bluuebunny/crossref_metadata_2025 huggingface dataset:
Superoxide mediates the toxicity of paraquat for Chinese hamster ovary cells.
Authors: Bagley A C , Krall J , Lynch R E
Published: May 2006

The roles of superoxide and H2O2 in the cytotoxicity of paraquat were assessed in Chinese hamster ovary cells. Neither catalase nor superoxide dismutase inhibited the loss of ability to form colonies when added to the medium. When introduced into the cells, superoxide dismutase but not catalase inhibited the toxicity of paraquat. That superoxide dismutase acted by its known catalytic action is shown by the loss of inhibition when the enzyme was inactivated by H2O2 before being introduced into the cells. The lack of inhibition by catalase, by dimethyl sulfoxide, and by desferoxamine suggests that the toxicity is not mediated by a reaction between H2O2 and superoxide to engender the hydroxyl radical. Exposure of Chinese hamster ovary cells to paraquat may be a suitable means to determine the effects of superoxide anion in cultured cells and the ways in which cells can resist this toxic action.
Activation of gene expression is adversely affected at high multiplicities of linked simian virus 40 enhancer.
Authors: Kumar R , Firak T A , Schroll C T , Subramanian K N
Published: May 2006

We have cloned multiple copies of 72-base-pair (bp) repeat transcriptional enhancer element from simian virus 40 in plasmid vectors upstream from the bacterial chloramphenicol acetyltransferase gene under the control of the simian virus 40 early promoter. Two copies of the 72-bp repeat provided efficient activation of gene expression. Increasing the number of linked 72-bp units to four substantially improved the activation of gene expression, but further addition of enhancers diminished the activation of gene expression proportionally to the number of enhancers added. Enhancer regions containing 10 or more copies of the 72-bp sequence were very inefficient in gene activation. Plasmids containing such expanded enhancer regions also competed less efficiently in vivo for trans-acting enhancer-binding factors. These effects are specific for the enhancer element and are not produced by reiterations of the 21-bp promoter element. Multiple enhancer units placed upstream do not interfere with the accuracy of mRNA initiation directed by the simian virus 40 early promoter in these plasmid constructs but do severely inhibit the initiation of replication at the neighboring simian virus 40 origin of replication that overlaps the early promoter region. These results are consistent with the hypothesis that the structural alterations induced in the DNA by a large number of copies of the enhancer are not favorable for the activation of a linked gene or for the binding of factors believed to mediate the enhancement effect.
The crystal structures of [Met5]enkephalin and a third form of [Leu5]enkephalin: observations of a novel pleated beta-sheet.
Authors: Griffin J F , Langs D A , Smith G D , Blundell T L , Tickle I J , Bedarkar S
Published: May 2006

The structures of [Met5]enkephalin (Tyr-Gly-Gly-Phe-Met) and [Leu5]enkephalin (Tyr-Gly-Gly-Phe-Leu) have been determined from single crystal x-ray diffraction data and refined to residuals of 0.100 and 0.092, respectively. The [Met5]enkephalin structure consists of dimers forming antiparallel beta-sheets extending in the monoclinic ac plane with 10.6 water molecules per dimer. The two molecules, related by pseudo two-fold axes, have similar backbone conformations and similar tyrosine and phenylalanine side-chain conformations. Both methionine residues are disordered and the disorder is different in the two independent molecules. Additional hydrogen bonds connect adjacent dimers to form infinite sheets normal to the b axis. The water molecules are found mainly in the interstices between the sheets. [Leu5]Enkephalin crystallizes as a monohydrate that is isomorphous with the [Met5]enkephalin structure with respect to the beta-sheet but different with respect to the tyrosine and phenylalanine side-chain conformations and water content. The peptide chains in both structures are fully extended and more nearly planar than pleated. The planes of the peptide chains in the dimers form an angle of 143.3 degrees with one another in [Met5]enkephalin and 156.0 degrees in [Leu5]enkephalin. This produces a zigzag pattern or pleat in the beta-sheets perpendicular to the direction of the peptide chains and, therefore, perpendicular to the normal beta-sheet pleat. The average repeat distance between Ni and Ni+2 in the peptide chains of both structures is 7.10 A, versus an ideal value of 6.68 A.
“Agroinfection,” an alternative route for viral infection of plants by using the Ti plasmid
Authors: Grimsley Nigel , Hohn Barbara , Hohn Thomas , Walden Richard
Published: May 2006

Most plant viruses are transmitted by insect vectors. We present an alternative method for the introduction of infectious viral DNA that uses the ability of
Agrobacterium
to transfer DNA from bacterial cells to plants. Cauliflower mosaic virus was chosen to develop this method because it is the best characterized plant DNA virus and can be introduced into plants via aphids, virus particles, viral DNA, or suitably treated cloned DNA. We show that systemic infection of turnips results from wounding and inoculation with strains of
Agrobacterium tumefaciens
in which more than one genome of cauliflower mosaic virus have been placed tandemly in the T-DNA of the tumor-inducing plasmid. Thus such constructions allow escape of the viral genome from the T-DNA once inside the plants. The combined use of the tumor-inducing plasmid and viral DNA opens the way to molecular biological approaches that are not possible with either system alone.
Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA.
Authors: Ashman C R , Jagadeeswaran P , Davidson R L
Published: May 2006

