bluuebunny/crossref_metadata_2025 huggingface dataset:
The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA.
Authors: Bhanot O S , Ray A
Published: May 2006

A bacteriophage phi X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing O6-methylguanine in a specific site was hybridized to a single-stranded region in gene G of phi X gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutant-containing infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, O6-methylguanine in replicating phi X DNA has a mutagenic frequency of 75%. When repair of O6 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.
Strategies for studying heterogeneous genetic traits in humans by using a linkage map of restriction fragment length polymorphisms.
Authors: Lander E S , Botstein D
Published: May 2006

Simple single-gene disorders in humans can be genetically mapped by using traditional methods of linkage analysis and increasingly abundant restriction fragment length polymorphisms (RFLPs). Many human diseases and traits, however, can be expected to be genetically heterogeneous (i.e., caused by any one of several genes), and traditional linkage analysis is much less effective in such circumstances. We present two methods, interval mapping and simultaneous search, designed to exploit the full power of a linkage map of the DNA markers. For the simplest situations, only 1/3 as many affected families are needed to map a heterogeneous trait by using these methods. Only 1/5-1/50 as many are needed to detect that genetic heterogeneity is present.
Nucleotide sequence of the yeast regulatory gene STE7 predicts a protein homologous to protein kinases.
Authors: Teague M A , Chaleff D T , Errede B
Published: May 2006

The nucleotide sequence of the STE7 gene from Saccharomyces cerevisiae has been determined. It is one of several regulatory genes implicated in the control of cell-type-specific gene expression in yeast. The predicted amino acid sequence shows homology to several members of the protein kinase family. These results suggest protein phosphorylation may play a key role in regulation leading to cell-type specialization in yeast.
Molecular detection of deletions involving band q14 of chromosome 13 in retinoblastomas.
Authors: Dryja T P , Rapaport J M , Joyce J M , Petersen R A
Published: May 2006

DNA fragments from a locus spanning 29 kilobases within chromosome band 13q14 detected deletions in 3 retinoblastomas out of 37 such tumors examined. Somatically occurring, homozygous deletions spanning at least 25 kilobases were detected in retinoblastomas from two unrelated patients. These deletions are bounded by the esterase D locus proximally. In a third patient, both tumor cells and leukocytes have a deletion of one chromosome 13 homolog, with one end of the deletion localized to a 1.55-kilobase fragment within the cloned region. It is likely that the cloned locus is within a few hundred kilobases of the retinoblastoma gene (i.e., the locus governing predisposition to such tumors) and that the deletions detected also involve the retinoblastoma gene. Further, it may be possible to base a successful approach to the isolation of the retinoblastoma gene on this assumed physical proximity of the two loci.
Ocular delayed hypersensitivity: a pathogenetic mechanism of chlamydial-conjunctivitis in guinea pigs.
Authors: Watkins N G , Hadlow W J , Moos A B , Caldwell H D
Published: May 2006

We used a naturally occurring, Chlamydia psittaci-caused eye disease in guinea pigs, guinea pig inclusion conjunctivitis, as an animal model to understand both the immune response and the pathogenesis of chlamydial eye infections. When instilled into the conjunctival sac of guinea pigs that had been previously infected and were immune, viable chlamydiae or a Triton X-100-soluble extract of them produced a short-lived (12-48 hr) eye disease indistinguishable clinically and histologically from that observed during primary chlamydial eye infection. The clinical and histologic findings were consistent with those of ocular delayed hypersensitivity. Ocular delayed hypersensitivity was induced by primary chlamydial infection at mucosal sites other than conjunctival, such as vaginal and intestinal. Preliminary characterization of the hypersensitivity allergen shows that it is heat sensitive and common to the genus Chlamydia. The allergen is apparently not surface-exposed on chlamydiae and requires viable but not replicating organisms for activity. Our observation should be useful in understanding pathogenetic mechanisms of Chlamydia trachomatis-caused infections in humans, in particular those that produce chronic inflammatory diseases, such as blinding trachoma and urogenital diseases.
From basic network principles to neural architecture: emergence of spatial-opponent cells.
Authors: Linsker R
Published: May 2006

The functional architecture of mammalian visual cortex has been elucidated in impressive detail by experimental work of the past 20-25 years. The origin of many of the salient features of this architecture, however, has remained unexplained. This paper is the first of three (the others will appear in subsequent issues of these Proceedings) that address the origin and organization of feature-analyzing (spatial-opponent and orientation-selective) cells in simple systems governed by biologically plausible development rules. I analyze the progressive maturation of a system composed of a few layers of cells, with connections that develop according to a simple set of rules (including Hebb-type modification). To understand the prenatal origin of orientation-selective cells in certain primates, I consider the case in which there is no external input, with the first layer exhibiting random spontaneous electrical activity. No orientation preference is specified to the system at any stage, and none of the basic developmental rules is specific to visual processing. Here I introduce the theory of "modular self-adaptive networks," of which this system is an example, and explicitly demonstrate the emergence of a layer of spatial-opponent cells. This sets the stage for the emergence, in succeeding layers, of an orientation-selective cell population.
Evidence against use of bacterial amino acid sequence data for construction of all-inclusive phylogenetic trees.
Authors: Meyer T E , Cusanovich M A , Kamen M D
Published: May 2006

