bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae.
Authors: Johnson L M , Kayne P S , Kahn E S , Grunstein M
Published: May 2006

Repression of transcription from the silent mating loci (HML alpha and HMRa) is essential for mating ability in Saccharomyces cerevisiae. This silencing is known to require at least five proteins (SIR1, SIR2, SIR3, SIR4, and histone H4) and is accompanied by a change in chromatin structure. We show here that four positions of histone H4 (N-terminal residues 16, 17, 18, and 19) are crucial to silencing. HML alpha and HMRa are efficiently repressed when these positions are occupied by basic amino acids but are derepressed when substituted with glycine. These results suggest that acetylation of Lys-16 would lead to derepression of the silent mating loci. Three strong extragenic suppressors of the latter H4 mutations were isolated and determined to be located in SIR3. These suppressors allow high mating efficiencies in cells expressing either wild-type H4 or H4 containing single amino acid substitutions. They did not allow efficient mating in a strain that contained an H4 N-terminal deletion. These results indicate that the SIR3 mutations do not bypass the requirement for the H4 N terminus but, rather, allow repression in the presence of a less than optimal H4 N terminus. This provides a link between one of the SIR proteins and a component of chromatin.
Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules.
Authors: Ruano G , Kidd K K , Stephens J C
Published: May 2006

We have developed a reliable method for the direct resolution of haplotypes or linkage phase from individuals who are multiply heterozygous in a given genomic region. The method is based on single-molecule dilution (SMD) of genomic template and amplification via biphasic polymerase chain reaction (booster PCR). We have verified the feasibility of the SMD method for a highly polymorphic region within the beta-globin cluster by analysis of triply heterozygous individuals of known haplotype. This approach should be useful in many studies in population or evolutionary genetics and in a variety of clinical settings.
Quantitative determination that one of two potential RNA-binding domains of the A protein component of the U1 small nuclear ribonucleoprotein complex binds with high affinity to stem-loop II of U1 RNA.
Authors: Lutz-Freyermuth C , Query C C , Keene J D
Published: May 2006

Many RNA-associated proteins contain a ribonucleoprotein (RNP) consensus octamer encompassed by a conserved 80 amino acid sequence, which we have termed an RNA recognition motif (RRM). RRM family members contain either one (class I) or multiple (class II) copies of this motif. We report here that a class II component of the U1 small nuclear RNP (snRNP), the A protein of U1 snRNP (U1snRNP-A), contains two RRMs (RRM1 and -2), yet has only one binding domain (RRM1) that interacts specifically with stem-loop II of U1 RNA. Quantitative analysis of binding affinities of fragments of U1snRNP-A demonstrated that an 86-amino acid polypeptide was competent to bind to U1 RNA with an affinity comparable to that of the full-length protein (Kd approximately 80 nM). The carboxyl-terminal RRM2 of U1snRNP-A did not bind to U1 RNA and may recognize an unidentified heterologous RNA. We propose that class II proteins may function as bridges between RNA components of RNP complexes such as the spliceosome.
Retroviral nucleocapsid protein-metal ion interactions: folding and sequence variants.
Authors: Green L M , Berg J M
Published: May 2006

Retroviral nucleocapsid proteins contain one or two proposed metal-binding sequences of the form Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys. Previously, we reported that an 18-amino acid peptide derived from the nucleocapsid protein of Rauscher murine leukemia virus (RMLV) binds metals such as Co2+ and Zn2+. We have now synthesized the entire nucleocapsid protein from RMLV. We report here that the protein also binds Co2+ and Zn2+ and does so with a higher affinity than does the peptide. Limited proteolysis and circular dichroism studies reveal that metal binding induces folding of the metal-binding domain and, perhaps, the regions adjacent to it but the remainder of the protein remains in a relatively unstructured state. In addition, we have synthesized sequence variants of the metal-binding domain that correspond to viral mutations reported in the literature. In many cases, the metal-binding properties of these peptides correlate with the observed biological activity, providing further evidence for the importance of metal binding to nucleocapsid function.
Spatial differentiation of the intestinal epithelium: analysis of enteroendocrine cells containing immunoreactive serotonin, secretin, and substance P in normal and transgenic mice.
Authors: Roth K A , Gordon J I
Published: May 2006

The mammalian intestinal epithelium undergoes continuous and rapid renewal of its four principal terminally differentiated cell types. These cells arise from multipotent stem cells located at or near the base of the crypts of Lieberkühn. The differentiation process is precisely organized along two spatial dimensions (axes)--from the crypt to the villus tip and from the duodenum to the colon. The enteroendocrine cell population provides a sensitive marker of the intestine's topologic differentiation. At least 15 different regionally distributed subsets have been described based on their principal neuroendocrine products. We have used immunocytochemical methods to characterize the spatial relationships of the serotonin-, secretin-, and substance P-containing enteroendocrine cell subsets in normal adult C57BL/6J x LT/Sv mice as well as in transgenic littermates that contain rat liver fatty acid-binding protein-human growth hormone fusion genes. Our results reveal precise spatial interrelationships between these populations and suggest a differentiation pathway that may involve the sequential expression of substance P, serotonin, and secretin.
Interferon-alpha selectively activates the beta isoform of protein kinase C through phosphatidylcholine hydrolysis.
Authors: Pfeffer L M , Strulovici B , Saltiel A R
Published: May 2006

