bluuebunny/crossref_metadata_2025 huggingface dataset:
Water structure of a hydrophobic protein at atomic resolution: Pentagon rings of water molecules in crystals of crambin
Authors: Teeter M. M.
Published: May 2006

The water structure has been analyzed for a model of the protein crambin refined against 0.945-Å x-ray diffraction data. Crystals contain 32% solvent by volume, and 77% of the solvent molecules have been located—i.e., 2 ethanol molecules and 64 water molecules with 10-14 alternate positions. Many water oxygen atoms found form chains between polar groups on the surface of the protein. However, a cluster of pentagonal arrays made up of 16 water molecules sits at a hydrophobic, intermolecular cleft and forms a cap around the methyl group of leucine-18. Several waters in the cluster are hydrogen-bonded directly to the protein. Additional closed circular arrays, which include both protein atoms and other water oxygen atoms, form next to the central cluster. This water array stretches in the
b
lattice direction between groups of three ionic side chains.
Self-organization in macromolecular systems: the notion of adaptive value.
Authors: Demetrius L
Published: May 2006

Self-organization in macromolecular systems refers to the transition from a random assembly of interacting oligomers to a system of stable heteropolymers. The concept of adaptive value describes the correlation between environmental variability and the variability in replication and mutation rates of the interacting oligomers. This paper describes a model of self-organization in macromolecular systems based on the concept of adaptive value. The equilibrium states of a set of interacting polymers are described by states that maximize the adaptive value. The analytic basis for this notion of equilibrium, which is called the adaptive value principle, is given and this principle is invoked to explain two examples of macromolecular self-assembly.
Search, encounter rates, and the evolution of anisogamy.
Authors: Cox P A , Sethian J A
Published: May 2006

We describe analytical and numerical models to study gamete encounters in two and three dimensions between differently sized gametes without assuming pre-existing mating types. Our results indicate that an isogamous population can be successfully invaded by a gametangium if it produces gametes of a different size. The existence of a low adaptive peak for isogamy and a much higher adaptive peak for anisogamy suggests that stochastic forces may be initially important in driving isogamy through the fitness saddle to anisogamy.
Patterns of polymorphism and linkage disequilibrium suggest independent origins of the human growth hormone gene cluster.
Authors: Chakravarti A , Phillips J A , Mellits K H , Buetow K H , Seeburg P H
Published: May 2006

Six restriction fragment length polymorphisms (RFLPs) detected in the human growth hormone-human chorionic somatomammotropin (hGH-hCS) gene cluster were studied in Mediterraneans, Northern Europeans, and American Blacks; the polymorphisms showed that, on the average, one of 500 bases in this cluster is variant. Haplotypes constructed for four of these RFLPs display strong nonrandom associations. However, the strongest associations were between RFLPs that are in homologous DNAs rather than between the physically closest RFLPs. From this and other evidence we argue that duplication of an ancestral hCS gene occurred at least twice, the second event being relatively recent. In other words, duplication of the hCS-L gene to produce the hCS-A gene occurred twice, so that hCS-A genes in humans may have independent origins. Our results imply that chromosomes with absent hCS genes (leading to hCS deficiency) may represent the nonduplicated ancestral unit rather than gene deletions.
Ca2+ causes active contraction of bile canaliculi: direct evidence from microinjection studies.
Authors: Watanabe S , Phillips M J
Published: May 2006

Cytoplasmic microinjection of Ca2+ triggers contraction of bile canaliculi in freshly isolated monolayer cultures of rat hepatocytes. Unseparated paired hepatocytes were used to demonstrate this motility-based phenomenon. Only one cell of the pair was injected, but fluorescein spread from the target cell to the opposite cell; also, the contractions were always uniform, equally involving both hepatocytes that form the canaliculus, indicating that communication exists between the cell pairs. Inhibitors of calmodulin and actin filaments, trifluoperazine and cytochalasin B, respectively, inhibited the Ca2+-induced contractions. Hence, the mechanism of contraction has features in common with actin-myosin based cytoplasmic motility behavior found in other non-muscle cells.
Determination of synaptic phenotype: insulin and cAMP independently initiate development of electrotonic coupling between cultured sympathetic neurons.
Authors: Kessler J A , Spray D C , Saez J C , Bennett M V
Published: May 2006

Electrotonic coupling between pairs of sympathetic neurons dissociated from superior cervical ganglia of neonatal rats is rare when cells are cultured for 2 weeks in a nutrient medium plus serum and is common when cells are cultured for the same period in serum-free defined medium. This defined medium is the same nutrient medium with five added factors (progesterone, transferrin, putrescine, insulin, and selenium). When added singly to serum-containing medium, insulin and, to a lesser extent, selenium promote the development of electrotonic and dye coupling. The insulin effect is obtained with doses as low as 0.01 microgram/ml and is maximal after exposures from 3 to 5 days. The incidence of electrotonic coupling is also enhanced by exposure of cells to dibutyryl cAMP. This effect is obtained with doses as low as 0.1 mM, is faster (being maximal at approximately equal to 12 hr exposure), and is prolonged in the presence of the phosphodiesterase inhibitor caffeine. Butyrate itself promotes coupling to a small extent, but cAMP involvement is confirmed by similar effects of other membrane permeant analogues. Endogenous levels of cAMP are significantly elevated in cultures grown in the defined medium but not in those in serum-containing medium to which insulin or selenium are added. We conclude that the promotion of coupling by cAMP and by insulin or selenium are independent. The development of coupling in the defined medium thus seems to be a consequence of the addition of promoting substances (insulin, selenium) and the removal of an inhibitory effect of serum on cAMP levels.
The possibility that the spectrum of intermediate two, seen in the course of reaction of flavoenzyme phenol hydroxylases, may be attributable to iminol isomers of a flavin-derived 6-arylamino-5-oxo(3H,5H)uracil.
Authors: Wessiak A , Noar J B , Bruice T C
Published: May 2006