A retroviral shuttle vector was constructed by introducing the Escherichia coli xanthine (guanine) phosphoribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, 1053-1062]. This vector was packaged into infectious virus which then was used to infect a hypoxanthine (guanine) phosphoribosyltransferase-deficient mouse cell line. Cell lines that expressed the gpt gene were isolated, and it was found that these cells contained a single integrated copy of the vector in a proviral form. Treatment of these cell lines with either ethyl methanesulfonate or BrdUrd produced a greater than 10-fold increase in the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact gpt genes have been recovered from a number of Sgur cell lines after COS cell fusion and introduced into E. coli as part of a plasmid. The complete DNA sequences of three mutant genes have been determined. Two of the mutant genes have a single base substitution, whereas the third has a 34-base-pair deletion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is that the mutations are induced in genes integrated into mammalian chromosomes rather than in genes existing as part of autonomously replicating plasmids.
HLA-DR2, -DR5, and DRw6 associated Dw subtypes correlate with HLA-DR beta and -DQ beta restriction fragment length polymorphisms.
Authors: Font M P , Gebuhrer L , Betuel H , Freidel C , Dausset J , Cohen D
Published: May 2006

DNAs extracted from peripheral blood leukocytes of 24 individuals, selected for their HLA-DR types, -DR2, -DR5, and -DRw6, were analyzed with four restriction enzymes, BamHI, EcoRV, HindIII, and Taq I, using the Southern technique. This panel includes 16 individuals with homozygous typing cells and 8 heterozygous individuals who carry rare Dw subtypes or unusual DR-DQ associations. Eighty-five polymorphic fragments were detected and assigned to the DR or DQ gene families according to their hybridization signals. Thirty-eight fragments (DR or DQ) were found to correlate with single DR or Dw specificities or rare associations such as DRw14-DQw3. Forty-two fragments correlated with the association of immunologically defined specificities. In total, these 85 fragments constituted 44 different patterns, each comprising 1-9 fragments. For each homozygous typing cell a combination of patterns was observed. Fourteen different combinations of 10-20 patterns were found among the 16 individuals with homozygous typing cells, showing that Dw18, Dw19, Dw9, and Dw5 are heterogeneous at the genomic level whereas only the Dw2 individuals tested here are identical.
Illegitimate recombination at the replication origin of bacteriophage M13.
Authors: Michel B , Ehrlich S D
Published: May 2006

Hybrids composed of phage M13 and plasmid pHV33 were used to study the formation of deletions in Escherichia coli. Eighty to ninety percent of the deletion endpoints were at the position of the nick introduced into the M13 replication origin by the phage gene II protein. This suggests the existence of a novel mechanism of illegitimate recombination.
DNA linkage analysis of X chromosome-linked chronic granulomatous disease.
Authors: Baehner R L , Kunkel L M , Monaco A P , Haines J L , Conneally P M , Palmer C , Heerema N , Orkin S H
Published: May 2006

Chronic granulomatous disease (CGD) is a disorder of phagocytes that is usually inherited as an X chromosome-linked trait. Previous family studies suggested that the CGD locus resides on the distal short arm (Xp22-Xpter). Using cloned, polymorphic DNA probes we have performed a linkage analysis within CGD families that suggests a more proximal location (Xp21). In addition, the CGD locus is proximal to the Duchenne muscular dystrophy locus and lies within a broad region of Xp in which recombination appears to be greater than anticipated on the basis of physical distance between markers. Regional localization of the X chromosome CGD locus should facilitate molecular cloning of the CGD gene and molecular dissection of the phagocyte oxidase system.
Chemically induced mutagenesis in a shuttle vector with a low-background mutant frequency.
Authors: Drinkwater N R , Klinedinst D K
Published: May 2006

We have developed a recombinant DNA shuttle vector that permits the molecular analysis of mutations induced in human cells by chemical or physical mutagens. The vector is able to replicate as a plasmid in Escherichia coli and in Epstein-Barr virus (EBV)-transformed human lymphoblastoid cell lines and contains the herpes simplex virus type 1 thymidine kinase gene (HSV tk) as the target for mutagenesis studies. After introduction of the vector into an EBV-transformed lymphoblastoid cell line (LCL-721) by electroporation, approximately equal to 2% of the transfected cells expressed the vector-encoded gene for hygromycin resistance. Plasmid DNA isolated from cells immediately after selection for hygromycin resistance (10 population doublings posttransfection) contained mutations in the HSV tk gene at a frequency of 6 X 10(-5). Treatment of plasmid-bearing LCL-721 cells with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of up to 15-fold in the frequency of mutations in the HSV tk gene. The dose-response for the induction of mutations in the plasmid-encoded gene closely paralleled that for the induction of mutations in the cellular gene for hypoxanthine (guanine) phosphoribosyltransferase.
Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocytes.
Authors: Hackett J , Bosma G C , Bosma M J , Bennett M , Kumar V
Published: May 2006