The early events that occur after interferon binds to discrete cell surface receptors remain largely unknown. Human leukocyte interferon (interferon-alpha) rapidly increases the binding of [3H]phorbol dibutyrate to intact HeLa cells (ED50 = 100 units/ml), a measure of protein kinase C activation, and induces the selective translocation of the beta isoform of protein kinase C from the cytosol to the particulate fraction of HeLa cells. The subcellular distribution of the alpha and epsilon isoforms is unaffected by interferon-alpha treatment. Activation of protein kinase C by phorbol esters mimics the inhibitory action of interferon-alpha on HeLa cell proliferation and down-regulation of protein kinase C blocks the induction of antiviral activity by interferon-alpha in HeLa cells. Increased phosphatidylcholine hydrolysis and phosphorylcholine production is accompanied by diacylglycerol production in response to interferon. However, inositol phospholipid turnover and free intracellular calcium concentration are unaffected. These results suggest that the transient increase in diacylglycerol, resulting from phosphatidylcholine hydrolysis, may selectively activate the beta isoform of protein kinase C. Moreover, the activation of protein kinase C is a necessary element in interferon action on cells.
Evidence for a heat shock transcription factor-independent mechanism for heat shock induction of transcription in Saccharomyces cerevisiae.
Authors: Kobayashi N , McEntee K
Published: May 2006

Transcription of the DNA damage-responsive gene, DDRA2, of Saccharomyces cerevisiae is activated by heat shock treatment as well as by mutagen/carcinogen exposure. Deletion analysis of upstream noncoding sequence indicated that sequences between approximately -190 and -140 base pairs were necessary for heat shock and DNA damage regulation of transcription. Fusion of this region to a CYC1-lacZ reporter gene demonstrated that the sequence between -202 and -165 base pairs was sufficient for basal level and heat shock-induced expression. This DNA sequence was unable to bind heat shock transcription factor as judged by binding competition experiments in vitro. These results indicate that yeast possesses a second, heat shock transcription factor-independent mechanism for activating transcription in response to thermal stress.
Interleukin 2 receptor beta chain expressed in an oligodendroglioma line binds interleukin 2 and delivers growth signal.
Authors: Okamoto Y , Minamoto S , Shimizu K , Mogami H , Taniguchi T
Published: May 2006

Interleukin 2 (IL-2) is a potent growth factor for T lymphocytes, playing a crucial role in the immune response. In view of the considerable evidence that the immunoregulatory cytokines (or lymphokines) also play a role in the growth and differentiation of cells in the central nervous system (CNS), we examined the operation of the IL-2 system in a cell line of CNS origin by expressing a cDNA encoding the beta chain of the human IL-2 receptor (IL-2R beta, a 75-kDa protein). When the cDNA was expressed in a human oligodendroglioma cell line, ONS-21, the IL-2R beta bound IL-2 with an affinity similar to that in lymphoid cells (Kd, approximately 2 nM). Furthermore, cell proliferation ([3H]thymidine incorporation) was stimulated by IL-2. These results demonstrate that the same cytokine receptor is functional in cells of the immune system and CNS and point to a molecular mechanism that is similar for growth-signal transduction between lymphoid and neural cells but that may be different in other cells, such as fibroblasts.
A specific defect in CD3 gamma-chain gene transcription results in loss of T-cell receptor/CD3 expression late after human immunodeficiency virus infection of a CD4+ T-cell line.
Authors: Willard-Gallo K E , Van de Keere F , Kettmann R
Published: May 2006

Sequential effects on cellular protein expression following human immunodeficiency virus (type 1) infection of a CD4+ T-cell line in vitro were investigated. Events in the human interleukin 2-dependent helper T-cell line WE17/10 are similar in several respects to the clinical progression in acquired immunodeficiency syndrome. WE17/10 cell infection is characterized by an extended period during which viral replication occurs without accompanying cytotoxicity and with a maximum 30% decrease in surface CD4. Cellular protein expression generally remains unaffected during this first phase of infection. However, after 2-3 months, a severe defect in the expression of the T-cell receptor/CD3 complex both on the cell surface and inside the cell becomes apparent. Other cell membrane markers, such as CD2 and CD25, remain constant throughout the course of infection; after its initial decrease, CD4 remains at 70% of control values. Lack of surface expression of the TCR/CD3 complex is correlated with a specific defect in transcription of the CD3 gamma-chain gene.
Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation.
Authors: Monsma F J , Mahan L C , McVittie L D , Gerfen C R , Sibley D R
Published: May 2006

