bluuebunny/crossref_metadata_2025 huggingface dataset:
The association of NADPH with the guanine nucleotide exchange factor from rabbit reticulocytes: a role of pyridine dinucleotides in eukaryotic polypeptide chain initiation.
Authors: Dholakia J N , Mueser T C , Woodley C L , Parkhurst L J , Wahba A J
Published: May 2006

The guanine nucleotide exchange factor (GEF) was purified to apparent homogeneity from postribosomal supernatants of rabbit reticulocytes by chromatography on DEAE-cellulose and phosphocellulose, fractionation by glycerol gradients, and chromatography on Mono S and Mono Q (Pharmacia). At the Mono S step GEF is isolated as a complex with the eukaryotic polypeptide chain initiation factor 2 (eIF-2) and is separated from this factor by column chromatography on Mono Q. An emission spectrum characteristic of a reduced pyridine dinucleotide was observed when GEF was subjected to fluorescence analysis. By both coupled enzymatic analysis and chromatography on reverse-phase or Mono Q columns, the bound dinucleotide associated with GEF was determined to be NADPH. The GEF-catalyzed exchange of eIF-2-bound GDP for GTP was markedly inhibited by NAD+ and NADP+. This inhibition was not observed in the presence of equimolar concentrations of NADPH. Similarly, the stimulation of ternary complex (eIF-2 X GTP X Met-tRNAf) formation by GEF in the presence of 1 mM Mg2+ was abolished in the presence of oxidized pyridine dinucleotide. These results demonstrate that pyridine dinucleotides may be directly involved in the regulation of polypeptide chain initiation by acting as allosteric regulators of GEF activity.
Mass extinctions: Sensitivity of marine larval types
Authors: Valentine James W. , Jablonski David
Published: May 2006

Developmental types of marine invertebrates may be divided into planktotrophs, which feed on suspended food items, and nonplanktotrophs, which do not feed but are supplied with nutrients (yolk) parentally; these may represent high mortality-fecundity and low mortality-fecundity strategies, respectively. Most versions of the bolide impact hypothesis of mass extinction propose occlusion of the sun by dust or smoke and severance of planktonic food chains for months or a few years, and this should select preferentially against planktotrophs. Yet among fossil prosobranch gastropods, planktotrophs survived the end-Cretaceous extinction equally as well as nonplanktotrophs. Indirect evidence suggests that end-Permian extinctions may have selected against planktotrophs but that the effect was prolonged over millions of years.
Cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients.
Authors: Wiley C A , Schrier R D , Nelson J A , Lampert P W , Oldstone M B
Published: May 2006

Dysfunction of the central nervous system (CNS) is a prominent feature of the acquired immune deficiency syndrome (AIDS). Many of these patients have a subacute encephalitis consistent with a viral infection of the CNS. We studied the brains of 12 AIDS patients using in situ hybridization to identify human immunodeficiency virus [HIV, referred to by others as human T-cell lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), AIDS-associated retrovirus (ARV)] nucleic acid sequences and immunocytochemistry to identify viral and cellular proteins. Nine patients had significant HIV infection in the CNS. In all examined brains, the white matter was more severely involved than the grey matter. In most cases the infection was restricted to capillary endothelial cells, mononuclear inflammatory cells, and giant cells. In a single case with severe CNS involvement, a low-level infection was seen in some astrocytes and neurons. These results suggest that CNS dysfunction is due to indirect effects rather than neuronal or glial infection.
Intranuclear appearance of the phosphorylated form of cytoskeleton-associated 350-kDa proteins in U1-ribonucleoprotein regions after growth stimulation of fibroblasts.
Authors: Sato C , Nishizawa K , Nakayama T , Nose K , Takasaki Y , Hirose S , Nakamura H
Published: May 2006

Cytoskeleton-associated 350-kDa and 80-kDa polypeptides, which were immunoprecipitated with polyclonal antibody against microtubule-associated protein 1 (MAP-1), were rapidly phosphorylated on mitogenic stimulation of quiescent fibroblasts with serum or growth factors. The enhanced phosphorylation was evident within 5 min and reached a maximum 2 hr after the stimulation. Phosphorylated MAP-1 analogues were first detected in the cytoplasm around the microtubule-organizing center and then in the nucleus by immunofluorescent staining with a monoclonal antibody that recognized the phosphorylated form of MAP-1. The monoclonal antibody reacted with the 350-kDa protein in immunoblot analysis and immunostained intranuclear speckles; both immunoreactions were abolished by treatment with alkaline or acid phosphatase. The nuclear speckles stained by the monoclonal antibody were also stained by anti-U1 small nuclear ribonucleoprotein antibodies on double immunofluorescence, suggesting that the stained regions are sites of maturation of messenger RNA. These results support the idea that part of the cytoskeleton-associated 350-kDa protein is phosphorylated and transferred to the nuclear region of mRNA modification as a common early process after growth stimulation.
Long-term expression of isomyosins and myoendocrine functions in ectopic grafts of atrial tissue.
Authors: Jockusch H , Füchtbauer E M , Füchtbauer A , Leger J J , Leger J , Maldonado C A , Forssmann W G
Published: May 2006

