bluuebunny/crossref_metadata_2025 huggingface dataset:
Retroviral transfer of genes into canine hemopoietic progenitor cells in culture: a model for human gene therapy.
Authors: Kwok W W , Schuening F , Stead R B , Miller A D
Published: May 2006

Amphotropic retroviral vectors containing either a mutant dihydrofolate reductase gene (DHFR) or the bacterial neomycin phosphotransferase gene (neo) were used to infect canine hemopoietic cells. We report successful transfer and expression of the DHFR and neo genes in canine hemopoietic progenitor cells (colony-forming units, granulocyte/macrophage) as measured by the ability of the viruses to confer resistance to either methotrexate or the aminoglycoside G418, respectively. Transfer was achieved in the absence of helper virus by using retrovirus packaging cell lines. Successful transfer of these genes into canine hemopoietic progenitor cells in vitro indicates the feasibility of gene transfer into canine marrow for autologous reconstitution. Studies of transfer of new genetic information into a large, outbred animal such as the dog will provide a preclinical model for future gene therapy in humans.
Cholinergic suppression: a postsynaptic mechanism of long-term associative learning.
Authors: Morielli A D , Matera E M , Kovac M P , Shrum R G , McCormack K J , Davis W J
Published: May 2006

Food avoidance learning in the mollusc Pleurobranchaea entails reduction in the responsiveness of key brain interneurons in the feeding neural circuitry, the paracerebral feeding command interneurons (PCNs), to the neurotransmitter acetylcholine (AcCho). Food stimuli applied to the oral veil of an untrained animal depolarize the PCNs and induce the feeding motor program (FMP). Atropine (a muscarinic cholinergic antagonist) reversibly blocks the food-induced depolarization of the PCNs, implicating AcCho as the neurotransmitter mediating food detection. AcCho applied directly to PCN somata depolarizes them, indicating that the PCN soma membrane contains AcCho receptors and induces the FMP in the isolated central nervous system preparation. The AcCho response of the PCNs is mediated by muscarinic-like receptors, since comparable depolarization is induced by muscarinic agonists (acetyl-beta-methylcholine, oxotremorine, pilocarpine), but not nicotine, and blocked by muscarinic antagonists (atropine, trifluoperazine). The nicotinic antagonist hexamethonium, however, blocked the AcCho response in four of six cases. When specimens are trained to suppress feeding behavior using a conventional food-avoidance learning paradigm (conditionally paired food and shock), AcCho applied to PCNs in the same concentration as in untrained animals causes little or no depolarization and does not initiate the FMP. Increasing the concentration of AcCho 10-100 times, however, induces weak PCN depolarization in trained specimens, indicating that learning diminishes but does not fully abolish AcCho responsiveness of the PCNs. This study proposes a cellular mechanism of long-term associative learning--namely, postsynaptic modulation of neurotransmitter responsiveness in central neurons that could apply also to mammalian species.
Inquiries into the structure-function relationship of ribonuclease T1 using chemically synthesized coding sequences.
Authors: Ikehara M , Ohtsuka E , Tokunaga T , Nishikawa S , Uesugi S , Tanaka T , Aoyama Y , Kikyodani S , Fujimoto K , Yanase K
Published: May 2006

The genes for ribonuclease T1 and its site-specific mutants were chemically synthesized and introduced to Escherichia coli. All enzymes were fusion products produced by joining the synthetic gene at specific restriction sites to the synthetic gene for human growth hormone in a plasmid containing the E. coli trp promoter. The fusion protein from this plasmid contained 66% of the amino-terminal sequences of the human growth hormone, which were recognizable immunologically. RNase T1 or its mutants were cleaved from the fusion protein with cyanogen bromide. The synthetic RNase T1 endowed with the revised wild-type triad Gly-Ser-Pro, residues 71-73, was fully functional, readily hydrolyzing pGpC bonds, whereas a mutant enzyme having the originally reported, erroneous triad Pro-Gly-Ser was totally inactive. Various amino acid substitutions were also introduced to the guanosine recognition region comprised of residues 42-45, Tyr-Asn-Asn-Tyr. Substitution of either of the tyrosine residues noted above with phenylalanine had no dramatic effect on the enzyme's function. Replacement of asparagine-43 with arginine or alanine also caused only a small change in the hydrolyzing activity--a mutant enzyme maintained greater than 50% of the wild-type activity. In sharp contrast, when aspartic acid or alanine was substituted for asparagine-44, the activity was dramatically reduced to a few percent of the wild-type activity.
Transcriptional regulation of steroid hydroxylase genes by corticotropin.
Authors: John M E , John M C , Boggaram V , Simpson E R , Waterman M R
Published: May 2006

Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P-450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues.
Cloning and sequencing of cDNA of bovine N-acetylglucosamine (beta 1-4)galactosyltransferase.
Authors: Narimatsu H , Sinha S , Brew K , Okayama H , Qasba P K
Published: May 2006