In order to clone the D1 dopamine receptor linked to adenylyl cyclase activation, the polymerase chain reaction was used with highly degenerate primers to selectively amplify a cDNA sequence from NS20Y neuroblastoma cell mRNA. This amplification produced a cDNA fragment exhibiting considerable sequence homology to guanine nucleotide-binding (G)-protein-coupled receptors that have been cloned previously. To characterize this cDNA further, a full-length clone was isolated from a rat striatal library by using the cDNA fragment as a probe. Sequence analysis of this cDNA clone indicated that it is indeed a member of the G-protein-coupled receptor family and exhibits greatest homology with the previously cloned catecholamine receptors. Northern blot analysis of various neural tissues revealed a transcript of approximately 4 kb that was predominantly located in the striatum with lesser amounts in the cortex and retina. In contrast, no mRNA was detected in the cerebellum, hippocampus, olfactory bulb, mesencephalon, or pituitary. In situ hybridization analysis also revealed a high abundance of mRNA in the striatum as well as in the olfactory tubercle. To establish the identity of this cDNA, we performed transient expression experiments in COS-7 cells. [3H]SCH-23390, a D1-selective radioligand, exhibited specific, saturable binding only in cells that were transfected with this cDNA. Competition binding analysis with a variety of dopaminergic ligands demonstrated a D1 dopaminergic pharmacology. In addition, dopamine as well as other D1-selective agonists stimulated cAMP accumulation in transfected COS-7 cells. We conclude that we have cloned a cDNA encoding the D1 dopamine receptor linked to the activation of adenylyl cyclase activity.
Interphotoreceptor retinoid-binding protein promotes rhodopsin regeneration in toad photoreceptors.
Authors: Okajima T I , Pepperberg D R , Ripps H , Wiggert B , Chader G J
Published: May 2006

Interphotoreceptor retinoid-binding protein (IRBP) has been hypothesized to function as an intercellular shuttle in the vertebrate eye, serving to transport retinoids between the retinal pigment epithelium (RPE) and photoreceptors in the process by which visual pigment is regenerated after photolysis. This hypothesis was tested in preparations utilizing the toad (Bufo marinus) eye and purified, initially ligand-free IRBP obtained from the bovine eye. Rod outer segments (ROS) or neural retinas were isolated and bleached, then incubated with native RPE (RPE-eyecup) in the presence or absence of IRBP. The amount of rhodopsin present after incubation was determined by spectrophotometric analysis and compared with that in control preparations receiving bovine serum albumin or Ringer's solution only. Supplementation with IRBP enhanced the formation of rhodopsin in both the ROS/RPE-eyecup and retina/RPE-eyecup preparations. Regeneration in ROS/RPE-eyecups receiving IRBP (1.8 nmol) increased in a roughly linear manner with the period of incubation (0-4 hr), at a rate of 0.44 nmol/hr. The extent of regeneration was graded with the quantities of IRBP and opsin introduced into the RPE-eyecup. With increasing amounts of IRBP (up to 5.2 nmol) or of initially available opsin (up to 15.6 nmol), the amount of rhodopsin formed (3-hr incubation) approached the same plateau value, about 2.5 nmol. Analysis of IRBP-supplemented Ringer's solution incubated in the RPE-eyecup showed 11-cis-retinal to be virtually the only retinoid withdrawn from the RPE. With large quantities of IRBP (3.2-9.2 nmol), the amount of 11-cis-retinal (2.7 +/- 0.5 nmol) withdrawn from the RPE during a 3-hr incubation was similar to the plateau value of rhodopsin formed in the ROS/RPE-eyecup. No 11-cis-retinal was observed in albumin-supplemented Ringer's solution (0.4-11.2 nmol of bovine serum albumin) or in Ringer's alone after similar incubation in the RPE-eyecup. The results suggest that an IRBP-mediated transfer of 11-cis-retinal from the RPE to the rods supports rhodopsin regeneration in vivo.
Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum.
Authors: Graybiel A M , Moratalla R , Robertson H A
Published: May 2006

Amphetamine and cocaine are stimulant drugs that act on central monoaminergic neurons to produce both acute psychomotor activation and long-lasting behavioral effects including addiction and psychosis. Here we report that single doses of these drugs induce rapid expression of the nuclear proto-oncogene c-fos in the forebrain and particularly in the striatum, an extrapyramidal structure implicated in addiction and in long-term drug-induced changes in motor function. The two drugs induce strikingly different patterns of c-fos expression in the striosome-matrix compartments and limbic subdivisions of the striatum, and their effects are pharmacologically distinct, although both are sensitive to dopamine receptor blockade. We propose that differential activation of immediate-early genes by psychostimulants may be an early step in drug-specific molecular cascades contributing to acute and long-lasting psychostimulant-induced changes in behavior.
Extinction and recolonization of local populations on a growing shield volcano.
Authors: Carson H L , Lockwood J P , Craddock E M
Published: May 2006

Volcanic action has resulted in the burial of the surfaces of Mauna Loa and Kilauea, Hawaii, by new lava flows at rates as high as 90% per 1000 years. Local populations of organisms on such volcanoes are continually being exterminated; survival of the species requires colonization of younger flows. Certain populations of the endemic Hawaiian species Drosophila silvestris exemplify such events in microcosm. Local populations at the base of an altitudinal cline were destroyed by two explosive eruptions within the last 2100 years. Natural recolonization restored the cline except for one young population that is genetically discordant with altitude.
Antigenic relationships among human rotaviruses as determined by outer capsid protein VP4.
Authors: Gorziglia M , Larralde G , Kapikian A Z , Chanock R M
Published: May 2006