A commonly held view of the mechanism of flavin mixed-function oxidases is that enzyme-bound 4a-hydroperoxyflavin (4a-FIHOOH) undergoes ring opening to provide a carbonyl oxide (IV), which, after transferring an oxene equivalent to substrate, yields a 6-arylamino-5-oxo(3H,5H)-uracil (I). The latter is then thought to undergo ring closure to form a 4a-hydroxyflavin (4a-FIHOH), which by loss of water yields flavin (scheme I). A close structural analogue of I (i.e., III) has been synthesized. Comparison of the spectra of III (and II), taken in solvents of widely differing dielectric constants and in a strongly basic medium, with those of the intermediate(s) observed to be formed in time between 4a-FlHOOH and 4a-FlHOH has shown that the enzyme-bound intermediate(s) does not resemble spectrally I nor its iminol tautomers.
Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast.
Authors: Karin M , Najarian R , Haslinger A , Valenzuela P , Welch J , Fogel S
Published: May 2006

Copper resistance in yeast is controlled by the CUP1 locus. The level of resistance is proportional to the copy number of this locus, which can be found in up to 15 tandemly iterated copies. To elucidate the molecular mechanisms controlling the amplification and expression of the CUP1, locus, we determined its full nucleotide sequence. We have also identified and mapped two transcription units within the basic amplification unit of CUP1 in laboratory yeast strains. One of those transcription units is inducible by copper and encodes a low molecular weight copper binding protein--copper chelatin. The increased production of chelatin, due to both gene amplification and induction of transcription, leads to increased resistance of yeast cells to copper ions.
Regional localization on the human X chromosome and polymorphism of the coagulation factor IX gene (hemophilia B locus).
Authors: Camerino G , Grzeschik K H , Jaye M , De La Salle H , Tolstoshev P , Lecocq J P , Heilig R , Mandel J L
Published: May 2006

Hemophilia B is an X-linked disease caused by a functional deficiency in coagulation factor IX. A cDNA clone corresponding to factor IX has been used to detect homologous sequences in the human genome. All DNA fragments hybridizing to the probe, under medium- or high-stringency conditions, are X-linked, and the patterns obtained suggest that a single large (greater than or equal to 20 kilobases) gene is detected. The gene has been mapped to the q26-q27 region of the long arm of the X chromosome by hybridization to DNA from a panel of human-mouse hybrid cell lines. A search for restriction fragment length polymorphisms using seven restriction enzymes has led to the detection of a Taq I polymorphism, with allelic frequencies of about 0.71 and 0.29. This genetic marker should be useful for the detection of carriers of the hemophilia B trait and for prenatal diagnosis in informative families and, more generally, for the establishment of a linkage map of the human X chromosome.
Na+-H+ exchange activity in renal brush border membrane vesicles in response to metabolic acidosis: The role of glucocorticoids.
Authors: Kinsella J , Cujdik T , Sacktor B
Published: May 2006

Amiloride-sensitive Na+ -H+ exchange activity in brush border membrane vesicles isolated from rat proximal tubule was increased in metabolic acidosis. The enhancement of exchange activity required an intact adrenal gland or glucocorticoid supplements. Ammonium and phosphate excretions were increased during acidosis and these were also largely dependent on an intact adrenal gland or glucocorticoid supplements. Amiloride-insensitive Na+ uptake and passive H+ permeability were not altered by acidosis or the glucocorticoid status of the animal. These findings are consistent with glucocorticoids having an important regulatory role in the kidney by orchestrating the proximal tubular adaptation to metabolic acidosis.
High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.
Authors: Cone R D , Mulligan R C
Published: May 2006

We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-psi-, lacks the psi site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-psi-, called psi-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected psi-AM cells remain helper-free, even after months in culture. psi-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of psi-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.
Nucleotide sequence of the 3' region of an infectious human T-cell leukemia virus type II genome.
Authors: Shimotohno K , Wachsman W , Takahashi Y , Golde D W , Miwa M , Sugimura T , Chen I S
Published: May 2006

The nucleic acid sequence of the 3' region of human T-cell leukemia virus type II (HTLV-II) proviral DNA was determined using a HTLV-II proviral clone that could be recovered as infectious, transforming virus. The sequence data indicate a region of unknown function of approximately equal to 1.6 kilobase pairs in the 3' region, analogous to the X region previously identified in human T-cell leukemia virus type I (HTLV-I). Three overlapping open reading frames are present in the X region of HTLV-II. One of these open reading frames, Xc, is most likely to encode a protein product, because it has greater predicted amino acid sequence homology (78%) with the X-IV region of HTLV-I and a greater percentage of its base differences with X-IV at the third nucleotide position of codons than do the other open reading frames. Sequences of the X-region that include the open reading frames are conserved in two deletion mutants of HTLV-II, which are associated with a subline of Mo cells with a decreased dependence on fetal bovine serum.
Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies.
Authors: Ross A H , Grob P , Bothwell M , Elder D E , Ernst C S , Marano N , Ghrist B F , Slemp C C , Herlyn M , Atkinson B
Published: May 2006

