bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene encoding the alpha chain of the T-cell receptor is moved immediately downstream of c-myc in a chromosomal 8;14 translocation in a cell line from a human T-cell leukemia.
Authors: Shima E A , Le Beau M M , McKeithan T W , Minowada J , Showe L C , Mak T W , Minden M D , Rowley J D , Diaz M O
Published: May 2006

The SKW-3 cell line, which was established from the malignant cells of a patient with T-cell chronic lymphocytic leukemia, is characterized by a translocation involving chromosome 8 (band q24) and chromosome 14 (band q11) [t(8;14)(q24;q11)]. To determine the position of the gene encoding the alpha chain of the T-cell receptor and of the human protooncogene myc (c-myc) in relation to the breakpoint junctions and to evaluate their possible role in the pathogenesis of T-cell neoplasia, we applied the techniques of in situ chromosomal hybridization and Southern blot analysis to SKW-3 cells. Our results indicate that the breakpoint on chromosome 14 at band q11 occurs close to a joining sequence of the gene encoding the alpha chain of the T-cell receptor. Additional rearrangements within the alpha-chain locus appear to split the variable region cluster. As a result of the rearrangements, the constant region of this gene, as well as some variable region segments, are translocated to chromosome 8, to the 3' side of the c-myc-coding exons. The identification of a breakpoint to the 3' side of c-myc suggests that this translocation is analogous to the variant (2;8) and t(8;22) translocations observed in the B-cell malignancies.
Restriction fragment length polymorphism caused by a deletion within the human c-abl gene (ABL).
Authors: Xu D Q , Galibert F
Published: May 2006

A restriction fragment length polymorphism at the human c-abl locus (ABL) has been detected in 67 unrelated individuals by agarose gel electrophoresis and Southern blot hybridization using 32P-labeled v-abl probes. This polymorphism is generated by the existence of two alleles, a and b, which are in Hardy-Weinberg equilibrium, with frequencies of 94.8% and 5.2%, respectively. The minor allele, b, is due to a deletion of about 500 base pairs in an intron located downstream of the codon for the phosphate-acceptor tyrosine residue of the c-abl gene product.
Meiosis in sesquidiploid hybrids of
Lycopersicon esculentum
and
Solanum lycopersicoides
Authors: Rick Charles M. , De Verna Joseph W. , Chetelat Roger T. , Stevens M. Allen
Published: May 2006

We have synthesized diploid hybrids between
Lycopersicon esculentum
and
Solanum lycopersicoides
and have converted them to allotetraploids. Two sesquidiploids, having two complements of the former parent and one of the latter, have been obtained by backcrossing the former parent with the alloploid. In meiosis of the sesquidiploid the
L. esculentum
chromosomes exhibit strong preferential pairing, consistently forming 12 bivalents, whereas the
S. lycopersicoides
chromosomes remain unpaired. This chromosomal comportment conforms with expectations based on meiosis of the 2
x
and 4
x
hybrids. Condensation of the
S. lycopersicoides
univalents is retarded in early diakinesis but their development appears normal at later stages. Presumably as a consequence of the orderly behavior of the
L. esculentum
bivalents and consequent contribution to each gamete, fertility of the sesquidiploids is higher than in
L. esculentum
autotriploids. The normally strict self-incompatibility is somewhat relaxed in the sesquidiploids. Extra
S. lycopersicoides
chromosomes can be transmitted from the sesquidiploid as pistillate parent, and the aneuploid progeny are viable. Establishment of alien addition races and their utilization to transmit desired genes from
S. lycopersicoides
to
L. esculentum
are anticipated.
Phospholipid methylation in starfish spermatozoa is linked to sperm chemoattraction.
Authors: Tezon J , Miller R L , Bardin C W
Published: May 2006

The mechanism whereby ovarian peptides cause sperm attraction was studied in the starfish. Phospholipid methylation and protein-O-carboxyl methylation, reactions linked to chemotactic responses in a variety of systems, were studied in starfish sperm. When sperm were preincubated with [methyl-3H]methionine and then exposed to the attractant, a rapid drop in radioactivity occurred in the phospholipid fraction. Methylated phospholipids decreased by 90% in the first 2 sec; however, no change was observed in endogenous methylation of protein carboxyl groups. The effect on phospholipid methylation was dose dependent, with a 40% reduction in radioactive phospholipids in sperm occurring with the minimal amount of attractant necessary to obtain a positive response in a sperm attraction bioassay. Attractants from species of starfish with little or no cross-reactivity in the bioassay had a limited effect on phospholipid methylation. The transmethylase inhibitor, homocysteine, caused a marked decrease in the accumulation of methylated phospholipids under basal conditions, which was correlated with as much as a 50-fold increase in sperm sensitivity to the attractant. The addition of chemoattractant resulted in a reduction in the amount of all individual methylated phospholipids, but the amount of phosphatidylmono[3H]methylethanolamine relative to the other methylated phospholipid decreased by a factor of 4 after stimulation. Homocysteine had the same effect. The reduction in methylated phospholipids by attractants suggests that phospholipid methylation is linked to the mechanism of action of these peptides. Methylation of phospholipids may play a role in the rapid desensitization of sperm cells to the attractant, which would be required for the orientation of the spermatozoa in the gradient of ovarian peptide.
Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene.
Authors: Earl P L , Jones E V , Moss B
Published: May 2006

