bluuebunny/crossref_metadata_2025 huggingface dataset:
An efficient deletion mutant packaging system for defective herpes simplex virus vectors: potential applications to human gene therapy and neuronal physiology.
Authors: Geller A I , Keyomarsi K , Bryan J , Pardee A B
Published: May 2006

We have previously described a defective herpes simplex virus (HSV-1) vector system that permits the introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli beta-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. We now report an efficient packaging system for HSV-1 vectors using a deletion mutant, D30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses beta-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.
Synergistic enhancement of globin gene expression by activator protein-1-like proteins.
Authors: Moi P , Kan Y W
Published: May 2006

DNA sequences corresponding to the four major DNase I hypersensitive sites upstream of the beta-globin gene cluster are essential for the achievement of high levels of globin gene expression and development regulation. In this study, we focused on one of these sites, hypersensitive site 2, which behaves as a powerful enhancer in transient expression and transgenic mouse experiments. We identified a tandem repeat of the activator protein 1 (AP-1) consensus sequence that binds AP-1-like proteins from nuclear extracts of K562 and HeLa cells. These proteins have the same binding properties as HeLa AP-1 but differ in the electrophoretic mobility and in functional assays. Transient-expression experiments in K562 of various deletion and point mutation constructs derived from hypersensitive site 2 indicate that the enhancer activity and the inducibility of a linked gamma-globin promoter are dependent upon the synergistic action of proteins bound to the tandem AP-1 repeat.
Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells.
Authors: Schmidhauser C , Bissell M J , Myers C A , Casperson G F
Published: May 2006

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.
In vitro assembly of infectious nucleocapsids of bacteriophage phi 6: formation of a recombinant double-stranded RNA virus.
Authors: Olkkonen V M , Gottlieb P , Strassman J , Qiao X Y , Bamford D H , Mindich L
Published: May 2006

A system is described for assembling infectious bacteriophage phi 6 nucleocapsids in vitro. Procapsids encoded by cDNA copies of genomic segment L in Escherichia coli were used to package and replicate viral RNA segments. The resulting filled particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein P8. The infected spheroplasts yielded infectious virions. A modified cDNA-derived RNA segment was inserted into virions by this method. The resulting infectious virions contained the same 4-base-pair deletion as the modified cDNA. These findings support the contention that the preformed procapsids are the "machine" that replicates the phi 6 genome, by showing that the cDNA-derived procapsids are competent to package and replicate RNA properly.
Thickness determination of biological samples with a zeta-calibrated scanning tunneling microscope.
Authors: Wang Z H , Hartmann T , Baumeister W , Guckenberger R
Published: May 2006

A single-tube scanning tunneling microscope has been zeta-calibrated by using atomic steps of crystalline gold and was used for measuring the thickness of two biological samples, metal-coated as well as uncoated. The hexagonal surface layer of the bacterium Deinococcus radiodurans with an open network-type structure shows thickness values that are strongly influenced by the substrate and the preparation method. In contrast, the thickness of the purple membrane of Halobacterium halobium with its densely packed less-corrugated structure exhibits very little variation in thickness in coated preparations and the values obtained are in good agreement with x-ray data.
Amplification of an ancestral mammalian L1 family of long interspersed repeated DNA occurred just before the murine radiation.
Authors: Pascale E , Valle E , Furano A V
Published: May 2006

Each mammalian genus examined so far contains 50,000-100,000 members of an L1 (LINE 1) family of long interspersed repeated DNA elements. Current knowledge on the evolution of L1 families presents a paradox because, although L1 families have been in mammalian genomes since before the mammalian radiation approximately 80 million years ago, most members of the L1 families are only a few million years old. Accordingly it has been suggested either that the extensive amplification that characterizes present-day L1 families did not occur in the past or that old members were removed as new ones were generated. However, we show here that an ancestral rodent L1 family was extensively amplified approximately 10 million years ago and that the relics (approximately 60,000 copies) of this amplification have persisted in modern murine genomes (Old World rats and mice). This amplification occurred just before the divergence of modern murine genera from their common ancestor and identifies the murine node in the lineage of modern muroid rodents. Our results suggest that repeated amplification of L1 elements is a feature of the evolution of mammalian genomes and that ancestral amplification events could provide a useful tool for determining mammalian lineages.
Mutational analysis of an archaebacterial promoter: essential role of a TATA box for transcription efficiency and start-site selection in vitro.
Authors: Reiter W D , Hüdepohl U , Zillig W
Published: May 2006