It has been proposed that phylogenetic trees, intended to show divergence of eukaryotic protein and nucleic acid sequences, be extended to include those from bacteria. However, we have compared the amino acid sequences of 18 of the most divergent mitochondrial cytochromes c with those of 18 bacterial cytochromes c2 and have found that the average percentage difference between these mitochondrial cytochromes c and cytochromes c2 was not significantly greater than that among the cytochromes c2 alone. The large discontinuities in physical-chemical properties recognized between the prokaryote and eukaryote cytochromes render it highly improbable that members of the two classes should be no more different from one another than members of either class alone, assuming that sequence differences can accurately reveal evolutionary divergence. Instead, we propose that divergent amino acid sequences approach a limit of change considerably less than for comparison of random sequences. This limit of change presumably is determined by the structure/function relationship. When two homologous protein sequences have reached such a limit, convergence or back-mutations and parallel mutations become as frequent as divergent mutations. As two diverging proteins approach this steady-state condition, sequence differences no longer reflect the numbers of mutations resulting in amino acid substitution and therefore species cannot be positioned on a phylogenetic tree. Insertions and deletions are less reversible than are amino acid substitutions and, provided they are well-documented, might be more reliable indicators of bacterial relationships. Nevertheless, we suggest that data available on bacterial protein sequences do not permit construction of all-inclusive phylogenetic trees. Comparisons of protein and rRNA trees suggest that similar restrictions apply to use of rRNA sequence data.
Transcriptional control of herpesvirus gene expression: gene functions required for positive and negative regulation.
Authors: Godowski P J , Knipe D M
Published: May 2006

We have used an in vitro nuclear run-off assay to measure the levels of transcription of specific herpes simplex virus genes at different times during a lytic infection. We analyzed the effects of inhibition of DNA replication and of defects in two herpes simplex virus regulatory proteins on the transcription of these genes. We present evidence that the transcription of the alpha ICP4 gene is negatively regulated during a lytic infection. The regulation of ICP4 gene transcription requires the beta protein ICP8 (where ICP = infected cell polypeptide). Transcription of the beta ICP8, gamma 1 ICP5, and gamma 2 glycoprotein C (gC) genes was dependent on ICP4, and transcription of the gamma 2gC gene was strongly inhibited when DNA replication was blocked. Defects in ICP8 also resulted in increased levels of transcription of the ICP4, ICP8, ICP5, and gC genes from parental viral genomes. Our results suggest that ICP8 may be important in maintaining the highly ordered cascade of viral gene expression.
Affinity purification of human tissue factor: interaction of factor VII and tissue factor in detergent micelles.
Authors: Guha A , Bach R , Konigsberg W , Nemerson Y
Published: May 2006

Tissue factor, a known initiator of blood coagulation, was found to be active in Triton X-100. A system consisting of tissue factor, factor VIIa, calcium ions, and coagulation factor X generated activated factor X at an appreciable rate. Based on this observation, we coupled human and bovine factor VII to a solid support. Each column bound tissue factor, solubilized in Triton X-100, in a species-specific manner. These interactions required calcium ions; when the columns were washed with detergent containing calcium ions, no tissue factor was eluted. When calcium ions were omitted from the eluant, tissue factor emerged as a sharp peak. Human tissue factor was extracted from an acetone brain powder into 2% Triton X-100. This extract, made 10 mM in CaCl2, was passed over a factor VII column. Human factor VII (1.2 mg) was coupled to 30 ml of Affi-Gel 15. This column bound approximately equal to 15 micrograms of human tissue factor. The eluted material was approximately equal to 25% pure. Final purification was achieved by gel filtration after chymotryptic digestion of contaminants. The tissue factor activity was stable to this treatment. The molecular weight determined by sodium dodecyl sulfate/PAGE (approximately equal to 46,000) was also unchanged by chymotrypsin. The final material was a single band on PAGE, demonstrated similar resistance to tryptic and chymotryptic digestion as bovine tissue factor, and had approximately the same specific coagulant activity as the previously purified bovine material. Tissue factor was also purified from human placenta, yielding a similar protein. A partial 28-residue sequence of the latter has been obtained.
Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida.
Authors: Schell M A
Published: May 2006

The in vivo transcription start sites of the nah and sal operons of the NAH7 plasmid were determined by S1 nuclease mapping and the nucleotide sequence surrounding these transcription start sites was determined. Since expression of both of these operons is coordinately controlled by the product of the transcriptional activator gene nahR, the sequences were compared to locate potential sites involved in common regulation. In the 100-base-pair region preceding transcription start sites of both operons, three regions of extensive homology were found and may be involved in nahR-mediated transcriptional control: between -80 and -60 with 81% homology; between -40 and -28 with 75% homology; between -1 and +15 with 70% homology. Comparison of the promoter sequences of nah and sal with the analogous sequences of the xylABC and xylDEFG operons of the TOL plasmid showed little homology between the 5' regions of these two sets of positively regulated hydrocarbon degradation operons. In addition, the transcription start site of the nahR regulatory gene was located and its promoter sequence was determined. The nahR promoter overlapped at the -35 position of the sal promoter; however, the nahR gene is transcribed in the opposite direction. Sequences similar to the consensus sequences of Escherichia coli promoters (at -35 and -10) were found in nah, sal, and nahR at the appropriate positions.
Bismuth-212-labeled anti-Tac monoclonal antibody: alpha-particle-emitting radionuclides as modalities for radioimmunotherapy.
Authors: Kozak R W , Atcher R W , Gansow O A , Friedman A M , Hines J J , Waldmann T A
Published: May 2006

