bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of Notch, a locus affecting neurogenesis in Drosophila melanogaster.
Authors: Artavanis-Tsakonas S , Muskavitch M A , Yedvobnick B
Published: May 2006

The Notch locus is one of the best characterized loci in Drosophila melanogaster in terms of its genetic structure and developmental effects. Mutations in this locus profoundly affect the differentiation of the early embryo. Using an inversion involving the Notch locus and previously cloned sequences, we have isolated chromosomal segments from the Notch region (3C7) encompassing 80 kilobases (kb) of DNA. Based on comparison between mutant and wild-type DNA, we have positioned cloned sequences within the Notch genetic map; furthermore, we have defined a region of approximately 40 kb within which the structural lesions correlating with all Notch alleles mapped to date appear to reside. We have examined the transcriptional activity of the cloned sequences during ontogeny and find a single size class of poly(A)+ RNA, 10.5 kb long, that is homologous to sequences within this 40-kb region. We conclude that DNA sequences belonging to the Notch locus have been cloned and that the 10.5-kb poly(A)+ RNA is essential for wild-type Notch function. We discuss these structural and transcriptional data in light of the existing genetic and developmental characterization of the Notch locus.
Action potentials and membrane ion channels in clonal anterior pituitary cells.
Authors: Adler M , Wong B S , Sabol S L , Busis N , Jackson M B , Weight F F
Published: May 2006

The electrophysiological properties of the mouse anterior pituitary cell line AtT-20/D16-16 were investigated with intracellular and patch-clamp techniques. Clonal AtT-20/D16-16 cells were found to be electrically excitable, with most cells exhibiting spontaneous bursting action potentials. The mean burst rates varied from 1.4 Hz at -55mV to 8.2 Hz at -25mV, showing an approximately linear frequency-current relationship in the low current range. The bursts consisted of one to several fast Na+ spikes superimposed on a slow pacemaker potential, followed by a Ca2+ spike and a Ca2+-sensitive afterhyperpolarization. Removal of either Na+ or Ca2+ from the bathing medium led to cessation of spontaneous activity and the appearance of arrhythmic firing patterns. Single channel recordings revealed the presence of Ca2+-dependent K+ channels with unitary conductances of approximately equal to 130 pS in physiological medium. These channels were activated by both intracellular Ca2+ and membrane depolarization. Addition of norepinephrine (10 microM) led to increases in burst frequency and beta-endorphin secretion mediated by activation of beta-adrenergic receptors. Our results, in conjunction with previous work, suggest that the Ca2+ that enters the cell during the burst may be involved in hormone secretion.
Regulation of the pro-opiomelanocortin mRNA levels in rat pituitary by dopaminergic compounds.
Authors: Chen C L , Dionne F T , Roberts J L
Published: May 2006

Dopamine-containing neurons directly innervate the intermediate lobe of the pituitary and dopaminergic compounds exert inhibitory effects on the secretion and the content of alpha-melanocyte-stimulating hormone and beta-endorphin in this tissue. In this study, we have investigated the effects of dopamine receptor agonists and antagonists on the level of pro-opiomelanocortin (POMC) mRNA in rat pituitary. RNAs isolated from neurointermediate pituitary (NIP) or anterior pituitary were spotted on nitrocellulose filters and the levels of POMC mRNA were quantified by hybridization to a POMC-specific complementary DNA probe coupled with autoradiography and densitometry. Administration of a dopamine receptor antagonist, haloperidol (2 mg/kg per day), to adult female rats resulted in a 3- to 5-fold increase in POMC mRNA level in the NIP. Treatment with the dopamine agonist 2-Br-alpha-ergocryptine (1 mg/kg per day) decreased significantly the content of POMC mRNA in the NIP. These drugs had no apparent effect on the POMC mRNA levels in the anterior pituitary. The effect of haloperidol and ergocryptine on POMC mRNA in the NIP is time- and dose-dependent. The elevation of POMC mRNA content in the NIP by haloperidol can be observed as early as 6 hr after treatment. These effects of dopaminergic compounds can also be demonstrated in adult male and ovariectomized female rats. The beta-endorphin content of the NIP, as measured by radioimmunoassay, and the de novo synthesis of POMC, as determined by radioactive amino acid labeling and two-dimensional gel electrophoresis analysis, also show negative regulation by dopaminergic compounds.
Decreased de novo synthesis of glomerular proteoglycans in diabetes: biochemical and autoradiographic evidence.
Authors: Kanwar Y S , Rosenzweig L J , Linker A , Jakubowski M L
Published: May 2006

The experimental model of streptozotocin-induced diabetes in rats was utilized to determine the biosynthetic and biochemical alterations in the proteoglycans of the glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) in diabetic nephropathy. Isolated kidneys from diabetic and control groups of animals were radiolabeled in an organ perfusion apparatus with [35S]sulfate of high specific activity (greater than 1,200 Ci/mmol; 1 Ci = 3.7 x 10(10) Bq) and processed for electron microscopic autoradiography, and the proteoglycans of the glomerular extracellular matrices were characterized. The results indicate that [35S]sulfate incorporation into glomerular extracellular matrices of diabetic animals was 30-40% less than that of the control group; however, no differences in the biochemical properties of the de novo synthesized proteoglycans from either group were observed. The relevance of the decreased de novo synthesis of sulfated proteoglycans of glomerular extracellular matrices is discussed in terms of increased glomerular permeability to plasma proteins and reduction in the glomerular filtration rate.
Structure and coding properties of a dominant Escherichia coli mutator gene, mutD.
Authors: Cox E C , Horner D L
Published: May 2006

