bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA amplification and neoplastic transformation mediated by a herpes simplex DNA fragment containing cell-related sequences.
Authors: Jariwalla R J , Tanczos B , Jones C , Ortiz J , Salimi-Lopez S
Published: May 2006

The transforming potential of the herpes simplex virus type 2 (HSV-2) BamHI fragment E (map position 0.533-0.583) encoding the 140-kDa ribonucleotide reductase was assayed by transfection in established Rat-2 cells. Foci of refractile, morphologically distinguishable cells were induced at lower efficiency and after a longer incubation period as compared to the human tumor oncogene EJ-Ha-ras. Focus-derived BamHI fragment E-transformed cell lines formed medium-to-large (0.1-0.25 mm) colonies in soft agar and were tumorigenic in immunocompetent syngeneic rats. Southern blot analysis of normal rat DNA after EcoRI digestion revealed specific DNA segments homologous to HSV-2 BamHI fragment-E DNA. In BamHI fragment E-transformed and tumor-derived lines, about 8- to 30-fold amplification was detected in a subset of the specific HSV-related DNA segments. In addition, extrachromosomal DNA was isolated from transformed cells by plasmid rescue and contained the left-hand 70% of HSV-2 Bam HI fragment E fused to rat DNA. These results indicate the presence in normal cells of nonrepetitive DNA segments, related to the transforming HSV-2 fragment, that can be targeted for genetic alterations associated with neoplastic transformation.
Proenkephalin mRNA in rat heart.
Authors: Howells R D , Kilpatrick D L , Bailey L C , Noe M , Udenfriend S
Published: May 2006

The distribution of preproenkephalin mRNA in rat tissues was investigated using a homologous cDNA probe for detection. The heart was found to contain larger amounts of the mRNA than any other tissue including brain, which heretofore had been considered the richest source. The identity of the message in heart was verified by hybridizing RNA blots with a synthetic oligodeoxynucleotide that recognizes a different region of the preproenkephalin mRNA sequence than does the cDNA probe. The preproenkephalin mRNA extracted from both heart and brain contained approximately equal to 1500 bases. Dissection of heart revealed that essentially all of the message is contained within the ventricles. In contrast to the large amounts of preproenkephalin mRNA in rat heart, the opioid peptide contents is only 3% of the amount in brain. The rat heart may be a useful model for the investigation of translation control of protein synthesis.
A preliminary three-dimensional structure of angiogenin.
Authors: Palmer K A , Scheraga H A , Riordan J F , Vallee B L
Published: May 2006

A preliminary three-dimensional structure of angiogenin has been computed, based on its homology to bovine pancreatic ribonuclease A. A standard-geometry structure of ribonuclease was first obtained from its x-ray coordinates. The fit of the backbone of angiogenin to that of ribonuclease was then optimized by taking account of amino acid deletions and by minimizing its conformational energy-plus-a-penalty distance function constraining its backbone to that of ribonuclease. Side-chain and backbone dihedral angles were allowed to vary throughout the cycles of energy minimization. In the last stages of minimization, the penalty distance function was removed. A low-energy structure resembling ribonuclease was obtained.
Long polypeptide 3
10
-helices at atomic resolution
Authors: Bavoso Alfonso , Benedetti Ettore , Di Blasio Benedetto , Pavone Vincenzo , Pedone Carlo , Toniolo Claudio , Bonora Gian Maria
Published: May 2006

The crystal-state preferred conformation of the terminally blocked homooctapeptide from the C
α,α
-dimethylated α-aminoisobutyric acid (Aib) residue,
p
BrBz-(Aib)
8
-OBu

t

, in which
p
BrBz is
para
-bromobenzoyl and OBu

t

is
tert
-butoxy, determined by x-ray diffraction analysis using direct methods, was found to be a 3
10
-helix stabilized by six consecutive intramolecular N—H....O=C hydrogen bonds of the C
10
-III (or III′) type. This is the first observation at atomic resolution of a regular 3
10
-helix longer than two complete turns. The solid-state structural analysis was extended to the terminally blocked, α-aminoisobutyric acid-rich octapeptide corresponding to the 2-9 sequence of the peptaibol antibiotics emerimicins III and IV,
p
BrBz-Aib
3
-L-Val-Gly-L-Leu-Aib
2
-OMe. Again, this peptide adopts a (right-handed) 3
10
-helical structure, although slightly distorted at the level of the L-leucine residue. The role of specific amino acid sequence and peptide main-chain length in stabilizing either the 3
10
- or the α-helical conformation and their possible implications on the nature of the channel formed by peptaibol antibiotics in the membrane are also briefly discussed.
Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration.
Authors: Moscatelli D , Presta M , Rifkin D B
Published: May 2006

A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 10(6)-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor.
Ethanol regulation of adenosine receptor-stimulated cAMP levels in a clonal neural cell line: an in vitro model of cellular tolerance to ethanol.
Authors: Gordon A S , Collier K , Diamond I
Published: May 2006

