bluuebunny/crossref_metadata_2025 huggingface dataset:
Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons.
Authors: Sethna P B , Hung S L , Brian D A
Published: May 2006

The genome of the porcine transmissible gastroenteritis coronavirus is a plus-strand, polyadenylylated, infectious RNA molecule of approximately 20 kilobases. During virus replication, seven subgenomic mRNAs are generated by what is thought to be a leader-priming mechanism to form a 3'-coterminal nested set. By using radiolabeled, strand-specific, synthetic oligodeoxynucleotide probes in RNA blot hybridization analyses, we have found a minus-strand counterpart for the genome and for each subgenomic mRNA species in the cytoplasm of infected cells. Subgenomic minus strands were found to be components of double-stranded replicative forms and in numbers that surpass full-length antigenome. We propose that subgenomic mRNA replication, in addition to leader-primed transcription, is a significant mechanism of mRNA synthesis and that it functions to amplify mRNAs. It is a mechanism of amplification that has not been described for any other group of RNA viruses. Subgenomic replicons may also function in a manner similar to genomes of defective interfering viruses to lead to the establishment of persistent infections, a universal property of coronaviruses.
The mouse homolog of the hst/k-FGF gene is adjacent to int-2 and is activated by proviral insertion in some virally induced mammary tumors.
Authors: Peters G , Brookes S , Smith R , Placzek M , Dickson C
Published: May 2006

The fibroblast growth factor-related protooncogenes, int-2 and hst/k-FGF, are within 17 kilobase pairs of one another on mouse chromosome 7 and are in the same transcriptional orientation. Approximately 70% of tumors induced in BR6 mice by mouse mammary tumor virus have proviral insertions adjacent to the int-2 gene. We find that the murine homolog of the hst/k-FGF gene can also be transcriptionally activated by the insertion of mouse mammary tumor virus DNA either upstream or downstream of the gene. In most tumors, only one of these adjacent genes is activated, but in some cases both genes are expressed. One of the hst-expressing tumors also has a virally activated int-3 gene. At least five distinct cellular genes (int-1, -2, -3, -4, and hst/k-FGF) can therefore contribute, either singly or in concert, to the development of histologically indistinguishable mammary tumors in mice infected by mouse mammary tumor virus.
Adenylate cyclases in yeast: a comparison of the genes from Schizosaccharomyces pombe and Saccharomyces cerevisiae.
Authors: Yamawaki-Kataoka Y , Tamaoki T , Choe H R , Tanaka H , Kataoka T
Published: May 2006

A Schizosaccharomyces pombe gene encoding adenylate cyclase has been cloned by cross-hybridization with the Saccharomyces cerevisiae adenylate cyclase gene. The protein encoded consists of 1692 amino acids and has adenylate cyclase activity that cannot be activated by the Sa. cerevisiae RAS2 protein. Sc. pombe cyclase has a high degree of homology (approximately 60%) with the catalytic domain of Sa. cerevisiae cyclase precisely mapped by a gene-deletion analysis. A 25-40% identity is observed throughout the middle segments of approximately 1000 residues of both cyclases, large parts of which are composed of repetitions of a 23-amino acid motif similar to those found in human glycoproteins, Drosophila chaoptin, and Toll gene product. However, a segment corresponding to the NH2-terminal 620 residues of Sa. cerevisiae cyclase appears lost from Sc. pombe cyclase, and the COOH-terminal 140 residues are not well conserved between the two yeast species. Deletions involving the COOH-terminal residues of Sa. cerevisiae cyclase cause loss of activation by the RAS2 protein. These results suggest that Sc. pombe cyclase may have lost the ability to interact with RAS proteins by the loss of a regulatory site.
The yeast STE12 protein binds to the DNA sequence mediating pheromone induction.
Authors: Dolan J W , Kirkman C , Fields S
Published: May 2006

The STE12 gene product of Saccharomyces cerevisiae is required for the transcription of two sets of cell-type-specific genes: the a-specific genes (active only in a cells) and the alpha-specific genes (active only in alpha cells). We show that radiolabeled STE12 protein, prepared by in vitro transcription and translation, is capable of forming complexes with unlabeled DNA fragments from two a-specific genes. Wild-type yeast, but not a ste12 mutant, produce a factor that forms complexes with labeled DNA from these same two genes. We use assays with yeast extracts to localize the binding site for the STE12-dependent activity. This site corresponds to the sequence identified as the pheromone induction element, which is responsible for increased transcription of genes when cells are exposed to alpha-factor or a-factor. Thus the STE12 protein may be an ultimate effector in the signal transduction pathway triggered by pheromone.
Serum- and oncoprotein-mediated induction of a gene with sequence similarity to the gene encoding carcinoembryonic antigen.
Authors: Klemenz R , Hoffmann S , Werenskiold A K
Published: May 2006