Tissue fragments of newborn rat atria were transplanted under the dorsal skin or into the bed of the anterior tibial muscle of nude mice. After 5-11 weeks, the grafts, which had reorganized into beating atrium-like structures, were analyzed and compared to ventricular tissue transplanted the same way. As revealed by monoclonal antibodies against alpha- and beta-type myosin heavy chains, atrial grafts retained a typical pattern of myosin expression distinct from that of ventricular grafts. The majority of ectopic atrial myocytes contained specific atrial granules in which cardiodilatin-immunoreactive material has been localized. Specific granules and cardiodilatin immunoreactivity were not found in myocytes of ventricular grafts. We conclude that the long-term maintenance of isomyosin expression and of the myoendocrine function of atrial tissue is largely independent of the anatomical environment.
Vasoactive intestinal peptide induces the synthesis of the cholesterol side-chain cleavage enzyme complex in cultured rat ovarian granulosa cells.
Authors: Trzeciak W H , Ahmed C E , Simpson E R , Ojeda S R
Published: May 2006

Vasoactive intestinal peptide (VIP) has been identified in ovarian nerves and stimulates steroid secretion from immature ovaries. To gain insight into its mechanism of action, the effect of VIP on the synthesis of the cholesterol side-chain cleavage enzyme complex was studied in ovarian granulosa cells from immature estrogen-primed rats. The cells were cultured for 48 hr in serum-free medium; the proteins were labeled with [35S]methionine; and the synthesis of cytochrome P-450, iron-sulfur protein, and NADPH:iron-sulfur protein reductase was evaluated by electrophoretic analysis after immunoisolation with polyclonal antibodies directed against the bovine adrenal enzymes. VIP at concentrations ranging from 0.001 to 1 microM stimulated 3- to 5-fold the synthesis of cytochrome P-450 and iron-sulfur protein. Peptide NH2-terminal histidine, COOH-terminal isoleucine, which has greater than 50% sequence homology of VIP, stimulated the synthesis of both proteins at approximately 50% of VIP effectiveness. Secretin, another member of the glucagon-secretin family of peptides, which has only 30% sequence homology to VIP, was without effect. Similar results were observed with the NADPH:iron-sulfur protein reductase. VIP-induced synthesis of the cholesterol side-chain cleavage enzyme complex was accompanied by a dose-related increase in cAMP accumulation and progestin formation. It is concluded that VIP regulates the synthesis of the ovarian cholesterol side-chain cleavage enzyme complex, which catalyzes the rate-limiting reaction in progesterone biosynthesis, and that the VIP effect is at least partially mediated through cAMP. It is suggested that a stimulatory action of VIP on the synthesis of ovarian progesterone may contribute to regulating the functional development of the ovary.
Yields of Soviet underground nuclear explosions at Novaya Zemlya, 1964-1976, from seismic body and surface waves
Authors: Sykes Lynn R. , Wiggins Graham C.
Published: May 2006

Surface and body wave magnitudes are determined for 15 U.S.S.R. underground nuclear weapons tests conducted at Novaya Zemlya between 1964 and 1976 and are used to estimate yields. These events include the largest underground explosions detonated by the Soviet Union. A histogram of body wave magnitude (m
b
) values indicates a clustering of explosions at a few specific yields. The most pronounced cluster consists of six explosions of yield near 500 kilotons. Several of these seem to be tests of warheads for major strategic systems that became operational in the late 1970s. The largest Soviet underground explosion is estimated to have a yield of 3500 ± 600 kilotons, somewhat smaller than the yield of the largest U.S. underground test. A preliminary estimation of the significance of tectonic release is made by measuring the amplitude of Love waves. The bias in m
b
for Novaya Zemlya relative to the Nevada test site is about 0.35, nearly identical to that of the eastern Kazakhstan test site relative to Nevada.
Molecular cloning of the neu gene: absence of gross structural alteration in oncogenic alleles.
Authors: Hung M C , Schechter A L , Chevray P Y , Stern D F , Weinberg R A
Published: May 2006

The neu gene is distantly related to the erbB gene and encodes a cell surface protein that appears to function as a growth factor receptor. To study the mechanisms that caused the conversion of the normal neu gene to an oncogenic allele, we have isolated molecular clones of the neu oncogene as well as a clone of the corresponding protooncogene. The transforming neu oncogene and the proto-neu gene clones exhibit identical restriction enzyme patterns. Amplification of the proto-neu gene in NIH 3T3 cells by means of cotransfection with a dihydrofolate reductase gene resulted in methotrexate-resistant colonies that produce high levels of normal neu-encoded p185 protein. In contrast to cells carrying low levels of the oncogene-encoded protein, these cells appeared normal. The results suggest that the lesion that led to activation of the neu gene is a minor change in DNA sequence and is apparently located in the protein-encoding region of the gene.
Epidermal growth factor-nonresponsive 3T3 variants do not contain epidermal growth factor receptor-related antigens or mRNA.
Authors: Schneider C A , Lim R W , Terwilliger E , Herschman H R
Published: May 2006

We have previously isolated three independent variants of Swiss 3T3 cells that are unable to generate a mitogenic response to epidermal growth factor (EGF). Each of the variants is unable to bind 125I-labeled EGF; each lacks a functional EGF receptor. We used an antiserum to murine EGF receptor to look for an EGF-receptor gene product in wild-type 3T3 cells and in the three EGF-nonresponsive variants. No cross-reactive material could be detected in any of the three variants, either in 125I-labeled cell extracts or in [35S]methionine metabolically labeled cells. 3T3 cells contained mRNA molecules homologous to a cDNA probe for the human EGF-receptor coding region. In contrast, no homologous RNA could be detected in any of the three variants. Analysis of genomic Southern blots of the DNA from 3T3 cells and the three EGF-nonresponsive variants indicated sequences from the EGF-receptor gene are present in the DNA of all four cell lines. These EGF-nonresponsive lines, which demonstrate proliferative responses to a variety of mitogens, will be ideal recipients for structure-function studies of the EGF receptor by transfection of the cloned gene.
The tumor promoter phorbol 12-myristate 13-acetate induces a program of altered gene expression similar to that induced by platelet-derived growth factor and transforming oncogenes.
Authors: Rabin M S , Doherty P J , Gottesman M M
Published: May 2006