Galactosyltransferases constitute a family of enzymes, each member of which transfers galactose from UDPgalactose to a specific acceptor molecule, generating a specific galactose-acceptor linkage. Two synthetic oligonucleotides, 27mer and 21mer, were synthesized, based on the amino acid sequences of two peptides derived from bovine milk N-acetylglucosaminide (beta 1-4)galactosyltransferase (EC 2.4.1.90), and used as hybridization probes to isolate cDNA clones for galactosyltransferase from a bovine mammary gland cDNA library. One of the plasmids, designated pLbGT-1, contains an insert of about 3.7 kilobases that hybridizes to both of the probes and encodes the amino acid sequences of five peptides obtained from bovine milk (beta 1-4)galactosyltransferase. A second plasmid, designated pLbGT-2, contains an insert of about 4.1 kilobases that hybridizes to only the 27mer and that encodes a polypeptide containing the sequence of the carboxyl-terminal 120 residues identical to the peptide encoded by pLbGT-1; the rest of the protein sequence, however, does not contain known sequences from bovine galactosyltransferase. The two cDNAs contain a 3'-untranslated region of about 2.7 kilobases that includes two copies of the Alu-equivalent sequences. pLbGT-1 and pLbGT-2 hybridize to mRNAs of various sizes obtained from the bovine and rat mammary gland and the human mammary tumor cell line MCF-7, with the longest mRNA from each species being around 4.5 kilobases. The results show that pLbGT-1 is a cDNA clone for bovine (beta 1-4)galactosyltransferase, and pLbGT-2 encodes a protein that is structurally and may be functionally related to transferases.
Phosphorylation of the rat hepatic polymeric IgA receptor.
Authors: Larkin J M , Sztul E S , Palade G E
Published: May 2006

In vivo labeling with [35S]cysteine has identified three transmembrane forms of the rat hepatic polymeric IgA receptor: (i) a 105-kDa core glycosylated precursor; (ii) a terminally glycosylated 116-kDa intermediate; and (iii) a mature 120-kDa form. In the current study we show that the 120-kDa form is phosphorylated. After in vivo labeling with [32P]orthophosphate, all receptor forms were immunoprecipitated from hepatic total microsomes (TM) (with an antireceptor antiserum), separated by NaDodSO4/PAGE, and detected by autoradiography. The 120-kDa form was selectively phosphorylated, whereas the 116- and 105-kDa forms incorporated no detectable 32P. To determine the topology of the phosphorylation sites, hepatic TM isolated from rats labeled in vivo with either [35S]cysteine or [32P]orthophosphate were treated with trypsin. TM were solubilized and receptors were immunoprecipitated from lysates. With increasing trypsin concentrations, the [35S]cysteine-labeled receptor triplet was degraded to a trypsin-resistant doublet of approximately 95 and 85 kDa, indicating that approximately 20 kDa was removed from the receptor endodomain by trypsin. The same treatment removed all detectable 32P from labeled receptors. Furthermore, no 32P was detected in the 80-kDa biliary form of the receptor. Serine was identified as the only phosphorylated residue in acid hydrolysates of 32P-labeled immunoprecipitated receptor. These findings indicate that (i) the 120-kDa form is the only phosphorylated species of the receptor; and (ii) the phosphorylated residues are serine(s) located in the endodomain of the protein.
Lowering extracellular Na+ concentration releases autocrine growth factors from renal epithelial cells.
Authors: Walsh-Reitz M M , Gluck S L , Waack S , Toback F G
Published: May 2006

Sodium influx is an important early signal during the onset of mitogenesis in many types of cells. From this observation, one would predict that a decrease in extracellular Na+ concentration might retard cell proliferation. We tested this prediction by exposing sets of cultures of monkey kidney epithelial cells (BSC-1 line) to medium with progressively reduced concentrations of Na+, and we measured the effect on cell multiplication. Unexpectedly, a reduction of the Na+ concentration from 155 mM (control) to 130 mM stimulated proliferation of epithelial cells but not of fibroblasts. Exposure of BSC-1 cells to low Na+ medium for 5 min was sufficient to commit them to accelerated growth. Further study revealed that the cells released two growth factors during this period: anionic proteins with apparent molecular weights of 6200 and 9000 whose properties differ from those of other known growth factors. Thus, a reduction in extracellular Na+ concentration apparently signaled the rapid release of autocrine growth factors that stimulate renal epithelial cell multiplication.
Homeo box gene expression in anterior and posterior regions of the Drosophila embryo.
Authors: Hoey T , Doyle H J , Harding K , Wedeen C , Levine M
Published: May 2006

The homeo box is a 180-base-pair coding sequence that has been implicated in the control of Drosophila development. A common feature of the nine previously reported homeo box genes is their involvement in the establishment of the segmentation pattern of the embryo. In this report we describe the isolation and properties of two additional homeo box genes, F90-2 and S67. Transcripts encoded by the two genes are detected in embryonic tissues that derive from regions near the anterior and posterior poles of the embryo, which are outside the limits of expression of known homeotic genes. These results suggest that at least some homeo box genes specify positional identity along the anterior-posterior body axis that is independent of the process of segmentation.
Mammary number and litter size in Rodentia: The “one-half rule”
Authors: Gilbert Avery Nelson
Published: May 2006