We have utilized a mouse mutant (C.B-17 scid) that lacks functional T and B lymphocytes to examine the relationship among transplantable progenitors of natural killer (NK) cells, T cells, and B cells. The NK-progenitor cells contained in the bone marrow were detected by their ability to generate mature NK cells, following transfer of bone marrow cells into NK cell-depleted and lethally irradiated mice. Regeneration of NK activity in the recipient mice was monitored by two different assays: the ability to rapidly clear infused YAC-1 cells in vivo and the ability of spleen cells to lyse YAC-1 cells in vitro. Recipients were also tested for the presence of mitogen-responsive T and B cells and for prethymocytes (thymus-repopulating cells). We found that the capacity of C.B-17 scid bone marrow cells to generate mature NK cells was equivalent to that of control C.B-17 bone marrow cells. The regenerated NK cells shared similar functional activity and surface phenotype. In contrast, bone marrow cells from C.B-17 scid mice failed to generate thymocytes and peripheral T and B cells. These data indicate that the transplantable NK-progenitor cells are not defective or deficient in C.B-17 scid mice and, therefore, are distinct from the transplantable progenitor(s) of T and B cells.
Lectin activity as a marker for Hodgkin disease cells.
Authors: Paietta E , Stockert R J , Morell A G , Diehl V , Wiernik P H
Published: May 2006

Treatment of cultured Hodgkin disease (HD) cells with neuraminidase results in decreased reactivity of monoclonal antibody VIM-D5 with its antigen, the X hapten, a fucosyl-N-acetyllactosamine. The other feature characteristic of HD cells is the expression of high levels of ectosialyltransferase activity. We present evidence for a cause-effect relationship between these two findings in that VIM-D5 antigenicity can be restored on neuraminidase-treated HD cells by modulating transferase activity. This can be interpreted in terms of a lectin activity of the ectosialyltransferase that binds the X hapten's desialylated galactosyl residues, thereby preventing antigen recognition by VIM-D5 antibody. This proposed mechanism is indistinguishable from the autoinhibition phenomenon described for another galactophilic binding protein, the hepatic binding protein (HBP), which binds its own terminal galactosyl residues following neuraminidase treatment. We establish a close relationship between the HD galactophilic binding site and HBP in that antiserum to HBP (i) inhibits the neuraminidase-induced loss of VIM-D5 antigenicity, (ii) blocks the binding of asialoglycoprotein to hepatocytes after being absorbed by and eluted from HD cells, and (iii) recognizes a single HD protein, which in its high level of expression is unique to HD cells. The presence of lectin activity in its classic sense on the surface of HD cells is confirmed by the erythrocyte-agglutinating ability of these cells. This lectin activity, which appears to be related to an ectosialyltransferase on the surface of HD cells, may serve as a marker for the abnormal cells characteristic of HD.
Location of the major antigenic sites involved in rotavirus serotype-specific neutralization.
Authors: Dyall-Smith M L , Lazdins I , Tregear G W , Holmes I H
Published: May 2006

Antigenic sites on the rotavirus major outershell glycoprotein were identified by using mutant viruses selected for resistance to neutralizing (serotype-specific) monoclonal antibodies. The glycoprotein genes from these mutants were sequenced to determine the position and nature of the resultant amino acid substitutions in the protein. Three regions (A, B, and C) were identified (amino acids 87-96, 145-150, and 211-223, respectively), of which region C appears to be the most important. A mutation in region C caused a 10-fold increase in resistance to neutralization by polyclonal antiviral antiserum. The results of this study, together with other data, indicate that the three-dimensional folding of the native protein is such that regions A and C are in close proximity.
Thomas-Fermi theory for atomic systems
Authors: Parr Robert G. , Ghosh Swapan K.
Published: May 2006

The classical Thomas-Fermi theory of the electrons in an atom is amended in a manner that produces continuity of the electron density ρ. This is done by imposing the constraint that ʃ
e

-2
kr

[unk]
2
ρ
d
r should be finite, with
k
determined by the nuclear cusp condition, followed or preceded by an optimum coordinate scaling. Electron densities and total energies are shown to be vastly improved.
A chemically explicit model for the molecular mechanism of the F1F0 H+-ATPase/ATP synthases.
Authors: Scarborough G A
Published: May 2006

A general hypothesis for the molecular mechanism of membrane transport based on current knowledge of protein structure and the nature of ligand-induced protein conformational changes has recently been proposed [Scarborough, G. A. (1985) Microbiol. Rev. 49, 214-231]. According to this hypothesis, the essential reaction undergone by all proteinaceous transport catalysts is a ligand-induced hinge-bending-type conformational change that results in the transposition of binding-site residues from access on one side of the membrane to access on the other side. Subsequent release and/or alteration of the ligand or ligands that induce the conformational change facilitates the converse conformational change, which returns the binding-site residues to their original position. With this simple cyclic ligand-dependent gating process as a central feature, biochemically orthodox mechanisms for virtually all known transporters are readily conceived. In this article, a chemically explicit model for the molecular mechanism of the F1F0 H+-ATPase/ATP synthases of mitochondria, bacteria, and chloroplasts, formulated within the guidelines of this general transport paradigm, is presented. At least three points of potential interest arise from this exercise. First, with the aid of the model, it is possible to visualize how energy transduction catalyzed by these enzymes might proceed, with no major events left unspecified. Second, explicit possibilities as to the molecular nature of electric field effects on the transport process are raised. And finally, it is shown that enzyme conformational changes, energy-dependent binding-affinity changes, and several other related phenomena as well, need not be taken as evidence of "action at a distance" or indirect energy coupling mechanisms, as is sometimes assumed, because such events are also integral features of the mechanism presented, even though all of the key reactions proposed for both ATP-driven proton translocation and proton translocation-driven ATP synthesis occur at the enzyme active site.
Cellular myc (c-myc) in fish (rainbow trout): its relationship to other vertebrate myc genes and to the transforming genes of the MC29 family of viruses.
Authors: Van Beneden R J , Watson D K , Chen T T , Lautenberger J A , Papas T S
Published: May 2006