We describe the molecular identification of a gene, designated T1, whose expression in mouse NIH 3T3 cells is strongly induced by the Ha-ras(EJ) and v-mos oncogenes and by serum. The T1 gene encodes a 38.5-kDa protein, as predicted from its primary sequence and shown by in vitro translation. The protein was processed at its amino terminus and extensively modified by N-linked glycosylation in vitro in the presence of microsomal vesicles. Sequence comparison of T1 with the MIPSX data base (Max-Planck-Institut für Biochemie, Martinsried/Munich) revealed similarity to the human carcinoembryonic antigen, a tumor marker which is overexpressed in colon adenocarcinomas and in fetal tissues. Considerable sequence similarity has also been observed to the short conserved region of other proteins which, like carcinoembryonic antigen, are encoded by members of the immunoglobulin gene superfamily.
Conservation of the sequence of islet amyloid polypeptide in five mammals is consistent with its putative role as an islet hormone.
Authors: Nishi M , Chan S J , Nagamatsu S , Bell G I , Steiner D F
Published: May 2006

Islet amyloid polypeptide (IAPP) is a 37-amino acid peptide found in the pancreatic amyloid deposits of type II (non-insulin-dependent) diabetic patients and insulinomas. We previously reported the nucleotide sequence of a human cDNA, which indicated that IAPP is a C-terminally amidated peptide derived by proteolytic processing of an 89-amino acid precursor. We now report the isolation of cDNA clones coding for cat, rat, mouse, and guinea pig IAPP precursors, obtained using the combination of "amplification of homologous DNA fragments" (AHF) and "rapid amplification of cDNA ends" (RACE). The predicted structure of IAPP precursors from these four mammals revealed that the IAPP moiety of each is derived from an 89- to 93-amino acid precursor by proteolytic processing and is likely to be amidated at the C terminus. The predicted amino acid sequence identities between the IAPP domains of these four mammals and human IAPP were 89% (cat), 84% (rat and mouse), and 78% (guinea pig). Within the IAPP domains, the N-terminal and C-terminal amino acid sequences are very highly conserved among the mammals, as is also the case with a structurally related neuropeptide, calcitonin-gene-related peptide (CGRP), suggesting that IAPP and CGRP interact with similar though not identical receptors. By contrast, the N- and C-terminal propeptides of the IAPP precursor show very little sequence conservation, which suggests that these regions do not represent additional biologically active molecules. Interspecies variations in the amino acid sequence of residues 20-29 of IAPP may account for the presence of amyloid deposits in the islets of humans and cats and their absence in rats and mice.
Nucleotide sequence and genomic organization of feline immunodeficiency virus.
Authors: Talbott R L , Sparger E E , Lovelace K M , Fitch W M , Pedersen N C , Luciw P A , Elder J H
Published: May 2006

An infectious molecular clone of the Petaluma strain of feline immunodeficiency virus (FIV) was isolated from a recombinant bacteriophage library containing genomic DNA prepared from FIV-infected Crandall feline kidney (CRFK) cells. The integrated provirus has a total length of 9472 base pairs. Three long open reading frames corresponding to GAG, POL, and ENV gene coding frames are evident. In addition, an open reading frame overlaps the 3' end of POL, in the region that encodes viral infectivity factor in the primate viruses. Several short open reading frames are present in the intergenic region between POL and ENV and within ENV, which may serve as exons for production of TAT and REV equivalents in FIV. Alignment of the predicted amino acid sequences of the FIV proteins with those of other lentiviruses indicates that FIV did not arise recently from any other characterized lentivirus.
A cyanobacterial mutant requiring the expression of ribulose bisphosphate carboxylase from a photosynthetic anaerobe.
Authors: Pierce J , Carlson T J , Williams J G
Published: May 2006

Ribulose bisphosphate carboxylase is essential for both photoautotrophic and photoheterotrophic growth of the cyanobacterium Synechocystis 6803. However, a mutant lacking cyanobacterial carboxylase could be obtained by replacing the natural carboxylase gene with the corresponding gene from Rhodospirillum rubrum, a photosynthetic anaerobe. This treatment produced an organism whose growth depended on the activity of the structurally and functionally dissimilar foreign carboxylase. As a further consequence of this mutagenic replacement, the mutant also lacked microscopically observable carboxysomes, the subcellular inclusion bodies in which the wild-type carboxylase naturally resides. The mutant, dependent on a carboxylase with an inferior relative specificity for CO2 versus O2 and apparently lacking carboxysomes, is extremely sensitive to the CO2/O2 ratio supplied during growth and is unable to grow at all in air. This response to the gas composition should prove useful for selection of various R. rubrum carboxylase mutants with altered specificities for CO2 and O2.
Identification and characterization of nuclear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells.
Authors: Nervi C , Grippo J F , Sherman M I , George M D , Jetten A M
Published: May 2006

Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion high-performance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and greater than 95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RAR alpha or RAR beta were enriched (20- to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high-affinity binding of RA and its benzoic acid analogs Ch55, Ch30, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of approximately 0.46 nM and 1400 +/- 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RAR alpha and RAR beta indicated that HL-60 cells contain predominantly transcripts encoded by the RAR alpha gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells.
Tissue-specific genetic variation in the level of mouse alcohol dehydrogenase is controlled transcriptionally in kidney and posttranscriptionally in liver.
Authors: Tussey L , Felder M R
Published: May 2006

