bluuebunny/crossref_metadata_2025 huggingface dataset:
The ras and myc oncogenes cooperate in tumor induction in many tissues when introduced into midgestation mouse embryos by retroviral vectors.
Authors: Compere S J , Baldacci P , Sharpe A H , Thompson T , Land H , Jaenisch R
Published: May 2006

Midgestation embryos were infected with replication-defective retroviral vectors that either transduced the myc oncogene, the ras oncogene, or both oncogenes simultaneously. The myc virus induced tumors in diverse organs at a very low frequency and with a long latency period, while approximately 20% of the mice derived from embryos infected with the ras virus developed tumors in the skin with a latency of 4-8 weeks. In contrast, infection of embryos with the ras/myc double oncogene virus resulted in 27% of the animals developing rapidly growing and malignant tumors in a great variety of tissues after a median latency period of 2-3 weeks. All tumors were of monoclonal origin, as shown by Southern analysis using the provirus as a molecular marker. Our results are consistent with the hypothesis that the ras and myc oncogenes cooperate in transforming cells, but that additional alterations are necessary for realization of the fully malignant phenotype. Our observations also suggest that a much wider range of cell types become targets for malignant transformation when the embryos are exposed to the myc and the ras oncogenes simultaneously than when exposed to the same oncogenes separately. Infection of mouse embryos with vectors carrying different oncogenes or oncogene combinations may be an efficient and rapid method for evaluating the spectrum of cell types at risk for malignant conversion following mutation of a protooncogene to a transforming gene.
Repair of the Escherichia coli chromosome after in vivo scission by the EcoRI endonuclease.
Authors: Heitman J , Zinder N D , Model P
Published: May 2006

We prepared a set of temperature-sensitive mutants of the EcoRI endonuclease. Under semipermissive conditions, Escherichia coli strains bearing these alleles form poorly growing colonies in which intracellular substrates are cleaved at EcoRI sites and the SOS DNA repair response is induced. Strains defective in SOS induction (lexA3 mutant) or SOS induction and recombination (recA56 and recB21 mutants) are not more sensitive to this in vivo DNA scission, whereas strains deficient in DNA ligase (lig4 and lig ts7 mutants) are extremely sensitive. We conclude that although DNA scission induces the SOS response, neither this induction nor recombination are required for repair. DNA ligase is necessary and may be sufficient to repair EcoRI-mediated DNA breaks in the E. coli chromosome.
An essential yeast protein, encoded by duplicated genes TIF1 and TIF2 and homologous to the mammalian translation initiation factor eIF-4A, can suppress a mitochondrial missense mutation.
Authors: Linder P , Slonimski P P
Published: May 2006

We describe the isolation and characterization of two previously undescribed genes, TIF1 and TIF2, from Saccharomyces cerevisiae. The protein-encoding sequences of the two genes are highly conserved, resulting in two completely identical proteins, whereas the flanking regions show no obvious homology. The two yeast proteins are highly similar to the translation initiation factor eIF-4A from mouse. Elevated gene dosage of TIF1 or TIF2 results in the suppression of a missense mutation in the mitochondrial oxi2 gene, which codes for subunit III of cytochrome-c oxidase, although the sequence of the Tif protein indicates its cytoplasmic localization. Inactivation of either gene by gene disruption has no effect on cell viability or on mitochondrial functions. However, simultaneous inactivation of both genes is lethal to the cell.
Comparison of the secondary structures of human class I and class II major histocompatibility complex antigens by Fourier transform infrared and circular dichroism spectroscopy.
Authors: Gorga J C , Dong A , Manning M C , Woody R W , Caughey W S , Strominger J L
Published: May 2006

We have examined the secondary structures of human class I and class II histocompatibility antigens in solution by Fourier transform infrared spectroscopy and circular dichroism in order to compare the relative amounts of alpha-helix, beta-sheet, and other structures, which are crucial elements in the comparison of the protein structures. Quantitation of infrared spectra of papain-solubilized HLA-A2, HLA-B7, and DR1 in phosphate buffer gave alpha-helix contents of 17%, 8%, and 10% and beta-sheet contents of 41%, 48%, and 53%, respectively. By circular dichroism, papain-solubilized HLA-A2, HLA-B7, and DR1 were also found to have comparable alpha-helix contents (e.g., 8%, 20%, and 17%, respectively). Circular dichroism analysis for beta-sheet gave 29% for papain-solubilized HLA-B7 and 42% for papain-solubilized DR1. The value for papain-solubilized HLA-A2 (74%) was anomalous. It is proposed that Trp-107 of HLA-A2, missing in both HLA-B7 and DR1, may be responsible for much of the anomaly. Due to the uncertainties inherent in quantitation of the amounts of secondary structures by both spectral methods, the differences in the contents of alpha-helix and beta-sheet in the three proteins are not considered significant. However, differences in the nature of the beta-sheet structures are suggested by infrared spectroscopy. These results provide physical evidence for an overall structure of class II antigens modeled on that of class I antigens.
Microbial glycolipids: possible virulence factors that scavenge oxygen radicals.
Authors: Chan J , Fujiwara T , Brennan P , McNeil M , Turco S J , Sibille J C , Snapper M , Aisen P , Bloom B R
Published: May 2006