Anti-Tac, a monoclonal antibody directed to the human interleukin 2 (IL-2) receptor, has been successfully conjugated to the alpha-particle-emitting radionuclide bismuth-212 by use of a bifunctional ligand, the isobutylcarboxycarbonic anhydride of diethylenetriaminepentaacetic acid. The physical properties of 212Bi are appropriate for radioimmunotherapy in that it has a short half-life, deposits its high energy over a short distance, and can be obtained in large quantities from a radium generator. Antibody specific activities of 1-40 microCi/microgram (1 Ci = 37 GBq) were achieved. Specificity of the 212Bi-labeled anti-Tac was demonstrated for the IL-2 receptor-positive adult T-cell leukemia line HUT-102B2 by protein synthesis inhibition and clonogenic assays. Activity levels of 0.5 microCi or the equivalent of 12 rad/ml of alpha radiation targeted by anti-Tac eliminated greater than 98% the proliferative capabilities of HUT-102B2 cells with more modest effects on IL-2 receptor-negative cell lines. Specific cytotoxicity was blocked by excess unlabeled anti-Tac but not by human IgG. In addition, an irrelevant control monoclonal antibody of the same isotype labeled with 212Bi was unable to target alpha radiation to cell lines. Therefore, 212Bi-labeled anti-Tac is a potentially effective and specific immunocytotoxic reagent for the elimination of IL-2 receptor-positive cells. These experiments thus provide the scientific basis for use of alpha-particle-emitting radionuclides in immunotherapy.
Estrogen formation and binding in the cerebral cortex of the developing rhesus monkey.
Authors: MacLusky N J , Naftolin F , Goldman-Rakic P S
Published: May 2006

These studies were undertaken to determine whether estrogen receptors and the microsomal enzyme system called the aromatase complex, which is responsible for conversion of androgen to estrogen, are present in the brain of the rhesus monkey during perinatal life. Four monkeys (three females--one fetus removed on day 153 of gestation and two infants, 5 and 6 days postnatal--and 1 male, 2 days postnatal) were studied. Cytosol estrogen receptors were detected in all brain regions examined. The apparent equilibrium dissociations constants for reaction of these sites with [3H]moxestrol were similar to those for uterine and pituitary cytosol estrogen receptors (0.3-1.1 nM). Within the brain, highest levels of binding were observed in the hypothalamus-preoptic area, with fairly even, lower concentrations throughout the cortical structures. Aromatase complex activity was detected in the majority of the tissue specimens. The highest levels of estrogen formation were observed in the hypothalamus. However, the amygdala, the hippocampus, and several of the cortex samples also contained measurable aromatase complex activity. Among the cortical samples, the highest levels of aromatase complex activity were found in regions of the association cortex (the dorsolateral-prefrontal, orbital-prefrontal, anterior cingulate, and parietal cortices). The lowest levels of aromatase activity were found in the somatosensory and motor cortices of the postnatal animals. These results suggest that locally-formed estrogen may be involved in the effects of circulating androgens on the developing primate neocortex.
Optical measurements of intracellular pH in single LLC-PK1 cells: demonstration of Cl-HCO3 exchange.
Authors: Chaillet J R , Amsler K , Boron W F
Published: May 2006

An optical method was used to continuously monitor intracellular pH (pHi) in single cultured LLC-PK1 cells. Rapidly growing or quiescent cells, attached to coverslips, were loaded with the pH-sensitive dye 4',5'-dimethyl-5(and -6)-carboxyfluorescein by exposing them to the dye's permeant precursor. pHi was calculated from the intracellular absorbance spectrum of the dye in a single cell. For cells incubated in HCO3(-)-free Ringer's solution, pHi recovered exponentially from acid loads applied by NH+4 prepulsing. Because the recovery was Na+-dependent and amiloride-sensitive, it was probably caused by Na-H exchange at the plasma membrane. In HCO3- Ringer's solution, external Cl- removal caused pHi to reversibly increase by approximately equal to 0.3. This pHi increase was substantially reduced by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or by conducting the Cl- removal in the nominal absence of HCO3-. Reducing [HCO3-]o from 25 to 5 mM at constant pCO2 (lowering pHo from 7.4 to 6.7) caused pHi to reversibly fall by approximately equal to 0.2. This pHi change was greatly diminished by DIDS, by removal of extracellular Cl-, or by performing the same pHo shift in the nominal absence of HCO3-. The pHi changes induced by altering [Cl-]o or pHo were not inhibited by Na+ removal. Our data indicate that LLC-PK1 cells possess a Na-independent Cl-HCO3 exchanger and that this transporter may be as important as the Na-H exchanger in determining pHi.
The 28-kDa vitamin D-dependent calcium-binding protein has a six-domain structure.
Authors: Hunziker W
Published: May 2006