Tissue-specific genetic variation in expression of the alcohol dehydrogenase, encoded by the Adh-1 gene, is found between C57BL/6J (B6) mice and B6.S congenic mice. B6.S mice contain a variant Adh-1 allele derived from a wild Danish strain in a B6 genetic background. B6 mice have nearly twice the alcohol dehydrogenase activity in liver but less than half the activity in kidney as B6.S mice. These tissue-specific genetic changes in alcohol dehydrogenase expression are manifest at the level of Adh-1-encoded mRNA. The regulatory site(s) involved act cis in both kidney and liver. These strains also differ in the extent to which androgen induces mRNA encoded by kidney Adh-1, with androgen increasing these levels 17-fold and 7.4-fold in the B6 and B6.S kidney, respectively. To identify the regulatory mechanism(s) underlying this strain variation in Adh-1 transcription in the B6 and B6.S kidney, liver, and androgen-induced kidney. For both uninduced and induced kidney, a difference in the transcription rate alone accounts for the strain difference in mRNA concentration. In contrast, because the Adh-1 transcription rate in liver does not differ significantly between B6 and B6.S mice, strain-specific variation in posttranscriptional regulation must be operative. Taken together these results indicate that the variation in Adh-1 expression between B6 and B6.S mice results from changes in both transcriptional and posttranscriptional control, and these controls are differentially operative in kidney and liver.
Fatal syndrome in mice engrafted with cells producing high levels of the leukemia inhibitory factor.
Authors: Metcalf D , Gearing D P
Published: May 2006

Cells of the murine hemopoietic cell line FDC-P1 were multiply infected with a retroviral construct containing cDNA encoding the leukemia inhibitory factor (LIF) to produce cells secreting high levels of LIF. Injection of these cells to unirradiated or irradiated syngeneic DBA/2 mice resulted in animals engrafted with LIF-producing cells in the marrow, spleen, and lymph nodes and with elevated serum LIF levels. These mice developed within 12-70 days a fatal syndrome characterized by cachexia, excess new bone formation, calcification in heart and skeletal muscle, pancreatitis, thymus atrophy, and abnormalities in the adrenal cortex and ovarian corpora lutea. Injection of mice with control FDC-P1 cells led to comparable organ engraftment, but the mice developed none of these lesions. The observations suggest that LIF may be a potent cachexia-inducing agent and may have marked effects on osteoblasts and calcium metabolism.
Osmoregulatory changes in myo-inositol transport by renal cells.
Authors: Nakanishi T , Turner R J , Burg M B
Published: May 2006

Renal medullary cells contain high concentrations of myo-inositol, sorbitol, betaine, and glycerophosphocholine, whose levels vary with urinary osmolality. Accumulation of these "compatible" organic osmolytes is believed to help the cells osmoregulate in response to the high extracellular osmolality that occurs as part of the urinary concentrating mechanism. MDCK cells (a line from dog kidney) were previously shown to accumulate myo-inositol in response to increased medium osmolality. We demonstrate here that this accumulation requires the presence of myo-inositol in the medium, implying that the myo-inositol is not synthesized by the cells but rather is transported into them from the extracellular solution. The MDCK cells contain sodium-dependent myo-inositol transporters. Relative to isotonic controls, sodium-dependent myo-inositol uptake is higher in cells exposed to increased osmolality either acutely (1-7 days) or chronically (greater than 1 year). Transport is further enhanced when the cells are cultured in myo-inositol-free medium. The transport has both high- and low-affinity components. The observed changes in transport involve changes in maximal velocity of the high-affinity component but not in its Km. We conclude that renal cells can osmoregulate by changing the number (or, less likely, the transport turnover rate) of functioning sodium-dependent myo-inositol transporters.
Point substitutions in Japanese alloalbumins.
Authors: Arai K , Madison J , Huss K , Ishioka N , Satoh C , Fujita M , Neel J V , Sakurabayashi I , Putnam F W
Published: May 2006

We have completed the structural study of five rare types of inherited albumin variants (alloalbumins) discovered in the Biochemical Genetics Study of 15,581 unrelated children in Hiroshima and Nagasaki. We have also identified the structural change in five other alloalbumin specimens detected during clinical electrophoresis of sera from Japanese living near Tokyo. Each of the five albumin variants from Nagasaki and Hiroshima has a single amino acid substitution. All of these substitutions differ, and none has been reported in non-Japanese populations. No instances of proalbumin variants or of albumin B (the most frequent alloalbumins in Caucasians) were detected in the children in Hiroshima and Nagasaki. However, one instance of a variant proalbumin and two examples of albumin B occurred in Japanese from the vicinity of Tokyo. In addition a previously unreported point substitution was found in albumin Tochigi, which is present in two unrelated persons from Tochigi prefecture. Four of the point mutations in the Japanese alloalbumins are in close proximity in a short segment of the polypeptide chain (residues 354-382) in which three additional point substitutions have been reported in diverse populations. These results, combined with earlier data, suggest that point substitutions are grouped in certain segments of the albumin molecule.
Colony formation from mesophyll protoplasts of a cereal, oat
Authors: Hahne Bettina , Fleck Jacqueline , Hahne Günther
Published: May 2006