Two important pathogens of developing countries, Mycobacterium leprae, the etiologic agent of leprosy, and Leishmania donovani, the protozoal parasite that causes kalaazar, persist in the human host primarily in mononuclear phagocytes. The mechanisms by which they survive in these otherwise highly cytocidal cells are presently unknown. Since the best understood cytocidal mechanism of these cells is the oxygen-dependent system that provides lethal oxidants including the superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH), and singlet oxygen (1O2), we sought specific microbial products of these organisms that might enable them to elude oxidative cytocidal mechanisms. Phenolic glycolipid I of M. leprae and lipophosphoglycan of L. donovani are unique cell-wall-associated glycolipids produced in large amounts by the organisms. In this study, phenolic glycolipid I derivatives and lipophosphoglycan were examined for their ability to scavenge potentially cytocidal oxygen metabolites in vitro. Electron spin resonance and spin-trapping indicate that phenolic glycolipid I derivatives and lipophosphoglycan are highly effective in scavenging hydroxyl radicals and superoxide anions. The results suggest that complex glycolipids and carbohydrates of intracellular pathogens that can scavenge oxygen radicals may contribute to their pathogenicity and virulence.
Information storage in the nervous system of Aplysia: specific proteins affected by serotonin and cAMP.
Authors: Eskin A , Garcia K S , Byrne J H
Published: May 2006

To identify proteins that may be involved in the induction of long-term changes in the nervous system, we investigated whether specific proteins in pleural sensory neurons of Aplysia were affected by procedures that mimic those used to produce long-term sensitization. Using two-dimensional PAGE, we found that exposure to serotonin (5-hydroxytryptamine, 5-HT) for 2 or 3 hr appeared to increase incorporation of labeled amino acids into one protein (P9) and decrease incorporation into two other proteins (P19 and P20). These effects of 5-HT were observed whether the labeled amino acid was leucine or methionine. The same proteins that were affected by 5-HT were also altered by the adenylate cyclase activator forskolin and by the 8-bromo and 8-benzylthio analogs of cAMP. Addition of Co2+ to 5-HT did not seem to affect the action of 5-HT on P9 and P20, but it did seem to block the effect of 5-HT on P19. However, the effect of analogs of cAMP on P9, P19, and P20 was not altered by inclusion of Co2+. A phorbol ester that activates protein kinase C did not appear to affect the proteins that were modified by 5-HT, but phorbol ester did appear to increase the amount of labeled amino acids incorporated into another protein (P24). To investigate the specificity of these effects for pleural ganglion neurons, we examined the effect of 3- and 6-hr treatments of 5-HT on proteins in the abdominal ganglion. 5-HT affected at least nine proteins in the abdominal ganglion. One of these proteins (P9) appeared to be the same as one altered by 5-HT in the pleural sensory neurons. However, the occurrence of some proteins and some effects of 5-HT were specific for one ganglion or the other. The identified proteins that were affected by both 5-HT and changes in cAMP may be involved in the induction of long-term changes in the nervous system of Aplysia.
Sodium channels in dendrites of rat cortical pyramidal neurons.
Authors: Huguenard J R , Hamill O P , Prince D A
Published: May 2006

The voltage-dependent properties that have been directly demonstrated in Purkinje cell and hippocampal pyramidal cell dendrites play an important role in the integrative capacities of these neurons. By contrast, the properties of neocortical pyramidal cell dendritic membranes have been more difficult to assess. Active dendritic conductances near sites of synaptic input would have an important effect on the input-output characteristics of these neurons. In the experiments reported here, we obtained direct evidence for the existence of voltage-dependent Na+ channels on the dendrites of neocortical neurons by using cell-attached patch and whole cell recordings from acutely isolated rat neocortical pyramidal cells. The qualitative and quantitative properties of dendritic and somatic currents were indistinguishable. Insofar as Na+ currents are concerned, the soma and primary apical dendrite can be considered as one relatively uniform compartment. Similar dendritic Na+ currents on dendrites in mature neurons would play an important role in determining the integrative properties of these cortical units.
A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes.
Authors: Saluz H , Jost J P
Published: May 2006

We describe a method that permits the study of the state of cytosine methylation and of in vivo protein-DNA interactions in higher eukaryotes. This powerful technique is applicable to any gene of interest at the single-copy level. To study DNA methylation, the total uncloned genomic DNA, digested with a restriction endonuclease is subjected to a cytosine-specific hydrazine reaction and chemical cleavage. The DNA fragments of interest are linearly amplified with Taq polymerase and a sequence-specific radioactivity labeled synthetic primer. Following amplification, the DNA fragments are separated on a sequencing gel that is directly autoradiographed. To study protein-DNA interactions in vivo, we use a similar method, except that the DNA of interest is isolated from cells treated either with dimethyl sulfate or UV light. The resolution power of this technique is demonstrated by two examples, which have been studied previously by the conventional methods of genomic sequencing and "footprinting."
Trypanothione is the primary target for arsenical drugs against African trypanosomes.
Authors: Fairlamb A H , Henderson G B , Cerami A
Published: May 2006