The region of the Escherichia coli chromosome coding for the mutD gene was cloned, mutD5 function resides on a 1.2-kilobase fragment coding for a 28-kilodalton (kDa) protein. A deletion end-point analysis shows that the presence of the 28-kDa protein is required for mutD5 function and suggests that the mutD functional region has sufficient capacity to code for a second polypeptide of approximately 20 kDa. Plasmids carrying the mutD5 and mut+ alleles both produce the 28-kDa species. The product of mutD5 is dominant when carried by single and multicopy plasmids. The product of mut+ is dominant only on multicopy plasmids. Thus, mutD5 exhibits negative complementation. We suggest that the 28-kDa protein participates in a multimeric structure, perhaps at the replication fork.
Intrinsic randomness and intrinsic irreversibility in classical dynamical systems
Authors: Courbage M. , Prigogine I.
Published: May 2006

We continue our previous work on dynamic “intrinsically random” systems for which we can derive dissipative Markov processes through a one-to-one change of representation. For these systems, the unitary group of evolution can be transformed in this way into two distinct Markov processes leading to equilibrium for either
t
→ + ∞ or
t
→ - ∞. To lift the degeneracy, we first formulate the second principle as a selection rule that is meaningful in intrinsically random systems. For these systems, this excludes a set of unrealizable states. As a result of this exclusion, permitted initial conditions correspond to a set of states that is not invariant through velocity inversion. In this way, the time-reversal symmetry of dynamics is broken and these systems acquire a new feature we may call “intrinsic irreversibility.” The set of admitted initial conditions can be characterized by an entropy displaying the amount of information necessary for their preparation. The initial conditions selected by the second law correspond to a finite amount of information, while the initial conditions that are rejected correspond to an infinite amount of information and are therefore “impossible.” We believe that our formulation permits a microscopic formulation of the second law of thermodynamics for well-defined classes of dynamical systems.
Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase.
Authors: Walter P , Gangloff J , Bonnet J , Boulanger Y , Ebel J P , Fasiolo F
Published: May 2006

The sequence of a 5-kilobase DNA insert containing the structural gene for yeast cytoplasmic methionyl-tRNA synthetase has been determined and a unique open reading frame of 2,253 nucleotides encoding a polypeptide chain of 751 amino acids (Mr, 85,500) has been characterized. The data obtained on the purified enzyme (subunit size, amino acid composition, and COOH-terminal sequence) are consistent with the gene structure. The protein sequence deduced from the nucleotide sequence reveals no obvious internal repeats. This protein sequence shows a high degree of homology with that of Escherichia coli methionyl-tRNA synthetase within a region that forms the putative methionyl adenylate binding site. This strongly suggests that both proteins derive from a common ancestor.
Affinity purification of bacteriophage T4 proteins essential for DNA replication and genetic recombination.
Authors: Formosa T , Burke R L , Alberts B M
Published: May 2006

The bacteriophage T4 helix-destabilizing protein, the product of gene 32, has been immobilized on an agarose matrix and used for affinity chromatography of lysates of T4-infected Escherichia coli cells. At least 10 T4-encoded early proteins and 3 or 4 host proteins are specifically retained by this gene 32 protein column. Nine of the T4 proteins have been identified as being involved in either DNA replication or genetic recombination. Notably, the T4 DNA polymerase (gene 43 protein) and two major proteins in the recombination pathway (the products of genes uvsX and uvsY) are specifically bound. On a preparative scale, the column is useful for purification of the bound proteins.
Orientation of a human leukocyte interferon molecule on its cell surface receptor: carboxyl terminus remains accessible to a monoclonal antibody made against a synthetic interferon fragment.
Authors: Arnheiter H , Ohno M , Smith M , Gutte B , Zoon K C
Published: May 2006

An 125I-labeled monoclonal antibody made against a synthetic 56-residue fragment of human leukocyte interferon (IFN) alpha 1 recognizes human, Escherichia coli-derived IFN alpha A bound to the surface of Madin-Darby bovine kidney cells. A major fraction of the antibody recognizes IFN specifically bound to the cells, because the number of bound antibody molecules corresponds to the number of cell-bound IFN molecules (as measured with radiolabeled ligand) and because the fraction of the IFN unspecifically bound to the cells is less than 10% of the total bound IFN. A synthetic carboxyl-terminal 16-residue IFN peptide, though not inhibiting binding of IFN to cells, inhibits binding of antibody to IFN. A recombinant IFN alpha A molecule with a carboxyl-terminal 13-residue deletion, though still able to compete for binding of IFN to cells, is not recognized by the antibody. Scatchard plot analysis of the binding data revealed apparent dissociation constants of 6.0 x 10(-10) M for the antibody-IFN interaction and of 4.0 x 10(-11) M for the IFN-cell receptor interaction. The antibody inhibits the binding of IFN to cells only weakly and neutralizes the antiviral activity of the ligand only when in a large molar excess. We conclude that the carboxyl-terminal 10-16 residues that are predicted from the cloned IFN cDNAs and that are present in some natural IFNs are not involved in binding to cells but are antigenic and hence exposed on the molecules' surface. That the carboxyl terminus is not directly involved in binding to cells is consistent with the observation that some IFNs with carboxyl-terminal deletions are biologically active.
Intracellular and extracellular processing of human apolipoprotein A-I: secreted apolipoprotein A-I isoprotein 2 is a propeptide.
Authors: Zannis V I , Karathanasis S K , Keutmann H T , Goldberger G , Breslow J L
Published: May 2006