By using a recently developed in vitro transcription assay, the 16S/23S rRNA-encoding DNA promoter from the archaebacterium Sulfolobus sp. B12 was dissected by deletion and linker substitution mutagenesis. The analysis of 5' and 3' deletion mutants defined a core promoter region between positions -38 and -2 containing all information for efficient and specific transcription. Further characterization of this region by linker substitution mutagenesis indicated two sequence elements important for promoter function--one located between positions -38 and -25 (distal promoter element) and the other one located between positions -11 and -2 (proximal promoter element). The distal promoter element encompassed the TATA-like "box A" element located approximately 26 nucleotides upstream of the majority of transcription start sites in archaebacteria (Archaeobacteria). All mutations within this box A motif virtually abolished promoter function. Complete inactivation of the proximal promoter element was dependent on extensive mutagenesis; this element is not conserved between archaebacterial promoters except for a high A + T content in stable RNA gene promoters from Sulfolobus. Mutants containing insertions or deletions between the distal and proximal promoter elements were only slightly affected in their transcription efficiency but displayed a shift in their major initiation site, retaining an essentially fixed distance between the distal promoter element and the transcription start site. Thus, efficient transcription and start-site selection were dependent on a conserved TATA-like sequence centered approximately 26 nucleotides upstream of the initiation site, a situation unlike that of eubacterial promoters but resembling the core structure of most eukaryotic RNA polymerase II (and some RNA polymerase III) promoters. This finding suggests a common evolutionary origin of these promoters consistent with the known similarities between archaebacterial and eukaryotic RNA polymerases.
Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor.
Authors: Hayashida K , Kitamura T , Gorman D M , Arai K , Yokota T , Miyajima A
Published: May 2006

Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, we obtained a homologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells--i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. We therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the alpha and beta subunits of the GM-CSF receptor, respectively.
Dopamine induces neurite retraction in retinal horizontal cells via diacylglycerol and protein kinase C.
Authors: Rodrigues P dos S , Dowling J E
Published: May 2006

Dopamine causes a significant retraction of neurites of bull-head catfish horizontal cells maintained in culture. The effects of dopamine are blocked by haloperidol and SCH 23390, a D1 antagonist, but not by sulpiride, a D2 antagonist. The dopamine-induced morphological changes were mimicked by SKF 38393, a D1 agonist, but not by quinpirole, a D2 agonist. Kainate also caused process retraction, but other neuroactive substances tested including glutamate, 5-hydroxytryptamine, N-methyl-D-aspartate, gamma-aminobutyric acid, and glycine caused only minor changes in neurite length. Cyclic AMP analogues do not induce neurite retraction in horizontal cells, indicating that this effect of dopamine is not mediated by cyclic AMP. However, a protein kinase C activator (phorbol 12-myristate 13-acetate) and synthetic diacylglycerol analogs (1-oleoyl-2-acetyl-sn-glycerol and dioctanoglycerol) caused marked neurite retraction. Their effects, as well as the dopamine-induced changes, were blocked by staurosporine, a potent protein kinase antagonist. The results suggest that dopamine causes neurite retraction by the activation of protein kinase C via diacylglycerol.
Isolation and amino acid sequences of squirrel monkey (Saimiri sciurea) insulin and glucagon.
Authors: Yu J H , Eng J , Yalow R S
Published: May 2006

It was reported two decades ago that insulin was not detectable in the glucose-stimulated state in Saimiri sciurea, the New World squirrel monkey, by a radioimmunoassay system developed with guinea pig anti-pork insulin antibody and labeled pork insulin. With the same system, reasonable levels were observed in rhesus monkeys and chimpanzees. This suggested that New World monkeys, like the New World hystricomorph rodents such as the guinea pig and the coypu, might have insulins whose sequences differ markedly from those of Old World mammals. In this report we describe the purification and amino acid sequences of squirrel monkey insulin and glucagon. We demonstrate that the substitutions at B29, B27, A2, A4, and A17 of squirrel monkey insulin are identical with those previously found in another New World primate, the owl monkey (Aotus trivirgatus). The immunologic cross-reactivity of this insulin in our immunoassay system is only a few percent of that of human insulin. Squirrel monkey glucagon is identical with the usual glucagon found in Old World mammals, which predicts that the glucagons of other New World monkeys would not differ from the usual Old World mammalian glucagon. It appears that the peptides of the New World monkeys have diverged less from those of the Old World mammals than have those of the New World hystricomorph rodents. The striking improvements in peptide purification and sequencing have the potential for adding new information concerning the evolutionary divergence of species.
Delayed reproduction and fitness in variable environments.
Authors: Tuljapurkar S
Published: May 2006

Many organisms delay the initiation of reproduction even though such delay is not adaptive in a constant environment. Theoretical arguments in this paper show that delaying reproduction can increase fitness in a sufficiently variable environment. This paper uses stochastic demography to analyze the fluctuating population structure produced by environmental uncertainty. The results explain previously puzzling features of life cycle delays observed in nature, predicting that populations of the same species living in environments of differing harshness can display different life history phenotypes, a number of distinct life history phenotypes can coexist neutrally within a single population, and genetic polymorphisms are easily maintained if heterozygotes have intermediate life history phenotypes.
Various regulatory sequences are deprived of their uniqueness by the universal rule of TA/CG deficiency and TG/CT excess.
Authors: Ohno S , Yomo T
Published: May 2006