The trypanosomatid metabolite N1,N8-bis-(glutathionyl)spermidine (trypanothione) has been demonstrated to form a stable adduct with the aromatic arsenical drug melarsen oxide [p-(4,6-diamino-s-triazinyl-2-yl)aminophenyl arsenoxide]. The stability constant of the melarsen-trypanothione adduct (Mel T) has been determined to be 1.05 x 10(7) M-1. When bloodstream Trypanosoma brucei are incubated with either melarsen oxide or the 2,3-dimercaptopropanol adduct of melarsen oxide (melarsoprol), Mel T is the only arsenical derivative detectable in acid-soluble extracts of the cells. Trypanothione may therefore be regarded as a primary target for aromatic arsenical derivatives against African trypanosomes. The selective toxic action of these compounds might arise through sequestration of intracellular trypanothione in the form of Mel T, or Mel T itself may be toxic within the cell. The latter possibility is illustrated by the finding that Mel T is an inhibitor of trypanothione reductase from T. brucei (Ki = 9.0 microM)--an enzyme that is central to the regulation of the thiol/disulfide redox balance in the parasite and absent from the host.
Structural basis for DNA bending.
Authors: Nadeau J G , Crothers D M
Published: May 2006

We report proton NMR studies on DNA oligonucleotides that contain A tracts of lengths known to produce various degrees of bending. Spectra of duplexes in the series 5'-(GGCAnCGG).(CCGTnGCC) (n = 3, 4, 5, 7, 9) reveal substantial structural changes within the An.Tn tract as its length is increased. Chemical-shift comparisons show that A tracts with fewer than about seven members do not contain regions of uniform [or poly(dA).poly(dT)-like] structure. Long An tracts (n greater than or equal to 7) appear to consist of an internal segment of homopolymeric conformation flanked by regions of transitional structure that occupy about four A.T pairs on the 5' side and two A.T pairs perhaps the directly adjacent G.C pair on the 3' side. In shorter duplexes (n less than 7), these two transitional regions overlap and an apparent mutual incompatibility causes length-dependent changes that are most pronounced near the 3' end. Throughout the series, there is a striking monotonic relationship between the location of an A.T pair in the A tract and the relative position of its ThyH3 resonance. The direction of the chemical-shift dispersion is opposite to that expected from consideration of ring-current effects alone; this discrepancy suggests a gradual decrease in ThyH3...N1Ade hydrogen-bond length as one moves from the 5' to the 3' end of the A tract and from short to long A tracts. Nuclear Overhauser effect measurements reveal that the interproton distances AdeH2...H1'Ade and AdeH2...H1'Thy vary along each A tract, except in the central regions of the longer ones where they are fairly constant and in good agreement with the poly(dA).poly(dT) structure proposed by Lipanov, A.A. & Chuprina, V. P. [(1987) Nucleic Acids Res. 15, 5833-5844]. This model features a substantial negative base-pair tilt, which has been suggested previously as the source of A-tract bending. In contrast, the nuclear Overhauser effect distances are inconsistent with at least one known crystallographic A-tract structure [DiGabriele, A. D., Sanderson, M. R. & Steitz, T. A. (1989) Proc. Natl. Acad. Sci. USA 86, 1816-1820], which lacks appreciable base-pair tilt.
Generation of fibrosarcomas in vivo by a retrovirus that expresses the normal B chain of platelet-derived growth factor and mimics the alternative splice pattern of the v-sis oncogene.
Authors: Pech M , Gazit A , Arnstein P , Aaronson S A
Published: May 2006

A retrovirus containing the entire human platelet-derived growth factor B-chain (PDGF-B) gene was constructed in order to investigate the in vivo biological activity of its encoded growth factor. When this virus was introduced into newborn mice, it reproducibly generated fibrosarcomas at the site of inoculation. Proviruses in each fibrosarcoma analyzed had lost 149 nucleotides downstream of the PDGF-B coding region. This deletion originated from an alternative or aberrant splice event that occurred within exon 7 of the PDGF-B gene and mimicked the v-sis oncogene. Thus, deletion of this region may be necessary for efficient retrovirus replication or for more potent transforming function. Evidence that the normal growth factor coding sequence was unaltered derived from RNase protection studies and immunoprecipitation analysis. Tumors were generally polyclonal but demonstrated clonal subpopulations. Moreover, tumor-derived cell lines became monoclonal within a few tissue culture passages and rapidly formed tumors in vivo. These findings argue that overexpression of the normal human PDGF-B gene product under retrovirus control can induce the fully malignant phenotype.
UACUAAC is the preferred branch site for mammalian mRNA splicing.
Authors: Zhuang Y A , Goldstein A M , Weiner A M
Published: May 2006

The conserved branch-site sequence UAC-UAAC is known to form base pairs with the complementary sequence GUAGUA in U2 small nuclear RNA (snRNA) during mRNA splicing in the yeast Saccharomyces cerevisiae. Although the GUAGUA element is conserved in mammalian U2 snRNA, mammalian branch sites conform only weakly to a YURAC consensus and can even be deleted without obvious effects on the efficiency of splicing in vivo. To understand why the GUAGUA element of U2 is conserved in evolution but the branch site is not, we have devised two different competitive assays for branch-site selection using the first intron of the human beta-globin gene. We find that a sequence resembling UACUAAC is the most efficient branch site for mammalian mRNA splicing both in vivo and in vitro. Our results suggest that in mammals U2 snRNA can form base pairs with the branch site and the interaction between U2 and the branch site can be augmented or replaced by an interaction between the spliceosome and some other element of the intron or exons, perhaps the conserved polypyrimidine tract located immediately upstream from the 3' splice site.
Differential expression of acetylcholine receptor mRNA in nuclei of cultured muscle cells.
Authors: Bursztajn S , Berman S A , Gilbert W
Published: May 2006