A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses.
Membrane tolerance to ethanol is rapidly lost after withdrawal: a model for studies of membrane adaptation.
Authors: Taraschi T F , Ellingson J S , Wu A , Zimmerman R , Rubin E
Published: May 2006

The structural properties of liver microsomes and erythrocytes obtained from rats that had been chronically administered ethanol were examined by electron spin resonance (ESR) following ethanol withdrawal for 1-10 days. Membranes obtained from control animals exhibited considerable molecular disordering upon the addition of ethanol in vitro (50-100 mM). Conversely, microsomal and erythrocyte membranes from alcoholic animals were resistant to this disordering by ethanol (membrane tolerance). These membrane properties were also apparent in lipid bilayers comprised of either total lipids or phospholipids isolated from the control and alcoholic animals. While several weeks of ethanol administration were required for both erythrocytes and microsomes to develop membrane tolerance, erythrocytes from alcoholic animals were disordered by ethanol in vitro after the animals had been withdrawn from ethanol for only 1 day. The same rapid loss of tolerance was observed in microsomes after 2 days of withdrawal. The same time course for the loss of tolerance was observed in lipid bilayers prepared from the total lipid and phospholipid extracts. No significant differences in the cholesterol/phospholipid ratio were observed between the microsomal or erythrocyte membranes isolated before and after withdrawal. Thus, alterations in the microsomal and erythrocyte phospholipids, and not cholesterol content, were responsible for conveying membrane tolerance. Membrane structural properties can be rapidly adjusted in a mammalian system in response to the withdrawal of the external membrane perturbant ethanol. The withdrawal model, which begins with established membrane tolerance and leads to rapid and complete loss of tolerance, provides a model to analyze the compositional changes responsible for this tolerance to disordering by ethanol.
Synthesis of U1 RNA in a DNA-dependent system from sea urchin embryos.
Authors: Morris G F , Price D H , Marzluff W F
Published: May 2006

A soluble extract prepared from blastula nuclei of sea urchin (Lytechinus variegatus) embryos accurately transcribes cloned sea urchin DNA. This extract synthesizes U1 RNA using a cloned U1 RNA gene as a template. The U1 RNA is initiated accurately, and a portion of the transcripts has the correct 3' end as judged by gel electrophoresis. Longer transcripts also are formed that extend at least 280 bases 3' of the gene, and some extend as far as 800 bases 3' of the gene. A template containing 203 bases 5' of the gene gave as efficient transcription as did the whole U1 gene. Accurate 3' end formation was obtained with a template extending only 34 bases 3' of the gene, but efficient 3' end formation required sequences between 34 and 67 bases 3' of the gene.
Linkage between ligand binding and the dimer-tetramer equilibrium in the Monod-Wyman-Changeux model of hemoglobin.
Authors: Edelstein S J , Edsall J T
Published: May 2006

G. Weber [(1984) Proc. Natl. Acad. Sci. USA 81, 7098-7102] has inferred that the Monod-Wyman-Changeux (MWC) model for ligand binding by hemoglobin would require (contrary to experimental evidence) that increased ligand binding must promote stabilization of alpha 2 beta 2 tetramers with respect to dissociation into alpha beta dimers. Reexamination of the MWC model, however, in the light of general linkage principles and the specific analysis by G. K. Ackers and M. L. Johnson [(1981) J. Mol. Biol. 147, 559-582] shows that the opposite relation must hold, in agreement with experiment. The T form of the tetramer, with low ligand affinity, must be destabilized and progressively dissociates into the high-affinity dimers, designated D, as ligand binding increases. Each ligand molecule bound shifts the standard Gibbs free energy delta G2T for the D-T equilibrium by approximately 3 kcal/mol in favor of the dimer. Thus, T must exist in (at least) five delta G levels of cooperative free energy as it becomes progressively destabilized by successive binding of ligand molecules. Dissociation of the R tetramer to dimers, in contrast, is independent of the amount of ligand bound, so long as dimers and R-state tetramers possess the same (high) affinity for ligand. While the intrinsic ligand-binding constants of the T and R states (KT and KR) remain unchanged throughout by the postulates of the model, the model should not be regarded as a strictly two-state system in view of the multiple free-energy levels indicated above. The present analysis gives approximate, though not precise, agreement with experimental findings on the dimer-tetramer equilibrium considered by Weber and provides a rationale for interpreting other recent experiments concerning this equilibrium.
Transforming growth factor alpha inhibits secretion of gastric acid.
Authors: Rhodes J A , Tam J P , Finke U , Saunders M , Bernanke J , Silen W , Murphy R A
Published: May 2006

Transforming growth factor alpha (TGF-alpha), a protein secreted by transformed cells and related to epidermal growth factor (EGF), was tested for its effects on gastric acid secretion. Guinea pig gastric mucosae were mounted in Ussing chambers and the rate of acid release was monitored by the pH-stat method. When administered prior to the secretagogue, TGF-alpha prevented the histamine-induced increase in the rate of acid secretion. Similarly, TGF-alpha caused a decrease in the rate of acid release in tissues that had already been stimulated with histamine. These data show that TGF-alpha inhibits gastric acid secretion in a manner similar to EGF and that the two growth factors share at least one physiological action unrelated to their mitogenic properties.
Construction of a map of the short arm of human chromosome 6.
Authors: Leach R , DeMars R , Hasstedt S , White R
Published: May 2006