We have isolated, cloned, and sequenced the rainbow trout (Salmo gairdneri) c-myc gene. The presumptive coding region of the trout c-myc gene shows extensive homology to the c-myc genes of chicken, mouse, and human. Comparison of nucleotide sequences reveals that human, mouse, chicken, and trout c-myc genes contain at least two coding exons, interrupted by introns of decreasing size of 1.38 kilobases (kb), 1.2 kb, 0.97 kb, and 0.33 kb, respectively. The exons are clearly delineated by donor-acceptor splice signals. The degree of nucleotide homology between trout, chicken, and human exon II is less than that observed for exon III. However, the greatest homology among these three genes is localized to two specific regions within exon II (myc boxes A and B). At the predicted amino acid level, fish c-myc shows considerable homology to vertebrate c-myc gene products. Trout c-myc is expressed in normal trout cells as a single 2.3-kb mRNA species, similar in size to other vertebrate transcripts.
Reduced hormone-stimulated adenylate cyclase activity in NIH-3T3 cells expressing the EJ human bladder ras oncogene.
Authors: Tarpley W G , Hopkins N K , Gorman R R
Published: May 2006

Recent studies have shown that the 21-kilodalton protein (p21) Ha-ras gene product shares sequence homology with and may exhibit biochemical properties similar to the mammalian guanine nucleotide-binding proteins. These data suggested that one of the biochemical functions of p21 in the vertebrate cell may be to regulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. We determined both in intact NIH-3T3 murine cells and in membranes isolated from these cells that the hormone-stimulated adenylate cyclase activity of cells expressing the EJ human bladder carcinoma oncogene (EJ-ras) is significantly reduced compared with control cells. Thus, the levels of cAMP measured in the EJ-ras-transformed cells by radioimmunoassay are reduced 78% and 93% after prostaglandin and isoproterenol stimulation, respectively, compared with the levels in control cells. Treatment of the EJ-ras-transformed cells with pertussis toxin or cholera toxin did not correct the alterations in adenylate cyclase activity. Cells expressing the normal human Ha-ras gene displayed intermediate levels of adenylate cyclase hormone sensitivity; these levels of adenylate cyclase activity were greater than those in the EJ-ras-transformed cells but lower than in control cells. Hormone-stimulated adenylate cyclase activities in cells transfected with Rous sarcoma virus DNA were similar to those in control cells. These data support the hypothesis that both the normal and mutated Ha-ras p21s are related to guanine nucleotide-binding proteins.
The neuronal identifier element is a cis-acting positive regulator of gene expression.
Authors: McKinnon R D , Shinnick T M , Sutcliffe J G
Published: May 2006

A middle-repetitive DNA element termed the identifier (ID) sequence, located in introns of postnatal-onset neuronal-specific genes, is transcribed in early postnatal rats by RNA polymerase III (Pol III) specifically in the brain. We show that these Pol III transcripts, although brain specific in vivo, are also expressed in immortalized rodent cell lines in culture. We demonstrate that the ID sequence can act as a positive regulator or enhancer of RNA polymerase II gene expression in cell lines that express these RNAs but not in primary cells in which Pol III transcription of the ID sequence is absent. Thus, ID elements may be positive regulators of postnatal-onset neuronal-specific gene expression.
Small-angle neutron scattering studies of chlorophyll micelles: Models for bacterial antenna chlorophyll
Authors: Worcester D. L. , Michalski T. J. , Katz J. J.
Published: May 2006

Micelles of hydrated chlorophyll a (P740), bacteriochlorophyll a (P865), bacteriochlorophyll c (P750), and pheophytin a prepared in organic media have been studied by small-angle neutron scattering to determine their shape, size, and mass per unit length. All of the micelles are hollow cylinders of well-defined size. The P740 and P750 cylinders are essentially monolayers of macrocycles crosslinked by water, probably in an arrangement similar to that of crystals of chlorophyll derivatives. The P865 micelle is more nearly a bilayer of macrocycles. We show that the curvature necessary to form cylinders probably results from intrinsic curvature of the five-coordinated chlorophyll macrocycle. Studies of P740 micelle formation and the disaggregating effects of another nucleophile (pyridine) are described. As the P750 micelles are nearly identical in size and optical spectra to the rod-shaped structures observed in chlorosomes, and the P865 micelles have optical properties very similar to the
in vivo
properties of the long-wavelength antenna of purple photosynthetic bacteria, we propose that features of the hydrated cylindrical micelles of these chlorophylls provide good models for antenna chlorophyll in photosynthetic bacteria.
Developing brain produces an angiogenesis factor.
Authors: Risau W
Published: May 2006

Embryonic chicken brain was analyzed for the presence of growth factors for capillary endothelial cells. A growth factor was found whose total and specific activities began to increase around the 12th day of development and reached a plateau around the 14th-16th day. A high specific activity was observed early in embryonic development at days 3 and 4. The growth factor was partially purified from extracts of 18-day-old embryonic chicken brain by heparin-Sepharose affinity chromatography where it eluted as a single peak of activity at 1.2-1.4 M sodium chloride. In chromatography on Sephacryl S200 gels, the activity eluted at a molecular size of 16- to 18-kDa. The growth factor is mitogenic for endothelial cells but not for smooth muscle or glial cells. In vivo assays using rabbit cornea and chicken chorioallantoic membrane demonstrated the angiogenic capacity of the chromatographed growth factor. The relationship between this early factor, the subsequent invasion of blood vessels into neural tissue, and the formation of the blood-brain barrier is discussed.
Chromosome constitution of polyploid wheats: Introduction of diploid wheat chromosome 4
Authors: Wazuddin M. , Driscoll C. J.
Published: May 2006