cDNA clones representing the VP4 gene of symptomatic human rotavirus strain KU (VP7 serotype 1) or DS-1 (VP7 serotype 2) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2) were constructed and inserted into a baculovirus expression vector under the control of the polyhedrin promoter. The resulting recombinants expressed the appropriate authentic VP4 rotavirus outer capsid protein. Guinea pigs immunized with these VP4 proteins developed antibodies that neutralized infectivity of the rotavirus from which the immunizing VP4 was derived. These antisera were then used in neutralization tests to define the extent and distribution of VP4 antigenic polymorphism among human rotaviruses. Three distinct serotypes and one subtype of the VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. For the most part, VP4 serotype segregated independently of VP7 serotype. Ten strains of human rotavirus that were associated with symptomatic infection and that exhibited VP7 serotype 1, 3, 4, or 9 specificity, each possessed a VP4 of the same serotype and subtype, designated VP4 serotype 1A. Both symptomatic human rotavirus strains with VP7 serotype 2 specificity were related by neutralization to the VP4 serotype 1A strains and were classified as a subtype of VP4 serotype 1--i.e., serotype 1B--since viruses of serotype 1A appeared to be prime strains. Four human rotavirus strains that were recovered from healthy infants in newborn nurseries in which virus transmission persisted over a long interval, belonged to VP7 serotype 1, 2, 3, or 4, but each strain possessed the same VP4 antigenic specificity that was designated VP4 serotype 2. Finally, a single strain of symptomatic human rotavirus of VP7 serotype 1 specificity possessed a unique VP4 that was provisionally classified as VP4 serotype 3 but this remains to be confirmed because neutralization tests were performed in only one direction. Among the 10 rotavirus strains whose VP4 gene was previously sequenced, there was complete concordance between assignment of VP4 serotype by neutralization and classification according to VP4 amino acid homology. Thus, rotaviruses that exhibited a VP4 amino acid homology of 89% or greater belonged to the same VP4 serotype and subtype as determined by neutralization. Finally, evidence was obtained that the serotype-specific domain is located on the VP8 subunit of VP4.
Dominant mutations causing alterations in acetyl-coenzyme A carboxylase confer tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides in maize.
Authors: Parker W B , Marshall L C , Burton J D , Somers D A , Wyse D L , Gronwald J W , Gengenbach B G
Published: May 2006

A partially dominant mutation exhibiting increased tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides was isolated by exposing susceptible maize (Zea mays) tissue cultures to increasingly inhibitory concentrations of sethoxydim (a cyclohexanedione). The selected tissue culture (S2) was greater than 40-fold more tolerant to sethoxydim and 20-fold more tolerant to haloxyfop (an aryloxyphenoxypropionate) than the nonselected wild-type tissue culture. Regenerated S2 plants were heterozygous for the mutant allele and exhibited a high-level, but not complete, tolerance to both herbicides. Homozygous mutant families derived by self-pollinating the regenerated S2 plants exhibited no injury after treatment with 0.8 kg of sethoxydim per ha, which was greater than 16-fold the rate lethal to wild-type plants. Acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2) is the target enzyme of cyclohexanedione and aryloxyphenoxypropionate herbicides. ACCase activities of the nonselected wild-type and homozygous mutant seedlings were similar in the absence of herbicide. ACCase activity from homozygous tolerant plants required greater than 100-fold more sethoxydim and 16-fold more haloxyfop for 50% inhibition than ACCase from wild-type plants. These results indicate that tolerance to sethoxydim and haloxyfop is controlled by a partially dominant nuclear mutation encoding a herbicide-insensitive alteration in maize ACCase.
Protein kinase C is present in human sperm: possible role in flagellar motility.
Authors: Rotem R , Paz G F , Homonnai Z T , Kalina M , Naor Z
Published: May 2006

We report the presence of protein kinase C (PKC) in ejaculated human sperm as revealed by enzymatic activity assay and indirect immunohistochemistry. PKC is localized in the equatorial segment and in the principal piece of the tail. Addition of phorbol 12-myristate 13-acetate resulted in increased flagellar motility that was blocked by known PKC inhibitors such as sphingosine, staurosporine, and 1-(5-isoquinoylinylsulfonyl)-2-methylpiperazine. A very good correlation (r = 0.9, P less than 0.001) was found between the percentage of PKC-stained sperm cells and motility. We propose that PKC is involved in the regulation of flagellar motility in human sperm.
Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells.
Authors: Brown D , Zhu X L , Sly W S
Published: May 2006

Rat carbonic anhydrase (CA) IV was purified by affinity chromatography and used to produce a specific antiserum in rabbits for immunolocalization studies in rat kidney. CA IV was localized in apical plasma membranes of the proximal convoluted tubule and the thick ascending limb of Henle. Both of these segments are involved in bicarbonate reabsorption in the rat. Immunofluorescent staining of the brush border was faint in the S1 segment, greatest in the S2 segment, and absent from the S3 segment of the proximal tubule. CA IV was also detected in the basolateral plasma membrane of proximal-tubule and thick-ascending-limb epithelial cells by immunofluorescence and immunoelectron microscopy. In the proximal tubule, an extracellular membrane CA had been previously suggested on the basis of electrophysiological studies. CA IV was not detected in intercalated cells of the collecting ducts. These cells contain, in contrast, abundant cytosolic CA II. Thus, the distribution of CA IV is quite distinct from that of CA II; it corresponds with the localization of an isoenzyme(s) that did not stain with antibodies against CA II but that was revealed by histochemical-staining procedures. We conclude that the apical CA IV is the luminal CA responsible for bicarbonate reabsorption in the proximal tubule and the thick ascending limb in the rat kidney. These studies also suggest that CA IV plays a role in bicarbonate transport across the basolateral plasma membrane in these two segments of the rat nephron.
Proximal tubular epithelial cells possess a novel 42-kilodalton guanine nucleotide-binding regulatory protein.
Authors: Zhou J , Sims C , Chang C H , Berti-Mattera L , Hopfer U , Douglas J
Published: May 2006

