bluuebunny/crossref_metadata_2025 huggingface dataset:
Cointegrate formation by Tn5, but not transposition, is dependent on recA.
Authors: Hirschel B J , Galas D J , Chandler M
Published: May 2006

We have studied the effect of the recA-dependent homologous recombination system of Escherichia coli on both Tn5-mediated cointegrate formation and Tn5 transposition. We demonstrate here that, whereas transposition of Tn5 is independent of the recA gene product (as has been shown by other workers), Tn5-mediated cointegrate formation is strongly dependent on recA. The structures of both the simple transposition products and the cointegrates formed in a recA- background seem to be the same as those produced in a recA+ background. These results provide strong evidence that Tn5 does not transpose via an obligate cointegrate intermediate and suggest that the recA effect on cointegrate formation is exerted during the process of transposition.
Functional origin of replication of pT181 plasmid DNA is contained within a 168-base-pair segment.
Authors: Khan S A , Adler G K , Novick R P
Published: May 2006

We have used a recently developed in vitro replication system from Staphylococcus aureus to determine the origin and direction of replication of pT181 plasmid DNA. The origin was located to within 168 base pairs by two methods: (i) sequential labeling of restriction endonuclease fragments after synchronous initiation in vitro in the presence of various amounts of dideoxy-TTP and (ii) by constructing in vitro deletions of pT181 DNA close to the origin of replication and testing for their ability to replicate in vitro pT181 plasmid was found to replicate unidirectionally and anticlockwise, as the map is conventionally drawn. The nucleotide sequence of the region containing the origin of replication has been determined and found to be partially or entirely contained within the coding sequence for the repC protein, which is uniquely required for pT181 plasmid replication. Preliminary evidence suggesting that pT181 replicates by a rolling circle mechanism is discussed.
Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells.
Authors: Horst J , Weckler C , Jacob E , Aulehla-Scholz C , Deppert W
Published: May 2006

The recombinant plasmid p102 based on pBR322 carrying approximately equal to 50% of the replicator proximal early region of simian virus 40 (SV40) DNA, including the viral origin of replication, has been constructed. It lacks a major part of the large tumor (T) antigen 3'-coding region, the T-antigen termination codon, and the polyadenylylation site. The plasmid was transferred together with the herpes simplex virus thymidine kinase (TK) gene as a selectable marker to mouse LTK- cells. TK+ cell clones were isolated and their high molecular weight DNAs were shown by DNA blotting and hybridization experiments to contain the SV40 DNA fragment from the recombinant. In some of these clones, heterogeneous expression of the SV40 DNA fragment could be detected by immunofluorescence while, in control experiments in which a plasmid containing the complete SV40 early DNA region was used, this extensive heterogeneity of T-antigen expression was not observed. RNA . DNA hybridization experiments showed that the SV40-specific RNA of those clones is polyadenylylated. The molecular weight of the T-antigen-related protein coded by p102 corresponded well to the expected coding capacity of the SV40 DNA fragment. Small tumor antigen was not expressed.
Monoclonal antibodies to 5'-triphospho-(2'-5')adenyladenosine oligonucleotides.
Authors: Cailla H , Le Borgne De Kaouel C , Roux D , Delaage M , Marti J
Published: May 2006

Thirteen monoclonal antibodies to ppp(A2'p5')nA oligonucleotides have been produced by fusing Y3 myeloma cells with spleen cells of rat hyperimmunized with A2'p5'A succinyl albumin as immunogen. 125I-labeled A2'p5'A succinyl tyrosine methyl ester was used as a labeled probe. Antibodies were detected by their ability to bind the labeled analog and were selected for their affinity for A2'p5'A. There were no significant differences between the properties of the monoclonal antibodies and of the antiserum. They discriminated between 2'-5' and 3'-5' phosphodiester bonds: they crossreacted poorly with (A3'p5')2A (crossreactivity ratio, greater than 10(4)) and even less with ATP an adenosine (crossreactivity ratio, greater than (6)). The affinity was high (Kd = 6 x 10(-12) M) for succinyl A2'p5'A, which is the best ligand, and also high for A2'p5'A (crossreactivity ratio, 3) and (A2'p5')2A, (A2'p5')3A, and (As'p5')4A (crossreactivity ratio, 7.5). The binding to triphosphorylated isomers, ppp(A2'p5)nA, was affected by the presence of the triphosphate groups, and the affinity increased as the length of the isomer increased (crossreactivity ratio, 10,000 for n = 1 to 200 for n = 4). Thermodynamic analysis of these data demonstrated that, in dephosphorylated isomers, the binding site of highest affinity was located at the 5'-OH end of the molecule whereas in phosphorylated isomers, the favorable binding sites were in the middle and at the 2'-OH end of the chain. Use of monoclonal antibodies of such a specificity together with the 125I-labeled 2-5A analog allows quantification of (A2'p5')nA directly and of ppp(A2'p5')nA after removal of the terminal phosphates by alkaline phosphatase treatment.
15N NMR study of a mixture of uniformly labeled tRNAs.
Authors: Gonnella N C , Birdseye T R , Nee M , Roberts J D
Published: May 2006

15N NMR spectra were taken of 15N-enriched tRNA extracted from bakers yeast; ammonium sulfate was used as a nitrogen source. The increase in the degree of denaturation of tRNA, which occurs with increase in temperature from 30 degrees C to 70 degrees C, resulted in no large changes in 15N chemical shifts at acidic and neutral pH but quite pronounced changes in proton-15N nuclear Overhauser effects.
Perturbations of enzymic uracil excision due to purine damage in DNA.
Authors: Duker N J , Jensen D E , Hart D M , Fishbein D E
Published: May 2006

