP-independent protein kinase when added directly to detergent extracts of differentiated 3T3-L1 adipocytes and humal placental membranes. The kinase is also activated by antibody to the insulin receptor and, to a lesser extent, by proinsulin. It catalyzes the phosphorylation of the 92,000-dalton component of the insulin receptor, histone, and casein; in each case, tyrosine is the principal amino acid modified. Under the conditions used to activate the kinase, insulin does not affect the rate of dephosphorylation of the receptor or of histone. The insulin-activated kinase is copurified with the human placental insulin receptor until the final elution from insulin-Sepharose. It remains to be established whether the kinase and the insulin receptor are separate molecules.
Extensive intragenic sequence homology in two distinct rat lens gamma-crystallin cDNAs suggests duplications of a primordial gene.
Authors: Moormann R J , den Dunnen J T , Bloemendal H , Schoenmakers J G
Published: May 2006

The nucleotide sequences of two different rat lens gamma-crystallin cDNA clones, pRL gamma 2 and pRL gamma 3, have been determined. pRL gamma 3 contains the complete coding information for a gamma-crystallin of 173 amino acids whereas pRL gamma 2 is incomplete in that it lacks the codons for the first three amino acids of a separate but very homologous gamma-crystallin of identical length. Both rat gamma-crystallins are homologous to the known amino acid sequence of bovine gamma-crystallin II which is only a single amino acid longer. The length of the region downstream the coding sequence to the A-A-T-A-A-A polyadenylylation signal sequence is 40 nucleotides in each clone. In pRL gamma 3 the poly(A) signal sequence is followed at 14 nucleotides by a remnant of the poly(A) tail which indicates that this clone contains a complete 3' noncoding region. pRL gamma 2 has only seven nucleotides following this signal sequence and no poly(A) tail, suggesting an incomplete 3' end. The cDNA clones show an overall nucleotide sequence homology of 85%. The mutual homology at the amino acid level is 73% whereas their amino acid homology with bovine gamma-crystallin II is about 70%. The nucleotide sequence of each clone also reveals a high intragenic homology and seems to be duplicated in itself. We suggest that the gamma-crystallin genes have arisen by multiple duplications of a primordial gene which consisted of about 120 nucleotides.
Recovery of myc-specific sequences by a partially transformation-defective mutant of avian myelocytomatosis virus, MC29, correlates with the restoration of transforming activity.
Authors: Ramsay G M , Enrietto P J , Graf T , Hayman M J
Published: May 2006

Avian myelocytomatosis virus MC29 transforms fibroblasts and macrophages in vitro. Recently we isolated three deletion mutants of MC29 that have a decreased ability to transform macrophages while retaining their capacity to transform fibroblasts. One of these mutants, MC29 td10H, on passage through chicken embryo cultures gave rise to a recovered virus MC29 10H B1, which has regained the ability to transform macrophages efficiently. Immunoprecipitation analysis of MC29 10H B1-infected cells revealed a 108,000-dalton gag-myc polyprotein as opposed to the 90,000-dalton protein of MC29 td10H or the 110,000-dalton polyprotein of wtMC29. Tryptic peptide mapping studies demonstrated that the 108,000-dalton protein had acquired v-myc peptides that were lost from the td10H 90,000-dalton polyprotein and two novel peptides. Restriction enzyme analysis of the MC29 10H B1 proviral DNA also showed that myc sequences had been acquired. These results suggest that MC29 td10H has recombined with c-myc sequences to generate a recovered virus, MC29 10H B1.
Leukemia-derived growth factor (non-interleukin 2) produced by a human malignant T lymphoid cell line.
Authors: Uittenbogaart C H , Fahey J L
Published: May 2006

A growth factor was found in the supernatants of MOLT-4f, a cell line derived from acute T lymphoblastic leukemia. This factor, which we designated leukemia-derived growth factor from MOLT-4f (LDGF-M4), is different from interleukin 2. LDGF-M4 has features of a polypeptide with a molecular weight in the range of 5,000-15,000, as indicated by gel diffusion chromatography. LDGF-M4 does stimulate MOLT-4f and at least two other T cell lines that do not respond to interleukin 2. Because MOLT-4f cells produce and respond to LDGF-M4, this factor may contribute to the independence of MOLT-4f and related T leukemia cell lines.
Enhancement of granulocyte-endothelial cell adherence and granulocyte-induced cytotoxicity by platelet release products.
Authors: Boogaerts M A , Yamada O , Jacob H S , Moldow C F
Published: May 2006

Complement-stimulated granulocytes adhere to and induce significant 51Cr release from endothelial cells in vitro. Platelets were stimulated to undergo release, and these release products significantly enhanced granulocyte-endothelial cell adherence and granulocyte-induced 51Cr release from endothelial cells. Platelet serotonin appeared to mediate these phenomena because serotonin antagonists blocked both the enhanced endothelial adherence and 51Cr release. In addition, added serotonin mimicked the effect seen with the stimulated platelets upon granulocyte--endothelial cell adherence and cytotoxicity completely. This enhancement appeared to be due to serotonin effects upon both the granulocyte and endothelial cells. These data suggest that a released platelet constituent might modulate in vivo granulocyte-endothelial cell interactions in clinical disorders.
Interaction of monoclonal antibodies with mammalian choline acetyltransferase.
Authors: Crawford G D , Correa L , Salvaterra P M
Published: May 2006