Differentiated leaf cells of gramineous plants, among them the cereals with their immense importance for human nutrition, are considered extremely recalcitrant to, if not incapable of, reentering the cell cycle. This recalcitrance is related to the poor wound response of the monocots—in contrast to most dicots—and the difficulties encountered in monocot tissue culture. We report here the highly reproducible induction of sustained divisions at high frequency (up to 95%) and colony formation from mesophyll protoplasts of a cereal, oat, demonstrating that—contrary to most earlier evidence—mesophyll cells of a gramineous plant have not irreversibly lost their potential for cell division.
Reversible inhibition of oligodendrocyte progenitor differentiation by a monoclonal antibody against surface galactolipids.
Authors: Bansal R , Pfeiffer S E
Published: May 2006

We have hypothesized that oligodendrocyte (OL) surface glycolipids, specifically galactocerebroside and sulfatide, play a role in the regulation of OL development by acting as sensors/transmitters of environment information. In support of this hypothesis we report here a reversible inhibition of OL progenitor cell differentiation by a monoclonal antibody [Ranscht mAb (R-mAb); Ranscht, B., Clapshaw, P. A. & Seifert, W. (1982) Proc. Natl. Acad. Sci. USA 79, 2709-2713] that reacts with these glycolipids. When isolated OL progenitors or mixed primary cultures are grown in the presence of the antibody, myelinogenic development is blocked in a dose-dependent manner at concentrations as low as 2 micrograms of IgG per ml. The inhibited cells express the OL progenitor markers O4 and vimentin but are negative for galactosylcerebroside, sulfatide, 2',3'-cyclic nucleotide 3'-phosphohydrolase, myelin basic protein, and myelin basic protein RNA expression. In contrast, the levels of total cellular protein and the expression of astrocytic glial fibrillary acidic protein in mixed cultures are not affected. Antibody-blocked cells have a distinctive morphology in which long, sparsely branched processes emanate from round cell bodies. Upon removing the perturbing antibody, the cells rapidly resume differentiation. Reverted mixed primary cultures, in which OL progenitors of several sequential developmental stages are present at the time of plating, differentiate more rapidly than control cultures, suggesting that the antibody-induced block results in a synchronization of developmental progression along the OL lineage by accumulating cells at the inhibition point. However, the normal temporal sequence of marker expression is maintained. Control studies with several other antibodies recognizing OL cell surface antigens, including HNK-1, neural cellular adhesion molecule (N-CAM), 1A9, anticholesterol, and O1, did not inhibit development. Since the inhibition occurs in highly enriched populations of OL progenitors, the inhibition does not involve cell-cell interactions between OLs and other cell types but concerns interactions of OLs with themselves, soluble factors, or OL extracellular matrix molecules and adhesion factors that provide essential environmental signals required for normal myelinogenic development.
Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers.
Authors: Kocher T D , Thomas W K , Meyer A , Edwards S V , Pääbo S , Villablanca F X , Wilson A C
Published: May 2006

With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments of mtDNA from more than 100 animal species, including mammals, birds, amphibians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nanogram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility of these primers offers opportunities for phylogenetic and population research.
Cis-acting elements determine the locus-specific shutoff of class I major histocompatibility genes in murine S49 lymphoma sublines.
Authors: Keeney J B , Hansen T H
Published: May 2006

Several tumors have been reported to down-regulate expression of their class I major histocompatibility molecules, potentially altering their immune recognition. To investigate this phenomenon, we are using various sublines isolated from the S49 lymphoma of the BALB/c mouse strain. These S49 tumor sublines were previously found to have shut off expression of their Kd, Dd, and/or Ld class I molecules in a locus-specific manner. Extensive Southern blot analyses indicated that there were no major chromosome aberrancies in these S49 sublines, and analyses of steady-state class I mRNA suggested that a form of transcriptional regulation was responsible for their variant class I expression. In this report, we characterize the nature of this locus-specific regulation of class I in S49 cells by producing somatic cell hybrids. Three phenotypically distinct S49 sublines were each fused to tumor cells with normal class I expression, and several of the resulting hybrids were analyzed. In every case, the class I molecules expressed by the hybrids were an exact composite of the two fusion partners. Thus, these fusions failed to rescue expression of the Kd, Dd, and/or Ld molecules shut off within each of the S49 tumor sublines. These findings indicate that this locus-specific shutoff of class I expression results from a cis-acting defect and not trans-acting factors. Because the analysis of each of three phenotypically different S49 cells implicated a form of cis-dominant regulation, we hypothesize that a common mechanism generating homologous mutations in class I genes is operative in S49 tumor cells.
Highly potent metallopeptide analogues of luteinizing hormone-releasing hormone.
Authors: Bajusz S , Janaky T , Csernus V J , Bokser L , Fekete M , Srkalovic G , Redding T W , Schally A V
Published: May 2006