Four DNA sequences that reveal restriction fragment length polymorphisms (RFLPs) on the short arm of human chromosome 6 have been identified. Two of these sequences were isolated from recombinant DNA libraries enriched for DNA from human chromosome 6, one was isolated as a subclone from a human DNA segment having homology to an HLA class I sequence, and one was isolated by virtue of its homology to part of the insulin gene. Genetic linkage was determined among these four polymorphic sequences and several genes known to be on chromosome 6: glyoxylase I (GLO1), HLA-DR alpha, HLA-DQ alpha, and HLA-B. Additionally, two of the four RFLPs were regionally localized by using 53 deletion cell lines that had been typed for HLA-A, -B, and -DR, and for glyoxylase I. A genetic map of the short arm of chromosome 6 has been constructed on the basis of linkage studies with the eight markers. The map spans a minimum of 60 recombinational units and will be useful in the study of HLA-associated diseases.
Biochemical and functional effects of prenatal and postnatal omega 3 fatty acid deficiency on retina and brain in rhesus monkeys.
Authors: Neuringer M , Connor W E , Lin D S , Barstad L , Luck S
Published: May 2006

Docosahexaenoic acid [22:6 omega 3; 22:6-(4,7,10,13,16,19)] is the major polyunsaturated fatty acid in the photoreceptor membranes of the retina and in cerebral gray matter. It must be obtained either from the diet or by synthesis from other omega 3 fatty acids, chiefly alpha-linolenic acid (18:3 omega 3). We tested the effect of dietary omega 3 fatty acid deprivation during gestation and postnatal development upon the fatty acid composition of the retina and cerebral cortex and upon visual function. Rhesus monkeys (Macaca mulatta) were fed semipurified diets very low in 18:3 omega 3 throughout pregnancy, and their infants received a similar diet from birth. A control group of females and their infants received a semipurified diet supplying ample 18:3 omega 3. In near-term fetuses and newborn infants of the deficient group, the 22:6 omega 3 content of phosphatidylethanolamine was one-half of control values in the retina and one-fourth in cerebral cortex. By 22 months of age, the content of 22:6 omega 3 in these tissues approximately doubled in control monkeys, but it failed to increase in the deficient group. Low levels of 22:6 omega 3 in the deficient animals' tissues were accompanied by a compensatory increase in longer-chain omega 6 fatty acids, particularly 22:5 omega 6. Functionally, the deficient animals had subnormal visual acuity at 4-12 weeks of age and prolonged recovery time of the dark-adapted electroretinogram after a saturating flash. Abnormally low levels of 22:6 omega 3 may produce alterations in the biophysical properties of photoreceptor and neural membranes that may underlie these functional impairments. The results of this study suggest that dietary omega 3 fatty acids are retina and brain.
Blood--brain barrier sodium/potassium pump: modulation by central noradrenergic innervation.
Authors: Harik S I
Published: May 2006

The active transport of Na+ and K+ across the blood--brain barrier by the membrane-bound enzyme Na+/K+-activated ATPase of brain microvessel endothelial cells has a major role in the maintenance of brain water and electrolyte homeostasis. To test whether the putative noradrenergic innervation of cerebral microvessels from the nucleus locus ceruleus contributes to the regulation of Na+/K+-ATPase activity of the blood--brain barrier, specific [3H]ouabain-binding studies were performed on cerebral microvessels and crude cortical membranes obtained from Wistar rats with unilateral 6-hydroxydopamine lesion of the nucleus locus ceruleus. Such lesion depleted norepinephrine by about 90% in the ipsilateral cerebral cortex without affecting the contralateral cortex. [3H]Ouabain binding to membranes of cerebral cortex and the cerebral microvessels was specific and saturable. The maximal ouabain-binding capacity in microvessels of the ipsilateral, norepinephrine-depleted, cerebral cortex was reduced by about 40%, without change in the affinity of binding. [3H]Ouabain binding to crude membrane fractions of the cerebral cortex was not significantly affected by locus ceruleus lesion. The results suggest that Na+/K+-ATPase activity of cerebral microvessels, and the consequent transport of Na+ and K+ across the blood--brain barrier, is modulated by noradrenergic innervation from the locus ceruleus.
Characterization of acetylated and acetolyzed glycoprotein high-mannose core oligosaccharides by fast-atom-bombardment mass spectrometry.
Authors: Tsai P K , Dell A , Ballou C E
Published: May 2006

Fast-atom-bombardment mass spectrometry has been applied to acetylated neutral and phosphorylated oligosaccharides from yeast glycoproteins and to their acetolysis products. Although acetylation increases the sample molecular weight and the complexity of the spectra, it also enhances the sensitivity of detection, is applicable to samples that contain salt, and is especially useful for analysis of phosphorylated derivatives. Acetylation by trifluoroacetic anhydride/glacial acetic acid is particularly convenient and can be done rapidly on a small amount of material. Acetolysis by acetic anhydride/glacial acetic acid/H2SO4 is done on the acetylated oligosaccharides, and the acetylated fragments are recovered by solvent extraction and immediately subjected to mass spectrometry. The methodology allows molecular weight determinations and sequence analysis by acetolysis to be carried out on a few micrograms of isolated oligosaccharide in a few hours.
Evidence for a common evolutionary origin of brain and pancreas cholecystokinin receptors.
Authors: Vigna S R , Thorndyke M C , Williams J A
Published: May 2006