Phage PBS-2 DNA, which contains uracil in place of thymine, was selectively damaged and then used as substrate for purified Bacillus subtilis uracil-DNA glycosylase. This enzyme releases uracil from DNA in a limited processive manner. Irradiation by ultraviolet light (greater than 305 nm) in the presence of isopropanol and a free radical photoinitiator introduced covalently bound 8-(2-hydroxy-2-propyl)purines into DNA. Methylation by dimethylsulfate yielded 7-methylguanine. Apurinic sites were produced by gentle heating of methylated DNA. Rates of enzymic release of uracil from DNA varied among these three substrates. The Vmax was markedly decreased for DNA containing 8-(2-hydroxy-2-propyl)purines and apurinic sites but was unaffected by the presence of larger quantities of 7-methylguanine. This suggests that certain types of damaged DNA moieties may decrease the capacity for uracil excision. Therefore, interference with enzymic excision of this potentially mutagenic base may constitute a common mechanism of action of the reaction products of several unrelated DNA damaging agents.
Localization of human immunoglobulin kappa light chain variable region genes to the short arm of chromosome 2 by in situ hybridization.
Authors: Malcolm S , Barton P , Murphy C , Ferguson-Smith M A , Bentley D L , Rabbitts T H
Published: May 2006

The genes for human immunoglobulin kappa light chains have been localized in normal lymphocyte and fibroblast chromosomes by in situ hybridization of probes from cloned DNA fragments of the kappa variable region locus. The localization was achieved by counting grains (after autoradiography) over chromosomes in a number of karyotypes. The variable region gene probes hybridized in a cluster on a region of the chromosome 2 short arm close to the centromere (2cen leads to p12). This location was confirmed in lymphocytes from a balanced translocation carrier 46XXt (2; 16) (q13; q22). Our results show that human kappa light chain genes are located in the region of the break point observed in specific chromosomal translocations associated with Burkitt lymphoma.
Transcriptional activity of the transposable element Tn10 in the Salmonella typhimurium ilvGEDA operon.
Authors: Blazey D L , Burns R O
Published: May 2006

Polarity of Tn10 insertion mutations in the Salmonella typhimurium ilvGEDA operon depends on both the location and the orientation of the Tn10 element. One orientation of Tn10 insertions in ilvG and ilvE permits low-level expression of the downstream ilvEDA and ilvDA genes, respectively. Our analysis of Salmonella ilv recombinant plasmids shows that this residual ilv expression must result from Tn10-directed transcription and does not reflect the presence of internal promoters in the ilvGEDA operon, as was previously suggested. The opposite orientation of Tn10 insertion in ilvE prevents ilvDA expression, indicating that only one end of Tn10 is normally active in transcribing adjacent genes. Both orientations of Tn10 insertion in ilvD exert absolute polarity on ilvA expression. Expression of ilvA is known to be dependent on effective translation of ilvD, perhaps reflecting the lack of a ribosome binding site proximal to the ilvA sequence. Therefore, recognition of the ability of Tn10 to promote transcription of contiguous genes in the ilvGEDA operon apparently requires the presence of associated ribosome binding sites.
Transposon Tn10 provides a promoter for transcription of adjacent sequences.
Authors: Ciampi M S , Schmid M B , Roth J R
Published: May 2006

Promoters located within the Tn10 insertion element cause transcription of "host" sequences adjacent to both ends of the inserted Tn10 element. These promoters are usually not observed in genetic experiments because their transcripts are efficiently terminated at nearby rho-dependent termination sites. The observations presented here provide an explanation for several confusing aspects of transposon behavior and suggest the possibility that many transposons possess promoters that have escaped detection for similar reasons.
Release of soluble immune complexes from immune adherence receptors on human erythrocytes is mediated by C3b inactivator independently of β1H and is accompanied by generation of C3c
Authors: Medof M. Edward , Prince Gregory M. , Mold Carolyn
Published: May 2006

Antigen·antibody complexes (Ag·Ab) prepared from
125
I-labeled bovine serum albumin and guinea pig anti-albumin were incubated at 37°C for 30 min with normal human serum diluted optimally for binding (1:16) and then with autologous erythrocytes (RBC). After washing, RBC-bearing antigen·antibody·complement complexes (Ag·Ab·C) were resuspended in serum reagents or solutions of purified complement components, and the kinetics of dissociation were analyzed. Ag·Ab·C dissociated in serum heated at 56°C for 30 min (SΔ30) but not in serum heated for 120 min (SΔ120). Dissociation in SΔ30 markedly decreased after adsorption with anti-C3b inactivator but not anti-β1H or anti-C4 binding protein (C4bp), and dissociation in SΔ120 markedly increased after addition of C3b inactivator. Hemolytic assays revealed that SΔ30 retained inactivator activity whereas SΔ120 lacked significant activity. Ag·Ab·C dissociated in the presence of purified inactivator or C3b but not β1H or C3. Dissociation was more rapid with inactivator than with C3b and occurred at 0°C as well as at 37°C. Treatment with inactivator inhibitor abolished dissociation in SΔ30; dissociation in inactivator deficient serum was markedly reduced. Addition of β1H did not enhance inactivator-mediated dissociation at limiting dilutions of inactivator, and adsorption of Ag·Ab·C with anti-β1H or preparation of Ag·Ab·C with serum adsorbed with anti-β1H did not diminish dissociation. After dissociation with inactivator, Ag·Ab·C were unchanged in size but were no longer able to bind to fresh RBC and gave enhanced binding to Raji and Daudi lymphoblastoid cells. NaDodSO
4
/polyacrylamide gel electrophoresis of Ag·Ab·C prepared with
125
I-labeled C3 revealed that, after binding to RBC, dissociation with inactivator was accompanied by generation of a C3 fragment the size of C3c. Preincubation of Ag·Ab·C with excess inactivator did not prevent subsequent binding of Ag·Ab·C to RBC but, immediately after binding, Ag·Ab·C dissociated rapidly. These findings indicate that C3b inactivator can release immune complexes from immune adherence receptors on human RBC, that release occurs independently of β1H, alters cell binding properties of immune complexes, and involves multiple cleavages of the C3b α′ chain, and that receptors in human RBC membrane are required for this C3b inactivator-mediated breakdown.
Left-handedness: association with immune disease, migraine, and developmental learning disorder.
Authors: Geschwind N , Behan P
Published: May 2006