We have recently proposed that the major secreted isoprotein form of human apolipoprotein A-I (designated apo A-I2) is modified extracellularly to become the predominant apo A-I form seen in plasma (designated apo A-I4). In the current report we demonstrate that the primary translation product of human apo A-I (designated apo A-I2p) has a 24-amino-acid NH2-terminal extension with a sequence of Met-Lys-Ala-Ala-Val-Leu-Thr-Leu-Ala-Val-Leu-Phe- Leu-Thr-Gly-Ser-Gln-Ala-Arg-His-Phe-Trp-Gln-Gln. The first 18 amino acids of this NH2-terminal extension are cleaved intracellularly by the signal peptidase, resulting in the formation of apo A-I2, which is the secreted form of apo A-I. Sequence analysis of apo A-I2 confirmed that it contains a hexapeptide extension at its NH2 terminus compared to apo A-I4. This observation demonstrates that apo A-I2 is a propeptide and that the apo A-I2 to apo A-I4 conversion involves the removal of the NH2-terminal hexapeptide of apo A-I2 by a protease in plasma, lymph, or both. Our findings indicate that apo A-I is synthesized as a prepropeptide, which undergoes intracellular and extracellular proteolysis to attain the major plasma apo A-I4 isoprotein form.
Soybean nodulin genes: Analysis of cDNA clones reveals several major tissue-specific sequences in nitrogen-fixing root nodules
Authors: Fuller Forrest , Künstner Peter W. , Nguyen Truyen , Verma Desh Pal S.
Published: May 2006

Plant gene products that could play a role in the process of symbiotic nitrogen fixation in leguminous plants were detected by screening a cDNA library prepared from soybean nodule poly(A)
+
RNA. About 13% of the 5,700 clones screened contained sequences detectable with a root cDNA probe while about 2,100 clones (37% of the library) contained sequences that were detectable only with nodule cDNA. Five unique sequence species, accounting for more than half of the 2,100 nodule-specific clones, were identified by cross-hybridization experiments. The most abundant species, represented by 860 clones, encodes the well-characterized protein, leghemoglobin (Lb). The other four species, designated NodA, NodB, NodC, and NodD, are represented by 350, 55, 61, and 6 clones, respectively. Each of these four species was found to be encoded by the plant nuclear genome at low copy number. The transcripts corresponding to the nodule-specific clones represented 12-15% (Lb), 6% (NodA), and 0.5-1.1% (NodB, NodC, and NodD) mole fraction of nodule polysomal mRNAs but could not be detected in root polysomal RNA. Hybrid-selection of nodule mRNAs by representative clones and
in vitro
translation indicated that polypeptides of
M
r
44,000, 27,000, 24,000, and 100,000-120,000 are encoded by NodA, NodB, NodC, and NodD sequences, respectively. These polypeptides reacted with antiserum prepared against total soluble nodule proteins suggesting that the cloned sequences encode nodule-specific proteins, nodulins.
Escherichia coli extract-catalyzed recombination in switch regions of mouse immunoglobulin genes.
Authors: Kataoka T , Takeda S , Honjo T
Published: May 2006

We have shown that Escherichia coli extracts catalyze recombination between mouse immunoglobulin mu and alpha genes inserted separately in lambda phage vectors carrying different genetic markers. Most of the recombination sites in the inserts are located in the switch regions of the heavy chain genes, as previously found in the expressed genes of myeloma cells. The recombination took place at relatively high frequency (10(-4)). The recombinational system in E. coli or lambda phage seems to prefer short nucleotide sequences similar to those used in the class switch recombination.
Selective inhibition of synthesis of enzymes for de novo fatty acid biosynthesis by an endotoxin-induced mediator from exudate cells.
Authors: Pekala P H , Kawakami M , Angus C W , Lane M D , Cerami A
Published: May 2006

An endotoxin-induced mediator from exudate cells markedly suppresses the activities of the key enzymes for de novo fatty acid biosynthesis--acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.2] and fatty acid synthetase--in differentiating 3T3-L1 murine preadipocytes. The loss in activity, at least in part, appears to be due to a specific effect on the synthesis of the enzymes, as determined by a decreased incorporation of [35S]methionine into immunoadsorbable acetyl-CoA carboxylase and fatty acid synthetase when the cells were exposed to the mediator. During this exposure, the radiolabeling of proteins with [35S]methionine in a particulate fraction was decreased by nearly 50% with little change in the soluble protein fraction. Sodium dodecyl sulfate/polyacrylamide gel analysis of the labeled protein indicated no major disturbances of protein synthesis in general; however, the syntheses of specific proteins in both the soluble and particulate fractions were enhanced or depressed. The present study demonstrates that endotoxin promotes the release of a mediator from exudate cells that regulates key anabolic activities in adipose cells.
Extraneuronal saxitoxin binding sites in rabbit myelinated nerve.
Authors: Ritchie J M , Rang H P
Published: May 2006