An evolutionary basis for the distinct forms of cholecystokinin (CCK) receptors in the mammalian brain and pancreas was examined. The brains and pancreases of ratfish, frog, snake, and chicken contained saturable, high-affinity binding sites for iodinated porcine CCK-33. In the ectothermic species, the brain and pancreas CCK receptors exhibited nearly the same relative specificities for various CCKs and gastrins. Sulfated CCK-8 and sulfated gastrin-17 were the most potent while their nonsulfated analogs and gastrin-4 were less potent. By contrast, in the chicken, the specificities of brain and pancreas CCK receptors closely resembled their mammalian counterparts. We conclude that brain and pancreas CCK receptors with new specificities for binding CCKs and gastrins evolved at the level of the divergence of endotherms (birds and mammals) from reptiles. We propose that the prior evolution of gastrin provided the selection pressure for these changes. The endotherm pancreas receptor arose in evolution by narrowing its requirement for the position of a sulfated tyrosine residue in its ligands from either the sixth or seventh position from the carboxyl terminus to the seventh position only. The endotherm brain receptor arose in evolution by losing its requirement for a sulfated ligand and by transferring its high-affinity binding domain from the tyrosine residue in the carboxyl termini of CCK and gastrin to the carboxyl-terminal tetrapeptide active site common to CCKs and gastrins.
A model for the RNA-catalyzed replication of RNA.
Authors: Cech T R
Published: May 2006

A shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena thermophila has enzymatic activity as a poly(cytidylic acid) polymerase [Zaug, A.J. & Cech, T.R. (1986) Science 231, 470-475]. Based on the known properties of this enzyme, a detailed model is developed for the template-dependent synthesis of RNA by an RNA polymerase itself made of RNA. The monomer units for RNA synthesis are tetra- and pentanucleotides of random base sequence. Polymerization occurs in a 5'-to-3' direction, and elongation rates are expected to approach two residues per minute. If the RNA enzyme could use another copy of itself as a template, RNA self-replication could be achieved. Thus, it seems possible that RNA catalysts might have played a part in prebiotic nucleic acid replication, prior to the availability of useful proteins.
Antigenic competition at the level of peptide-Ia binding.
Authors: Babbitt B P , Matsueda G , Haber E , Unanue E R , Allen P M
Published: May 2006

We examined the direct binding of a hen egg white lysozyme peptide, HEL(46-61), to membrane I-Ak (protein encoded in the A locus of the I region) molecules in the presence of detergent. A number of synthetic peptide derivatives, which did not stimulate our T-cell reactive hybridomas, competed for the binding of HEL(46-61) to I-Ak and also inhibited the functional presentation of HEL(46-61). Inhibitors included a peptide lacking a tyrosine at position 53 and a peptide corresponding to the autologous lysozyme peptide. Presentation was examined with cells or with supported planar phospholipid membranes bearing only I-Ak and HEL(46-61). Other peptides that did not compete for the binding did not inhibit functional presentation. We concluded that the binding of an immunogenic peptide to I-A is critical for presentation, that the I-A molecule does not discriminate between autologous and foreign related determinants but does recognize structurally different peptides. Our evidence suggests that our immunogenic peptide bears noncontiguous amino acids critical for contact I-A binding interspersed with amino acids critical for interaction with T cells.
Isolation of human mdr DNA sequences amplified in multidrug-resistant KB carcinoma cells.
Authors: Roninson I B , Chin J E , Choi K G , Gros P , Housman D E , Fojo A , Shen D W , Gottesman M M , Pastan I
Published: May 2006

The ability of tumor cells to develop simultaneous resistance to structurally different cytotoxic drugs constitutes a major problem in cancer chemotherapy. It was previously demonstrated that multidrug-resistant Chinese hamster cell lines contain an amplified, transcriptionally active DNA sequence designated mdr. This report presents evidence that multidrug-resistant sublines of human KB carcinoma cells, selected for resistance to either colchicine, vinblastine, or Adriamycin (doxorubicin), display amplification of two different DNA sequences homologous to the hamster mdr gene. Segments of the human mdr DNA sequences, designated mdr1 and mdr2, have been cloned. mdr1 sequences were amplified in all of the highly drug-resistant sublines and were expressed as a poly(A)+ RNA species of 4.5 kilobases that was detected in the resistant cells but not in the parental cell line. No expression of mdr2 sequences was detected. mdr2 sequences were coamplified with mdr1 in some of the multidrug-resistant sublines and, in two independently derived cell lines, underwent very similar rearrangements. The data suggest that the mdr1 gene is involved in multidrug resistance in human cells.
Hepatocellular carcinoma in ground squirrels persistently infected with ground squirrel hepatitis virus.
Authors: Marion P L , Van Davelaar M J , Knight S S , Salazar F H , Garcia G , Popper H , Robinson W S
Published: May 2006