The universal rule of TA/CG deficiency-TG/CT excess endures the extremely high mutation rate of a retrovirus (human immunodeficiency virus type 1) as well as methylation of CAG rather than CG in a plant (maize). Among the consistently abundant nucleotide oligomers, there are two complementary pairs of palindromic nucleotide pentamers containing TG and CA. Out of the CAGTG and CACTG pair emerged the heptameric pair for the long-distance recombination of immunoglobulin genes, CACAGTG and CACTGTG. Reflecting their origin, these heptamers are found everywhere in all DNA, and a substantial fraction of them are accompanied by nonameric components properly spaced from them. It appears that, were the recombination event not confined to B cells, results of illegitimate recombinations might be disastrous. The other pentameric pair is TGCAT and ATGCA. Out of this pair emerged the complementary pair of transcription enhancer decamers: TNATTTGCAT for immunoglobulin light chains and ATGCAAATNA for immunoglobulin heavy chains. Again reflecting their origin, these decamers are found everywhere in all DNA and some genes--for example, in the 3' flanking region of immunoglobulin heavy chain constant region--are accompanied by a downstream "TATA box." It seems that even with regard to the productively recombined immunoglobulin genes, misinitiation of enhanced transcription is a real possibility.
Nontumorigenic squamous cell carcinoma line converted to tumorigenicity with methyl methanesulfonate without activation of HRAS or MYC.
Authors: Milo G E , Shuler C , Kurian P , French B T , Mannix D G , Noyes I , Hollering J , Sital N , Schuller D , Trewyn R W
Published: May 2006

Plasticity of human tumor populations could account for the reason why many tumorigenic human cell lines lose this feature when grown in culture. Methyl methanesulfonate (MMS) was used to convert premalignant squamous cell carcinoma (SCC) cell line SCC-83-01-82 to a malignant phenotype. The MMS-treated SCC-83-01-82 cells (MMS-SCC-83-01-82) produced progressively growing tumors in 5 of 11 splenectomized BALB/c nude mice within 3-5 months. A cell line, designated SCC-83-01-82 CA, was established in vitro from one of the mouse tumors and was repassaged successively. This SCC-83-01-82 CA cell line was aggressively tumorigenic. A tumor greater than or equal to 2.0 cm in size was present within a month, as opposed to the 3-5 months required for the tumors produced by the MMS-SCC-83-01-82 cells. Examination of frozen cross sections by in situ hybridization revealed that focal areas of the tumor produced by the MMS-SCC-83-01-82 cells expressed MYC and HRAS mRNA. However, by the third passage in vivo, the levels of expression of the corresponding genes in the mouse tumors were undetectable. Blot-hybridization analysis of the RNA from the MMS-SCC-83-01-82 cells and the subsequently derived tumors and cells did not indicate any consistent overexpression of MYC, HRAS, or KRAS. Restriction fragment length polymorphism analysis of both MYC and HRAS genes revealed neither rearrangement nor amplification of MYC nor point mutation in the 11th or 12th codon of HRAS. The data suggest that alterations in MYC and HRAS were not directly involved in either the initial transformation or MMS-induced tumorigenic conversion of the SCC-83-01-82 cell line. Persistence of tumorigenicity after reisolation of the MMS-converted premalignant SCC-83-01-82 cells did not disappear immediately following the treatment with MMS.
Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates Ca2+/calmodulin-independent activity.
Authors: Waxham M N , Aronowski J , Westgate S A , Kelly P T
Published: May 2006

We have examined the role of Thr-286 autophosphorylation in the autoregulation of Ca2+/calmodulin-dependent protein kinase II. Using site-directed mutagenesis, we have substituted alanine or serine for Thr-286, or isoleucine for Arg-283, in the 50-kDa subunit of the kinase and expressed each protein in bacteria. Activation and autophosphorylation of all four enzymes were stringently dependent on Ca2+/calmodulin, indicating that neither Arg-283 nor Thr-286 is an absolute requirement for the pseudosubstrate inhibition of the enzyme. Autophosphorylation of the Ile-283 or Ala-286 enzyme generated little, if any, Ca2+/calmodulin-independent kinase activity, unlike the parent (Thr-286) or Ser-286 enzyme. The enzymes expressed in bacteria are predominantly monomeric, indicating that the generation of Ca2+/calmodulin-independent activity does not require the cooperative interactions of subunits normally present in the brain holoenzyme.
Two versions of the gene encoding the 41-kilodalton subunit of the telomere binding protein of Oxytricha nova.
Authors: Hicke B J , Celander D W , MacDonald G H , Price C M , Cech T R
Published: May 2006