Muscle cells in vitro and in vivo are multinucleated and express acetylcholine receptors (AcChoRs). On innervated cells, the AcChoRs form clusters which lie under the nerve terminals. However, noninnervated cells in culture also express clusters of AcChoR. Both in vivo and in vitro the AcChoR clusters appear to be associated with clusters of nuclei. We have used in situ hybridization to determine whether all the nuclei in cultured chicken embryo myotubes are equally active in expressing the AcChoR alpha subunit message. Cells were hybridized with 35S-labeled probes that contained either both an exon and an intron region or only exon sequences. Control cultures were hybridized with a labeled actin DNA probe or poly(U). The hybrids were detected by emulsion autoradiography; simultaneously, the nuclei were visualized with bisbenzamide. Cells hybridized with the intron/exon probe showed a striking preferential silver grain localization in and around some of the myotube nuclei, whereas those hybridized with the exon probe gave a rather homogeneous grain distribution in the cytoplasm. These results show that myotube nuclei possess differential activation capacities for the expression of AcChoR alpha subunit mRNA and that this difference is due to differential rates of transcription.
A host-encoded DNA-binding protein promotes termination of plasmid replication at a sequence-specific replication terminus.
Authors: Sista P R , Mukherjee S , Patel P , Khatri G S , Bastia D
Published: May 2006

We have purified approximately 6600-fold an approximately 40-kDa protein (Ter protein) encoded by Escherichia coli that specifically binds to two sites at the 216-base-pair replication terminus (tau) of the plasmid R6K. Chemical footprinting experiments have shown that the Ter protein binds to two 14- to 16-base-pair sequences that exist as inverted repeats in the tau fragment. Site-directed mutagenesis of one of the terminus sequences (tau R) resulted in a mutant tau R that failed to bind to the Ter protein. The same mutant terminus also failed to terminate DNA replication in vivo. These experiments strongly suggest that the interaction of the Ter protein with tau sequences plays an essential role in the termination of DNA replication, specifically at tau.
Activation of the Ki-ras protooncogene in spontaneously occurring and chemically induced lung tumors of the strain A mouse.
Authors: You M , Candrian U , Maronpot R R , Stoner G D , Anderson M W
Published: May 2006

The strain A mouse has a high incidence of spontaneous lung tumors and is susceptible to lung tumor induction by chemical carcinogens. By utilizing transfection assay, Southern blot analysis, and DNA amplification techniques, we have detected an activated Ki-ras gene in the DNAs of both spontaneously occurring and chemically induced lung tumors of strain A mice. The point mutations in the spontaneous lung tumors were in both codon 12 (60%) and codon 61 (30%). In contrast, 100% of the mutations in the Ki-ras gene detected in methylnitrosourea-induced lung tumors and 93% of the mutations in the Ki-ras genes detected in benzo[a]pyrene-induced lung tumors were in codon 12, whereas 90% of the mutations in the Ki-ras genes detected in ethyl carbamate-induced lung tumors were in codon 61. The selectivity of mutations in the Ki-ras oncogene observed in chemically induced tumors, as compared to spontaneous tumors, suggests that these chemicals directly induce point mutations in the Ki-ras protooncogene. These data indicate that the strain A mouse lung tumor model is a very sensitive system to detect the ability of chemicals to activate the Ki-ras protooncogene in lung tissue.
Single-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated
Authors: Blank A. , McKeon Thomas A.
Published: May 2006

Single-strand-preferring nucleases (EC 3.1.30.1) selectively cleave internucleotide bonds in single-stranded regions of predominantly duplex DNA and DNA·RNA hybrids and extensively degrade denatured DNA and RNA. The functions of single-strand-preferring nuclease in plants are unknown. We have monitored this nuclease activity in flag leaves of wheat (
Triticum aestivum
L. cv. Chinese Spring) undergoing natural senescence and in primary leaves of wheat seedlings undergoing dark-induced senescence. In falg leaves, nuclease activity remained at basal levels during the first 2 weeks after anthesis, while chlorophyll content increased to a maximum. Nuclease activity then rose in concert with a decline in chlorophyll, reaching a 16-fold elevation at 5 weeks post-anthesis, when 53% of the chlorophyll had been lost. When 8-day-old wheat seedlings were induced to senesce by placing them in darkness, nuclease activity rose without apparent lag, reaching a 13-fold elevation in 7 days, when 61% of the chlorophyll had been lost. The increase in nuclease activity was reversible upon reexposure of seedlings to light, a decline beginning without apparent lag. Reversibility was complete for plants that had been held in darkness for 5 days, with activity returning to the control level in 2 days. These senescence-related changes in nuclease activity, measured in conventional assays, were consistent with concomitant analysis by activity staining of sodium dodecyl sulfate/polyacrylamide gels. We conclude that an increase in single-strand-preferring nuclease activity is closely associated with wheat leaf senescence and that nuclease activity is subject to negative photoregulation.
Basic fibroblast growth factor is a substrate for protein phosphorylation and is phosphorylated by capillary endothelial cells in culture.
Authors: Feige J J , Baird A
Published: May 2006