Treatment of mouse NIH 3T3 cells with the phorbol ester tumor promoter, phorbol 12-myristate 13-acetate, results in altered transcription of several genes as measured in nuclear run-off experiments. The first set of genes, whose altered transcription occurs rapidly in the absence of protein synthesis, is typified by induction of c-myc and c-fos and decreased transcription of alpha 2 type I procollagen. This work demonstrates the existence of a second class of genes whose rapidly increased transcription requires prior protein synthesis, which is represented by the gene encoding a secreted lysosomal protein, MEP. Similar induction of MEP RNA is seen after treatment with platelet-derived growth factor or transformation with Kirsten sarcoma virus.
RNA polymerase II transcription terminates at a specific DNA sequence in a HeLa cell-free reaction.
Authors: Baek K H , Sato K , Ito R , Agarwal K
Published: May 2006

Recently, we identified a specific DNA sequence from the gastrin gene that regulates RNA polymerase II transcription termination in vivo. In the studies presented here, we examined the processing and termination activity of this sequence in vitro. When present in an in vitro synthesized RNA, this sequence (U9A2U5AU4AU4AU5) does not serve as an RNA processing signal on incubation with HeLa whole-cell extract. However, transcription of template DNA in HeLa whole-cell extract does terminate near the 5' end of this sequence. Nuclease S1 and exonuclease VII mapping of the 3' region of the in vitro synthesized RNAs confirm these results. The termination activity of the sequence A9T2A5TA4TA4TA5 is independent of the distance from the promoter and of the nature of the DNA template (linear vs. circular). The termination activity of the sequence shows a strong orientation dependence. These results strongly suggest that the termination activity of this cis-acting element is modulated by a trans-acting cellular factor. The unique structural feature of this sequence, a 10.5-base-pair inverted repeat, may determine the specificity of interaction of the trans-acting factor with the cis-acting element, resulting in accurate termination of transcription.
Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions.
Authors: Wolfbauer G , Glick J M , Minor L K , Rothblat G H
Published: May 2006

A possible mechanism for the formation of smooth muscle foam cells in the atherosclerotic lesion was explored. Cultured macrophages (J774 cell line) were induced to form cytoplasmic cholesteryl ester inclusions by exposure to acetylated low density lipoprotein in the presence of cholesterol-rich phospholipid dispersions. The macrophages were disrupted by brief sonication, and the inclusions were isolated by flotation. When these inclusions were placed in direct contact with cultured smooth muscle cells, cellular uptake of the inclusions in a time- and dose-dependent manner was observed. Light and electron microscopy indicated the presence of lipid inclusions throughout the cytoplasm of the cells. Uptake of inclusion lipid by the smooth muscle cells was inhibited by several metabolic inhibitors, indicating that the process is dependent on metabolic activity. A modest but significant hydrolysis of the cholesteryl ester was observed, showing that the stored cholesteryl esters are metabolically available.
Two DR beta allelic series defined by exon II-specific synthetic oligonucleotide genomic hybridization: a method of HLA typing?
Authors: Le Gall I , Millasseau P , Dausset J , Cohen D
Published: May 2006

Comparisons of exon II HLA-DR beta sequences have shown that nucleotide variations are principally clustered within the following three regions: V1 (amino acid 8-15), V2 (25-32), and V3 (70-77). V1, V2, and V3-derived 24-mers have been synthesized, the DR beta sequences coming from DR1, DR3, Drw6, DR4, DR5, and DRw53 haplotypes. Each oligonucleotide was hybridized to Pvu II-digested DNA samples from 13 HLA genotyped families; therefore, 52 haplotypes have been investigated. Six polymorphic Pvu II fragments were detected, constituting two allelic series probably corresponding to the beta 1 and beta 2 locus of the DR region. The first series (beta 1) comprises a minimum of nine alleles while the second series (beta 2), which is less polymorphic, comprises at least four alleles. Certain patterns correlate perfectly with certain DR specificities, whereas other patterns define new subdivisions as in DR3 and DRw6 haplotypes. Although it appears that some mismatches do not always prevent hybridization in the conditions used in this work, this method will provide in many instances a convenient tool for HLA-DR typing.
Localization and comparative nucleotide sequence analysis of the transforming domain in herpes simplex virus DNA containing repetitive genetic elements.
Authors: Jones C , Ortiz J , Jariwalla R J
Published: May 2006

