bluuebunny/crossref_metadata_2025 huggingface dataset:
Conversion of placental alkaline phosphatase from a phosphatidylinositol-glycan-anchored protein to an integral transmembrane protein.
Authors: Berger J , Micanovic R , Greenspan R J , Udenfriend S
Published: May 2006

Placental alkaline phosphatase (PLAP) is normally anchored to the plasma membrane of cells by a phosphatidylinositol-glycan anchor after removal of a carboxyl-terminal peptide from the nascent enzyme. To investigate the signals required for this processing we constructed a chimeric cDNA. The latter was designed to code for a truncated precursor form of PLAP, containing the phosphatidylinositol-glycan attachment site but incapable of any form of membrane attachment, fused to a carboxyl-terminal peptide of vesicular stomatis virus glycoprotein. Expression of the PLAP-vesicular stomatis virus glycoprotein chimeric cDNA in transfected COS cells produced an enzymatically active protein that was attached to the plasma membrane, with the PLAP domain on the outer surface. Assays for the presence of phosphatidylinositol-glycan attachment proved negative, whereas an antibody assay confirmed the presence of the vesicular stomatis virus glycoprotein carboxyl-terminal peptide, leading to the conclusion that the truncated PLAP is attached to the cells by the membrane-spanning domain of the vesicular stomatis virus glycoprotein. In light of previous findings on carboxyl-terminal requirements of PLAP these studies suggest that an essential signal for correct sorting between transmembrane insertion and phosphatidylinositol-glycan attachment resides in the cytoplasmic domain.
Increased capacity for glutathione synthesis enhances resistance to radiation in Escherichia coli: a possible model for mammalian cell protection.
Authors: Moore W R , Anderson M E , Meister A , Murata K , Kimura A
Published: May 2006

A strain of Escherichia coli, enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase activities by recombinant DNA techniques, is more resistant to the lethal effects of gamma-irradiation than is the corresponding wild strain. Although the gene-enriched strain has higher glutathione levels than the wild strain, the observed radioresistance appears to be associated with the increased capacity of the gene-enriched strain to synthesize glutathione when irradiated rather than to the cellular levels of glutathione per se. Thus, resistance was abolished in the presence of buthionine sulfoximine, a selective inactivator of gamma-glutamylcysteine synthetase that decreases glutathione synthesis but that does not act directly to lower cellular glutathione levels. Conclusions drawn from studies on this E. coli model system may have relevance to protection of mammalian cells by glutathione.
A stretch-activated K+ channel sensitive to cell volume.
Authors: Sackin H
Published: May 2006

The role of K+ channels in cell osmoregulation was investigated by using the patch-clamp technique. In cell-attached patches from Necturus proximal tubule, the short-open-time K+ channel at the basolateral membrane could be stretch-activated by pipette suction, where a negative pressure of 6 cm H2O (588.6 Pa) was sufficient to increase the open probability of the channel by a factor of 4.0 +/- 0.8 (n = 7 tubules). A 50% reduction in bath osmolarity increased cell volume by 66 +/- 10% and increased the K+-channel open probability by a factor of 5.8 +/- 1.4 (n = 7) in the same cell-attached patches that were activated by pipette suction. A kinetic analysis indicates one open state and at least two closed states for this epithelial K+ channel. Both suction and swelling shorten the longest time constant of the closed-time distribution by a factor of 3, without significant effect on either the mean open time or the shorter closed-state time constant. The similar effect of suction and swelling is consistent with the hypothesis that stretch-activated K+ channels mediate the increase in macroscopic K+ conductance that occurs during osmoregulation of amphibian proximal tubules. Calculations based on a simple model indicate that small increments in cell volume could produce statistically significant increases in K+-channel activity.
Epinephrine induces changes in the subcellular distribution of the inhibitory GTP-binding protein Gi alpha-2 and a 38-kDa phosphorylated protein in the human platelet.
Authors: Crouch M F , Winegar D A , Lapetina E G
Published: May 2006

By using antibodies specific for alpha subunits of inhibitory GTP-binding proteins (Gi alpha polypeptides) to probe Western blots of whole platelet protein, we detected Gi alpha-2 as the predominant Gi alpha species present in platelets. The subcellular compartmentalization of distinct Gi alpha-2-immunoreactive polypeptides coupled to thrombin and alpha 2-adrenergic receptors was examined in Triton X-100 platelet lysates prepared by highspeed centrifugation. This treatment permitted separation of the Triton-insoluble membrane skeleton from Triton-soluble cell components. In cells treated with either alpha-thrombin or epinephrine, we observed that a greater proportion of Gi alpha-2 was localized in the Triton-soluble fraction than in the Triton-insoluble fraction. Pertussis toxin was found to catalyze ADP-ribosylation of Gi alpha-2 in whole platelets. In thrombin-stimulated cells, this activity was confined to the Triton-soluble fraction and was markedly lower than that of unstimulated cells. Epinephrine, on the other hand, promoted translocation of a portion of the pertussis toxin-sensitive Gi alpha-2 from the Triton-soluble fraction to the Triton-insoluble fraction. In addition, epinephrine stimulated translocation of a phosphorylated protein of approximately 38 kDa that was not ADP-ribosylated by pertussis toxin. This protein expressed immunoreactivity with the general Gi alpha antiserum AS/7 but not with the Gi alpha-2 antiserum LE/3. These findings suggest a role for specific localization of Gi alpha proteins in epinephrine-induced platelet responses.
Nucleocytoplasmic transport of ribosomes in a eukaryotic system: is there a facilitated transport process?
Authors: Khanna-Gupta A , Ware V C
Published: May 2006