Litter size and mammary number in the mammalian order Rodentia show a significant positive correlation. Mean litter size is typically one-half the number of available mammaries, while maximum litter size approximates mammary number. Similar relationships are found in the families Muridae, Cricetidae, and Sciuridae. The relationship of litter size to mammary number is significantly different between the arboreal and terrestrial squirrels, and between the hystricomorph and nonhystricomorph rodents. Mammary number may have operated as a selective constraint on litter size over evolutionary time.
Forskolin increases the rate of acetylcholine receptor desensitization at rat soleus endplates.
Authors: Middleton P , Jaramillo F , Schuetze S M
Published: May 2006

We have studied the function of acetylcholine (AcCho) receptors (AcChoRs) in rat soleus endplates before and after exposing the muscles to forskolin, a potent activator of adenylate cyclase. AcChoR function was tested by recording the membrane depolarization evoked by pulses of ionophoretically applied AcCho. Brief (2 msec) AcCho pulses delivered at 7 Hz evoked constant responses at untreated endplates. In contrast, after 10-100 microM forskolin was added to the bath, responses to similar pulse trains fell by as much as 80% within 1 sec. AcCho sensitivity recovered completely in less than 1 min after the pulses were stopped but fell again when the pulses were resumed. Similarly, longer (1 sec) ionophoretic AcCho pulses evoked roughly constant responses at control endplates, but after forskolin treatment the depolarization fell by one-half within less than 200 msec. These results indicate that forskolin increases the rate at which AcChoRs desensitize when exposed to agonist. Focal extracellular recordings showed that 20-100 microM forskolin also increased the decay rate of miniature endplate currents, indicating that forskolin may decrease AcChoR channel open time. Inhibitors of cAMP phosphodiesterase increased the potency of forskolin. When used alone, these inhibitors had effects similar to those of forskolin but smaller. Patch-clamp experiments indicated that forskolin at 100 microM may also interact with AcChoR channels directly, but at 20 microM this effect is negligible. Therefore, it is likely that the forskolin effects were mediated primarily by increased levels of intracellular cAMP.
Amino acid sequence of mammalian elongation factor 2 deduced from the cDNA sequence: homology with GTP-binding proteins.
Authors: Kohno K , Uchida T , Ohkubo H , Nakanishi S , Nakanishi T , Fukui T , Ohtsuka E , Ikehara M , Okada Y
Published: May 2006

Complementary DNA clones, pHEW1 and pRE2, coding for hamster and rat polypeptide chain elongation factor 2 (EF-2), respectively, were isolated and sequenced. It was shown that the cDNA insert in pHEW1 contains a 2574-base-pair open reading frame coding for an 857-amino acid polypeptide with Mr 95,192, excluding the initiation methionine. Comparative studies of sequence homology among EF-2 and several GTP-binding proteins show that five regions in the amino-terminal position of EF-2, corresponding to about 160 amino acids, show homology with GTP-binding proteins, including protein synthesis elongation and initiation factors, mammalian ras proteins, and transducin. The carboxyl-terminal half of EF-2 contains several regions that have 34-75% homology with bacterial elongation factor G. These results suggest that the amino-terminal region of EF-2 participates in the GTP-binding and GTPase activity whereas the carboxyl-terminal region interacts with ribosomes. Finally, the sequence provides direct evidence that diphthamide (2-[3-carboxy-amido-3-(trimethylammonio)propyl]histidine), the site of ADP-ribosylation by diphtheria toxin, is produced by post-translational modification of a histidine residue in the primary translational product.
Adenovirus type 12 early region 1A proteins repress class I HLA expression in transformed human cells.
Authors: Vasavada R , Eager K B , Barbanti-Brodano G , Caputo A , Ricciardi R P
Published: May 2006

The adenovirus type 12 (Ad12) early region 1A (E1A) gene is thought to play a major role in repressing class I major histocompatibility complex expression in transformed rodent cells. However, since transformation by adenovirus requires both E1A and E1B genes, it has not been demonstrated whether the Ad12 E1A gene acts alone or synergistically with the E1B gene to accomplish this effect. Moreover, it is not known whether the repression of class I antigen synthesis by Ad12-transforming gene products occurs only in rodent cells. We show that the Ad12 E1A gene, in the absence of the E1B gene, is capable of greatly reducing the levels of class I HLA antigens and mRNAs in primary human cells transformed by the E1A gene of Ad12 and the large tumor antigen (T-antigen) gene of BK virus; control cells transformed by BK virus T-antigen gene alone or the highly related simian virus 40 T-antigen gene showed no apparent alteration in class I HLA expression. Human recombinant interferon gamma was able to restore synthesis of class I HLA antigens in transformed cells that produced Ad12 E1A proteins, indicating that these cells were not deficient for class I genes. These results strongly indicate that the Ad12 E1A proteins modulate class I gene expression by similar mechanisms in both transformed rodent and human cells.
Pancreatic B cells possess defense mechanisms against cell-specific toxicity.
Authors: Pipeleers D , Van de Winkel M
Published: May 2006