The 7.5-kilobase BamHI E fragment (BamHI-E) of herpes simplex virus type 2 (HSV-2) DNA (map position 0.533-0.583) encodes the 144-kDa subunit of ribonucleotide reductase and induces the neoplastic transformation of immortalized cell lines. To define the minimal transforming region of BamHI-E, a series of subclones were constructed that spanned the entire fragment. These subclones were assayed for focus formation in Rat-2 cells. Removal of the promoter region from the viral 144-kDa-protein gene left the transforming activity of DNA clones intact. A 481-bp Pst I-Sal I subclone of BamHI-E was capable of inducing focus formation and tumorigenic conversion. The nucleotide sequence of this fragment and the colinear nontransforming region of HSV-1 DNA was determined and compared. Striking differences were detected in the structure and organization of repeated sequence elements. Specifically, transforming HSV-2 DNA contains multiple regions of alternating purines and pyrimidines, G + C-rich sequences that are potential binding sites for transcription factor Sp1, and insertion-like sequence elements that are interrupted by base substitutions in nontransforming HSV-1 DNA. These results define a distinct transforming domain in HSV-2 DNA composed of repetitive elements implicated in gene rearrangement and activation.
Identification of the 5'-flanking regulatory region responsible for the difference in transcriptional control between mouse complement C4 and Slp genes.
Authors: Nonaka M , Kimura H , Yeul Y D , Yokoyama S , Nakayama K , Takahashi M
Published: May 2006

To elucidate the molecular basis underlying the difference in the mode of gene expression between mouse complement C4 (constitutive) and sex-limited protein (Slp) (testosterone-regulated), we compared nucleotide sequences and transcriptional regulatory activities of the 5'-flanking regions of these two genes. Although the two sequences showed a high degree of overall homology (95%) up to 1.9 kilobases (kb) upstream from the transcription initiation site, the Slp sequence lacked a 31-nucleotide segment containing ACACCC repeats and a 60-nucleotide segment containing ACAC repeats, which are present, respectively, 1.6 kb and 200 base pairs (bp) upstream from the transcription initiation site of the C4 gene. When assayed in human hepatoma-derived HepG2 cells, the 1.8-kb 5'-flanking DNA fragment of the C4 gene demonstrated strong transcriptional activity, whereas the corresponding DNA fragment of the Slp gene showed only negligible activity. By progressive deletion experiments, it was shown that the difference in the constitutive transcriptional activity of the C4 and Slp genes was accounted for by the presence or absence of the positive regulatory domain located between 1700 bp and 400 bp upstream of the transcription initiation site.
Possible involvement of RAS-encoded proteins in glucose-induced inositolphospholipid turnover in Saccharomyces cerevisiae.
Authors: Kaibuchi K , Miyajima A , Arai K , Matsumoto K
Published: May 2006

Incubation of yeast Saccharomyces cerevisiae at very low (0.02%) glucose levels led to arrest of the cell cycle at the G0/G1 phase. Readdition of glucose to these "starved" yeast resulted in cell proliferation. In glucose-starved yeast, glucose stimulated 32P incorporation into phosphatidic acid, phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate but not into phosphatidylethanolamine and phosphatidylcholine. Preincubation of yeast with [3H]inositol and subsequent exposure to glucose resulted in rapid formation of [3H]inositol monophosphate and [3H]inositol trisphosphate, presumably derived from phosphatidylinositol and phosphatidylinositol bisphosphate. Under similar conditions, glucose elicited both efflux and influx of Ca2+ in yeast. Glucose-induced 32P incorporation into inositolphospholipids and formation of [3H]inositol phosphates were more pronounced in RAS-related mutants such as ras1, ras1 ras2 bcy1, and RAS2Val19 than in the wild-type strain. These results strongly suggest that glucose stimulates inositolphospholipid turnover, Ca2+ mobilization, and subsequent cell proliferation in a manner similar to that of growth factors with mammalian cells, and that RAS-encoded proteins are involved in regulation of this glucose-induced inositolphospholipid turnover in yeast.
17 beta-estradiol regulates and v-Ha-ras transfection constitutively enhances MCF7 breast cancer cell interactions with basement membrane.
Authors: Albini A , Graf J , Kitten G T , Kleinman H K , Martin G R , Veillette A , Lippman M E
Published: May 2006

The interaction of a line of human breast carcinoma cells (MCF7) with basement membrane components, particularly laminin, was altered by exposure of the cells to estrogen as well as by transfection of the cells with the v-Ha-ras oncogene. In both cases, the cells show a greater ability to attach to a laminin substrate, to migrate to laminin, to grow in the presence of a basement membrane matrix, and to cross barriers of reconstituted basement membrane. These responses were associated with an increase in the expression of laminin receptors. It is postulated that the increase in the invasive behavior of the cells treated with estrogen or transfected with v-Ha-ras is related to the increased number of laminin receptors and their interaction with laminin. Estrogen had no discernible effect on the v-Ha-ras transfected cells. It appears that in the MCF7 cells, the malignant phenotype is under hormonal control and that this control is bypassed after v-Ha-ras transfection.
Improved flow cytometric analysis of leukocyte subsets: simultaneous identification of five cell subsets using two-color immunofluorescence.
Authors: Horan P K , Slezak S E , Poste G
Published: May 2006