The nerve growth factor (NGF) receptor was characterized by using a new series of anti-receptor monoclonal antibodies (MAbs). These MAbs (i) showed significantly greater reactivity with a melanoma cell line expressing higher levels of NGF receptor, (ii) inhibited the binding of 125I-labeled NGF to its receptor, and (iii) immunoprecipitated both metabolically labeled and 125I-labeled NGF affinity-labeled receptor. These experiments defined the receptor as a 75-kDa cell-surface protein. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of human nevi, melanomas, neurofibromas, a pheochromocytoma, and peripheral nerves. Uniform staining of the cytoplasm suggests that, in addition to cell-surface NGF receptors, there is a population of intracellular receptors.
Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method.
Authors: Collins F S , Weissman S M
Published: May 2006

The principle of a DNA cloning procedure that directionally generates genomic DNA fragments 50-2000 kilobases away from an initial probe is presented. The method depends on partial digestion of high molecular weight genomic DNA and subsequent ligation at very low concentration to generate covalent DNA circles. A library of the junction fragments from these circles can then be constructed. Biological or physical selection of the junction pieces can be achieved by incorporating a marker DNA fragment into the covalent circles. A 45-kilobase cosmid fragment has been successfully used to test the procedure. At appropriately low ligation concentrations (0.8 micrograms/ml), approximately equal to 90% of the ligated DNA is present as monomeric circles. Larger DNA fragments will require reducing the DNA concentration as the inverse square root of the DNA length. A suppressor tRNA gene has been tested as the selectable marker gene. Ligation of the digested circles into an amber-mutated lambda phage and propagation in a sup- host allows only the phage that contain junction fragments to produce plaques. Potential applications of this approach, such as mapping of complex genetic loci or moving from a linked gene toward a gene of interest, are presented and discussed.
Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chromatography: identity with pituitary fibroblast growth factor.
Authors: Gospodarowicz D , Cheng J , Lui G M , Baird A , Böhlent P
Published: May 2006

Brain and pituitary fibroblast growth factors (FGF) have been purified to apparent homogeneity from crude tissue extracts by a three-step procedure, including salt precipitation, ion-exchange chromatography, and heparin-Sepharose affinity chromatography. Brain and pituitary FGF have similar amino acid compositions and are indistinguishable with respect to molecular weight (16,000 by polyacrylamide gel electrophoresis), retention behavior in reversed-phase high-performance liquid chromatography, and recognition by antibodies directed against the amino-terminal sequence of pituitary FGF. Brain FGF preparations purified by heparin-Sepharose contain, in addition to the major FGF molecular species, at least two additional forms of the growth factor, which appear to be very similar by all the above criteria, except for retention in high-performance liquid chromatography.
Simian virus 40 DNA replication in vitro.
Authors: Li J J , Kelly T J
Published: May 2006

Soluble extracts prepared from monkey cells (COS-1 or BSC-40) infected with simian virus 40 (SV40) catalyze the efficient replication of exogenously added plasmid DNA molecules containing the cloned SV40 origin of replication. Extracts prepared from uninfected monkey cells also support origin-dependent replication in vitro but only in the presence of added SV40 large tumor (T) antigen. Very little DNA synthesis is observed when the cloned viral origin contains a 4-base-pair deletion mutation known to abolish SV40 DNA replication in vivo or when the parental plasmid vector lacking SV40 sequences is employed as template. The in vitro replication reaction proceeds via branched intermediates (theta structures) that resemble in vivo replication intermediates. Replication is sensitive to aphidicolin but relatively resistant to dideoxythymidine triphosphate. The product of the reaction consists of covalently closed circular DNA molecules that contain full-length daughter strands hydrogen bonded to the parental template. These observations support the conclusion that replication in the in vitro system closely resembles SV40 DNA replication in vivo. The system provides a biochemical assay for the replication activity of SV40 T antigen and should also facilitate the purification and functional characterization of cellular proteins involved in DNA replication.
Functions of purified E1A protein microinjected into mammalian cells.
Authors: Krippl B , Ferguson B , Rosenberg M , Westphal H
Published: May 2006

We have purified the human type C adenovirus E1A 13S mRNA gene product, expressed in Escherichia coli, and demonstrate that the protein exhibits genuine viral functions upon microinjection into mammalian cells. We show that the E1A protein activates expression of the adenovirus E2A gene and induces expression from the major late transcription unit of the adenovirus E1A deletion mutant, H5dl312. We use this functional assay to examine the stability of E1A protein microinjected into cells and find that E1A exhibits full function for at least 18 hours after its injection. In addition, the purified E1A protein was used to generate a high-titer monospecific rabbit antiserum. This antiserum was used to detect and localize E1A proteins within adenovirus-infected cells as well as within microinjected cells. The E1A protein is found to rapidly and quantitatively localize to the cell nucleus following microinjection into the cell cytoplasm. Thus, nuclear localization is an intrinsic property of the E1A polypeptide. The ability of the E1A protein to localize to the cell nucleus and to induce expression from the H5dl312 major late transcription unit is shown to be highly heat stable.
Lens transglutaminase selects specific beta-crystallin sequences as substrate.
Authors: Berbers G A , Feenstra R W , van den Bos R , Hoekman W A , Bloemendal H , de Jong W W
Published: May 2006