Monoclonal antibodies selective for rat brain choline acetyltransferase (acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6) were prepared by standard techniques. Five cell lines were isolated from spleen cell-SP/2 hybrids by repetitive cloning with a screening method that used the intrinsic activity of the enzyme. All cell lines secrete immunoglobulin of mouse subclass IgGl, and none inhibit the enzyme activity directly. The size of antibody-enzyme immune complexes formed with different pairs of the monoclonal antibodies was determined by gel filtration with HPLC. By comparing the elution position of choline acetyl-transferase activity after incubation with paired monoclonal antibodies, the spatial relationship of antibody binding domains relative to each other can be defined and classified as independent, mutually exclusive, or overlapping. Immune complexes in excess of Mr 600,000 were formed by some pairs of antibodies with the antigen, indicating independent binding domains on the enzyme. In one case, the paired antibodies formed an immune complex of only Mr 300,000, indicating that they bound in a mutually exclusive fashion. In most cases, pairs of antibodies reacted with the enzyme to give simultaneously both higher and lower Mr immune complexes. We conclude that all five antibodies bind to a relatively localized region of the enzyme surface. Antibodies were screened for usefulness as immunohistochemical markers of choline acetyltransferase-containing neurons by using the indirect immunoperoxidase method. One antibody intensely stains cell bodies of motor neurons and processes in a selective manner in the rat spinal cord and brain stem by using aldehyde-fixed tissue; the remaining antibodies do not react with fixed tissue.
Sequence of terminal regions of cowpox virus DNA: arrangement of repeated and unique sequence elements.
Authors: Pickup D J , Bastia D , Stone H O , Joklik W K
Published: May 2006

One terminal EcoRI fragment of the genome of cowpox virus (CPV) strain Brighton red has been cloned in plasmid pBR325, and the nucleotide sequence of the 2,725-base-pair Sal I fragment corresponding to that at the end of the viral genome has been determined. The fragment consists of three unique sequence regions flanking two sets of repeated sequence. The repeated sequence sets are composed of four types of subunits, the majority of which are arranged in higher-order repeat units. The subunits are themselves closely related; two are subsets of a third, whereas the fourth is a recombinant of the first two. The fragment possesses no long open reading frames (maximal coding potential, 65 amino acids). The sequence of this CPV DNA Sal I fragment is compared with that of the corresponding fragment of vaccinia virus WR DNA [Baroudy, B. M., Venkatesan, S. & Moss, B. (1982) Cell 28, 315-324; Venkatesan, S., Baroudy, B. M. & Moss, B. (1981) Cell 25, 805-813]. Two of the unique sequence regions of the two viruses are related to the extent of 96%, and the third contains at least one sequence of 112 residues that is 98% homologous. As for the repeated sequence sets, those of vaccinia virus are composed of only two, rather than four, types of subunit, one of which is identical to one of the CPV subunits, whereas the other differs from another CPV subunit by only three mismatches and one deletion. However, the arrangement of subunits in the two viruses is different, that in vaccinia virus DNA being simpler. Both subunits as well as repeat units probably arose as a result of unequal crossover.
Inhibitory monoclonal antibody to calf thymus RNA polymerase II blocks formation of enzyme-DNA complexes.
Authors: Carroll S B , Stollar B D
Published: May 2006

Monoclonal antibodies were prepared against purified calf thymus RNA polymerase II (RNA nucleotidyltransferase, EC 2.7.7.6). Two distinct antibodies were identified on the basis of their ability to bind RNA polymerase II in a solid-phase assay and to remove enzyme activity from solution in complexes precipitated by anti-immunoglobulin antibodies. One monoclonal antibody directly inhibited enzyme activity in solution. Detailed study of the mechanism of enzyme inhibition by this antibody revealed that it prevented the formation of enzyme-DNA complexes and had no effect on chain elongation. Inhibition of DNA binding and catalytic activity depended on preincubation of antibody with enzyme and was irreversible. Without preincubation, the enzyme readily bound to DNA in the presence of antibody, indicating that the binding of the free enzyme to DNA is faster than that of the antibody to the enzyme. In a competitive immunoassay enzyme-DNA complexes were much less reactive with antibody than was free enzyme, suggesting that DNA either blocks the antigenic site or alters the enzyme's conformation, preventing antibody recognition. These antibodies may serve as useful reagents for analysis of RNA polymerase structure and function.
A DNA-mediated transformation system for Dictyostelium discoideum.
Authors: Hirth K P , Edwards C A , Firtel R A
Published: May 2006

We have established a transformation system for Dictyostelium discoideum. The transformation vector contains the protein coding region of the Tn5 neomycin resistance gene fused to the proposed promoter of the Dictyostelium actin 8 gene; the vector also contains a sequence that acts as an autonomously replicating sequence (ars) in yeast. Using this vector, we can transform Dictyostelium vegetative amoebae to be resistant to aminoglycoside G418 at a frequency of between 10(-6) and 10(-4) of the input cells. The transformed cell lines are stable and contain vector sequences integrated within chromosomal DNA.
Chemical identity of the acrasin of the cellular slime mold Polysphondylium violaceum.
Authors: Shimomura O , Suthers H L , Bonner J T
Published: May 2006