Macronuclear chromosomes of the ciliated protozoan Oxytricha nova terminate with a single-stranded (T4G4)2 overhang. The (T4G4)2 telomeric overhang is tenaciously bound by a protein heterodimer. We have cloned and sequenced the gene encoding the 41-kDa subunit of this telomere binding protein. The predicted amino acid sequence comprises two distinct regions, a carboxyl-terminal two-thirds that is 23% lysine and bears similarity to histone H1 and an amino-terminal one-third containing a hydrophobic stretch of about 15 amino acids. Two macronuclear versions of the gene differ in nucleotide sequence at several positions, but the derived polypeptides differ only at a single position, Ser-110 or Ala-110. Both versions harbor a small intron. The existence of this intron demonstrates that, despite the elimination of 95% of the micronuclear genome from the developing macronucleus, at least some noncoding DNA is retained during macronuclear development of hypotrichous ciliates.
Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport.
Authors: Koo E H , Sisodia S S , Archer D R , Martin L J , Weidemann A , Beyreuther K , Fischer P , Masters C L , Price D L
Published: May 2006

In the brains of aged humans and cases of Alzheimer disease, deposits of amyloid in senile plaques are located in proximity to nerve processes. The principal component of this extracellular amyloid is beta/A4, a peptide derived from a larger amyloid precursor protein (APP), which is actively expressed in brain and systemic organs. Mechanisms that result in the proteolysis of APP to form beta/A4, previously termed beta-amyloid protein, and the subsequent deposition of the peptide in brain are unknown. If beta/A4 in senile plaques is derived from neuronally synthesized APP and deposited at locations remote from sites of synthesis, then APP must be transported from neuronal cell bodies to distal nerve processes in proximity to deposits of amyloid. In this study, using several immunodetection methods, we demonstrate that APP is transported axonally in neurons of the rat peripheral nervous system. Moreover, our investigations show that APP is transported by means of the fast anterograde component. These findings are consistent with the hypothesis of a neuronal origin of beta/A4, in which amyloid is deposited in the brain parenchyma of aged individuals and cases of Alzheimer disease. In this setting, we suggest that APP is synthesized in neurons and delivered to dystrophic nerve endings, where subsequent alterations of local processing of APP result in deposits of brain amyloid.
Additional polymorphisms at marker loci D9S5 and D9S15 generate extended haplotypes in linkage disequilibrium with Friedreich ataxia.
Authors: Fujita R , Hanauer A , Sirugo G , Heilig R , Mandel J L
Published: May 2006

The gene for Friedreich ataxia (FA), a severe recessive neurodegenerative disease, has previously been shown to be tightly linked to the polymorphic markers D9S15 and D9S5 on human chromosome 9. In addition, the observation of linkage disequilibrium suggested that D9S15 is within 1 centimorgan (cM) of the disease locus, FRDA. Although D9S5 did not show recombination with FRDA, its localization was less precise (0-5 cM) due to its lower informativeness. We have now identified additional polymorphisms at both marker loci. Two cosmids spanning 50 kilobases around D9S5 were isolated, and a probe derived from one of them detects an informative three-allele polymorphism. We have found a highly polymorphic microsatellite sequence at D9S15 which is rapidly typed by the DNA polymerase chain reaction. The polymorphism information contents at the D9S5 and D9S15 loci have been increased from 0.14 to 0.60 and from 0.33 to 0.74, respectively. With the additional polymorphisms the lod (log10 odds ratio) score for the D9S15-FRDA linkage is now 48.10 at recombination fraction theta = 0.005 and for D9S5-FRDA, the lod score is 27.87 at theta = 0.00. We have identified a recombinant between D9S15 and FRDA. However, due to the family structure, it will be of limited usefulness for more precise localization of FRDA. The linkage disequilibrium previously observed between D9S15 and FRDA is strengthened by analysis of the haplotypes using the microsatellite polymorphism, while weaker but significant disequilibrium is found between D9S5 and FRDA. Extended haplotypes that encompass D9S5 and D9S15 show a strikingly different distribution between chromosomes that carry the FA mutation and normal chromosomes. This suggests that both marker loci are less than 1 cM from the FRDA gene and that a small number of mutations account for the majority of FA cases in the French population studied. D9S5 and D9S15 are thus excellent start points to isolate the disease gene.
Molecular cloning of a cDNA encoding a major pathogenic domain of the Heymann nephritis antigen gp330.
Authors: Pietromonaco S , Kerjaschki D , Binder S , Ullrich R , Farquhar M G
Published: May 2006