We have examined the kinetics of the process by which ribosomes are exported from the nucleus to the cytoplasm using Xenopus laevis oocytes microinjected into the germinal vesicle with radiolabeled ribosomes or ribosomal subunits from X. laevis, Tetrahymena thermophila, or Escherichia coli. Microinjected eukaryotic mature ribosomes are redistributed into the oocyte cytoplasm by an apparent carrier-mediated transport process that exhibits saturation kinetics as increasing amounts of ribosomes are injected. T. thermophila ribosomes are competent to traverse the Xenopus nuclear envelope, suggesting that the basic mechanism underlying ribosome transport is evolutionarily conserved. Microinjected E. coli ribosomes are not transported in this system, indicating that prokaryotic ribosomes lack the "signals" required for transport. Surprisingly, coinjected small (40S) and large (60S) subunits from T. thermophila are transported significantly faster than individual subunits. These observations support a facilitated transport model for the translocation of ribosomal subunits as separate units across the nuclear envelope whereby the transport rate of 60S or 40S subunits is enhanced by the presence of the partner subunit. Although the basic features of the transport mechanism have been preserved through evolution, other aspects of the process may be mediated through species-specific interactions. We hypothesize that a species-specific nuclear 40S-60S subunit association may expedite the transport of individual subunits across the nuclear envelope.
Complete amino acid sequence of a human monocyte chemoattractant, a putative mediator of cellular immune reactions.
Authors: Robinson E A , Yoshimura T , Leonard E J , Tanaka S , Griffin P R , Shabanowitz J , Hunt D F , Appella E
Published: May 2006

In a study of the structural basis for leukocyte specificity of chemoattractants, we determined the complete amino acid sequence of human glioma-derived monocyte chemotactic factor (GDCF-2), a peptide that attracts human monocytes but not neutrophils. The choice of a tumor cell product for analysis was dictated by its relative abundance and an amino acid composition indistinguishable from that of lymphocyte-derived chemotactic factor (LDCF), the agonist thought to account for monocyte accumulation in cellular immune reactions. By a combination of Edman degradation and mass spectrometry, it was established that GDCF-2 comprises 76 amino acid residues, commencing at the N terminus with pyroglutamic acid. The peptide contains four half-cystines, at positions 11, 12, 36, and 52, which create a pair of loops, clustered at the disulfide bridges. The relative positions of the half-cystines are almost identical to those of monocyte-derived neutrophil chemotactic factor (MDNCF), a peptide of similar mass but with only 24% sequence identity to GDCF. Thus, GDCF and MDNCF have a similar gross secondary structure because of the loops formed by the clustered disulfides, and their different leukocyte specificities are most likely determined by the large differences in primary sequence.
Quantitative imaging of free and total intracellular calcium in cultured cells.
Authors: Chandra S , Gross D , Ling Y C , Morrison G H
Published: May 2006

Techniques of fluorescence and ion microscopies were combined to study the free [Ca2+] and total Ca in NIH 3T3 fibroblast and L6 rat myoblast cells. Free Ca2+ measurements with the Ca2+ indicator fura-2 and digital imaging reveal an inhomogeneous distribution of free cytoplasmic Ca2+ in both cell lines. Fura-2 also reveals a difference in free Ca2+ activity between the nucleus and cytoplasm of cells. Ion microscopic observations on sister cells show that total Ca in the cytoplasm is also inhomogeneously distributed and that mean cytoplasmic levels of total Ca are higher than levels in the nuclei. In the nuclei of NIH 3T3 cells, the mean free [Ca2+] and total [Ca] were 110 +/- 30 nM and 225 +/- 43 microM, respectively, while regions in the cell cytoplasm contained up to 490 +/- 270 nM free [Ca2+] and 559 +/- 184 microM mean total [Ca]. Intracellular total Ca was greater than 3 orders of magnitude higher than intracellular free Ca2+ in either nuclear or cytoplasmic compartments. Perinuclear cytoplasmic regions in 3T3 cells contained higher free and total Ca than the cell nucleus. Loading of cells with fura-2 did not modify the subcellular distribution of total K, Na, Ca, or Mg. This combination of two powerful ion imaging techniques provides a comparison between free and total calcium in cells and introduces a different approach for examining the role of this important element in cell physiology.
Comparison of a human T-cell lymphotropic virus type I strain from cerebrospinal fluid of a Jamaican patient with tropical spastic paraparesis with a prototype human T-cell lymphotropic virus type I.
Authors: Sarin P S , Rodgers-Johnson P , Sun D K , Thornton A H , Morgan O S , Gibbs W N , Mora C , McKhann G , Gajdusek D C , Gibbs C J
Published: May 2006

The isolation and characterization of a human T-cell lymphotropic retrovirus related to human T-cell lymphotropic virus type I (HTLV-I) from cerebrospinal fluid of a Jamaican patient with tropical spastic paraparesis is described. The virus isolate is a typical type C retrovirus as seen by electron microscopy and is related to prototype HTLV-I isolated from patients with adult T-cell leukemia but is not identical to this prototype HTLV-I as seen by restriction enzyme mapping.
Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum.
Authors: Radolf J D , Norgard M V , Schulz W W
Published: May 2006