The aggregation chemoattractant (or acrasin) of Polysphondylium violaceum, a species of cellular slime mold that does not respond chemotactically to cAMP, has been identified. It was extracted and purified from aggregating amoebae, then analyzed for amino acid composition and by IR and mass spectrometry. The active molecule is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethyl ester (Mr, 327), which we have named glorin. The compound has been synthesized and shows normal chemotactic activity with the amoebae of P. violaceum.
Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8.
Authors: Tschopp J , Podack E R , Müller-Eberhard H J
Published: May 2006

The ultrastructure of the membrane attack complex (MAC) of complement had been described as representing a hollow cylinder of defined dimensions that is composed of the proteins C5b, C6, C7, C8, and C9. After the characteristic cylindrical structure was identified as polymerized C9 [poly(C9)], the question arose as to the ultrastructural identity and topology of the C9-polymerizing complex C5b-8. An electron microscopic analysis of isolated MAC revealed an asymmetry of individual complexes with respect to their length. Whereas the length of one boundary (+/- SEM) was always 16 +/- 1 nm, the length of the other varied between 16 and 32 nm. In contrast, poly(C9), formed spontaneously from isolated C9, had a uniform tubule length (+/- SEM) of 16 +/- 1 nm. On examination of MAC-phospholipid vesicle complexes, an elongated structure was detected that was closely associated with the poly(C9) tubule and that extended 16-18 nm beyond the torus of the tubule and 28-30 nm above the membrane surface. The width of this structure varied depending on its two-dimensional projection in the electron microscope. By using biotinyl C5b-6 in the formation of the MAC and avidin-coated colloidal gold particles for the ultrastructural analysis, this heretofore unrecognized subunit of the MAC could be identified as the tetramolecular C5b-8 complex. Identification also was achieved by using anti-C5 Fab-coated colloidal gold particles. A similar elongated structure of 25 nm length (above the surface of the membrane) was observed on single C5b-8-vesicle complexes. It is concluded that the C5b-8 complex, which catalyzes poly(C9) formation, constitutes a structure of discrete morphology that remains as such identifiable in the fully assembled MAC, in which it is closely associated with the poly(C9) tubule.
Secretin and vasoactive intestinal peptide acutely increase tyrosine 3-monooxygenase in the rat superior cervical ganglion
Authors: Ip Nancy Y. , Ho Calvin K. , Zigmond Richard E.
Published: May 2006

We have previously shown that stimulation of the preganglionic cervical sympathetic trunk leads to an acute increase in tyrosine hydroxylase (TyrOHase) activity in the rat superior cervical ganglion. This increase appears to be mediated in part by acetylcholine and in part by a second neurotransmitter. As a first step in an attempt to determine the identity of this noncholinergic transmitter, we have examined the ability of a number of neuropeptides to increase ganglionic TyrOHase activity
in vitro
. Secretin and vasoactive intestinal peptide (VIP) both stimulated TyrOHase activity, whereas angiotensin II, bombesin, bradykinin, cholecystokinin octapeptide, glucagon, insulin, luteinizing hormone-releasing hormone, [D-Ala
2
, Met
5
]enkephalinamide, motilin, neurotensin, somatostatin, and substance P produced no effects. Secretin produced a significant increase in TyrOHase activity at 1 nM and a maximal elevation at 0.1 μM. VIP produced a significant increase at 0.1 μM and a near maximal effect at 10 μM. Although secretin was about 2 orders of magnitude more potent than VIP, it produced a significantly smaller maximal increase in enzyme activity. Incubation of ganglia with both secretin (10 μM) and VIP (10 μM) produced an increase in TyrOHase activity that was not significantly different from that produced by VIP alone. The stimulatory effects of secretin and VIP were reversible within minutes after removal of the peptides. Neither incubation of intact ganglia with the cholinergic antagonists hexamethonium and atropine nor prior decentralization of ganglia altered the response to the peptides. Thus, the data demonstrate that secretin and VIP acutely increase TyrOHase activity in the superior cervical ganglion and suggest that they produce this effect by acting directly on ganglionic neurons. It remains to be determined whether secretin or VIP or a related peptide is released during preganglionic nerve firing and whether one or more of these peptides is responsible for the noncholinergic elevation of TyrOHase activity produced by preganglionic nerve stimulation.
Production and characterization of monoclonal antibodies against the "brain-specific" proteins 14-3-2 and S-100.
Authors: Haan E A , Boss B D , Cowan W M
Published: May 2006