We report an experimental study designed to test the following hypothesis derived from clinical observations: There is an elevated frequency in left-handed individuals and in their families of immune disease, migraine, and developmental learning disorders. In two separate investigations the frequency of these conditions was compared in strongly left-handed subjects and in strongly right-handed controls. In each of the investigations we found markedly higher frequencies of immune disease in the left-handers than in the right-handers. The rate of learning disabilities was also much higher in the left-handers than in the right-handers in both investigations. In a second study the frequency of left-handedness was compared in patients with migraine or immune disease and in general population control subjects free of these disorders. There was a higher frequency of left-handedness in patients with migraine and myasthenia gravis than in controls. We present a brief outline of a hypothesis that may account for an increased frequency of immune disease in left-handers and in their families.
Efficient photochemical formation of 1 alpha-hydroxyprevitamin D3.
Authors: Dauben W G , Phillips R B , Jefferies P
Published: May 2006

Control of the band width of the irradiating light between 280 and 310 nm and of the temperature of the reaction permits formation of 1 alpha-hydroxyprevitamin D3 in yields of over 80%, at 40-60% conversion of the 1 alpha-hydroxyprovitamin D3. The procedures developed permit doubling the photochemical yield of the 1 alpha-hydroxyvitamin D3 compounds which are the major circulatory forms of vitamin D3.
[LeuB24]insulin and [AlaB24]insulin: altered structures and cellular processing of B24-substituted insulin analogs.
Authors: Assoian R K , Thomas N E , Kaiser E T , Tager H S
Published: May 2006

We have used insulin analogs having leucine or alanine substitutions at positions B24 and B25 to examine the structural basis for insulin binding and insulin metabolism by isolated rat hepatocytes. Apparent receptor binding affinities for the analogs were in the order insulin greater than [LeuB24]insulin greater than [LeuB25]insulin = [AlaB24]insulin. Incubation of the corresponding 125I-labeled peptides with hepatocytes followed by analysis of the cell-associated products showed that [125I]iodoinsulin and [125I]iodo-[LeuB25]insulin were processed to a peptide intermediate which appeared as an ascending shoulder on the peak of cell-associated hormone during gel filtration; similar incubations using [125I]iodo-[LeuB24]insulin or [125I]iodo-[AlaB24]insulin failed to yield detectable amounts of the intermediate. In addition, assessment of the structures of insulin and the three insulin analogs by tyrosine radioiodination showed that [LeuB24]insulin and [AlaB24]insulin maintain similar solution conformations which differ from the conformations taken by insulin and [LeuB25]insulin. We conclude that (a) alterations in side-chain bulk at position B24 result in long-range structural perturbations in the insulin molecule, (b) these structural alterations lead to an altered cellular processing of the two B24 insulin analogs, and (c) the selectivity of this processing arises from events subsequent to ligand-receptor recognition.
Viroids and prions.
Authors: Diener T O , McKinley M P , Prusiner S B
Published: May 2006

Viroids are small "naked" infectious RNA molecules that are pathogens of higher plants. The potato spindle tuber viroid (PSTV) is composed of a covalently closed circular RNA molecule containing 359 ribonucleotides. The properties of PSTV were compared with those of the scrapie agent, which causes a degenerative neurological disease in animals. PSTV was inactivated by ribonuclease digestion, psoralen photoadduct formation, Zn2+ -catalyzed hydrolysis, and chemical modification with NH2OH. The scrapie agent resisted inactivation by these procedures, which modify nucleic acids. The scrapie agent was inactivated by proteinase K and trypsin digestion, chemical modification with diethylpyrocarbonate, and by exposure to phenol, NaDodSO4, KSCN, or urea. PSTV resisted inactivation by these procedures, which modify proteins. Earlier evidence suggested that the scrapie agent is smaller than PSTV. Its small size seems to preclude the presence of a genome coding for the protein(s) of a putative capsid. The properties of the scrapie agent distinguish it from both viroids and viruses and have prompted the introduction of the term "prion" to denote a small proteinaceous infectious particle that resists inactivation by procedures that modify nucleic acids.
Isolation and identification of a biologically active peptide derived from the CH3 domain of human IgG1.
Authors: Morgan E L , Hugli T E , Weigle W O
Published: May 2006