Vitamin D-dependent 28-kDa calcium-binding protein (CaBP28) cDNA clones were isolated from a chicken intestinal library. The nucleotide sequence analysis of the CaBP28 cDNA shows an open reading frame of 786 nucleotides, coding for a 262-amino acid 30.167-kDa protein. Interestingly, the protein contains six repeats of a domain with the feature of a calcium-binding site. In two of the six domains, oxygen-containing amino acids important for the positioning of calcium are absent, suggesting that these two sites have lost their calcium-binding capability and might have adopted a new function in evolution. In the chicken intestine, three different sized species of CaBP28 mRNA (2.0, 2.8, and 3.1 kilobases) are detected. Primer extension and S1 nuclease mapping show that the three CaBP28 mRNA species share a common 5' end but differ in the length of their 3' noncoding sequence. A similar triplet of CaBP28 mRNAs is identified in the rat kidney by the chicken probe, showing an interspecies conservation of the CaBP28. In the rat intestine, however, no CaBP28 mRNA could be detected. Instead, a vitamin D-dependent 9-kDa CaBP (CaBP9) is expressed, with an mRNA size of approximately equal to 0.7 kilobase that does not cross-hybridize with the CaBP28 probe. This indicates that the CaBP28 and CaBP9 are the product of two independent genes.
Activation of complement by immunoglobulin M is impaired by the substitution serine-406----asparagine in the immunoglobulin mu heavy chain.
Authors: Shulman M J , Pennell N , Collins C , Hozumi N
Published: May 2006

We have isolated and analyzed the DNA encoding the mu heavy chain constant region of a mutant IgM that is defective in initiating complement-dependent cytolysis. By assaying the expression of mu-chain genes that were constructed in vitro from mutant and wild-type gene segments, we have mapped the mutation into a 555-base-pair segment that spans part of the third and fourth constant region domains. In this segment there is one nucleotide change, such that the mutant mu-chain gene encodes asparagine rather than the normal serine at amino acid position 406 in the third constant domain. We have used site-directed mutagenesis to introduce a comparable mutation into the normal mu-chain gene and confirmed that this substitution causes the production of IgM with the original mutant phenotype. Evidence is also provided that the serine-406----asparagine substitution might cause the mutant mu chain to be abnormally glycosylated.
Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells.
Authors: Bachmann M , Mayet W J , Schröder H C , Pfeifer K , Meyer zum Büschenfelde K H , Müller W E
Published: May 2006

Monoclonal antibodies were raised against homogeneous Ro and La antigens, two proteins associated with Ro and La ribonucleoproteins (RNPs). The specificity of the monoclonal antibodies was proven by immunoblot analysis and by immunoprecipitation. The anti-Ro antibody reacted with a Mr 95,000 protein in a mouse lymphoma cell extract and with a Mr 60,000 polypeptide in extracts from human spleen. The anti-La antibody recognized a Mr 50,000 polypeptide in the mouse L5178y cell extract. The two monoclonal antibodies precipitated RNPs that contained the typical RNA species of Ro or La RNPs. The localization of Ro and La antigen was performed by direct immunofluorescence microscopy. It was found that the anti-Ro antibody reacted with a fibrous network that behaves like cytokeratin, one of the intermediate filament systems. The anti-La antibody reacted with nuclear structures that gave a speckled-type pattern.
Differential effects of transforming growth factor type beta on the growth and function of adrenocortical cells in vitro.
Authors: Hotta M , Baird A
Published: May 2006

Transforming growth factor type beta (TGF-beta) suppresses basal as well as corticotropin (ACTH)-stimulated steroid formation by bovine adrenocortical cells in culture. The effect is dose dependent and is not accompanied by any change in adrenocortical cell growth. The minimum effective dose of TGF-beta is 4 X 10(-13) M (10 pg/ml), and maximal inhibition is observed at a concentration of 4 X 10(-11) M (1 ng/ml). A 16- to 20-hr incubation with TGF-beta is required to decrease steroidogenesis, and 12-18 hr are required before cells treated with TGF-beta recover complete responsiveness to corticotropin. Increases in cAMP mediated by corticotropin, forskolin, and isobutylmethylxanthine are not modified by the addition of TGF-beta; thus adenylate cyclase activity is unaffected by TGF-beta. Although TGF-beta inhibits the formation of all of the delta 4-steroids measured (including cortisol, corticosterone, aldosterone, and androstenedione), its effect can be completely reversed by the addition of 25-hydroxycholesterol, pregnenolone, or progesterone to the cells. In contrast, the addition of low density lipoprotein has no effect suggesting that TGF-beta targets the conversion of cholesterol precursors to cholesterol. The results demonstrate a highly potent effect of TGF-beta on the differentiated function of the adrenocortical cell. The inhibition of steroidogenesis can be dissociated from any effect on cell proliferation, and it occurs distal to the formation of cAMP but proximal to the formation of cholesterol. The results suggest that in the adrenal, TGF-beta or TGF-beta-like proteins may be playing an important role in modifying the differentiated state of the adrenocortical cell.
Toward a physical map of the genome of the nematode
Caenorhabditis elegans
Authors: Coulson Alan , Sulston John , Brenner Sydney , Karn Jonathan
Published: May 2006