We have raised mouse hybridomas that secrete monoclonal antibodies against bovine brain-specific proteins 14-3-2 and S-100, and we have characterized the antibodies by immunoperoxidase and immunofluorescence methods in sections and in tissue cultures of rat brain. One monoclonal antibody to 14-3-2 (E8.F9) has been found to react strongly with bovine 14-3-2 and with rat neuron-specific enolase in an enzyme-linked immunosorbent assay (ELISA) and to react weakly with rat nonneuronal enolase. This pattern of specificity is reflected in strong neuronal labeling and occasional weak glial labeling in immunocytochemical preparations. After appropriate tissue fixation, E8.F9 could be shown to be localized primarily to the cytoplasm of neurons; with less adequate fixation nuclear labeling was also seen. A monoclonal antibody to the calcium binding protein S-100 (G12.B8) reacted strongly with bovine S-100 in an ELISA and with the major protein bands in electrophoretically separated S-100. In immunocytochemical preparations G12.B8 labeled the cytoplasm of astrocytes. Both antibodies are of the IgG1 subclass. Because of its specificity, the antibody against the S-100 protein promises to be useful as an immunological marker for astrocytes in the adult animal and in mature tissue cultures of brain cells. Although it has been thought that the generally low levels and relatively late appearance of S-100 during ontogeny may restrict its usefulness as a marker for developing astrocytes, preliminary immunocytochemical evidence indicates that G12.B8 selectively labels radial glial cells and astrocytes or astrocyte precursors as early as, or even earlier than, antibodies against the glial fibrillary acidic protein. The antibody against neuron-specific enolase is likely to be of limited use as a neuronal marker because of its crossreactivity with nonneuronal enolase.
Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.
Authors: Domdey H , Wiebauer K , Kazmaier M , Müller V , Odink K , Fey G
Published: May 2006

Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end of C3 mRNA. The length of C3 mRNA was determined to be 5,100 +/- 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the mature C3 beta chain was predicted to be Ile-Pro-Met-Tyr-Ser-Ile-Ile-Thr-Pro-Asn-Val-Leu-Arg-Leu-Glu. This sequence is in good agreement with the reported amino acid sequences of human and guinea pig C3 beta chains. These data position the C3 beta subunit to the NH2-terminal portion of the precursor C3 molecule (pro-C3) and establish the order of subunits in pro-C3 to be NH2-beta-alpha-COOH. In addition, the cDNA sequence indicates that an NH2-terminal extension peptide precedes the beta chain in pro-C3. The amino acid sequence of the mouse C3a fragment and its flanking regions was determined. The data indicate the presence of four arginine residues located between the COOH terminus of the C3 beta and the NH2 terminus of the C3 alpha subunits in pro-C3. The coding sequences of the amino acids that constitute the internal thioester domain in C3 were determined. Unexpectedly, the glutamyl residue that has been shown to participate in the thioester bond in native C3 was found to be encoded as a glutamine.
Monoclonal antibodies against the Drosophila nervous system.
Authors: Fujita S C , Zipursky S L , Benzer S , Ferrús A , Shotwell S L
Published: May 2006

A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.
Evidence for multiple effects of vitamin D3 on calcium absorption: response of rachitic chicks, with or without partial vitamin D3 repletion, to 1,25-dihydroxyvitamin D3.
Authors: Wasserman R H , Brindak M E , Meyer S A , Fullmer C S
Published: May 2006

The effects of vitamin D3 or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or both, on the relationship among calcium absorption, vitamin D-induced calcium-binding protein (CaBP), and phospholipid metabolism were examined. When 1,25(OH)2D3 was injected intracardially into D3-deficient chicks, both the stimulation of calcium absorption and the induction of the synthesis of CaBP occurred 2-4 hr later. When 1,25(OH)2D3 was injected into chicks partially repleted with D3, an earlier increase in calcium absorption was observed without a significant change in the concentration of CaBP already present in the duodenal mucosa. Other early events were an increased uptake of calcium by the intestinal tissue and an alteration in phospholipid metabolism. These and other observations support the proposal that at least two phases of calcium absorption are influenced by 1,25(OH)2D3--permeation of calcium across the brush border, and transfer of calcium through and out of the cell. The first phase responds more rapidly to 1,25(OH)2D3 than does the second phase, correlates with changes in phospholipid metabolism, and might not be dependent on de novo protein synthesis. The second phase correlates with CaBP synthesis and therefore is dependent on protein synthesis. Either the first phase or the second phase can constitute the limiting step in calcium absorption.
Chronometric cosmology and fundamental fermions
Authors: Segal I. E.
Published: May 2006

It is proposed that the fundamental fermions of nature are modeled by fields on the chronometric cosmos that are not precisely spinors but become such only in the nonchronometric limit. The imbedding of the scale-extended Poincaré group in the linearizer of the Minkowskian conformal group defines such fields, by induction.
Expression of transforming region of Moloney murine sarcoma virus in Escherichia coli as a fusion protein with small tumor antigen of polyoma virus.
Authors: Donoghue D J , Hunter T
Published: May 2006

Bacterial expression of the transforming region of Moloney murine sarcoma virus, designated mos, was obtained as a fusion protein with a portion of the small tumor antigen of polyoma virus. This was accomplished by fusing the entire mos open reading frame, encoding a 41,000-dalton protein, with a plasmid that expresses a beta-galactosidase-polyoma fusion protein under lac operon control. The resulting plasmid directed synthesis of the predicted polyoma antigen-sarcoma virus fusion protein of 59,000 daltons. This protein was immunoprecipitated by an anti-polyoma tumor antigen antiserum that recognized polyoma determinants at the NH2 terminus of the hybrid protein. This protein was also immunoprecipitated by an antiserum directed against a synthetic peptide containing the 12 COOH-terminal amino acids encoded by the mos open reading frame. This work confirms the existence of a long open reading frame in the mos gene and resolves a discrepancy between different nucleotide sequences for its COOH-terminal coding region.
High-resolution NMR studies of transmembrane cation transport: use of an aqueous shift reagent for 23Na.
Authors: Pike M M , Simon S R , Balschi J A , Springer C S
Published: May 2006