Heymann nephritis is an experimental autoimmune disease in rats that is characterized by accumulation of immune deposits (IDs) in kidney glomeruli. The disease is initiated by the binding of circulating antibodies to a membrane glycoprotein, gp330, which is a resident protein of clathrin-coated pits on glomerular epithelial cells (podocytes). We have defined a domain representing about 10% of gp330 that appears to be responsible for the formation of stable glomerular IDs. A cDNA clone (clone 14) was isolated from a rat kidney cDNA expression library by screening with IgG eluted from glomeruli of rats in early stages (3 days) of passive Heymann nephritis. The clone 14 cDNA contains an open reading frame encoding the C-terminal 319 amino acids of gp330. The predicted amino acid sequence contains four internal repeats of 11 amino acids, which are also found in the putative ligand-binding region of carbohydrate-binding lectin-like receptors. An antibody raised against the clone 14 fusion protein recognized gp330 by immunoblotting and induced formation of subepithelial IDs typical of passive Heymann nephritis when injected into normal rats. When the clone 14 fusion protein was used to immunize rats, subepithelial IDs of active Heymann nephritis were found after 12 weeks. No IDs were formed by active or passive immunization of rats with fusion proteins derived from other regions of gp330. These results demonstrate that clone 14 encodes a region of gp330 responsible for antibody binding and ID formation in vivo.
Magnesium dietary intake modulates blood lipid levels and atherogenesis.
Authors: Altura B T , Brust M , Bloom S , Barbour R L , Stempak J G , Altura B M
Published: May 2006

In this study, we have examined the effects of variation in dietary Mg on the atherogenic process. Oral supplementation of rabbits fed a high cholesterol diet (1% or 2%) with the Mg salt magnesium aspartate hydrochloride (Magnesiocard) (i) lowers the level of serum cholesterol and triglycerides in normal (25-35%) as well as atherosclerotic (20-40%) animals and (ii) attenuates the atherosclerotic process markedly. In addition, we found that dietary deficiency of Mg augments atherogenesis markedly and stimulates (or activates) macrophages of the reticuloendothelial system. Evidence is presented to indicate that the hypercholesterolemic state may cause the loss of Mg from soft tissues to the serum, thereby masking an underlying Mg deficiency.
Fractionation of heparin by chromatography on a tissue plasminogen activator-Sepharose column.
Authors: Andrade-Gordon P , Strickland S
Published: May 2006

Heparin stimulates the activity of tissue plasminogen activator (t-PA) and binds to t-PA. To study this interaction, a complex between t-PA and N-acetylated heparin was formed and then linked to Sepharose. This procedure selectively links the t-PA to the column because the acetylated heparin has no free amino groups. The procedure also protects the heparin-binding site(s) on the enzyme during coupling to the matrix. The t-PA column separates heparin into two fractions, one with low affinity for t-PA and one with high affinity. Both fractions of heparin effectively accelerate inactivation of thrombin by antithrombin III. However, the fractions differ in their ability to stimulate t-PA: the low-affinity heparin has no effect on the activity of t-PA, whereas the high-affinity heparin enhances this activity. These heparin fractions will be useful in characterizing the biochemical basis and physiological consequences of the heparin--t-PA interaction.
Physical mapping of a low-copy DNA sequence in rye (Secale cereale L.).
Authors: Gustafson J P , Butler E , McIntyre C L
Published: May 2006

A 900-base-pair (bp) sequence from a cDNA clone of the rye endosperm-storage-protein gene Sec-1 was labeled with biotin and hybridized to Secale cereale 'Blanco' chromosomes. Hybridization was seen on the satellite part of the short arm of chromosome 1R, where the Sec-1 gene has been genetically mapped, in approximately 8.5% of the cells analyzed. The clone cross-hybridized at a lower frequency to rye chromosome arms 1R (long) (4.4%) and 2R (short) (2.6%), where two additional rye storage-protein loci, Sec-3 and Sec-2, respectively, have been mapped. A fourth hybridization site was observed on the short arm of chromosome 6R at a frequency of 3.0%. The cross-hybridization is attributed to a combination of residual sequence homology between the protein loci and the low-stringency conditions used in in situ hybridization. In situ hybridization mapping, in combination with chromosome walking by using molecular techniques, is suggested as an excellent approach to physical mapping of chromosomes.
Pahhph-5: a mouse mutant deficient in phenylalanine hydroxylase.
Authors: McDonald J D , Bode V C , Dove W F , Shedlovsky A
Published: May 2006