A phosphorylated basic fibroblast growth factor (FGF) can be detected in extracts of bovine capillary endothelial cells and human hepatoma cells. Accordingly, human basic FGF contains consensus sequences that account for its phosphorylation on Thr-112 by the catalytic subunit of the cAMP-dependent protein kinase A (PK-A) and on Ser-64 by the calcium- and phospholipid-dependent protein kinase C (PK-C). A kinetic analysis of both of these reactions revealed that basic FGF is among the better substrates for these enzymes. Although the kinase responsible for the phosphorylation in vivo has not yet been identified, we examined the effects of phosphorylation on the biological activity, heparin-binding capacity, and receptor-binding capacity of phosphorylated basic FGF. No effects of phosphorylation were observed when the mitogen was phosphorylated by PK-C. In contrast, when basic FGF was phosphorylated in the receptor-binding domain with PK-A, the growth factor was 3-8 times better at displacing radiolabeled basic FGF in the radioreceptor assay. No effects were seen on the binding of this FGF to immobilized heparin or cell-associated glycosaminoglycans, suggesting that this phosphorylation modifies the affinity of basic FGF for its receptor. Biological assays for basic FGF failed to identify differences between the phosphorylated and unphosphorylated forms of recombinant basic FGFs presumably because of the presence of ectophosphatases and the experimental conditions of proliferation and mitogenic assays (37 degrees C, 24-96 hr). Because the relative affinity of basic FGF for its receptor and cell-associated glycosaminoglycans may regulate its activity, the identification of a modified form of basic FGF may be of particular importance in understanding the mechanisms that regulate its biological activity, bioavailability, and processing to and from the extracellular matrix.
Tumor-specific loss of 11p15.5 alleles in del11p13 Wilms tumor and in familial adrenocortical carcinoma.
Authors: Henry I , Grandjouan S , Couillin P , Barichard F , Huerre-Jeanpierre C , Glaser T , Philip T , Lenoir G , Chaussain J L , Junien C
Published: May 2006

We have compared constitutional and tumor genotypes in nine cases of hereditary Wilms tumor (WT) and in three unrelated cases of familial adrenocortical carcinoma (ADCC). Since susceptibility to these tumors can be observed in malformation syndromes associated with a constitutional deletion of band 11p13 (WT) and with a constitutional duplication of band 11p15.5 (WT, ADCC), we investigated these two candidate regions by using 11p polymorphic markers. As expected, somatic chromosomal events, resulting in a loss of heterozygosity limited to region 11p15.5, were observed in the tumor of two familial cases of adrenocortical carcinoma. Surprisingly, however, analysis of the WT of two patients with a constitutional deletion of band 11p13, associated with aniridia, genitourinary abnormalities, and mental retardation (WAGR syndrome), revealed a loss of heterozygosity limited to region 11p15.5. These data therefore suggest that observation of a specific loss of heterozygosity may not necessarily point to the site of the initial germinal mutation. Together with previous similar observations of a loss of heterozygosity limited to 11p15.5 in breast cancer and in rhabdomyosarcoma, our data suggest that region 11p15.5 may carry a non-tissue-specific gene that could be involved in genetic predisposition, in tumor progression, or in both.
Virus-induced delayed-type hypersensitivity reaction is sequentially mediated by CD8+ and CD4+ T lymphocytes.
Authors: Moskophidis D , Lehmann-Grube F
Published: May 2006

After subcutaneous inoculation into the hind foot of a mouse, the lymphocytic choriomeningitis (LCM) virus multiplies locally, attaining 10(7)-10(8) mouse infectious units per g of tissue; elimination commences around day 7. About 1 day earlier, the foot begins to swell, which is regarded as a delayed-type hypersensitivity (DTH) reaction. To answer the question of whether the local inflammatory response is involved in virus clearance, we needed to known what cells mediate both these phenomena. With three different procedures--namely, depletion in vivo of defined cells by treatment of mice with monoclonal antibodies ("serologic surgery"), adoptive immunization with negatively selected cells, and adoptive immunization with cells from mice differing at the major histocompatibility gene complex--it is shown that the LCM virus-induced local DTH reaction consists of two phases that are sequentially mediated by (first) class I-restricted cytotoxic/suppressive CD8+ and (second) class II-restricted helper/inducer CD4+ T lymphocytes. In contrast, for virus elimination only the former subset of T lymphocytes was found to be needed. Thus, an association may exist between the CD8+ cell-mediated component of the local DTH response and control of the infection, but the CD4+ cell-mediated part appears to be of doubtful antiviral relevance.
Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.
Authors: Fong Y , Rosenbaum M , Tracey K J , Raman G , Hesse D G , Matthews D E , Leibel R L , Gertner J M , Fischman D A , Lowry S F
Published: May 2006

