bluuebunny/crossref_metadata_2025 huggingface dataset:
Base-pair substitution hotspots in GAG and GCG nucleotide sequences in Escherichia coli K-12 induced by cis-diamminedichloroplatinum (II).
Authors: Brouwer J , van de Putte P , Fichtinger-Schepman A M , Reedijk J
Published: May 2006

Cell killing and mutation induction by cis- and trans-Pt(NH3)2Cl2 in Escherichia coli were examined by studying forward mutagenesis in the lacI gene in cells with different repair capacities. Survival experiments showed that repair-proficient cells were slightly more sensitive for the cis isomer than for the trans isomer, whereas repair-deficient RecA and UvrB cells were extremely sensitive only for the cis compound. cis-Pt(NH3)2Cl2 induced mutagenesis in both wild-type cells and RecA cells but not in UvrB cells; whereas no detectable mutagenesis was induced by treatment with the trans compound. Examination of the nature of the mutations induced by cis-Pt(NH3)2Cl2, by using the LacI system, revealed that base-pair substitutions leading to nonsense mutants are only induced in wild-type cells, suggesting that the intact products of both the uvrB and the recA gene are necessary for the repair responsible for this type of mutagenesis. Investigation of the nonsense mutants reveals that 70% of these mutations result from GC leads to TA or GC leads to AT substitutions at sites where the guanine is part of a GAG or GCG sequence. These results are discussed in relation to existing theories on the interaction between Pt compounds and DNA. A model for Pt--DNA adducts, leading to base-pair substitutions, is proposed.
Alternate pathways of DNA replication: DNA polymerase I-dependent replication.
Authors: Niwa O , Bryan S K , Moses R E
Published: May 2006

We have previously shown that some Escherichia coli [derivatives of strain HS432 (polA1, polB100, polC1026)] can replicate DNA at a restrictive temperature in the presence of a polCts mutation and that such revertants contain apparent DNA polymerase I activity. We demonstrate here that this strain of E. coli becomes temperature-resistant upon the introduction of a normal gene for DNA polymerase I or suppression of the polA1 nonsense mutation. Such temperature-resistant phenocopies become temperature-sensitive upon introduction of a temperature-sensitive DNA polymerase I gene. Our results confirm that DNA replication is DNA polymerase I-dependent in the temperature-resistant revertants, indicating that an alternative pathway of replication exists in E. coli. HS432 contains a transducible locus (which we term pcbA) that can support an alternate pathway in other E. coli strains, so the effect of suppression of polCts is a general one.
A common mutant EcoRI restriction endonuclease site in the 5' flanking portion of the human alpha-globin gene.
Authors: Beutler E , Kuhl W , Johnson C
Published: May 2006

A mutant EcoRI endonuclease restriction site has been identified in 3 of 37 Black subjects and in 2 sibs of one of these persons. This mutation was not encountered in 13 Whites. It is located approximately 6 kilobases "inside" the normal site in the 5' flanking sequence of the alpha-globin chain complex. The shortened alpha-globin gene-bearing segment produced in the EcoRI digest produces a restriction map similar to that observed for the common alpha-globin gene deletion observed in the Black population. However, the restriction map with BamHI is normal, confirming that all four alpha loci are present.
Cellular immunity of mice to Leishmania donovani in vitro: lymphokine-mediated killing of intracellular parasites in macrophages.
Authors: Chang K P , Chiao J W
Published: May 2006

Leishmania donovani, an intracellular protozoan, causes kala-azar by parasitizing the macrophages of its mammalian host. Outbred NCS and CD-1 mice develop immunity to this parasite. This immunity was demonstrable when supernatant fluids from cultured splenic lymphocytes were added to infected macrophages. Only the lymphokine preparations from infected mice showed significant leishmanicidal activity. Mice receiving multiple inocula were more potent producers of leishmanicidal lymphokines than were those receiving single inocula. The expression of leishmanicidal activity in our system required continuous presence of the lymphokine preparation and was independent of trypsin- or neuraminidase-sensitive receptors of the macrophages. Light and electron microscopy revealed that, in the presence of lymphokines, macrophages appeared to be "activated," and intracellular leishmanias developed specific subcellular lesions in the kinetoplast-mitochondria. A time-course study showed that cultivation of the lymphocytes for 1 1/2 days completed the release of their leishmanicidal lymphokines which were heat-labile molecules larger than 50,000 daltons.
Two alpha heavy chain disease proteins with different genomic deletions demonstrate that nonexpressed alpha heavy chain genes contain methylated bases.
Authors: Dackowski W , Morrison S L
Published: May 2006

Two independently arising alpha heavy chain mutants have been found to synthesize heavy chains with CH1 deletions of approximately equal extent. Both were isolated from heavy chain-producing variants of the mouse myeloma W3129 and demonstrate that it is possible to arrive at the heavy chain disease phenotype by the pathway H + L leads to H leads to delta H. Analysis of genomic DNA by digestion with restriction endonucleases followed by molecular hybridization showed that one mutant (delta 37) had a deletion of approximately 0.2 kilobase and the second mutant (delta 15) had a deletion of approximately 0.5 kilobase. Mouse myeloma cells contain several alpha chain alleles but only one is expressed; the presence of the deletion in delta 37 and delta 15 made it possible to identify the restriction fragments from the expressed allele. Analysis of the fragments produced after cleavage with an isoschizomeric pair of restriction enzymes, Msp I and Hpa II, indicated that, in the W3129 cell line and its variants, the unexpressed alpha alleles contain methylated bases. The influence of methylation on gene expression remains to be elucidated.
Na and K dependence of the Na/K pump in cystic fibrosis fibroblasts.
Authors: Reznik V M , Schneider J A , Mendoza S A
Published: May 2006