Flow cytometric analysis of human peripheral blood leukocytes has typically been achieved by staining multiple aliquots of the same sample with fluorescent reagents specific for cell subsets of interest. Spectrally discrete fluorochrome tags have been developed for applications in which identification of multiple subsets (e.g., T and B cells) or of subsets not uniquely identified by a single reagent (e.g., activated T cells) requires use of multiple reagents per aliquot. Extension of this approach to more than two reagents per aliquot has led to multicolor methods requiring dual laser excitation and complex instrumentation. We describe an alternative two-color method using commercially available reagents that allows simultaneous identification of five discrete immune cell subsets using only a single excitation source. The technique uses dilution of commercial fluorochrome-labeled reagents with competing unlabeled reagents to selectively produce discrete fluorescence intensity profiles for cell subsets that would otherwise display overlapping or indistinguishable profiles when stained with reagents bearing the same fluorochrome. For example, the fluorescence intensity of phycoerythrin-labeled helper T (Th) cells can be adjusted to be distinct from that of phycoerythrin-labeled suppressor T (Ts) cells. Extending this technique to two colors, we have used a combination of seven different monoclonal antibodies to simultaneously quantify Th, Ts, B cells, natural killer cells, and monocytes in a single aliquot. An additional advantage of this approach is the ability to more accurately quantify "null" cells. Adjustment of fluorescence intensity profiles of different cell subsets by this method is applicable to flow cytometric analysis of a wide variety of cell types. The technique significantly extends the analytical capacity of flow cytometry without significantly increasing the complexity of the instrumentation required.
Properties of a mouse ribosomal protein promoter.
Authors: Dudov K P , Perry R P
Published: May 2006

The three mouse ribosomal protein genes that have been analyzed to date, although evolutionarily unrelated, exhibit some remarkably similar sequence features at their 5' ends, some of which are shared by other genes in the "housekeeping" category. In order to appreciate the significance of these features, we have examined the transient expression of the genes encoding ribosomal proteins L32 and S16, as well as that of a set of 5'-deletion mutants of the L32 gene, in transfected primate cells. Our results reveal two important characteristics of the elements that positively regulate L32 transcription: they do not extend more than 36 nucleotides upstream of the major transcriptional initiation site, and they are insensitive to the presence of cis-linked simian virus 40 enhancer elements. The relevance of these findings to the general design features of housekeeping gene promoters is discussed.
Anti-beta-endorphin immunoglobulin G in humans.
Authors: Roy B F , Rose J W , McFarland H F , McFarlin D E , Murphy D L
Published: May 2006

Human IgG specific for beta-endorphin was identified by enzyme-linked immunoabsorbent assay and isolated by affinity chromatography. From a sample of 27 subjects, three individuals with major depression demonstrated plasma IgG highly reactive with human beta-endorphin, while four other subjects (two with depression and two randomly selected blood donors) had intermediate reactivity. The recognition site for beta-endorphin was retained by F(ab')2 fragments.
Oxidation of reduced menaquinone by the fumarate reductase complex in Escherichia coli requires the hydrophobic FrdD peptide.
Authors: Cecchini G , Thompson C R , Ackrell B A , Westenberg D J , Dean N , Gunsalus R P
Published: May 2006

Plasmids carrying cloned segments of the frd operon of Escherichia coli have been used in genetic complementation studies to identify two independent mutants defective in the frdD gene, which encodes the hydrophobic FrdD polypeptide of the fumarate reductase complex. Mutations in the frdA and frdB genes have also been mapped by this technique. One of the FrdD peptide mutants, DW109 (frdD-109), showed that fumarate reductase was not as tightly bound to the membrane in this mutant. In addition, the mutation in the FrdD peptide caused an almost total loss of the ability of the enzyme to oxidize either menaquinol-6, a physiological donor for fumarate reduction, or reduced benzyl viologen. However, the mutation did not impair the ability of the membrane-bound fumarate reductase complex to function with succinate as substrate, as evidenced by unchanged turnover numbers for phenazine methosulfate and 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone (a quinone analogue) reductase activities. These data establish the essential role of the FrdD polypeptide both in the interaction of the enzyme with reduced menaquinone and thus in anaerobic respiration with fumarate as electron acceptor, and in binding the enzyme to the membrane.
Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta.
Authors: Chung B C , Matteson K J , Voutilainen R , Mohandas T K , Miller W L
Published: May 2006

Conversion of cholesterol to pregnenolone is mediated by P450scc [cholesterol, reduced-adrenal-ferrodoxin: oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.67]. RNA from several human adrenal samples was translated in vitro and immunoprecipitated with anti-bovine P450scc, indicating that P450scc mRNA represents about 0.5% of human adrenal mRNA in normal, hypertrophied, and malignant adrenals. A 1626-base-pair human adrenal P450scc cDNA was cloned in bacteriophage lambda gt10. Primer extension data indicated P450scc mRNA is about 1850 bases long and that all adrenal P450scc mRNA has the same 5' end. A full-length clone containing 1821 bases was obtained from a human testis cDNA library to yield the complete sequence. The encoded human preP450scc contains 521 amino acids with a molecular weight of 60189.65. The testis and adrenal sequences were identical; the human cDNA and amino acid sequences are 82% and 72% homologous, respectively, with the bovine sequences. P450scc cDNA was used to probe DNA from a panel of mouse-human somatic cell hybrids, showing that the single human P450scc gene lies on chromosome 15. The human P450scc gene is expressed in the placenta in early and midgestation; primary cultures of placental tissue indicate P450scc mRNA accumulates in response to cyclic AMP.
Monoclonal antibodies increase intracellular Ca2+ in sea urchin spermatozoa.
Authors: Trimmer J S , Schackmann R W , Vacquier V D
Published: May 2006