A Ca2+-dependent transglutaminase (EC 2.3.2.13) has been demonstrated in the eye lenses of several mammalian species [Lorand, L., Hsu, L. K. M., Siefring, G. E., Jr., & Rafferty, N. S. (1981) Proc. Natl. Acad. Sci. USA 78, 1356-1360]. Using [3H]methylamine as a convenient probe for transglutaminase activity, we have explored the action of this enzyme in the bovine eye lens. We could characterize the glutamine residues acting as acyl-donor sites in three beta-crystallin chains, which are the only substrates for lens transglutaminase among the various lens-specific structural proteins, the crystallins. A single glutamine was found to bind [3H]methylamine in each of these three chains: glutamine -9 in beta Bp (beta B2), glutamine -21 in beta B3, and glutamine -23 or -24 in beta A3. The four glutamines are all located in the NH2-terminal regions, which presumably extend from the compact two-domain structure of the beta-crystallin chains. It was, moreover, established that several components of the lens cytoskeleton are substrates for transglutaminase.
Photomorphogenesis in Physarum: induction of tubulins and sporulation-specific proteins and of their mRNAs.
Authors: Putzer H , Verfuerth C , Claviez M , Schreckenbach T
Published: May 2006

Sporangiophore formation in Physarum plasmodia starts about 10 hr after photoinduction. It is characterized by the induction of two tubulins and of at least 15 major sporangiophore morphogenetic proteins. In vitro translation of extracted mRNA revealed that differential gene expression is based on a highly synchronous temporal program of loss of plasmodial and induction of sporulation-specific mRNA species. Using a cloned cDNA encoding part of a sporangiophore morphogenetic protein from Physarum as a probe it was found that the induction of the complementary mRNA activity is due to the induction of the mRNA itself. The results suggest that light induces, with a lag phase of about 10 hr, the transient activation of sporulation-specific genes.
src- and fps-containing avian sarcoma viruses transform chicken erythroid cells.
Authors: Kahn P , Adkins B , Beug H , Graf T
Published: May 2006

We report here that several oncogene-transducing avian sarcoma virus strains, namely Rous sarcoma virus (src), Fujinami sarcoma virus (fps), and PRCII (fps), transform avian erythroid cells in vitro and in vivo. The src- and fps-transformed erythroblasts grow in vitro for 20-30 generations, require special growth conditions, and tend to differentiate spontaneously. In these properties, they resemble erythroid cells transformed with the erbB-containing H strain of avian erythroblastosis virus (AEV-H) but differ from those transformed with AEV-ES4 (erbA, erbB), which grow under standard culture conditions and rarely differentiate spontaneously. Erythroblasts transformed with viruses carrying temperature-sensitive mutations in the src or fps oncogene and then shifted to the nonpermissive temperature in the presence of anemic serum (as a source of an erythropoietin-like factor) differentiate terminally into erythrocytes. These results demonstrate that several members of the src gene family other than erbB have the capacity to transform erythroid cells.
Introduction of a selectable gene into different animal tissue by a retrovirus recombinant vector.
Authors: Stuhlmann H , Cone R , Mulligan R C , Jaenisch R
Published: May 2006

The potential use of retrovirus vectors to transduce foreign genetic information into cells of different tissues of an animal was explored by introducing a recombinant genome carrying the Eco gpt gene into postimplantation mouse embryos. To obviate the need for preparing concentrated virus stocks, psi 2-2-5 cells producing the replication-defective murine sarcoma virus (MSV)-gpt virus were microinjected directly into embryos. The psi 2-2-5 cells were mixed with cells producing replication-competent Moloney murine leukemia virus (Mo-MuLV) to facilitate spread of the vector. A high percentage of the manipulated embryos continued to develop without disturbance and were analyzed either prior to birth or as adults for expression of both helper and Eco gpt virus. Microinjection of as few as 10 Mo-MuLV-producing cells resulted in viremia of greater than 50% of the embryos or adults, 25%-30% of which produced MSV-gpt recombinant virus in a variety of organs including thymus, spleen, lung, kidney, and brain. The fraction of vector-producing cells, however, was 3 to 5 orders of magnitude lower than that of helper-virus-producing cells. Our results demonstrate that a selectable gene can be introduced by retroviral vectors into animals and can be expressed in a wide variety of different somatic tissues.
Reduction of scattering to an invariant finite displacement in an ambient space-time
Authors: Segal I. E.
Published: May 2006