A 24-amino acid residue peptide has been isolated from the Fc region of a human IgG1 myeloma protein. The peptide has associated with it the same ability to induce murine B cells to polyclonally secrete antibody as does the intact Fc fragment. Amino acid composition of the peptide indicates that on a mole/mole basis the isolated peptide is identical to that published for residues 335-358 in the Eu IgG1 sequence. This peptide corresponds roughly to the first 24 amino acids of the CH3 domain. It is believed that the immunoregulatory properties that have been ascribed to the Fc fragment are associated with this peptide.
Autoantibodies occurring in two different rheumatic diseases react with the same nuclear ribonucleoprotein particle.
Authors: Douvas A S
Published: May 2006

Patients affected with systemic lupus erythematosus and mixed connective tissue disease produce antibodies directed against two nuclear antigens, Sm and nuclear ribonucleoprotein (nRNP), respectively. The two antigens exhibit a relationship of partial identity in serologic assays, but the molecular basis of this relationship was not understood. This report describes the isolation of a nRNP particle containing both nRNP and Sm antigens. The particle was isolated by sucrose density gradient centrifugation of a rat liver nuclear extract followed by anti-nRNP affinity chromatography of a 14S gradient fraction. The protein moiety of the isolated particle consisted primarily of two polypeptides, P13 (Mr, 13,000) and P30 (Mr, 30,000). The immunoreactivity of P13 and P30 was demonstrated directly by transfer of these proteins from gels to nitrocellulose paper, followed by immunoautoradiography. Anti-nRNP sera reacted only with P30, whereas anti-Sm sera reacted with P13. Anti-nRNP sera were previously found to react with P13, but only in the presence of RNA [Douvas, A. S., Stumph, W. E., Reyes, P. R. & Tan, E. M. (1979) J. Biol. Chem. 254, 3608--3616]. From these observations it was concluded that P13 is the Sm antigen. The precipitating nRNP antigen is composed of P30--RNA complexes or P13--RNA complexes, with a RNA-independent reaction occurring with P30. The partial identity between Sm and nRNP antigens can be explained on the basis of a common reactivity to the P30--P13--RNA particle, with anti-Sm sera capable of binding additionally to RNA-free P13.
Metabolic fuel and amino acid transport into the brain in experimental diabetes mellitus.
Authors: McCall A L , Millington W R , Wurtman R J
Published: May 2006

We used the Oldendorf brain uptake index method to study the blood-brain barrier transport of several metabolic substrates in diabetes. Glucose transport into the brain was decreased by 1/3 in rats with moderate diabetes induced by prior injection of streptozotocin (65 mg/kg of body weight). The transports of mannose and the poorly metabolized hexoses 2-deoxyglucose and 3-O-methylglucose were similarly reduced. Likewise, brain glucose transport was decreased in rats with alloxan-induced diabetes. These alterations in brain hexose influx appeared to be related to chronic (1-2 days) hyperglycemia rather than to insulin-lack per se. Thus, starvation of the diabetic rats for 48 hr restored both the plasma glucose concentration and brain hexose transport to normal. Conversely, the substitution of 10% sucrose for their drinking water both increased plasma glucose and decreased hexose transport in insulin-treated diabetic rats. The 45% decrease in maximal glucose transport rate observed and the uniformity of diminished hexose transport probably imply a decrease in the number of available high-affinity transport carriers at the blood-brain barrier. This defect was specific for hexoses in that the transports of neutral and basic amino acids and of beta-hydroxybutyrate were not similarly affected. These results suggest that chronic hyperglycemia decreases the number of hexose carrier molecules available at the blood-brain barrier. Such an adaptation could operate to decrease the net flux of glucose into the brain during sustained hyperglycemia. It also may explain the abnormal sensitivity to abrupt blood glucose lowering in patients with diabetes mellitus.
Cell-specific differences in O6-alkylguanine DNA repair activity during continuous exposure to carcinogen.
Authors: Swenberg J A , Bedell M A , Billings K C , Umbenhauer D R , Pegg A E
Published: May 2006

The activity of the alkyl acceptor protein (AAP) responsible for repair of DNA containing the promutagenic lesion O6-alkylguanine was determined in hepatocytes and nonparenchymal cells (NPC) obtained from livers of control rats and rats exposed to hepatocarcinogens that primarily induce vascular or hepatocellular neoplasms. Basal levels of AAP activity were found to be 4-5 times higher in hepatocytes than in NPC. Exposure to 1,2-dimethylhydrazine or diethylnitrosamine produced a 2- to 3-fold enhancement of this activity in hepatocytes after exposure for as little as 3 days. The enhanced hepatocyte activity persisted throughout a 28-day exposure to 1,2-dimethylhydrazine. In contrast, AAP activity in NPC was decreased during the first week of exposure to 1,2-dimethylhydrazine and subsequently returned to control levels. No enhancement of AAP was apparent in the NPC. These and related data suggest that enhancement of this activity in rat hepatocytes is a response to cell proliferation. In contrast, the data clearly demonstrate that neither increased cell replication nor the presence of O6-alkylguanine was capable of enhancing AAP activity in NPC. Cellular differences in the repair of O6-alkylguanine appear to be a critical mechanism responsible for cell specificity in chemical carcinogenesis by alkylating agents.
Translocation of immunoglobulin VH genes in Burkitt lymphoma.
Authors: Erikson J , Finan J , Nowell P C , Croce C M
Published: May 2006