A technique for digital characterization and comparison of DNA fragments, using restriction enzymes, is described. The technique is being applied to fragments from the nematode
Caenorhabditis elegans
(
i
) to facilitate cross-indexing of clones emanating from different laboratories and (
ii
) to construct a physical map of the genome. Eight hundred sixty clusters of clones, from 35 to 350 kilobases long and totaling about 60% of the genome, have been characterized.
Oral immunization and protection of raccoons (Procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine.
Authors: Rupprecht C E , Wiktor T J , Johnston D H , Hamir A N , Dietzschold B , Wunner W H , Glickman L T , Koprowski H
Published: May 2006

Animal rabies control has been frustrated by the existence of multiple wildlife reservoirs and the lack of efficacious oral vaccines. In this investigation, raccoons fed a vaccinia-rabies glycoprotein recombinant virus in a sponge bait developed rabies virus-neutralizing antibody (0.6-54.0 units) and resisted street rabies virus infection 28 and 205 days after feeding. Additional raccoons immunized by oral infusion with attenuated antigenic variants of rabies virus strains CVS-11 and ERA failed to develop rabies virus-neutralizing antibody. This work demonstrates the feasibility of a recombinant virus vaccine containing the rabies glycoprotein gene for immunization of raccoons, and possibly other wildlife, to obtain long-term protection against rabies.
In vivo evidence that cGMP is the second messenger for atrial natriuretic factor.
Authors: Huang C L , Ives H E , Cogan M G
Published: May 2006

cGMP generation has been associated with many of the vascular and endocrine actions of atrial natriuretic factor (ANF) in vitro. To examine the role of cGMP as a second messenger for the renal hemodynamic action of ANF in vivo, we measured glomerular filtration rate (GFR) and cGMP concentration in systemic artery, renal vein, and urine as well as in Bowman's space and end-proximal tubule (by free-flow micropuncture) after administration of ANF. ANF increased GFR by 45% and simultaneously induced a greater than 5-fold increase of cGMP concentration in glomerular ultrafiltrate (Bowman's space) when compared to controls. There was no significant increase in either systemic artery or renal vein cGMP concentration. Thus, the source of increased Bowman's space cGMP is not from the blood via filtration but rather from either glomerular mesangial or epithelial cells, which are not in direct contact with the circulation. Although a small amount of tubular handling of cGMP occurred along the length of the nephron, the augmented cGMP production from the glomerulus accounted for most of the 10- to 12-fold higher urinary cGMP excretion observed after ANF administration. Intrarenal arterial infusion of dibutyryl cGMP, but not dibutyryl cAMP, increased GFR in a dose-dependent fashion (from 10 to 1000 microM) by a mechanism similar to that of ANF--an increase in glomerular hydraulic pressure. Thus, ANF markedly stimulated glomerular production of cGMP, which coincided with a marked increase in GFR. Since dibutyryl cGMP itself was capable of increasing GFR, cGMP is the likely second messenger for ANF in vivo.
Primary structure of blood coagulation factor XIIIa (fibrinoligase, transglutaminase) from human placenta.
Authors: Takahashi N , Takahashi Y , Putnam F W
Published: May 2006

We have determined the primary structure of human placental factor XIIIa, an enzyme [fibrinoligase, transglutaminase, fibrin-stabilizing factor, EC 2.3.2.13 (protein-glutamine:amine gamma-glutamyltransferase)] that forms intermolecular isopeptide bonds between fibrin molecules as the last step in blood coagulation. Placental factor XIIIa is an unglycosylated polypeptide chain of 730 amino acid residues (Mr = 83,005) that appears to be identical to the a subunit of the plasma zymogen factor XIII. Ca2+-dependent activation of factor XIIIa by thrombin removes a blocked amino-terminal peptide and unmasks a reactive thiol group at Cys-314. A second specific cleavage after Lys-513 by thrombin inactivates factor XIIIa and produces an amino-terminal 56-kDa fragment and a 24-kDa fragment. The amino acid sequence of factor XIIIa is unique and does not exhibit internal homology, but its active center is similar to that of the thiol proteases. The probable Ca2+-binding site of factor XIIIa has been identified by homology to the high-affinity sites in calmodulins. Knowledge of the primary structure of factor XIIIa will aid elucidation of the mechanism of its enzymatic action and that of the many tissue transglutaminases of which it is the prototype. This will also facilitate production of factor XIIIa by recombinant DNA technology for use in treatment of congenital factor XIII deficiencies and in the postoperative healing of wounds.
Two mammalian heat shock proteins, HSP90 and HSP100, are actin-binding proteins.
Authors: Koyasu S , Nishida E , Kadowaki T , Matsuzaki F , Iida K , Harada F , Kasuga M , Sakai H , Yahara I
Published: May 2006

