bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the production of secretory proteins: Intracellular degradation of newly synthesized “defective” collagen
Authors: Berg Richard A. , Schwartz Martin L. , Crystal Ronald G.
Published: May 2006

Confluent cultures of human fetal lung fibroblasts degrade approximately 10% of their newly synthesized collagen within the cell prior to secretion. This basal level of intracellular degradation could not be inhibited by colchicine or cytochalasin B, inhibitors of microtubular and microfilament function, respectively, or by
N
α
-
p
-tosyl-L-lysine chloromethyl ketone, chloroquine, or NH
4
Cl, inhibitors of lysosomal enzymes. In contrast, cells in early logarithmic growth degrade approximately 30% of their newly synthesized collagen. This enhanced degradation of collagen in rapidly growing cells could be suppressed by inhibitors of lysosomal proteases and partially inhibited by disrupters of microtubular and microfilament function. A significant proportion of the collagen synthesized by these cultures contained prolyl residues that were incompletely hydroxylated. Because such collagen is “defective” (i.e., not capable of assuming a triple helical conformation), the results suggest that enhanced intracellular degradation may be a mechanism by which cells control the quality of collagen they produce. To test this hypothesis, confluent cells were incubated with the proline analog
cis
-4-hydroxyproline; such cells demonstrated enhanced collagen degradation that could be inhibited by agents that interfere with lysosomal, microtubular, or microfilament function. Because collagen containing
cis
-4-hydroxyproline cannot form a perfect triple helix, the data are consistent with the concept that defective collagen is recognized by cells and degraded prior to secretion. Thus, the proportion of newly synthesized collagen that undergoes intracellular degradation seems to be modulated, in part, by the conformation of the collagen molecule. Intracellular proteolysis may represent a means by which collagen-producing cells regulate the quality and quantity of collagen available for extracellular function. Although the exact mechanism of intracellular collagen degradation is unknown, the data presented here are consistent with a role for lysosomal proteases in this process.
Mammalian epidermal growth factor promotes plant growth
Authors: Dyer Melvin I.
Published: May 2006

Application of mouse submaxillary gland epidermal growth factor to young sorghum seedlings at low concentrations (≈0.4-4 μM) increased shoot growth significantly over 3- and 6-day periods. The effects were dose dependent.
Germ-line reinsertions of AKR murine leukemia virus genomes in Akv-1 congenic mice.
Authors: Rowe W P , Kozak C A
Published: May 2006

Congenic mouse strains NIH,Akv-1 and NIH,Akv-2 carry the two high ecotropic virus-inducing loci of the AKR mouse on the NIH Swiss genetic background. Progeny tests of animals in three separate congenic families show that both Avk-1 and Akv-2 are stably transmitted as classical mendelian loci in these mice. However, during the process of inbreeding, additional chromosomal viral loci were detected in several NIH.Akv-1 sublines. These loci appeared only in the progeny of virus-positive females. They segregate with mendelian ratios, are unlinked to markers on chromsome 7 near Akv-1, and are phenotypically expressed as high-virus-inducing loci. The generation of new loci for viurs induction, no doubt resulting from the rare germ-line reintegration of the endogenous ectropic provirus, represents a unique form of gene duplication and rearrangement.
Induction of lymphoma in athymic mice: a model for study of the human disease.
Authors: Beattie G , Baird S , Lannom R , Slimmer S , Jensen F C , Kaplan N O
Published: May 2006

A murine lymphoma, designated L1, was produced in immunologically deficient nude mice after chronic antigenic stimulation by infection with the pinworms Aspiculuris tetraptera and Syphacia obvelata. In vivo, L1 involves primarily the spleen and lymph nodes, with infiltration of liver, kidney, and bone marrow also observed. It is characterized by large clusters of B cells and null cells, and by rare T cells. The lymphoma cells express murine leukemia virus antigens (gp70 and p30) on the surface. L1 can be passaged successfully both in vivo and in vitro. The lymphoblasts that proliferate in vitro are null, but injecton back into the mouse produces a similar pattern of B cells, null cells, and occasional T cells as seen in the mouse-to-mouse transfers. Infectious viruses have been isolated from L1 cells and from tissue culture supernates and have been identified as a B-tropic murine leukemia virus and a xenotropic virus. The possibilities of this model for studying the etiology of human lymphoma are discussed.
Solubilization of hemoglobin S by other hemoglobins.
Authors: Benesch R E , Edalji R , Benesch R , Kwong S
Published: May 2006

The polymerization of mixtures of Hb S with hemoglobins A, A2, and F has been investigated by analysis of the proportions of S and non-S hemoglobin both in the supernate and in the pellet after centrifugation. In all cases the non-S hemoglobin was incorporated into the polymer even in the absence of hybrids in the order A > A2 > F. The solubility of Hb S is substantially increased by the other hemoglobins, especially by Hb F, which would account for its antisickling effect. It appears that the excluded volume effect of the other hemoglobin on Hb S is largely counterbalanced by the solubilizing effect arising from the interaction between the two hemoglobins in solution. The ability of hybrid hemoglobins to gel was demonstrated directly with tetramers in which alpha beta s dimers were covalently linked to alpha beta A, alpha delta A2, and alpha gamma F dimers.
Unique long-acting antiglucocorticoid in whole and broken cell systems.
Authors: Simons S S , Thompson E B , Johnson D F
Published: May 2006

