bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a class of nonformylated Enterococcus faecalis-derived neutrophil chemotactic peptides: the sex pheromones.
Authors: Sannomiya P , Craig R A , Clewell D B , Suzuki A , Fujino M , Till G O , Marasco W A
Published: May 2006

Bacteria produce a heterogeneous mixture of neutrophil chemotactic agents in culture filtrates. Formylmethionyl peptides have been shown to comprise a significant portion of the chemotactic activity in bacterial culture filtrates; however, not all of the chemotactic agents in bacterial culture filtrates are formylated peptides. To examine whether nonformylated peptides derived from bacteria could act as chemotactic agents, we studied several nonformylated hepta- and octapeptide Enterococcus faecalis-derived sex pheromones, their modified derivatives, and their competitive inhibitors for activation of rat peritoneal neutrophils. Several of these peptides, in particular cAM373 and cPD1, proved to be potent chemotactic agents in submicromolar concentrations as well as inducers of lysosomal granule enzyme secretion. Moreover, the more biologically active peptides were able to compete with fMet-Leu-[3H]Phe for binding to the formyl peptide receptor. These studies demonstrate that the formylmethionyl moiety may be an absolute requirement only for the binding of di- and tripeptides to the formyl peptide receptor. Larger peptides that may have or that may allow for additional contact points between the peptide and receptor may require N-formylation only relatively. Indeed, by removing this structural restraint, the formyl peptide receptor may interact with an unlimited number of peptide fragments of both infectious and host origins to then modulate neutrophil responses to infection and inflammation.
BCL2-mediated tumorigenicity of a human T-lymphoid cell line: synergy with MYC and inhibition by BCL2 antisense.
Authors: Reed J C , Cuddy M , Haldar S , Croce C , Nowell P , Makover D , Bradley K
Published: May 2006

A gene-transfer approach was used to explore the function of the BCL2 (B-cell lymphoma/leukemia 2) gene in a human T-cell line, Jurkat. Though stable introduction of a BCL2 expression plasmid into Jurkat T cells was by itself insufficient, the combined transfer of BCL2 and MYC genes markedly enhanced the tumorigenicity of these cells in athymic mice. Moreover, a BCL2 antisense expression plasmid ablated tumor formation by Jurkat cells, providing further evidence that this oncogene contributes to the regulation of the in vivo growth of these human T lymphocytes. In addition to their influence on tumor formation, BCL2 sense and antisense expression plasmids increased and decreased, respectively, the in vitro survival of Jurkat T cells in serum-free medium. These observations extend to T cells the finding of synergy of BCL2 with MYC previously reported for B cells and provide evidence that BCL2 can regulate the growth of human T cells.
Enhanced c-myc gene expression during forelimb regenerative outgrowth in the young Xenopus laevis.
Authors: Géraudie J , Hourdry J , Vriz S , Singer M , Méchali M
Published: May 2006

Analysis of the expression of the c-myc protooncogene has been carried out in the forelimb regenerate of the Xenopus laevis froglet. Northern blot hybridization analysis revealed the presence of a 2.5-kilobase c-myc transcript in the regenerate forelimb at a level at least 7-fold more than the one found in nonregenerating forelimbs or stumps of regenerating forelimbs. In situ hybridization analyses confirmed the relative abundance of c-myc RNA in the regenerate forelimb and provided evidence of spatial localization of high levels of c-myc RNA in specific cell layers. The deepest layers of the wound epithelium of epidermal origin showed a strong signal, whereas virtually no c-myc RNA was detected in the outermost layers. Labeling was also observed in mesenchymal cells of the blastema where it was relatively evenly distributed. This pattern of c-myc RNA in the regenerate might indicate that the expression of c-myc plays a role in the regulation of the continued proliferation of specific cells of the regenerate, whereas repression of this gene in the epidermis correlates with terminal differentiation of keratinocytes.
Introduction of site-specific mutations into the genome of influenza virus.
Authors: Enami M , Luytjes W , Krystal M , Palese P
Published: May 2006

We succeeded in rescuing infectious influenza virus by transfecting cells with RNAs derived from specific recombinant DNAs. RNA corresponding to the neuraminidase (NA) gene of influenza A/WSN/33 (WSN) virus was transcribed in vitro from plasmid DNA and, following the addition of purified influenza virus RNA polymerase complex, was transfected into MDBK cells. Superinfection with helper virus lacking the WSN NA gene resulted in the release of virus containing the WSN NA gene. We then introduced five point mutations into the WSN NA gene by cassette mutagenesis of the plasmid DNA. Sequence analysis of the rescued virus revealed that the genome contained all five mutations present in the mutated plasmid. The ability to create viruses with site-specific mutations will allow the engineering of influenza viruses with defined biological properties.
Regulation of beta-cell glucose transporter gene expression.
Authors: Chen L , Alam T , Johnson J H , Hughes S , Newgard C B , Unger R H
Published: May 2006