23Na NMR studies of large unilamellar vesicles of egg lecithin in salt solutions are reported. A shift reagent, the dysprosium nitrilotriacetate ion Dy[N(CH2CO2)3]3-2 has been used to distinguish between 23Na+ inside and outside the vesicles. When both are present and the shift reagent is present on only one side, two clearly distinct resonances are observed. Creation of a Na+ concentration gradient and subsequent catalysis of passive transport induced by the introduction of gramicidin can be monitored easily by using the relative intensities of the two resonances. We report the observation of transport both out of and into vesicles.
Increase in apparent compressibility of cytochrome c upon oxidation.
Authors: Eden D , Matthew J B , Rosa J J , Richards F M
Published: May 2006

The apparent molal adiabatic compressibilities of ferri- and ferrocytochrome c have been determined from measurements of density and sound velocity. The values found were +2.99 X 10(-8) and -2.40 X 10(-8) cm5 mol-1 dyne-1 for the ferri and ferro forms, respectively. Experiments were performed on identical solutions containing either the oxidized or reduced form of protein. Solutions of ferricytochrome c were found to have significantly greater adiabatic compressibility than equivalent solutions of ferrocytochrome c at 25 degrees C and pH 7.15. The remarkable similarity of the three-dimensional structures of the ferri and ferro proteins [Takano, T. & Dickerson, R.E. (1980) Proc. Natl. Acad. Sci. USA 77, 6371-6375] strongly suggests that this difference in compressibility is due to an increase in volume fluctuations within ferricytochrome c relative to the ferro form rather than a change in equilibrium structure or hydration. Such a difference in the dynamic properties of the structures is consistent with both the crystallographic thermal B factors and the observed increase in amide hydrogen exchange kinetics when ferrocytochrome c is oxidized. The relative magnitude of the root mean square volume fluctuations is approximated from an ideal solution treatment of the compressibility data and yields a ratio of delta Vrms (ferri cyt c)/ delta Vrms (ferro cyt c) = 1.3.
Synthesis of low molecular weight heat shock peptides stimulated by ecdysterone in a cultured Drosophila cell line.
Authors: Ireland R C , Berger E M
Published: May 2006

Treatment of Schneider's line 3 Drosophila cells with the steroid hormone ecdysterone rapidly stimulated the synthesis and accumulation of the polypeptide previously designated p7 [Berger, E. M., Ireland, R. C. & Wyss, C. (1980) Somatic Cell Genet. 6, 119-129]. In this report, p7 is identified as the 23,000-dalton heat shock polypeptide (hsp23). In addition to hsp23, the synthesis of the low molecular weight heat shock polypeptides hsp22, hsp26, and hsp27 was also stimulated by ecdysterone, although to different extents. Hybridization of a nick-translated genomic clone containing the hsp23 gene to a total RNA blot showed that ecdysterone stimulation of hsp23 synthesis was the result of an increase in the hsp23 RNA content of S3 cells. We detected no effect of the hormone on the synthesis of heat shock polypeptides hsp68, hsp70, and hsp83.
Assignment of human alpha 1-antitrypsin to chromosome 14 by somatic cell hybrid analysis.
Authors: Darlington G J , Astrin K H , Muirhead S P , Desnick R J , Smith M
Published: May 2006

Human alpha 1-antitrypsin ( alpha-1-AT;Pi) production was analyzed in 11 primary mouse hepatoma-human lymphoid cell hybrids and in 14 secondary rat hepatoma-human fetal liver fibroblast hybrids. The presence of human alpha-1-AT was determined by Laurell immunoelectrophoresis of concentrated and isotopically labeled supernatant medium. Human alpha-1-AT production segregated in the mouse-human hybrids concordantly with human purine nucleoside phosphorylase and with chromosome 14. All rat-human hybrids that were alpha-1-AT positive were also positive for human purine nucleoside phosphorylase and chromosome 14. Our study demonstrated the usefulness of rodent hepatoma cell hybrids for mapping human liver-specific genes because differentiated functions are expressed despite the fact that the human parental cells did not express these functions. Our study also showed that human alpha-1-AT gene product can be processed for secretion in the rodent hepatoma cellular environment. The mouse-human hybrids showed that no other human chromosome carries genes necessary for processing or secretion of human alpha-1-AT in the hybrid cell milieu.
Cell-free translation and partial characterization of mRNA coding for enkephalin-precursor protein.
Authors: Dandekar S , Sabol S L
Published: May 2006