Changes in intracellular free Ca2+ ([Ca2+]i) of sea urchin (Strongylocentrotus purpuratus) spermatozoa were measured using the fluorescent Ca2+ indicators fura-2 and indo-1. The intracellular pH (pHi) of sperm was also determined. The fucose sulfate-rich glycoconjugate component of egg jelly induced increases in [Ca2+]i and pHi in sperm and induced the acrosome reaction. Monoclonal antibodies (mAbs) to external domains of a 210-kDa glycoprotein of the sperm plasma membrane induced a 23-fold increase in [Ca2+]i (vs. 9-fold for fucose sulfate-rich glycoconjugate), but the mAbs did not cause the pHi to increase and did not induce the acrosome reaction. When the mAb treatment which induced an increase in [Ca2+]i was combined with an NH4Cl treatment, which increased the pHi, the acrosome reaction was induced. mAb-induced increases in [Ca2+]i were dependent on millimolar concentrations of extracellular Ca2+ and were reversed by placing sperm in Ca2+-free seawater or by chelating Ca2+ with EGTA. The mAb-induced [Ca2+]i increase was sensitive to the pH of the seawater, although mAb binding was not. The data show that increased [Ca2+]i and pHi are necessary for induction of the acrosome reaction and suggest that the 210-kDa protein may play a role in regulating Ca2+ entry into the spermatozoan. These mAbs make it possible to separate the increase in [Ca2+]i from the increase in pHi and may be useful in the elucidation of the regulatory role of Ca2+ in sperm physiology.
Transgenesis by means of blastocyst-derived embryonic stem cell lines.
Authors: Gossler A , Doetschman T , Korn R , Serfling E , Kemler R
Published: May 2006

This study demonstrates that blastocyst-derived embryonic stem cells (ES cells) can be used as a vehicle for transgenesis. The method is nearly as efficient as other methods, and the introduced neomycin phosphotransferase (neo) gene is stably transmitted through several generations with no apparent loss in G418 resistance. An important factor contributing to the efficiency of this process is the rigorous selection, before blastocyst injection, of genetically transformed cells for in vitro developmental pluripotency. One of the advantages of the ES cell route to transgenesis is that it provides investigators with the opportunity to screen for the desired genetic alterations before reintroducing the ES cells into the animal.
The cloning, genetic mapping, and expression of the constitutive sucrose synthase locus of maize
Authors: McCarty Donald R. , Shaw Janine R. , Hannah L. Curtis
Published: May 2006

Two differentially expressed genes encode isoenzymes of sucrose synthase in
Zea mays
. A clone of the shrunken 1 (
Sh1
) locus, the structural gene for the major endosperm form of sucrose synthase, was used to isolate a genomic clone of constitutive sucrose synthase (
Css
), the structural gene for the isoenzyme expressed in embryo and other tissues. The
Css
clone was positively identified by RNA blot analysis of RNA from wild type and a
sh1
deletion stock and by analysis of the
in vitro
translation product of hybrid-selected mRNA. Southern blot analysis of DNA from monosomic plants derived from an
r-x1
stock, coupled with restriction fragment length polymorphism mapping, placed the
Css
gene 32 map units from
Sh1
on chromosome 9. In seedling tissues, Css mRNA is present at higher levels than Sh1 mRNA. Expression of both
Sh1
and
Css
in root tissue is enhanced by anaerobic conditions, although
Css
is induced to a lesser extent than is
Sh1
. Thus,
Css
appears to be expressed constitutively, whereas
Sh1
is expressed at high levels only in response to specific developmental and environmental stimuli.
Structural differences in a single gene encoding the V kappa Ser group of light chains explain the existence of two mouse light-chain genetic markers.
Authors: Boyd R T , Goldrick M M , Gottlieb P D
Published: May 2006

Two phenotypic markers of mouse immunoglobulin kappa light chains, the IB-peptide marker and the Ef1a isoelectric focusing marker, are expressed by the C58/J, AKR/J, RF/J, and PL/J strains (called expressor strains) but not by BALB/c and most inbred strains. Expression is linked to the kappa light-chain locus and the Lyt-2/Lyt-3 genes on chromosome 6. Light chains bearing these markers belong to a group of variable region kappa chain (V kappa) regions called V kappa Ser, which has a serine amino terminus and a framework 1 region not observed to date among BALB/c light chains. Southern hybridization of genomic DNA with a V kappa Ser-specific cDNA probe has demonstrated a single strongly hybridizing DNA fragment in all strains of mice tested. Characteristic restriction enzyme polymorphisms define the V kappa Ser alleles of expressor (Igk-VSera) and nonexpressor (Igk-VSerb) strains. In the present study, the unrearranged V kappa Ser gene and its flanking regions from an expressor (C.C58) and nonexpressor (BALB/c) strain have been cloned and their nucleotide sequences determined. The C.C58 V kappa Ser gene isolated (the Igk-VSera allele) was shown to code for the two phenotypic markers described. While the nucleotide sequence of the BALB/c coding region (the Igk-VSerb allele) shows 97% identity with the C.C58 gene, single nucleotide substitutions lead to structural changes in the encoded protein which render it IB-negative and Ef1a-negative. These differences alone can explain the failure of strains containing the BALB/c allele to express these kappa-chain phenotypic markers. Also, the BALB/c gene contains a single substitution in a conserved octamer sequence approximately equal to 100 nucleotides upstream of the coding region, which could affect its expression. Finally, the C.C58 allele contains a BAM5/R repetitive DNA element approximately equal to 1200 nucleotides upstream of the coding regions that is not present in BALB/c. This element gives rise to the EcoRI and BamHI restriction enzyme polymorphisms, which distinguish the Igk-VSera and Igk-VSerb alleles.
Substance P receptors in primary cultures of cortical astrocytes from the mouse.
Authors: Torrens Y , Beaujouan J C , Saffroy M , Daguet de Montety M C , Bergström L , Glowinski J
Published: May 2006