The acute and chronic neurologic effects of ethanol appear to be due to its interaction with neural cell membranes. Chronic exposure to ethanol induces changes in the membrane that lead to tolerance to the effects of ethanol. However, the actual membrane changes that account for tolerance to ethanol are not understood. We have developed a model cell culture system, using NG108-15 neuroblastoma-glioma hybrid cells, to study cellular tolerance to ethanol. We have found that adenosine receptor-stimulated cAMP levels increased markedly upon acute exposure to ethanol. However, the cells became tolerant to ethanol, since chronically treated cells required ethanol to maintain normal adenosine-stimulated cAMP levels. Moreover, the cells appeared to be dependent on ethanol, as evidenced by reduced adenosine-stimulated cAMP levels in the absence of ethanol. Recovery occurred after ethanol was withdrawn. These cellular changes appear to parallel the clinical events of acute ethanol intoxication, tolerance, and dependence.
Genetic complexity of host-selection behavior in
Drosophila
Authors: Jaenike John
Published: May 2006

Genetic variation for settling responses to different types of food and for oviposition behavior exists within natural populations of
Drosophila tripunctata
. After flies from two isofemale strains and F
2
derived from crosses between them were released in the wild and recaptured at either mushroom or tomato baits, the oviposition preferences of females for these foods were determined in the laboratory. Whereas settling behavior and oviposition-site preference were negatively correlated in the parental strains, there was no association of these behaviors in the F
2
generation. Furthermore, lines produced by crossing these two strains followed by inbreeding did not manifest such a negative correlation. Together, these results indicate that two important aspects of host selection are under independent genetic control. These findings caution against using a single component of the process of host selection as indicative of the probability that a particular resource will be utilized in the field, and they bear on the question of how speciation based on genetic variation for host selection may be accomplished.
Localization of the gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A synthase to human chromosome 5.
Authors: Leonard S , Arbogast D , Geyer D , Jones C , Sinensky M
Published: May 2006

A series of hybrids between primary human cells and a Chinese hamster somatic cell mutant (Mev-1), defective in expression of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase [(S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA-acetylating, EC 4.1.3.5], has been prepared that complements the mutant defect. A technique based on differential sensitivity of this enzyme activity to inhibition by magnesium ion is described that allows the discrimination of expression of human and hamster HMG-CoA synthase in these hybrids. The results indicate a structural gene defect in expression of HMG-CoA synthase activity in Mev-1 cells. Segregation of human chromosomes that do not possess the complementing marker have allowed the assignment of human HMG-CoA synthase activity to chromosome 5. This is the second demonstrably transcriptionally regulated enzyme of cholesterologenesis to be assigned to chromosome 5, the other being HMG-CoA reductase.
Transforming activity of human papillomavirus type 16 DNA sequence in a cervical cancer.
Authors: Tsunokawa Y , Takebe N , Kasamatsu T , Terada M , Sugimura T
Published: May 2006

A genomic DNA sample from cervical cancer tissue, containing human papillomavirus (HPV) type 16, was found to induce malignant transformation of NIH 3T3 cells when it was tested by transfection assays using the calcium phosphate coprecipitation technique. The primary and secondary transformants contained the HPV type 16 DNA sequences and human specific Alu family sequences. To the best of our knowledge, it has not been reported previously that HPV type 16 DNA sequences in total genomic DNA from a cervical cancer have transforming activity.
A newly identified hepatitis B type virus in tree squirrels.
Authors: Feitelson M A , Millman I , Halbherr T , Simmons H , Blumberg B S
Published: May 2006

Virus-associated particles have been isolated from the livers of three common gray tree squirrels (Sciurus carolinensis pennsylvanicus) that have histological evidence of hepatitis. Two of these livers were also positive by orcein staining, suggesting the presence of surface antigen in the cytoplasm of hepatocytes. Fractionation of these particles by CsCl density equilibrium gradient centrifugation and assay of the fractions for surface antigen, core antigen, and DNA polymerase activities demonstrate the presence of all three at an approximate density peak of 1.27. Electron microscopic examination of purified virus preparations showed spherical particles with a mean diameter of 25 nm. Initial characterization of the DNA polymerase product by gel electrophoresis showed a single DNase I sensitive band, migrating slightly faster than the woodchuck hepatitis virus DNA polymerase product. The presence of apparently cross-reacting antibodies was demonstrated by purified hepatitis B surface and/or core antigens binding to some squirrel sera in solid phase assays. Infected tree squirrels appear to lack detectable antigen in their sera. These results suggest that the tree squirrels studied are chronic carriers of a hepatitis B type virus. The host-virus interaction described herein may be useful in understanding the chronic carrier state associated with hepatitis B in man.
Gene conversion in a cytochrome P-450 gene family.
Authors: Atchison M , Adesnik M
Published: May 2006