Insulin-dependent diabetes develops when more than 90% of the insulin containing B cells are destroyed. The present study investigates whether the target B cells can counteract the damaging effects of cytotoxic substances. Purified islet cells were first exposed for 3-10 min to t-butylhydroperoxide, alloxan, streptozotocin, or B-cell surface antibodies plus complement, then cultured for 20 hr before the percent of dead cells was counted. t-Butylhydroperoxide destroyed all islet cell types whereas the three other agents exerted a dose-dependent toxicity upon islet B cells only. The survival of drug- and complement-treated cells varied with the culture conditions present between the initial cellular attack and the moment of cell death. For the four B-cell toxic agents tested, an increase in medium glucose following any of these treatments reduced the percent of dead cells. This protective effect was not observed with galactose or fructose, nor could it be induced in islet non-B cells; it was additive to the protective action glucose induced during preincubation of the cells prior to their exposure to certain cytotoxic agents such as alloxan. Nicotinamide also enhanced the survival of drug-treated B cells, irrespective of the damaging compound. The vitamin was most effective when applied immediately after the initial drug or complement treatment; it also protected islet non-B cells--in contrast to glucose. The present in vitro study has led to the recognition of defense mechanisms in pancreatic B cells. Physiologic compounds such as glucose and nicotinamide were found to stimulate islet B cells to counteract the damaging effects of B-cell toxic conditions. It is conceivable that the events involved in this protection are implicated in the pathogenesis and/or prevention of insulin-dependent diabetes.
Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.
Authors: Bradley M O , Kraynak A R , Storer R D , Gibbs J B
Published: May 2006

These studies have compared the ability of NIH 3T3 cells containing different ras oncogenes to form tumor nodules in the lungs of nude mice after tail vein injection. The genes studied include the normal cellular and bladder tumor ras genes, recombinant viral/cellular ras genes, recombinant yeast/mammalian ras genes, and a constructed gene with yeast RAS1 sequences significantly modified by deletions and an oncogenic mutation. The results show that NIH 3T3 cells containing these genes readily form lethal tumor nodules in the lungs of nude mice after tail vein injection. No control NIH 3T3 cells formed lung tumors within 66 days. Although there were some quantitative differences in the potencies of the various lines, the striking conclusion is that NIH 3T3 cells transformed by either normal or activated mammalian ras genes form approximately equal numbers of experimental lung metastases. In addition, cells transformed by a significantly modified yeast RAS1 gene containing a purposefully introduced oncogenic mutation were also equally active in this assay. The amount of p21 (the 21-kDa protein encoded by ras), as measured by immunoprecipitation, was approximately the same in the parent lines before injection as in the tumors recovered after injection. This result indicates that there is no selection for metastatic sublines containing larger quantities of p21. Transfection of EJ bladder tumor ras DNA into NIH 3T3 cells followed by injection 3 days later into the tail veins of nude/beige mice indicated that the EJ ras gene can confer a metastatic phenotype within 3.5 cell generations without selection or clonal growth in vitro. Thus, the biochemical changes initiated after introduction of the c-Ha-ras gene into NIH 3T3 cells result in the almost immediate acquisition of phenotypes necessary for experimental metastasis.
Discrimination of muscle and neuronal Na-channel subtypes by binding competition between [3H]saxitoxin and mu-conotoxins.
Authors: Moczydlowski E , Olivera B M , Gray W R , Strichartz G R
Published: May 2006

The effect of two mu-conotoxin peptides on the specific binding of [3H]saxitoxin was examined in isolated plasma membranes of various excitable tissues. mu-Conotoxins GIIIA and GIIIB inhibit [3H]saxitoxin binding in Electrophorus electric organ membranes with similar KdS of approximately equal to 50 X 10(-9) M in a manner consistent with direct competition for a common binding site. GIIIA and GIIIB similarly compete with the majority (80-95%) of [3H]saxitoxin binding sites in rat skeletal muscle with KdS of approximately 25 and approximately 140 X 10(-9) M, respectively. However, the high-affinity saxitoxin sites in lobster axons, rat brain, and rat heart are virtually insensitive to GIIIA concentrations up to 10 microM. These results and previously published data suggest that three Na-channel subtypes can be distinguished on the basis of toxin pharmacology: Na channels of skeletal muscle and Electrophorus electroplax have high affinity for mu-conotoxins and tetrodotoxin, neuronal Na channels have low affinity for mu-conotoxins and high affinity for tetrodotoxin, while heart Na channels and a similar subtype also found in denervated muscle have low affinity for both mu-conotoxin and tetrodotoxin.
Potential proteolytic activity of human plasma fibronectin.
Authors: Keil-Dlouha V , Planchenault T
Published: May 2006

Evidence is presented that fibronectin (FN) polypeptide chain contains a latent proteinase. Human plasma FN was cleaved with cathepsin D into three main fragments: 140-kDa and 70-kDa single-chain and 140-kDa double-chain polypeptides. Their separation was achieved according to their affinity for heparin-Sepharose. A single-chain 140-kDa fragment (H-1) was eluted in the first peak. This peptide corresponds to the already described fragment that originates from the central part of FN; it contains a low-affinity heparin-binding site, one free SH group, and a cell-binding site. After reduction and further purification by preparative polyacrylamide gel electrophoresis, this fragment revealed a spontaneous decomposition, which could be attributed to proteolytic degradation. The subfragments, ranging from 25 to 95 kDa, yielded the same proteolytically active doublet of 28-30 kDa when tested by NaDodSO4/polyacrylamide gel electrophoresis in a gel containing copolymerized gelatin or fibrinogen. The proteolytic activity was inhibited by specific SH proteinase inhibitors. The proteinase forms a labeled complex after its incubation with 125I-labeled cystatin. Neither FN, cathepsin D, nor any products from previous purification steps were proteolytically active under the conditions of the assay. It was suggested that the same fragment may also yield an inhibitor, since structural analogies were found between the cell-binding region of FN and SH proteinase inhibitors.
Tubulin: a factor necessary for the synthesis of both Sendai virus and vesicular stomatitis virus RNAs.
Authors: Moyer S A , Baker S C , Lessard J L
Published: May 2006