The Na and K dependence of the Na/K pump was measured in skin fibroblasts from patients with cystic fibrosis and age/sex-matched controls. Under basal conditions, there was no difference between control and cystic fibrosis cells in protein per cell, intracellular Na and K content, or Na/K pump activity (measured as ouabain-sensitive 86Rb uptake). There was no difference in the Na dependence of the Na/K pump between cystic fibrosis cells and control cells. In cells from patients with cystic fibrosis, the Na/K pump had a significantly lower affinity for K (Km = 1.6 mM) when compared to normals (Km = 0.9 mM). This difference was demonstrated by using two independent experimental designs.
Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.
Authors: Hendy G N , Kronenberg H M , Potts J T , Rich A
Published: May 2006

We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of double-stranded DNA into the Pst I site of plasmid pBR322 using the poly(dG) . poly(dC) homopolymer extension technique. Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA. Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' noncoding region, the entire coding region of 345 nucleotides, and the entire 3' noncoding region of 348 nucleotides of the mRNA. The coding sequence predicts the previously unknown "pre" amino acid sequence and clarifies the hormone's amino acid sequence, which has been disrupted. The 5' noncoding region contains an AUG codon followed by a UGA stop codon before the authentic initiator codon. The 3' noncoding region is 120 nucleotides longer than in bovine preproparathyroid mRNA and contains two A-A-U-A-A-A sequences, potential signals for polyadenylation.
Amino acid sequence of the protein inhibitor of mitochondrial adenosine triphosphatase.
Authors: Frangione B , Rosenwasser E , Penefsky H S , Pullman M E
Published: May 2006

The complete amino acid sequence of the mitochondrial ATPase inhibitor peptide was determined. The molecule contains 84 residues of which 40 are charged amino acids that occur in clusters along the chain. A section of the chain, located at the COOH-terminal end, contains several duplicated regions, the most prominent of which are pentapeptides. This section of the chain also contains all of the five histidines present in the molecule. Some of the physicochemical properties of the protein and an improved purification procedure are described.
Prolactin-like activity of anti-prolactin receptor antibodies on casein and DNA synthesis in the mammary gland.
Authors: Djiane J , Houdebine L M , Kelly P A
Published: May 2006

Prolactin receptors were partially purified from rabbit mammary gland membranes by using an affinity chromatography technique. Antibodies against this prolactin receptor preparation were obtained in guinea pig and sheep. Both antisera were able to inhibit the binding of 125I-labeled ovine prolactin to rabbit mammary gland membranes. When added to culture media of rabbit mammary explants, the anti-prolactin receptor antiserum inhibited the capacity of prolactin to initiate casein synthesis and casein mRNA accumulation as a function of the antiserum concentration. However, in the absence of prolactin, both antisera (guinea pig and sheep) at moderate concentrations were capable of mimicking prolactin action on casein gene expression and on DNA synthesis. At higher concentrations, the anti-prolactin receptor antibodies inhibited their own actions. Several characteristics of the prolactin effect were also observed with the anti-prolactin receptor antibody: the stimulatory effect of the antibody was amplified by glucocorticoids; colchicine, which was capable of blocking prolactin action, also prevented the induction by the antibody. Lysosomotropic agents, which do not interfere with prolactin action, did not alter the response observed with the antibody. These results indicate that an anti-prolactin receptor antibody can mimic two major actions of prolactin obtained in mammary explant culture and suggests that the prolactin molecule is not required beyond the initial binding to its receptor.
Primary step in the bacteriorhodopsin photocycle: photochemistry or excitation transfer?
Authors: El-Sayed M A , Karvaly B , Fukumoto J M
Published: May 2006

The absorption polarization of the first intermediate (K610) formed at room temperature in the proton-pumping photochemical cycle of bacteriorhodopsin (bR) shows a strong correlation with the polarization direction of the photolyzed parent molecule. The results suggest that, unlike other photosynthetic systems, excitation transfer does not take place prior to the primary photochemical change in bR. These observations together with the previously observed circular dichroism and the polarization temperature dependence are discussed in terms of the exciton structure and the nature of the absorption bandwidths (i.e., homogeneous vs. inhomogeneous) of the bR monomers within the trimer structure.
High-frequency cotransfer of the transformed phenotype and a tumor-specific transplantation antigen by DNA from the 3-methylcholanthrene-induced Meth A sarcoma of BALB/c mice.
Authors: Hopkins N , Besmer P , DeLeo A B , Law L W
Published: May 2006