We have produced cell hybrids between mouse myeloma cells, which do not produce immunoglobulin chains, and Burkitt lymphoma cells (Daudi), which express surface IgM. Daudi Cells carry a reciprocal chromosome translocation between chromosomes 8 and 14, described as (8;14)(q24;q32). The hybrids were studied for the expression of human immunoglobulin chains and human isozyme markers, for the presence of human chromosomes, and for the presence of the human genes for heavy chain variable regions (VH) and mu and gamma chain constant (C) regions. The results indicate that the expressed mu chain gene is on normal chromosome 14 in Daudi cells. We have also determined that the chromosome 14 involved in the translocation (14q+) carries the gene for C mu and C gamma 1-4 and probably several genes for the variable region (V). Certain hybrids had lost both the chromosomes 14 but had retained the abnormal chromosome 8 (8q-) that carries the terminal end of the long arm of chromosome 14. These hybrids were studied for the presence of human VH, C mu,, and C gamma DNA sequences, and the results indicated that the hybrid cells with the 8q- chromosome contained VH genes that not C genes. Therefore, we conclude that, in the Daudi Burkitt lymphoma, the break in chromosome 14 occurred within the chromosome segment containing V region genes. As a result of the translocation some of these VH genes became associated with chromosome 8. It is possible that the expression of malignancy in Burkitt lymphoma is caused by immunoglobulin V region gene translocation resulting in activation of a gene on the long arm of human chromosome 8.
Two-fluid theory and thermodynamic properties of liquid mixtures: Application to simple mixtures of nonelectrolytes
Authors: Brandani V. , Prausnitz J. M.
Published: May 2006

In this last of three articles describing a two-fluid theory of liquid mixtures, the important result is a derivation of local compositions to describe nonrandomness (clustering) of mixing. The model is applied to the system methane/tetrafluoromethane. Although it appears that the two-fluid theory with local compositions may provide an improvement over random-mixing theories, it is likely that the use of local compositions overestimates the effect of nonrandomness.
Escherichia coli host factor required specifically for the phi X174 stage III reaction: in vitro identification and partial purification.
Authors: Wolfson R , Eisenberg S
Published: May 2006

A cell-free extract prepared from phi X174-infected Escherichia coli cells sustained in vitro synthesis of viral DNA (stage III reaction) when supplemented with fraction II from uninfected cells. The reaction was dependent upon deoxyribonucleoside triphosphate, ATP, added phi X174 replicative form I DNA template, and the fraction II from uninfected cells. This reaction differed from the stage II reaction (semiconservative replication of duplex replicative form DNA) by the production of stable viral protein-DNA complexes sensitive to anti-phi X174 antiserum. Three types of protein-DNA complexes were identified, 50S, 92S, and a 114S complex that cobanded in CsCl and cosedimented in neutral sucrose gradients with a phi X174 phage marker. The sensitivity of these complexes to anti-phi X174 antiserum and Staphylococcus aureus provided a relatively rapid biochemical assay for direct measurement of the amount of DNA synthesized by the stage III reaction. With this assay, an E. coli factor (SIII) required specifically for the synthesis of viral protein-DNA complexes was identified and purified 200-fold from uninfected E. coli cells. The partially purified SIII factor was required for the synthesis of DNA and viral protein-DNA complexes in the phi X174-infected cell extracts and could not be replaced by rep protein, single-strand binding protein, or DNA polymerase III holoenzyme.
Calmodulin stimulation of 45Ca2+ transport and protein phosphorylation in cholinergic synaptic vesicles.
Authors: Rephaeli A , Parsons S M
Published: May 2006

Cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica exhibit ATP-dependent uptake of 45Ca2+ that is stimulated by exogenous calmodulin. ATP-independent uptake also occurs, but it is only weakly stimulated by calmodulin. Saturating calmodulin decreased the Michaelis constant for ATP-dependent 45Ca2+ uptake from 52 +/- 0.4 to 12 +/- 0.2 microM and increased the maximal velocity from 3.4 +/- 0.3 to 5.2 +/- 0.5 nmol/mg of protein per min. The dose-response curve for calmodulin-dependent stimulation showed a maximal increase of 3.5-fold in the uptake rate; 0.2 microM calmodulin gave half-maximal stimulation. The activity of the vesicle-associated ATPase was unaffected. Incubation of vesicles with [gamma-32P]ATP and Ca2+ resulted in phosphorylation of four polypeptides of molecular weights about 64,000, 58,000, 54,000, and 41,000 when calmodulin was added. Vesicles that were previously phosphorylated and purified exhibited 2-fold enhanced ATP-independent uptake of 45Ca2+. Cyclic AMP could not substitute for calmodulin. The calcium transport system of the cholinergic synaptic vesicle is regulated by a calcicalmodulin-dependent protein kinase that is vesicle-associated.
Molecular cloning of mRNA sequences for cardiac alpha- and beta-form myosin heavy chains: expression in ventricles of normal, hypothyroid, and thyrotoxic rabbits.
Authors: Sinha A M , Umeda P K , Kavinsky C J , Rajamanickam C , Hsu H J , Jakovcic S , Rabinowitz M
Published: May 2006