Tubulin acts as a positive transcription factor for in vitro RNA synthesis by two different negative-strand viruses: Sendai virus, a paramyxovirus; vesicular stomatitis virus (VSV), a rhabdovirus. A monoclonal antibody directed against beta-tubulin completely inhibited not only mRNA synthesis and RNA replication catalyzed in vitro by extracts of cells infected with either virus but also mRNA synthesis by detergent-disrupted purified virions. The synthesis of both a leader-like RNA and the NP mRNA directed by detergent-disrupted purified Sendai virions was shown to be totally dependent on the addition of purified tubulin. The addition of purified tubulin, although not required, also stimulated mRNA synthesis directed by detergent-disrupted VSV virions 2- to 7-fold. Finally, there appears to be an association between tubulin and the L protein of VSV, since both monoclonal and polyclonal anti-tubulin antisera specifically immunoprecipitated not only tubulin but also the L protein of two different VSV serotypes from the soluble protein fraction of infected cells.
Complete nucleotide and deduced amino acid sequence of bovine phenylethanolamine N-methyltransferase: partial amino acid homology with rat tyrosine hydroxylase.
Authors: Baetge E E , Suh Y H , Joh T H
Published: May 2006

We report here the isolation of a cDNA clone containing the full coding region of bovine phenylethanolamine N-methyltransferase (PNMTase, EC 2.1.1.28, S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase). The complete nucleotide sequence of the cDNA has been determined, and the amino acid sequence of PNMTase deduced. Cultured cells transfected with an expression vector containing this cDNA produced high levels of PNMTase enzymatic activity. Antibodies specific for tyrosine hydroxylase [EC 1.14.16.2, tyrosine 3-monooxygenase; L-tyrosine, tetrahydrobiopterine: oxygen oxidoreductase (3-hydroxylating)], the first enzyme in the catecholamine pathway, possess a striking affinity for the PNMTase protein synthesized in vitro. Comparison of the deduced amino acid sequence of bovine PNMTase to rat tyrosine hydroxylase reveals that PNMTase shares significant homology with tyrosine hydroxylase and supports previous protein and immunological data suggesting that the catecholamine biosynthetic enzymes are structurally related.
Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein.
Authors: Tullius T D , Dombroski B A
Published: May 2006

A method has been developed for making "footprints" of proteins bound to DNA. The hydroxyl radical, generated by reduction of hydrogen peroxide by iron(II), is the reagent used to cut the DNA. Hydroxyl radical breaks the backbone of DNA with almost no sequence dependence, so all backbone positions may be monitored for contact with protein. In addition to defining the DNA sequence in contact with the protein, hydroxyl radical footprints embody structural information about the DNA-protein complex. For example, hydroxyl radical footprints of the bacteriophage lambda repressor and Cro protein show directly that these proteins are bound to only one side of the DNA helix. Additional contacts of lambda repressor and Cro protein with DNA, not observed by other chemical footprinting methods, are revealed by hydroxyl radical footprinting.
Yeast gene required for spindle pole body duplication: homology of its product with Ca2+-binding proteins.
Authors: Baum P , Furlong C , Byers B
Published: May 2006

Saccharomyces cerevisiae strains bearing temperature-sensitive alleles of the cell division cycle gene CDC31 are specifically defective in duplication of the spindle pole body, the microtubule-organizing center of yeast. To define the function encoded by CDC31 more fully, we have isolated genomic clones of the gene by selection for complementation of a temperature-sensitive allele. The locus from which the clone was derived was marked by integration of a nutritional marker and found by meiotic mapping to cosegregate with CDC31. The polypeptide sequence of the open reading frame in the CDC31 gene was determined and compared with the sequences of other known proteins. This revealed significant homology with the calmodulins and other members of the Ca2+-binding protein family. On the basis of comparison with these related proteins, it is evident that the CDC31 gene product has at least two binding sites for Ca2+ and is also homologous with other regions of the calmodulin sequence. We propose that Ca2+ fluxes within the yeast cell play a key role in spindle pole body duplication and consequently in the organization of the microtubule arrays.
Single nisoldipine-sensitive calcium channels in smooth muscle cells isolated from rabbit mesenteric artery.
Authors: Worley J F , Deitmer J W , Nelson M T
Published: May 2006

Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.
Bacterial lipopolysaccharides, phorbol myristate acetate, and zymosan induce the myristoylation of specific macrophage proteins.
Authors: Aderem A A , Keum M M , Pure E , Cohn Z A
Published: May 2006