The changes in binding of 3H-labeled saxitoxin (STX) to rabbit sciatic nerve during axonal regeneration (after nerve crush) and during axonal degeneration (after nerve section) were measured and compared with the corresponding changes in the sciatic nerves of other mammals (rat, guinea pig, and cat). In the rabbit and rat, regeneration after nerve crush is associated with a 2- to 4-fold increase in STX binding capacity, consistent with the known corresponding increase in the number of nodes of Ranvier in regenerating nerve. Furthermore, consistent with the disappearance of nodes that occurs with Wallerian degeneration, nerve section leads to a disappearance of all, or most, of the STX binding in rat and guinea pig nerve, similar to that previously found for cat nerve. However, in the rabbit, nerve section leads to a large maintained increase in STX binding. Intraneural injection of diphtheria toxin, which is known to damage Schwann cells and which causes an increase in STX binding in intact nerves, abolishes the binding in cut nerves. It is suggested that the increased binding in cut nerves is to nonneuronal sites situated on the surface membrane of the Schwann cells, which have greatly proliferated in number as axonal degeneration has progressed. The reason for the difference between rabbits and other species and the possibility that the binding sites of rabbit Schwann cells represent functional sodium channels remain to be investigated.
Template requirements for the initiation of adenovirus DNA replication.
Authors: Challberg M D , Rawlins D R
Published: May 2006

The first step in the replication of the adenovirus genome is the covalent attachment of the 5'-terminal nucleotide, dCMP, to the virus-encoded terminal protein precursor (pTP). This reaction can be observed in vitro and has been previously shown to be dependent upon either viral DNA or linearized plasmid DNA containing viral terminal sequences. Plasmids containing deletions or point mutations within the viral terminal sequence were constructed by site-directed mutagenesis. In the case of linear double-stranded templates, pTP-dCMP formation required sequences located within the first 18 base pairs of the viral genome. This sequence contains a segment of 10 base pairs that is conserved in all human adenovirus serotypes. Point mutations within the conserved segment greatly reduced the efficiency of initiation, while a point mutation at a nonconserved position within the first 18 base pairs had little effect. Single-stranded DNAs can also support pTP-dCMP formation in vitro. In contrast to the results obtained with duplex templates, experiments with a variety of single-stranded templates, including phage M13-adenovirus recombinants, denatured plasmids, and synthetic oligodeoxynucleotides, failed to reveal any requirements for specific nucleotide sequences. With single-stranded templates containing no dG residues, the specific deoxynucleoside triphosphate requirements of the initiation reaction were altered.
Construction and characterization of extrachromosomal probes for mutagenesis by carcinogens: site-specific incorporation of O6-methylguanine into viral and plasmid genomes.
Authors: Green C L , Loechler E L , Fowler K W , Essigmann J M
Published: May 2006

Organic synthesis and recombinant DNA technology were used to situate a putatively premutagenic DNA lesion, O6-methylguanine (O6MeGua), at a specific location in the genomes of two bacterial viruses, M13mp8 and phi X174, and of the bacterial plasmid pBR322. In each genome the first guanine residue in the unique recognition sequence for restriction endonuclease Pst I (5'-C-T-G-C-A-G-3') was replaced with O6MeGua. This was accomplished by ligating a chemically synthesized tetranucleotide, 5'-pTpm6GpCpA-3', into a circular, genome-length heteroduplex in which the four internal nucleotides of the Pst I recognition site had been removed from one strand of the DNA double helix (ligation yield, approximately equal to 50%). It was established that the tetranucleotide was located specifically at the Pst I site and that the presence of O6MeGua rendered the ligation product resistant to cleavage by Pst I. Sensitivity of the genome to Pst I was restored upon treatment with purified Escherichia coli O6MeGua DNA-methyltransferase, a repair protein that removes the methyl group from DNA-bound O6MeGua. This result, in combination with other data, showed unambiguously that O6MeGua was incorporated with high yield into the Pst I recognition sequence.
Cytosolic Ca2+-dependent neutral proteinases from rabbit liver: activation of the proenzymes by Ca2+ and substrate.
Authors: Pontremoli S , Melloni E , Salamino F , Sparatore B , Michetti M , Horecker B L
Published: May 2006

Two neutral Ca2+-dependent proteinases, differing in molecular size, have been isolated from rabbit liver. Both are recovered as inactive proenzymes that can be converted to the active forms by high (0.1-1.0 mM) concentrations of Ca2+ in the absence of substrate or, in the presence of a protein substrate, by low (1-5 microM) concentrations of Ca2+. The activated proteinases required only 1-5 microM Ca2+ for maximal activity. Substrates hydrolyzed were denatured globin, globin, casein, and to a lesser extent, several extracellular proteins; no digestion was observed with several intracellular cytosolic enzymes tested. Only those proteins that served as substrates were capable of promoting conversion of the proenzymes to the active low-Ca2+-requiring proteinases.
Two populations of prelysosomal structures transporting asialoglycoproteins in rat liver.
Authors: DeBanne M T , Bolyos M , Gauldie J , Regoeczi E
Published: May 2006

Analyses by differential centrifugation of liver homogenates from rats that had received 131I-labeled asialoorosomucoid showed that, 1 min after injection, most of the intracellular ligand was associated with a particle that did not sediment at 2.5 X 10(5) g-min. However, by 10 min, undigested ligand became associated with a particle that did sediment at this speed. On analytical ultracentrifugation in sucrose gradients, both kinds of particles exhibited low densities (1.11-1.13 g X ml-1). In contrast to asialoorosomucoid, 125I-labeled asialotransferrin type 3, under noncatabolic conditions, remained largely confined to the nonsedimenting particle regardless of the duration of the study. Induction of catabolism of asialotransferrin was accompanied by the appearance of the ligand in the sedimentable particle. The nonsedimentable particle was separated by immunoadsorption from other subcellular particles contained in the low-density subcellular fraction. The adsorbant , prepared by immobilizing purified antibodies to the Gal/GalN-specific lectin from rat liver on coated polyacrylamide beads, removed 75-80% of the asialoorosomucoid and transferrin binding capacities present, together with a similar portion of the radioligands tested (asialoorosomucoid, asialotransferrin type 3, and human diferric transferrin). Significantly, the sialytransferase activity remained unadsorbed. From these findings, the nonsedimentable particle appears to be involved in the transport of ligands destined to such diverse fates as exocytosis or lysosomal degradation. The sedimentable particle, on the other hand, seems to represent a link between the first particle and the lysosome.
Rapid internalization of the transferrin receptor in K562 cells is triggered by ligand binding or treatment with a phorbol ester.
Authors: Klausner R D , Harford J , van Renswoude J
Published: May 2006