Chromosome 4 of diploid wheat (chromosome
d4
) is not present in hexaploid wheat. This chromosome has been added to hexaploid wheat and observed not to pair meiotically with its 21 chromosomes. Also, chromosome
d4
compensates for Cornerstone male sterility, which involves a recessive mutation in chromosome arm 4AS. Chromosome
d4
has been separately substituted for chromosomes 4A and 4B. These two substituted hexaploid chromotypes have the entire genome of diploid wheat and may have agricultural significance. An alternative hypothesis of the evolution of polyploid wheats is proposed that involves the loss of chromosome
d4
and the retention of two versions of chromosome 4B at the early tetraploid stage.
The right border region of pTiT37 T-DNA is intrinsically more active than the left border region in promoting T-DNA transformation.
Authors: Jen G C , Chilton M D
Published: May 2006

Deletions of border regions of T-DNA on Agrobacterium Ti plasmid or mini-T plasmid have shown the right border region of pTiT37 T-DNA to be more active than the left border region in promoting T-DNA transformation. In this study we examine the possibility that the apparent difference in activity of left and right border regions may be due to position or orientation differences between the left and right borders with respect to the transferred onc genes. We have constructed eight similar single-border mini-T plasmids that contain a left border segment or a right border segment of pTiT37 T-DNA at various positions with either orientation with respect to the onc genes. We assayed the plant tumor-inducing activity of these mini-T plasmids in Agrobacterium tumefaciens LBA4404 containing the virulence helper plasmid pAL4404. Regardless of the position and orientation of the border-containing segment in the mini-T plasmid, mini-T plasmids with the right border segment were highly virulent, whereas those with the left border segment were only weakly so. These results indicate that the difference in transformational activity between the left and right border regions is intrinsic and not an effect of position or orientation with respect to the onc genes. The pattern of the mini-T plasmid sequences integrated into the plant genome suggests that T-DNA transformation involves the directional transfer of a linear intermediate bounded by the border repeats.
Purification, characterization, and antitumor activity of nonrecombinant mouse tumor necrosis factor.
Authors: Haranaka K , Carswell E A , Williamson B D , Prendergast J S , Satomi N , Old L J
Published: May 2006

Mouse tumor necrosis factor (TNF) was purified from serum through a series of steps, and each step was monitored for L-cell cytotoxicity in vitro and tumor-necrotizing activity in vivo. The two activities copurified and could not be dissociated. Purified mouse TNF has a specific activity of 2.2 X 10(7) (L-cell assay in the absence of actinomycin D) and 1 microgram causes necrosis of the standard TNF-sensitive sarcoma Meth A. TNF has a Mr of 39,000 +/- 2000 by gel filtration and a Mr of 16,000-18,000 by NaDodSO4/PAGE. Both molecular weight forms display cytotoxic and necrotizing activities. TNF has a pI of 3.9 and is destroyed by trypsin, protease, elastase, and alpha-chymotrypsin but not by neuraminidase or papain. These characteristics of nonrecombinant mouse TNF clearly resemble those of recombinant human and mouse TNF.
Demonstration of clonable alloreactive host T cells in a primate model for bone marrow transplantation.
Authors: Reisner Y , Ben-Bassat I , Douer D , Kaploon A , Schwartz E , Ramot B
Published: May 2006

The phenomenon of marrow rejection following supralethal radiochemotherapy was explained in the past mainly by non-T-cell mechanisms known to be resistant to high-dose irradiation. In the present study a low but significant number of radiochemoresistant-clonable T cells was found in the peripheral blood and spleen of Rhesus monkeys following the cytoreductive protocol used for treatment of leukemia patients prior to bone marrow transplantation. More than 95% of the clonable cells are concentrated in the spleen 5 days after transplant. The cells possess immune memory as demonstrated by the generation of alloreactive-specific cytotoxicity. The present findings suggest that host-versus-graft activity may be mediated by alloreactive T cells. It is hoped that elimination of such cells prior to bone marrow transplantation will increase the engraftment rate of HLA-nonidentical marrow in leukemia patients.
Herpes simplex virus immediate early infected-cell polypeptide 4 binds to DNA and promotes transcription.
Authors: Beard P , Faber S , Wilcox K W , Pizer L I
Published: May 2006

In herpes simplex virus (HSV)-infected cells, there is a sequential expression of viral genes. In vivo experiments have implicated the Mr 175,000 immediate early protein ICP4 (infected-cell polypeptide 4) in the regulation of viral RNA synthesis, but the mechanism whereby ICP4 regulates transcription of viral genes is at present unknown. In this report we describe experiments with an in vitro transcription system and a purified preparation of ICP4 (estimated 5% of total protein). Using DNA from the HSV glycoprotein D gene (gD) as the template, we have observed that specific binding occurs between ICP4 and DNA sequences adjacent to the gD gene promoter and ICP4 stimulates initiation of transcription from the gD gene. The degree of stimulation depends on the amount of ICP4 present in the incubation. The kinetics of RNA synthesis demonstrate that the protein acts at the initiation step of transcription. These results identify ICP4 as a viral transcription factor whose presence on DNA facilitates the formation of transcription complexes.
Mechanism for food avoidance learning in the central pattern generator of feeding behavior of
Pleurobranchae californica
Authors: London Jill A. , Gillette Rhanor
Published: May 2006