A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous refeeding. A 6-hr methionyl growth hormone infusion achieved steady-state serum levels comparable to normal physiologic peaks and was associated with a significant increase in limb amino acid uptake, without a change in body amino acid oxidation. Myosin heavy-chain mRNA levels, measured by quantitative dot blot hybridization, were also significantly elevated after growth hormone administration. The data indicate that methionyl growth hormone can induce intracellular amino acid accrual and increased levels of myofibrillar protein mRNA during hospitalized nutritional support and suggest growth hormone to be a potential therapy of lean body wasting.
Atriopeptin inhibition of thrombin-mediated changes in the morphology and permeability of endothelial monolayers.
Authors: Baron D A , Lofton C E , Newman W H , Currie M G
Published: May 2006

To determine the role of endothelial atriopeptin (AP) receptors, we examined the effect of AP-III on the morphology and macromolecular permeability of monolayer cultures of bovine aortic endothelial cells. AP-III alone (10(-9)-10(-6) M) had no observable effect on the morphology of the monolayers or their permeability to 125I-labeled albumin. However, incubation of the endothelial monolayers with AP-III (10(-8)-10(-6) M) antagonized thrombin-induced (1 unit/ml) cell-shape change and the formation of intercellular gaps. AP-III also opposed the effect of thrombin on the distribution of actin filaments in the endothelial cytoskeleton. Further, thrombin caused a 2-fold increase in monolayer permeability to 125I-labeled albumin, which was abolished by 10(-8)-10(-6) M AP-III pretreatment. Taken together with the findings that AP-III exhibited specific and saturable binding in these cells, these data suggest that AP regulates endothelial permeability through a receptor-mediated process.
Phorbol ester induces photoreceptor-specific degeneration in a Drosophila mutant.
Authors: Minke B , Rubinstein C T , Sahly I , Bar-Nachum S , Timberg R , Selinger Z
Published: May 2006

In the retinal degeneration B (rdgB) mutant of Drosophila, the major class of photoreceptors degenerate when the fly is raised in the light for several days; raising the fly in the dark largely prevents the degeneration. Thus, the rdgB is a conditional mutant that requires the operation of some stages of the phototransduction cascade to express its characteristic phenotype. We report here experiments that examine the ability of chemical agents to mimic light by causing photoreceptor-specific degeneration in the dark. Application of a specific activator of protein kinase C, phorbol ester, to eyes of rdgB flies led to a degeneration of the photoreceptors that was indistinguishable from that caused by light: both light and phorbol ester-induced degeneration were characterized by (i) selective degeneration of one class of photoreceptors; (ii) a unique pattern of degeneration; and (iii) the appearance of light-induced regenerative spikes at early stages of degeneration. Application of phorbol ester to the eyes of wild-type flies had no effect. We suggest that light or phorbol ester activates a protein kinase C and results in a sustained or excessive phosphorylation of proteins in the rdgB mutant, leading to photoreceptor degeneration. Furthermore, the results are consistent with identification of the rdgB gene product as a phosphoprotein phosphatase that is nonfunctional or absent in the mutant.
Mammalian dihydroorotase: nucleotide sequence, peptide sequences, and evolution of the dihydroorotase domain of the multifunctional protein CAD.
Authors: Simmer J P , Kelly R E , Rinker A G , Zimmermann B H , Scully J L , Kim H , Evans D R
Published: May 2006

Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA. The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetase-DHO-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus.
Defective chlorophyll a/b-binding protein genes in the genome of a homosporous fern.
Authors: Pichersky E , Soltis D , Soltis P
Published: May 2006

The majority of homosporous ferns have a chromosome number that is severalfold greater than that of diploid seed plants. These fern species have therefore been generally considered to be of polyploid origin. Enzyme electrophoretic investigations have demonstrated, however, that within fern genera, species having the lowest chromosome numbers (n = 27-52) have the number of isozymes typical of diploid seed plants; there is no isozyme evidence for polyploidy of these plants. We have constructed a genomic DNA library from Polystichum munitum (n = 41), a homosporous fern, and have screened the library for sequences homologous to the chlorophyll a/b-binding (CAB) protein genes of higher plants. The majority of the sequences isolated and characterized by nucleotide sequence determination represent defective CAB genes. This result is in contrast to the situation in the genomes of diploid angiosperms, where most, and sometimes all, copies of the CAB gene family represent functional members. Several hypotheses could explain the existence of multiple defective CAB genes in P. munitum. (i) The defective CAB genes are the result of "gene silencing" following polyploidy. (ii) P. munitum has not gone through a polyploidization event, but several, and perhaps the majority, of its CAB genes have mutated to a nonfunctional state (a phenomenon not yet observed in any of the genomes of non-fern plants so far examined). (iii) Some defective CAB genes have been specifically amplified in the genome of P. munitum.
Specific high molecular weight mRNAs induced by associative learning in Hermissenda.
Authors: Nelson T J , Alkon D L
Published: May 2006