It has been postulated that a glucose transporter of beta cells (GLUT-2) may be important in glucose-stimulated insulin secretion. To determine whether this transporter is constitutively expressed or regulated, we subjected conscious unrestrained Wistar rats to perturbations in glucose homeostasis and quantitated beta-cell GLUT-2 mRNA by in situ hybridization. After 3 hr of hypoglycemia (glucose at 29 +/- 5 mg/dl), GLUT-2 and proinsulin mRNA signal densities were reduced by 25% of the level in control rats. After 4 days (blood glucose at 57 +/- 7 mg/dl vs. 120 +/- 10 mg/dl in saline-infused control rats), GLUT-2 and proinsulin mRNA densities were reduced by 85% and 65%, respectively (P = 0.001). After 12 days (glucose at 54 +/- 8 mg/dl), GLUT-2 mRNA signal density was undetectable whereas proinsulin mRNA was reduced by 51%. After 12 days of hypoglycemia, the Km for 3-O-methyl-D-glucose transport in isolated rat islets, normally 18-20 mM, was 2.5 mM. This provides functional evidence of a profound reduction of high Km glucose transporter in beta cells. In contrast, GLUT-2 was only slightly reduced by hypoglycemia in liver. To determine the effect of prolonged hyperglycemia, we also infused animals with 50% (wt/vol) glucose for 5 days (glucose at 200 +/- 50 mg/dl). Hyperglycemic clamping increased GLUT-2 mRNA by 46% (P = 0.001) whereas proinsulin mRNA doubled (P = 0.001). We conclude that GLUT-2 expression in beta cells, but not liver, is subject to regulation by certain perturbations in blood glucose homeostasis.
PRP5: a helicase-like protein required for mRNA splicing in yeast.
Authors: Dalbadie-McFarland G , Abelson J
Published: May 2006

A 96-kDa protein predicted by the DNA sequence of the Saccharomyces cerevisiae PRP5 gene contains a domain that bears a striking resemblance to a family of RNA helicases characterized by the conserved amino acid sequence Asp-Glu-Ala-Asp (D-E-A-D). Previous work indicated that the product of the PRP5 gene is required for splicing and that spliceosome assembly does not occur in its absence. However, its precise role in splicing and the nature of its biochemical activity remained unknown. To examine the role of PRP5 in splicing, we cloned the gene by complementation of a temperature-sensitive mutation and determined its DNA sequence. We discuss here the possible roles for an RNA helicase in splicing and for the activity of the PRP5 protein.
Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule.
Authors: Ozawa M , Ringwald M , Kemler R
Published: May 2006

We have recently found that the cytoplasmic region of the cell adhesion molecule uvomorulin associates with three proteins named catenin alpha, beta, and gamma. Here we show by analysis of various mutant uvomorulin polypeptides expressed in mouse L cells that this association is mediated by a specific domain in the cytoplasmic region. A specific recognition site for catenins is located in a 72-amino acid domain. Interestingly, 69 of the 72 amino acid residues are encoded by a single exon of the uvomorulin gene. To demonstrate the direct interaction between catenins and the 72-amino acid domain, cDNA constructs composed of H-2Kd cDNA and various 3' sequences of uvomorulin were expressed in L cells. Chimeric proteins between H-2Kd and the 72-amino acid domain of uvomorulin were shown, by immunoprecipitation with anti-H-2Kd antibodies, to complex with catenin alpha, beta, and gamma. Catenins connect uvomorulin to cytoskeletal structures. We provide biochemical evidence for an association of the uvomorulin-catenin complex with actin bundles. Our results suggest that catenin alpha plays a key role in the association with actin filaments, whereas catenin beta binds more directly to the cytoplasmic region of uvomorulin. In cell aggregation assays with transfected cells expressing normal or mutant uvomorulin, the adhesive function was expressed only when uvomorulin was associated with catenins. From these results we conclude that the cytoplasmic anchorage of uvomorulin is of major biological importance.
Overexpression of Cu-Zn superoxide dismutase in Drosophila does not affect life-span.
Authors: Seto N O , Hayashi S , Tener G M
Published: May 2006

Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (Cu-Zn SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. The Cu-Zn SOD gene (Sod) cloned from Drosophila melanogaster was introduced via P element-mediated transformation into the germ line. Homozygous lines carrying additional copies of the Sod gene were recovered and characterized. Increases in Sod transcripts and enzyme activity were observed in the transformed lines, indicating that all of the sequence information required for gene expression is contained on the inserted gene fragment. The effects of additional SOD on oxygen free radical metabolism and longevity were investigated. Additional SOD did not markedly affect oxygen metabolism or longevity.
Identification of an additional member of the protein-tyrosine-phosphatase family: evidence for alternative splicing in the tyrosine phosphatase domain.
Authors: Matthews R J , Cahir E D , Thomas M L
Published: May 2006

Protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48) have been implicated in the regulation of cell growth; however, to date few tyrosine phosphatases have been characterized. To identify additional family members, the cDNA for the human tyrosine phosphatase leukocyte common antigen (LCA; CD45) was used to screen, under low stringency, a mouse pre-B-cell cDNA library. Two cDNA clones were isolated and sequence analysis predicts a protein sequence of 793 amino acids. We have named the molecule LRP (LCA-related phosphatase). RNA transfer analysis indicates that the cDNAs were derived from a 3.2-kilobase mRNA. The LRP mRNA is transcribed in a wide variety of tissues. The predicted protein structure can be divided into the following structural features: a short 19-amino acid leader sequence, an exterior domain of 123 amino acids that is predicted to be highly glycosylated, a 24-amino acid membrane-spanning region, and a 627-amino acid cytoplasmic region. The cytoplasmic region contains two approximately 260-amino acid domains, each with homology to the tyrosine phosphatase family. One of the cDNA clones differed in that it had a 108-base-pair insertion that, while preserving the reading frame, would disrupt the first protein-tyrosine-phosphatase domain. Analysis of genomic DNA indicates that the insertion is due to an alternatively spliced exon. LRP appears to be evolutionarily conserved as a putative homologue has been identified in the invertebrate Styela plicata.
Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in Escherichia coli.
Authors: Jakubowski H
Published: May 2006

Previous in vitro studies have established a pre-transfer proofreading mechanism for editing of homocysteine by bacterial methionyl-, isoleucyl-, and valyl-tRNA synthetases. The unusual feature of the editing is the formation of a distinct compound, homocysteine thiolactone. Now, two-dimensional TLC analysis of 35S-labeled amino acids extracted from cultures of the bacterium Escherichia coli reveals that the thiolactone is also synthesized in vivo. In E. coli, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 109 molecules of methionine incorporated into protein in vivo. These results not only directly demonstrate that the adenylate proofreading pathway for rejection of misactivated homocysteine operates in vivo in E. coli but, in general, establish the importance of error-editing mechanisms in living cells.
The priA gene encoding the primosomal replicative n' protein of Escherichia coli.
Authors: Lee E H , Masai H , Allen G C , Kornberg A
Published: May 2006

The Escherichia coli gene encoding protein n' has been isolated and named priA for primosomal protein A. Protein n' is absolutely required for the conversion of single-stranded phi X174 DNA to the duplex replicative form in an in vitro-reconstituted system. The gene maps to 88.7 minutes on the chromosome adjacent to the cytR locus. Soluble protein extracts from cells harboring the priA gene on a multicopy plasmid contained 45-fold more n' replication activity than wild-type extracts. Enhanced overproduction of greater than 1000-fold was achieved by replacing the natural Shine-Dalgarno sequence with that of the phage T7 phi 10 gene and placing this priA under the control of the T7 phage promoter and RNA polymerase. The priA sequence reveals a 732-amino acid open reading frame and a nucleotide-binding consensus site consistent with the size and ATPase activity of the purified protein. The gene for protein n has been named priB and the putative gene for protein n", priC.
Increase in T-type calcium current in atrial myocytes from adult rats with growth hormone-secreting tumors.
Authors: Xu X P , Best P M
Published: May 2006

Growth hormone (GH) has pronounced effects on protein synthesis and cell growth in cardiac muscle from adult animals, although the mechanism of its action is not understood. Because Ca2+ has been implicated as a regulator of mitogenic processes in a number of tissues, we investigated whether GH affects the transmembrane movement of Ca2+ through voltage-activated channels of cardiac myocytes. Atrial and ventricular myocytes were isolated from adult rats with GH-secreting tumors and studied electrophysiologically by using patch-clamp techniques. Tumor-bearing rats re-enter an active growth phase and double their body weight over age-matched controls 8 weeks after introduction of the tumor. Atrial myocytes from tumor-bearing animals showed a 3-fold increase in the density of T-type Ca2+ current compared with cells from control animals, although the voltage dependency of activation and inactivation of T-type current was not altered. The increase in T-current density of atrial myocytes preceded by at least a week any measurable change in heart weight, body weight, or myocyte size. L-type Ca2+ currents in atrial and ventricular cells were not affected. The results suggest that a tumor-derived growth factor, most likely GH, can cause a specific enhancement of T-type Ca2+ current in atrial myocytes.
The genetic defect in Cockayne syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA.
Authors: Venema J , Mullenders L H , Natarajan A T , van Zeeland A A , Mayne L V
Published: May 2006

Cells from patients with Cockayne syndrome (CS) are hypersensitive to UV-irradiation but have an apparently normal ability to remove pyrimidine dimers from the genome overall. We have measured the repair of pyrimidine dimers in defined DNA sequences in three normal and two CS cell strains. When compared to a nontranscribed locus, transcriptionally active genes were preferentially repaired in all three normal cell strains. There was no significant variation in levels of repair between various normal individuals or between two constitutively expressed genes, indicating that preferential repair may be a consistent feature of constitutively expressed genes in human cells. Neither CS strain, from independent complementation groups, was able to repair transcriptionally active DNA with a similar rate and to the same extent as normal cells, indicating that the genetic defect in CS lies in the pathway for repair of transcriptionally active DNA. These results have implications for understanding the pleiotropic clinical effects associated with disorders having defects in the repair of DNA damage. In particular, neurodegeneration appears to be associated with the loss of preferential repair of active genes and is not simply correlated with reduced levels of overall repair.
Rectification of acetylcholine-elicited currents in PC12 pheochromocytoma cells.
Authors: Ifune C K , Steinbach J H
Published: May 2006