Although persistent infection with hepatitis B virus and woodchuck hepatitis virus has been associated with development of hepatocellular carcinoma in the host, little has been known of such an association with ground squirrel hepatitis virus (GSHV), which is closely related to the woodchuck virus. Colonies of GSHV-infected and -uninfected Beechey ground squirrels were observed for tumors for a period of 5 years. Tumors developed in seven squirrels after a minimum of 2.4 years of observation per animal; each of the seven animals was over 4 years old when the tumor was detected. The predominant type of tumor was hepatocellular carcinoma, which appeared in 2 of 28 GSHV-bearing animals studied and in 1 of 23 squirrels with antibody to the virus. No hepatocellular carcinoma appeared in 24 GSHV marker-free squirrels. Integrated GSHV DNA was found in the hepatocellular carcinoma tissue of the one carrier animal examined, paralleling the frequent findings of integrated hepatitis B and woodchuck hepatitis viral DNA in human and woodchuck hepatocellular carcinoma. Although the incidence of liver carcinoma reported here in carrier ground squirrels is neither as great as that in carrier woodchucks nor statistically different from the incidence in noncarrier squirrels, the data presented suggest that persistent infection with GSHV may also be associated with hepatocellular carcinoma.
Modulation of granule cell migration by a glia-derived protein.
Authors: Lindner J , Guenther J , Nick H , Zinser G , Antonicek H , Schachner M , Monard D
Published: May 2006

Cultured explants from early postnatal mouse cerebellum were used to examine the influence of a 43-kDa glia-derived neurite-promoting factor (GdNPF) on the migration of [3H]thymidine-labeled granule cell neurons. GdNPF, which is a potent serine protease inhibitor, significantly reduced the extent of granule cell migration in a dose-dependent manner. This effect could be neutralized by addition of thrombin, which binds GdNPF. Other protease inhibitors such as aprotinin, hirudin, soybean trypsin inhibitor, leupeptin, 6-aminocaproic acid, and D-Phe-Pro-ArgCH2Cl do not show this inhibitory effect. These results demonstrate that a glia-derived protein can regulate the migration of postmitotic neurons, an important cellular event in the development of the nervous system.
Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication.
Authors: Nicosia A , Perugini M , Franzini C , Casagli M C , Borri M G , Antoni G , Almoni M , Neri P , Ratti G , Rappuoli R
Published: May 2006

Pertussis toxin, a protein composed of five different subunits (S1, S2, S3, S4, and S5), is the major virulence factor of Bordetella pertussis. We have cloned and sequenced a DNA fragment of 4.7 kilobases that contains the genes coding for the five subunits. The genes are clustered within 3.2 kilobases in the following order: S1, S2, S4, S5, and S3. A sequence closely resembling Escherichia coli promoters is found only before the S1 gene, and a possible termination signal is present at the end of the S3 gene, which suggests that the pertussis toxin genes are organized in a single operon. A possible Shine-Dalgarno sequence is present before the S1 gene but not before the other four genes that 8-12 nucleotides upstream from the ATG codon show a new consensus sequence, 5'TCC(T)GG3', possibly involved in the regulation of translation. We have also found sequence homology between the S2 and S3 genes and their protein products indicating that gene duplication played a major role in the evolution of pertussis toxin.
Membrane-dependent coagulation reaction is independent of the concentration of phospholipid-bound substrate: fluid phase factor X regulates the extrinsic system.
Authors: Forman S D , Nemerson Y
Published: May 2006

Negatively charged phospholipids accelerate blood coagulation; one suggestion is that the binding of the gamma-carboxyglutamic acid-containing zymogens to these lipid surfaces increases their effective concentration as substrates. Alternatively, the charged phospholipids could enhance the direct interaction of substrate with the catalytic complex, which is localized at the membrane surface. We distinguished the alternatives by using prothrombin fragment 1 to compete with the substrate for membrane binding sites without interfering with the direct enzyme-substrate interaction. In a tissue factor-factor VIIa system containing neutral phospholipids (to which the substrate does not bind), prothrombin fragment 1 had no significant effect on factor X activation (Km, 877 +/- 111 nM and 791 +/- 103 nM, with and without prothrombin fragment 1, respectively). In contrast, in a system containing 30% phosphatidylserine, prothrombin fragment 1 displaced phospholipid-bound factor X, increasing the free factor X concentration and the reaction velocity in all 22 determinations. As the velocity increases correlated only with the free factor X concentration, we conclude that the free factor X concentration controls this reaction velocity. The Km in the 30% phosphatidylserine system, calculated using free factor X concentration, was 41 +/- 5 nM and 63 +/- 9 nM, with and without prothrombin fragment 1, respectively. Thus, the negatively charged lipids decreased the intrinsic Km by over 90%. The methodology employed should be applicable to ligand-receptor systems in which ligand binds nonspecifically to the membrane surface.
Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein.
Authors: Smotkin D , Wettstein F O
Published: May 2006

Human papillomavirus type 16 DNA and RNA were characterized in the cervical cancer-derived CaSki cell line, which contains only integrated DNA, and in a cervical cancer, which contains predominantly plasmid DNA. In both, a major RNA can code for the early open reading frame E7 and a minor one can code for E6. The cervical cancer, but not the CaSki cell line, contains a minor RNA that can code for an intact E2 protein, and this may relate to the continued presence of plasmid DNA. The RNA mapping data suggest that the poly(A)+ RNA is transcribed from a minor fraction of the several hundred gene copies present, and in the cervical cancer these genomes appear to be integrated. The E7 protein has been identified in CaSki cells and the prevalence of its mRNA suggests a possible function in progression to, or long-term maintenance of, the malignant state.
Tissue-specific control of rat malic enzyme activity and messenger RNA levels by a high carbohydrate diet.
Authors: Dozin B , Rall J E , Nikodem V M
Published: May 2006