We transformed BALB/3T3 mouse cells with cellular DNA extracted from the Meth A sarcoma, a 3-methylcholanthrene-induced tumor of BALB/c mice, and asked whether foci arising in the transfection possess the previously defined Meth A tumor-specific transplantation antigen (TSTA). Five of eight foci selected from one experiment possessed Meth A TSTA. DNA extracted from one of the five TSTA-positive clones was used in secondary rounds of transfection transformation. Four out of five foci tested from the secondary transfections possessed Meth A TSTA. These results suggest that in the Meth A sarcoma a transforming gene and a genetic determinant of TSTA are intimately related: they may be identical or very closely linked; alternatively, a particular transforming gene might induce the expression of a particular TSTA. Another possible explanation for these results is that the cotransfer of certain cellular genes by DNA transfection is considerably higher than predicted from the limited studies presently available.
Structural relationship and posttranslational modification of stage-specific proteins synthesized during early preimplantation development in the mouse.
Authors: Van Blerkom J
Published: May 2006

The synthesis of stage-related proteins characteristic of meiotic maturation and early preimplantation development occurs in the absence of significant transcription. Previous work indicated that some of the stage-related proteins typical of the early postfertilization period are synthesized in unfertilized oocytes at the same time that they are detected in fertilized eggs. This observation has led to the suggestion that protein synthesis in newly fertilized eggs is regulated by an intrinsic developmental program initiated during the resumption of meiosis (meiotic maturation) and supported in part by previously untranslated mRNA. It also has been proposed that the rapid and complex changes in protein synthesis that characterize this period may involve differential gene expression or selective protein degradation, or both. To date, cell-free translation of oocyte RNA has not demonstrated the existence of a sizeable population of preformed mRNA that could support the observed changes in protein synthesis. I have tested the notion that the apparent changes in protein synthesis during early development in the mouse may be derived from families of proteins related both in amino acid sequence and posttranslational modification. The findings show that many changes in protein synthetic patterns related to early development after fertilization are independent of fertilization and involve the posttranslational modification of proteins with identical or very similar primary structures. The results are discussed with respect to current interpretations of quantitative and qualitative changes in protein synthesis during early mammalian development as they relate to differential gene expression and presumed activation of preformed mRNA.
Proline and glucose transport by renal membranes from dogs with spontaneous idiopathic Fanconi syndrome.
Authors: Medow M S , Reynolds R , Bovee K C , Segal S
Published: May 2006

The uptake of proline and glucose by renal brush border membrane vesicles isolated from Basenji dogs exhibiting a spontaneous Fanconi syndrome was examined. The magnitude of the 1-min, Na+-gradient uptake of proline (0.02 mM) and glucose (1.0 mM) by vesicles from affected dog kidney was lower than normal. Concentration-dependent 15-sec uptake in vesicles from normal and affected dogs reveals a two-component transport system for proline and glucose. Kinetic analysis shows altered Km and Vmax values for both proline and glucose transport in the affected dogs. The data suggest that, in this model of Fanconi syndrome, the defective renal reabsorption of proline and glucose is associated with an alteration of luminal transport system.
Interaction between auditory and motor activities in an avian song control nucleus.
Authors: McCasland J S , Konishi M
Published: May 2006

Discrete telencephalic nuclei HVc (hyperstriatum ventrale, pars caudale) and RA (nucleus robustus archistriatalis) have been implicated by lesion studies in the control of vocalization in songbirds. We demonstrate directly the role of HVc in vocalization by presenting neuronal recordings taken from HVc of singing birds. Intracellular recordings from anesthetized birds have shown that many neurons in HVc respond to auditory stimuli. We confirm this result in the extracellular recordings from awake-behaving birds and further demonstrate responses of HVc neurons to playback of the bird's own song. The functional significance of these responses is not yet clear, but behavioral studies show that auditory feedback plays a crucial role in the development of normal song. We show that the song-correlated temporal pattern of neural activity persists even in the deaf bird. Furthermore, we show that in the normal bird, the activity pattern correlated with production of certain song elements can be clearly distinguished from the pattern of auditory responses to the same song elements. This result implies that an interaction occurs in HVc of the singing bird between motor and auditory activity. Through experiments involving playback of sound while the bird is singing, we show that the interaction consists of motor inhibition of auditory activity in HVc and that this inhibition decays slowly over a period of seconds after the song terminates.
Transfer of synaptic vesicle antigens to the presynaptic plasma membrane during exocytosis.
Authors: von Wedel R J , Carlson S S , Kelly R B
Published: May 2006

We have utilized immunofluorescence techniques to look for synaptic vesicle antigens on the plasma membrane of resting and active nerve terminals. Rabbit antiserum was raised against purified cholinergic synaptic vesicles from the electric organ of Narcine brasiliensis, a marine electric ray. Antibodies to synaptic vesicles were shown to bind selectively to nerve terminals in cryostat sections of frog nerve-muscle preparations. Binding was demonstrated indirectly by using fluorescein-labeled goat anti-rabbit antibodies. Structures in cross sections that bound antiserum were identified as nerve terminals because of their size, shape, and position and because they coincided with sites that bound rhodamine-conjugated alpha-bungarotoxin and had acetylcholine esterase activity. Presumably, sectioning gave antibodies access to binding sites within the nerve terminal. However, when antibodies to synaptic vesicles were added to the bathing medium of intact neuromuscular preparations prior to sectioning, antibody binding was marginal or undetectable, suggesting that few vesicle antigens were normally accessible on the outer surface of resting nerve terminals. When intact preparations were stimulated to release their vesicular acetylcholine by the addition of 1 mM LaCl3, antibody binding to the intact nerve terminals became striking. These findings suggest that the synaptic vesicle membrane and the synaptic terminal plasma membrane differ in composition. They also provide further support for the exocytotic hypothesis of neurotransmitter release, which predicts that vesicle markers should be exposed on the outside of nerve terminals when vesicles fuse with the plasma membrane during stimulation.
alpha-Globulin sequences are located in a region of early-replicating DNA in murine erythroleukemia cells.
Authors: Furst A , Brown E H , Braunstein J D , Schildkraut C L
Published: May 2006