The proximal tubule of the kidney represents an important location where adenylate cyclase regulates salt and water transport; yet a detailed characterization of the distribution and classification of guanine nucleotide-binding protein (G protein) and adenylate cyclase is lacking. We used purified brush border (20-fold) and basolateral membranes (14-fold) to characterize parathyroid hormone- and G protein-regulated adenylate cyclase and G-protein distribution. Adenylate cyclase was predominantly localized to basolateral membranes, while the 46-kDa alpha subunit of the stimulatory G protein (Gs) was 2-fold higher in brush border membranes than in basolateral membranes. The alpha subunit of the inhibitory G protein (Gi; 41 kDa) was equally distributed on immunoblotting but was 2-fold higher in brush border membranes than in basolateral membranes on radiolabeling with pertussis toxin. A 42-kDa cholera toxin substrate that cross-reacted with antisera to the common alpha subunit of G proteins and to Gs on immunoblotting and that was not immunoprecipitated with two Gi antisera was the most abundant alpha subunit and comprised approximately 1% of the total membrane proteins. These observations suggest that G proteins are important regulators of proximal tubular transport independent of adenylate cyclase.
Identification and characterization of a regulated promoter element in the epidermal growth factor receptor gene.
Authors: Hudson L G , Thompson K L , Xu J , Gill G N
Published: May 2006

We have identified a 36-base-pair proximal element (-112 to -77 relative to the AUG translation initiation codon) in the epidermal growth factor receptor 5' region that functions as a promoter; mediates inductive responses to epidermal growth factor, phorbol 12-myristate 13-acetate, and cyclic AMP; and acts in an orientation-independent manner. This region functions as an enhancer when transferred to a heterologous promoter containing a TATA box. Mutations within the 36-base-pair region alter function as assayed by reporter gene expression in recipient cells. A protein has been identified that demonstrates appropriate binding specificity to mutant DNA sequences that correlates with promoter activity observed in vivo. On the basis of DNA binding characteristics and size, the identified protein appears distinct from several previously identified transcription factors known to bind to G+C-rich regions.
Mutagenesis of essential functional residues in acetylcholinesterase.
Authors: Gibney G , Camp S , Dionne M , MacPhee-Quigley K , Taylor P
Published: May 2006

The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence of a catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrate-inhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.
Do voltage-dependent K+ channels require Ca2+? A critical test employing a heterologous expression system.
Authors: Armstrong C M , Miller C
Published: May 2006

Removal of Ca2+ from the solution bathing neurons is known in many cases to alter the gating properties of voltage-dependent K+ channels and to induce a large, nonselective "leak" conductance. We used a heterologous expression system to test whether the leak conductance observed in neurons is mediated by voltage-dependent K+ channels in an altered, debased conformation. Voltage-dependent K+ channels were expressed in an insect cell line infected with a recombinant baculovirus carrying the cDNA for Drosophila Shaker "A-type" K+ channels. These expressed channels respond to low Ca2+ identically to voltage-dependent K+ channels in native neuronal membranes; upon removal of external Ca2+, Shaker K+ currents disappear and are replaced by a steady, nonselective leak conductance. However, control cells devoid of Shaker channels were free of any voltage-dependent conductances and did not generate a leak when external Ca2+ was removed. These results show that Ca2+ is essential for proper function of voltage-dependent K+ channels and is required to stabilize the native conformations of these membrane proteins.
Circular single-stranded RNA replicon in Saccharomyces cerevisiae.
Authors: Matsumoto Y , Fishel R , Wickner R B
Published: May 2006

Circular RNA replicons have been reported in plants and, in one case, in animal cells. We describe such an element in yeast. In certain yeast strains, a 20S RNA species appears on transfer of cells to acetate medium. This phenotype shows cytoplasmic (non-Mendelian) inheritance and the 20S RNA is associated with 23-kDa protein subunits as a 32S particle. We demonstrate that yeast 20S RNA is an independent replicon with no homology to host genomic, mitochondrial, or 2-microns plasmid DNA or to the L-A, L-BC, or M1 double-stranded RNA viruses of yeast. The circularity of the 20S RNA is shown by the apparent absence of 3' and 5' ends, by two-dimensional gel electrophoresis, and by electron microscopy. Replication of yeast 20S RNA proceeds through an RNA-RNA pathway, and a 10,000-fold amplification occurs on shift to acetate medium. The copy number of 20S RNA is also reduced severalfold by the SKI gene products, a host antiviral system that also lowers the copy numbers of yeast double-stranded RNA viruses. Yeast 20S RNA and the hepatitis delta virus show some similarities.
Loss of heterozygosity on chromosomes 17 and 18 in breast carcinoma: two additional regions identified.
Authors: Cropp C S , Lidereau R , Campbell G , Champene M H , Callahan R
Published: May 2006