Two high molecular weight heat shock proteins, HSP90 (Mr, 90,000) and HSP100 (Mr, 100,000), were separately purified from extracts of cultured cells of a mouse lymphoma cell line, L5178Y. Both of the HSPs exist in homodimeric form under physiological conditions. Their physicochemical properties are quite similar to each other. Each of the purified HSPs was shown to coprecipitate with rabbit skeletal muscle actin under actin-polymerizing conditions. Both HSP90 and HSP100 increased the low-shear viscosity of filamentous actin solutions in a dose-dependent manner, which suggests that these HSPs cross-link actin filaments. Although some molecular properties and the effects described above on actin solution of HSP90 and HSP100 resemble those of alpha-actinin, the HSPs were distinguished from alpha-actinin by various means, including visualization of molecular shapes by electron microscopy with the aid of the low-angle rotary shadowing technique. Immunofluorescence staining by specific antisera against HSP90 revealed that HSP90 was localized in ruffling membranes in addition to the cytoplasmic space.
Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins.
Authors: Morgan R W , Christman M F , Jacobson F S , Storz G , Ames B N
Published: May 2006

Hydrogen peroxide treatment induces the synthesis of 30 proteins in Salmonella typhimurium. Five of these proteins are also induced by heat shock, including the highly conserved DnaK protein. The induction of one of these five proteins by heat shock is dependent on oxyR, a positive regulator of hydrogen peroxide-inducible genes, while the induction of the other four by heat shock is oxyR independent. Five of the 30 hydrogen peroxide-inducible proteins have been identified, and their structural genes have been mapped. Other stresses such as nalidixic acid, ethanol, or cumene hydroperoxide treatment also induce subsets of the 30 hydrogen peroxide-inducible proteins as well as additional proteins. Hydrogen peroxide-inducible proteins are shown to be largely different from those proteins induced by aerobiosis. In addition, the expression of the katG (catalase) gene is shown to be regulated by oxyR at the level of mRNA.
Proliferation-sensitive nuclear phosphoprotein "dividin" is synthesized almost exclusively during S phase of the cell cycle in human AMA cells.
Authors: Celis J E , Nielsen S
Published: May 2006

Studies of the polypeptides synthesized by normal and transformed human cultured cells under a variety of physiological conditions have revealed a basic 54-kDa protein NEPHGE 10a, whose rate of synthesis is sensitive to changes in the rate of cell proliferation. This nuclear phosphoprotein, which we have termed "dividin" (present only in populations of cells committed to divide), is synthesized almost exclusively during the S phase of the cell cycle of transformed human amnion cells (AMA). Dividin synthesis is first detected late in G1 near the G1/S transition border, reaches a maximum late in S phase, and declines thereafter. As expected for an S phase-specific protein, no detectable synthesis of dividin was observed in growth-arrested normal human cultured cells of epithelial and fibroblast origin. These findings suggest a role for this protein in events leading to DNA replication and cell division.
Psoralens potentiate ultraviolet light-induced inhibition of epidermal growth factor binding.
Authors: Laskin J D , Lee E , Laskin D L , Gallo M A
Published: May 2006

The psoralens, when activated by ultraviolet light of 320-400 nm (UVA light), are potent modulators of epidermal cell growth and differentiation. Previously, we reported that, in mammalian cells, these compounds bind to specific saturable high-affinity cellular receptor sites. In the present studies, we demonstrate that binding of psoralens to their receptors followed by UVA light activation is associated with inhibition of epidermal growth factor (EGF) receptor binding. Inhibition of EGF binding, which required UVA light, was rapid and dependent on the dose of UVA light (0.5-2.0 J/cm2), as well as the concentration of psoralens (10 nM to 1 microM). Higher doses of UVA light (2.0-6.0 J/cm2) by themselves were also inhibitory, indicating that psoralens potentiate the UVA-induced inhibition of EGF binding. A number of biologically active analogs of psoralen, including 8-methoxypsoralen, 5-methoxypsoralen, and 4,5',8-trimethylpsoralen, when activated by UVA light, were found to be inhibitors of binding. Inhibition of EGF binding by psoralens was observed in a variety of human and mouse cell culture lines known to possess psoralen receptors. In the epidermal-derived line PAM 212, at least two populations of receptors with different affinities for EGF were found. Psoralens and UVA light selectively inhibited binding to the higher-affinity EGF receptors, an effect analogous to that of the phorbol ester tumor promoters. As observed with phorbol esters, photoactivated psoralens appeared to inhibit EGF binding by an indirect mechanism. These data demonstrate that the psoralens and UVA light have direct biological effects on cell-surface membranes. Since EGF is a growth-regulatory peptide, the ability of psoralens and UVA light to inhibit EGF binding may underlie the biologic effects of these agents in the skin.
Speciation by monobrachial centric fusions
Authors: Baker Robert J. , Bickham John W.
Published: May 2006

Fixation of centric fusions in natural populations often encounters minimal meiotic problems due to the ability of trivalents to segregate normally; therefore, little sterility barrier is achieved between a founder population and the parental stock. However, a strong sterility barrier can develop between different founder populations fixed for centric fusions that are monobrachially homologous in the resulting biarmed chromosomes (one arm is homologous but the other is nonhomologous). Hybridization through secondary contact then results in complex multivalents, which encounter problems in segregation and produce unbalanced gametes. Speciation mediated by centric fusions is a peripatric speciation model that does not postulate populational phenomena atypical of those characteristic of most mammals. The model appears applicable to a diversity of mammalian taxa such as bats of the
Rhogeessa tumida-parvula
complex, shrews of the
Sorex araneus
complex, and rodents of the
Mus musculus
and
Rattus rattus
complexes.
Molecular cloning and primary nucleotide sequence analysis of a distinct human immunodeficiency virus isolate reveal significant divergence in its genomic sequences.
Authors: Desai S M , Kalyanaraman V S , Casey J M , Srinivasan A , Andersen P R , Devare S G
Published: May 2006