The biosynthesis of the protein precursor of [Met]enkephalin (Tyr-Gly-Gly-Phe-Met) was studied using cell-free translation systems to characterize the enkephalin-precursor gene product and mRNA. Proteins obtained by translation of bovine adrenal medullary mRNA in the presence of [35S]methionine were digested with trypsin and carboxypeptidase B. The resultant peptides were immunoprecipitated with anti[Met]enkephalin serum and subsequently analyzed by reverse phase HPLC. [35S][Met]Enkephalin (up to 0.2 fmol/micrograms mRNA, identified by antigenic specificity and chromatographic mobility, was isolated from peptides obtained from proteins whose synthesis was dependent upon adrenal medullary mRNA. Both trypsin and carboxy-peptidase B were required to generate [35S][Met]enkephalin from translation products. The largest protein containing the [35S]-[Met]enkephalin sequence synthesized in the wheat germ system has a Mr, of 31,000 +/- 1000, determined by NaDodSO4/polyacrylamide gel electrophoresis. Adrenal medullary mRNA coding for protein containing the [35S][Met]enkephalin sequence was resolved into a major fraction of 1450 +/- 150 nucleotides and a minor fraction of 47000 +/- 450 nucleotides, as determined by agarose gel electrophoresis in the presence of methylmercuric hydroxide. It is proposed that the major enkephalin-precursor gene product of adrenal medulla is a protein of Mr approximately 31,000.U
An interaction between gramicidin and the sigma subunit of RNA polymerase.
Authors: Fisher R , Blumenthal T
Published: May 2006

Gramicidin, a peptide antibiotic produced by Bacillus brevis, inhibits initiation of transcription by RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). We show here that the presence of gramicidin causes an increase in the rate of cleavage of the sigma subunit of Escherichia coli RNA polymerase by trypsin, although it does not alter the cleavage rate of any of the core subunits. Furthermore, whereas isolated sigma is cleaved much faster than is sigma in holoenzyme, gramicidin substantially decreases the trypsin cleavage rate of isolated sigma. Inhibition of RNA polymerase activity by gramicidin in consistent with a sigma-specific effect: the antibiotic is a strong inhibitor of transcription of T7 phage DNA, which requires sigma for activity, but it has little effect on transcription of sigma-independent templates, such as poly(dA-dT).poly)dA-dT) and calf thymus DNA. These results are discussed in light of the hypothesized role for gramicidin in the initiation of sporulation of B. brevis.
Molecular cloning and nucleotide sequence of full-length of cDNA coding for porcine gastrin.
Authors: Yoo O J , Powell C T , Agarwal K L
Published: May 2006

We have cloned in EScherichia coli a cDNA copy of mRNA coding for the porcine antral mucosal hormone preprogastrin. Full-length double-stranded cDNA was synthesized and inserted into the Pst I endonuclease site in plasmid pBR322 by using a homopolymeric extension technique. A partial cDNA clone was used as a probe to identify a complete cDNA clone in a cDNA library by colony hybridization. Four positive clones were isolated, one of which corresponded to porcine preprogastrin mRNA. The nucleotide sequence of the cDNA insert (602 nucleotides) revealed 312 nucleotides in the entire mRNA coding region, 61 nucleotides in the 5' untranslated region, 86 nucleotides in the 3'untranslated region, and a poly(A) tail of 86 nucleotides. Gastrin is located near the carboxyl end of preprogastrin and is flanked at both its amino and carboxyl ends by a pair of basic amino acid residues. The presence of glycine and a pair of basic amino acid residues adjacent in the carboxyl-terminal phenylalanine of gastrin indicates that the glycine and a pair of basic amino acid residues may be required for the enzymatic amidation of phenylalanine to phenylalanine amide.
On the enzymic mechanism of oxidative phosphorylation.
Authors: Green D E , Vande Zande H
Published: May 2006

Oxidative phosphorylation, like substrate-level phosphorylation, involves oxidative conversion of inorganic phosphate to a reactive species followed by interaction of this species with enzyme-bound ADP to form enzyme-bound ATP. The reactive species in a phosphoryl ester in substrate-level phosphorylation and phosphonium ion of orthophosphate in oxidative phosphorylation. The coupled synthesis is mediated by a combination of two classical enzymes in substrate-level phosphorylation and by a set of energy-coupled enzymes in oxidative phosphorylation. The full range of experimental evidence supporting this proposed enzymic mechanism of oxidative phosphorylation is presented as well as the rationalization of phenomena that hitherto have eluded explanation.
Oriented properties of the chlorophylls: Electronic absorption spectroscopy of orthorhombic pyrochlorophyllide
a
-apomyoglobin single crystals
Authors: Boxer Steven G. , Kuki Atsuo , Wright Karen A. , Katz Bradley A. , Xuong Nguyen H.
Published: May 2006

The orientations of the transition dipole moments in chlorophyll (Chl) are among the most useful spectroscopic properties for determining macromolecular architecture in photosynthetic complexes; however, the relationships between these orientations and the Chl molecular geometry are unknown. In order to solve this problem, we have prepared single crystals of the synthetic 1:1 complex between pyrochlorophyllide
a
and apomyoglobin. The protein crystallizes readily in the orthorhombic (
B
) form, space group
P
2
1
2
1
2
1
, and the unit cell dimensions are determined to be within 0.5% of those for native MetMb crystals of the same type. These green crystals are highly dichroic, and the strong absorption along the crystallographic
a
axis in the

Q
y

band is red-shifted by about 9 nm, relative to the corresponding feature in a solution of the protein. Although the crystal structure for native Mb in this space group has not been determined, the direction cosines of the heme normal relative to the crystal axes have been measured. By using these values, an appropriate trigonometric analysis, and the measured polarized single-crystal spectra, the orientation of the Chl transition dipole moment for the

Q
y

transition can be specified relative to the crystal axes. With the completion of the protein crystal structure, this result will lead directly to the orientations of the optical transition dipole moments relative to the molecular geometry. The effects of vibronic coupling and the protein environment on the absorption properties of Chl are discussed in detail.
ESR studies of O2 uptake by Chinese hamster ovary cells during the cell cycle.
Authors: Lai C S , Hopwood L E , Hyde J S , Lukiewicz S
Published: May 2006