The biological properties of cortisol 21-mesylate (CM), an alkylating derivative of cortisol, were investigated in a line of rat hepatoma tissue culture (HTC) cells. CM appears to bind to glucocoticoid receptors in cell-free extracts because CM inhibits the specific binding of [3H]dexamethasone. However, in whole cells CM not only fails to induce the enzyme tyrosine aminotransferase (TyrATase) but also inhibits the induction of TyrATase by dexamethasone. Thus CM is an antiglucocorticoid. This is not caused by cell death, because CM is relatively nontoxic up to concentrations of 10 microM. The concentration of CM needed for half maximal inhibition of TyrATase induction is an order of magnitude lower than that predicted from the apparent cell-free affinity of CM for the glucocorticoid receptors of HTC cells, which suggests that the cell-free binding data does not reflect an equilibrium situation. In fact, the reactive alpha-keto mesylate group was intentionally incorporated into cortisol in hopes of obtaining a steroid capable of undergoing irreversible reactions. When HTC cells were preincubated with either CM or the reversible antiglucocorticoid progesterone and then washed to remove free steroid, only the CM-treated cells failed to show subsequent induction of TyrATase by dexamethasone. Furthermore, preincubation of HTC-cell cytosol with CM blocked approximately 75% of the subsequent exchange binding of [3H]dexamethasone to glucocorticoid receptor sites. Thus, the actions of CM in whole and broken cells either require an exceptionally long time for reversal or are not reversible. Together, these results indicate that CM is a unique antagonist and could be an irreversible antiglucocorticoid in vitro.
In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.
Authors: Zipursky S L , Reinberg D , Hurwitz J
Published: May 2006

The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and Escherichia coli rep protein, DNA-binding protein, and DNA polymerase III elongation system; (iii) support replication of duplexes catalyzed by the phi X174 A protein and extracts of E. coli.
Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose.
Authors: Thomas P S
Published: May 2006

A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently.
Nucleotide sequence analysis of the transforming region and large terminal redundancies of Moloney murine sarcoma virus.
Authors: Reddy E P , Smith M J , Canaani E , Robbins K C , Tronick S R , Zain S , Aaronson S A
Published: May 2006

The sequence of the transforming region of the Moloney murine sarcoma virus genome has been determined by using molecularly cloned viral DNA. This region, 3.6 to 5.8 kilobase pairs from the left end of the molecule, contains the entire cellular insertion (src) sequence as well as helper viral sequences including the large terminal repeat (LTR). On the viral RNA strand, a long (1224 bases) open reading frame commenced to the left of the src-helper virus junction and terminated at a point 58 nucleotides into helper viral sequences to the right of src. Possible promoter and acceptor splice signals were detected in helper viral sequences upstream from this open reading frame. On the antiviral RNA strand, several promoter-like sequences, including one within the src region itself, were identified. However, no open reading frame downstream from these promoters was detected in the antiviral RNA strand. The LTR was found to contain promoter-like sequences as well as LTR was found to contain promoter-like sequences as well as mRNA capping and polyadenylylation signals. In addition, it possessed an 11-base inverted terminal repeat at each end. Thus, the structure of the Moloney murine sarcoma virus genome with an LTR at each end resembles that of prokaryotic transposable elements.
Anchorage-independent muscle cell differentiation
Authors: Puri Emilio C. , Caravatti Mario , Perriard Jean-Claude , Turner David C. , Eppenberger Hans M.
Published: May 2006

Cells from embryonic chicken muscle were cultivated in serum-free medium. After two days, the suspended cells (almost all of which were nondividing myoblasts) were subcultured in serum-containing medium, either in gelatincoated tissue culture dishes (to promote reattachment) or in bacteriological dishes (to prevent reattachment). The extent of fusion was high in both suspended and reattached cultures. Newly synthesized proteins from day-5 cultures were resolved by two-dimensional electrophoresis and detected by autoradiography. Not only were the same protein species synthesized, but also the relative intensities of the spots corresponding to known muscle-specific proteins as well as the patterns of the many unidentified spots were similar. Synthesis of creatine kinase subunits B and M at different times was determined. In both suspended and reattached cells there was, as expected for differentiating myogenic cells, a marked increase by day 5 in the ratio of M to B subunits synthesized. Immunofluorescent staining with antibodies against an M-line protein with
M
r
165,000 revealed myofibrils partially wound about the nuclei of suspended cells; these became strung out in the axis of the cell as reattached cells elongated. The synthesis of muscle proteins, their assembly into myofilaments, and formation of well-organized myofibrils is evidently not anchorage dependent. However, proper alignment of parallel arrays of myofibrils in register does appear to require cell attachment to substrate.
Wide distribution of immunoreactive renin in nerve cells of human brain.
Authors: Slater E E , Defendini R , Zimmerman E A
Published: May 2006