Associative conditioning of Hermissenda crassicornis has been demonstrated to result in long-term changes in the potassium currents IA and ICa2(+)-K+ in photoreceptor neurons in the eye and to increase mRNA levels in the eye 2- to 3-fold. mRNA isolated from Hermissenda trained with paired light and rotation stimuli was labeled with [3H]acetic anhydride, while mRNA from naive animals or from animals subjected to random light and rotation stimuli was labeled with [14C]acetic anhydride. The labeled RNA was combined and separated by agarose gel electrophoresis. The overall size distribution of labeled mRNA was shifted to longer chain lengths in the paired group. In addition, the 3H/14C ratios were markedly increased for 21 distinct size bands, indicating increased mRNA of specific chain lengths in the paired group. Increases in the same size bands were also observed with mRNA labeled in vivo with 32Pi. This indicates that associative learning in Hermissenda results in a specific induction of a distinct set of at least 21 mRNAs, rather than in a generalized increase in synthesis of all mRNA, thus resembling in some respects a differentiation-like response.
A neutral amino acid change in segment IIS4 dramatically alters the gating properties of the voltage-dependent sodium channel.
Authors: Auld V J , Goldin A L , Krafte D S , Catterall W A , Lester H A , Davidson N , Dunn R J
Published: May 2006

Sodium channels encoded by the rat IIA cDNA clone [Auld, V. J., Goldin, A. L., Krafte, D. S., Marshall, J., Dunn, J., Catterall, W. A., Lester, H. A., Davidson, N. & Dunn, R. J. (1988) Neuron 1, 449-461] differ at seven amino acid residues from those encoded by the rat II cDNA [Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H. & Numa, S. (1986) Nature (London) 320, 188-192]. When expressed in Xenopus oocytes, rat IIA channels display a current-voltage relationship that is shifted 20-25 mV in the depolarizing direction relative to channels expressed from rat II cDNA or rat brain poly(A)+ mRNA. By modifying each variant residue in rat IIA to the corresponding residue in rat II, we demonstrate that a single Phe----Leu substitution at position 860 in the S4 segment of domain II is sufficient to shift the current-voltage relationship to that observed for channels expressed from rat brain poly(A)+ RNA or rat II cDNA. Rat genomic DNA encodes leucine but not phenylalanine at position 860, indicating that the phenylalanine at this position in rat IIA cDNA likely results from reverse transcriptase error.
Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin.
Authors: Paruchuri D K , Seifert H S , Ajioka R S , Karlsson K A , So M
Published: May 2006

We recently identified a set of mammalian cell receptors for Neisseria gonorrhoeae that are glycolipids. These receptors, lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriosylceramide [GalNAc( beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], were shown to be specifically bound by a gonococcal outer membrane protein distinct from pilin and protein II. Here we report the isolation of the gene encoding the gangliotetraosylceramide-binding adhesin from a N. gonorrhoeae MS11 gene bank in Escherichia coli. Transposon mutagenesis studies in E. coli indicate that the adhesion is a protein with a molecular mass of 36,000 Da. The gene encoding the 36-kDa protein is duplicated in MS11 since two transposon insertions were required to abolish expression of the gene in this bacterium. This protein is present on the surface of the gonococcus and is not associated with the pilus.
Tumor trapping of 5-fluorouracil: in vivo 19F NMR spectroscopic pharmacokinetics in tumor-bearing humans and rabbits.
Authors: Wolf W , Presant C A , Servis K L , el-Tahtawy A , Albright M J , Barker P B , Ring R , Atkinson D , Ong R , King M
Published: May 2006

The pharmacokinetics of 5-fluorouracil (5FU) were studied in vivo in patients with discrete tumors and in rabbits bearing VX2 tumors by using 19F NMR spectroscopy. The human studies were conducted in a 1.5-T Magnetom magnetic resonance imager (Siemens), and the rabbit studies were conducted in a 4.7-T GE/Nicolet 33-cm bore magnet. Free 5FU was detected in the tumors of four of the six patients and in all VX2 tumors but not in normal rabbit tissues. No other metabolites were seen in these tumors, contrary to the extensive catabolism we had previously documented using 19F NMR spectroscopy in both human and animal livers. The tumor pool of free 5FU in those human tumors that trapped 5FU was determined to have a half-life of 0.4-2.1 hr, much longer than expected and significantly longer than the half-life of 5FU in blood (5-15 min), whereas the half-life of trapped 5FU in the VX2 tumors ranged from 1.05 to 1.22 hr. In this initial experience, patient response to chemotherapy may correlate with extent of trapping free 5FU in the human tumors. These studies document that NMR spectroscopy is clinically feasible in vivo, allows noninvasive pharmacokinetic analyses at a drug-target tissue in real time, and may produce therapeutically important information at the time of drug administration. Demonstration of the trapping of 5FU in tumors provides both a model for studying metabolic modulation in experimental tumors (in animals) and a method for testing modulation strategies clinically (in patients).
Structural and biochemical properties of cloned and expressed human and rat steroid 5 alpha-reductases.
Authors: Andersson S , Russell D W
Published: May 2006

The microsomal enzyme steroid 5 alpha-reductase is responsible for the conversion of testosterone into the more potent androgen dihydrotestosterone. In man, this steroid acts on a variety of androgen-responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. Here we describe the isolation, structure, and expression of a cDNA encoding the human steroid 5 alpha-reductase. A rat cDNA was used as a hybridization probe to screen a human prostate cDNA library. A 2.1-kilobase cDNA was identified and DNA sequence analysis indicated that the human steroid 5 alpha-reductase was a hydrophobic protein of 259 amino acids with a predicted molecular weight of 29,462. A comparison of the human and rat protein sequences revealed a 60% identity. Transfection of expression vectors containing the human and rat cDNAs into simian COS cells resulted in the synthesis of high levels of steroid 5 alpha-reductase enzyme activity. Both enzymes expressed in COS cells showed similar substrate specificities for naturally occurring steroid hormones. However, synthetic 4-azasteroids demonstrated marked differences in their abilities to inhibit the human and rat steroid 5 alpha-reductases.
Base changes at position 792 of Escherichia coli 16S rRNA affect assembly of 70S ribosomes.
Authors: Santer M , Bennett-Guerrero E , Byahatti S , Czarnecki S , O'Connell D , Meyer M , Khoury J , Cheng X , Schwartz I , McLaughlin J
Published: May 2006