We have isolated cDNA clones from thyrotoxic (pMHC alpha) and normal (pMHC beta) adult rabbit hearts. Restriction map analysis and DNA sequence analyses show that, although there is strong homology between overlapping regions of the two clones, they are distinctly different. The two clones exhibited 78-83% homology between the derived amino acid sequences and those determined by direct amino acid sequence analysis of rabbit fast skeletal muscle myosin heavy chains. The clones specify a segment of the myosin heavy chain corresponding to subfragment 2 and the COOH-terminal portions of subfragment 1. Nuclease S1 mapping was used to compare transcription of the two clones with expression of the alpha and beta forms of myosin heavy chains in the ventricles of thyrotoxic, hypothyroid (propylthiouracil-treated), and normal rabbits. Thyrotoxic ventricles contained only pMHC alpha transcripts whereas hypothyroid ventricles contained exclusively pMHC beta transcripts. These data correlate well with the presence of alpha- and beta-form myosin heavy chains. In the normal young adult rabbit, pMHC beta transcripts predominate, agreeing with the known beta form/alpha form ratio of 4:1. We therefore conclude that pMHC alpha and pMHC beta contain sequences of the alpha- and beta-form myosin heavy chain genes, respectively.
Structure of the mitochondrial F1 ATPase at 9-A resolution.
Authors: Amzel L M , McKinney M , Narayanan P , Pedersen P L
Published: May 2006

The soluble portion (F1 ATPase) of the mitochondrial ATP-synthesizing system is a multisubunit enzyme of molecular weight 380,000. It is composed of five different subunits, alpha, beta, gamma, and epsilon. The subunit stoichiometry is not known but there are strong suggestions that it is alpha 3 beta 3 gamma delta epsilon. We have determined the three-dimensional structure of the F1 ATPase of rat liver mitochondria to 9-A resolution by using x-ray diffraction techniques. The molecule appears to be formed by two equivalent halves, each formed by three regions of approximately equal size. These regions form a distorted hexagonal or octahedral arrangement. None of the regions form closed symmetrical trimers in the complex. It is proposed that, if the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, the major subunits exist in at least two different environments in the complex. In this arrangement, the different copies of the major subunits are functionally not equivalent. This observation appears to offer a natural explanation of the complicated binding and labeling data of F1 ATPases.
Repeat array in Epstein-Barr virus DNA is related to cell DNA sequences interspersed on human chromosomes.
Authors: Heller M , Henderson A , Kieff E
Published: May 2006

The third internal repeat (IR3) simple repeat array in Epstein-Barr virus (EBV) DNA has a high degree of homology to a reiterated component of cell DNAs. 32P-Labeled human or mouse DNAs hybridize to the IR3 sequence on Southern blots of viral DNA. EBV IR3 probe identifies many restriction enzyme fragments on Southern blots of human and mouse DNAs that have extensive homology to IR3. Cytological hybridization shows that IR3 is homologous to at least one region on each human chromosome except the Y chromosome.
Recent duplication and germ-line diversification of rat immunoglobulin kappa chain gene joining segments.
Authors: Burstein Y , Breiner A V , Brandt C R , Milcarek C , Sweet R W , Warszawski D , Ziv E , Schechter I
Published: May 2006

Sequence determination of the joining segment gene (J) cluster in the kappa chain (J kappa) in the embryonic context demonstrates that rat genome contains seven J kappa gene segments that expanded from an ancestral cluster of five J kappa genes. The rat J segments are separated by about 300 base pairs (bp) and are flanked 5' by the presumed variable region (V)/J recombination signal sequence and 3' by the RNA splicing signal. Two of the J gene segments designated J2A and J2B and their 5'-flanking spacer DNA bear striking homology to J2 and its 5'-flanking spacer. Thus, the unit of duplication was the entire J kappa coding region and 5' noncoding spacer (345 bp). The duplication probably occurred as two separate unequal crossing-over (UXO) events. The first UXO event can be confined to recombination within an identical stretch (14 bp long) located at the 3' ends of the coding regions of J1 and J2. The second event could involve a longer segment (372 bp) of tight homology generated by the first UXO event, thus increasing the probability of repeated expansion of the same DNA segment. The sequence homology among the rat duplicated segments (98-99%) is larger than the homology between the corresponding rat and mouse segments (89%), showing that the rat J kappa gene expansion must have occurred after rat and mouse divergence 10 X 10(6) yr ago. We estimate that the first and second UXO events occurred 2 X 10(6) and 1 X 10(6) yr ago, respectively. J3 of rat and mouse share the same mutation (G leads to C) in the RNA splicing signal that presumably inactivates J3. This mutation preceded divergence of the two species. A mutation in the first nucleotide of codon 96 has occurred in both duplicated segments, the only position along 345 bp where J2, J2A, and J2B differ from each other. This results in three different amino acids at position 96 not present in any other J kappa. These mutations are physiologically significant because they diversify the third complementarity-determining region (CDR3) and, thus, may reflect selective pressure to increase antibody diversity. The germ-line diversification of CDR3 was exercised within the last 1-2 X 10(6) yr.
Glanzmann thrombasthenia: deficient binding of von Willebrand factor to thrombin-stimulated platelets.
Authors: Ruggeri Z M , Bader R , de Marco L
Published: May 2006