In an effort to evaluate data on genomic relatedness among the various human immunodeficiency viruses (HIVs), we have molecularly cloned a virus isolate designated HIV (CDC-451). Preliminary characterization of the HIV (CDC-451) clone indicated that the restriction enzyme map was distinct from those of other known HIV isolates. Analysis of the primary nucleotide sequence of the regions encoding the structural proteins and comparison with sequences known for other HIV isolates indicated substantial differences for HIV (CDC-451). The sequences encoding the group-specific antigen gene, although they showed some variation, were conserved to a greater extent than were those encoding envelope proteins. In the envelope gene sequences, most of the changes (up to 24.5% divergence) were located in the amino-terminal region encoding a glycoprotein with a Mr of 120,000. The carboxyl-terminal region, encoding a protein of Mr 41,000, was more highly conserved. The variation in the sequences encoding envelope proteins may have important implications for the antigenic properties and/or pathogenicity of the disease and for its detection and ultimate eradication.
Vasopressin-mediated inhibition of atrial natriuretic factor-stimulated cGMP accumulation in an established smooth muscle cell line.
Authors: Nambi P , Whitman M , Gessner G , Aiyar N , Crooke S T
Published: May 2006

Rat thoracic aortic smooth muscle cells (line A10, ATCC CRL 1476) display a high density of atrial natriuretic factor (ANF) receptors. ANF stimulated the accumulation of cGMP in these cells in a time- and dose-dependent fashion. These cells are known to display a high density of vasopressin receptors of the vascular V1 subtype. These vasopressin receptors mediate inhibition of isoproterenol-stimulated cAMP accumulation and stimulation of inositol phosphate accumulation and calcium fluxes. Addition of [8-arginine]vasopressin ([Arg8]VP) to these cells inhibited ANF-stimulated cGMP accumulation. Inhibition of cGMP accumulation was dependent on the concentration of [Arg8]VP, with half-maximal and maximal effects occurring at 0.4 and 10 nM, respectively. [Arg8]VP did not have significant effects on basal cGMP levels. The inhibition by [Arg8]VP appears to be mediated by V1 receptors, since the V2 renal receptor agonist [1-desaminocysteine,8-D-arginine]vasopressin was ineffective. Also, the selective V1 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-(O-methyltyrosine),8-arginine]vasopressin and the mixed V1/V2 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-(O-ethyl-D-tyrosine),4-valine,8-arginine]vasopressin blocked the [Arg8]VP-mediated effect, whereas the selective V2 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-D-isoleucine,4-valine,8-arginine]vasopressin was minimally effective. These data show that in rat aortic smooth muscle cells, V1 receptors are negatively coupled to guanylate cyclase. These data also suggest that the vasoconstrictor activity of [Arg8]VP might involve inhibition of ANF-receptor-mediated vascular relaxation through inhibition of cGMP accumulation in addition to its effects on isoproterenol-mediated cAMP accumulation and inositol phosphate accumulation and calcium fluxes.
Purification and characterization of the amiloride-sensitive sodium channel from A6 cultured cells and bovine renal papilla.
Authors: Benos D J , Saccomani G , Brenner B M , Sariban-Sohraby S
Published: May 2006

The amiloride-binding Na+ channel protein of high electrical resistance epithelia was solubilized and purified from cultured A6 toad kidney cells and bovine renal papilla. Purification was assessed by enrichment in [3H]methylbromoamiloride specific binding. Chromatography of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilized plasma membrane vesicles on agarose-immobilized wheat-germ agglutinin provided a 130-fold enrichment of the amiloride-binding component compared to the cell homogenate. Further purification was achieved by either amiloride-affinity chromatography or size-exclusion HPLC. When the HPLC and amiloride affinity-purified material was injected into a second higher molecular weight exclusion HPLC column, only a single peak with Mr 800,000 was found. Further HPLC separation of the Mr 800,000 material at low ionic strength resolved two peaks with apparent Mrs 800,000 and 700,000. Only the 700-kDa component displayed specific [3H]methylbromoamiloride binding activity. The final binding specific activity achieved was 1300 pmol/mg of protein, corresponding to 91% homogeneity of the protein.
U6 small nuclear RNA is transcribed by RNA polymerase III.
Authors: Kunkel G R , Maser R L , Calvet J P , Pederson T
Published: May 2006

A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.
Direct evidence for the existence of a neutrophil-derived platelet activator (neutrophilin).
Authors: Chignard M , Selak M A , Smith J B
Published: May 2006