Freeze fracture and deep etching were used to investigate the ultrastructural basis for the observation that anti-treponemal antibodies bind poorly to the surface of virulent Treponema pallidum. Fractures of T. pallidum outer membranes contained scarce, uniformly sized intramembranous particles (IMPs). IMPs on the convex faces often appeared to form linear arrays that wound in spirals about the organism. In contrast to the outer membrane, IMPs of the cytoplasmic membrane were randomly distributed, numerous, and heterogeneous in size. In Escherichia coli and T. pallidum cofractures, IMPs of the E. coli outer membranes were densely packed within the concave fracture faces, while the T. pallidum fractures were identical to the experiments lacking the E. coli internal controls. Outer membranes of two representative nonpathogenic treponemes, Treponema phagedenis biotype Reiter and Treponema denticola, contained numerous IMPs, which segregated preferentially with the concave halves. Examination of apposed replicas and deep-etched specimens indicated that at least some of the IMPs extend through the T. pallidum outer membrane and are exposed on the surface of the organism. The outer membrane of intact T. pallidum appears to contain a paucity of integral membrane proteins that can serve as targets for specific antibodies. These findings appear to represent an unusual parasitic strategy for evasion of host humoral defenses.
Identifying determinants of folding and activity for a protein of unknown structure.
Authors: Bowie J U , Sauer R T
Published: May 2006

We have generated an extensive genetic map of functionally allowed and/or structurally allowed amino acid substitutions in Arc repressor, a DNA binding protein of unknown structure. Analysis of the allowed substitution patterns identifies residues that are likely to be involved in protein function and identifies side chains that play important structural roles, including residues likely to form the hydrophobic core. The identities of approximately one-third of the residues in Arc repressor are functionally important, about one-half are structurally important, and the remainder are unimportant for either structure or function. The patterns of obligatory hydrophobic positions permit strong predictions of secondary structure.
Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers.
Authors: Blaustein R O , Koehler T M , Collier R J , Finkelstein A
Published: May 2006

The three separate proteins that make up anthrax toxin--protective antigen (PA), edema factor (EF), and lethal factor (LF)--act in binary combinations to produce two distinct reactions in experimental animals: edema (PA + EF) and death (PA + LF). PA is believed to interact with a membrane receptor, and after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol. PA can be separated, after mild trypsinolysis, into two fragments, PA65 (65 kDa) and PA20 (20 kDa). We demonstrate that trypsin-cleaved PA is capable of forming cation-selective channels in planar phospholipid bilayer membranes and that this activity is confined to the PA65 fragment; PA20, LF, and EF are devoid of channel-forming activity. These PA65 channels exhibit pH-dependent and voltage-dependent activity--a property reminiscent of the channels formed by the two-chain proteins diphtheria, tetanus, and botulinum toxins.
Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets.
Authors: Ferrell J E , Martin G S
Published: May 2006

We have previously shown that a number of platelet proteins become phosphorylated at tyrosine residues in response to platelet-activating agents. Here we present two lines of evidence implicating a platelet integrin, glycoprotein IIb-IIIa, in the regulation of a specific subset of these tyrosine phosphorylations. (i) Two peptides that inhibit the binding of fibrinogen and other ligands to gpIIb-IIIa, Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val, also inhibited the thrombin-induced tyrosine phosphorylation of this subset of proteins. The tetrapeptide Arg-Gly-Glu-Ser, which does not inhibit fibrinogen binding, did not inhibit thrombin-stimulated tyrosine phosphorylation. (ii) Platelets lacking gpIIb-IIIa (from a subject with Glanzmann thrombasthenia) did not undergo this subset of tyrosine phosphorylation in response to thrombin, although other serine, threonine, and tyrosine phosphorylations proceeded normally. These findings suggest a role for tyrosine-specific protein phosphorylation in integrin-mediated cell-matrix recognition.
Deregulated expression of human c-jun transforms primary rat embryo cells in cooperation with an activated c-Ha-ras gene and transforms rat-1a cells as a single gene.
Authors: Schütte J , Minna J D , Birrer M J
Published: May 2006

While the ability of the retroviral oncogene V-jun to transform chicken cells led to its discovery, the oncogenic potential of its cellular homologue, c-jun, which encodes a transcription factor, is unknown. We isolated a 1070-base-pair cDNA clone containing the unmutated entire open reading frame of c-jun from a human small cell lung cancer line. This cDNA as well as a 5.6-kilobase normal human genomic DNA fragment containing the c-jun gene were placed under the control of retroviral long terminal repeats and introduced into primary rat embryo cells (RECs), with or without other oncogenes, and into an immortal rat fibroblast cell line, Rat-1a, as a single gene. In Rat-1a cells the expression of human c-jun mRNA was associated with the ability to clone in soft agarose and form tumors in nude mice. When the c-jun cDNA or genomic DNA constructs were introduced into RECs, no foci of transformed cells were seen with c-jun alone or c-jun cotransfected with deregulated c-myc or L-myc protooncogenes. However, cotransfection of the c-jun constructs with an activated human c-Ha-ras gene led to foci of transformed cells which gave rise to immortalized cell lines that cloned in soft agarose and formed tumors in nude mice. Furthermore, formation of foci of transformed RECs by the c-jun/ras combination was augmented 3-fold by the tumor promoter phorbol 12-tetradecanoate 13-acetate. We conclude that deregulated expression of human c-jun can participate in malignant transformation of normal mammalian cells.
Lipoproteins alter the catalytic behavior of the platelet-activating factor acetylhydrolase in human plasma.
Authors: Stafforini D M , Carter M E , Zimmerman G A , McIntyre T M , Prescott S M
Published: May 2006