Metal complexes related to the cytotoxic complexes cisplatin [cis-diamminedichloroplatinum(II)] and transbis(salicylaldoximato)copper(II) were incorporated into suitably modified luteinizing hormone-releasing hormone (LH-RH) analogues containing D-lysine at position 6. Some of the metallopeptides thus obtained proved to be highly active LH-RH agonists or antagonists. For instance, SB-40, a PtCl2-containing metallopeptide in which platinum is coordinated to an N epsilon-(DL-2,3-diaminopropionyl)-D-lysine residue [D-Lys(DL-A2pr] at position 6, showed 50 times higher LH-releasing potency than the native hormone. SB-95, [Ac-D-Nal(2)1,D-Phe(pCl)2, D-Pal(3)2, Arg5,D-Lys[DL-A2pr(Sal2Cu)]6,D-Ala10]LH-RH, where Nal(2) is 3-(2-naphthyl)alanine, Pal(3) is 3-(3-pyridyl)alanine, and copper(II) is coordinated to the salicylideneimino moieties resulting from condensation of salicylaldehyde with D-Lys(DL-A2pr)6, caused 100% inhibition of ovulation at a dose of 3 micrograms in rats. Most metallopeptide analogues of LH-RH showed high affinities for the membrane receptors of rat pituitary and human breast cancer cells. Some of these metallopeptides had cytotoxic activity against human breast cancer and prostate cancer cell lines in vitro (this will be the subject of a separate paper on cytotoxicity evaluation). Such cytostatic metallopeptides could be envisioned as targeted chemotherapeutic agents in cancers that contain receptors for LH-RH-like peptides.
Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes.
Authors: Grossman R M , Krueger J , Yourish D , Granelli-Piperno A , Murphy D P , May L T , Kupper T S , Sehgal P B , Gottlieb A B
Published: May 2006

Psoriasis is a common papulosquamous skin disease. The histopathology is characterized by epidermal hyperplasia and inflammation. Recent studies suggest that keratinocyte proliferation and inflammation in psoriasis are manifestations of the same underlying pathological process. Interleukin 6 (IL-6), a cytokine that is a major mediator of the host response to tissue injury and infection, is produced by both keratinocytes and leukocytes in culture. IL-6 expression was studied in psoriatic plaques by immunoperoxidase staining with two different polyclonal anti-recombinant IL-6 antisera and by in situ nucleic acid hybridization with IL-6 cRNA probes. Epidermal and dermal cells in active psoriatic plaques from 35 psoriasis patients stained heavily for IL-6 as compared with nonlesional skin and with plaques after treatment with antimetabolic and antiinflammatory agents. Absorption of the anti-recombinant IL-6 antisera with purified fibroblast-derived IL-6 or with recombinant IL-6, but not bovine serum albumin, removed the immunostaining. Increased levels of IL-6 were detected in the plasma of patients with active psoriasis (mean 3 ng/ml) by using two different bioassays. IL-6 production by proliferating keratinocytes was suggested by IL-6-specific immunostaining in cultured normal and psoriatic keratinocytes and by the detection of mRNA specific for IL-6 in psoriatic epidermis by in situ hybridization. IL-6 stimulated the proliferation of cultured, normal human keratinocytes as assessed by two different assays. Thus, IL-6 could directly contribute to the epidermal hyperplasia seen in psoriatic epithelium as well as affect the function of dermal inflammatory cells.
Intracerebroventricular infusion of interleukin 1 rapidly decreases peripheral cellular immune responses.
Authors: Sundar S K , Becker K J , Cierpial M A , Carpenter M D , Rankin L A , Fleener S L , Ritchie J C , Simson P E , Weiss J M
Published: May 2006

Low doses (50-200 pg or 3.1-12.4 fmol) of interleukin 1 (IL-1) infused into the brain of rats produced rapid suppression of various cellular immune responses in peripheral lymphocytes of rats. Fifteen minutes after infusion of purified IL-1 beta into the lateral ventricle, natural killer cell activity, response to phytohemagglutinin stimulation, and interleukin 2 production were markedly suppressed in lymphocytes isolated from blood and spleen. These effects were due to infusion of IL-1 into brain since they did not occur when IL-1 was infused into the cisterna magna (essentially posterior to brain) or was injected intraperitoneally. Effects of IL-1 in brain could be blocked by simultaneous infusion of alpha-melanocyte-stimulating hormone, which is known to block the biological actions of IL-1. To stimulate release of endogenous IL-1 in brain, lipopolysaccharide was infused; this produced similar effects as IL-1, and these effects also were blocked by alpha-melanocyte-stimulating hormone. At longer intervals after infusion of IL-1 and lipopolysaccharide (3, 6, and 24 hr), immune responses returned to baseline or remained suppressed; i.e., "rebound" immunopotentiation did not occur. Finally, IL-1 infusion suppressed cellular immune responses in adrenalectomized animals, thereby showing that the effects of central IL-1 on peripheral cellular immune responses were, at least in part, independent of the stimulatory effect of IL-1 on secretion of adrenal hormones. These results indicate a link from brain to peripheral immune responses by means of action of a cytokine acting in the brain.
Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation: evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells.
Authors: Koike T , Martin D P , Johnson E M
Published: May 2006