The mRNAs encoding the two major phenobarbital-inducible forms of cytochrome P-450 of rat liver, P-450b and P-450e, are remarkably similar (98% homologous) in nucleotide sequence, but the distribution of differences within them is not random. While the 5' halves (approximately equal to 1 kilobase) appear to be identical, there are 36 divergent residues in the remaining sequences of the two mRNAs, with 14 differences residing in two short highly divergent segments, which in the P-450e gene are located within exon 7. DNA sequence analysis of portions of a number of P-450b/e-related genes provides strong evidence that at least one of the short divergent segments is the result of a recent gene conversion event between an ancestor to the cytochrome P-450e gene and a related donor P-450 gene of unknown function. The sequence data also suggest that extensive gene conversion has occurred within all the members of this gene family in the region including exons 7 and 8 and the intron between them, with a resultant homogenization of those sequences relative to other portions of the genes. Genomic Southern blotting analysis demonstrates that the presence of an apparent "constant" region in the 5' halves of the P-450b and P-450e mRNAs does not reflect a rearrangement in somatic cells of a germ-line DNA configuration. It is therefore proposed that it, too, is a consequence of a very recent gene conversion event between ancestors of the genes encoding both proteins or of an unequal crossing-over between them. On the basis of these and other data we propose that gene conversion represents an important evolutionary mechanism for the generation of related cytochrome P-450 isozymes in which regions of extraordinary sequence similarity and dissimilarity are intermixed. The gene conversion mechanism would account for some of the overlaps in substrate specificities of distantly related P-450s as well as for substantial differences in catalytic properties between closely related members of the same P-450 protein family.
Alteration of adenyl dinucleotide metabolism by environmental stress.
Authors: Baker J C , Jacobson M K
Published: May 2006

Exposure of cultured mammalian cells to a variety of conditions that induce the synthesis of stress proteins, including hyperthermia, ethanol, cadmium, and arsenite resulted in an increased cellular content of adenyl dinucleotides including diadenosine tetraphosphate (Ap4A). Exposure to other agents that cause metabolic perturbations not known to induce the synthesis of stress proteins, such as cyclohexamide, cytosine arabinoside, hydroxyurea, and ultraviolet irradiation did not alter the content of these nucleotides. It is proposed that these unique nucleotides may mediate adaptive responses of mammalian cells to environmental stress.
Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor beta: a proposed model for indirect mitogenesis involving autocrine activity.
Authors: Leof E B , Proper J A , Goustin A S , Shipley G D , DiCorleto P E , Moses H L
Published: May 2006

Treatment of quiescent cultures of mouse embryo-derived AKR-2B cells with transforming growth factor beta resulted in an early induction of c-sis mRNA. The increase in c-sis mRNA was followed by a corresponding increase in protein similar to platelet-derived growth factor (PDGF) in the culture medium. In addition, PDGF-regulated genes (c-fos and c-myc) were stimulated by transforming growth factor beta with delayed kinetics relative to that seen in other cell systems with direct PDGF stimulation. A model is proposed in which the monolayer mitogenicity of transforming growth factor beta is mediated by the induction of c-sis and PDGF and the subsequent autocrine stimulation of c-fos, c-myc, and other PDGF-inducible genes.
Components of microtubular structures in Saccharomyces cerevisiae.
Authors: Pillus L , Solomon F
Published: May 2006

Most studies of cytoskeletal organelles have concentrated on molecular analyses of abundant and biochemically accessible structures. In many of the classical cases, however, the nature of the system chosen has precluded a concurrent genetic analysis. The mitotic spindle of the yeast Saccharomyces cerevisiae is one example of an organelle that can be studied by both classical and molecular genetics. We show here that this microtubule structure also can be examined biochemically. The spindle can be isolated by selective extractions of yeast cells by using adaptations of methods successfully applied to animal cells. In this way, microtubule-associated proteins of the yeast spindle are identified.
Common evolutionary origin of hepatitis B virus and retroviruses.
Authors: Miller R H , Robinson W S
Published: May 2006

Hepatitis B virus (HBV), although classified as a double-stranded DNA virus, has been shown recently to replicate by reverse transcription of an RNA intermediate. Also, the putative viral polymerase has been found to share amino acid homology with reverse transcriptase of retroviruses. Using computer-assisted DNA and protein sequence analyses, we examined the genomes of 13 hepadnavirus isolates (nine human, two duck, one woodchuck, and one ground squirrel) and found that other conserved regions of the hepadnavirus genome share homology to corresponding regions of the genomes of type C retroviruses and retrovirus-like endogenous human DNA elements. Specifically, the most highly conserved sequence of the HBV genome, positioned at or near the initiation site for first-strand HBV DNA synthesis, is homologous over 67 nucleotides to the U5 region, a comparable region in retrovirus long terminal repeats. Within a highly conserved (i.e., 90%) 16-nucleotide sequence a heptanucleotide sequence CCTTGGG is 97% homologous between 27 virus isolates. Also, we found that the highly conserved HBV core, or nucleocapsid, protein shares 41% homology over 98 amino acids with the carboxyl-terminal region of the p30 gag nucleocapsid protein of type C retroviruses. In both cases, as with the previously reported polymerase homology, HBV is most homologous to the murine leukemia/sarcoma retroviruses. Further analysis revealed additional similarities between hepadnavirus and retroviral genomes. Taken together, our results suggest that HBV and retroviruses have a common evolutionary origin, with HBV arising through a process of deletion from a retrovirus, or retrovirus-like, progenitor.
Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major.
Authors: Beverley S M , Ellenberger T E , Cordingley J S
Published: May 2006