Binding sites for substance P were labeled on intact cortical glial cells from newborn mice in primary culture using 125I-labeled Bolton-Hunter-labeled substance P. Maximal specific binding (95% of total binding) was reached after 2-3 weeks in culture. The binding was saturable, reversible, and temperature dependent. Scatchard and Hill analysis revealed a single population of noninteracting high-affinity binding sites (Kd, 0.33 nM; Bmax, 14.4 fmol per dish). Competition studies made with tachykinins and substance P analogues indicated that the characteristics of the 125I-labeled Bolton-Hunter labeled substance P binding sites on glial cells were identical to those on rat brain synaptosomes. 125I-labeled Bolton-Hunter labeled substance P binding sites were visualized by autoradiography, and differences in the intensity of labeling were seen among astrocytes. Substance P was found to stimulate phosphatidylinositol turnover; the EC50 value (0.36 nM) was identical to the IC50 value (0.38 nM) determined in binding studies. 125I-labeled Bolton-Hunter labeled substance P binding sites were also found on astrocytes derived from other brain structures and from the spinal cord of mice.
Activation of transcription by the bacteriophage 434 repressor.
Authors: Bushman F D , Ptashne M
Published: May 2006

Bacteriophage 434 encodes a repressor that, like bacteriophage lambda repressor, both activates and represses transcription. As in the lambda chromosome, a region of the 434 chromosome, called the right operator, contains three repressor binding sites (OR1, OR2, and OR3) that mediate these effects on two adjacent promoters. We now show that a part of the 434 repressor, the amino-terminal domain, activates leftward transcription when bound to OR2. We show that 434 repressor bound to OR2 closely approaches (touches) RNA polymerase bound to the leftward promoter. Model building based on ethylation interference and other experiments suggests that in three cases, those involving lambda repressor, 434 repressor, and bacteriophage P22 repressor, and in spite of differences in detailed arrangements, transcription is activated by a contact between the repressor and the same part of RNA polymerase.
The mRNA coding for the secreted protease transin is expressed more abundantly in malignant than in benign tumors.
Authors: Matrisian L M , Bowden G T , Krieg P , Fürstenberger G , Briand J P , Leroy P , Breathnach R
Published: May 2006

Transin RNA is a 1.9-kilobase RNA transcript induced by oncogenes in rat embryo fibroblast cell lines. We show that RNA species complementary to a cloned transin cDNA are present in mouse skin squamous cell carcinomas induced by a classical initiation-promotion protocol but not in premalignant, benign papillomas or in normal epidermis. A single application of a tumor promoting phorbol ester to normal epidermis elicits a transient increase in these RNA levels. Transin RNA encodes a secreted protease, an activity consistent with a functional role for enhanced expression of transin RNA in the progression of benign, encapsulated tumors to malignant, invasive carcinomas.
Fluid phase endocytosis by cultured rat hepatocytes and perfused rat liver: implications for plasma membrane turnover and vesicular trafficking of fluid phase markers.
Authors: Scharschmidt B F , Lake J R , Renner E L , Licko V , Van Dyke R W
Published: May 2006

Hepatocytes take up a variety of ligands via receptor-mediated endocytosis, yet little is known regarding either the volume of fluid or the amount of membrane internalized via endocytosis in liver cells. In these studies, we have utilized radiolabeled inulin to characterize fluid phase endocytosis by rat hepatocytes in primary culture and perfused rat liver. Uptake of inulin by cultured hepatocytes was nonlinear with time, occurring most rapidly during the first 2 min. Inulin uptake and efflux in cultured hepatocytes and inulin uptake by perfused rat liver were kinetically compatible with the entry of inulin into a rapidly (t1/2, 1-2 min) turning-over (presumably endosomal) compartment that exchanged contents with the extracellular space and comprised approximately 3% of hepatocyte volume, as well as entry into and concentration of inulin within slowly (t1/2, greater than 1 hr) turning-over storage compartments. Based on inulin uptake, it is estimated that cultured hepatocytes endocytosed the equivalent of 20% or more of their volume and 5 or more times their plasma membrane surface area each hour. Neither chloroquine (1 mM) nor taurocholate (200 microM) affected inulin handling by cultured cells, whereas colchicine (10 microM) inhibited transfer to storage compartments by greater than 50%. In conjunction with our previous observations, the present findings suggest that inulin endocytosed across the basolateral membrane is largely (congruent to 80%) regurgitated back into plasma, with smaller amounts transported to intracellular storage compartments (congruent to 18%) or to bile (congruent to 2%). Transport of inulin via these pathways is unaffected by taurocholate and does not require vesicle acidification, whereas intact microtubular function is required for transfer to storage compartments or biliary secretion.
Evolutional and ecological implications of the properties of deep-sea barophilic bacteria
Authors: Yayanos A. Aristides
Published: May 2006

The rate of reproduction of deep-sea bacteria from six different capture depths between 1957 and 10,476 meters was studied as a function of temperature and pressure. The results showed the following: the true deep-sea bacteria of different depths have several characteristics, presumably evolutionally derived, distinguishing them from each other and from bacteria of atmospheric-pressure environments; pressure plays a significant role in determining the distribution of oceanic life; and pressure-adapted bacteria are easily recovered from and ubiquitous in the deep ocean. Organisms evolving in habitats of different temperatures and pressures need to be studied to understand the physical limits of life, the distribution of life within the earth and its oceans, the role of organisms in organic diagenesis and petroleum formation, and the possible existence of life on and within other planets.
Mitochondrial DNA analysis of hybridization between sympatric white-tailed deer and mule deer in west Texas.
Authors: Carr S M , Ballinger S W , Derr J N , Blankenship L H , Bickham J W
Published: May 2006