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro; these cells die upon acute deprivation of NGF. We studied the effects of agents that cause membrane depolarization on neuronal survival after NGF deprivation. High-K+ medium (greater than or equal to 33 mM) prevented cell death; the effect of K+ was dose-dependent (EC50 = 21 mM). The protection by high K+ was abolished either by withdrawal of extracellular Ca2+ or by preloading the cells with a Ca2+ chelator. The involvement of Ca2+ flux across membranes in high-K+ saving of NGF-deprived neurons was also supported by experiments using Ca2+-channel antagonists and agonists. The Ca2+ antagonists nimodipine and nifedipine effectively blocked the survival-promoting effect of high K+. The Ca2+ agonists Bay K 8644 and (S)-202-791 did not by themselves save neurons from NGF deprivation but did strongly augment the effect of high K+; EC50 was shifted from 21 mM to 13 mM. These data suggest that dihydropyridine-sensitive L-type Ca2+ channels play a major role in the high-K+ saving. The depolarizing agents choline (EC50 = 1 mM) and carbamoylcholine (EC50 = 1 microM), acting through nicotinic cholinergic receptors, also rescued NGF-deprived neurons. The saving effect of nicotinic agonists was not blocked by withdrawal of extracellular Ca2+ but was counteracted by a chelator of intracellular Ca2+, suggesting the possible involvement of Ca2+ release from internal stores. Based on these findings we propose a "Ca2+ set-point hypothesis" for the degree of trophic-factor dependence of sympathetic neurons in vitro.
Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii.
Authors: Fernández E , Schnell R , Ranum L P , Hussey S C , Silflow C D , Lefebvre P A
Published: May 2006

The nitrate reductase structural gene of Chlamydomonas reinhardtii has been isolated from a genomic library by using a nitrate reductase cDNA probe from barley. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-1. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binding domains of tobacco and Arabidopsis thaliana nitrate reductases and human NADH cytochrome b5 reductase. The identity of the cloned gene as nitrate reductase was confirmed by its ability to complement a nit-1 mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, significantly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.
Multistep pathway for replication-dependent nucleosome assembly.
Authors: Fotedar R , Roberts J M
Published: May 2006

We have used cell-free DNA replication to study the relationship between DNA replication and chromatin assembly. As others have reported, we find that DNA replication facilitates nucleosome assembly. We show here that replication-dependent nucleosome assembly occurs in at least two steps. The first step requires replicating DNA; the second step occurs after replication has been completed and is promoted by a nuclear extract. Consistent with this multistep model, we observe that the replicated simian virus 40 minichromosome is organized into a repeating array of DNA-protein particles that are structurally distinct from mature nucleosomes. These particles may be precursors in a pathway of nucleosome assembly since in the second, replication-independent step the nuclear extract converts this nascent chromatin into nucleosomes.
Calcium buffer injections block fucoid egg development by facilitating calcium diffusion.
Authors: Speksnijder J E , Miller A L , Weisenseel M H , Chen T H , Jaffe L F
Published: May 2006

The polarity of fucoid eggs is fixed either when tip growth starts or a bit earlier. A steady flow of calcium ions into the incipient tip is thought to establish a high calcium zone that is needed for its localization and formation. To test this hypothesis, we have injected seven different 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-type calcium buffers into Pelvetia eggs many hours before tip growth normally starts. Critical final cell concentrations of each buffer prove to block outgrowth (as well as cell division) for up to 2 weeks. This critical inhibitory concentration is lowest for two buffers with dissociation constants or Kd values of 4-5 x 10(-6) M and increases steadily as the buffers' Kd values shift either below or above this optimal value to ones as low as 4 x 10(-7) M or as high as 9.4 x 10(-5) M. To analyze these results, we have derived an equation (based on the concept of facilitated diffusion) for the effects of diffusable calcium buffers on steady-state calcium gradients. The data fit this equation quite well if it is assumed that cytosolic free calcium at the incipient tip is normally kept at about 7 microM and, thus, far above the general cytosolic level.
The sphere organelle contains small nuclear ribonucleoproteins.
Authors: Gall J G , Callan H G
Published: May 2006