By use of the indirect peroxidase-antiperoxidase complex immunocytochemical technique, antibody to purified human renal renin was applied to formalin-fixed paraffin sections of human cadaver brain. Immune reaction products were observed in most nerve cells in all areas of the brain examined; staining was limited to the soma and proximal dendrites. These experiments have confirmed the presence of a renin-like substance in central nervous tissue and suggest a more generalized function for "brain renin" than was previously anticipated.
Biosynthesis of vitamin B12: nature of the volatile fragment generated during formation of the corrin ring system.
Authors: Mombelli L , Nussbaumer C , Weber H , Müller G , Arigoni D
Published: May 2006

It is shown with the help of radiolabeled substrates that biological formation of the corrin ligand system of cobyrinic acid is linked with the release of an equivalent of acetic acid from a hydroporphinoid precursor in which the C-20 atom has undergone methylation prior to its excision from the macrocycle.
Specific phospholipids are required to reconstitute adenylate cyclase solubilized from rat brain.
Authors: Hebdon G M , LeVine H , Sahyoun N E , Schmitges C J , Cuatrecasas P
Published: May 2006

Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was solubilized from a rat brain homogenate with sodium deoxycholate. This solubilized preparation had no detectable enzymic activity with either Mg-ATP or Mn-ATP as substrate. The activity could be restored by addition of either nonionic detergent or certain specific phospholipids. Maximal restoration of enzyme activity was obtained with Triton X-100, L-alpha-phosphatidylcholine, L-alpha-lysophosphatidylcholine, phosphatidyl-N-monomethylethanolamine, or sphingomyelin. Activity was only partially restored by phosphatidylethanolamine (40-60%) or phosphatidyl-N,N-dimethylethanolamine (10-20%). Other phospholipids tested, including phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid, could not restore enzyme activity but, instead, could inhibit the stimulation of enzyme activity by phosphatidylcholine. The restoration of activity by L-alpha-phosphatidylcholine was inhibited by cholesterol at concentrations above 33 mol %, although this effect was not observed with three different esters of cholesterol. These studies suggest a possible specific role of phospholipids in modulating adenylate cyclase activity.
A model of clonal attenuation.
Authors: Prothero J , Gallant J A
Published: May 2006

Two formal models of clonal attenuation [Kirkwood, T. B. L. & Holliday, R. (1975) J. Theor. Biol. 53, 481-496; Shall, S. & Stein, W. D. (1979) J. Theor. Biol. 76, 219-231] are considered in the light of recent data on the changing distribution of replicative potential among individual cultured fibroblasts on subcloning. The experimental data [Smith, J. R., Pereira-Smith, O. & Good, P. I. (1977) Mech. Ageing Dev. 6, 283-286] are shown to contradict both models. A new model, compatible with the subcloning data, is proposed. This model involves a gradual increase in the probability of commitment during cell growth in culture and a small number (about seven) of divisions following commitment. The gradual increase in commitment probability is shown to be compatible with the gradual accumulation of a gene product subject to autogenous regulation.
Embryonic erythroid differentiation in the human leukemic cell line K562.
Authors: Rutherford T , Clegg J B , Higgs D R , Jones R W , Thompson J , Weatherall D J
Published: May 2006

K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.
Serologically distinguishable alterations in the molecular specificity of cell cohesion during morphogenesis in
Dictyostelium discoideum
Authors: Wilcox David K. , Sussman Maurice
Published: May 2006

Cells of the mutant strain JC-5 of the slime mold
Dictyostelium discoideum
exhibit at a specific, late developmental stage a temperature-sensitive morphogenetic defect associated with the loss of cell cohension. We show that at the restrictive temperature, the loss of cohesion and attendant dispersal of multicellular aggregates is associated with the disappearance or sequestration of a plasma membrane-bound moiety capable of reacting with and, hence, absorbing cohesion-blocking Fab. At the permissive temperature, the maintenance of cohesiveness past the critical stage or the recovery of lost cohesiveness is correlated with the presence or reappearance of the Fab-reactive moiety.