Magnetic interactions between dissolved oxygen and nitroxide radical spin probes lead to broadening of the ESR lines. We have used a closed-chamber method based on this property to determine the maximum rate of O2 uptake per cell (Vmax per cell) in cultured mammalian cells. A suitable spin probe and a cell suspension are mixed in an aerated medium, and the rate of disappearance of dissolved O2 is measured. The effects of temperature, pH, and microwave power on the determination of dissolved oxygen in solution were studied. For asynchronous Chinese hamster ovary cells, oxygen uptake is 3.8 X 10(7) oxygen molecules per cell per sec and appears to be enzymatically limited at oxygen concentrations greater than 10 microM. About 5-10 X 10(5) cells were used for each measurement, making it possible to study mitotically synchronized cells. Using this method, we have found that Vmax per unit cell volume changes during the cell cycle of Chinese hamster ovary cells from a minimum in mitosis to maxima in both G1 and late S phases. Advantages and limitations of spin probes for studying the O2 uptake of intact cells are discussed.
Appearance of crystalloid endoplasmic reticulum in compactin-resistant Chinese hamster cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
Authors: Chin D J , Luskey K L , Anderson R G , Faust J R , Goldstein J L , Brown M S
Published: May 2006

We have developed a line of Chinese hamster ovary cells with a 500-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the membrane-bound enzyme that controls cholesterol synthesis. This line, designated (UT-1, was obtained by stepwise adaptation of cells to growth in increasing concentrations of compactin, a competitive inhibitor of reductase. Reductase accounts for approximately 2% of total cell protein in UT-1 cells, as calculated from enzyme specific activity and by immunoprecipitation of reductase after growth of cells in [35S]methionine. After solubilization in the presence of the protease inhibitor leupeptin and electrophoresis in NaDodSO4/polyacrylamide gels, reductase subunits from UT-1 cells were visualized by immunoblotting as a single band (Mr = 62,000). To accommodate the increased amounts of reductase, UT-1 cells developed marked proliferation of tubular smooth endoplasmic reticulum (ER) membranes, as revealed by immunofluorescence and electron microscopy. The ER tubules were packed in crystalloid hexagonal arrays. When UT-1 cells were incubated with low density lipoprotein, reductase activity was suppressed by 90% in 12 hr and the crystalloid ER disappeared. UT-1 cells should be useful for studies of the regulation of reductase and also for studies of the synthesis and degradation of smooth ER.
Autosomal dosage compensation Drosophila melanogaster strains trisomic for the left arm of chromosome 2.
Authors: Devlin R H , Holm D G , Grigliatti T A
Published: May 2006

Drosophila melanogaster individuals trisomic for an entire chromosome arm can survive to late stages of pupal development. We have examined the levels of five enzymes whose structural genes are located on the left arm of chromosome 2 both in trisomy 2L and in diploid strains. In trisomies, three distally mapping loci showed compensated levels of expression close to that observed in the diploid strains. Analysis of electrophoretic variants of a compensated locus revealed that all three alleles are active in trisomies. The two proximally located loci displayed dose-dependent levels of expression. Therefore, at the level of the individual gene, autosomal compensation appears to be an all-or-none phenomenon. Furthermore, the compensatory response may be regionally distributed along the chromosome arm. The presence of both autosomal and sex-linked dosage compensation prompts us to speculate that there phenomenon are similar homeostatic mechanisms that modulate gene expression both in euploid and aneuploid genomes.
Primary structure analysis of the major internal protein p24 of human type C T-cell leukemia virus
Authors: Oroszlan Stephen , Sarngadharan M. G. , Copeland Terry D. , Kalyanaraman V. S. , Gilden Raymond V. , Gallo Robert C.
Published: May 2006

A human type C retrovirus [human T-cell leukemia (lymphoma) virus; HTLV], recently isolated from young adult patients with cutaneous T-cell lymphoma or leukemia, was not detectably related to other known animal retroviruses in molecular hybridization studies, by comparison of reverse transcriptase and the major core protein p24. The p24 core protein was purified to homogeneity. The amino acid composition, the COOH-terminal amino acid, and the NH
2
-terminal amino acid sequence of the first 25 residues of this major internal structural protein were determined. These results were then compared to the known structure of the internal core protein of other retroviruses. The compositional data reveal that HTLV p24 is chemically distinct from p30-p24 of other animal retroviruses, in agreement with the earlier immunological analyses. However, HTLV p24 shares the common NH
2
-terminal proline and COOH-terminal leucine of all mammalian type C viral p30s. In addition, like bovine leukemia virus (BLV), HTLV lacks the common prolylleucylarginine tripeptide and the larger conserved region found near the NH
2
terminus of the other mammalian type C viral p30s. Alignment of the amino acid sequence of HTLV p24 with previously determined sequences of other retrovirus proteins, including BLV p24, reveals statistically significant sequence homology only to BLV. The results reported here demonstrate that HTLV p24 is related to but chemically distinct from the major core protein of other retroviruses. Similarly, previous results showed that there was no immunological crossreactivity of the p24 protein and reverse transcriptase of HTLV with other retroviruses, including BLV, and no nucleic acid sequence homology. However, the present results, combined with the common size of the p24 and reverse transcriptase, suggest that HTLV may be closer to BLV than any other known retrovirus.
Organizational principles in the peripheral sympathetic nervous system: Subdivision by coexisting peptides (somatostatin-, avian pancreatic polypeptide-, and vasoactive intestinal polypeptide-like immunoreactive materials
Authors: Lundberg J. M. , Hökfelt T. , Änggård A. , Terenius L. , Elde R. , Markey K. , Goldstein M. , Kimmel J.
Published: May 2006