Platelet-activating factor (PAF) has been implicated as a mediator of inflammation, allergy, shock, and thrombosis. A specific degradative enzyme, PAF acetylhydrolase (EC 3.1.1.47), is found in plasma and could regulate the concentration of PAF in blood. In plasma, 70% of the PAF acetylhydrolase is found with low density lipoprotein (LDL), and the remainder is in high density lipoprotein (HDL). In previous studies we found that with subsaturating concentrations of PAF the activity in LDL seemed to be the relevant one; e.g., depletion of LDL slowed degradation of PAF, while removal of HDL accelerated the degradation slightly. We have pursued this observation by using plasma from humans with lipoprotein mutations. In abetalipoproteinemia, all of the PAF acetylhydrolase activity was in HDL, whereas in Tangier disease all of the activity was in LDL. In both conditions the total activity measured in an optimized assay was normal or increased. However, when we measured the t1/2 of PAF in plasma, we found that it was prolonged in subjects with abetalipoproteinemia compared to normal controls. Conversely, the t1/2 in Tangier plasma was shortened. We next demonstrated that the PAF acetylhydrolase in HDL was recognized by an antibody to the enzyme purified from LDL, establishing that the enzyme in the two particles is the same protein. Finally, we inactivated the PAF acetylhydrolase in isolated lipoprotein particles and then reconstituted them with enzyme from the opposite particle. The reconstituted particles were used to measure the t1/2 of PAF, and we again found that the LDL particle was more efficient. We conclude that the lipoprotein environment of the PAF acetylhydrolase markedly influences its catalytic behavior. This may be important in pathophysiology and will complicate attempts to assess the role of this enzyme in such circumstances.
Colorimetric detection of specific DNA segments amplified by polymerase chain reactions.
Authors: Kemp D J , Smith D B , Foote S J , Samaras N , Peterson M G
Published: May 2006

The polymerase chain reaction (PCR) procedure has many potential applications in mass screening. We describe here a general assay for colorimetric detection of amplified DNA. The target DNA is first amplified by PCR, and then a second set of oligonucleotides, nested between the first two, is incorporated by three or more PCR cycles. These oligonucleotides bear ligands: for example, one can be biotinylated and the other can contain a site for a double-stranded DNA-binding protein. After linkage to an immobilized affinity reagent (such as a cloned DNA-binding protein, which we describe here) and labeling with a second affinity reagent (for example, avidin) linked to horseradish peroxidase, reaction with a chromogenic substrate allows detection of the amplified DNA. This amplified DNA assay (ADA) is rapid, is readily applicable to mass screening, and uses routine equipment. We show here that it can be used to detect human immunodeficiency virus sequences specifically against a background of human DNA.
Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector.
Authors: Fuerst T R , Fernandez M P , Moss B
Published: May 2006

Cis- and trans-acting elements of the Escherichia coli lac operon were transferred to vaccinia virus and used to regulate gene expression. A recombinant virus that constitutively expresses a modified lac repressor gene (lacI) was constructed. We calculated that each infected cell contained approximately 2 x 10(7) active repressor molecules (and 1-2 x 10(4) copies of the vaccinia virus genome). A strong vaccinia-virus late promoter was modified by insertion of the lac operator (lacO) at various positions. The ability of each modified promoter to regulate expression of beta-galactosidase was tested by transient assays in cells infected with wild-type or lacI-containing vaccinia virus. Placement of the lacO just downstream of the conserved TAAAT sequence of a late promoter was consistent with a minimal effect on basal expression and good repressibility, whereas basal expression was severely inhibited when lacO overlapped or preceded the TAAAT motif. A single recombinant vaccinia virus containing lacI and the beta-galactosidase gene under control of the optimal lacO promoter was constructed. In the absence of inducer, cells infected with this double recombinant virus synthesized little or no detectable beta-galactosidase. Addition of isopropyl beta-D-thiogalactoside restored expression to greater than 20% of the unrepressed level. This inducible vector system has potential applications for expression of heterologous and homologous genes.
Pertussis toxin induces structural changes in G alpha proteins independently of ADP-ribosylation.
Authors: Ribeiro-Neto F A , Rodbell M
Published: May 2006