The scattering transformation
S
for a wave equation in Minkowski space M
0
is reducible (rigorously in the classical case, necessarily partially heuristically in the nonlinear quantum case) to the action of a distinguished finite transformation ζ in the ambient universal cosmos M. M
0
is invariantly imbedded in M, relative to any given point of observation, and the space-like surfaces
x
0
=
s
in M
0
converge as
s
→ ±∞ to finite light cones C
±
in M. The generator ζ of the infinite cyclic center of the connected group of all casuality-preserving transformations in M (isomorphic to
SU
(2,2)/
Z
2
) carries C
-
into C
+
and acts on solutions of relativistic wave equations as
S
, in an invariant bundle formulation. The establishment of
S
is simplified, the symmetry and regularity properties of
S
are enhanced, the scope of the scattering concept is extended to important equations such as those of Yang-Mills (lacking an invariant separation into free and interaction components), and the treatment of bound and scattering states is more unified.
Identification and characterization of a new transcriptional termination factor from Escherichia coli.
Authors: Briat J F , Chamberlin M J
Published: May 2006

We have identified and partially purified an activity from Escherichia coli that enhances transcription termination at the bacteriophage T7 early terminator when cloned on the plasmid pAR1707. The factor also causes the transcript to be terminated at a site several nucleotides earlier than in its absence. The resulting 3' OH ends of the transcripts are identical to those found in vivo by S1 nuclease mapping. From this we conclude that the factor we have identified is probably responsible for determination of the 3' OH ends of T7 RNA transcripts in vivo. This factor does not act by processing a preformed RNA transcript, nor is it replaced by rho protein or nusA or nusB proteins. Therefore, it appears to be a new transcription termination factor, and we have designated it "tau factor." Elucidation of its role in transcription in E. coli will depend on its purification to homogeneity and further studies of its properties.
Blockade of autocrine stimulation in simian sarcoma virus-transformed cells reverses down-regulation of platelet-derived growth factor receptors.
Authors: Garrett J S , Coughlin S R , Niman H L , Tremble P M , Giels G M , Williams L T
Published: May 2006

The viral (v)-sis oncogene encodes a protein (p28sis) that is structurally homologous to platelet-derived growth factor (PDGF). We have shown that simian sarcoma virus (SSV)-transformed cells containing the v-sis oncogene release a Mr 20,000 substance that is recognized by antisera to synthetic peptide sequences contained in p28sis. Medium conditioned by SSV-transformed cells competes with 125I-labeled PDGF for specific PDGF receptor sites, initiates DNA synthesis, and stimulates tyrosine phosphorylation of the PDGF receptor when added to normal cells. When normal cells are co-cultured with SSV-transformed cells, the PDGF receptors of the normal cells are down-regulated by factors released from the transformed cells. Thus, SSV-transformed cells release material that is functionally similar to PDGF. We have used anti-phosphotyrosine antibodies to purify PDGF receptors and to detect PDGF-stimulated receptors in normal cells. SSV-transformed cells have no PDGF receptors detectable by these antibodies or by 125I-labeled PDGF binding studies. However, when SSV-transformed cells are exposed to suramin, a compound that blocks binding of PDGF to its receptors, the receptors reappear on the cell surface and within 8 hr are present at the same levels as in control cells. These "new" receptor sites can be phosphorylated in response to PDGF. Thus, the absence of PDGF receptors in SSV-transformed cells is due to down-regulation of the receptors by an autocrine mechanism that can be blocked by suramin.
Simian virus 40 T antigen is required for viral excision from chromosomes.
Authors: Miller J , Bullock P , Botchan M
Published: May 2006

We describe experiments that show that simian virus 40 (SV40) T antigen is required for viral excision from host chromosomes at some point prior to or during the homologous recombination events that create circular wild-type virus. Two recombinant SV40-pBR322 plasmids were constructed such that homologous recombination across similar-sized but different duplications of SV40 would reconstitute wild-type viral DNA. One plasmid (pSVED) was constructed such that the duplication separates the viral early T-antigen promoter from the coding sequences; the other recombinant (pSVLD) contains a duplication of the late viral sequences and thus maintains a complete T-antigen gene. These plasmids were individually established in Rat 2 cells via cotransformation with the herpes virus Tk gene. Both classes of cell lines contained integrated tandem arrays of the plasmids and yielded equivalent levels of infectious virus after cell fusions with COS-7 cells; however, only the T+ lines yielded virus after cell fusion with CV-1 cells. These results are consistent with the notion that viral excision is initiated by T-antigen-mediated in situ replication of viral DNA as proposed in the "onion skin" model. In contrast, both plasmids yielded infectious virus when transiently introduced via transfection into CV-1 cells. This latter finding is discussed in terms of the possible induction of cellular repair and recombination pathways evoked by the introduction of damaged DNA into the nucleus.
Long-term cultured B lymphocytes and their precursors reconstitute the B-lymphocyte lineage in vivo.
Authors: Kurland J I , Ziegler S F , Witte O N
Published: May 2006

Long-term in vitro cultured B lymphocytes and their precursors were tested for their ability to reconstitute a functional B-cell lineage in vivo. Continuous in vitro production of cells representing early stages of the B-lymphocyte lineage originates from a bone marrow cell type that can be distinguished from a multipotential hematopoietic stem cell. Progenitors of the long-term cultured B-lymphoid cells are recoverable in vitro after in vivo passage into lethally irradiated mice. Long-term cultured B-lymphoid cells and their progenitors reconstituted the B-lymphocyte lineage and restored normal humoral immunity in genetically defective mice. Cultured B-lymphoid cells did not cause lymphoid tumors in vivo in irradiated syngeneic mice. These findings indicate that multipotential hematopoietic stem cells are not directly required for the continued production of functionally competent B lymphocytes in vitro, and they suggest the existence of a B lymphoid progenitor cell with in vitro, and possibly in vivo, stem cell-like qualities.
Antigen presentation by supported planar membranes containing affinity-purified I-Ad.
Authors: Watts T H , Brian A A , Kappler J W , Marrack P , McConnell H M
Published: May 2006