Food-avoidance conditioning in the mollusk
Pleurobranchaea
results in suppression of the feeding response to food stimuli. In conditioned animals, identified interneurons of the central pattern generator (CPG) for feeding behavior, the Int-2s, respond to a food stimulus with greater and more long-lasting excitation than controls. Enhanced Int-2 responsiveness to food stimuli is associated with markedly heightened Int-2 excitability. Sustained activity in the Int-2s arrests motor output of the oscillatory CPG in the protraction/retraction movement cycle of feeding through tonic excitation of a population of retractor interneurons and inhibition of protractors. The CPG locus of the learning mechanism is permissive of sensory excitation of alternative behavior and leaves the possibility open for release of the suppressed behavior in a fully aroused state.
Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid.
Authors: Tokino T , Murotsu T , Matsubara K
Published: May 2006

Mini-F plasmid encodes a protein, E protein, that is indispensable for its autonomous replication. We have constructed a plasmid that overproduces the E protein and have purified the protein to apparent homogeneity. Using nitrocellulose filter binding and nuclease digestion assays, we demonstrated that the E protein binds to three unique regions of the mini-F DNA sequence: the replication origin (ori2) and an incompatibility locus (incB), another incompatibility locus (incC), and the promoter for the E gene. These binding sites have a common 8-base-pair sequence. These findings suggest the direct role of the E protein in initiation of mini-F replication and copy number control. They are also in line with the in vivo evidence that the incompatibility phenotype caused by incB and incC DNA is due to titration of a factor(s) indispensable for replication and that the production of the E initiator protein of the mini-F plasmid is under autoregulatory control.
An atypical insulin receptor with high affinity for insulin-like growth factors copurified with placental insulin receptors.
Authors: Jonas H A , Newman J D , Harrison L C
Published: May 2006

Insulin receptors purified from human placenta by sequential affinity chromatography on wheat germ lectin-agarose and insulin coupled to 1,1'-carbonyldiimidazole-activated agarose (CDI-agarose) retained full binding activity but bound a greater than predicted amount of 125I-labeled insulin-like growth factor I (IGF-I). IGF-I and multiplication-stimulating activity (MSA; the rat homologue of IGF-II) were equipotent in displacing either 125I-labeled IGF-I or 125I-labeled MSA from the purified receptors; insulin was 5-15 times more potent. Competitive binding studies indicated that this IGF binding activity could not be explained by cross-reaction with classical insulin receptors or by coelution of IGF-I or IGF-II receptors. Instead, it was due to a minor population of discrete atypical insulin receptors (6-18% total insulin receptors) with moderately high affinity (Kd = 2-4 X 10(-9) M) for IGF-I and MSA. These receptors were not an artifact of insulin-CDI-agarose chromatography, since they were present in wheat germ lectin-agarose-purified preparations and could also be purified from insulin-succinyldiaminodipropylamino-agarose. Affinity labeling with 125I-labeled MSA revealed that these atypical receptors had the same binding subunit (Mr 140,000) as classical insulin and IGF-I receptors. They displayed intermediate reactivity with polyclonal and monoclonal antibodies to the insulin and IGF-I receptors. It is therefore likely that insulin receptors purified by immunoadsorption would also contain atypical insulin receptors. The finding of more than one type of insulin receptor might relate to the slight variations in the cDNA nucleotide sequences and the multiple mRNA species reported for the insulin receptor [Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758].
Carbon source regulation of RAS1 expression in Saccharomyces cerevisiae and the phenotypes of ras2- cells.
Authors: Breviario D , Hinnebusch A , Cannon J , Tatchell K , Dhar R
Published: May 2006

Transcriptional analysis of the yeast RAS genes in different culture conditions suggests that the inability of ras2 mutants to grow in nonfermentable carbon sources results from the regulation of RAS1 mRNA expression. The amount of RAS1 mRNA is significantly repressed in cultures grown on the nonfermentable carbon sources ethanol and acetate. As a result, low RAS function should be expressed under these conditions in a ras2 mutant. This can explain the inability of ras2- cells to grow on nonfermentable carbon sources. This interpretation is supported by the finding that an extragenic suppressor of ras2- (sra6-15), which restores growth on ethanol or acetate, also leads to an increase in the amount of RAS1 mRNA under these conditions. The sra6-15 mutation does not alter the level of RAS1 mRNA in cells grown on glucose. The pattern of transcriptional regulation described for the RAS1 gene is not shared by RAS2, indicating differential control of the functionally homologous yeast RAS genes at the level of gene expression.
Analysis of the phorbol ester pharmacophore on protein kinase C as a guide to the rational design of new classes of analogs.
Authors: Wender P A , Koehler K F , Sharkey N A , Dell'Aquila M L , Blumberg P M
Published: May 2006