The time of replication in the S phase of regions of the mouse genome including the alpha-globin genes was determined in the murine erythroleukemia cell line transformed by Friend virus. Cells grown for short times in the presence of BrdUrd were fractionated into synchronous populations by centrifugal elutriation. The DNA was cleaved by restriction endonucleases, and fragments containing bromouracil (BrU-DNA) were isolated in density gradients of Cs2SO4. BrU-DNA fractions replicated during selected S-phase intervals were subjected to electrophoresis in agarose gels, transferred to diazobenzyloxymethyl-paper, and hybridized to an alpha-globin probe. Reconstruction experiments using a cloned mouse EcoRI fragment including one alpha-globin gene demonstrated that the extent of hybridization provides an accurate measurement that the extent of hybridization provides an accurate measurement of the concentrations of specific fragments in a DNA sample. The alpha-globin fragments were detected primarily in the BrU-DNA replicated during early S phase (approximately the first quarter of S). This result was confirmed in other synchrony experiments and by Cot analysis. (Cot is the initial concentration of DNA in mol of nucleotide per liter multiplied by the time in sec.) The temporal replication of mouse satellite sequences, already known from previous studies, was used as an internal control for cell synchrony. To show that the globin sequences were not lost from the cells in late S phase during isolation of the DNA, we quantitated th alpha-globin fragments in BrU-DNA prepared from a mixture of cells in early and late S phase. The results demonstrate that the alpha-globin gene regions in these cells are replicated during early S phase.
Immunohistological demonstration of respiratory syncytial virus antigens in Paget disease of bone.
Authors: Mills B G , Singer F R , Weiner L P , Holst P A
Published: May 2006

Respiratory syncytial virus antisera have been found to produce a positive immunohistologic response in osteoclasts in bone sections or in cells cultured from Paget disease lesions in 12 out of 12 patients tested. These experiments were carefully controlled by several means. Use of experimentally infected cells served as positive controls. Adsorption of antisera on human bone powder and KB cells did not remove the specific immunologic stain, but adsorption of the antisera by the virus did. Negative results were also obtained in osteoclasts of patients with primary or secondary hyperparathyroidism. In addition, negative results in specimens of Paget disease were found with antisera to measles; parainfluenza 1, 2, and 3; influenza A, B and C; rubella; and herpes simplex. These results are consistent with the hypothesis that the nuclear and cytoplasmic inclusions in the osteoclasts of Paget disease are a result of viral activity.
A multilocus estimator of mating system parameters in plant populations
Authors: Shaw D. V. , Kahler A. L. , Allard R. W.
Published: May 2006

A multilocus method of estimating mating system parameters in populations is presented that recovers information from classification over multiple loci that single-locus estimators do not detect. It is shown that the multilocus estimator provides a standard of reference (null hypothesis) that can be useful in analyzing the effects of factors such as population heterogeneity and post-mating zygotic selection on the transmission of genetic information at the population level.
Extended x-ray absorption fine structure of copper in cytochrome
c
oxidase: Direct evidence for copper—sulfur ligation
Authors: Scott Robert A. , Cramer Stephen P. , Shaw Robert W. , Beinert Helmut , Gray Harry B.
Published: May 2006

The copper x-ray fluorescence excitation spectrum of cytochrome
c
oxidase (ferrocytochrome
c
:oxygen oxidoreductase, EC 1.9.3.1) has been recorded in the 245—270 K range. The beat pattern observed in the extended x-ray absorption fine structure can be accounted for only by postulating a combination of sulfur and nitrogen (or oxygen) ligands to the copper. The average Cu—S distance is 2.27 ± 0.02 Å and the average Cu—N (or Cu—O) distance is 1.97 ± 0.02 Å. The amplitudes require
ca
, 1-1.5 sulfurs and 2 nitrogens (or oxygens) per copper. The distribution of sulfur ligands between Cu
A
and Cu
B
sites is not known, although there is some evidence that two sulfur atoms are bound to Cu
A
.
Synthesis of [alpha-methyltyrosine-4]angiotensin II: studies of its conformation, pressor activity, and mode of enzymatic degradation.
Authors: Khosla M C , Stachowiak K , Smeby R R , Bumpus F M , Piriou F , Lintner K , Fermandjian S
Published: May 2006

Modifications in angiotensin II and its antagonistic peptides that should have increased in vivo half-lives but not reduced biological activity were studied by determining the effect of alpha-methylation of the tyrosine in position 4. [alpha-Methyltyrosine-4]angiotensin II, synthesized by the solid-phase procedure, showed 92.6 +/- 5.3% pressor activity of angiotensin II. Incubation with alpha-chymotrypsin for 1 hr indicated absence of degradation although, under the same conditions, angiotensin II was completely degraded to two components. Comparison of the 1H NMR spectra in aqueous solution and the circular dichroism spectra in trifluoroethanol of angiotensin II and [alpha-methyltyrosine-4]angiotensin II suggested that alpha methylation of the tyrosine residue in angiotensin II does not lead to major changes in the overall solution conformation. These results are in contrast to those obtained with N-methylation in position 4, which drastically reduced the biological activity and produced remarkable changes in the peptide backbone and a severe limitation in rotational freedom of the side chains in tyrosine. Thus, it may be possible to synthesize potent angiotensin II analogs that have greater resistance to enzymatic degradation by alpha-methylation in position 4 (or 5) and simultaneous suitable modification at the NH2 and COOH termini.
Complete amino acid sequence of the Fc region of a human delta chain.
Authors: Shinoda T , Takahashi N , Takayasu T , Okuyama T , Shimizu A
Published: May 2006