I-Ad, purified from A20-1.11 cells by affinity chromatography, was incorporated into supported planar membranes by incubation of I-Ad-containing phospholipid vesicles with clean glass coverslips. Such planar membranes present a peptide digest of ovalbumin to the ovalbumin-specific, I-Ad-restricted T-cell hybridoma 3DO-54.8, resulting in the antigen-specific release of interleukin 2. However, when the same material was provided in the form of small unilamellar vesicles, no response was obtained. Antigen presentation by the I-Ad-containing planar membranes was inhibited by the monoclonal antibody MKD6 (anti-I-Ad) but not by the antibody 10-2.16 (anti-I-Ak). The antibody GK1.5, which recognizes the T-cell surface antigen L3T4, was also inhibitory. In contrast to the results with purified I-Ad, crude membrane preparations from A20-1.11 cells were effective in antigen presentation in both planar and vesicular forms.
The neurula stage mouse embryo in control of neuroblastoma.
Authors: Podesta A H , Mullins J , Pierce G B , Wells R S
Published: May 2006

The purpose of this study was to determine whether the neurula stage mouse embryo can regulate tumor formation of C-1300-3 neuroblastoma cells. Five neuroblastoma cells were injected into the second somite of neurula stage embryos, and their ability to form tumors was tested, 24 hr later, by transplanting the portion of the embryo containing the cancer cells into the testes of adult mice. Only one-third the number of tumors was obtained in comparison with controls in which (i) five neuroblastoma cells were injected into blocks of liver tissue that were then transplanted into the testes of adult animals or (ii) five C-1300-3 neuroblastoma cells were injected directly into the testes. When five C-1300-3 cells were injected into somites, which had been dissected from embryos, and the injected somites were placed in animals, significantly fewer tumors were obtained in relationship with controls. Although it is not known whether the neuroblastoma cells are induced to differentiate or are killed by the embryonic tissue, the effect appeared to be specific because the tumor-forming ability of L1210 leukemia, B-16 melanoma, embryonal carcinoma 247, and a parietal yolk sac carcinoma was unaffected by somites.
Analysis of Sendai virus mRNAs with cDNA clones of viral genes and sequences of biologically important regions of the fusion protein.
Authors: Hsu M , Choppin P W
Published: May 2006

cDNA clones representing five of the genes of Sendai virus (P, HN, NP, F, and M) were isolated and used to identify the viral mRNAs by hybridization. Five mRNAs that were monocistronic transcripts of these genes were identified. A sixth transcript, which was identified on the basis of size and of hybridization to viral RNA but not to the cDNA of the other five genes, is thought to represent the message for the L protein. In addition, polycistronic transcripts of the NP and P genes and of the M and F genes were also found. The latter establishes the position of the F gene adjacent to the M gene; these results confirm and extend the previously reported partial gene order of the virus. Nucleotide sequences and derived amino acid sequences of two biologically important regions of the F protein--approximately 25% of F proximal to its COOH terminus and the region spanning the site of the proteolytic cleavage that activates the fusion activity of the protein--are presented. The F protein has an unusually large "cytoplasmic domain" of 42 amino acids beyond the hydrophobic region by which it is anchored in the viral membrane. A single possible trypsin cleavage site was found at the junction of the F1 and F2 polypeptides, and 26 hydrophobic amino acids extend from this cleavage site at the NH2 terminus of the F1 polypeptide.
Sequence of the initiation factor IF2 gene: unusual protein features and homologies with elongation factors.
Authors: Sacerdot C , Dessen P , Hershey J W , Plumbridge J A , Grunberg-Manago M
Published: May 2006

The gene for protein synthesis initiation factor IF2 in Escherichia coli, infB, is located downstream from nusA on the same operon. We sequenced about 3 kilobases of DNA beginning within nusA and including the entire infB structural gene plus another 392 bases downstream. This region contains no obvious strong promoter signals, but a possible transcriptional termination or pausing site occurs downstream from infB. The putative initiator codon for IF2 alpha (97,300 daltons) is AUG; that for IF2 beta (79,700 daltons) is GUG, located 471 bases downstream in the same reading frame. The codon usage for IF2 is typical of other highly expressed proteins in E. coli and suggests that IF2 mRNA is efficiently translated. IF2 alpha contains two adjacent regions (residues 104-155 and 167-214) that are rich in alanine and charged amino acids and that show striking periodicities in their sequences. These regions may alternate between flexible and helical conformations, thereby drawing together the NH2-terminal and COOH-terminal globular domains of the factor as IF2 interacts with ribosomes or tRNA. Certain regions of the DNA and protein sequences of IF2 share strong homologies with elongation factor EF-Tu and lesser homology with EF-G. In particular, a region of EF-Tu implicated in GTP binding contains sequences and secondary structure that are conserved in IF2. The homologies indicate that the genes for IF2 and the elongation factors are derived at least in part from a common ancestor.
Phorbol esters modulate insulin receptor phosphorylation and insulin action in cultured hepatoma cells.
Authors: Takayama S , White M F , Lauris V , Kahn C R
Published: May 2006