We demonstrate stimulus-dependent incorporation of exogenously added [3H]myristic acid into specific macrophage proteins. In control unstimulated cells an 18-kDa protein is the major acylated species. In cells incubated with bacterial lipopolysaccharide (LPS), or its monoacyl glucosamine phosphate derivative, fatty acid is incorporated into proteins with molecular mass of 68 kDa and a doublet of approximately 42-45 kDa. Phorbol 12-myristate 13-acetate (PMA) or a phagocytic stimulus (zymosan) promotes the acylation of a similar array of proteins. However, PMA and zymosan also promote the myristoylation of unique proteins of 92 and 50 kDa. The fatty acid associated with each of the acylated proteins is myristic acid. The myristate is probably linked to the proteins through amide bonds, since it is not released by treatment with hydroxylamine. Palmitate and arachidonate are not incorporated into proteins in the same manner. Temporal analysis revealed that LPS-induced proteins are myristoylated by 30 min, while the 50-kDa protein myristoylated in response to PMA is labeled later. Most myristoylated proteins appear to be associated with the membrane fraction. Macrophages from C3H/HeJ mice, which do not respond to LPS, do not show any LPS-dependent protein acylation. Interestingly, zymosan and PMA induce the myristoylation of the 50-kDa protein in C3H/HeJ macrophages, but not the acylation of the 68-kDa and 42-kDa doublet species. We suggest that myristoylation of specific proteins is an intermediary in the capacity of LPS, PMA, and zymosan to alter macrophage functions such as arachidonic acid metabolism.
Covalent linkage of ribonuclease S-peptide to microinjected proteins causes their intracellular degradation to be enhanced during serum withdrawal.
Authors: Backer J M , Dice J F
Published: May 2006

The amino-terminal 20 amino acids are required for microinjected ribonuclease A (RNase A) to be taken up by lysosomes and degraded at an enhanced rate during serum withdrawal. We used water-soluble carbodiimides to covalently attach the RNase S-peptide (residues 1-20) to [3H]RNase S-protein (residues 21-124) at unspecified locations. We then measured catabolism of the [3H]S-protein-S-peptide conjugate after its microinjection into human diploid fibroblasts. The attached S-peptide caused the degradation of S-protein to be enhanced 2-fold in the absence of serum. Control experiments showed that degradation of [3H]RNase S-protein remained unresponsive to serum after conjugation with the inactive fragment, RNase S-peptide (residues 1-10). Covalent attachment of RNase S-peptide had a similar effect on the catabolism of two other proteins. Degradation rates of microinjected 125I-labeled lysozyme and 125I-labeled insulin A chain are normally unresponsive to serum withdrawal. However, breakdown rates of microinjected 125I-labeled lysozyme-S-peptide and 125I-labeled insulin A chain-S-peptide conjugates were increased 2-fold during serum deprivation. We suggest that RNase S-peptide acts as a "single sequence" that directs cytosolic proteins to lysosomes through a pathway that is activated by deprivation conditions.
Adenovirus early region 1A protein increases the number of template molecules transcribed in cell-free extracts.
Authors: Leong K , Berk A J
Published: May 2006

Protein encoded by adenovirus early region 1A (E1A) stimulates transcription from adenovirus promoters in vivo. Here we show that this effect can be observed in vitro. In a run-off transcription assay from the adenovirus serotype 2 (Ad2) major late promoter, extracts prepared 20 hr postinfection were 5-15 times more active than mock-infected-cell extracts prepared in parallel. Similar results were observed for in vitro transcription from the protein IX and E3 adenovirus promoters, whereas a 2-fold increase was observed for the human beta-globin promoter. The increased activities of infected-cell extracts did not depend on the expression of viral late proteins or the small E1A-encoded proteins but did require expression of the large E1A protein. These results are consistent with the large E1A protein stimulating transcription in vitro as it does in vivo. By limiting in vitro transcription to one initiation per template, we found that the higher activity of an infected-cell extract was due to an increase in the number of templates transcribed. These results suggest that the large E1A protein either increases the number of active transcription factors in infected cells or facilitates the interaction of cellular transcription factors with promoter DNA.
11-Dehydrothromboxane B2: a quantitative index of thromboxane A2 formation in the human circulation.
Authors: Catella F , Healy D , Lawson J A , FitzGerald G A
Published: May 2006