The current-voltage (I-V) relationship for acetylcholine-elicited currents in the rat pheochromocytoma cell line PC12 is nonlinear. Two voltage-dependent processes that could account for the whole-cell current rectification were examined, receptor channel gating and single receptor channel permeation. We found that both factors are involved in the rectification of the whole-cell currents. The voltage dependence of channel gating determines the shape of the I-V curve at negative potentials. The single-channel I-V relationship is inwardly rectifying and largely responsible for the characteristic shape of the whole-cell I-V curve at positive potentials. The rectification of the single-channel currents is produced by the voltage-dependent block of outward currents by intracellular Mg2+ ions.
Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain.
Authors: Brünger A T , Milburn M V , Tong L , deVos A M , Jancarik J , Yamaizumi Z , Nishimura S , Ohtsuka E , Kim S H
Published: May 2006

Normal RAS proteins play a key role of molecular switch in the transduction of the growth signal from extracellular to intracellular space. The state of the switch is "on" when GTP is bound and "off" when GDP is bound to the protein. The crystal structure of a complex between a nonhydrolyzable GTP analog and the catalytic domain of a RAS protein has been determined by a rotation-translation search method. The orientations and positions of four independent molecules have been determined using a single molecule as a probe in the search. The crystal structure reveals that the gamma phosphate of the GTP analog induces extensive conformational changes on two loop regions of the protein.
Separate Cl- conductances activated by cAMP and Ca2+ in Cl(-)-secreting epithelial cells.
Authors: Cliff W H , Frizzell R A
Published: May 2006

We studied the cAMP- and Ca2(+)-activated secretory Cl- conductances in the Cl(-)-secreting colonic epithelial cell line T84 using the whole-cell patch-clamp technique. Cl- and K+ currents were measured under voltage clamp. Forskolin or cAMP increased Cl- current 2-15 times with no change in K+ current. The current-voltage relation for cAMP-activated Cl- current was linear from -100 to +100 mV and showed no time-dependent changes in current during voltage pulses. Ca2+ ionophores or increased pipette Ca2+ increased both Cl- and K+ currents 2-30 times. The Ca2(+)-activated Cl- current was outwardly rectified, activated during depolarizing voltage pulses, and inactivated during hyperpolarizing voltage pulses. Addition of ionophore after forskolin further increased Cl- conductance 1.5-5 times, and the current took on the time-dependent characteristics of that stimulated by Ca2+. Thus, cAMP and Ca2+ activate Cl- conductances with different properties, implying that these second messengers activate different Cl- channels or that they induce different conductive and kinetic states in the same Cl- channel.
Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells.
Authors: Cushing S D , Berliner J A , Valente A J , Territo M C , Navab M , Parhami F , Gerrity R , Schwartz C J , Fogelman A M
Published: May 2006

After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2- to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human glioma U-105MG cell line. Antibody that had been prepared against cultured baboon smooth muscle cell chemotactic factor (anti-SMCF) did not inhibit monocyte migration induced by the potent bacterial chemotactic factor f-Met-Leu-Phe. However, anti-SMCF completely inhibited the monocyte chemotactic activity found in the media of U-105MG cells, EC, and SMC before and after exposure to MM-LDL. Moreover, monocyte migration into the subendothelial space of a coculture of EC and SMC that had been exposed to MM-LDL was completely inhibited by anti-SMCF. Anti-SMCF specifically immunoprecipitated 10-kDa and 12.5-kDa proteins from EC. Incorporation of [35S]methionine into the immunoprecipitated proteins paralleled the monocyte chemotactic activity found in the medium of MM-LDL stimulated EC and the levels of MCP-1 mRNA found in the EC. We conclude that (i) SMCF is in fact MCP-1 and (ii) MCP-1 is induced by MM-LDL.
Generation of transgenic mice with elevated blood pressure by introduction of the rat renin and angiotensinogen genes.
Authors: Ohkubo H , Kawakami H , Kakehi Y , Takumi T , Arai H , Yokota Y , Iwai M , Tanabe Y , Masu M , Hata J
Published: May 2006