This moiety is absent or sterically incapable of reaction with Fab preparations at an earlier developmental stage in either mutant or wild-type cells—i.e., at a time when they have just entered into multicellular aggregates. Conversely, a serologically distinguishable membrane-bound moiety present in the early mutant or wild-type cells, whose reaction with homologous Fab also precludes their cohesion, is absent or serologically unreactive in either mutant or wild-type cells that are at comparable late developmental stages. We conclude that the cohesive moiety responsible for initiation of cell aggregates is supplanted by or transformed into a serologically distinct, cohesive complex responsible for the maintenance of the aggregate's integrity through the later stages of development.
Cmu gene rearrangement of mouse immunoglobulin genes in normal B cells occurs on both the expressed and nonexpressed chromosomes.
Authors: Nottenburg C , Weissman I L
Published: May 2006

We have examined the organization of heavy-chain immunoglobulin genes on both the expressed and nonexpressed chromosomes of normal B lymphocytes from allotype heterozygous (BALB/c X C57BL/J)F1 mice. The C mu genes of BALB/c mice are on 12.4-kilobase EcoRI and 13.1-kilobase Kpn I restriction fragments, whereas those of C57BL/J mice are on 13.6-kilobase EcoRI and 14.3-kilobase Kpn I restriction fragments, allowing the examination of rearrangements on each chromosome independently. B lymphocytes from spleen and Peyer's patches expressing both IgD and IgM of the BALB/c allotype were isolated with a fluorescence-activated cell sorter. EcoRI and Kpn I restriction digests were hybridized with a C mu gene-containing probe. The C mu gene is present on both chromosomes. DNA rearrangements occur on both the expressed and nonexpressed chromosome within the 3.6-kilobase Kpn I/EcoRI restriction fragment containing the joining (JH) gene locus. We conclude that allelic exclusion of heavy-chain immunoglobulin gene expression is not mediated by JH-region DNA rearrangement of the expressed chromosome only. In contrast, analysis of the C kappa gene region from the same sorted B-cell DNA reveals a substantial quantity of germ-line context DNA. We also demonstrate that the deletions observed on the Eco RI fragment containing the C mu gene in myeloma cells and in C mu gene-containing recombinant DNAs do not usually occur in normally differentiating B lymphocytes and are likely to be confined to myeloma tumor cells.
Monoclonal antibodies against antigens displayed on a progressively growing mammary tumor.
Authors: Tax A , Manson L A
Published: May 2006

We have produced lymphocyte hybridomas between mouse myeloma cells and either spleen cells of C3H/f/C57BL/6 mice bearing the Mm5mt/c1 tumor-producing murine mammary tumor virus (MMTV) or spleen cells from Fisher rats inoculated with the same tumor. Two classes of hybridoma-secreted monoclonal antibodies were obtained. In the first class are IVC11, IIIA1, and VE7, each of which precipitated a 52,000-dalton protein from 125I-labeled purified preparations of MMTV and [3H]glucosamine-labeled Mm5mt/c1 cell extracts. A second class of monoclonal antibodies, represented by IC12 and IIIB5, reacted specifically with C3H MMTV-secreting cells in radioimmunoassays (RIA) with whole cells and did not precipitate proteins with labeled virus or metabolically labeled Mm5mt/c cell extracts. IC12 and IIB5 could be distinguished from each other in a RIA in which antigen discs were prepared from a membrane preparation of Mm5mt/c1 cells. In this assay, IC12 antibody was blocked from reacting with discs precoated with goat anti-MMTV, and IIB5 antibody was not.
A brain membrane protein similar to the rat src gene product.
Authors: Scheinberg D A , Strand M
Published: May 2006

We report the purification to homogeneity of a 20,000-dalton, transformation-related, rat cell membrane protein. This protein, p20, was originally identified in preparations of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus. The chromatographically purified p20 was an acidic hydrophobic protein, capable of specifically binding GTP (dissociation constant = 15 microM). This nucleotide binding property and other previously reported characteristics were similar to properties ascribed to the Harvey sarcoma virus src gene product. p20 also appeared similar to this src gene product when immunoprecipitates of both proteins were directly compared by one- and two-dimensional NaDodSO4 gel electrophoreses. However, the proteins were not identical, because their tryptic maps differed. Using a competition radioimmunoassay, we have measured the concentration of p20 in cells, viruses, and rat tissues: p20 was not encoded by rat sarcoma viruses because it was increased only slightly after Kirsten sarcoma virus transformation of rat cells and was not increased in nonrat cells transformed by the Kirsten or Harvey sarcoma virus. Remarkably, of 10 rat tissues examined, p20 was found predominantly in brain, specifically in the membranes.
Identification of a third type of lambda light chain in mouse immunoglobulins.
Authors: Azuma T , Steiner L A , Eisen H N
Published: May 2006

A third type of mouse lambda chain was identified in the course of examining light chains from four myeloma proteins and two monoclonal antibodies. On the basis of their antigenic properties and a characteristic COOH-terminal tryptic peptide, the light chains from these immunoglobulins had previously been provisionally identified as lambda 2 chains. However, four of these chains, designated now as lambda 3 (or lambda III), were found to share constant region features that clearly distinguish them from the other types of mouse lambda chains (lambda 1 and lambda 2). The amino acid sequence of more than 90% of the lambda 3 constant region (from position 120 to the COOH-terminus) was determined. In this region the lambda 3 sequence differs from the lambda 2 constant region at 5 positions and from the lambda 1 constant region at about 30 positions.
Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.
Authors: Kit S , Otsuka H , Qavi H , Hazen M
Published: May 2006