We have determined the nucleotide sequence of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) gene of the protozoan parasite Leishmania major (dihydrofolate reductase, EC 1.5.1.3 and thymidylate synthase, EC 2.1.1.45). The DHFR-TS protein is encoded by a single 1560-base-pair open reading frame within genomic DNA, in contrast to vertebrate DHFRs or mouse and phage T4 TSs, which contain intervening sequences. Comparisons of the DHFR-TS sequence with DHFR and TS sequences of other organisms indicate that the order of enzymatic activities within the bifunctional polypeptide chain is DHFR followed by TS, the Leishmania bifunctional DHFR-TS evolved independently and not through a phage T4-related intermediate, and the rate of evolution of both the DHFR and TS domains has not detectably changed despite the acquisition of new functional properties by the bifunctional enzyme. The Leishmania gene is 86% G+C in the third codon position, in contrast to genes of the parasite Plasmodium falciparum, which exhibit an opposite bias toward A+T. The DHFR-TS locus is encoded within a region of DNA amplified in methotrexate-resistant lines, as previously proposed.
The primate retina contains two types of ganglion cells, with high and low contrast sensitivity.
Authors: Kaplan E , Shapley R M
Published: May 2006

Previously, we discovered that the broadband cells in the two magnocellular (large cell) layers of the monkey lateral geniculate nucleus (LGN) are much more sensitive to luminance contrast than are the color-sensitive cells in the four parvocellular (small cell) layers. We now report that this large difference in contrast sensitivity is due not to LGN circuitry but to differences in sensitivity of the retinal ganglion cells that provide excitatory synaptic input to the LGN neurons. This means that the parallel analysis of color and luminance in the visual scene begins in the retina, probably at a retinal site distal to the ganglion cells.
Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.
Authors: Fong D , Calhoun D H , Hsieh W T , Lee B , Wells R D
Published: May 2006

The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonucleotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this clone encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with approximately equal to 880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome.
Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.
Authors: Neumann D , Barchan D , Safran A , Gershoni J M , Fuchs S
Published: May 2006

Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.
Loss of perforated synapses in the dentate gyrus: morphological substrate of memory deficit in aged rats.
Authors: Geinisman Y , de Toledo-Morrell L , Morrell F
Published: May 2006

Most, but not all, aged rats exhibit a profound deficit in spatial memory when tested in a radial maze--a task known to depend on the integrity of the hippocampal formation. In this study, animals were divided into three groups based on their spatial memory capacity: young adult rats with good memory, aged rats with impaired memory, and aged rats with good memory. Memory-impaired aged animals showed a loss of perforated axospinous synapses in the dentate gyrus of the hippocampal formation in comparison with either young adults or aged rats with good memory. This finding suggests that the loss of perforated axospinous synapses in the hippocampal formation underlies the age-related deficit in spatial memory.
Homologous down-regulation of the insulin receptor is associated with increased receptor biosynthesis in cultured human lymphocytes (IM-9 line).
Authors: Rouiller D G , Gorden P
Published: May 2006

Cultured IM-9 lymphocytes were preincubated with 1 microM insulin, a condition resulting in a 56% reduction in cell surface insulin receptors. Cellular proteins were then metabolically labeled, and the radioactivity incorporated into the insulin proreceptor and receptor mature subunits was measured over a 4-hr chase period. As early as 30 min of chase, incorporation into the proreceptor was 28 +/- 6% higher in down-regulated cells than in control cells (mean +/- SEM, P less than 0.05). By 1 hr of chase, the difference reached 41 +/- 14% for the proreceptor and 84 +/- 28% for the alpha subunit (P less than 0.01); values returned to normal by 2 hr. At 4 hr of chase, labeling of the alpha subunit of down-regulated cells was diminished 36 +/- 9% below control (P less than 0.05). The increased biosynthetic rate of the proreceptor was more prominent when the chase medium contained 25 microM monensin, an inhibitor of processing of the proreceptor into mature subunits. Similar effects occurred whether [3H]mannose or [3H]lysine was used as biosynthetic marker. The effect was specific for the insulin receptor. These data demonstrate that insulin receptor homologous down-regulation is associated with increased proreceptor biosynthesis and processing into mature subunits. This might represent a cellular mechanism compensating for insulin-induced receptor loss.
Single-channel recordings demonstrate that cGMP opens the light-sensitive ion channel of the rod photoreceptor.
Authors: Matthews G
Published: May 2006