The role of the renin-angiotensin system in blood pressure control and in the development of hypertension was investigated by generating transgenic mice carrying the rat renin or angiotensinogen gene or both genes under the control of the mouse metallothionein I promoter. The systolic blood pressure was significantly elevated in transgenic mice carrying both transgenes but was maintained normally in those bearing either of the transgenes. The transgene was effectively and properly transcribed to form the mature mRNA in the transgenic mice. The production of rat renin and angiotensinogen in the transgenic mice carrying the corresponding transgene was also verified by immunoanalyses of these proteins. Furthermore, the specific angiotensin-converting enzyme inhibitor captopril was effective in reducing the elevated blood pressure of the hypertensive transgenic mice. These results indicate that the combined action of the exogenous rat renin and angiotensinogen is responsible and necessary for elevation of blood pressure in the hypertensive transgenic mice.
A macrophage mRNA selectively induced by gamma-interferon encodes a member of the platelet factor 4 family of cytokines.
Authors: Farber J M
Published: May 2006

In order to identify novel mediators synthesized in activated macrophages, a cDNA library was prepared from cultures of the mouse macrophage cell line RAW 264.7 that had been treated with lymphokine-rich conditioned medium from mitogen-stimulated mouse spleen cells. Differential plaque hybridization identified a cDNA, designated m119, that detected a 1.6-kilobase mRNA that accumulated in response to gamma-interferon (IFN-gamma) but not in response to other macrophage activators, including IFN-alpha, IFN-beta, and lipopolysaccharide. The mRNA encoded a predicted protein of Mr 14,461 containing a 21-amino acid signal peptide. The primary structure of the predicted protein indicated that it is a member of a recently described family of cytokines related to platelet factor 4, including Gro/melanoma growth stimulatory activity and neutrophil-activating peptide/interleukin 8. The selective induction of the m119 mRNA by IFN-gamma that the predicted m119 protein mediates a macrophage activity regulated by IFN-gamma. The m119 protein may be a cytokine that affects the growth, movement, or activation state of cells that participate in immune and inflammatory responses. It is proposed that the gene encoding this protein be called mig, for monokine induced by gamma interferon.
Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 6-phosphate, AMP, and magnesium.
Authors: Ke H M , Zhang Y P , Lipscomb W N
Published: May 2006

The crystal structure of fructose-1,6-bisphosphatase (EC 3.1.3.11) complexed with fructose 6-phosphate, AMP, and Mg2+ has been solved by the molecular replacement method and refined at 2.5-A resolution to a R factor of 0.215, with root-mean-square deviations of 0.013 A and 3.5 degrees for bond lengths and bond angles, respectively. No solvent molecules have been included in the refinement. This structure shows large quaternary and tertiary conformational changes from the structures of the unligated enzyme or its fructose 2,6-bisphosphate complex, but the secondary structures remain essentially the same. Dimer C3-C4 of the enzyme-fructose 6-phosphate-AMP-Mg2+ complex twists about 19 degrees relative to the same dimer of the enzyme-fructose 2,6-bisphosphate complex if their C1-C2 dimers are superimposed on one another. Nevertheless, many interfacial interactions between dimers of C1-C2 and C3-C4 are conserved after quaternary structure changes occur. Residues of the AMP domain (residues 6-200) show large migrations of C alpha atoms relative to barely significant positional changes of the FBP domain (residues 201-335).
Differential regulation of interleukin 4 and interleukin 5 gene expression: a comparison of T-cell gene induction by anti-CD3 antibody or by exogenous lymphokines.
Authors: Bohjanen P R , Okajima M , Hodes R J
Published: May 2006

Murine T helper type 2 clones were stimulated with immobilized anti-CD3 antibody or with recombinant lymphokines to compare the expression of T-cell activation genes induced by these stimuli. Immobilized anti-CD3 antibody, recombinant interleukin 2 (IL-2), and recombinant interleukin 4 (IL-4) all induced proliferation of the T helper type 2 clones 10-5-17 and D10. Proliferation of these clones induced by anti-CD3 antibody was completely inhibited by cyclosporine A, whereas cyclosporine A had little effect on proliferation induced by recombinant IL-2 or recombinant IL-4. Both immobilized anti-CD3 antibody, and recombinant IL-2 induced the expression of the protooncogenes c-myc and c-myb. Immobilized anti-CD3 antibody also induced expression of the lymphokine genes IL-4, interleukin 5 (IL-5), and granulocyte-macrophage colony-stimulating factor. In contrast, recombinant IL-2 induced IL-5 mRNA expression but did not induce detectable expression of IL-4 or granulocyte-macrophage colony-stimulating factor mRNA. Likewise, recombinant IL-4 induced expression of IL-5 but not IL-4 mRNA. Thus, the IL-4 and IL-5 genes appear to be differentially regulated after stimulation with recombinant lymphokines. Effects of cyclosporine A and the protein synthesis inhibitors cycloheximide and anisomycin on IL-4 and IL-5 gene expression suggest that these genes are activated by different pathways after anti-CD3 stimulation. Cyclosporine A completely inhibited anti-CD3-induced expression of IL-4 mRNA but not of IL-5 mRNA, and protein-synthesis inhibitors completely inhibited induction of IL-5 mRNA but not of IL-4 mRNA. Together, our data show that T-cell receptor-mediated and lymphokine receptor-mediated signals induce different patterns of lymphokine gene expression and provide strong evidence that the IL-4 and IL-5 genes are differently regulated.
Ca2+/calmodulin-dependent protein kinase II: localization in the interphase nucleus and the mitotic apparatus of mammalian cells.
Authors: Ohta Y , Ohba T , Miyamoto E
Published: May 2006