Treatment of human K562 cells with 4 beta-phorbol 12-myristate 13-acetate (PMA) resulted in an approximately 50% reduction in cell surface transferrin receptors within 30-45 min as judged by binding of both ligand and anti-receptor antibody. The affinity of the remaining surface receptors for diferric transferrin appeared to be unaltered. The time-dependent loss in transferrin receptors was also dependent upon PMA concentration, with a half-maximal effect observed at approximately 1 nM. The kinetic parameters for the binding, internalization, intracellular residency, and recycling of 125I-labeled transferrin were unchanged by PMA treatment, as were the rate and extent of internalization of anti-receptor antibody. Moreover, despite the decrease in surface receptors, uptake of 59Fe from transferrin proceeded at a rate comparable to that seen in untreated cells. Accounting for this observation was the fact that ligand induced a reduction in surface receptors in untreated but not PMA-treated cells. Quantitative immunoprecipitation of transferrin receptors from surface-iodinated K562 cells revealed that little receptor internalization occurred in untreated cells in the absence of ligand, but internalization of ligand-occupied receptors in these cells was readily detected. In contrast, PMA treatment resulted in the rapid internalization of surface receptors irrespective of occupancy. Thus, binding of ligand appeared to trigger the internalization of receptors that were relatively static in their unoccupied state, and a signal for receptor internalization was also provided by PMA treatment. The possibility that this signal involves phosphorylation of the transferrin receptor is discussed.
Expression of the transformation-sensitive protein "cyclin" in normal human epidermal basal cells and simian virus 40-transformed keratinocytes.
Authors: Celis J E , Fey S J , Larsen P M , Celis A
Published: May 2006

A cell population highly enriched in human epidermal basal cells has been obtained and characterized by using antibodies specific for various cell types in the epidermis. Quantitative two-dimensional gel electrophoretic analysis (isoelectric focusing) of [35S]methionine-labeled polypeptides from basal cells and simian virus 40-transformed keratinocytes showed that the basal cells synthesize very low amounts (less than 0.02% of the total protein) of the nuclear, transformation-sensitive protein cyclin as compared to the transformed cells, which synthesize this protein constitutively (0.15% of the total protein). Very low levels of cyclin were observed in total human epidermis, and preliminary studies of two basaliomas have shown a significant synthesis of this protein in these tumors. Immunofluorescence studies using antibodies to proliferating cell nuclear antigen that immunoprecipitate cyclin confirmed the above observations at least in the case of the cultured cells. Taken together, these results support the notion that cyclin may be a central component of the pathway(s) that controls cell proliferation.
Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.
Authors: Helseth D L , Veis A
Published: May 2006

The proteolytic removal of the extension COOH-terminal propeptide from procollagen has been examined in vitro. A crude enzyme activity was identified in a whole-chicken-embryo extract that acted at acid pH and appeared to be similar to one identified previously [Davidson, J. M., McEneany , L. S. G. & Bornstein , P. (1979) Eur. J. Biochem. 100, 551-558]. This activity was inhibitable by pepstatin but not by leupeptin, suggesting that it might be cathepsin D. Cathepsin D was purified 907-fold from chicken livers by affinity chromatography on pepstatin-aminohexyl-Sepharose 4B and was found to remove the COOH propeptides from procollagen. At pH 6.0, the site of cleavage appeared to shift from the COOH telopeptide to the COOH telopeptide/propeptide junction, based upon the difference in electrophoretic migration of the cleavage products, although determining the actual cleavage site will require end-group analysis. A model for the involvement of cathepsin D in the in vivo processing of procollagen is presented.
Lampbrush loop-specific protein of Drosophila hydei.
Authors: Hulsebos T J , Hackstein J H , Hennig W
Published: May 2006

By immunofluorescence techniques and protein blotting experiments we have shown that an antiserum specifically reacts with a Mr 80,000 protein (the "Ps protein") in the lampbrush loop " pseudonucleolus " in spermatocyte nuclei of Drosophila hydei. Comparative studies of X/Y and X/0 testes indicate that the gene encoding the Ps protein is not located on the Y chromosome but on an autosome or the X chromosome. The Ps protein is tissue specific. It is likely to be a rather conserved protein since the antigenic determinant recognized by the antiserum could be detected in the spermatocyte nuclei of a number of other Drosophila species. For those species with prominent Y chromosomal lampbrush loops, it could be shown that the cross-reaction is, as in D. hydei, associated with a specific Y chromosomal loop.
T-cell-mediated cytotoxicity against autologous malignant melanoma: analysis with interleukin 2-dependent T-cell cultures.
Authors: Knuth A , Danowski B , Oettgen H F , Old L J
Published: May 2006