A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability to incorporate [3H]dThd into DNA and an HSv-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TK+ phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame.
Direct visualization of receptors for thyrotropin-releasing hormone with a fluorescein-labeled analog.
Authors: Halpern J , Hinkle P M
Published: May 2006

Thyrotropin-releasing hormone (TRH) binds to specific receptors on GH4C1 pituitary tumor cells. A fluorescently labeled analog of TRH was synthesized by coupling pGlu-His-ProNH(CH2)6NH2 to fluorescein isothiocyanate. The fluorescein-labeled peptide (FL-TRH) stimulated prolactin synthesis and release by GH4C1 cultures and bound to TRH receptors with an apparent Kd of 400 nM. Binding of FL-TRH to unfixed, viable GH4C1 cells was followed by fluorescence microscopy. After incubation with 1.4 microM FL-TRH for 1 hr at 37 degrees C, the surface of all cells was fluorescent and patches of intense fluorescence were evident. Control cultures incubated with FL-TRH and excess TRH were not fluorescent, and a line of pituitary tumor cells which lacks TRH receptors displayed little fluorescence after incubation with FL-TRH. When GH4C1 cells were incubated with FL-TRH for 1 hr at 37 degrees C and then with excess TRH for an additional 1 hr, the fluorescence associated with the cells was diminished to control levels. The results demonstrate that the fluorescein-labeled peptide labels specific TRH receptors.
Characterization of angiotensin receptors on bovine adrenal fasciculata cells.
Authors: Vallotton M B , Capponi A M , Grillet C , Knupfer A L , Hepp R , Khosla M C , Bumpus F M
Published: May 2006

We have further characterized angiotensin receptors on bovine adrenal fasciculata cells whose presence was previously demonstrated by the intrinsic agonistic activity of angiotensin II (AII), dex-Asp1-AII, angiotensin I (AI), and des-ASp1-AI on steroidogenesis. The specific binding of AII and des-Asp1-AII labeled with 125I to dispersed bovine fasciculata cells was studied. For both peptides, a single class of binding sites accounted for the data with a mean (+/- SEM) Ka value of 0.23 +/- 0.123 X 10(8) liters/mol for AII and 0.68 X 10(8) liters/mol for des-Asp1-AII. The concentration at which unlabeled AII and des-Asp1-AII displaced 50% of the tracers (Kd) was similar to that at which they induced half-maximal stimulation of steroidogenesis (Kact). For AI and des-Asp1-AI, Kd greater than Kact. Analogs of AII or des-Asp1-AII with antagonistic properties upon steroidogenesis competed also with binding of the tracers. Corticotropin (ACTH) did not inhibit binding. Although ACTH stimulated the formation of cyclic AMP, none of the angiotensins with intrinsic activity did so. Calcium, but not potassium, appeared to potentiate the steroidogenic activity of AII. These data suggest that there is a single class of receptors for angiotensins and analogs in zona fasciculata. These receptors show characteristics that differentiate them from ACTH receptors in zona fasciculata or angiotensin receptors in zona glomerulosa cells.
Localized binding of [3H]muscimol to synapses in chicken retina.
Authors: Yazulla S , Brecha N
Published: May 2006

Binding sites for [3H]muscimol, an analogue of gamma-aminobutyric acid (GABA) were localized in the synaptic layers of chicken retina by light microscopic and electron microscopic autoradiography. Light microscopic autoradiography of cryostat sections incubated in [3H]muscimol or [3H]GABA revealed identical binding patterns: a band over the inner plexiform layer (IPL) and a band over the outer plexiform layer (OPL). This binding pattern differed from the uptake pattern for [3H]GABA: labeling over horizontal, amacrine, and ganglion cell bodies as well as very intense labeling over lamina 5 in the proximal IPL. Statistical analysis of electron microscopic autoradiography data from the IPL indicated that only amacrine synapses bind [3H]muscimol (i.e., make GABAergic synapses). Processes of amacrine, bipolar, or ganglion cells can be postsynaptic to these amacrine synapses. The highest concentration of synapses binding [3H]muscimol occurred in laminae 2 and 4 of the IPL and not in lamina 5 as might be expected from the density of [3H]GABA uptake. In the OPL, [3H]muscimol binding occurred over specialized junctions proximal to photoreceptor terminals. In cone receptor terminals, [3H]muscimol binding was suspected near horizontal cell dendrite/receptor terminal membranes lateral to the synaptic ribbon, supporting the hypothesis that horizontal cells are involved in a GABAergic feedback loop with cone terminals. We conclude that the synaptic binding pattern provides a more accurate concept of GABAergic synaptic interaction than does the uptake pattern for [3H]GABA because the two patterns in the IPL are not related.
Loss of chromosomal high mobility group proteins HMG1 and HMG2 when mouse neuroblastoma and Friend erythroleukemia cells become committed to differentiation.
Authors: Seyedin S M , Pehrson J R , Cole R D
Published: May 2006