The loss of heterozygosity (LOH) at specific regions of the human genome in tumor DNA is recognized as evidence for a tumor-suppressor gene located within the corresponding region of the homologous chromosome. Restriction fragment length polymorphism analysis of a panel of primary human breast tumor DNAs has led to the identification of two additional regions on chromosomes 17q and 18q that frequently are affected by LOH. Deletions of each of these regions have a significant correlation with clinical parameters that are associated with aggressive breast carcinomas. Previous restriction fragment length polymorphism analysis of this panel of tumors has uncovered several other frequently occurring mutations. LOH on chromosome 18q frequently occurs in tumors with concomitant LOH of loci on chromosomes 17p and 11p. Similarly, tumors having LOH on 17q also have LOH on chromosomes 1p and 3p. This suggests that certain combinations of mutations may collaborate in the development and malignant progression of breast carcinomas.
Evidence that insulin and isoprenaline activate the cGMP-inhibited low-Km cAMP phosphodiesterase in rat fat cells by phosphorylation.
Authors: Degerman E , Smith C J , Tornqvist H , Vasta V , Belfrage P , Manganiello V C
Published: May 2006

Incubation of intact rat fat cells with maximally effective concentrations of insulin (1 nM, 12 min) or isoprenaline (300 nM, 3 min) increased particulate cGMP- and cilostamide-inhibited, low-Km cAMP phosphodiesterase (cAMP-PDE) activity by about 50% and 100%, respectively. In 32P-labeled cells, these agents induced serine 32P-phosphorylation of a 135-kDa particulate protein and, to a variable and lesser extent, a 44-kDa protein, which were selectively immunoprecipitated by anti-cAMP-PDE, as analyzed by SDS/PAGE and autoradiography. In the absence of hormonal stimulation, little phosphorylation was detected (less than 10% of that with the hormones). The two phosphoproteins were identified as cAMP-PDE or a closely related molecule (in the case of the 44-kDa species, perhaps a proteolytic fragment) since (i) amounts of 32P in the immunoprecipitated 135-kDa protein paralleled enzyme inactivation, (ii) prior incubation of the anti-cAMP-PDE with the pure rat or bovine enzyme selectively blocked the immunoprecipitation of the phosphoproteins, (iii) 135- and 44-kDa proteins reacted with the anti-cAMP-PDE on Western immunoblots, and (iv) the two phosphoproteins copurified with cAMP-PDE activity through DEAE-Sephacel chromatography and were isolated by highly selective affinity chromatography on cilostamide-agarose. Thus, in fat cells, catecholamine- and insulin-induced activation of the cAMP-PDE may be mediated via phosphorylation by cAMP-dependent protein kinase and an insulin-activated serine protein kinase, respectively.
Regulation of interleukin 3 mRNA expression in mast cells occurs at the posttranscriptional level and is mediated by calcium ions.
Authors: Wodnar-Filipowicz A , Moroni C
Published: May 2006

Interleukin 3 (IL-3) is transiently produced by murine bone marrow-derived mast cells in response to antigen stimulation of the high-affinity immunoglobulin E receptors. We have studied the postreceptor signaling pathways involved in regulating expression of the IL-3 gene in the murine mast cell line PB-3c. Large amounts of IL-3 mRNA accumulated after exposure of cells to calcium ionophore A23187, a reagent that increases intracellular Ca2+. Phorbol 12-myristate 13-acetate, which stimulates protein kinase C, did not induce IL-3 mRNA accumulation, although it did potentiate the effect of A23187. Nuclear run-on analysis showed that the IL-3 gene is constitutively transcribed in unstimulated cells and that treatment with A23187 and/or phorbol ester has no influence on its transcription rate. The effect of A23187 was found to be due to stabilization of the IL-3 mRNA. In cells maintained in the presence of A23187 the IL-3 mRNA was stable during 3 hr of incubation with actinomycin D, whereas removal of A23187 under the same conditions resulted in rapid degradation of the mRNA. These results indicate that control of expression of the IL-3 gene in mast cells is primarily at the posttranscriptional level and that the Ca2(+)-dependent signal-transduction pathway plays an important role in this process. Synthesis of granulocyte/macrophage colony-stimulating factor mRNA in response to A23187 and phorbol ester was found to be subject to both transcriptional and posttranscriptional regulation.
DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history.
Authors: Yuhki N , O'Brien S J
Published: May 2006

The major histocompatibility complex (MHC) is a multigene complex of tightly linked homologous genes that encode cell surface antigens that play a key role in immune regulation and response to foreign antigens. In most species, MHC gene products display extreme antigenic polymorphism, and their variability has been interpreted to reflect an adaptive strategy for accommodating rapidly evolving infectious agents that periodically afflict natural populations. Determination of the extent of MHC variation has been limited to populations in which skin grafting is feasible or for which serological reagents have been developed. We present here a quantitative analysis of restriction fragment length polymorphism of MHC class I genes in several mammalian species (cats, rodents, humans) known to have very different levels of genetic diversity based on functional MHC assays and on allozyme surveys. When homologous class I probes were employed, a notable concordance was observed between the extent of MHC restriction fragment variation and functional MHC variation detected by skin grafts or genome-wide diversity estimated by allozyme screens. These results confirm the genetically depauperate character of the African cheetah, Acinonyx jubatus, and the Asiatic lion, Panthera leo persica; further, they support the use of class I MHC molecular reagents in estimating the extent and character of genetic diversity in natural populations.
Immunoglobulin switch circular DNA in the mouse infected with Nippostrongylus brasiliensis: evidence for successive class switching from mu to epsilon via gamma 1.
Authors: Yoshida K , Matsuoka M , Usuda S , Mori A , Ishizaka K , Sakano H
Published: May 2006