In human plasma, 11-dehydrothromboxane (TX) B2 is a major long lived metabolite (t1/2 45 min) formed from infused TXB2, the hydration product of biologically active TXA2. Plasma concentrations of TXB2 itself are readily confounded by ex vivo platelet activation and, theoretically, an enzymatic derivative of this compound, not subject to formation in whole blood, would more accurately reflect TXA2 formation in vivo. To address this hypothesis, we developed a sensitive assay for both 11-dehydro-TXB2 and TXB2, using capillary gas chromatography/negative-ion chemical ionization mass spectrometry. We established that whole blood possesses a minor capacity to form 11-dehydro-TXB2, attributable to nonenzymatic formation in erythrocytes. However, the nonenzymatic formation of 11-dehydro-TXB2 was not a practical limitation to its use as an index of TX biosynthesis. Blood was drawn from healthy volunteers (i) via an indwelling catheter at the time of insertion and at 30, 60, 90, 180, and 240 min thereafter and (ii) via separate venipunctures at 0 time and at 90 and 240 min thereafter. Plasma TXB2 drawn via the catheter at baseline (66 +/- 63 pg/ml) was substantially greater than the maximal estimate of endogenous TXB2 (1-2 pg/ml) in plasma [Patrono, C., Ciabattoni, G., Pugliese, F., Perruci, A., Blair, I. A. & FitzGerald, G. A. (1986) J. Clin. Invest. 77, 590-594] and increased in magnitude and variance over time (339 +/- 247 pg/ml at 240 min). By contrast, 11-dehydro-TXB2 did not change significantly in the sequential catheter samples or in the samples drawn by separate venipuncture. Basal plasma concentrations in volunteers were depressed by pretreatment with 325 mg of aspirin. Furthermore, the range of concentrations in patients with severe atherosclerosis in whom urinary 2,3-dinor-TXB2 was increased was significantly higher (5-50 pg/ml, P less than 0.01) than in healthy subjects (0.9-1.8 pg/ml). Concentrations of 11-dehydro-TXB2 were increased in patients who had recently suffered a pulmonary embolism to a greater extent than either the 11-dehydro-13,14-dihydro-15-keto-TXB2 or the 2,3-dinor-TXB2 metabolites in plasma. These results indicate that plasma TXB2 is readily confounded by platelet activation ex vivo. Measurement of enzymatic metabolites of TXB2 minimizes this problem. The 11-dehydro metabolite is the most appropriate analytic target to detect phasic release of TXA2 in the human circulation, such as might occur in human syndromes of platelet activation.
Down-regulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific binding sequence within a receptor cDNA clone.
Authors: Okret S , Poellinger L , Dong Y , Gustafsson J A
Published: May 2006

A cDNA clone for the rat glucocorticoid receptor (GR) was used to study mechanisms of GR mRNA regulation. Treatment of rat hepatoma culture cells with 0.5 microM dexamethasone caused a small, initial increase in the GR mRNA level after 6 hr as well as a 50% to 95% reduction of the GR mRNA level after 24 hr of incubation when studied by RNA blot hybridization. After 72 hr, the initial GR mRNA level was restored. The down-regulation of GR mRNA levels appears to be independent of protein synthesis, since it also was observed in the presence of cycloheximide. However, cycloheximide caused a 4-fold increase in intracellular levels of GR mRNA. Using an immunoprecipitation assay, we could demonstrate that the GR specifically interacts with a GR cDNA clone, which represents a 2.6-kilobase fragment of the 3' nontranslated region of the GR mRNA. Nuclease protection experiments indicate the presence of several internal GR-binding regions in the above fragment.
Identification and characterization of the NMYC gene product in human neuroblastoma cells by monoclonal antibodies with defined specificities.
Authors: Ikegaki N , Bukovsky J , Kennett R H
Published: May 2006

Increased N-myc (now designated NMYC in human gene nomenclature) gene expression has been detected at the transcriptional level in certain types of neoplasms. As yet, the N-myc gene product has not been identified. To detect and characterize the N-myc gene product, we have developed monoclonal antibodies against the putative N-myc gene product made in Escherichia coli as a fusion protein. The antibodies that recognize the N-myc-specific regions were selected on the basis of their reactivities to different portions of the fusion protein. These monoclonal antibodies detect a pair of closely migrating polypeptides of 60 and 63 kDa in nuclear fractions of human neuroblastoma cells. The relative levels of the polypeptides are roughly proportional to the level of N-myc transcripts present in a panel of neuroblastoma lines. These two polypeptides have a half-life of approximately equal to 35 min, and they are indistinguishable from each other by their epitopic profiles.
Use of monosomics to map cloned DNA fragments in maize
Authors: Helentjaris Tim , Weber David F. , Wright Scott
Published: May 2006

Monosomic maize (
Zea mays
L.) plants were generated using the
r-X1
deficiency system, and the monosomy was confirmed both genetically and cytologically. Genomic DNAs prepared from a group of plants, each monosomic for one chromosome, were digested with restriction enzymes, electrophoresed in agarose gels, and blotted onto nylon membranes. Hybridization of labeled cloned DNA fragments to these blots proved efficient in assigning each fragment to the chromosome from which it originated. Cloned DNA has previously contributed to loci detection through the use of the restriction fragment length polymorphisms (RFLPs), these loci subsequently being arranged into linkage groups by segregation analysis. In this study, these linkage groups were assigned to specific chromosomes, facilitating the construction of a linkage map for maize containing 112 RFLP loci. An additional 35 loci were also assigned to chromosomes by this method; however, the linkage relationships of these loci to other RFLP loci on each chromosome remains undetermined.
Molecular analysis of the DNA sequences involved in the transcriptional regulation of the phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.
Authors: Bergman L W , McClinton D C , Madden S L , Preis L H
Published: May 2006

The expression of the PHO5 gene of Saccharomyces cerevisiae is transcriptionally regulated in response to the level of inorganic phosphate present in the growth medium. We have identified, by DNA deletion analysis, the sequences (upstream activator sequences) that mediate this response. The sequence 5' CTGCACAAATG 3' is present in two copies located within a 60-base-pair region. The presence of a single copy of the sequence is sufficient for the phosphate-mediated transcriptional response. In addition, a DNA fragment that contains two copies of this sequence will act to repress transcription of a CYC1-lacZ fusion when placed either upstream or downstream of the CYC1 activator sequence.
Patients with IgA nephropathy have circulating anti-basement membrane antibodies reacting with structures common to collagen I, II, and IV.
Authors: Cederholm B , Wieslander J , Bygren P , Heinegård D
Published: May 2006