The effect of the tumor-promoting agent phorbol 12-myristate 13-acetate (PMA) on insulin receptors and insulin action was studied in rat hepatoma cells in culture. PMA (0.1-1.0 micrograms/ml) did not affect insulin binding either acutely or chronically but inhibited insulin stimulation of glycogen synthase and tyrosine aminotransferase. PMA (1 microgram/ml) stimulated the phosphorylation of the beta subunit of insulin receptor purified from [32P]phosphate-labeled Fao cells by 1.3-fold in the absence of insulin. In contrast, insulin-stimulated phosphorylation in the presence of PMA was reduced. Phosphoamino acid analysis of the beta subunit after PMA stimulation revealed an increase of both phosphoserine and phosphothreonine residues, whereas insulin stimulated primarily phosphorylation of tyrosine and serine residues. Insulin stimulation of cells after PMA treatment revealed a decrease in phosphotyrosine when compared to cells stimulated by insulin alone. Tryptic peptide mapping of the beta subunit by a two-dimensional chromatographic/electrophoretic separation revealed nine phosphopeptides from the cells treated with PMA. Insulin stimulated phosphorylation at six new sites in the receptor, three of which appeared to be similar to those in PMA-treated cells. This report shows that phorbol esters stimulate insulin receptor phosphorylation, inhibit insulin-induced receptor phosphorylation and insulin action, and suggest a physiologic relation between insulin action and the calcium-activated and phospholipid-dependent protein kinase C.
A noncholinergic site-directed monoclonal antibody can impair agonist-induced ion flux in Torpedo californica acetylcholine receptor.
Authors: Donnelly D , Mihovilovic M , Gonzalez-Ros J M , Ferragut J A , Richman D , Martinez-Carrion M
Published: May 2006

We have employed several monoclonal antibodies (mAbs) directed against several regions of the acetylcholine receptor (AcChoR) to assist in the determination of the antigenic structure of this multisubunit glycoprotein and to better understand molecular events involved in the impairment of neuromuscular transmission in the autoimmune disease myasthenia gravis. Among three mAbs shown to block agonist-induced ion fluxes, mAb 371A is a putative probe of an ion channel domain(s) of the AcChoR. It appears to bind to an antigenic determinant whose structure is maintained upon treatment with sodium dodecyl sulfate, the stoichiometry of binding being of one mAb per alpha-bungarotoxin binding site. Binding of mAb 371A to the AcChoR does not affect binding of cholinergic agonists or antagonists (carbamoylcholine and d-tubocurarine) or neurotoxins (alpha-bungarotoxin) or the ability of membrane-bound AcChoR to undergo reversible sensitization-desensitization affinity transitions. However, this mAb inhibits agonist-induced thallium (T1+) influx into AcChoR-rich membrane vesicles, as measured on a millisecond time scale by means of a rapid kinetics "stopped-flow/fluorescence quenching" technique. The stoichiometry of inhibition by bound mAb 371A coincides with that for maximal binding.
Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics.
Authors: Saghai-Maroof M A , Soliman K M , Jorgensen R A , Allard R W
Published: May 2006

Spacer-length (sl) variation in ribosomal RNA gene clusters (rDNA) was surveyed in 502 individual barley plants, including samples from 50 accessions of cultivated barley, 25 accessions of its wild ancestor, and five generations of composite cross II (CCII), an experimental population of barley. In total, 17 rDNA sl phenotypes, made up of 15 different rDNA sl variants, were observed. The 15 rDNA sl variants comprise a complete ladder in which each variant differs in length from adjacent variants by approximately equal to 115 nucleotide pairs. Studies of four rDNA sl variants in an F2 population showed that these variants are located at two unlinked loci, Rrn1 and Rrn2, each with two codominant alleles. Using wheat-barley addition lines, we determined that Rrn1 and Rrn2 are located on chromosomes 6 and 7, respectively. The nonrandom distribution of sl variants between loci suggests that genetic exchange occurs much less frequently between than within the two loci, which demonstrates that Rrn1 and Rrn2 are useful as new genetic markers. Frequencies of rDNA sl phenotypes and variants were monitored over 54 generations in CCII. A phenotype that was originally infrequent in CCII ultimately became predominant, whereas the originally most frequent phenotype decreased drastically in frequency, and all other phenotypes originally present disappeared from the population. We conclude that the sl variants and/or associated loci are under selection in CCII.
High-level production of biologically active human alpha 1-antitrypsin in Escherichia coli.
Authors: Courtney M , Buchwalder A , Tessier L H , Jaye M , Benavente A , Balland A , Kohli V , Lathe R , Tolstoshev P , Lecocq J P
Published: May 2006