The complete amino acid sequence of an Fc-like fragment designated Fc delta (t) and obtained by limited proteolysis with trypsin of an intact myeloma IgD protein (NIG-65) has been determined. The fragment contains 226 amino acid residues and has a molecular weight of 32,000 per monomeric unit. It has three glucosamine oligosaccharides at asparagine residues 68, 159, and 210. Of these, glucosamine-159 is characteristic of the delta chain and has no counterpart position in any of the other classes. On the other hand, glucosamine-68 is shared by gamma, mu, and epsilon, and glucosamine-210 is shared by alpha and mu. Although the Fc delta (t) has the common framework structure of immunoglobulins, its sequence has many individual characteristics when its two domains are compared separately with the counterpart domain of other heavy chains. Such comparison has shown that the two Fc domains of the delta chain should be placed in an independent branch in topology; for all the other classes, the Fc domains are paired well with their counterparts. The comparison has also shown that there are three prominent gaps by which each domain can be divided into two homologous halves. For each class of immunoglobulin, a moderate degree of internal homology exists between the first half and the second half of each domain of the Fc, suggesting that the primordial gene may have coded for a unit about the size of a half domain. Based on this observation together with sequence comparisons, a possible genetic mechanism is proposed for the origin and evolution of the genes for immunoglobulin domains.
Complete amino acid sequence of a 50,000-dalton fragment of human ceruloplasmin.
Authors: Dwulet F E , Putnam F W
Published: May 2006

The complete amino acid sequence has been determined for a 50,000-dalton fragment that is an internal segment of the single polypeptide chain of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F4) contains 405 amino acid residues, has one glucosamine-containing carbohydrate unit, and, together with the 19,000-dalton fragment that follows it, accounts for the carboxyl-terminal half of the molecule. Fragment Cp F4 has a very nonuniform distribution of certain amino acid residues, which show a high potential to be adjacent to or one residue separated from a similar amino acid. This is most pronounced for acid and amide residues (65% clustered), aromatic residues (56% clustered), and basic residues (41% clustered). In addition, there is a long-range clustering of proline residues at the amino- and carboxyl-terminal 60 residues (50% clustered). Also, there are a number of short repeated segments of sequence. Calculations based on parameters predictive of secondary structure folding patterns indicate that the 50,000-dalton fragment has a low content of alpha-helix and is predominantly beta-sheet, beta-turn, and random in structure. Limited enzymatic cleavage of human ceruloplasmin to yield 67,000-, 50,000-, and 19,000-dalton fragments occurs at specific exposed sites of random structure in between domain-like regions.
Isolation and characterization of a cloned DNA sequence associated with the murine
Ah
locus and a 3-methylcholanthrene-induced form of cytochrome
P
-450
Authors: Negishi Masahiko , Swan David C. , Enquist Lynn W. , Nebert Daniel W.
Published: May 2006

Using partially purified mouse liver 23S mRNA known to be associated with the
Ah
locus and 3-methylcholanthrene-induced cytochrome
P
1
-450, we synthesized double-stranded cDNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) and the Klenow A fragment of
Escherichia coli
DNA polymerase I. The double-stranded cDNA was inserted into pBR322 plasmid DNA by
Pst
I cleavage and homopolymeric “tailing” and cloned in
E. coli
LE392. Clone 46 hybridized with [
32
P]cDNA made from 23S mRNA from “
Ah
-responsive” C57BL/6N mice but did not hybridize with similarly prepared [
32
P]cDNA from “
Ah
-nonresponsive” DBA/2N mice. Clone 30 was positive, and clone 7 was negative, with both C57BL/6N and DBA/2N [
32
P]cDNA probes; these two clones were therefore used as “positive” and “negative” control clones, respectively. By translation-arrest experiments, clone 46 DNA and clone 30 DNA were shown to be associated with anti-
P
1
-450- and anti-albumin-precipitable material, respectively. By agarose gel electrophoresis of
Pst
I digests, the clone 46 DNA insert was shown to be 1100 base pairs in total length and to contain one internal
Pst
I site. The cDNA made from total mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mice hybridized to the two fragments of
Pst
I-digested DNA from clone 46, whereas similarly prepared cDNA from 3-methylcholanthrene-treated DBA/2N and control C57BL/6N and DBA/2N mice did not. Of 11 restriction endonucleases used, two (
Pst
I and
Xba
I) had sites within the clone 46 DNA insert. After hybridization of clone 46
32
P-labeled nick-translated DNA to
Eco
RI fragments from A/HeJ mouse genomic DNA and fractionation by RPC-5 chromatography and gel electrophoresis, only one positive band (3-4 kilobase pairs appeared. These data demonstrate conclusively that pBR322 clone 46 DNA is associated with mRNA controlled by the murine
Ah
locus, presumably the structural gene encoding 3-methylcholanthrene-induced
P
1
-450.
Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.
Authors: Shoji S , Parmelee D C , Wade R D , Kumar S , Ericsson L H , Walsh K A , Neurath H , Long G L , Demaille J G , Fischer E H , Titani K
Published: May 2006