The diterpene diester phorbol 12-myristate 13-acetate and the alkaloid teleocidin B are structurally unrelated natural products that display similar potent irritant and tumor-promoting activities. Computer modeling of these and other structural classes of tumor promoters show a marked similarity in the relative positions of certain heteroatoms and hydrophobic groups. For phorbol this mapping consists of the C-4, C-9, and C-20 hydroxyl groups as well as a hydrophobic region filled by a long-chain acyl functionality attached to either the C-12 or the C-13 positions. Diacylglycerols, thought to be the endogenous activators of the major phorbol ester receptor protein kinase C likewise fit this model in a stereospecific fashion. As an initial test of the utility of the model, members of a new and simplified class of activators were synthesized that possess the predicted essential structural features. These compounds all inhibited specific phorbol ester binding to protein kinase C, albeit with low affinity (10-60 microM); further analysis of one derivative, decylhydroxylindole, confirmed that the inhibition of phorbol ester binding was competitive. This same derivative inhibited epidermal growth factor binding in intact Swiss 3T3 cells and studies with another derivative showed phosphorylation of a 40-kDa protein in platelets. Both of these in vivo responses are characteristic of phorbol esters.
Structure of a bovine gene for P-450c21 (steroid 21-hydroxylase) defines a novel cytochrome P-450 gene family.
Authors: Chung B C , Matteson K J , Miller W L
Published: May 2006

P-450c21, a cytochrome P-450 enzyme [steroid 21-monooxygenase (steroid 21-hydroxylase), EC 1.14.99.10], mediates the 21-hydroxylation of glucocorticoid and mineralocorticoid hormones in the adrenal gland. The complete sequence of a bovine P-450c21 gene shows it is 3447 base pairs long and contains 10 exons. The intron/exon organization and encoded amino acid sequence indicate that P-450c21 represents a unique family of genes in the P-450 gene superfamily. Primer extension and S1 nuclease protection experiments identified several cap sites for initiation of transcription; the principal cap site produces mRNA with a 5' untranslated region only 11 bases long. S1 nuclease protection experiments confirm that P-450c21 is actively expressed in the adrenal and the testis, an organ not known to secrete 21-hydroxylated steroids.
Two related but distinct forms of the Mr 36,000 tyrosine kinase substrate (calpactin) that interact with phospholipid and actin in a Ca2+-dependent manner.
Authors: Glenney J
Published: May 2006

A method was devised that allows the identification of proteins related to the Mr 36,000 tyrosine kinase substrate calpactin based on their ability to interact with actin and phospholipid in a calcium-dependent manner. Two distinct proteins, detected in human A431 cells and fibroblasts, were resolved by two-dimensional gel electrophoresis. One of these proteins (calpactin I) appears identical to the Mr 34,000-39,000 substrate of the pp60src tyrosine kinase and the second (calpactin II) reacts with antibodies to the Mr 35,000 substrate of the epidermal growth factor receptor. Both proteins interact with phospholipid and actin, are rather basic, and share structural and antigenic determinants. A major difference between the two proteins is noted in their state of association with the Mr 10,000 light chain; i.e., calpactin I is associated with the light chain while calpactin II is not.
Analog "neuronal" networks in early vision.
Authors: Koch C , Marroquin J , Yuille A
Published: May 2006

Many problems in early vision can be formulated in terms of minimizing a cost function. Examples are shape from shading, edge detection, motion analysis, structure from motion, and surface interpolation. As shown by Poggio and Koch [Poggio, T. & Koch, C. (1985) Proc. R. Soc. London, Ser. B 226, 303-323], quadratic variational problems, an important subset of early vision tasks, can be "solved" by linear, analog electrical, or chemical networks. However, in the presence of discontinuities, the cost function is nonquadratic, raising the question of designing efficient algorithms for computing the optimal solution. Recently, Hopfield and Tank [Hopfield, J. J. & Tank, D. W. (1985) Biol. Cybern. 52, 141-152] have shown that networks of nonlinear analog "neurons" can be effective in computing the solution of optimization problems. We show how these networks can be generalized to solve the nonconvex energy functionals of early vision. We illustrate this approach by implementing a specific analog network, solving the problem of reconstructing a smooth surface from sparse data while preserving its discontinuities. These results suggest a novel computational strategy for solving early vision problems in both biological and real-time artificial vision systems.
Determination of the molecularity of the colicin E1 channel by stopped-flow ion flux kinetics.
Authors: Bruggemann E P , Kayalar C
Published: May 2006

A fluorescence technique that measures fast ion fluxes across liposome membranes was used to determine the molecularity of the colicin E1 channel. The rate of flux of Tl+ (used as a K+ analogue) into large unilamellar vesicles was measured by its ability to quench the fluorescence of a membrane-impermeable fluorophore entrapped in the vesicles. The dependence of Tl+ flux rate on the concentration of ionophore in the vesicle suspension reveals the molecularity of the ionophore. The method is demonstrated with two ionophores, valinomycin and gramicidin, whose molecularities are known to be one and two, respectively. The molecularity of the colicin E1 channel was determined to be one. This method can be used to study the properties of any ionophore that mediates K+ flux.
Differential response to the cytopathic effects of human T-cell lymphotropic virus type III (HTLV-III) superinfection in T4+ (helper) and T8+ (suppressor) T-cell clones transformed by HTLV-I.
Authors: De Rossi A , Franchini G , Aldovini A , Del Mistro A , Chieco-Bianchi L , Gallo R C , Wong-Staal F
Published: May 2006