Indirect immunofluorescence was used to determine the distribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in rat embryo fibroblast 3Y1 cells, rat C6 glioma cells, and human epidermoid carcinoma KB cells. During interphase at growing phase, CaM kinase II was localized diffusely in the cytoplasm and in the nucleus. In the nucleus, the enzyme was localized within the whole nuclear matrix in which the enzyme was specially concentrated in nucleoli. During mitosis, CaM kinase II was found to be a dynamic component of the mitotic apparatus, particularly present at microtubule-organizing centers. In metaphase and anaphase, CaM kinase II was observed at centrosomes and between the spindle poles. During telophase, CaM kinase II was condensed as a bright fluorescent dot at the midzone of the intercellular bridge between two daughter cells, while tubulin was found at each side of the midbody. Colchicine, a microtubule inhibitor, disorganized the tubulin- and CaM kinase II specific fluorescent structure of mitotic 3Y1 cells. In cold-treated cells, CaM kinase II was localized predominantly at centrosomes. The localization of CaM kinase II in the cell nucleus and the mitotic apparatus suggests that the enzyme may play a role in the cell cycle progression of mammalian cells.
A biological price of antibiotic resistance: major changes in the peptidoglycan structure of penicillin-resistant pneumococci.
Authors: Garcia-Bustos J , Tomasz A
Published: May 2006

Pneumococcal strains with greatly elevated levels of resistance to penicillin have by now been described with increasing frequency worldwide. The mechanism of antibiotic resistance in these strains involves the molecular remodeling of cell wall synthetic enzymes (penicillin binding proteins). We have now analyzed the peptidoglycan structures of 10 penicillin-susceptible and 10 penicillin-resistant clinical isolates (4 of intermediate and 6 of high level resistance) with a high-resolution HPLC technique. Cell wall peptidoglycan of the susceptible strains contained monomeric and oligomeric forms of primarily (70% or more) linear stem peptides with the sequence of L-Ala-D-iGln-L-Lys-D-Ala (where iGln is isoglutamine). In contrast, the major peptide species (70% or more) of resistant cell walls were abnormal branched-stem peptides carrying Ala-Ser or Ala-Ala dipeptides on the epsilon-amino groups of the stem peptide lysine residues. The structural alteration in the peptidoglycan was not related to serotype, date, or site of isolation but showed strong correlation with penicillin resistance and was cotransformed with high-level penicillin resistance during genetic transformation. We suggest that the remodeling of the active site of penicillin binding proteins in the resistant bacteria, which results in the reduced affinity for penicillin, also changes the substrate preference of these enzymes for the more hydrophobic branched peptides (instead of linear peptides) for cell wall synthesis.
Cytologic assessment of nuclear and cytoplasmic O-linked N-acetylglucosamine distribution by using anti-streptococcal monoclonal antibodies.
Authors: Turner J R , Tartakoff A M , Greenspan N S
Published: May 2006

Recent studies have demonstrated the existence of single O-linked N-acetylglucosamine (O-GlcNAc) residues on cytoplasmic and nuclear glycoproteins. Labeled lectin and enzymatic techniques have been used to identify O-GlcNAc-bearing proteins, but no antibodies generally reactive with such O-linked GlcNAc moieties have been described. We have previously characterized monoclonal antibodies (mAbs) specific for the GlcNAc residues of streptococcal group A carbohydrate, which is composed of a polyrhamnose backbone with GlcNAc side chains. We now report that these mAbs recognize O-GlcNAc-bearing proteins. By immunofluorescence, the mAbs reacted strongly with the nuclear periphery and nucleoplasm of mammalian cells and stained the cytoplasm less intensely. The distribution was not consistent with labeling of the endoplasmic reticulum, Golgi complex, or plasma membrane. Furthermore, the staining pattern of a mutant cell line, which retains terminal GlcNAc residues on many N-linked glycans, was indistinguishable from that of wild-type cells. Nuclear and cytoplasmic staining were inhibited by free GlcNAc and were completely abolished by galactosylation of terminal GlcNAc residues. Indirect ELISA demonstrated GlcNAc- and galactosylation-inhibitable binding of the mAbs to a 65-kDa human erythrocyte cytosolic protein known to contain O-GlcNAc. Thus, these mAbs react with O-GlcNAc without apparent influence of peptide determinants, do not show detectable binding to N- or O-glycans, and, therefore, represent a valuable tool for the study of O-GlcNAc moieties. In addition, these mAbs provide the first cytologic analysis of the distribution of O-GlcNAc residues throughout the nucleus and the cytoplasm of mammalian cells.
Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.
Authors: Funk C D , Furci L , FitzGerald G A
Published: May 2006