Chromosomal high mobility group (HMG) proteins HMG1 and HMG2 from mouse neuroblastoma cells and Friend erythroleukemic cells were analyzed by acetic acid/urea/polyacrylamide gel electrophoresis. Compared to rapidly growing cells, levels of HMG1 and HMG2 were decreased in mouse neuroblastoma cells that had been induced to differentiate by serum deprivation. This comparison revealed a reciprocal relationship between these HMG proteins and H10, a histone known to be in higher concentrations in nondividing cells. When cell growth was inhibited by means of density inhibition, however, HMG1 and -2 levels were not affected in either HeLa or mouse neuroblastoma cells, even though H10 did not accumulate. This observation establishes that HMG1 and -2 contents are not correlated with mitotic rate per se. Treatment of mouse neuroblastoma by sodium butyrate, which stops cell division without commitment to differentiation, had no effect on the level of HMG1 and -2. However, the level was decreased by dibutyryl cyclic AMP and dimethyl sulfoxide treatments, which, like serum deprivation, induced irreversible morphological differentiation in the neuroblastoma cells. Moreover, induction of differentiation (hemoglobin synthesis) in Friend erythroleukemic cells by dimethyl sulfoxide showed a decrease in the contents of HMG1 and -2. These observations suggest that preferential loss of HMG1 and -2 in mouse neuroblastoma and Friend erythroleukemia cells may be related to commitment of these cells to differentiation.
Escherichia coli factor Y sites of plasmid pBR322 can function as origins of DNA replication.
Authors: Zipursky S L , Marians K J
Published: May 2006

The Escherichia coli replication factor Y, in conjunction with other genetically undefined E. coli replication factors and the gene products of the E. coli dnaB, dnaC, and dnaG loci, is involved in de novo primer formation on the phi X174 (+) single-strand circular DNA template [(+) ss(c)DNA]. The participation of factor Y in this series of reactions is correlated with its phi X174 (+) ss(c)-specific DNA-dependent ATPase activity. Recently two factor Y effector DNA segments of the plasmid pBR322 have been identified in close proximity to the plasmid origin of DNA replication. We report here that insertion of these factor Y sites into the filamentous phage f1R229 (+) ss(c)DNA confers upon it the ability to be converted to RF DNA in vitro through a rifampicin-resistant dnaB, dnaC, and dnaG gene product-dependent pathway. Our data suggest that factor Y effector sites can function as origins of DNA replication.
Expression of early adenovirus genes requires a viral encoded acidic polypeptide.
Authors: Ricciardi R P , Jones R L , Cepko C L , Sharp P A , Roberts B E
Published: May 2006

Host-range mutants of adenovirus 5 that contain a defect in region E1A (0-4.5 units) fail to replicate in HeLa cells and to transform rodent cells. In HeLa cells, these mutants synthesize only the two RNAs from E1A that share the same 5' and 3' termini but differ in length by the amount of internal sequence removed by splicing. RNA from wild-type virus, selected by hybridization to DNA from region E1A, translates into polypeptides of Mr 51,000 and 48,000 that are highly acidic in isoelectric focusing gels. These acidic Mr 51,000 and Mr 48,000 polypeptides are encoded by the longer and shorter E1A RNAs, respectively. Two of the host-range mutants, H5hr1 and H5hr2, fail to synthesize the Mr 51,000 polypeptide but do produce the Mr 48,000 polypeptide and a novel polypeptide thought to be a truncated portion of the Mr 51,000 polypeptide. H5hr1 and H5hr2 are hypothesized to have termination codons in sequences found only in RNA encoding the Mr 51,000 polypeptide. This prediction is verified for H5hr1 by DNA sequence analysis. The other three host-range mutants (H5hr3-5) synthesize both acidic polypeptides and are predicted to be missense. These results strongly imply that the Mr 51,000 polypeptide, alone or in combination with the Mr 48,000 polypeptide, is needed to regulate expression of adjacent viral genes during the early phase of adenovirus infection.
Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture.
Authors: Laszlo A , Radke K , Chin S , Bissell M J
Published: May 2006

We have investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the src gene-associated protein kinase, the 34- to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response to TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation of other cellular phosphotyrosine-specific kinases. We have shown previously that temperature-sensitive RSV-infected cells at nonpermissive temperature demonstrate an increased sensitivity to TPA treatment [Bissell, M. J., Hatie, C. & Calvin, M. (1979) Proc. Natl. Acad. Sci. USA 76, 348-352]. Our present results indicate that this is not due to reactivation of the phosphorylating activity of the defective src gene product or to its leakiness, and they lend support to the notion of multistep viral carcinogenesis.
Herpesvirus ateles and herpesvirus saimiri transform marmoset T cells into continuously proliferating cell lines that can mediate natural killer cell-like cytotoxicity.
Authors: Johnson D R , Jondal M
Published: May 2006