Sympathetic ganglia and some peripheral tissues of adult guinea pig and cat were analyzed by the indirect immunofluorescence technique with antisera to catecholamine-synthesizing enzymes and some peptides. In the guinea pig, noradrenergic neurons could be subdivided into three populations containing respectively (
i
) somatostatin-like immunoreactive material, (
ii
) avian pancreatic polypeptide (APP)-like immunoreactive material, and (
iii
) apparently only noradrenaline (NA; norepinephrine). A fourth population of sympathetic neurons was nonadrenergic and contained vasoactive intestinal polypeptide (VIP)-immunoreactive material. In the cat many noradrenergic neurons with APP and some without this peptide were seen, but no somatostatin-immunoreactive neurons were observed. Also a population of non-adrenergic, presumably cholinergic, neurons containing a VIP-like peptide was observed. These neuron populations seemed to innervate different tissues with some target specificity. For example, in the nasal mucosa of the cat, nerves containing NA/APP-like immunoreactive material (called NA/APP nerves) were found around small arteries and arterioles, whereas venules and sinusoids were surrounded by nerves containing only NA (called NA nerves). Also in the submandibular salivary gland of the cat, the NA/APP nerves surrounded arteries and arterioles, whereas NA nerves were seen in relation to acini and ducts. The sympathetic (cholinergic) VIP-containing neurons innervated blood vessels and exocrine tissue in the cat sweat glands. In the coeliac-superior mesenteric ganglion complex of the guinea pig and cat, a dense network of VIP-immunoreactive fibers was seen preferentially around noradrenergic ganglionic cell bodies lacking APP-immunoreactive material. Thus, adult peripheral sympathetic neurons can be subdivided into several categories on the basis of specific peptides. These subdivisions may innervate specific targets and may receive peptide-specific neuronal inputs.
Molecular dynamics of an α-helical polypeptide: Temperature dependence and deviation from harmonic behavior
Authors: Levy Ronald M. , Perahia David , Karplus Martin
Published: May 2006

The mean square amplitudes of atomic fluctuations for a polypeptide (decaglycine) α-helix evaluated from molecular dynamics simulations at seven temperatures between 5 and 300 K are compared with analytic harmonic results and with experimental values. Above 100 K the harmonic approximation significantly underestimates the amplitudes of the displacements. Analysis of the time dependence of the fluctuations shows that low-frequency modes (<75 cm
-1
) dominate the atomic fluctuations and that there is a contribution with a very long relaxation time (>10 ps). Quantum corrections to the amplitude of the fluctuations are found to be small above 50 K. The mean square amplitudes obtained from the molecular dynamics simulations are compared with the values derived from x-ray temperature (Debye-Waller) factors for metmyoglobin (80, 250, and 300 K) and ferrocytochrome
c
(300 K).
Site-specific cleavage/packaging of herpes simplex virus DNA and the selective maturation of nucleocapsids containing full-length viral DNA
Authors: Vlazny Donald A. , Kwong Ann , Frenkel Niza
Published: May 2006

Defective genomes present in serially passaged herpes simplex virus (HSV) stocks have been shown to consist of tandemly arranged repeat units containing limited sets of the standard virus DNA sequences. Invariably, the HSV defective genomes terminate with the right (S component) terminus of HSV DNA. Because the oligomeric forms can arise from a single repeat unit, it has been concluded that the defective genomes arise by a rolling circle mechanism of replication. We now report on our studies of defective genomes packaged in viral capsids accumulating in the nuclei and in mature virions (enveloped capsids) translocated into the cytoplasm of cells infected with serially passaged virus. These studies have revealed that, upon electrophoresis in agarose gels, the defective genomes prepared from cytoplasmic virions comigrated with nondefective standard virus DNA (
M
r
100 × 10
6
). In contrast, DNA prepared from capsids accumulating in nuclei consisted of both full-length defective virus DNA molecules and smaller DNA molecules of discrete sizes, ranging in
M
r
from 5.5 to 100 × 10
6
. These smaller DNA species were shown to consist of different integral numbers (from 1 to approximately 18) of defective genome repeat units and to terminate with sequences corresponding to the right terminal sequences of HSV DNA. We conclude on the basis of these studies that (
i
) sequences from the right end of standard virus DNA contain a recognition signal for the cleavage and packaging of concatemeric viral DNA, (
ii
) the sequence-specific cleavage is either a prerequisite for or occurs during the entry of viral DNA into capsid structures, and (
iii
) DNA molecules significantly shorter than full-length standard viral DNA can become encapsidated within nuclear capsids provided they contain the cleavage/packaging signal. However, capsids containing DNA molecules significantly shorter than standard virus DNA are not translocated into the cytoplasm.