The cytotoxic reactivity of lymphocytes for autologous melanoma cells was studied in a group of 13 melanoma patients. No cytotoxicity was observed with lymphocytes freshly isolated from peripheral blood or with lymphocytes cocultured for 7 days with autologous melanoma cells. Growth of lymphocytes previously sensitized with autologous melanoma in vitro in interleukin 2 (IL-2)-containing medium, however, resulted in cytotoxic reactivity for autologous melanoma in 7/13 patients. The reactivity of IL-2-dependent lymphocytes for autologous melanoma was particularly striking in one patient (A.V.) who has had an unexpectedly favorable clinical course and, because of their consistently high reactivity, AV lymphocytes were selected for detailed specificity analysis. After 2-3 weeks in culture in IL-2-containing medium, AV lymphocytes were cytolytic for autologous melanoma cells but not autologous Epstein-Barr virus-transformed B cells, autologous fibroblasts, or allogeneic tumor targets. Specificity of autologous melanoma reactivity was confirmed by competitive inhibition assays. The IL-2-dependent AV lymphocytes formed rosettes with sheep erythrocytes and expressed OKT 3 and Ia antigens. After longer periods of culture, AV lymphocytes were found to react with a wider range of target cells, and repeated attempts to isolate cultures with restricted reactivity to autologous melanoma by resensitization with autologous melanoma and limiting-dilution techniques were unsuccessful. The restricted reactivity of early cultures could be preserved, however, in frozen storage, but shifted again toward broader reactivity after several weeks in culture. The recognition of cytotoxic T cells with initial restricted reactivity for autologous melanoma suggests reinvestigation of the question of specific cellular immunity to human cancer.
Intramural mechanism of esophageal peristalsis: roles of cholinergic and noncholinergic nerves.
Authors: Crist J , Gidda J S , Goyal R K
Published: May 2006

We examined the role of peripheral cholinergic and noncholinergic mechanisms in esophageal peristalsis. Intramural nerve elements in rings of circular muscle from six different levels of the opossum esophagus were stimulated transmurally so as to cause neurally mediated muscle contractions. Stimulus frequency was varied from 2 to 40 Hz. An increase in stimulus frequency caused an increase in latencies of contractions in rings from distal esophageal sites and a decrease in latencies in rings from proximal sites. This resulted in a marked slowing of the calculated peristaltic speed. Increasing stimulus frequency also caused an increase in duration and amplitude of contractions. These effects were reversed by atropine (0.1 microM), suggesting that higher stimulus frequencies recruited more cholinergic nerves. In the presence of atropine, increasing the stimulus frequency caused an increase in latencies of contraction at all sites, suggesting that increasing stimulation frequency applied to noncholinergic nerves causes an increase in latencies of contraction at all sites. The results of this study indicate that both noncholinergic and cholinergic nerves play a role in the peripheral mechanism of esophageal peristalsis. Cholinergic nerve stimulation reduces the latency and enhances the amplitude and duration of contractions seen with noncholinergic nerve stimulation alone. The influence of cholinergic innervation is most prominent proximally and decreases distally along the smooth muscle portion of the esophagus. This peripherally located gradient of cholinergic innervation plays an important role in determining the speed and amplitude of esophageal peristalsis.
Neophobia in the foraging-site selection of a neotropical migrant bird: An experimental study
Authors: Greenberg Russell
Published: May 2006

I hand-raised chestnut-sided warblers (
Dendroica pensylvanica
) in a room with eight experimental microhabitats; the microhabitats were removed after 6 weeks. I then measured the response of the warblers to the eight “natal” and eight “novel” microhabitats in two experiments conducted 2 and 4 months after removal. Chestnut-sided warblers responded with decreased feeding latency (neophobia) and a greater preference for foraging at the natal microhabitats. I suggest that an ontogenetic increase in neophobia restricts chestnut-sided warblers to foraging at microhabitats most similar to those experienced as juveniles.
The [poky] mutant of Neurospora contains a 4-base-pair deletion at the 5' end of the mitochondrial small rRNA.
Authors: Akins R A , Lambowitz A M
Published: May 2006

[ poky ] and other group I extranuclear mutants of Neurospora crassa are characterized by gross deficiencies of mitochondrial small ribosomal subunits and small (19S) rRNA. Blot-hybridization and other experiments suggest that the 19S rRNA (2.0 kilobases) is synthesized via precursors that contain 5'-end extensions. The ratio of precursors to mature rRNA is higher in [ poky ] and other group I mutants than in wild type, indicating that the defect involves impaired processing and/or instability of 19S rRNA. [ poky ] and other group I mutants contain a 4-base-pair deletion in the coding sequence for the mitochondrial small rRNA, just downstream from what would normally be the 5' end of the rRNA. This deletion apparently results in synthesis of aberrant 19S rRNAs that are missing 38-45 nucleotides from their 5' ends. We propose that the 4-base-pair deletion is the primary defect in [ poky ] and other group I extranuclear mutants.
Isolation of the yeast INO1 gene: located on an autonomously replicating plasmid, the gene is fully regulated.
Authors: Klig L S , Henry S A
Published: May 2006