Herpesvirus ateles (HVA) and herpesvirus saimiri (HVS) have the capacity to transform cotton-topped marmoset T lymphocytes into continuously proliferating cell lines that retain some functions associated with cell-mediated immunity. In the present paper, we demonstrate that HVA/HVS-transformed T cell lines are cytotoxic in a short-term 51Cr release assay and that this killing resembles killing by marmoset natural killer (NK) cells. The relationship between NK cells and HVA/HVS-transformed killer cell lines is discussed in view of present knowledge of the origin and function of the NK system. It is suggested that the described cytotoxic cell lines may be useful for further defining cellular cytotoxicity with regard to cell surface recognition, regulatory events, and lytic mechanisms.
Monoclonal antibody against a Burkitt lymphoma-associated antigen.
Authors: Wiels J , Fellous M , Tursz T
Published: May 2006

A monoclonal antibody, referred to as 38.13, was obtained by fusing murine myeloma cells with Lewis rat splenocytes sensitized with Daudi cells (human Burkitt lymphoma containing Epstein--Barr virus genome but lacking HLA-A, -B, and -C and beta 2-microglobulin molecules at the cell surface). 38.13 antibody was demonstrated to be a rat IgM. By complement-dependent microcytotoxicity and indirect immunofluorescence assays, 38.13 antibody was shown to react specifically with cells derived from Burkitt tumors, including both Epstein--Barr virus genome-carrying and Epstein--Barr virus-negative Burkitt lymphoma. By contrast, Epstein--Barr virus-containing lymphoblastoid cell lines derived from normal B lymphocytes were not recognized by 38.13 antibody. Fresh malignant cells from patients affected with various lymphoproliferative disorders were negative, except 4/8 having abdominal Burkitt-like lymphomas. Normal lymphocytes from peripheral blood, spleen, lymph node, tonsil, and bone marrow and mitogen (phytohemagglutinin, pokeweed mitogen, and concanavalin A)-activated blasts were also negative. Thus, 38.13 antibody apparently recognized a Burkitt-associated antigen that is not related to Epstein--Barr virus. The pattern of reactivity of 38.13 antibody with various Burkitt lymphoma cells appeared quite heterogenous and some Burkitt cells were consistently negative. 38.13 antibody thus defines a subset of Burkitt lymphomas.
Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin.
Authors: Suggs S V , Wallace R B , Hirose T , Kawashima E H , Itakura K
Published: May 2006

We have synthesized two sets of 15-base-long oligodeoxyribonucleotides corresponding to all possible coding sequences for a small portion of human beta 2-microglobulin. Labeled oligonucleotides were used as hybridization probes to screen bacterial clones containing cDNA sequences primed with oligo(dT) and inserted into the plasmid vector pBR322. One beta 2-microglobulin cDNA clone was detected in the 535 bacterial plasmid clones that were screened. The clone has been characterized by blotting and nucleotide sequence analysis. The cloned beta 2-microglobulin sequence contains 217 base pairs of the 3' untranslated region of the mRNA and 328 base pairs (97%) of the coding region.
Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones.
Authors: Mumford R A , Pierzchala P A , Strauss A W , Zimmerman M
Published: May 2006

A porcine kidney microsomal metalloendopeptidase has been enriched 3900-fold. Gel filtration on a calibrated Toyo-Soda G-3000 SW column indicated an appropriate molecular weight for the endopeptidase of 88,000 +/- 2000. The purified enzyme is inhibited by a number of synthetic inhibitors of thermolysin. The endopeptidase hydrolyzes the succinyl (Suc)-containing fluorogenic peptide substrate Suc-Ala-Ala-Phe-(7-amino-4-methylcoumarin) at the Ala-Phe position with a Km of 2.9 X 10(-4) M. The endopeptidase also hydrolyzes a variety of peptides including corticotropin, substance P, angiotensin I and II, neurotensin, somatostatin, bradykinin, and the renin tetradecapeptide substrate. The endopeptidase hydrolyzes both [Leu]- and [Met]enkephalin at the Gly-Phe bond.
Carbohydrate modifications of the high mobility group proteins.
Authors: Reeves R , Chang D , Chung S C
Published: May 2006

This paper reports the results of numerous biochemical analyses which indicate that the "high mobility group" proteins (HMGs) of mouse and bovine cells are bona fide glycoproteins and can, in addition, be modified by poly(ADP-ribose) addition in vitro. The sugars N-acetylglucosamine, mannose, galactose, glucose, fucose, and one unknown sugar (possibly xylose) have been identified in purified preparations of HMGs 14 and 17. Furthermore, the fucose-specific lectin Ulex europeus agglutinin I bound both to the isolated HMGs and to monomer nucleosomes containing HMGs released from "active chromatin" by micrococcal nuclease digestion. Selective alkaline borohydride reductive cleavages of the HMGs suggested that the oligosaccharide prosthetic groups are primarily bound to these proteins by N-glycosidic linkages. The unexpected finding that the HMGs contain covalently bound complex carbohydrate moieties allows for a potentially great amount of variability and specificity in these proteins that may have important biological implications.
Repair of O6-ethylguanine in DNA by a chromatin fraction from rat liver: transfer of the ethyl group to an acceptor protein.
Authors: Mehta J R , Ludlum D B , Renard A , Verly W G
Published: May 2006