Patch-clamp recordings were made from outer segments of single dark-adapted rod photoreceptors from toad retina. When patch pipettes were filled with a solution free of divalent cations, inward current through individual light-sensitive ion channels was sufficiently large to allow single-channel recording. Channel activity was suppressed by illumination within the normal response range of the dark-adapted rod. When illumination was restricted to a portion of the outer segment, the effect of light on channel activity was spatially localized to the illuminated region. Hyperpolarization of the recorded patch did not reduce the frequency of channel opening, indicating that the suppression of channel activity by light was due to illumination itself and not to light-induced hyperpolarization of the rod. After recording single light-sensitive channel activity in the intact cell, the patch of membrane was detached to form an excised, inside-out patch. Cyclic GMP applied to the intracellular face of the excised patch then opened a channel that appeared to be the same as the light-sensitive channel recorded earlier from the same membrane in the intact, functioning rod. This provides direct evidence that cyclic GMP acts as an internal transmitter that opens the light-sensitive channel of the vertebrate photoreceptor.
Gene induction and repression by salt treatment in roots of the salinity-sensitive Chinese Spring wheat and the salinity-tolerant Chinese Spring ×
Elytrigia elongata
amphiploid
Authors: Gulick Patrick , Dvořák Jan
Published: May 2006

An artificial amphiploid from a cross between salinity-sensitive bread wheat cultivar Chinese Spring and highly tolerant
Elytrigia elongata
(Host) Nevski (=
Agropyron elongatum
Host) shows enhanced salinity tolerance relative to Chinese Spring. Poly(A)
+
RNA was isolated from roots, expanding leaves, and old leaves from amphiploid and Chinese Spring plants prior to and after acclimation to high levels of NaCl in solution cultures. Two-dimensional gel electrophoresis of the
in vitro
translation products was used to compare these mRNA populations. The amphiploid had 10 mRNA species induced or enhanced and 8 species repressed in root tissue during acclimation to saline growth conditions. These 18 transcripts affected by salt treatment were also detected in wheat roots, but only 4 of these were similarly regulated. In Chinese Spring the acclimation to saline stress resulted in a marked change in the level of expression of 34 transcripts in root tissue; of these, 26 were detected in the amphiploid and only 6 were regulated as in the amphiploid. No differences were seen in gene expression between salt-treated and control plants in leaves and meristematic crowns and unexpanded leaves of the amphiploid.
Cotranslational insertion of selenocysteine into formate dehydrogenase from Escherichia coli directed by a UGA codon.
Authors: Zinoni F , Birkmann A , Leinfelder W , Böck A
Published: May 2006

The structural gene (fdhF) for the 80-kDa selenopolypeptide of formate dehydrogenase (formate:benzyl viologen oxidoreductase, EC 1.2.--.--) from Escherichia coli contains an in-frame UGA codon at amino acid position 140 that is translated. Translation of gene fusions between N-terminal parts of fdhF with lacZ depends on the availability of selenium in the medium when the hybrid gene contains the UGA codon; it is independent of the presence of selenium when an fdhF portion upstream of the UGA position is fused to lacZ. Transcription does not require the presence of selenium in either case. By localized mutagenesis, the UGA codon was converted into serine (UCA) and cysteine (UGC and UGU) codons. Each mutation relieved the selenium dependency of fdhF mRNA translation. Selenium incorporation was completely abolished in the case of the UCA insertion and was reduced to about 10% when the UGA was replaced by a cysteine codon. Insertion of UCA yielded an inactive fdhF gene product, while insertion of UGC and UGU resulted in polypeptides with lowered activities as components in the system formerly known as formate hydrogenlyase. Altogether the results indicate that the UGA codon at position 140 directs the cotranslational insertion of selenocysteine into the fdhF polypeptide chain.
Identification and synthesis of a recognition signal for the attachment of glycosaminoglycans to proteins.
Authors: Bourdon M A , Krusius T , Campbell S , Schwartz N B , Ruoslahti E
Published: May 2006

Comparison of the amino acid sequences of three different proteoglycan core proteins reveals a 12-amino acid sequence that is about 50% homologous among these proteoglycans. In each of the proteoglycans, this sequence surrounds the serine-glycine dipeptide in which the serine is known or presumed to be substituted with a chondroitin/dermatan sulfate glycosaminoglycan chain. Peptides containing this sequence from two proteoglycans were examined for their ability to serve as acceptors for xylosyltransferase, the enzyme that begins the assembly of glycosaminoglycan chains. Those peptides corresponding to amino acid sequences known to contain glycosaminoglycan-substituted serine residues in the protein were efficient xylosyltransferase acceptors, whereas peptides from sequences with no glycosaminoglycan-substituted serine residues were not. Amino acid substitutions at four critical sites in the acceptor peptides showed that single substitutions could completely abolish acceptor activity or greatly reduce it. The results suggest that the proteoglycan recognition consensus sequence for the attachment of glycosaminoglycans to core proteins consists of acidic amino acids closely followed by the tetrapeptide Ser-Gly-Xaa-Gly, where Xaa is any amino acid. The signal appears to be contained in the primary sequence information. In this regard it resembles a number of other signals for protein processing and intracellular routing.
Isolation of a cytochrome
aa
3
gene from
Bradyrhizobium japonicum
Authors: O'Brian Mark R. , Maier Robert J.
Published: May 2006