The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, we have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.
Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin D-deficient and vitamin D-replete chickens: a 44Ca stable isotope study.
Authors: Chandra S , Fullmer C S , Smith C A , Wasserman R H , Morrison G H
Published: May 2006

The intestinal absorption of calcium includes at least three definable steps; transfer across the microvillar membrane, movement through the cytosolic compartment, and energy-dependent extrusion into the lamina propria, Tracing the movement of calcium through the epithelium has been hampered by lack of suitable techniques and, in this study, advantage was taken of ion microscopy in conjunction with cryosectioning and use of the stable isotope 44Ca to visualize calcium in transit during the absorptive process. The effect of vitamin D, required for optimal calcium absorption, was investigated. Twenty millimolar 44Ca was injected into the duodenal lumen in situ of vitamin D-deficient and vitamin D-replete chickens. At 2.5, 5.0, and 20.0 min after injection, duodenal tissue was obtained and processed for ion microscopic imaging. At 2.5 min. 44Ca was seen to be concentrated in the region subjacent to the microvillar membrane in tissue from both groups. At 5.0 and 20.0 min, a similar pattern of localization was evident in D-deficient tissues. In D-replete tissues, the distribution of 44Ca became more homogenous, indicating that vitamin D increased the rate of transfer of Ca2+ from the apical to the basolateral membrane, a function previously ascribed to the vitamin D-induced calcium-binding protein (28-kDa calbindin-D). Quantitative aspects of the calcium absorptive process were determined in parallel experiments with the radionuclide 47Ca. Complementary information on the localization of the naturally occurring isotopes of calcium (40Ca) and potassium (39K) is also described.
Feedforward excitation of the hippocampus by afferents from the entorhinal cortex: redefinition of the role of the trisynaptic pathway.
Authors: Yeckel M F , Berger T W
Published: May 2006

For the past 3 decades, functional characterizations of the hippocampus have emphasized its intrinsic trisynaptic circuitry, which consists of successive excitatory projections from the entorhinal cortex to the dentate gyrus, from granule cells of the dentate to the CA3/4 pyramidal cell region, and from CA3/4 to the CA1/2 pyramidal cell region. Despite unequivocal anatomical evidence for a monosynaptic projection from entorhinal to CA3 and CA1/2, few in vivo electrophysiological studies of the direct pathway have been reported. In the experiments presented here, we stimulated axons of entorhinal cortical neurons in vivo and recorded evoked single unit and population spike responses in the dentate, CA3, and CA1 of hippocampus, to determine if pyramidal cells are driven primarily via the monosynaptic or trisynaptic pathways. Our results show that neurons within the three subfields of the hippocampus discharge simultaneously in response to input from a given subpopulation of entorhinal cortical neurons and that the initial monosynaptic excitation of pyramidal cells then is followed by weaker excitatory volleys transmitted through the trisynaptic pathway. In addition, we found that responses of CA3 pyramidal cells often precede those of dentate granule cells and that excitation of CA3 and CA1 pyramidal cells can occur in the absence of granule cell excitation. In total, these results argue for a different conceptualization of the functional organization of the hippocampus with respect to the propagation of activity through its intrinsic pathways: input from the entorhinal cortex initiates a two-phase feedforward excitation of pyramidal cells, with the dentate gyrus providing feedforward excitation of CA3, and with both the dentate and CA3 providing feedforward excitation of CA1.
DNA octamer element can confer E1A trans-activation, and adenovirus infection results in a stimulation of the DNA-binding activity of OTF-1/NFIII factor.
Authors: Chellappan S P , Nevins J R
Published: May 2006

Adenovirus E1A-dependent trans-activation of viral transcription involves the utilization and alteration of multiple sequence-specific transcription factors. Cellular genes are also activated by E1A, one example being the immunoglobulin heavy-chain locus when assayed by transfection into fibroblast cells. We have explored the basis for the E1A-dependent activation of this cellular transcription unit. We demonstrate that the ATTTGCAT ("octamer") element found in the heavy-chain enhancer and promoter is a target for E1A trans-activation since this sequence can confer inducibility to the normally unresponsive simian virus 40 early promoter. In addition, adenovirus infection stimulates the DNA-binding activity of the ubiquitous octamer-specific factor, OTF-1, and we presume that this is the basis for the stimulation of transcription. Although there are no octamer elements in the adenovirus genome that are known to be important for transcription, there are octamer elements in the viral terminal repeat sequences. These elements bind the NFIII factor and are important for the initiation of DNA replication. Since the NFIII factor has been shown to be identical to OTF-1, we suggest that the stimulation of OTF-1/NFIII activity during an adenovirus infection may be important for efficient initiation of adenovirus DNA synthesis.
Retinoblastoma growth suppressor and a 300-kDa protein appear to regulate cellular DNA synthesis.
Authors: Howe J A , Mymryk J S , Egan C , Branton P E , Bayley S T
Published: May 2006