Pertussis toxin catalyzes ADP-ribosylation of a family of GTP-binding proteins (G alpha proteins) involved in signal transduction. It is thought that this activity is responsible for the attenuating effects of the toxin on the actions of a number of hormones and neurotransmitters. By utilizing specific antisera for detecting on electrophoretic transfer blots (Western blots) alpha proteins that are subject to ADP-ribosylation, it was found that treatment of these proteins with pertussis toxin resulted in shifts in their electrophoretic mobility and marked enhancement of their immunoreactivity compared to untreated proteins. No changes in mobility or immunoreactivity with specific antisera were observed with beta subunits of G proteins. Both effects on alpha proteins required the same ingredients, including detergents, ATP, and sulfhydryl reducing agents, that other studies have shown are required for activation of the ADP-ribosylating activity of pertussis toxin. However, NAD+, the substrate for ADP-ribosylating activity, was not required. Moreover, inhibition of the ADP-ribosylating activity by 50 mM nicotinamide failed to block the NAD-independent effects of the toxin. These findings indicate that the toxin induces structural changes in alpha proteins independently of its ADP-ribosylating activity and raise the possibility that these structural changes are primary to ADP-ribosylation and causative of many of the biological effects of pertussis toxin.
Structural and functional division into two domains of the large (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2.
Authors: Kirchhausen T , Nathanson K L , Matsui W , Vaisberg A , Chow E P , Burne C , Keen J H , Davis A E
Published: May 2006

The clathrin-associated protein complex 2 (AP-2 complex) is a group of proteins associated with clathrin-coated vesicles and believed to interact with cytoplasmic domains of receptors found in the plasma membrane. AP-2 was purified as an assembly of several polypeptide chains (alpha, beta, AP50, and AP17), of which only the alpha and beta chains (100-115 kDa) show significant heterogeneity. We have obtained cDNA clones for two distinct rat brain beta chains. We have also studied the domain organization of bovine brain AP-2 complexes by selective proteolysis. Results of these studies show that the alpha and beta chains have a similar two-domain organization. Their amino-terminal domains are relatively invariant whereas their carboxyl-terminal domains are variable in both sequence and length. We propose that the variable domains select receptors for inclusion in coated vesicles.
Allozymes of glucose-6-phosphate isomerase differentially modulate pentose-shunt metabolism in the sea anemone Metridium senile.
Authors: Zamer W E , Hoffmann R J
Published: May 2006

We tested the hypothesis that kinetic differences among allelic variants of glucose-6-phosphate isomerase (GPI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) from the sea anemone Metridium senile differentially modulate glucose metabolism at the glycolysis-pentose-shunt branch point. Fractional contribution of pentose shunt and absolute flux of glucose in glycolysis were measured in fasted or fed anemones acclimated to 5 degrees C or 15 degrees C. When fed, anemones of genotype Gpiss routed a greater fraction of glucose through the shunt than did Gpiff anemones; the effect was more pronounced at 5 degrees C than at 15 degrees C. This confirms predictions from kinetic and population data and is consistent with thermal selection maintaining the variation. Relative levels of shunt metabolism increased at 5 degrees C, compared with 15 degrees C, in fed anemones regardless of genotype, but the proportion of glucose metabolized by the pentose shunt was unchanged by temperature in fasted anemones. Glucose flux through the shunt was constant at approximately 5 pmol.mg-1.hr-1 in fed anemones at 5 degrees C and 15 degrees C and in fasted anemones at 15 degrees C, indicating apparently near-perfect thermal acclimation of the absolute flux of glucose through the shunt in fed, but not in fasted, anemones. Rates of glucose oxidation and flux through the shunt in freshly collected anemones were similar to those of anemones fed and acclimated at 15 degrees C in the laboratory. If these differences affect organismal-level processes, Gpi variation could contribute to Darwinian fitness in thermally varying environments.
Synthesis of germ-line gamma 1 immunoglobulin heavy-chain transcripts in resting B cells: induction by interleukin 4 and inhibition by interferon gamma.
Authors: Berton M T , Uhr J W , Vitetta E S
Published: May 2006

Interleukin 4 (IL-4) induces the expression of IgG1 and IgE in lipopolysaccharide-stimulated B cells. Previous studies have suggested that heavy-chain class switching may be regulated by increasing the accessibility of specific switch regions to switch recombinases. In this study, we have used an RNase protection assay to demonstrate that IL-4 induces expression of germ-line gamma 1 transcripts in B cells within 4 hr of culture; induction is dose-dependent and is inhibited by interferon gamma. IL-4 alone is capable of inducing the expression of germ-line gamma 1 transcripts in small, resting B cells, but lipopolysaccharide enhances expression. The germ-line transcripts are the same size (1.8 and 3.4 kilobases) as the secreted and membrane forms of the functional gamma 1 mRNAs and presumably result from the splicing of an upstream switch-region exon(s) to the gamma 1 constant-region exon(s). These data strongly support the "accessibility" model for the regulation of isotype switching and suggest that lymphokines such as IL-4 may direct specific switch events by transcriptional activation of the corresponding switch regions.
Amyloid protein and neurofibrillary tangles coexist in the same neuron in Alzheimer disease.
Authors: Grundke-Iqbal I , Iqbal K , George L , Tung Y C , Kim K S , Wisniewski H M
Published: May 2006

In Alzheimer disease, paired helical filaments accumulate in the neuron, and amyloid fibers are found in the extracellular space in the neuropil and brain vessels. Amyloid and paired helical filaments are morphologically distinct. Although messenger RNA that encodes the amyloid has also been shown in several tissues, including brain, the intracellular expression of the protein has not been observed. By using monoclonal antibodies to a synthetic amyloid beta peptide, the present study demonstrates that amyloid reactivity is present in both Alzheimer patients and normal individuals in different types of neurons, including the neurons with the neurofibrillary tangles, but not in the tangle itself.
Relationship of insulin-like growth factor II gene expression in muscle to synaptogenesis.
Authors: Ishii D N
Published: May 2006