The Saccharomyces cerevisiae gene, INO1 , encoding the highly regulated enzyme, myo-inositol-1-phosphate synthase [1L-myo-inositol-1-phosphate lyase (isomerizing), EC 5.5.1.4], was isolated by genetic complementation. The cloned sequence was shown to complement two independent IN01 alleles ( ino1 -5 and ino1 -13). One of these mutants ( ino1 -5) fails to make any material that is crossreactive with antibody to the wild-type inositol-1-phosphate synthase. The cloned DNA restored not only inositol prototrophy to this mutant but also its ability to make material crossreactive with anti-inositol-1-phosphate synthase antibody. The sequence on an integrative plasmid was also shown to recombine with the INO1 locus, thereby confirming its genetic identity. The DNA was subcloned and used for Southern blot analysis, revealing that the cloned DNA (5.4 kilobases long) represents a unique sequence in the yeast genome. Inositol-1-phosphate synthase was fully regulated when its gene was located extrachromosomally on the autonomously replicating plasmid. In cells ( ino1 -) containing the cloned INO1 gene on a high-copy-number plasmid, the enzyme was fully repressible. Furthermore, the gene product was not expressed when the plasmid was transferred into a strain containing an ino4 mutation, which also prevents expression of chromosomal copies of INO1 . These results establish that the intact structural gene and associated regulatory components have been isolated and that positioning of the gene in its normal chromosomal location is not required for full regulation of inositol-1-phosphate synthase.
Removal of UV light-induced pyrimidine-pyrimidone(6-4) products from Escherichia coli DNA requires the uvrA, uvrB, and urvC gene products.
Authors: Franklin W A , Haseltine W A
Published: May 2006

Ultraviolet light induces the formation of cyclobutane pyrimidine dimers and pyrimidine- pyrimidone (6-4) photoproducts in cellular DNA. In Escherichia coli, the uvrA, uvrB, and uvrC genes are necessary for excision of cyclobutane dimers. To determine whether the uvrABC gene products are required for (6-4) product removal from DNA, a sensitive HPLC assay was developed that allows the separation and quantitation of both types of photoproducts. Both the T T cyclobutane dimer and the T-C(6-4) product were completely removed from the DNA after 2 hr of repair in a wild-type strain. Both products were also removed in the wild-type strain in the presence of chloramphenicol, an inhibitor of protein synthesis. No decrease in the amount of either T T cyclobutane dimer or of T-C(6-4) products was observed in strains that were deficient in any one of the three uvr gene products under similar conditions. We conclude the uvrABC enzyme complex is required for excision of (6-4) photoproducts from E. coli DNA.
Numbers of specific types of neuron in layer IVab of cat striate cortex.
Authors: Solnick B , Davis T L , Sterling P
Published: May 2006

Layer IVab of the visual cortex (area 17) of the cat contains about 51,400 neurons per mm3, including about 400-1200 per mm3 of each of three categories of neuron believed from previous work to represent discrete types. Each type forms about 0.5-1.5% of all the IVab neurons, which suggests that the total number of types in this layer might be much greater than previously supposed, perhaps as many as 50 or more. From their densities and estimates of their dendritic fields, we calculate that each type completely "covers" layer IVab in the tangential plane but only by a small factor (1.3-4.2).
Nonenzymic ADP-ribosylation of specific mitochondrial polypeptides.
Authors: Hilz H , Koch R , Fanick W , Klapproth K , Adamietz P
Published: May 2006

The apparent NAD:protein ADP-ribosyl transferase activity of mitochondria and submitochondrial particles from beef heart and rat liver is simulated by a reaction sequence that consists of an enzymic hydrolysis of NAD to ADP-ribose (ADP-Rib) by NAD glycohydrolase(s) and a nonenzymic ADP-ribosylation of acceptor proteins by the free ADP-Rib formed. The nonenzymic ADP-ribosylation of mitochondrial proteins showed two pH optima and exhibited the same remarkable selectivity as the reaction with NAD. The predominant acceptor in beef heart mitochondria was a 30-kDa protein, whereas in mitochondrial extracts of rat liver a 50-55 kDa polypeptide served as an acceptor. No authentic ADP-Rib transferase activity could be detected even when free ADP-Rib was trapped by NH2OH. Once formed, the mitochondrial ADP-Rib conjugates were resistant to hydroxylamine. NH2OH-resistant mono(ADP-Rib)-protein conjugates as found in most cells may also be products of nonenzymic ADP-ribosylation. In mouse tissues, their amounts relate to protein and NAD contents, and they increase specifically and reversibly in the hypothyroid status. Furthermore, intact rat liver mitochondria contain a mono(ADP-Rib)-polypeptide (50-55 kDa) that appeared to be identical with the polypeptide reacting with ADP-Rib in vitro.
Isolation of human DNA sequences that bind to nuclear factor I, a host protein involved in adenovirus DNA replication.
Authors: Gronostajski R M , Nagata K , Hurwitz J
Published: May 2006

Nuclear factor I is a 47,000-dalton protein isolated from human HeLa cells that is required for the in vitro replication of adenovirus DNA. This protein was previously shown to bind specifically to nucleotides 17-48 of the left-hand terminus of cloned adenovirus serotype 5 DNA. An in vitro assay for DNA sequences that compete with adenovirus DNA for the binding of nuclear factor I has been developed. With this assay, we have shown specific binding of human DNA sequences to nuclear factor I. Using the DNA binding activity of nuclear factor I, we have isolated and cloned segments of human DNA that bind tightly to this protein. One nuclear factor I binding site is present about every 100,000 base pairs in the HeLa cell genome. The binding of these DNA molecules to nuclear factor I resembles the binding of cloned adenovirus DNA to the protein and is resistant to high ionic strength. The isolation of DNA sequences from HeLa cells that bind specifically to nuclear factor I suggests that this protein interacts with host DNA in vivo.
Membrane protein damage and repair: Selective loss of a quinone-protein function in chloroplast membranes
Authors: Kyle D. J. , Ohad I. , Arntzen C. J.
Published: May 2006