Bradyhizobium japonicum
strain LO501 is a Tn
5
-induced mutant that does not express the terminal oxidase cytochrome
aa
3
(cytochrome-
c
oxidase, EC 1.9.3.1). Two and one-half kilobase pairs of LO501 genomic DNA that flanks the transposon was isolated and used as a hybridization probe to obtain the wild-type gene from a cosmid library. Two subcloned fragments from two of the isolated cosmids were ligated into broad host range vectors, and restriction maps of these fragments were generated. The resultant plasmids, pCA1 and pBL33, each contained DNA homologous to that mutated in strain LO501. The two plasmids were each introduced into strain LO501 by conjugal transfer, and it was found that pCA1, but not pBL33, complemented the oxidase mutant. The transconjugant strain LO501[pCA1] expressed wild-type levels of cytochrome
aa
3
, as discerned spectrophotometrically, and had restored
N,N,N′,N′
-tetramethyl-
p
-phenylenediamine oxidase activity. Furthermore, the frequency of complementation of LO501 cells that received pCA1 by conjugation was 1.0, demonstrating that pCA1 complemented the mutant in trans. The results show that pCA1 contains the entire wild-type gene that was mutated in strain LO501, and this gene is required for cytochrome
aa
3
expression.
A family of short, interspersed repeats is associated with tandemly repetitive DNA in the human genome.
Authors: Mermer B , Colb M , Krontiris T G
Published: May 2006

A family of short, interspersed repeats in the human genome, designated the Mst II family, is described. The canonical structure of the repeat consists of a 220-base-pair (bp) left arm joined to a 160-bp right arm by a 39-bp junction sequence. The right arm is absent in some isolates. Some homology with the "O" and "THE" (transposon-like element) families of repeats was observed, suggesting that the Mst II elements could be a subgroup of a SINE superfamily. The 39-bp junction sequence is tandemly repeated in one of our clones. The association of tandemly repetitive sequences with Mst II elements or the putative superfamily is probably nonrandom; a search of DNA sequence data bases revealed that approximately 80 bp of the Mst II left arm occurs immediately adjacent to the tandem repeat that comprises the human homologue to the BK virus enhancer. The fortuitous occurrence of a gene duplication event involving an Mst II repeat has allowed us to estimate a mutation rate for human DNA.
Gene tandem-mediated selection of coliphage λ-receptive
Agrobacterium, Pseudomonas
, and
Rhizobium
strains
Authors: Ludwig Robert A.
Published: May 2006

Agrobacterium, Pseudomonas
, and
Rhizobium
spp. have been made receptive to coliphage λ. To achieve this, recombinant (pTROY) plasmids carrying a constitutive
Escherichia coli lamB
gene encoding the λ receptor and expressed from an insertion sequence 3 (IS
3
) promoter were introduced into various bacteria. Because the wild-type λ receptor was not expressed in these bacteria, a procedure called the
lamB
gene tandem protocol was used to select
lamB
alleles that expressed the λ receptor. This gene tandem protocol may have general use in adapting genes with nonselectable traits to different organisms.
Agrobacterium, Pseudomonas
, and
Rhizobium
strains carrying pTROY41613, which encoded a gene tandem-selected λ receptor, could be quantitatively transduced with λ-packaged cosmids. With the ability to confer λ receptivity on organisms, phage λ now becomes a general DNA delivery agent.
Complete suppression of in vivo growth of human leukemia cells by specific immunotoxins: nude mouse models.
Authors: Hara H , Seon B K
Published: May 2006

In this study, immunotoxins containing monoclonal anti-human T-cell leukemia antibodies are shown to be capable of completely suppressing the tumor growth of human T-cell leukemia cells in vivo without any overt undesirable toxicity. These immunotoxins were prepared by conjugating ricin A chain (RA) with our monoclonal antibodies, SN1 and SN2, directed specifically to the human T-cell leukemia cell surface antigens TALLA and GP37, respectively. We have shown that these monoclonal antibodies are highly specific for human T-cell leukemia cells and do not react with various normal cells including normal T and B cells, thymocytes, and bone marrow cells. Ascitic and solid human T-cell leukemia cell tumors were generated in nude mice. The ascitic tumor was generated by transplanting Ichikawa cells (a human T-cell leukemia cell line) i.p. into nude mice, whereas the solid tumor was generated by transplanting s.c. MOLT-4 cells (a human T-cell leukemia cell line) and x-irradiated human fibrosarcoma cells into x-irradiated nude mice. To investigate the efficacy of specific immunotoxins in suppressing the in vivo growth of the ascitic tumor, we divided 40 nude mice that were injected with Ichikawa cells into four groups. Each group of 10 mice was injected with one of the following mixtures: 40 micrograms of purified control mouse IgG [IgG1(kappa)] (group 1), 40 micrograms of control RA conjugate (group 2), 20 micrograms of purified SN1 antibody [IgG1(kappa)] and 20 micrograms of purified SN2 antibody [IgG1(kappa)] (group 3), or 20 micrograms of SN1-RA and 20 micrograms of SN2-RA (group 4). Mice in groups 1 and 2 formed large ascitic tumors, and died 5.8-7.0 weeks after the transplantation. Group 3 mice also formed large ascitic tumors and died 6.4-7.8 weeks after the transplantation. However, none of the mice in group 4 that were treated with SN1-RA and SN2-RA showed any signs of a tumor or undesirable toxic effects for the 20 weeks that they were followed after the transplantation; these mice were indistinguishable from healthy control nude mice that were not injected with Ichikawa cells. Treatment with SN1-RA plus SN2-RA completely suppressed solid tumor growth in 4 of 10 nude mice carrying solid tumors and partially suppressed the tumor growth in the remaining 6 nude mice. These results strongly suggest that SN1-RA and SN2-RA may be useful for clinical treatment.
Interferon gamma induces lung colonization by intravenously inoculated B16 melanoma cells in parallel with enhanced expression of class I major histocompatibility complex antigens.
Authors: Taniguchi K , Petersson M , Höglund P , Kiessling R , Klein G , Kärre K
Published: May 2006