It has recently been shown that patients with IgA nephropathy have circulating IgA antibodies against extracts of human glomerular basement membrane. The present study extends those observations and demonstrates by using ELISA and immunoblotting techniques that patients with IgA nephropathy have circulating IgA antibodies against collagen IV alpha chains. The antigenicity of the alpha chains could be destroyed by digestion with collagenase, which indicates that the antigenic site(s) is located on the triple helical part of collagen IV. Furthermore, it was shown by inhibition tests that the IgA antibodies are directed against epitopes also present in collagen I and II isolated after pepsin digestion.
Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides.
Authors: Davis B D , Chen L L , Tai P C
Published: May 2006

Among the pleiotropic effects of aminoglycosides, their irreversible uptake and their blockade of initiating ribosomes have appeared to explain their bactericidal action, while the contributions of translational misreading and membrane damage and the mechanism of that damage have remained uncertain. We now present evidence that incorporation of misread proteins into the membrane can account for the membrane damage. The bactericidal action thus appears to result from the following sequence, in which each step is essential: slight initial entry of the antibiotic; interaction with chain-elongating ribosomes, resulting in misreading; incorporation of misread protein into the membrane, creating abnormal channels; increased (and irreversible) entry through these channels, and hence increased misreading and formation of channels; and, finally, blockade of initiating ribosomes. This mechanism can account for several previously unexplained observations: that streptomycin uptake requires protein synthesis during, but not after, the lag before the membrane damage; that streptomycin-resistant cells, which fail to take up streptomycin, can do so after treatment by another aminoglycoside; and that puromycin at moderate concentrations accelerates streptomycin uptake, while high concentrations (which release shorter chains) prevent it. In addition, puromycin, prematurely releasing polypeptides of normal sequence, also evidently creates channels, since it is reported to promote streptomycin uptake even in streptomycin-resistant cells. These findings imply that normal membrane proteins must be selected not only for a hydrophobic anchoring surface, but also for a tight fit in the membrane.
Flow rates and reaction rates in the Galapagos Rise spreading center hydrothermal system as inferred from
228
Ra/
226
Ra in vesicomyid clam shells
Authors: Turekian Karl K. , Cochran J. Kirk
Published: May 2006

The
228
Ra/
226
Ra ratios in a previously dated vesicomyid clam shell were used to determine that seawater was in contact with mid-oceanic-ridge basalt glass for 22-45 years prior to arrival to the surface at 350°C at the Galapagos Rise Spreading Center. The minimum rate of reaction for the 45-year sojourn time, based on a water/rock ratio of 2.8 derived from
226
Ra concentrations, is 8 g of basalt altered per kg of seawater per year.
Relationship between the pharmacological activity of antitumor drugs Ametantrone and mitoxantrone (Novatrone) and their ability to condense nucleic acids.
Authors: Kapuscinski J , Darzynkiewicz Z
Published: May 2006

Ametantrone (HAQ) and mitoxantrone (DHAQ) are structurally similar antitumor drugs of the anthracenedione class. The cytostatic, cytotoxic, and antitumor activities of these drugs are different, with DHAQ being 10-100 times more potent, per molar basis. Both drugs are strong intercalators and intercalative modes of binding are suspected as relevant to their pharmacological activity. No significant differences, however, that could explain the differences in pharmacological activity are observed in their intercalative properties with respect to base specificity and binding affinity. A correlation, however, is evident between their potency and ability to condense nucleic acids inasmuch as DHAQ condenses nucleic acids at concentrations that are lower by a factor of 5-40 than those of HAQ and these effects can be observed at their pharmacological concentrations. The condensation is base- and sugar-specific and the long purine sequences of single-stranded RNA are the most sensitive. Electron microscopy of L1210 cells exposed a short time (90 min) to 0.21-21 microM DHAQ reveals segregation of nucleoli; the segregated granular portion shows increased electron opacity. In some preparations patchy areas of nuclear chromatin characterized by increased electron opacity can be seen. The results are compatible with the possibility that pharmacological effects of these antitumor drugs could involve condensation of nucleic acids, primarily of RNA in nucleoli.
Pks, a raf-related sequence in humans.
Authors: Mark G E , Seeley T W , Shows T B , Mountz J D
Published: May 2006

A human fetal liver cDNA library was screened at reduced hybridization stringency for v-raf-related sequences. In addition to the expected c-raf-1 cDNA, a second sequence was isolated. Comparison of the second gene (pks) to the other raf-related sequences revealed nucleotide homologies of 71%. The predicted amino acid sequence of the kinase domain is sufficiently similar to that of v-raf to suggest that pks may encode a polypeptide that exhibits serine/threonine kinase activity. The expression of pks mRNA (2.7 kilobases long) is elevated in peripheral blood mononuclear cells isolated from two patients with angioimmunoblastic lymphadenopathy with dysproteinemia, a disease in which autoantibodies are produced following the lymphoproliferative activation of B cells. Analysis of somatic cell hybrids for segregation of the pks locus revealed the presence of an additional locus closely related to the pks sequence.