The complete amino acid sequence of the 349-residue catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle is presented. The sequence of the subunit (Mr 40,580 including phosphate groups at threonine-196 and serine-337) was derived largely by automated Edman degradation of nine fragments generated from the carboxymethylated protein by cleavage of methionyl bonds with cyanogen bromide. These fragments were aligned along the polypeptide chain by analysis of methionine-containing tryptic peptides isolated from protein radiolabeled in vitro by [14C]methyl exchange at methionyl residues. The molecule contains only two cysteinyl residues, at positions 198 and 342. It is relatively polar, containing clusters of cationic residues toward the amino terminus and anionic residues towards the carboxyl terminus. Predictions of secondary structure suggest the presence of three major domains with approximately half of the residues occurring in alpha-helices and 12% in beta-strands.
Release of 7-methylguanine residues from alkylated DNA by extracts of Micrococcus luteus and Escherichia coli.
Authors: Laval J , Pierre J , Laval F
Published: May 2006

Cell extracts from Micrococcus luteus release both free 3-methyladenine and free 7-methylguanine from alkylated DNA. The glycosylase activity responsible for the liberation of 7-methylguanine is not 3-methyladenine-DNA glycosylase, which, when purified, does not liberate it. Furthermore, the heat inactivation rates of the two enzymatic activities are different. The release of 7-methylguanine by chemical depurination of ethanol-soluble oligonucleotides has been ruled out. A similar activity releasing 7-methylguanine is also found in Escherichia coli.
Antifolate-induced misincorporation of deoxyuridine monophosphate into DNA: inhibition of high molecular weight DNA synthesis in human lymphoblastoid cells.
Authors: Sedwick W D , Kutler M , Brown O E
Published: May 2006

In vitro exposure of a human lymphoblastoid cell line (WIL-2) to the antifolate metoprine (DDMP), when followed by the addition of exogenous deoxyuridine, led to intracellular accumulation of deoxyuridine triphosphate (dUTP) and incorporation of deoxyuridine monophosphate (dUMP) into DNA. When newly synthesized DNA was extracted from DDMP-treated cells that had been labeled with deoxyuridine for up to 3 min, most of the DNA synthesized was no larger than 4 S on alkaline sucrose gradients. In contrast, the predominant form of newly synthesized alkali-stable DNA in cells not treated with drug was larger than 4 S. Abnormal progression of DNA synthesis, degradation of newly synthesized DNA, or both occurred as a delayed consequence of DDMP treatment in the absence of exogenous deoxyuridine when thymidine was used to label DNA of DDMP-treated stability of antifolate-induced misincorporation of dUMP into DNA was not elucidated, it was clear that antifolates can directly perturb the quality as well as the quantity of DNA synthesized by drug-treated cells.
Ca2+- and calmodulin-stimulated endogenous phosphorylation of neurotubulin.
Authors: Burke B E , DeLorenzo R J
Published: May 2006

Ca2+ plays a major role in the functional use of tubulin in brain and other tissues. It activates an endogenous tubulin kinase system in brain cytosol, tubulin, and presynaptic nerve terminal fractions prepared from rat brain. Activation of the Ca2+ tubulin kinase system was modulated by the Ca2+ receptor protein calmodulin. The concentrations of Ca2+ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 microM and 0.4 micrograms, respectively. Ca2+ -calmodulin tubulin kinase activity was very unstable after death, and procedures were developed to stabilize the activity of this enzyme system. Evidence is presented demonstrating that the Ca2+ -calmodulin tubulin kinase system is distinct from the previously described cyclic AMP-Mg2+ tubulin kinase. The results suggest that Ca2+- and calmodulin-stimulated phosphorylation of tubulin may be a major biochemical mechanism modulating some of calcium's effects on tubulin and may play a significant role in mediating some of calcium's actions on cell functions.
Alkyl-metal and aryl-metal bond chemistry in coordinately unsaturated polynuclear metal complexes
Authors: Schmidt G. F. , Muetterties E. L. , Beno M. A. , Williams Jack M.
Published: May 2006

A new class of coordinately unsaturated polynuclear rhodium and iridium alkyl, benzyl, and aryl derivatives of the form [RM(1,5-cyclooctadiene)]
x
have been prepared by the reaction of the organolithium reagent with the cyclooctadienemetal chlorides at -78°C. The x-ray crystal structure of [μ-CH
3
Rh (1,5-cyclooctadiene)]
2
is reported. The analogous iridium dimer decomposes by an initial sequence of α-hydrogen abstraction and then reductive elimination of hydrogen to give [μ-CH
2
Ir(1,5-cyclooctadiene)]
2
. Formed in high yield by the decomposition of the ethylrhodium complex was [HRh(C
8
H
11
)]
4
, a tetrahedral cluster with face-bridging hydride ligands. Also discussed are the reactivities of the benzyl and phenyl derivatives. Unique reaction pathways for C—H bond activation and scission in this chemistry are delineated.
Systematic punctuation of eukaryotic DNA by A+T-rich sequences.
Authors: Moreau J , Matyash-Smirniaguina L , Scherrer K
Published: May 2006