A striking correlation between insulin-like growth factor II (IGF-II) gene expression and turnover of neuromuscular synapses was observed. The IGF-II gene was expressed at a high level in fetal rat hind limb muscles prior to the developmental formation of synapses and increased while polyneuronal innervation accumulated. Thereafter, there was a selective down-regulation of IGF-II mRNAs that was exactly coincident with the postnatal time course for elimination of superfluous synapses. The hypothesis that innervation might provide a signal suppressing IGF-II gene expression was tested. Upon transection of the sciatic nerve, there was up-regulation of IGF-II mRNA content in muscle. This up-regulation was selective and correlated with the capacity of denervated muscle to accept reinnervation. These results suggest that the IGF-II gene may play a role in the development and turnover of synapses.
Brain phospholipase C isozymes: differential mRNA localizations by in situ hybridization.
Authors: Ross C A , MacCumber M W , Glatt C E , Snyder S H
Published: May 2006

mRNAs for isozymes of phospholipase C (PLC) were localized in rat brain by in situ hybridization with oligonucleotide probes for PLC isozymes I, II, and III of Rhee's group [Suh, P.-G., Ryu, S. H., Moon, K. H., Suh, H. W. & Rhee, S. G. (1988) Proc. Natl. Acad. Sci. USA 85, 5419-5423 and (1988) Cell 54, 161-169], and isozyme I of Bennett and Crooke [Bennett, C. F., Balcarek, J. M., Varrichio, A. & Crooke, S. T. (1988) Nature (London) 334, 268-270], which we designate PLC-A. The isozymes displayed different localizations. PLC-A mRNA was highest in the mitral cell layer of the olfactory bulb, choroid plexus, hippocampus and dentate gyrus, magnocellular hypothalamic nuclei, rostral raphe nuclei, and cerebellar Purkinje cells. PLC-I was highest in the internal granular cell layer of the olfactory bulb, cerebral cortex, caudate, nucleus of the lateral olfactory tract, reticular nucleus of thalamus, hippocampus and dentate gyrus, and granule cell layer of the cerebellum. PLC-II had a more widespread distribution, with relatively high levels in the internal granular layer of the olfactory bulb, hippocampus and dentate gyrus, and cerebellar Purkinje and granule cells. PLC-III label was low throughout the brain. These distributions suggest selective coupling of individual PLC isozymes with particular postsynaptic receptors. PLC-A may be preferentially associated with 5-hydroxytryptamine 1C receptors, vasopressin V1 receptors, and a subtype of glutamate receptors. PLC-I may be linked to muscarinic m1 and m3 receptors as well as other receptors. The distribution of PLC-II mRNA resembles that of src protooncogene, with which it displays sequence homology.
cAMP and forskolin decrease gamma-aminobutyric acid-gated chloride flux in rat brain synaptoneurosomes.
Authors: Heuschneider G , Schwartz R D
Published: May 2006

The effects of the cyclic nucleotide cAMP on gamma-aminobutyric acid-gated chloride channel function were investigated. The membrane-permeant cAMP analog N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate inhibited muscimol-induced 36Cl- uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent manner (IC50 = 1.3 mM). The inhibition was due to a decrease in the maximal effect of muscimol, with no change in potency. Similar effects were observed with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and the phosphodiesterase inhibitor isobutylmethylxanthine. The effect of endogenous cAMP accumulation on the gamma-aminobutyric acid-gated Cl- channel was studied with forskolin, an activator of adenylate cyclase. Under identical conditions, in the intact synaptoneurosomes, forskolin inhibited muscimol-induced 36Cl- uptake and generated cAMP with similar potencies (IC50 = 14.3 microM; EC50 = 6.2 microM, respectively). Surprisingly, 1,9-dideoxyforskolin, which does not activate adenylate cyclase, also inhibited the muscimol response, suggesting that forskolin and its lipophilic derivatives may interact with the Cl- channel directly. Indeed, forskolin inhibition of muscimol-induced 36Cl- uptake was extremely rapid (within 5 sec), preceding the accumulation of sufficient levels of cAMP. After 5 min, a slower phase of inhibition was seen, similar to the time course for cAMP accumulation. The data suggest that gamma-aminobutyric acid (GABAA) receptor function in brain can be regulated by cAMP-dependent phosphorylation.
Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone.
Authors: Nagai R , Zarain-Herzberg A , Brandl C J , Fujii J , Tada M , MacLennan D H , Alpert N R , Periasamy M
Published: May 2006