In euthyroid rats fed a high carbohydrate fat-free diet for 10 days, the mass of cellular malic enzyme mRNA in liver is increased 7- to 8-fold above the basal level. Malic enzyme activity is stimulated to the same extent. This effect does not result from an increase either in the transcriptional activity of the malic enzyme gene, as determined by nuclear run-off transcription assay, or in the content of intranuclear malic enzyme RNA sequences. Mathematical modeling shows that this increase in cytoplasmic mRNA is compatible with retarded degradation of cytoplasmic mRNA. Regulation of malic enzyme by carbohydrates is liver-specific, since no response is observed in the following nonhepatic tissues: brain, heart, spleen, kidney, testis, and lung. Furthermore, the amplitude of the response in liver depends on the thyroid state of the animals, being lower (by a factor of approximately 4) in hypothyroidism and higher (12- to 15-fold) when normal animals are injected simultaneously with a daily dose of 15 micrograms of triiodothyronine per 100 g of body weight for 10 days. Since thyroid hormones regulate liver malic enzyme synthesis predominantly at the nuclear level and carbohydrates at the cytoplasmic level, the additive effect of triiodothyronine and a high carbohydrate diet on the activity of malic enzyme is readily explicable.
DNA differences found between Africanized and European honeybees.
Authors: Hall H G
Published: May 2006

The harmful en masse introduction of Africanized honeybees into the United States will occur within 5 years. Possible means of control are dependent on a reliable way to distinguish the Africanized bees from the extant European bees. Current means of identification are inadequate. Reported here are the encouraging initial results to distinguish the bees by their nuclear DNA. With 9 restriction enzymes and 16 probes, six genetic differences have been found among three samples of European bees from California. Twelve additional differences were detected between the European samples and a sample of Africanized bees from Costa Rica.
Mutation affecting the expression of immunoglobulin variable regions in the rabbit.
Authors: Kelus A S , Weiss S
Published: May 2006

We have found a variant of the allotype allele a2 in the rabbit, which presumably arose by mutation, that segregates as expected for an allele at the a locus. This allele is called "ali" and the corresponding rabbit strain is called "Alicia." In heterozygous animals (ali/a1 and ali/a3) the concentration of a2 molecules is lower by a factor of 1000 than in standard a2/a2 homozygotes. In homozygous ali/ali individuals the a2 concentration varies with age--i.e., very low in young rabbits and higher in older ones--but it never reaches normal levels. The low level of a2 is compensated by increased amounts of a-negative molecules. Southern blot analysis did not reveal any gross changes in the intron between JH and C mu (joining region of immunoglobulin heavy chain and constant region of immunoglobulin mu chain) or in the number of VH gene segments encoding a locus specificities. We suggest that the ali phenotype is due to a mutation in a control element.
N
2
-Succinylated intermediates in an arginine catabolic pathway of Pseudomonas aeruginosa
Authors: Jann Alfred , Stalon Victor , Wauven Corinne Vander , Leisinger Thomas , Haas Dieter
Published: May 2006

Arginine-nonutilizing (
aru
) mutants of
Pseudomonas aeruginosa
strain PAO converted L-arginine to
N
2
-succinylarginine or
N
-succinylglutamate, which were identified by high-voltage electrophoresis and HPLC. Addition of aminooxyacetate, an inhibitor of pyridoxal phosphate-dependent enzymes, to resting cells of the wild-type PAO1 in arginine medium led to the accumulation of
N
2
-succinylornithine. Enzyme assays with crude
P. aeruginosa
extracts established the following pathway: L-arginine + succinyl-CoA →
N
2
-succinylarginine →
N
2
-succinylornithine →
N
_succinylglutamate 5-semialdehyde →
N
-succinylglutamate → succinate + glutamate. Succinyl-CoA may be regenerated from glutamate via 2-ketoglutarate. L-Arginine induced the enzymes of the pathway, and succinate caused catabolite repression. Purified
N
2
-acetylornithine 5-aminotransferase (
N
2
-acetyl-L-ornithine: 2-oxoglutarate aminotransferase, EC 2.6.1.11), an arginine biosynthetic enzyme, efficiently transaminated
N
2
-succinylornithine; this explains the enzyme's dual role in arginine biosynthesis and catabolism. The succinylarginine pathway enables
P. aeruginosa
to utilize arginine efficiently as a carbon source under aerobic conditions, whereas the other three arginine catabolic pathways previously established in
P. aeruginosa
fulfill different functions.
Dynamics of the B-to-Z transition in supercoiled DNA.
Authors: Pohl F M
Published: May 2006