Previous work has suggested that oncogenic transformation by the E1A gene products of adenovirus type 5 may be mediated through interactions with at least two cellular proteins, the 105-kDa product of the retinoblastoma growth suppressor gene (p105-Rb) and a 300-kDa protein (p300). By using viral mutants, we now show that the induction of cellular DNA synthesis in quiescent cells by E1A differs from transformation in that E1A products induce synthesis if they are able to bind to either p105-Rb or p300, and only mutant products that bind to neither are extremely defective. These results suggest that p105-Rb and p300 (or cellular proteins with similar E1A-binding properties) provide parallel means by which DNA synthesis can be regulated.
Cloning and expression of rat liver CTP: phosphocholine cytidylyltransferase: an amphipathic protein that controls phosphatidylcholine synthesis.
Authors: Kalmar G B , Kay R J , Lachance A , Aebersold R , Cornell R B
Published: May 2006

CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) is a key regulatory enzyme in the synthesis of phosphatidylcholine in higher eukaryotes. This enzyme can interconvert between an inactive cytosolic form and an active membrane-bound form. To unravel the structure of the transferase and the mechanism of its interaction with membranes, we have cloned a cytidylyltransferase cDNA from rat liver by the oligonucleotide-directed polymerase chain reaction. Transfection of the rat clone into COS cells resulted in a 10-fold increase in cytidylyltransferase activity and content. The activity of the transfected transferase was lipid-dependent. The central portion of the derived protein sequence of the rat clone is highly homologous to the previously determined yeast cytidylyltransferase sequence [Tsukagoshi, Y., Nikawa, J. & Yamashita, S. (1987) Eur. J. Biochem. 169, 477-486]. The rat protein sequence lacks any signals for covalent lipid attachment and lacks a hydrophobic domain long enough to span a bilayer. However, it does contain a potential 58-residue amphipathic alpha-helix, encompassing three homologous 11-residue repeats. We propose that the interaction of cytidylyltransferase with membranes is mediated by this amphipathic helix lying on the surface with its axis parallel to the plane of the membrane such that its hydrophobic residues intercalate the phospholipids.
A human cellular sequence implicated in trk oncogene activation is DNA damage inducible.
Authors: Ben-Ishai R , Scharf R , Sharon R , Kapten I
Published: May 2006

Xeroderma pigmentosum cells, which are deficient in the repair of UV light-induced DNA damage, have been used to clone DNA-damage-inducible transcripts in human cells. The cDNA clone designated pC-5 hybridizes on RNA gel blots to a 1-kilobase transcript, which is moderately abundant in nontreated cells and whose synthesis is enhanced in human cells following UV irradiation or treatment with several other DNA-damaging agents. UV-enhanced transcription of C-5 RNA is transient and occurs at lower fluences and to a greater extent in DNA-repair-deficient than in DNA-repair-proficient cells. Southern blot analysis indicates that the C-5 gene belongs to a multigene family. A cDNA clone containing the complete coding sequence of C-5 was isolated. Sequence analysis revealed that it is homologous to a human cellular sequence encoding the amino-terminal activating sequence of the trk-2h chimeric oncogene [Kozma, S. C., Redmond, S. M. S., Xiao-Chang, F., Saurer, S. M., Groner, B. & Hynes, N. E. (1988) EMBO J. 7, 147-154]. The presence of DNA-damage-responsive sequences at the 5' end of a chimeric oncogene could result in enhanced expression of the oncogene in response to carcinogens.
Isolation and characterization of a Chinese hamster ovary cell line deficient in fatty alcohol:NAD+ oxidoreductase activity.
Authors: James P F , Rizzo W B , Lee J , Zoeller R A
Published: May 2006

We have isolated a mutant Chinese hamster ovary cell line that is defective in long-chain fatty alcohol oxidation. The ability of the mutant cells to convert labeled hexadecanol to the corresponding fatty acid in vivo was reduced to 5% of the parent strain. Whole-cell homogenates from the mutant strain, FAA.1, were deficient in long-chain fatty alcohol:NAD+ oxidoreductase (FAO; EC 1.1.1.192) activity, which catalyzes the oxidation of hexadecanol to hexadecanoic acid, although the intermediate fatty aldehyde was formed normally. A direct measurement of fatty aldehyde dehydrogenase showed that the FAA.1 strain was defective in this component of FAO activity. FAA.1 is a two-stage mutant that was selected from a previously described parent strain, ZR-82, which is defective in ether lipid biosynthesis and peroxisome assembly. Because of combined defects in ether lipid biosynthesis and fatty alcohol oxidation, the ability of the FAA.1 cells to incorporate hexadecanol into complex lipids was greatly impaired, resulting in a 60-fold increase in cellular fatty alcohol levels. As the FAO deficiency in FAA.1 cells appears to be identical to the defect associated with the human genetic disorder Sjögren-Larsson syndrome, the FAA.1 cell line may be useful in studying this disease.
Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice.
Authors: Korfhagen T R , Glasser S W , Wert S E , Bruno M D , Daugherty C C , McNeish J D , Stock J L , Potter S S , Whitsett J A
Published: May 2006

Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5'-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.