Sympatric populations of white-tailed deer and mule deer (Odocoileus virginianus and Odocoileus hemionus, respectively) on a west Texas ranch share a common mitochondrial DNA restriction map genotype. Phylogenetic analysis indicates that this genotype is more characteristic of O. virginianus than of O. hemionus. The genotype of west Texas deer differs from that of O. virginianus from South Carolina by five mutational events (1.3% sequence divergence), whereas it differs from that of O. hemionus from California by 17 events (5.5% divergence). We suggest that interspecies hybridization has occurred, primarily between mule deer bucks and white-tailed deer does, with preferential absorption of hybrid offspring into the mule deer gene pool. Introgressive hybridization may be involved in ongoing displacement of mule deer by white-tailed deer in west Texas.
Evidence for reactive oxygen species inducing mutations in mammalian cells.
Authors: Hsie A W , Recio L , Katz D S , Lee C Q , Wagner M , Schenley R L
Published: May 2006

We have studied the mutagenicity (by selecting for mutants resistant to 6-thioguanine) and cytotoxicity (by determining cellular cloning efficiency) of physical and chemical agents in Chinese hamster ovary (CHO) cells, clone CHO-K1-BH4 (K1-BH4), and its radiation-hypersensitive transformant, AS52. AS52 cells contain a single functional copy of a bacterial gene, the xanthine/guanine phosphoribosyltransferase (gpt) gene instead of its mammalian equivalent, the hypoxanthine/guanine phosphoribosyltransferase (hprt) gene. We found that x-ray and neutron irradiations are equally toxic to both cell types; however, these physical agents are approximately equal to 10 times more mutagenic to AS52 cells than to K1-BH4 cells. Our earlier studies using Southern blot analysis showed that x-irradiation produces mostly or exclusively deletion mutations in both cell types. If reactive oxygen species mediate the mutagenic effects of radiations and chemicals, then radiomimetic compounds such as streptonigrin and bleomycin, which exert their biological effects via reactive oxygen species, and oxidizing compounds such as potassium superoxide and hydrogen peroxide should elicit a similar differential mutagenic response in both cell types. On the other hand, agents such as ethyl methanesulfonate, ICR 191, and UV light, which do not produce reactive oxygen species, should not elicit differential mutagenicity. Our results fulfill such predictions. The apparent hypermutability of AS52 cells probably results from a higher recovery of multilocus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants.
Suppression of in vivo polyclonal IgE responses by monoclonal antibody to the lymphokine B-cell stimulatory factor 1.
Authors: Finkelman F D , Katona I M , Urban J F , Snapper C M , Ohara J , Paul W E
Published: May 2006

The lymphokine B-cell stimulatory factor 1 (BSF-1) has been shown to greatly enhance the differentiation of lipopolysaccharide-activated B cells into IgG1- and IgE-secreting cells in vitro. To determine whether in vivo IgG1 and IgE antibody responses are BSF-1 dependent, the ability of a monoclonal rat IgG1 anti-BSF-1 antibody, 11B11, to affect polyclonal IgG1 and IgE production in mice infected with the nematode parasite Nippostrongylus brasiliensis or injected with a purified goat antibody to mouse IgD was studied. 11B11-containing ascites fluid or purified 11B11 strongly inhibited IgE production in both systems but did not affect IgG1 production, while control ascites or normal rat IgG1 had no IgE-inhibitory activity. These results indicate an important physiologic role for BSF-1 in the generation of IgE antibody responses and suggest means for limiting the production of antibodies responsible for allergic reactions without inhibiting protective antibody responses.
Morphological alterations in endothelial cells from human aorta and umbilical vein induced by forskolin and phorbol 12-myristate 13-acetate: a synergistic action of adenylate cyclase and protein kinase C activators.
Authors: Antonov A S , Lukashev M E , Romanov Y A , Tkachuk V A , Repin V S , Smirnov V N
Published: May 2006

The morphological effects on human endothelial cells of phorbol 12-myristate 13-acetate (PMA) and of agents that increase intracellular cAMP concentration were studied. The adenylate cyclase activator forskolin (10 microM), the cyclic nucleotide phosphodiesterase inhibitor methylisobutylxanthine (100 microM), dibutyryl-cAMP (10 microM), histamine (10 microM), and PMA (0.1 microM) significantly altered the morphology of human aortic and umbilical vein endothelial cells in primary cultures. These effects reached a maximum 40-80 min after the effector addition and became negligible 30-60 min after its removal. PMA and forskolin were strongly synergistic in altering endothelial cell morphology. All the effects of cAMP-elevating compounds and of PMA were abolished completely by 1 microM colchicine. In explants taken from human adult or child aortas, forskolin and PMA produced alterations in endothelial morphology qualitatively identical to those observed in endothelial cell cultures. Endothelium in these preparations closely resembled that found in zones of expected altered hemodynamic stresses of human aorta. Our data suggest that the morphology of endothelium in vivo may be regulated by separate or synergistic action of hormone-dependent adenylate cyclase and of inositol phospholipid turnover systems and might be important for maintenance of endothelial monolayer integrity under normal physiological and pathological conditions.