Treatment of H-2-deficient nonmetastatic B16 melanoma cells with physiological doses of interferon gamma (IFN-gamma) reduced cellular growth in vitro but induced a shift to the lung-colonizing phenotype as assessed after intravenous injection of the treated cells. As little as 1 antiviral unit of recombinant IFN-gamma per ml induced B16 cells to form 3-40 pulmonary metastases in each injected mouse, whereas a 1000-fold higher concentration of IFN-beta was required to see similar effects. IFN-gamma may induce cell-surface molecules that contribute to the metastatic ability of the tumor cells. The efficient enhancement of metastatic ability after IFN-gamma treatment of the B16 cells was paralleled by an increased H-2 antigen expression and decreased sensitivity to natural killer cells. The experiments support the idea that metastasis may not depend exclusively on stable genetic changes or heterogeneity within a tumor population but may be also influenced through the modulation of the phenotype by physiological or pharmacological agents. The results are also discussed with regard to the role of different effector cells in tumor cell clearance and in relation to lymphokine-based strategies for therapy.
Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line.
Authors: Drucker D J , Philippe J , Mojsov S , Chick W L , Habener J F
Published: May 2006

Insulin secretion is controlled by a complex set of factors. Although blood glucose levels serve as the major stimulus of insulin secretion in mammals, insulin release is also modulated by amino acids, catecholamines, glucagon, and other, intestinal hormones. The identification of factors that modulate insulin production has engendered much interest because of their potential importance in the altered dynamics of insulin secretion in response to glucose characteristic of maturity-onset diabetes mellitus. Decoding of the glucagon gene has uncovered two additional glucagon-like peptides encoded in proglucagon, the polypeptide precursor of glucagon. One of these peptides, glucagon-like peptide I, is processed from proglucagon in two forms, of 31 and 37 amino acids. We report that the smaller of the two glucagon-like peptides potently increases cAMP levels, insulin mRNA transcripts, and insulin release in cultured rat insulinoma cells. These results indicate that glucagon-like peptide I may be a physiologic modulator of insulin gene expression.
Characterization of five partial deletions of the factor VIII gene.
Authors: Youssoufian H , Antonarakis S E , Aronis S , Tsiftis G , Phillips D G , Kazazian H H
Published: May 2006

Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. By using cloned DNA probes, we have characterized the following five different partial deletions of the factor VIII gene from a panel of 83 patients with hemophilia A: (i) a 7-kilobase (kb) deletion that eliminates exon 6; (ii) a 2.5-kb deletion that eliminates 5' sequences of exon 14; (iii) a deletion of at least 7 kb that eliminates exons 24 and 25; (iv) a deletion of at least 16 kb that eliminates exons 23-25; and (v) a 5.5-kb deletion that eliminates exon 22. The first four deletions are associated with severe hemophilia A. By contrast, the last deletion is associated with moderate disease, possibly because of in-frame splicing from moderate disease, possibly because of in-frame splicing from adjacent exons. None of those patients with partial gene deletions had circulating inhibitors to factor VIII. One deletion occurred de novo in a germ cell of the maternal grandmother, while a second deletion occurred in a germ cell of the maternal grandfather. These observations demonstrate that de novo deletions of X-linked genes can occur in either male or female gametes.
Effector and suppressor circuits of the immune response are activated in vivo by different mechanisms.
Authors: Okamoto H , Kripke M L
Published: May 2006

The application of fluorescein isothiocyanate (FITC) onto the skin of mice induces a contact hypersensitivity immune response. Lymph nodes draining the skin painted with FITC contain fluorescent cells that induce contact hypersensitivity to FITC when injected into normal mice. The antigen-presenting cells responsible for activating the effector pathway of the contact hypersensitivity response express Ia histocompatibility determinants and are resistant to inactivation with gamma-radiation. Exposing the skin to low doses of UV radiation (280-320 nm) before the application of FITC suppresses the contact hypersensitivity response to FITC. Cells present in the draining lymph nodes of these mice induce suppressor T lymphocytes when injected into normal recipients. The inducer cells in the draining lymph nodes are Thy 1+, Ia- and are inactivated by gamma-radiation. These studies demonstrate that different mechanisms are involved in the in vivo activation of effector and suppressor immune responses, and they suggest that the mode of initial antigen presentation determines which immunologic circuit will be activated in response to a contact-sensitizing antigen.
Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells.
Authors: Westermark P , Wernstedt C , Wilander E , Hayden D W , O'Brien T D , Johnson K H
Published: May 2006