We have characterized immunoglobulin switch circular DNA in mice infected with the nematode parasite Nippostrongylus brasiliensis. Two kinds of circular DNA were identified in the lymph nodes as excision products of switch recombination of immunoglobulin heavy-chain constant region (CH) genes. One is a recombinant between C mu and C gamma 1 (gamma 1 circle), and the other is a recombinant between C gamma 1 and C epsilon (epsilon circle). In the epsilon circle, a short piece of switch mu (S mu) sequence was inserted between S epsilon and S gamma 1 sequences. The inserted S mu sequence could be a trace of the preceding switch from C mu to C gamma 1. These findings indicate that parasitic infection can induce class switch recombinations in a successive manner, first from C mu to C gamma 1, and then from C gamma 1 to C epsilon.
Placental transfer of essential fatty acids in humans: venous-arterial difference for docosahexaenoic acid in fetal umbilical erythrocytes.
Authors: Ruyle M , Connor W E , Anderson G J , Lowensohn R I
Published: May 2006

Docosahexaenoic acid [22:6(n-3); 22:6(4,-7,10,13,16,19) (DHA)] is required in quantity by the developing nervous system of the fetus. This need could be met through synthesis of DHA from linolenic acid in the fetus or through placental transfer of DHA directly. To study the placental transfer of n-3 fatty acids, we obtained umbilical and maternal blood samples from 26 healthy women and infants at parturition and measured the fatty acid composition and content of both plasma and erythrocytes. A striking finding was a considerable venous-arterial difference for DHA in the umbilical erythrocytes as a proportion of total fatty acids and in absolute concentration. This difference of 2.2 micrograms per billion erythrocytes was 6 times larger than the difference in fetal plasma, when the plasma and erythrocyte concentrations were normalized to whole blood. Most other erythrocyte fatty acids showed a similar trend. In umbilical plasma, significant venous-arterial differences were found for 16:0, 16:1, 18:2, and total saturated fatty acids. There was a similar trend for most other plasma fatty acids. Compared with maternal blood, fetal plasma and erythrocytes had higher levels of 20:4 and DHA and lower levels of 18:2 and 18:3(n - 3) fatty acids as a proportion of total fatty acids. These results suggest that erythrocytes play a major role in the necessary transport of the essential fatty acid DHA into the fetus.
Endogenous neurotransmitter activates N-methyl-D-aspartate receptors on differentiating neurons in embryonic cortex.
Authors: Blanton M G , Lo Turco J J , Kriegstein A R
Published: May 2006

Before synapses form in embryonic turtle cerebral cortex, an endogenous neurotransmitter activates N-methyl-D-aspartate (NMDA) channels on neurons in the cortical plate. Throughout cortical development, these channels exhibit voltage-dependent Mg2+ blockade and are antagonized by D-2-amino-5-phosphonovaleric acid, a selective NMDA receptor antagonist. The activation in situ of these nonsynaptic NMDA channels demonstrates a potential physiological substrate for control of early neuronal differentiation.
Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery.
Authors: Oliver D B , Cabelli R J , Dolan K M , Jarosik G P
Published: May 2006

Escherichia coli azi mutants, whose growth is resistant to millimolar concentrations of sodium azide, were among the earliest E. coli mutants isolated. Genetic complementation, mapping, and DNA sequence analysis now show that these mutations are alleles of the secA gene, which is essential for protein export across the E. coli plasma membrane. We have found that sodium azide is an extremely rapid and potent inhibitor of protein export in vivo and that azi mutants are more resistant to such inhibition. Furthermore, SecA-dependent in vitro protein translocation and ATPase activities are inhibited by sodium azide, and SecA protein prepared from an azi mutant strain is more resistant to such inhibition. These studies point to the utility of specific inhibitors of protein export, such as sodium azide, in facilitating the dissection of the function of individual components of the protein export machinery.
Adult T-cell leukemia-derived factor/thioredoxin, produced by both human T-lymphotropic virus type I- and Epstein-Barr virus-transformed lymphocytes, acts as an autocrine growth factor and synergizes with interleukin 1 and interleukin 2.
Authors: Wakasugi N , Tagaya Y , Wakasugi H , Mitsui A , Maeda M , Yodoi J , Tursz T
Published: May 2006