Mutant mice exhibiting heritable hyperphenylalaninemia have been isolated after ethylnitrosourea mutagenesis of the germ line. We describe one mutant pedigree in which phenylalanine hydroxylase activity is severely deficient in homozygotes and reduced in heterozygotes while other biochemical components of phenylalanine catabolism are normal. In homozygotes, injection of phenylalanine causes severe hyperphenylalaninemia and urinary excretion of phenylketones but not hypertyrosinemia. Severe chronic hyperphenylalaninemia can be produced when mutant homozygotes are given phenylalanine in their drinking water. Genetic mapping has localized the mutation to murine chromosome 10 at or near the Pah locus, the structural gene for phenylalanine hydroxylase. This mutant provides a useful genetic animal model affected in the same enzyme as in human phenylketonuria.
Activation and blocking of neuronal nicotinic acetylcholine receptor reconstituted in Xenopus oocytes.
Authors: Bertrand D , Ballivet M , Rungger D
Published: May 2006

Neuronal nicotinic acetylcholine receptor of the alpha 4/non-alpha (alpha 4/n alpha) type was reconstituted in Xenopus oocytes after nuclear injection of cDNA expression vectors. Functional neuronal receptor was only formed when the two subunits alpha 4 and n alpha were coinjected, neither alpha 4 nor n alpha alone being effective. Responses to bath application of acetylcholine (AcCho) have been measured in voltage clamp. AcCho doses as low as 10 nM induce currents of up to 50 nA. Dose-response studies indicate a Kd of about 0.77 x 10(-6) M and a Hill coefficient of 1.5, thus predicting more than one AcCho binding site per receptor molecule. The current-voltage relationship of AcCho-induced currents presents a strong inward rectification. Responses to AcCho were compared to those of three other agonists: L-nicotine, carbachol, and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Sensitivities to AcCho, nicotine, and DMPP are quite similar. Sensitivity to carbachol is much lower, but the currents are otherwise indistinguishable from those induced by AcCho. Five AcCho antagonists--neuronal bungarotoxin (kappa-bungarotoxin), tubocurarine (TC), hexamethonium bromide (Hex), decamethonium bromide (Dec), and mecamylamine (Mec)--have been tested. Neuronal bungarotoxin has no effect on the alpha 4/n alpha channel, whereas 2.5 microM TC reduces by half the current peak evoked by 1 microM AcCho. The block by TC is independent of membrane voltage. By contrast, the block of AcCho-induced currents by Hex or Dec is strongly voltage dependent, suggesting that these substances enter the channel. The block by Mec is detectable at concentrations as low as 100 nM when applied together with 1 microM AcCho and is voltage independent. Hex, Dec, and Mec are effective only when AcCho is present. While the effects of all other agents are fully reversible, the Mec block is persistent.
GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)2Cys6 binuclear cluster.
Authors: Pan T , Coleman J E
Published: May 2006

The DNA-binding domain of the transcription factor GAL4, consisting of the 62 N-terminal residues and denoted GAL4(62*), contains a Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys motif, which has been shown previously to bind two Zn(II) or Cd(II) ions. Binding of Zn(II) or Cd(II) is essential for the recognition by GAL4 of the specific palindromic DNA sequence to which it binds upstream of genes for galactose-metabolizing enzymes, the UASG sequence. On the basis of the 113Cd NMR chemical shifts of the two bound 113Cd(II) ions, we propose a binuclear cluster model for this Zn(II)-binding subdomain. 1H-113Cd heteronuclear multiple-quantum NMR spectroscopy and phase-sensitive double-quantum filtered 1H correlation spectroscopy of the 112Cd(II)- and 113Cd(II)-substituted GAL4(62*) derivatives provide direct evidence that the two bound 113Cd(II) ions are coordinated only by the six cysteine residues, two of which form bridging ligands between the two 113Cd(II) ions. The latter can be identified from the pattern of 1H-113Cd J coupling. Thus a binuclear metal ion cluster rather than a "zinc finger" is formed by the six cysteine residues of the GAL4 DNA-binding domain. This model can be directly applied to eight other fungal transcription factors which have been shown to contain similarly spaced Cys6 clusters. 1H NMR spectra of apo-GAL4(62*) suggest conformational fluctuation of the metal-binding subdomain upon removal of Zn(II) or Cd(II). Both Cd(II)2- and Zn(II)2-containing species of GAL4 can be formed, and the similar 1H NMR spectra suggest similar conformations.
Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin.
Authors: Monaco L , Murtagh J J , Newman K B , Tsai S C , Moss J , Vaughan M
Published: May 2006

ADP-ribosylation factors (ARFs) are approximately 20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known ARFs. Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone psi ARF 4, a putative ARF pseudogene, from a human genomic library in lambda phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A)+ RNA the cDNA corresponding to the expressed homolog of psi ARF 4, referred to as human ARF 4. It appears that psi ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.
Molecular and genetic characterization of a radiation-induced structural rearrangement in mouse chromosome 2 causing mutations at the limb deformity and agouti loci.
Authors: Woychik R P , Generoso W M , Russell L B , Cain K T , Cacheiro N L , Bultman S J , Selby P B , Dickinson M E , Hogan B L , Rutledge J C
Published: May 2006