Incubation of O6-[3H]ethylguanine-containing DNA with a rat liver chromatin fraction resulted in a decrease in the O6-ethylguanine content of the DNA. Analysis of the products of this reaction showed that the ethyl group had been transferred from the O6-ethylguanine to a protein acceptor. When the incubation mixture was separated on a cesium chloride gradient, the radioactivity removed from O6-ethylguanine appeared in a low-density band. This material has been isolated and subjected to trypsin digestion and high-pressure liquid chromatography analysis; it was sensitive to trypsin and the digest contained new high-pressure liquid chromatography peaks characteristic of oligopeptides. Radioactive peaks from the trypsin digestion have been digested further to the amino acid level and have been shown to contain S-[3H]ethylcysteine. Thus, we conclude that the repair activity in rat liver chromatin removes the ethyl group from O6-ethylguanine and transfers it to a cysteine moiety contained in an acceptor protein.
Modelling the unusual temperature dependence of atomic displacements in proteins by local nonharmonic potentials.
Authors: Gavish B
Published: May 2006

Recent x-ray studies of metmyoglobin at different temperatures [Frauenfelder, H. Petsko, G. A. & Tsernoglou, D. (1979) Nature (London) 280, 558-563] show that the dependence of the mean square displacements of individual atoms on the absolute temperature T can be described analytically by T mu, in which the parameter mu undergoes local variations in the range -2 to +20. This strong deviation from harmonic behavior mu = 1 is modelled here, in the classical limit, by local nonharmonic potentials, in which a particle spends part of its time in one or more weakly bound states (traps) and the rest of it as an almost free particle in a restricted volume (cavity). Analytic calculations of the mean square atomic displacement in such cavity--traps potentials for a simplified three-dimensional geometry yield the following results: A parametrically described T mu behavior characterizes a transition region in temperature below which the particle is "trapped" (solid-like) and above which the particle is "free" (liquid-like). The magnitude of mu increases with an increase in the ratio of cavity volume to trap volume. Mu greater than 0 type of behavior can be obtained by a single trap and mu less than 0 by two symmetric traps. A comparison is made between the predictions of the model and the experimental results, and the elucidation of the model parameters from x-ray data is discussed.
Morphologic study of the internalization of a lysosomal enzyme by the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells.
Authors: Willingham M C , Pastan I H , Sahagian G G , Jourdian G W , Neufeld E F
Published: May 2006

The binding and internalization of a model lysosomal enzyme, beta-galactosidase, was visualized by use of rabbit anti-beta-galactosidase and goat anti-rabbit IgG; the second antibody was labeled with rhodamine or fluorescein (for detection by fluorescence) or with horseradish peroxidase (for electron microscopy). Chinese hamster ovary cells were incubated with beta-galactosidase at 4 degrees C, and then were washed and sequentially incubated in the cold with the two antibodies. The beta-galactosidase was found primarily in coated pits. The binding of the enzyme was completely inhibited by 5 mM mannose 6-phosphate. After the reaction with enzyme and antibodies, the cells were warmed to 37 degrees C; within 1 minute, the beta-galactosidase--antibody complex had begun to move to uncoated vesicles (receptosomes). After 8 min, the beta-galactosidase--antibody complex was seen in receptosomes near tubular elements in the Golgi/GERL area, within such tubular elements and at times, in vesicular elements that may correspond to coated structures of the GERL system. After 15 min, the enzyme--antibody complex was found in lysosomes near the Golgi/GERL are and a half-hour later it was in lysosomes distributed throughout the cytoplasm. Double-label experiments using beta-galactosidase and gold/alpha 2-macroglobulin showed the presence of the two ligands in the same coated pits and receptosomes. Thus, the pathway for internalization of beta-galactosidase via the mannose 6-phosphate receptor is similar to the pathway established for other ligands such as low density lipoprotein and alpha 2-macroglobulin.
Transmission electron microscopic method for gene mapping on polytene chromosomes by in situ hybridization.
Authors: Wu M , Davidson N
Published: May 2006

A transmission electron microscope method for gene mapping by in situ hybridization to Drosophila polytene chromosomes has been developed. As electron-opaque labels, we use colloidal gold spheres having a diameter of 25 nm. The spheres are coated with a layer of protein to which Escherichia coli single-stranded DNA is photochemically crosslinked. Poly(dT) tails are added to the 3' OH ends of these DNA strands, and poly(dA) tails are added to the 3' OH ends of a fragmented cloned Drosophila DNA. These probe--dA strands are hybridized in situ to polytene chromosome squashes. Gold spheres are linked to the hybridized probe--dA strands by A.T base pairing. The sphere positions relative to the chromosome bands can be observed by transmission electron microscopy. The method shows low background and high resolution.