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by the failure of platelets to aggregate in response to almost all stimuli. However, thrombasthenic platelets will aggregate with bovine and porcine von Willebrand factor (vWF) and will show normal ristocetin-induced binding and aggregation in the presence of human vWF. In contrast, we now report that the specific binding of vWF to the thrombin-stimulated platelets was less than 20% of normal in three patients with Glanzmann thrombasthenia. Analysis of binding isotherms was based on the assumption of one class of binding sites for vWF on the platelet membrane. Double-reciprocal plots were used to calculate maximal binding at saturation and apparent dissociation constant (Kd). In nine normals, 2.82 +/- 0.64 micrograms (+/- SD) of vWF bound to 10(8) platelets at saturation, with Kd (+/- SD) = 3.65 +/- 1.23 micrograms/ml. In two patients with thrombasthenia binding was markedly decreased and did not approach saturation. In the third patient, binding at saturation corresponded to 0.21 micrograms per 10(8) platelets, with Kd = 3.93 micrograms/ml. These findings suggest that mechanisms underlying the vWF-platelet interaction are incompletely reflected in ristocetin-dependent assay systems. Moreover, these results, in addition to those previously reported for fibronectin, suggest that the platelet defect in Glanzmann thrombasthenia is not limited to decreased binding of fibrinogen but involves several glycoproteins that are known to interact with platelets.
A single domain of human prostatic acid phosphatase shows antibody-mediated restoration of catalytic activity.
Authors: Choe B K , Dong M K , Walz D , Gleason S , Rose N R
Published: May 2006

By limited proteolysis with mouse submaxillaris protease, human prostatic acid phosphatase (EC 3.1.3.2) was cleaved into three fragments, Sp1, Sp2, and Sp3, which individually had no enzymatic activity. One of the fragments, Sp3, regained enzymatic activity after interaction with rabbit antibody to prostatic acid phosphatase. The Sp3 fragment was purified and characterized as to its molecular weight, amino acid composition, and carbohydrate content. The Sp3 fragment behaved like the parent molecule in L(+)-tartrate affinity and in trapping of a phosphoryl intermediate. The same Sp3 fragment also bears the most prominent antigenic determinants. This evidence suggest that Sp3 is the enzymatically active domain of prostatic acid phosphatase.
Regulation of phosphorylation of proteins I, IIIa, and IIIb in rat neurohypophysis in vitro by electrical stimulation and by neuroactive agents.
Authors: Tsou K , Greengard P
Published: May 2006

The state of phosphorylation of proteins I, IIIa, and IIIb--neuron-specific phosphoproteins--was studied in neurosecretory endings of the neurohypophysis in vitro. Brief periods (a few seconds) of electrical stimulation caused large increases in the state of phosphorylation of all three proteins. The three proteins were dephosphorylated within 1 min after termination of the stimulation. High potassium, 8-bromo-cAMP, and dopamine also stimulated the phosphorylation of the three proteins. The effect of dopamine was blocked by the dopamine antagonist fluphenazine. Peptide mapping of protein I revealed that electrical stimulation or high potassium increased the state of phosphorylation of two regions of the molecule, whereas 8-bromo-cAMP and dopamine increased the state of phosphorylation of only one of these regions.
Sprouting from chicken embryo dorsal root ganglia induced by nerve growth factor is specifically inhibited by affinity-purified antiganglioside antibodies.
Authors: Schwartz M , Spirman N
Published: May 2006

The involvement of gangliosides in processes related to nerve regeneration and sprouting has been demonstrated recently. The type of interaction by which gangliosides may influence neuronal sprouting was investigated in the present work. Affinity-purified rabbit anti-GM1 antibodies were found to block the sprouting from dorsal root ganglia (DRG) of chicken embryo induced by nerve growth factor (NGF). Only a moderately inhibitory effect was produced by antibodies directed to GM2, suggesting a specific interaction between NGF and GM1. In spite of the low inhibitory effect of anti-GM2 antibodies on sprouting, their binding to dissociated DRG cells was found to be higher than that of anti-GM1 antibodies. These results emphasize the high resolution of the system and suggest that the interaction of NGF with GM1 is involved in the process of sprouting.
Isolation, characterization, and purification to homogeneity of a rat brain protein (GABA-modulin).
Authors: Guidotti A , Konkel D R , Ebstein B , Corda M G , Wise B C , Krutzsch H , Meek J L , Costa E
Published: May 2006

gamma-Aminobutyric acid (GABA)-modulin is a brain neuropeptide that appears to modulate specific high-affinity (20 nM) GABA recognition sites in brain. When added to crude synaptic membranes this peptide inhibits binding of [3H]GABA to the high-affinity site and prevents facilitation of [3H]diazepam binding elicited by GABA. GABA-modulin has been purified to homogeneity by ammonium sulfate precipitation, gel chromatography, and reverse-phase HPLC. Homogeneity was confirmed by a variety of means, including chromatography under four different HPLC conditions, two different polyacrylamide gel electrophoreses, and end group analysis. Purified GABA-modulin contains approximately 126 amino acids and has a molecular weight of 16,500. The GABA-modulin molecule contains an abundance of hydrophilic basic residues, and neither cysteine nor GABA is present. End group analyses of GABA-modulin showed that histidine is the free COOH terminus and the NH2 terminus is blocked. GABA-modulin specifically blocked both [3H]GABA binding to synaptic membranes (IC50, 0.5 microM) and GABA-stimulated [3H]diazepam binding; the binding of [3H]GABA to low-affinity sites was not affected.
Thalamic inputs to cytochrome oxidase-rich regions in monkey visual cortex.
Authors: Livingstone M S , Hubel D H
Published: May 2006