Human neutrophils and platelets were loaded with the intracellular calcium indicator fura-2. The chemotactic peptide N-formyl-Met-Leu-Phe (fMet-Leu-Phe) induced a rapid elevation of cytosolic free calcium in cytochalasin B-treated neutrophils but failed to increase the cytosolic calcium in platelets. On the other hand, when unloaded neutrophils were incubated together with autologous fura-2-loaded platelets, fMet-Leu-Phe stimulated a 6-fold increase in platelet cytosolic calcium subsequent to a brief lag. Parallel experiments demonstrated that the addition of fMet-Leu-Phe to neutrophil/platelet incubates also elicited platelet aggregation and serotonin release. Platelet activation showed a positive correlation with the concentration of fMet-Leu-Phe added to the mixed cell population. Cell-free supernatants prepared from fMet-Leu-Phe-stimulated neutrophils were capable of inducing platelet calcium mobilization, aggregation, and secretion. The amount of platelet-activating material present in the supernatant was proportional to the number of activated neutrophils. Preincubation of platelets with BN 52021, acetylsalicylic acid, SQ-29,548, or hirudin did not modify the aggregation response induced by the supernatant collected from fMet-Leu-Phe-activated neutrophils, suggesting that the material was not 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), arachidonic acid, thromboxane A2, or thrombin. Pretreatment of the neutrophil supernatant with an ADP (creatine phosphate/creatine phosphokinase) or a superoxide/peroxide (superoxide dismutase/catalase) scavenging system also had no effect on aggregation or secretion, indicating that these substances did not participate in platelet activation. The biological activity present in the neutrophil supernatant was destroyed by heat and inactivated by treatment with phenylmethylsulfonyl fluoride, indicating that it is a protein and most probably an enzyme with serine protease activity. These data provide the direct observation of secondary signal transmission to platelets following primary activation of neutrophils. We propose the name neutrophilin for the neutrophil-derived mediator.
In vitro modification of human immunodeficiency virus infection by granulocyte-macrophage colony-stimulating factor and gamma interferon.
Authors: Hammer S M , Gillis J M , Groopman J E , Rose R M
Published: May 2006

The ability of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and gamma interferon (IFN-gamma) to modify human immunodeficiency virus (HIV; also called HTLV-III/LAV) infection in the monocytic cell line U-937 was examined. When added to persistently infected cell cultures, GM-CSF at 30-300 units per ml produced maximal reductions in reverse transcriptase activity of 37-55% 10-14 days after its addition, whereas IFN-gamma produced reductions of 64-68% 10-17 days after addition. When used prior to acute HIV infection and maintained in the cell culture system, these cytokines reduced reverse transcriptase activity 90-100% and nearly eliminated viral antigen expression but did not prevent return of productive infection after their removal. These results indicate that, in a monocyte model of HIV infection, GM-CSF and IFN-gamma substantially restrict HIV expression and that these cytokines deserve further evaluation as therapeutic alternatives in HIV-related disorders.
Cloning of T4 gene 32 and expression of the wild-type protein under lambda promoter PL regulation in Escherichia coli.
Authors: Shamoo Y , Adari H , Konigsberg W H , Williams K R , Chase J W
Published: May 2006

Bacteriophage T4 gene 32 encodes a single-stranded DNA binding protein required for T4 DNA replication, recombination, and repair. Previous attempts at cloning gene 32 have failed due to a presumably deleterious effect on host cell viability. In addition, overexpression of gene 32 would be expected to be limited by the autoregulatory ability of the gene 32 product g32P. A repetitive A + T-rich sequence flanking the ribosome binding site of gene 32 has been implicated in this translational regulation. To circumvent these problems, the wild-type gene for g32P has been reconstructed in M13 using restriction fragments from T4 g32am453 and synthetic oligodeoxynucleotides so that it no longer includes its native promoter and putative autoregulatory region. The g32am453 codon TAG was changed back to TGG as in wild-type gene 32 using site-directed oligodeoxynucleotide mutagenesis. In vectors containing the lambda leftward promoter PL, gene 32 is overexpressed with the resulting transcripts being depressed at g32P concentrations that repress the wild-type gene 32 transcripts.
Satellite tobacco ringspot virus RNA: A subset of the RNA sequence is sufficient for autolytic processing
Authors: Buzayan Jamal M. , Gerlach Wayne L. , Bruening George
Published: May 2006

The satellite RNA of tobacco ringspot virus depends upon tobacco ringspot virus for its replication and source of coat protein. The satellite RNA reduces virus accumulation and the severity of virus-induced symptoms. Repetitive sequence, dimeric, and higher forms of the satellite RNA are known to autolytically process to form biologically active monomeric RNA of 359 nucleotide residues [Prody, G. A., Bakos, J. T., Buzayan, J. M., Schneider, I. R. & Bruening, G. (1986)
Science
231, 1577-1580], with a 5′-hydroxyl and a 2′,3′-cyclic phosphodiester as the new terminal groups. We show here that transcripts of full-length and truncated DNA clones of the satellite RNA sequence also process in a nonenzymic reaction. One such transcript was an RNA that has about one-fourth of the satellite RNA sequence, representing the 3′-terminal and 5′-terminal portions of monomeric RNA joined in the junction that is cleaved in dimeric RNA. This RNA autolytically processed more efficiently than molecules with a larger proportion of the satellite RNA nucleotide sequence.