We show by immunofluorescence microscopy of amphibian oocyte nuclei that small nuclear ribonucleoproteins (snRNPs) occur in lampbrush chromosome loops, in a few dozen extrachromosomal organelles previously described as "spheres," and in thousands of smaller granules. Spheres are variable in size (up to approximately 20 microns in diameter in the newt Notophthalmus and approximately 10 microns in the frog Xenopus) and are easily distinguishable from nucleoli by morphology and composition. Spheres occur both free in the nucleoplasm and attached to specific chromosome loci, the sphere organizers. Oocyte nuclei of a cricket and a spider contain essentially similar organelles, suggesting that spheres may be common throughout the animal kingdom. We suggest that spheres play a role in the assembly of snRNP complexes for the nucleus comparable to the way that nucleoli assemble ribosomal RNP complexes for the cytoplasm.
A strategy to detect and isolate an intron-containing gene in the presence of multiple processed pseudogenes.
Authors: Davies B , Feo S , Heard E , Fried M
Published: May 2006

We have devised a strategy that utilizes the polymerase chain reaction (PCR) for the detection and isolation of intron-containing genes in the presence of an abundance of processed pseudogenes. The method depends on the genomic DNA sequence between the PCR primers spanning at least one intron in the gene of interest, resulting in the generation of a larger intron-containing PCR product in addition to the smaller PCR product amplified from the intronless pseudogenes. A unique intron probe isolated from the larger PCR product is used for the detection of intron-containing clones from recombinant DNA libraries that also contain pseudogene clones. This method has been used successfully for the selective isolation of an intron-containing rat L19 ribosomal protein gene in the presence of multiple pseudogenes. Analysis of a number of mammalian ribosomal protein multigene families by PCR indicates that they all contain only a single gene with introns.
cGMP induces phase shifts of a mammalian circadian pacemaker at night, in antiphase to cAMP effects.
Authors: Prosser R A , McArthur A J , Gillette M U
Published: May 2006

The suprachiasmatic nuclei (SCN) of mammals contain a circadian clock that synchronizes behavioral and physiological rhythms to the daily cycle of light and darkness. We have been probing the biochemical substrates of this endogenous pacemaker by examining the ability of treatments affecting cyclic nucleotide-dependent pathways to induce changes in the phase of oscillation in electrical activity of rat SCN isolated in brain slices. Our previous work has shown that daytime treatments that stimulate cAMP-dependent pathways induce phase shifts of the SCN pacemaker in vitro but treatments during the subjective night are without effect. In this study we report that the phase of SCN oscillation is reset by treatments that stimulate cGMP-dependent pathways, but only during the subjective night. Thus, the nocturnal period of SCN sensitivity to cGMP is in antiphase to the diurnal period of sensitivity to cAMP. These results suggest that cAMP and cGMP affect the SCN pacemaker through separate biochemical pathways intrinsic to the SCN. These studies provide evidence that changing biochemical substrates within the SCN circadian clock may underlie some aspects of differential temporal sensitivity of mammals to resetting stimuli.
Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli.
Authors: Ohara O , Dorit R L , Gilbert W
Published: May 2006

The genomic sequencing procedure is applied to the direct sequencing of uncharacterized regions of bacterial DNA by a "multiplex walking" approach. Samples of bulk Escherichia coli DNA are cut with various restriction enzymes, subjected to chemical sequencing degradations, run in a sequencing gel, and transferred to nylon membranes. When a labeled oligomer is hybridized to a membrane, a sequence ladder appears wherever the probe lies near a restriction cut. New probes, based on sequence that lies beyond other restriction sites, are then synthesized, and the membranes are reprobed to reveal new sequence. Repeated cycles of oligomer probe synthesis and subsequent reprobing permit rapid sequence walking along the genome. This oligomer walking technique was used to sequence the pyruvate kinase (EC 2.7.1.40) gene in E. coli without resorting to cloning or to library construction. The sequenced region was amplified by the polymerase chain reaction and subsequently transcribed and translated using both in vivo and in vitro systems, and the resultant gene product characterized to show that the gene encodes the type I isoform of pyruvate kinase.
Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro.
Authors: Dingwall C , Ernberg I , Gait M J , Green S M , Heaphy S , Karn J , Lowe A D , Singh M , Skinner M A , Valerio R
Published: May 2006

tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays. tat does not bind detectably to antisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-loop structures, or TAR DNA.
Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.
Authors: Curtin P T , Liu D P , Liu W , Chang J C , Kan Y W
Published: May 2006

Several lines of evidence suggest that erythroid-specific DNase I hypersensitive sites (HS) located far upstream of the human beta-globin gene are important in regulating beta-globin gene expression. We used the polymerase chain reaction technique to amplify and clone an 882-base-pair DNA fragment spanning one of these HS, designated HSII, which is located 54 kilobases upstream of the beta-globin gene. The cloned HSII fragment was linked to a human beta-globin gene in either the genomic (HSII-beta) or antigenomic (HSII-beta) orientation. These two constructs and a beta-globin gene alone (beta) were injected into fertilized mouse eggs, and expression was analyzed in liver and brain from day-16 transgenic fetuses. Five of 7 beta-transgenic fetuses expressed human beta-globin mRNA, but the level of expression per gene copy was low, ranging from 0.93 to 22.4% of mouse alpha-globin mRNA (average 9.9%). In contrast, 11 of 12 HSII-beta transgenic fetuses expressed beta-globin mRNA at levels per gene copy ranging from 31.3 to 336.6% of mouse alpha-globin mRNA (average 139.5%). Only three fetuses containing intact copies of the HSII-beta construct were produced. Two of three expressed human beta-globin mRNA at levels per gene copy of 179.2 and 387.1%. Expression of human beta-globin mRNA was tissue-specific in all three types of transgenic fetuses. These studies demonstrate that a small DNA fragment containing a single erythroid-specific HS can stimulate high-level human beta-globin gene expression in transgenic mice.
Deletion of the cytoplasmic region of the CD3 epsilon subunit does not prevent assembly of a functional T-cell receptor.
Authors: Transy C , Moingeon P , Stebbins C , Reinherz E L
Published: May 2006