We isolated six human T-cell lymphotropic virus type I (HTLV-I)-transformed T-cell clones carrying the phenotypic markers of helper and suppressor T cells. Five of the transformed T-cell clones produced infectious HTLV-I, but one (clone 55) contained a defective provirus and was therefore not competent for viral replication. To test whether there is interference between HTLV-I and the cytopathic virus HTLV-III in infection and/or their biological effects, we superinfected these T-cell clones with HTLV-III. The recipient cells that we used displayed either the OKT4 or the OKT8 membrane antigens (helper or suppressor phenotype, respectively). The superinfection was successful in all cases, regardless of phenotype of the recipient cells and status of viral production. Both HTLV-III and HTLV-I were expressed by the infected cell lines containing complete HTLV-I proviruses, as demonstrated by electron microscopy and immunofluorescence. However, only HTLV-III in the virus mixture obtained from the culture supernatants was transmitted to the human neoplastic T-cell line H9. The nonproducer clone 55 did not express HTLV-I upon superinfection with HTLV-III. HTLV-III exerted its cytopathic effect on all but one of the superinfected T-cell clones 15-20 days after infection. The exception, clone 67, is also the only cell clone that expresses the phenotypic marker of suppressor T lymphocytes (OKT8); the other clones carry the OKT4 antigen, correlated with helper functions. The virus released from the superinfected clone 67 is cytopathic for fresh peripheral and umbilical-cord blood lymphocytes, suggesting that cellular factors, rather than a genetic change in the virus, may be responsible for the lack of cytopathic effect of HTLV-III on the suppressor T-cell clone 67.
Regulated gene expression in transfected primary chicken erythrocytes.
Authors: Hesse J E , Nickol J M , Lieber M R , Felsenfeld G
Published: May 2006

We describe a method for studying transient gene expression in primary avian erythroid cells that involves controlled osmotic shock, followed by DNA transfection using DEAE-dextran. Cells treated in this way reproducibly express high levels of chloramphenicol acetyltransferase (CAT) when transfected with a plasmid having the cat gene coupled to an appropriate viral promoter. An observed correlation between levels of CAT expression and extent of hemoglobin release during controlled shock makes it possible to choose optimum conditions for expression in erythroid cells at various stages of embryonic development. Using these techniques, we have investigated the effect on CAT expression of fusing to the cat gene various portions of the chicken adult beta-globin (beta A) gene. We show that in 9-day or 12-day embryonic erythrocytes, the promoter activity of the 5' flanking region of the beta A gene (in the absence of any viral promoters) is strongly stimulated by a downstream sequence, located in the region 110-588 base pairs on the 3' side of the poly(A) signal, that acts as an enhancer. Its activity is reduced in 5-day embryonic cells and absent in primary chicken fibroblasts and mouse L cells, suggesting that this transient expression system will be useful in studying developmentally regulated globin gene expression.
Rhizobium meliloti genes required for nodule development are related to chromosomal virulence genes in Agrobacterium tumefaciens
Authors: Dylan T. , Ielpi L. , Stanfield S. , Kashyap L. , Douglas C. , Yanofsky M. , Nester E. , Helinski D. R. , Ditta G.
Published: May 2006

Symbiotically essential genes have been identified in
Rhizobium meliloti
that are structurally and functionally related to chromosomal virulence (
chv
) genes of
Agrobacterium tumefaciens
. Homologous sequences also exist in the genomes of other fast-growing rhizobia including
Rhizobium trifolii, Rhizobium leguminosarum
, and
Rhizobium phaseoli
. In
Agrobacterium
, the
chvA
and
chvB
loci are known to be essential for oncogenic transformation of dicotyledonous plants and for attachment to plant cells [Douglas, C. J., Staneloni, R. J., Rubin, R. A. & Nester, E. W. (1985)
J. Bacteriol.
64, 102-106], and the
chvB
locus has been implicated in the production of (1→2)-β-glucan, a unique exopolysaccharide component [Puvanesarajah, V., Schell, F. M., Stacey, G., Douglas, C. J. & Nester, E. W. (1985)
J. Bacteriol.
164, 102-106]. Site-directed transposon insertion mutants in the
chvA
and
chvB
-equivalent regions of
R. meliloti
are symbiotically defective. Mutants in the
chvB
-equivalent region have been examined in detail and have been found to induce the formation of nodule-like structures on alfalfa that are devoid of bacteroids, lack infection threads, and cannot fix nitrogen. Such mutants fluoresce normally in the presence of Calcofluor, a histochemical stain for β-linked polysaccharides, and produce normal amounts of total exopolysaccharide. The
Rhizobium
loci have been designated
ndv
because of their requirement for nodule development.
Successful liver allografts in mice by combination with allogeneic bone marrow transplantation.
Authors: Nakamura T , Good R A , Yasumizu R , Inoue S , Oo M M , Hamashima Y , Ikehara S
Published: May 2006

Successful liver allografts were established by combination with allogeneic bone marrow transplantation. When liver tissue of BALB/c (H-2d) or C57BL/6J (H-2b) mice was minced and grafted under the kidney capsules of C3H/HeN (H-2k) mice, it was rejected. However, when C3H/HeN mice were irradiated and reconstituted with T-cell-depleted BALB/c or BALB/c nu/nu bone marrow cells, or with fetal liver cells of BALB/c mice, they accepted both donor (stem-cell)-type (BALB/c) and host (thymus)-type (C3H/HeN) liver tissue. Assays for both mixed-lymphocyte reaction and induction of cytotoxic T lymphocytes revealed that the newly developed T cells were tolerant of both donor (stem-cell)-type and host (thymus)-type major histocompatibility complex determinants. We propose that liver allografts combined with bone marrow transplantation should be considered as a viable therapy for patients with liver disease such as liver cirrhosis and hepatoma.