In primate primary visual cortex, staining for cytochrome oxidase reveals a regular array of blob-like structures, most prominent in layers II and III but also present in layers V and VI. In an attempt to learn more about the input to these blobs, we injected the lateral geniculate bodies of macaques and squirrel monkeys with [3H]proline or horseradish peroxidase and looked in the cortex for transported label. As expected, label was present in layers IVa, IVc alpha, IVc beta, and VI. In addition, both methods revealed an array of puffs deep in layer III. Seen in tangential sections, the puffs precisely matched the cytochrome blobs. These results indicate a projection from the lateral geniculate body to the blob regions deep in layer II/III, either indirect via layer IV or more likely direct. In area 18 stained for cytochrome oxidase, we also observed complex banding patterns; these were remarkably similar to the pattern found after [3H]proline or horseradish peroxidase injection and were also similar to the pattern produced with 2-deoxyglucose labeling after stimulation with vertical or horizontal stripes; the proline and peroxidase labels probably represent a projection from the pulvinar to area 18.
DNA between variable and joining gene segments of immunoglobulin kappa light chain is frequently retained in cells that rearrange the kappa locus.
Authors: Van Ness B G , Coleclough C , Perry R P , Weigert M
Published: May 2006

A systematic analysis of the fate of the DNA between kappa chain variable (V kappa) and joining (J kappa) genes in cells that have rearranged kappa loci was carried out. The DNA from a variety of kappa-producing plasmacytomas, lambda-producing hybridomas, and kappa-expressing lymphocytes was digested, fractionated by size, and analyzed with two probes containing sequences 5' of J kappa. In 13 of 28 plasmacytomas examined the rearrangement of V kappa and J kappa appears to be accompanied by loss of DNA upstream of J kappa. However, in the rest of the plasmacytomas one or more upstream sequences are retained in a new context. In 9 of 12 lambda-producing hybridomas (which frequently rearrange both kappa loci) one or more upstream segments were detected. These unique fragments were probably generated by a recombination event near or at the J kappa region. The extent to which the region between V and J is maintained in kappa-expression lymphocytes was also measured. Most (76%) of the region upstream of J kappa is retained in the population, even though 68% of the kappa loci are rearranged. In order to explain how these upstream elements occur in some, but not all, cell lines, and the significant occurrence in the lymphocyte population, we propose a model in which a step in V--J joining involves mitotic recombination by unequal sister chromatid exchange.
Purification of a lymphoid cell line product with leukocyte inhibitory factor activity.
Authors: Meshulam D H , Blair H E , Wong B L , Charm S , Minowada J , Rocklin R E
Published: May 2006

Leukocyte inhibitory factor (LIF), a lymphokine that inhibits the random and directed migration of polymorphonuclear (PMN) leukocytes, was purified from a human non-T, non-B leukemia cell line (Reh). From 10 liters of serum-free supernatant, 1.3 microgram of protein with LIF activity was obtained by the sequential use of affinity chromatography with concanavalin A-Sepharose, hydrophobic chromatography with hexylagarose, and gel filtration chromatography. The specific activity of LIF recovered represented an 80,000-fold purification over that of the initial crude serum-free supernatants, and the preparation at that point was estimated to be 80--90% pure. To both assess the purity of the preparation and provide a further purification step, Reh LIF activity recovered by the above procedures was subjected to isoelectric focusing. One major stainable protein band was identified; its isoelectric point was pH 5.4--5.5. Gels run in parallel for recovery of biologic activity revealed only one region (pH 5.4--5.5) with ability to inhibit PMN leukocyte migration. Iodination of Reh LIF resulted in a loss of biologic activity, but isoelectric focusing of this material revealed one major 125I-labeled band (pH 5.1) and several minor bands. The coincidence of biologic LIF activity with one stainable protein band as identified by isoelectric focusing implies that the final product may be homogeneous.
Allelic exclusion of immunoglobulin expression is not caused by somatic segregation
Authors: Wabl Matthias R. , Tenkhoff Maria
Published: May 2006

We have investigated the karyotype of immunoglobulin-producing cells in heterozygous animals. Using a karyotypic marker for one homolog of a chromosome carrying immunoglobulin genes, we established that immunoglobulin-producing cells are heterozygous with respect to this chromosome. Therefore, allelic exclusion of immunoglobulin expression cannot be caused by somatic chromosome segregation.
A covalent linkage between daunorubicin and proteins that is stable in serum and reversible by lysosomal hydrolases, as required for a lysosomotropic drug-carrier conjugate: in vitro and in vivo studies.
Authors: Trouet A , Masquelier M , Baurain R , Deprez-De Campeneere D
Published: May 2006

Daunorubicin (DNR) has been conjugated to succinylated serum albumin by an amide bond joining the amino group of the drug and a carboxyl side chain of the protein either directly or with the intercalation of a peptide spacer arm varying from one to four amino acids. During in vitro incubation with lysosomal hydrolases, intact DNR could be released extensively only from conjugates prepared with a tri- or tetrapeptide spacer arm. These latter conjugates remained very stable in the presence of serum. When tested in vivo against the intraperitoneal form of L1210 leukemia, the conjugates in which DNR was linked to serum albumin directly or via one amino acid were completely inactive but the conjugate with a dipeptide spacer arm was not more active than free DNR. In parallel with the in vitro studies, the best therapeutic results were obtained with the conjugates formed with tri- and tetrapeptidic spacer arms; they were much more active than DNR, inducing a high percentage of long-term survivors. Thus, use of a tri- or tetrapeptide spacer arm is essential to obtain DNR-protein conjugates that remain stable in serum and from which DNR can be released through the action of lysosomal hydrolases. The in vivo results suggest, moreover, that these conjugates are endocytosed by L1210 cells and that DNR is released intracellularly after digestion by lysosomal enzymes. This conjugation method can be applied to other drugs possessing a free amino group and to various potential carriers, such as antibodies, polypeptide hormones, and glycoproteins, that have amino or carboxyl side chains.