Amyloid deposits localized to the islets of Langerhans are typical of non-insulin-dependent human diabetes mellitus and of diabetes mellitus in adult cats. Amyloid deposits also commonly occur in insulin-producing pancreatic tumors. We have purified a major protein--insulinoma or islet amyloid polypeptide (IAPP)--from human and cat islet amyloid and from amyloid of a human insulinoma. IAPP from human insulinoma contained 37 amino acid residues and had a theoretical molecular mass of 3850 Da. The amino acid sequence is unique but has greater than 40% identity with the human calcitonin gene-related peptide. A partial amino acid sequence of cat islet IAPP corresponding to positions 1-27 of human insulinoma IAPP was identical to the human IAPP except for substitutions in three positions. An antiserum raised to a synthetic human insulinoma IAPP-(7-17) undecapeptide showed specific immunohistochemical reactivity with human and cat islet amyloid and with islet B cells. The significance of this pancreatic neuropeptide-like protein is unknown, but it is suggested that it may exert an important endocrine regulatory effect.
Rod phosphorylation favors folding in a catch muscle myosin.
Authors: Castellani L , Cohen C
Published: May 2006

Myosin from a molluscan catch muscle is unusual in being phosphorylated in the rod by an endogenous heavy chain kinase. The overall structure of the molecule resembles that of other muscle myosins, although the tail is somewhat longer (approximately equal to 1700 A). At low ionic strength the unphosphorylated molecules associate in filaments that display a striking axial repeat of 145 A. Phosphorylation of the rod enhances myosin solubility in the range of NaCl between 0.05 and 0.15 M. Depending on the ionic strength and the counterions present, the soluble species corresponds to an antiparallel folded dimer (15 S) or to a folded monomer (10 S). Unphosphorylated myosin can also be partially solubilized into folded monomers by addition of ATP in 0.15 M NaCl. A similar molecular folding has also been observed in smooth muscle and nonmuscle myosins that depends, however, on the state of phosphorylation of the light chains in the myosin head. We discuss these results in relation to possible mechanisms for control of catch contraction.
Voltage dependence of K
+
channels in guard-cell protoplasts
Authors: Schroeder Julian I. , Raschke Klaus , Neher Erwin
Published: May 2006

Stomatal pores in leaves enable plants to regulate the exchange of gases with their environment. Variations of the pore aperture are mediated by controlled changes of potassium salt concentrations in the surrounding guard cells. The voltage-dependent gating of K
+
-selective channels in the plasma membrane (plasmalemma) of cell-wall-free guard cells (protoplasts) was studied at the molecular level in order to investigate the regulation of K
+
fluxes during stomatal movements. Inward and outward K
+
currents across the plasmalemma of guard cells were identified by using the whole-cell configuration of the patch-clamp technique. Depolarizations of the membrane potential from a holding potential of -60 mV to values more positive than -40 mV produced outward currents that were shown to be carried by K
+
. Hyperpolarizations elicited inward K
+
currents. Inward and outward currents were selective for K
+
over Na
+
and could be partially blocked by exposure to extracellular Ba
2+
. In cell-attached and excised membrane patches, previously identified K
+
-selective single channels in guard cells were studied. Averaging of single-channel currents during voltage pulses resulted in activation and deactivation kinetics that were similar to corresponding kinetics of inward and outward currents in whole cells, showing that K
+
-selective channels were the molecular pathways for the K
+
currents recorded across the plasmalemma of single guard-cell protoplasts. Estimates demonstrate that K
+
currents through the voltage-gated K
+
channels recorded in whole guard cells can account for physiological K
+
fluxes reported to occur during stomatal movements in leaves.
Contribution of a p75 interleukin 2 binding peptide to a high-affinity interleukin 2 receptor complex.
Authors: Tsudo M , Kozak R W , Goldman C K , Waldmann T A
Published: May 2006

There are at least two forms of cellular receptors for interleukin 2 (IL-2); one with a very high affinity and the other with a lower affinity. We identified a non-Tac IL-2 binding peptide with a relative molecular weight of 75,000 (p75). Cell lines bearing either the p55 Tac or the p75 peptide alone manifested low-affinity IL-2 binding, whereas a cell line bearing both peptides manifested both high- and low-affinity receptors. After the internalization of labeled IL-2 through high-affinity receptors, the p75 peptide could not be detected by cross-linking studies. Furthermore, fusion of cell membranes from low-affinity IL-2 binding cell lines bearing the Tac peptide alone with membranes from a cell line bearing the p75 peptide alone generated hybrid membranes bearing high-affinity receptors. These results suggest a multichain model for the high-affinity IL-2 receptor in which high-affinity receptors would be expressed when both Tac and p75 IL-2 binding peptides are present and associated in a receptor complex.