The T-cell receptor (TCR) is a molecular complex comprised of a clonally restricted, immunoglobulin-like heterodimer (Ti), responsible for specific antigen recognition, and a set of monomorphic polypeptide CD3 subunits, termed gamma, delta, epsilon, zeta, and eta, presumed to be involved in transmembrane signaling events. To investigate the role of the CD3 epsilon subunit in signal transduction, we have transfected a murine hybridoma T-cell line with either wild-type or variant human CD3 epsilon cDNA that encodes a protein lacking 49 of the 55 cytoplasmic amino acid residues. Both wild-type and truncated CD3 epsilon human proteins assemble with endogenous murine CD3/Ti subunits to form functional surface TCRs: Anti-human CD3 epsilon monoclonal antibodies bind exclusively to these chimeric TCRs and trigger interleukin 2 production from the murine cells. Thus, the CD3 epsilon cytoplasmic domain is not required for assembly of the multimeric TCR. Furthermore, it is dispensable for the transduction of a stimulus delivered to the external part of the molecule, suggesting that interaction between the transmembrane and/or external regions of the other TCR chains is a prerequisite for transmembrane signaling.
Primarily chronic progressive and relapsing/remitting multiple sclerosis: two immunogenetically distinct disease entities.
Authors: Olerup O , Hillert J , Fredrikson S , Olsson T , Kam-Hansen S , Möller E , Carlsson B , Wallin J
Published: May 2006

HLA class II gene polymorphism was investigated in 100 patients with clinically definite multiple sclerosis (MS) by restriction fragment length polymorphism analysis of Taq I-digested DNA using DRB, DQA, and DQB cDNA probes. Twenty-six patients had primarily chronic progressive MS and 74 had relapsing/remitting MS. The latter group included patients with a secondary progressive evolution of symptoms. Both clinical forms of MS were found to be associated with the DRw15,DQw6 haplotype. In addition, primarily chronic progressive MS was positively associated with the DQB1 restriction fragment pattern seen in DR4,DQw8, DR7,DQw9, and DRw8, DQw4 haplotypes, as well as negatively associated with the Taq I DQB1 allelic pattern corresponding to the serological specificity DQw7. Relapsing/remitting MS was positively associated with the DQB1 allelic pattern observed in the DRw17,DQw2 haplotype. These three DQB1 alleles are in strong negative linkage disequilibria with DRw15. The two susceptibility markers of each clinical form of MS act additively in determining the genetic susceptibility, as the relative risks for individuals carrying both markers roughly equal the sum of respective risks. Different alleles of the DQB1 locus defined by restriction fragment length polymorphisms contribute to susceptibility and resistance to primarily chronic progressive MS as well as to susceptibility to relapsing/remitting MS. The observed immunogenetic heterogeneity between the different clinical forms of MS favors the hypothesis that primarily chronic progressive MS and relapsing/remitting MS are two distinct disease entities.
Golgi apparatus in chick skeletal muscle: changes in its distribution during end plate development and after denervation.
Authors: Jasmin B J , Cartaud J , Bornens M , Changeux J P
Published: May 2006

In the course of studies about the cellular and molecular mechanisms of motor end plate formation, the distribution of the Golgi apparatus (GA) has been investigated by immunofluorescence methods in chick skeletal muscle in primary culture and in innervated muscles of 15-day-old chicks. By using a monoclonal antibody directed against the GA, we confirmed the known distribution of the GA in myogenic cells: a juxtanuclear polarized organization in myoblasts and a perinuclear nonpolarized distribution in myotubes. In contrast, the innervated anterior latissimus dorsi muscle of "young adult" chicks displayed a focal distribution of GA that appeared restricted to areas located underneath the motor end plates identified by alpha-bungarotoxin fluorescent labeling of the acetylcholine receptor. Five days after denervation of anterior latissimus dorsi muscle, a striking reorganization and expansion of the GA was observed. The GA now showed a perinuclear distribution in close association with every nucleus of the muscle fibers as observed in myotubes. The focal distribution of the GA in innervated muscle fibers and its remodeling upon denervation are interpreted in terms of a model of local synthesis, processing, and routing of acetylcholine receptor to the end plate and of regulation of these processes by functional motor innervation.