A loss of electron transport capacity in chloroplast membranes was induced by high-light intensities (photoinhibition). The primary site of inhibition was at the reducing side of photosystem II (PSII) with little damage to the oxidizing side or to the reaction center core of PSII. Addition of herbicides (atrazine or diuron) partially protected the membrane from photoinhibition; these compounds displace the bound plastoquinone (designated as Q
B
), which functions as the secondary electron acceptor on the reducing side of PSII. Loss of function of the 32-kilodalton Q
B
apoprotein was demonstrated by a loss of binding sites for [
14
C]atrazine. We suggest that quinone anions, which may interact with molecular oxygen to produce an oxygen radical, selectively damage the apoprotein of the secondary acceptor of PSII, thus rendering it inactive and thereby blocking photosynthetic electron flow under conditions of high photon flux densities.
The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: macrophages of endocrine organs.
Authors: Hume D A , Halpin D , Charlton H , Gordon S
Published: May 2006

Macrophages of endocrine organs have been identified by immunohistochemical localization of the macrophage-specific antigen F4/80. F4/80+ cells line vascular sinuses and capillaries in anterior and posterior pituitary, adrenal cortex, corpus luteum, parathyroid, pineal gland, and islets of Langerhans. In testis approximately 20% of interstitial cells are F4/80+. F4/80+ cells infiltrate corpus luteum in increased numbers during luteolysis.
Fibrinopeptide A binds Gly-Pro-Arg-Pro.
Authors: Root-Bernstein R S , Westall F C
Published: May 2006

The tetrapeptide Gly-Pro-Arg-Pro inhibits fibrinogen aggregation, probably by binding to the same sites used during initiation of fibrin formation. The Gly-Pro-Arg-Pro binding sites have not yet been identified. However, their possible sequence and locations have been predicted on the basis of the amino acid pairing hypothesis. One of these predicted sites is on fibrinopeptide A. We report here that nuclear magnetic resonance studies indicate that Gly-Pro-Arg-Pro binds to fibrinopeptide A with a binding constant, K, of ca. 10(4) per mol. We also report results of 19 related peptide combinations used as controls.
Recombinant human gamma-interferon induces human monocyte polykaryon formation.
Authors: Weinberg J B , Hobbs M M , Misukonis M A
Published: May 2006

Monocyte or macrophage polykaryons (MP) are seen in different tissues in various inflammatory states and in normal bone (osteoclasts). The factors controlling the formation and the function of MP are not completely understood. This study was designed to evaluate the effects of the lymphokine gamma-interferon (IFN-gamma) on human monocyte function in vitro. Purified recombinant IFN-gamma [20-200 units/ml (0.1-1.0 nM)] caused the appearance of MP in cultures of normal human monocytes cultured in 10% unheated autologous serum. The MP were noted by as early as 36 hr of culture with fusion indices of 40%-60% and up to 160 nuclei per cell. The effect was seen with both recombinant IFN-gamma and natural IFN-gamma produced by Staphylococcal enterotoxin A-stimulated lymphocytes, but IFN-alpha (leukocyte-derived and recombinant) and IFN-beta did not induce MP formation. The activity of the IFN-gamma was destroyed by heating at 56 degrees C for 4 hr, incubating at pH 2 for 3 hr, or incubating with antibody against IFN-gamma. Populations of monocytes incubated 3 days with 100 units of IFN-gamma per ml (0.5 nM) had enhanced capacity to produce H2O2 in response to phorbol 12-myristate 13-acetate and increased content of acid phosphatase and plasminogen activator. As determined by autoradiography, the MP did not incorporate [3H]dThd into their nuclei. Thus, the IFN-gamma appears to induce MP formation by a process of monocyte fusion, and to "activate" monocytes, as judged by various parameters.
The spectral domain of multivariate harmonizable processes
Authors: Rao M. M.
Published: May 2006

The spectral or frequency domain of a multivariate weakly harmonizable process is a vector space of vector functions that are integrable relative to a positive definite matrix bimeasure. It has a norm derived from a Gramian inner product. The completeness property of this space, which is needed for many applications, has been open for some years. An affirmative solution and an application to optimal least-squares estimation of a weakly harmonizable signal are presented here.
Rapid induction of a specific nuclear mRNA precursor by thyroid hormone.
Authors: Narayan P , Liaw C W , Towle H C
Published: May 2006

Administration of thyroid hormone to the thyroidectomized rat results in a rapid and dramatic increase in the relative amount of hepatic mRNA coding for spot 14--a translational product with an approximate Mr of 17,500 and isoelectric point of 4.9. We have now isolated a cDNA clone containing sequences homologous to this thyroid hormone-responsive mRNA. Two distinct mRNA species that differed by 200 nucleotides in length were found to be capable of hybridizing to the cDNA probe. Both mRNA species were proportionally elevated in relative concentration in rats with increasing plasma levels of thyroid hormone. The earliest change in the levels of mature mRNA occurred at 20 minutes following thyroid hormone treatment of the thyroidectomized rat. Analysis of nuclear RNA revealed a single higher molecular weight species that was homologous to spot 14 mRNA. An increase in the nuclear level of this putative precursor occurred by 10 minutes following thyroid hormone administration, a time preceding the earliest change in mature mRNA. Thus, thyroid hormone appears to act at least in part at a nuclear level in altering the cellular concentration of this mRNA species. The rapidity of this change suggests that it may reflect a direct response to the binding of thyroid hormone to its nuclear receptor.