A cDNA clone containing the complete human alpha 1-antitrypsin sequence was isolated from a human liver cDNA bank by screening with a chemically synthesized oligonucleotide probe. DNA sequences encoding the alpha 1-antitrypsin mature polypeptide were inserted into an Escherichia coli expression vector that allows transcription from the efficient leftward promoter of bacteriophage lambda (PL) and initiation of translation at the lambda cII gene ribosome-binding site. This construction resulted in the induction of a 45-kilodalton protein at a level of approximately 15% of total cell protein. The polypeptide produced was recognized by antisera raised against human alpha 1-antitrypsin protein and displayed normal biological activity in an in vitro antielastase assay.
Variant antigen genes of Trypanosoma brucei: genomic alteration of a spliced leader orphon and retention of expression-linked copies during differentiation.
Authors: Parsons M , Nelson R G , Stuart K , Agabian N
Published: May 2006

Variant surface glycoprotein (VSG) gene expression in Trypanosoma brucei involves not only the sequential activation of individual VSG genes during mammalian bloodstream stage antigenic variation but also the regulation of gene expression during cyclic transmission through alternate mammalian and insect hosts. In the bloodstream stage, transcriptional activation of many VSG genes is correlated with the appearance of an additional copy of the gene in a novel genomic location, the expression-linked copy. The parasite loses the ability to synthesize VSG during differentiation from mammalian bloodstream to insect procyclic stage. Five different bloodstream populations were individually converted to procyclic forms. In each case, the procyclic cells retained the expression-linked copy of the bloodstream parent, and it remained in the same immediate genomic context. Transcripts homologous to the VSG structural gene exon were found in bloodstream stage RNA but not in procyclic RNA. Nevertheless, transcripts containing sequences homologous to the VSG mRNA spliced leader were abundant in both procyclic and bloodstream stage cells. When the genomic organization of sequences homologous to the VSG leader was examined, a specific alteration correlated with procylic differentiation was found. These data are discussed in light of biological studies on antigenic variation.
Enzyme with dual lipoxygenase activities catalyzes leukotriene A4 synthesis from arachidonic acid.
Authors: Shimizu T , Rådmark O , Samuelsson B
Published: May 2006

When arachidonic acid was incubated with homogenates of potato tubers, two isomers of 6-trans-leukotriene B4, epimeric at C-12, were formed in addition to the major product, (5S-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HPETE). To elucidate the mechanism of biosynthesis of the dihydroxy-acids, the lipoxygenase from the potato tubers was purified to apparent homogeneity by a combination of conventional chromatographic procedures and high-performance liquid chromatography equipped with a chromatofocusing column (Mono-P). The purified lipoxygenase acted on arachidonic acid and bishomo-gamma-linolenic acid to yield (5S)-hydroperoxy- and (8S)-hydroperoxyicosanoids, respectively. Furthermore, the purified enzyme converted 5-HPETE to leukotriene A4, with the presence of the epoxide intermediate being demonstrated by 18O2 experiments, methanol trapping, as well as further conversion to leukotriene B4 by the purified leukotriene A4 hydrolase. Several experiments, including those with lipoxygenase inhibitors, heat treatment, and competitive inhibition, indicated that both the 5-lipoxygenase and leukotriene A4 synthase activities resided in the same protein and that the formation of leukotriene A4 from 5-HPETE was catalyzed by the 8-lipoxygenase activity of the enzyme.
Evidence that the major postsynaptic density protein is a component of a Ca2+/calmodulin-dependent protein kinase.
Authors: Kelly P T , McGuinness T L , Greengard P
Published: May 2006

Polypeptides of Mr 50,000 and 60,000 in isolated synaptic junctions have been compared to polypeptides of corresponding molecular weight in Ca2+/calmodulin-dependent protein kinase II. The polypeptides of corresponding molecular weight from the two preparations were shown by several criteria to be indistinguishable. These criteria included 125I-labeled tryptic/chymotryptic peptide patterns, 32P-labeled proteolytic peptide maps, and crossreactivity on immunoblots using polyclonal and monoclonal antibodies. Furthermore, studies examining the phosphorylation of substrate proteins, by the endogenous synaptic junction kinase and by Ca2+/calmodulin-dependent protein kinase II, indicated that the two enzymes have similar substrate specificities. Since the Mr 50,000 polypeptide present in synaptic junctions is known to be the major postsynaptic density protein, the present results indicate that the major postsynaptic density protein is a component of Ca2+/calmodulin-dependent protein kinase II.
Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP.
Authors: Hoess R H , Abremski K
Published: May 2006

The interaction between the P1 recombinase protein Cre and the DNA site at which it acts, loxP, has been studied by using nuclease protection techniques. The region of DNA protected by Cre against nuclease attack by DNase I or neocarzinostatin is a 34-base-pair (bp) region containing two 13-bp inverted repeats separated by an 8-bp spacer region. These protected sequences have previously been shown to be required for efficient Cre-mediated recombination at loxP. The results of the above protection experiments suggest (i) that no more than 34 bp may be required for loxP recombination and (ii) that the asymmetry of loxP recombination is due to the 8-bp spacer sequence. With neocarzinostatin, a specific nucleotide within the 8-bp spacer region is not protected. This nucleotide is located in a 2-bp sequence shown to be involved in a loxP crossover event, suggesting that this region remains exposed after Cre binding. Protection experiments have also been done with loxP sites that have either the left or right inverted repeat deleted. The nuclease protection pattern of these sites reveals that each loxP site consists of two binding domains for Cre, each being composed of one 13-bp inverted repeat and the contiguous 4 bp of the 8-bp spacer region.