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e., quantitative and/or qualitative reorganization during development and in response to stress. Recent studies indicate that SR Ca2+ uptake function is altered in adaptive cardiac hypertrophy and failure. The molecular basis (genetic and phenotypic) for these changes is not understood. In an effort to determine the underlying causes of these changes, we characterized the rabbit cardiac Ca2+-ATPase phenotype by molecular cloning and ribonuclease A mapping analysis. Our results show that the heart muscle expresses only the slow-twitch SR Ca2+-ATPase isoform. Second, we quantitated the steady-state mRNA levels of two major SR Ca2+ regulatory proteins, the Ca2+-ATPase and phospholamban, to see whether changes in mRNA content might provide insight into the basis for functional modification in the SR of hypertrophied hearts. In response to pressure overload hypertrophy, the relative level of the slow-twitch/cardiac SR Ca2+-ATPase mRNA was decreased to 34% of control at 1 week. The relative Ca2+-ATPase mRNA level increased to 167% of control after 3 days of treatment with thyroid hormone. In contrast, in hypothyroid animals, the relative Ca2+-ATPase mRNA level decreased to 51% of control at 2 weeks. The relative level of phospholamban mRNA was decreased to 36% in 1-week pressure overload. Hyperthyroidism induced a decrease to 61% in the phospholamban mRNA level after 3 days of treatment, while hypothyroidism had virtually no effect on phospholamban mRNA levels. These data indicate that the expression of SR Ca2+-ATPase and phospholamban mRNA may not be coordinately regulated during myocardial adaptation to different physiological conditions.
Multigene families for anthracycline antibiotic production in Streptomyces peucetius.
Authors: Stutzman-Engwall K J , Hutchinson C R
Published: May 2006

Hybridization of polyketide synthase genes from heterologous Streptomyces sp. led to the identification of four unlinked regions of DNA from Streptomyces peucetius that contain genes that encode the production of the same or closely related metabolites, some of which are intermediates of the daunorubicin pathway. DNA fragments from each region that hybridized with the heterologous polyketide synthase genes were hybridized with each other, but very little sequence similarity was observed even though at least two of the regions have similar (if not identical) functions in metabolite production. Some regions, however, do have sequence similarity with other anthracycline-producing Streptomyces sp.
Transfection of fetal rat intestinal epithelial cells by viral oncogenes: establishment and characterization of the E1A-immortalized SLC-11 cell line.
Authors: Emami S , Mir L , Gespach C , Rosselin G
Published: May 2006

Intestinal epithelial cells from 19-day-old rat fetuses underwent electropermeabilization and were successfully transfected by three recombinant plasmids containing the cloned oncogenes from the human adenovirus type 2 early region E1A (SLC-11 cells) and polyoma virus and simian virus 40 large T tumor antigens (SLC-21 and SLC-41 cells). SLC-11 cells were propagated for 21 months in culture (current passage, 76; doubling time, 17 hr) and were immortalized by E1A, as shown by RNA transfer blot (Northern blot) analysis and indirect immunofluorescence of the nuclear oncoproteins. These cells were not tumorigenic in either athymic nude mice or syngeneic Wistar rats and showed a nearly normal karyotype with minimal chromosomal changes. The immortalized epithelial cell line SLC-11 retained several of the phenotypes observed in the parent cells of the intestinal mucosa, including cytoplasmic villin, cytokeratins, enkephalinase, and cell surface receptors sensitive to vasoactive intestinal peptide. It is concluded that immortal SLC-11 cells are a suitable model for studying the proliferation and differentiation of epithelial intestinal cells and analyzing cancer progression in the gastrointestinal tract.
Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications.
Authors: Kaul D K , Fabry M E , Nagel R L
Published: May 2006

To understand the role of sickle cell adherence to the vascular endothelium in the pathophysiology of sickle cell anemia (SS) vasoocclusion, we have carried out a microcirculatory study utilizing the ex vivo mesocecum vasculature of the rat. A single bolus of washed oxy-normal (AA) erythrocytes or oxy-SS cells (unseparated or density-defined SS cell classes) was infused. Hemodynamic monitoring and intravital microscopic observations of the microvascular flow revealed higher peripheral resistance for SS erythrocytes and adherence of these cells exclusively to the venular endothelium but rare or no adherence of AA cells. The extent of adhesion was inversely correlated with venular diameters (r = -0.812; P less than 0.00001). The adhesion of SS erythrocytes is density-class dependent: reticulocytes and young discocytes (SS1) greater than discocytes (SS2) greater than irreversible sickle cells and unsicklable dense discocytes (SS4). Selective secondary trapping of SS4 (dense cells) is found in postcapillary venules where deformable SS cells are preferentially adhered. We conclude that in the oxygenated condition, vasoocclusion can be induced by two events: (i) random precapillary obstruction by a small number of SS4 cells; (ii) increased adhesion of SS1 and SS2 cells in the immediate postcapillary venules. A combination of precapillary obstruction, adhesion in postcapillary venules, and secondary trapping of dense cells may induce local hypoxia, increased polymerization of hemoglobin S, and rigidity of SS erythrocytes, thereby extending obstruction to nearby vessels.
Co-release of acetylcholine and gamma-aminobutyric acid by a retinal neuron.
Authors: O'Malley D M , Masland R H
Published: May 2006