Isodenaturation of avian and mammalian DNA of Mr greater than 2.5 X 10(8) in 85% (vol/vol) formamide led to the observation by electron microscopy of A+T-rich zones ("AT-rich linkers") of 300-3000 base pairs which are distributed over the entire genome in a characteristic pattern. Linkers of mean length 800 base pairs are found either isolated or in clusters of about four to six linkers of more heterogeneous size separated on average by 2500 base pairs (in duck DNA). In between clusters, single linkers segment the DNA at distances of 10 to more than 100 kilobase pairs, with a majority in the range of 10-30 kilobase pairs. An analogous organization of linkers is found in rat and mouse DNA. The internal organization of the clusters varies, however, in a fashion that might be related to the large amount of light satellite DNA in the mouse and its apparent absence in rat and avian DNA. It is possible to fragment the DNA under appropriate conditions by the single-strand-specific nuclease S1 at the site of these A+T-rich zones and to obtain, on alkaline sucrose gradients, a bimodal pattern of DNA fragments of the size corresponding to the pattern observed by electron microscopy. The implications of this observation for DNA organization, chromatin structure, units of transcription and replication, and possible targets of A+T-specific drugs are discussed.
Nucleotide sequence of a ribosomal RNA gene intron from slime mold Physarum polycephalum.
Authors: Nomiyama H , Sakaki Y , Takagi Y
Published: May 2006

The Physarum polycephalum 26S ribosomal RNA gene contains two intervening sequences (introns). The DNA sequence of one of these introns was analyzed together with that of its flanking regions (exons). In addition, the nucleotide sequence of the corresponding region of the reverse transcript of 26S rRNA was determined, and from comparisons of both sequences the precise location and size of the intron were determined. Our findings, when compared with those for Tetrahymena and Chlamydomonas rRNA gene introns, led to the conclusion that certain characteristics exist near the ends of these introns. (i) An exon ends in T at the exon/intron junction and an intron ends in G at the intron/exon junction in all cases. (ii) For each intron, direct repeats several nucleotides long are present 5 to approximately 30 nucleotides upstream from both the exon/intron and intron/exon junctions.
Role of GTP hydrolysis in microtubule treadmilling and assembly.
Authors: Margolis R L
Published: May 2006

GTP hydrolysis accompanies addition of tubulin to microtubules. I find that hydrolysis is a requirement for the opposite-end assembly/disassembly of microtubules and consequent subunit treadmilling from one end to the other of the polymer. Neither GDP nor guanosine 5'-[beta, gamma-imido]triphosphate allows or participates in the treadmilling reaction. Therefore, there is a requirement for hydrolysis in the addition of subunits to the favored assembly end of the microtubule. Podophyllotoxin, an assembly inhibitory drug, "caps" the microtubule assembly end, preventing subunit loss from that site to equilibrium. Continued hydrolysis of GTP is required to maintain the podophyllotoxin cap. A corollary of this finding is that GTP hydrolysis is required for cap formation. Microtubules assembled in GTP enter a metastable state when all remaining GTP is hydrolyzed. This state is characterized by its ability to maintain indefinitely a subunit/polymer distribution ratio that is arbitrary and that can be altered at will by brief chilling or by addition of small amounts of GTP. This metastable state is labile to podophyllotoxin. Use of podophyllotoxin allows measurement of the microtubule treadmilling rate; use of podophyllotoxin in the absence of GTP allows measurement of the overall rate of dimer dissociation from the microtubule. Measurement of these rates has permitted determination of the efficiency with which adding dimers incorporate into the microtubule treadmill and are not lost to assembly end equilibrium. The efficiency varies with GTP concentration for unknown reasons, being high at 0.1 mM GTP and low at higher GTP concentrations.
Structural and functional domains of the Rous sarcoma virus transforming protein (pp60src).
Authors: Levinson A D , Courtneidge S A , Bishop J M
Published: May 2006

The transforming protein (pp60src) of the Rous sarcoma virus (RSV) is a phosphoprotein with the enzymatic ability to phosphorylate tyrosine in protein substrates. Previous work has indicated that the bulk of pp60src may be attached to the plasma membrane of infected cells. In an effort to better understand the mechanism by which pp60src induces the neoplastic phenotype, we have characterized further the attachment of pp60src to the plasma membrane, and we have identified separate molecular domains that are responsible for the attachment to membranes and for the protein kinase activity. Our results indicate that pp60src may be an integral membrane protein that is nevertheless synthesized on soluble polyribosomes. Subsequent to its synthesis, the protein attaches to plasma membrane without concomitant cleavage of a signal polypeptide. The amino-terminal quarter (or some portion thereof) of pp60src anchors the protein to the plasma membrane by forces that can be disrupted only with detergents. By contrast, protein kinase activity is located in the carboxyl-terminal half of the molecule. It appears that pp60src is designed on the one hand for tethering to the plasma membrane and on the other hand for enzymatic activity beyond the confines of the membrane. The fact that pp60src is but one of at least four different viral transforming proteins located on the plasma membrane implies that neoplastic transformation may commonly originate in events that occur at the periphery of the cell.
Down regulation of specific binding of [20-3H]phorbol 12,13-dibutyrate and phorbol ester-induced differentiation of human promyelocytic leukemia cells.
Authors: Solanki V , Slaga T J , Callaham M , Huberman E
Published: May 2006

Binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDB) to intact human promyelocytic leukemia cells susceptible (HL-60) or resistant (R-35) to phorbol ester-induced differentiation was characterized. Specific binding of [3H]PDB to both HL-60 and R-35 cells at 37 degrees C reached a maximum within 15-20 min. Maximal specific [3H]PDB binding to HL-60 cells was followed by a decline (down regulation) of radioactivity. This down regulation was temperature dependent, because no loss of radiolabel occurred by 1 hr at 4 degrees C. The down regulation of bound [3H]PDB seen in HL-60 cells at 37 degrees C was not observed with R-35 cells. Prior exposure of the HL-60 cells but not of R-35 cells to 1 microM phorbol 12-myristate 13-acetate for 90 min at 37 degrees C caused a marked reduction in the specific binding of [3H]PDB. When [3H]PDB binding was carried out at 4 degrees C, [3H]PDB bound to both cell types in a rapid, specific, and reversible manner. At equilibrium, HL-60 and R-35 cells were found to contain almost the same number of binding sites, which had dissociation constants of about 50 nM, indicating that the failure of R-35 cells to undergo PDB-induced differentiation was not associated with any change in the affinity or in the number of [3H]PDB binding sites. These results indicate that the down regulation of specific [3H]PDB binding may be a crucial early event in the control of phorbol ester-induced terminal differentiation in HL-60 cells. Furthermore, we suggest that such down regulation may be involved in other cellular and biochemical effects of phorbol diester tumor promoters.
Depurination causes mutations in SOS-induced cells.
Authors: Schaaper R M , Loeb L A
Published: May 2006

Introduction of apurinic sites into phi X174 am3 DNA leads to loss of biological activity when measured in a transfection assay. For single-stranded DNA, approximately one apurinic site constitutes a lethal hit; for double-stranded (RFI) DNA, approximately 3.5 hits per strand are lethal. When the reversion frequency of am3 DNA is measured, no increase due to depurination is observed above the background level. However, a large increase in reversion frequency is observed when the same DNA is assayed by using spheroplasts derived from bacteria previously exposed to UV light. The results suggest that apurinic sites are impediments to a replicating DNA polymerase; however, nucleotides can be incorporated opposite these sites under SOS-induced conditions. We estimate the frequency of mutagenesis per apurinic site to be less than 1 in 1400 in normal spheroplasts and 1 in 100 in SOS-induced spheroplasts.
Antigen-presenting cell function in induction of helper T cells for cytotoxic T-lymphocyte responses: evidence for antigen processing.
Authors: Weinberger O , Herrmann S , Mescher M F , Benacerraf B , Burakoff S J
Published: May 2006

We demonstrate that splenic adherent cells (SACs) play an active role in the presentation of H-2Kk antigen for an alloreactive cytotoxic T-lymphocyte (CTL) response. If antigen is incubated with SACs for 12 hr, they will provide maximal stimulation and present the antigen in the context of their Ia molecules. UV irradiation of these SACs, prior to the 12-hr incubation with H-2Kk antigen, abrogates this stimulatory capacity. Macrophage-bound antigen is not sufficient for stimulation of a response; a second signal is required as well, that, in our system, is provided by phorbol myristic acetate. The SACs are involved in the activation of helper T cells; however, they are not required for presentation of antigen to the precytotoxic T-lymphocyte, which requires two signals for activation, one provided by antigen and the other by a T-cell-derived helper factor.
Molecular weight of the membrane C5b-9 complex of human complement: characterization of the terminal complex as a C5b-9 monomer.
Authors: Bhakdi S , Tranum-Jensen J
Published: May 2006

The hydrodynamic properties of the detergent-solubilized, terminal membrane complex of serum complement components C5-C9 [C5b-9(m)] were studied to obtain an estimate of its molecular weight. In a solution of Triton X-100/deoxycholate, the protein complex binds 17% Triton X-100 and 11% deoxycholate by weight. The sedimentation coefficient of the protein-detergent complex is 26 S as determined by sucrose density gradient ultracentrifugation, and gel filtration indicated a molecular radius of 11 nm. It was ascertained by electron microscopy that these hydrodynamic parameters apply to mono-dispersed C5b-9(m) complexes, which were observed as nonaggregated, hollow protein cylinders and were identical to the complement "lesions" formed on target membranes. The calculated molecular weight of the protein-detergent complex is approximately 1,286,300 to which the protein moiety contributes approximately 1,000,000. The results indicate that the C5b-9(m) complex formed on biological membranes is a monomer entity of the C5-C9 complement components.
Production of hybridomas secreting monoclonal antibodies to the human leukocyte interferons.
Authors: Staehelin T , Durrer B , Schmidt J , Takacs B , Stocker J , Miggiano V , Stähli C , Rubinstein M , Levy W P , Hershberg R , Pestka S
Published: May 2006

Thirteen monoclonal antibodies to human leukocyte interferon have been obtained. They exhibit different patterns of binding to purified leukocyte interferon species that are consistent with the structural multiplicity of the human leukocyte interferons. These antibodies will be useful as probes into the structure of the human leukocyte interferons, for their purification, and for rapid assay of leukocyte interferon.