Interleukin 1 (IL-1) has been obtained from the Epstein-Barr virus-infected B-lymphoblastoid cell line 3B6 and shown to be involved in autocrine growth of 3B6 B cells. Independently, adult T-cell leukemia-derived factor (ADF) was purified from human T-lymphotropic virus I-infected leukemic T-cell line (ATL-2) and reported as an interleukin 2 (IL-2) receptor-inducing factor. We have previously reported the same molecular mass, pI, and NH2-terminal amino acid sequence for both 3B6-derived IL-1 and ADF. cDNA cloning of ADF demonstrated high homology with the prokaryotic disulfide reducing enzyme thioredoxin. We show here that ADF and 3B6-derived IL-1 are identical. By RNA blot, 3B6 and ATL-2 cells were shown to contain high levels of 0.6-kilobase mRNA corresponding to ADF. Such message was not detected in resting peripheral blood lymphocytes but could be weakly induced by lymphocyte activation. Antibodies have been raised against synthetic peptides corresponding to the NH2 terminus and the COOH terminus of ADF. Immunoblotting and sequential immunoprecipitation with these antibodies revealed the same 13-kDa protein in 3B6 and ATL-2 cells. Recombinant ADF could sustain growth of 3B6 and ATL-2 cells at low cellular concentration without fetal calf serum; ADF, thus, appears involved in their autocrine growth. Similarly, recombinant ADF could enhance growth of other B-cell lines, including the Epstein-Barr virus-negative Burkitt lymphoma line BL41 and the lymphoblastoid cell lines CRAG8, CRB95, and 1G8. Finally, recombinant ADF exhibits marked synergism with other cytokines, such as IL-1 and IL-2, allowing virally infected lymphocytes to respond to suboptimal amounts of a variety of growth factors.
Mutations of the Gs alpha-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis.
Authors: Weinstein L S , Gejman P V , Friedman E , Kadowaki T , Collins R M , Gershon E S , Spiegel A M
Published: May 2006

Affected members of most kindreds with Albright hereditary osteodystrophy have a partial deficiency of functional Gs, the guanine nucleotide-binding protein that stimulates adenylyl cyclase. By use of the polymerase chain reaction to amplify genomic fragments with the attachment of a high-melting G + C-rich region (GC clamp) and analysis of these fragments by denaturing gradient gel electrophoresis, heterozygous mutations in the Gs alpha-subunit gene were found in two kindreds. These included a G----C substitution at the donor splice junction of intron 10 and a coding frameshift created by a single base deletion within exon 10. The findings illustrate the heterogeneity of genetic defects in Albright hereditary osteodystrophy and the usefulness of the polymerase chain reaction-denaturing gradient gel electrophoresis method to search rapidly for mutations in a large candidate gene.
The recombinant immunotoxin anti-Tac(Fv)-Pseudomonas exotoxin 40 is cytotoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia.
Authors: Kreitman R J , Chaudhary V K , Waldmann T , Willingham M C , FitzGerald D J , Pastan I
Published: May 2006

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin containing the heavy and light variable regions of the anti-Tac monoclonal antibody fused to a mutant form of Pseudomonas exotoxin (PE). Anti-Tac binds to the p55 subunit of the human interleukin 2 (IL-2) receptor, and anti-Tac(Fv)-PE40 kills human or monkey cell lines that contain either the intact IL-2 receptor or its p55 subunit alone. To assess the usefulness of anti-Tac(Fv)-PE40 in treatment of IL-2 receptor-positive leukemia, we tested peripheral blood mononuclear cells from six patients with adult T-cell leukemia. In each of the six patients, anti-Tac(Fv)-PE40 was extremely cytotoxic to the malignant cells. Metabolic activity and sensitivity of the fresh cells improved when a small amount of IL-2 (10 units per ml) was present during incubation. The toxin concentration necessary to inhibit protein synthesis by 50% after 16-hr incubation of cells with immunotoxin varied from 1.6 to 16 ng/ml (2.5-25 x 10(-11) M). In every case, binding was by means of the Tac antigen because anti-Tac(Fv)-PE40 cytotoxicity was prevented by adding excess anti-Tac antibody. Moreover, anti-Tac alone or an inactive mutant of anti-Tac(Fv)-PE40 without ADP-ribosylation activity had very little cytotoxic activity. Peripheral blood mononuclear cells from normal controls, from a patient with Tac-negative leukemia, and from adult T-cell leukemia patients without significant peripheral blood involvement were not sensitive to anti-Tac(Fv)-PE40. These results indicate that anti-Tac(Fv)-PE40 is a potent cytotoxin against adult T-cell leukemia cells in vitro and warrants clinical testing.
Gating current associated with inactivated states of the squid axon gating channel.
Authors: Bekkers J M , Forster I C , Greeff N G
Published: May 2006

Sodium (Na) channel gating currents were measured in squid (Loligo forbesi) axons to study transitions among states occupied by the Na channel when it is inactivated. These measurements were made at high temporal resolution with a low-noise voltage clamp. The inactivation-resistant gating current, I(g,inact), could be separated into a very fast (tau = 5-25 mus) and a slower (tau = 40-200 mus) component over a wide range of test potentials (-140 mV to 80 mV) and for three different starting potentials (-70 mV, 0 mV, and 50 mV). The time constants for these components plotted against test potential lay on two bell-shaped curves; the time constants at any particular test potential did not depend on the starting potential. Both components had charge-voltage curves that saturated between -150 mV and 50 mV. A fast spike, similar to the fast component of I(g, inact), was also apparent in recordings of the fully recovered total "on" gating current. I(g, inact)(fast) and I(g, inact)(slow) could not together be described by the simplest possible model, a linear three-state scheme; however, I(g, inact)(fast) could be modeled by a two-state scheme operating in parallel with other gating processes. I(g, inact)(slow) and the gating current due to recovery from inactivated states into resting states could together be well described by a three-state scheme. This lends support to models in which a pair of inactivated states are connected by a single voltage-dependent step to the resting states of the Na system.