Molecular characterization of mutations in the mouse, particularly those involving agent-induced major structural alterations, is proving to be useful for correlating the structure and expression of individual genes with their function in the whole organism. Here we present the characterization of a radiation-induced mutation that simultaneously generated distinct alleles of both the limb deformity (ld) and agouti (a) loci, two developmentally important regions of chromosome 2 normally separated by 20 centimorgans. Cytogenetic analysis revealed that an interstitial segment of chromosome 17 (17B- 17C; or, possibly, 17A2-17B) had been translocated into the distal end of chromosome 2, resulting in a smaller-than-normal chromosome 17 (designated 17del) and a larger form of chromosome 2 (designated 2(17). Additionally, a large interstitial segment of the 2(17) chromosome, immediately adjacent and proximal to the insertion site, did not match bands 2E4-2H1 at corresponding positions on a normal chromosome 2. Molecular analysis detected a DNA rearrangement in which a portion of the ld locus was joined to sequences normally tightly linked to the a locus. This result, along with the genetic and cytogenetic data, suggests that the alleles of ld and a in this radiation-induced mutation, designated ldIn2 and ajIn2, were associated with DNA breaks caused by an inversion of an interstitial segment in the 2(17) chromosome.
Proposed regulatory pathway encoded by the nodV and nodW genes, determinants of host specificity in Bradyrhizobium japonicum.
Authors: Göttfert M , Grob P , Hennecke H
Published: May 2006

Bradyrhizobium japonicum is the root nodule endosymbiont of soybean (Glycine max), mung bean (Vigna radiata), cowpea (Vigna unguiculata), and Siratro (Macroptilium atropurpureum). We report the characteristics of a nodulation-gene region of B. japonicum that contributes only marginally to the bacterium's ability to nodulate soybean but is essential for the nodulation of the three alternative hosts. This DNA region consists of two open reading frames designated nodV and nodW. The predicted amino acid sequences of the NodV and NodW proteins suggest that they are members of the family of two-component regulatory systems, which supports the hypothesis that NodV responds to an environmental stimulus and, after signal transduction, NodW may be required to positively regulate the transcription of one or several unknown genes involved in the nodulation process. It seems likely that all host plants produce the necessary signal, whereas host specificity may be brought about by the product(s) of the gene(s) activated by NodW.
Coinfection of insect cells with recombinant baculovirus expressing pp60v-src results in the activation of a serine-specific protein kinase pp90rsk.
Authors: Vik T A , Sweet L J , Erikson R L
Published: May 2006

A recombinant baculovirus was constructed for the production of the serine-specific protein kinase, pp90rsk (where rsk is ribosomal S6 kinase), in insect cells. The Xenopus pp90rsk expressed in the infected cells had nearly undetectable enzyme activity in contrast to the same enzyme coproduced with the v-src oncogene product pp60v-src. The transforming gene product pp60v-src very effectively activated pp90rsk, whereas the products of c-src and the myristoylation-minus nontransforming virus NY315 were markedly less effective. Only a fraction of the total pp90rsk population was activated, and it could be partially separated from unactivated protein by ion-exchange chromatography. When compared to the unactivated form, the activated enzyme displayed about a 4000-fold increase in the capacity to phosphorylate the ribosomal protein S6. The enhanced enzymatic activity appeared to be due to phosphorylation of pp90rsk.
Genomic organization of mouse Fc gamma receptor genes.
Authors: Kulczycki A , Webber J , Soares H A , Onken M D , Thompson J A , Chaplin D D , Loh D Y , Tillinghast J P
Published: May 2006

We have isolated and characterized the gene coding for the mouse Fc receptor that is termed Fc gamma RIIa. The gene contains five exons and spans approximately 9 kilobases. Unlike most members of the immunoglobulin gene superfamily, this gene utilizes multiple exons to encode its leader peptide. The first exon encodes the hydrophobic region of the signal sequence; the second exon, which contains only 21 base pairs, encodes a segment of the signal peptidase recognition site; and the beginning of the third exon encodes the predicted site of peptidase cleavage. The third and fourth exons each code for immunoglobulin-like extracellular domains. The fifth exon encodes the hydrophobic transmembrane domain and the cytoplasmic tail. Partial characterization of the Fc gamma RIIb gene indicates that it also contains multiple leader exons, including a 21-base-pair exon and two exons coding for homologous immunoglobulin-like extracellular domains. However, the Fc gamma RIIb gene uses four exons to encode its intracytoplasmic region. Analysis using contour-clamped homogeneous electric field (CHEF) gels indicates that the Fc gamma RIIa and Fc gamma RIIb genes are linked within 160 kilobases on mouse chromosome 1.
Genes that can be mutated to unmask hidden antigenic determinants in the cuticle of the nematode Caenorhabditis elegans.
Authors: Politz S M , Philipp M , Estevez M , O'Brien P J , Chin K J
Published: May 2006