Rabbit retinas were vitally stained with 4',6-diamidino-2-phenylindole (DAPI), a fluorescent compound that selectively accumulates within the cholinergic amacrine cells. The retinas were then incubated in vitro in the presence of radioactive gamma-aminobutyric acid (GABA) and autoradiographed. The cells that accumulated DAPI were found to accumulate GABA, confirming immunohistochemical evidence that the cholinergic amacrine cells contain GABA. Incubation of retinas in the presence of elevated concentrations of K+ caused them to release acetylcholine and GABA, and autoradiography showed depletion of radioactive GABA from the cholinergic amacrine cells. This indicates that the cholinergic amacrine cells can secrete acetylcholine and GABA. Retinas were double-labeled with [14C]GABA and [3H]acetylcholine, allowing simultaneous measurement of their release. The release of [14C]GABA was found to be independent of extracellular Ca2+. Radioactive GABA synthesized endogenously from [14C]glutamate behaved the same way as radioactive GABA accumulated from the medium. In the same experiments the simultaneously measured release of [3H]acetylcholine was strongly Ca2+-dependent, indicating that the releases of acetylcholine and GABA are controlled by different mechanisms. Synaptic vesicles immunologically isolated from double-labeled retinas contained much [3H]acetylcholine and little or no [14C]GABA. These results suggest that the cholinergic amacrine cells release acetylcholine primarily by vesicle exocytosis and release GABA primarily by means of a carrier.
Human recombinant lipocortin 1 has acute local anti-inflammatory properties in the rat paw edema test.
Authors: Cirino G , Peers S H , Flower R J , Browning J L , Pepinsky R B
Published: May 2006

Human recombinant lipocortin 1 has been tested for anti-inflammatory activity in a conventional model of acute inflammation. Microgram amounts of the protein, locally administered, inhibited edema of the rat paw when induced by subplantar injections of carrageenin: the ED50 was 10-20 micrograms per paw, and inhibition (maximum of 60-70%) was not dependent upon an intact adrenal cortex. Doses of lipocortin that produced approximately 50% inhibition in the carrageenin test were inactive against edema elicited by bradykinin, serotonin, platelet-activating factor-acether, or dextran, whereas edema caused by Naja mocambique venom phospholipase A2 was strongly inhibited by lipocortin. The protein inhibited edema when rats were pretreated with agents that depleted mast-cell amines, kininogen, or polymorphonuclear leukocytes prior to initiation of the carrageenin edema but had no inhibitory action when rats were pretreated with the dual cyclooxygenase/lipoxygenase inhibitor BW 755C. These results demonstrate that human recombinant lipocortin has potent local anti-inflammatory activity, probably through selectively interfering with eicosanoid generation. Lipocortin is relatively ineffective against edema caused by mast-cell degranulation or kinins, except when degranulation is caused by phospholipase A2.
Pasteurella multocida toxin: potent mitogen for cultured fibroblasts.
Authors: Rozengurt E , Higgins T , Chanter N , Lax A J , Staddon J M
Published: May 2006

Native Pasteurella multocida toxin (PMT) is shown to be an extremely potent mitogen for Swiss 3T3 fibroblasts. Half-maximal stimulation of DNA synthesis was obtained at concentrations of 1 and 2 pM for recombinant PMT (rPMT) and PMT, respectively. The degree of rPMT-induced DNA synthesis was comparable to that elicited by 10% fetal bovine serum and, moreover, was observed in the complete absence of other factors. Cell proliferation was also enhanced by rPMT. The toxin was also a potent mitogen for BALB/c and NIH 3T3 cells, 3T6 cells, and tertiary mouse embryo or human fibroblasts. The mitogenic activity of rPMT was heat-labile. A polyclonal antiserum to PMT inhibited DNA synthesis when added early, but not late, during treatment of the Swiss 3T3 cells with rPMT. A similar time-dependent action of methylamine was also observed. Furthermore, transient exposure of the cells to rPMT at 37 degrees C, but not at 4 degrees C, resulted in a stimulation of DNA synthesis. Thus, toxin action may require cell entry and processing via an acidic compartment. The toxin, at mitogenic concentrations, caused a large increase in the production of inositol phosphates. In contrast, rPMT did not increase the intracellular concentration of cyclic AMP in Swiss 3T3 cells. The basis of rPMT action may afford a unique insight into molecular signaling events involved in the control of cell proliferation.
Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies.
Authors: Georges E , Bradley G , Gariepy J , Ling V
Published: May 2006

P-glycoprotein is a highly conserved membrane protein shown to be overexpressed in many multidrug-resistant tumor cell lines. P-glycoprotein is encoded by a small gene family in mammalian cells. Class I and II isoforms cause multidrug resistance, whereas class III does not. In this report, we have characterized three P-glycoprotein-specific monoclonal antibodies (mAbs) by high-resolution epitope mapping with a series of hexapeptides. mAb C494 is gene specific, binding to a sequence present only in the class I isoform of hamster and human. The mAb C32 recognizes a sequence conserved in hamster class I and II isoforms but not in class III isoforms. In contrast, the mAb C219 recognizes a highly conserved amino acid sequence found in all P-glycoprotein isoforms characterized to date. These mAbs were used to reveal differential expression and specific localization of the three P-glycoprotein isoforms in hamster tissues by immunohistochemical staining and competition with epitope-specific peptides. Colonic epithelial cells expressed predominantly the class I isoform in a polarized manner, adrenal cortical cells expressed predominantly the class II isoform, whereas a small percentage of skeletal muscle fibers expressed the class III isoform of P-glycoprotein. These findings suggest that the P-glycoprotein isoforms have distinct physiological roles associated with specialized cell functions.
Nonminimum phase non-Gaussian autoregressive processes.
Authors: Breidt F J , Davis R A , Lii K S , Rosenblatt M
Published: May 2006

The structure of non-Gaussian autoregressive schemes is described. Asymptotically efficient methods for the estimation of the coefficients of the models are described under appropriate conditions, some of which relate to smoothness and positivity of the density function f of the independent random variables generating the process. The principal interest is in nonminimum phase models.