The sequence (dC-dG)16, inserted into the polylinker of plasmid pUC8, adopts a left-handed Z-DNA conformation at "natural" supercoil density. The radioactively labeled monoclonal antibody Z-D11, which has a very high affinity for this DNA conformation, provides a convenient sensitive tool to measure selectively the amount of Z-DNA. Chloroquine reversibly changes the supercoil density of plasmid DNA and thereby the equilibrium between right- and left-handed double-helical DNA. The time-dependent formation or disappearance of Z-DNA was measured by using the antibody either as a fast indicator of Z-DNA or as an additional effector of the B-to-Z equilibrium. In the middle of the transition, a relaxation time of about 1 hr is observed in 0.1 M NaCl at 22 degrees C. The kinetic data are compatible with an all-or-none transition between the two conformations. The overall rate constant for Z-DNA formation, kBZ, decreases with the square of the chloroquine concentration, while the reverse one, kZB, increases with about the fourth power.
Enhanced expression of the bacterial chloramphenicol acetyltransferase gene in mouse cells cotransfected with synthetic polynucleotides able to form Z-DNA.
Authors: Banerjee R , Grunberger D
Published: May 2006

Recent studies have demonstrated that the left-handed, Z-DNA conformation is favored in polymers containing alternating purine/pyrimidine sequences that can exist in vivo and may play a role in gene expression. On the basis of this assumption, we have studied the effect of various cotransfected polynucleotides on the transient expression of the chloramphenicol acetyltransferase (CAT) gene in thymidine kinase-deficient murine L cells. Cotransfections were performed by calcium phosphate coprecipitation of CAT gene plasmids with various polymers, and the CAT enzymatic activity was measured in cell lysates after 48 hr. About 2- to 10-fold stimulation of CAT gene expression was observed when the cells were cotransfected with 10 micrograms (per 10-cm culture dish) of plasmid pSV2cat, which contains simian virus 40 (SV40) promoter and enhancer sequences, and 2-10 micrograms of polymers that can form Z-DNA, such as poly(dG-m5dC) X poly(dG-m5dC) or poly(dG-dC) X poly(dG-dC), as compared to transfection with pSV2cat alone. Further, enhanced CAT gene expression was also observed when cotransfections were performed with these polymers and two other plasmid vectors, one containing the SV40 promoter but no enhancer and the other lacking any SV40 regulatory sequences. However, poly(dA-dC) X poly(dG-dT), which can form Z-DNA, did not induce any stimulation. Similarly, no or very little stimulation was observed after cotransfection of pSV2cat with either poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT), which do not adopt the Z conformation. These results suggest that certain polynucleotides may enhance transcription of the CAT gene.
Clustered arrangement of keratin intermediate filament genes.
Authors: Powell B C , Cam G R , Fietz M J , Rogers G E
Published: May 2006

We report here that component members of the keratin intermediate filament (IF) type I and type II gene families of sheep are closely linked but apparently the two families are not. Nine genes, accounting for up to half of the keratin IF gene repertoire, were mapped in four cosmid clones and the linkage between the genes ranged from several kilobases to 20 kilobases. In one cosmid, three tandem type I genes had the same transcriptional arrangement and were regularly spaced. In another cosmid, tandem genes encoding type II keratins were identified and, surprisingly, a solitary exon was discovered in the intergene region between the two type II genes. In a normal gene this exon encodes one of the most conserved amino acid regions of IF proteins, the C-terminal end of the alpha-helical core. Homologous C-terminal protein subdomains were encoded by two wool keratin type II genes and we suggest that this arrangement may also exist in the other wool keratin type II genes.
Partitioning of long-chain alcohols into lipid bilayers: implications for mechanisms of general anesthesia.
Authors: Franks N P , Lieb W R
Published: May 2006

Alcohols act as anesthetics only up to a certain chain length, beyond which their biological activity disappears. Although the molecular nature of general anesthetic target sites remains unknown, presently available data support the hypothesis that this "cutoff" in anesthetic activity could be due to a corresponding cutoff in the absorption of long-chain alcohols into lipid-bilayer portions of nerve membranes. To test this hypothesis, we have developed an extremely sensitive biological assay, based on inhibition of the light-emitting firefly luciferase reaction, which is capable of measuring lipid-bilayer/buffer partition coefficients K for very lipid soluble compounds. Contrary to the hypothesis and reported data, we find a strictly linear increase in log(K) as the chain length increases [delta(delta G0)CH2 = - 3.63 kJ/mol] for the primary alcohols from decanol to pentadecanol, with no hint of a cutoff. The fact that alcohols continue to partition into lipid bilayers long after their biological activity has ceased is consistent with the view that the primary target sites in general anesthesia are proteins rather than the lipid-bilayer portions of nerve membranes.
Transition from metaphase to anaphase is accompanied by local changes in cytoplasmic free calcium in Pt K2 kidney epithelial cells.
Authors: Ratan R R , Shelanski M L , Maxfield F R
Published: May 2006

We have used a Ca2+-sensitive dye, fura-2, to investigate the role of Ca2+ during mitosis in Pt K2 epithelial cells. The concentration of cytoplasmic free calcium, [Ca2+]i, increased 2-fold between metaphase and anaphase. Digital image analysis revealed two patterns of [Ca2+]i localization during anaphase. In half of the anaphase cells, the increase in [Ca2+]i was greatest in the region near the spindle poles and decreased radially. In the other anaphase cells, there was a ring of high [Ca2+]i in the cytoplasm, surrounding an area of low [Ca2+]i in the spindle midzone. Although the reason for the different patterns is not known, peak [Ca2+]i in both cases was sufficient to maintain a 2- to 6-fold gradient in [Ca2+]i from the polar region to the midzone. [Ca2+]i gradients may thus regulate spindle microtubule equilibria and directed chromosome movement during mitosis.
A gene deletion ending within a complex array of repeated sequences 3' to the human beta-globin gene cluster.
Authors: Henthorn P S , Mager D L , Huisman T H , Smithies O
Published: May 2006