Rabbit antisera directed against a mixture of proteins solubilized from the wild-type adult Caenorhabditis elegans cuticle were used to isolate mutants, induced by ethyl methanesulfonate treatment, that exhibit alterations in surface antigenicity by immunofluorescence. Genetic mapping and complementation data for four such mutations define two genes, srf-2(I) and srf-3(IV). The mutant phenotypes observed by immunofluorescence appear to result from unmasking of antigenic determinants that are normally hidden in the wild-type cuticle. In support of this hypothesis, surface radioiodination experiments indicate that components labeled on the wild-type surface are missing or less readily labeled on the surface of srf-2 and srf-3 mutants.
A glutathione conjugate of hepoxilin A3: formation and action in the rat central nervous system.
Authors: Pace-Asciak C R , Laneuville O , Su W G , Corey E J , Gurevich N , Wu P , Carlen P L
Published: May 2006

Incubation of (8R)- and (8S)-[1-14C]hepoxilin A3 [where hepoxilin A3 is 8-hydroxy-11,12-epoxyeicosa-(5Z,9E,14Z)-trienoic acid] and glutathione with homogenates of rat brain hippocampus resulted in a product that was identified as the (8R) and (8S) diastereomers of 11-glutathionyl hepoxilin A3 by reversed-phase high performance liquid chromatographic comparison with the authentic standard made by total synthesis. Identity was further confirmed by cleavage of the isolated product with gamma-glutamyltranspeptidase to yield the corresponding cysteinylglycinyl conjugate that was identical by reversed-phase high performance liquid chromatographic analysis with the enzymic cleavage product derived from the synthetic glutathionyl conjugate. The glutathionyl and cysteinylglycinyl conjugate are referred to as hepoxilin A3-C and hepoxilin A3-D, respectively, by analogy with the established leukotriene nomenclature. Formation of hepoxilin A3-C was greatly enhanced with a concomitant decrease in formation of the epoxide hydrolase product, trioxilin A3, when the epoxide hydrolase inhibitor trichloropropene oxide was added to the incubation mixture demonstrating the presence of a dual metabolic pathway in this tissue involving hepoxilin epoxide hydrolase and glutathione S-transferase processes. Hepoxilin A3-C was tested using intracellular electrophysiological techniques on hippocampal CA1 neurons and found to be active at concentrations as low as 16 nM in causing membrane hyperpolarization, enhanced amplitude and duration of the post-spike train afterhyperpolarization, a marked increase in the inhibitory postsynaptic potential, and a decrease in the spike threshold. These findings suggest that these products in the hepoxilin pathway of arachidonic acid metabolism formed by the rat brain may function as neuromodulators.
Mutation eliminating mitochondrial leader sequence of methylmalonyl-CoA mutase causes muto methylmalonic acidemia.
Authors: Ledley F D , Jansen R , Nham S U , Fenton W A , Rosenberg L E
Published: May 2006

Methylmalonyl-CoA mutase (EC 5.4.99.2) is a mitochondrial matrix enzyme whose activity is deficient in the inherited disorder methylmalonic acidemia. Previous studies on primary fibroblast cell lines from patients with methylmalonic acidemia have delineated a variety of biochemical phenotypes underlying this disorder. One cell line with primary mutase apoenzyme deficiency exhibited a particularly unusual phenotype; it expressed an abnormally small and unstable immunoreactive protein, which was not imported by mitochondria. We now report cloning and sequencing of the cDNA encoding this mutant protein. The mutation is a single base change, a cytosine----thymine transition, which introduces an amber termination codon at position 17 within the mitochondrial leader sequence. The immunoreactive protein produced by these cells reflects translation from AUG codons downstream from this termination codon and, hence, lacks a mitochondrial leader peptide. This mutation represents a complex prototype for a class of mutations in which absence of the mitochondrial targeting sequence leads to absence of a functioning gene product.
Vitamin B12S-promoted model rearrangement of methylmalonate to succinate is not a free radical reaction.
Authors: Choi G Y , Choi S C , Galan A , Wilk B , Dowd P
Published: May 2006

To probe for free radical intermediates in the model methylmalonate to succinate rearrangements promoted by vitamin B12s, a model series with a pentenyl side chain radical trap has been devised. The control free radical, generated by tri-n-butyltin hydride treatment of bromomethyl-pentenylmalonate thioester, undergoes rapid cyclization to the six-membered ring, and, as anticipated, no succinate rearrangement product is detected. By contrast when the bromide is treated with vitamin B12s, little cyclized product is observed; the major product is the pentenyl succinate. This result demonstrates that the latter rearrangement does not follow a free radical pathway.