To investigate the function of base 792 of 16S rRNA in 30S ribosomes of Escherichia coli, the wild-type (adenine) residue was changed to guanine, cytosine, or uracil by oligonucleotide-directed mutagenesis. Each base change conferred a unique phenotype on the cells. Cells containing plasmid pKK3535 with G792 or T792 showed no difference in generation time in LB broth containing ampicillin, whereas cells with C792 exhibited a 20% increase in generation time in this medium. To study the effect on cell growth of a homogeneous population of mutant ribosomes, the mutations were cloned into the 16S rRNA gene on pKK3535 carrying a spectinomycin-resistance marker (thymine at position 1192), and the cells were grown with spectinomycin. Cells containing G792 or C792 showed 16% and 56% increases in generation time, respectively, and a concomitant decrease in 35S assimilation into proteins. Cells with T792 did not grow in spectinomycin-containing medium. Maxicell analyses indicated decreasing ability to form 70S ribosomes from 30S subunits containing guanine, cytosine, or uracil at position 792 in 16S rRNA. It appeared that C792-containing 30S ribosomes had lost the ability to bind initiation factor 3.
Reciprocal expression of human ETS1 and ETS2 genes during T-cell activation: regulatory role for the protooncogene ETS1.
Authors: Bhat N K , Thompson C B , Lindsten T , June C H , Fujiwara S , Koizumi S , Fisher R J , Papas T S
Published: May 2006

The expression of the protooncogenes ETS1 and ETS2 has been studied in purified human T cells activated either by cross-linking of the T-cell receptor-CD3 complex on their cell surface or by direct stimulation with phorbol esters and ionomycin. Our results show that resting T cells express high levels of ETS1 mRNA and protein, while expression of ETS2 is undetectable. Upon T-cell activation, ETS2 mRNA and proteins are induced, while ETS1 gene expression decreases to very low levels. Late after stimulation, ETS1 mRNA is reinduced and maintained at a high level, while ETS2 gene expression decreases to undetectable levels. Therefore, it appears that in human T cells, ETS2 gene products are associated with cellular activation and proliferation, while ETS1 gene products are preferentially expressed in a quiescent state.
In vitro activation of heat shock transcription factor DNA-binding by calcium and biochemical conditions that affect protein conformation.
Authors: Mosser D D , Kotzbauer P T , Sarge K D , Morimoto R I
Published: May 2006

The transcription of heat shock genes in response to physiological stress requires activation of heat shock transcription factor (HSF). Although the transcriptional response is most commonly induced by temperature elevation, the biochemical events involved in HSF activation in vivo can also be triggered at normal physiological temperatures by chemicals that inhibit metabolic processes. We have used a HeLa cell-free system in which HSF DNA-binding is activated by conditions that affect protein conformation, including increasing concentrations of hydrogen ions, urea, or nonionic detergents. Treatment with calcium ions also results in a concentration- and time-dependent activation of HSF in vitro. Pretreatment with each of these biochemical conditions reduces the temperature dependence for HSF activation in vitro. These results suggest that HSF is activated either directly by undergoing a conformational change or indirectly through interactions with unfolded proteins.
Lipid biophysics of water loss through the skin.
Authors: Potts R O , Francoeur M L
Published: May 2006

The regulation of water loss through the skin is a poorly understood but crucial process in maintaining terrestrial life-forms. In mammalian skin, the outermost layer, called the stratum corneum (SC), is rate-limiting to water loss. We have evaluated temperature-dependent changes in water vapor permeability and infrared spectra of porcine SC. In particular, we have analyzed the infrared absorption peaks due to the extracellular lipids of the SC. These results show a remarkable correlation between water permeability and the frequency of the C-H stretching vibrations over a broad range of temperature. Since the spectral changes reflect an increased number of alkyl gauche conformers, these results suggest that water permeability is dependent upon the hydrocarbon-chain disorder of SC lipids.
Escherichia coli thymidylate synthase: amino acid substitutions by suppression of amber nonsense mutations.
Authors: Michaels M L , Kim C W , Matthews D A , Miller J H
Published: May 2006

By using site-directed oligonucleotide mutagenesis, amber nonsense stop codons (5'-TAG-3') have been introduced at 20 sites in the Escherichia coli thymidylate synthase gene. By transforming the thyA mutant plasmids into 13 strains, each of which harbor different amber suppressor tRNAs, we were able to generate over 245 amino acid substitutions in E. coli thymidylate synthase (EC 2.1.1.45). Growth characteristics of these mutants have been studied, yielding a body of information that includes some surprising results in light of the recently published crystal structure of the enzyme.