A DNA fragment containing the deletion junction region from an Indian individual with a type of hereditary persistence of fetal hemoglobin has been cloned. Using a probe isolated from this deletion-spanning clone, we located the 3' breakpoint of the deletion in normal DNA to a region 30 kilobase pairs (kb) downstream of the beta-globin gene. The deletion removes 48.5 kb of DNA. Sequences of the deletion junction and of the normal DNA surrounding the 3' breakpoint were determined and compared to the previously determined sequence of the normal DNA surrounding the 5' breakpoint. This comparison shows that the deletion was the result of a nonhomologous recombinational event, although there is a 5-base-pair (bp) region of local homology between the normal DNAs at their breakpoints. The 5' deletion breakpoint occurs in the Alu family repeat 3' to the A gamma-globin gene. The 3' breakpoint is located within a region that contains the following: a portion of an L1 (Kpn I) repeat, a perfect 160-bp palindrome, and a set of 41-bp direct repeats that are found elsewhere in the human genome. A variation in restriction fragment lengths was observed in this region in one family.
Two types of calcium channels in guinea pig ventricular myocytes.
Authors: Mitra R , Morad M
Published: May 2006

In cardiac muscle, Ca2+ plays a key role in regulation of numerous processes, including generation of the action potential and development of tension. The entry of Ca2+ into the cell is regulated primarily by voltage-gated channels in the membrane. Until recently, it was felt that only one type of Ca2+ channel existed in cardiac ventricular muscle. Experiments reported here suggest that in isolated guinea pig ventricular myocytes, there are two distinct types of Ca2+ channels with markedly different activation thresholds, inactivation kinetics, and sensitivities to inorganic and organic Ca2+ channel blockers. The channels were also distinguished based on their response to increased frequency of clamping such that the current through the low-threshold channel decreased while that through the high-threshold channel increased. In a few cells, the current through both channels was enhanced by isoproterenol, a beta-adrenergic agonist, but only the high-threshold channel was enhanced by the Ca2+-channel agonist Bay K 8644. Thus, isolated guinea pig ventricular myocytes appear to have two types of Ca2+ channels distinguished by various criteria.
Discrete wave mechanics: An introduction
Authors: Wall Frederick T.
Published: May 2006

Discrete wave mechanics is formulated for particles in one-dimensional systems by use of a simple finite difference equation. The solutions involve wave vectors (instead of wave functions) as well as a newly defined “wave vector energy.” In the limit, as
c
→ ∞, the treatment reduces to that of Schrödinger's wave mechanics. Specific calculations are made for completely free particles as well as for particles confined to a one-dimensional box. The results exhibit a striking compatibility with relativistic considerations. The wave vectors show properties that can be identified with particles and anti-particles—each possess identical probability distributions with energies that add up to zero.
Algebraic K-theory of spaces stratified fibered over hyperbolic orbifolds
Authors: Farrell F. T. , Jones L. E.
Published: May 2006

Among other results, we rationally calculate the algebraic K-theory of any discrete cocompact subgroup of a Lie group G, where G is either O(
n
, 1), U(
n
, 1), S
p
(
n
, 1), or F
4
, in terms of the homology of the double coset space Γ\G/K, where K is a maximal cocompact subgroup of G. We obtain the formula K

n

(ZΓ) [unk] [unk] ≅ [unk]

i
=0

∞
H

i

(Γ\G/K; [unk]

n
-
i

), where [unk]

j

is a stratified system of Q vector spaces over Γ\G/K and the vector space [unk]

j

(Γ
g
K) corresponding to the double coset Γ
g
K is isomorphic to K

J

(Z(Γ [unk]
g
K
g
-1
)) [unk] Q. Note Γ [unk]
g
K
g
-1
is a finite subgroup of Γ. Earlier, a similar formula for discrete cocompact subgroups Γ of the group of rigid motions of Euclidean space was conjectured by F. T. Farrell and W. C. Hsiang and proven by F. Quinn.
Inhibition of gene expression in plant cells by expression of antisense RNA
Authors: Ecker Joseph R. , Davis Ronald W.
Published: May 2006

Due to the paucity of mutations in biochemical pathways in plants, an alternative approach to classical genetics was tested by expressing antisense RNA in plant cells. A series of plasmids was constructed with the bacterial gene for chloramphenicol acetyltransferase (EC 2.3.1.28) linked in either the sense or antisense orientation to several different plant gene promoters. Various ratios of sense and antisense chloramphenicol acetyltransferase gene plasmids were introduced into plant protoplasts by electric field-mediated DNA transfer (“electroporation”) and the level of expression in each combination was monitored by chloramphenicol acetyltransferase assays. Transcription of antisense RNA was found to effectively block the expression of target genes. Thus, the observation that antisense RNA inhibits gene expression in bacteria and animal systems has been extended to the plant kingdom. Antisense RNA techniques have immediate practical applications in both basic research and in plant genetic engineering.