bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast recombination: the association between double-strand gap repair and crossing-over.
Authors: Orr-Weaver T L , Szostak J W
Published: May 2006

In previous experiments, we have used yeast transformation to study the recombinogenic repair of double-strand breaks and gaps. A plasmid containing a double-strand gap within sequences homologous to the yeast genome integrates efficiently by crossing-over. During the process of integration, the double-strand gap is repaired, using chromosomal information as a template. This repair reaction results in the transfer of genetic information from one DNA duplex to another and is therefore a pathway for gene conversion. Because meiotic gene conversion is associated with a high frequency (up to 50%) of crossing-over, we wished to determine the degree of association of double-strand gap repair with crossing-over. Only the class of repair events resulting in crossovers (plasmid integration) were detected in our earlier experiments with nonreplicating plasmids. In this paper, we describe the outcome of double-strand gap repair in plasmids that are capable of autonomous replication and therefore allow recovery of both crossover and noncrossover products. After the correct repair of a double-strand gap, we recover approximately equal numbers of integrated and nonintegrated plasmids. Thus, gene conversion by double-strand gap repair can occur either with or without crossing-over, and it is similar in this respect to meiotic gene conversion. Circularization of linear plasmid DNA by ligation is also observed, suggesting that yeast has an additional repair pathway for double-strand breaks that is independent of recombination. Gap repair on replicating plasmids permits rapid cloning of chromosomal alleles.
Naturally occurring cytotoxic human antibodies recognize H-2-controlled murine lymphocyte antigens.
Authors: Iványi P , Leupers T , van Mourik P
Published: May 2006

Human sera contain cytotoxic naturally occurring (CyNa) antibodies which discriminate between lymph node cells from mice differing only at the H-2 complex. Sera from three healthy subjects (normal human sera, NH sera) and one serum from a patient with multiple sclerosis reacted with cells expressing Db, Kd, Kk, and Kp molecules, respectively. However, the following observations suggested that the binding specificity of these CyNa antibodies is to antigens that are distinct from the classical H-2 antigens: (i) the NH sera did not contain cytotoxic anti-HLA antibodies, (ii) redistribution (capping) of H-2 antigens did not induce resistance to lysis for CyNa antibodies, and (iii) individual variation was demonstrated in the expression of the murine lymphocyte antigens detected by the human CyNa antibodies. The reason for this variation appeared to be different for individual NH serum. A maternal effect influenced the expression of the murine lymphocyte antigen detected by one NH serum (anti-H-2b). The differences detected by another NH serum (anti-H-2p) appeared to be inherited, as shown by progeny testing. We hypothesize that the human CyNa antibodies may be directed against antigens controlled or modified by murine viruses (milk borne or endogenous), whose expression is under the influence of the H-2 complex, and that their production might have been stimulated by the products of human genes homologous to murine viruses.
Cyclic AMP-dependent constitutive expression of gal operon: use of repressor titration to isolate operator mutations.
Authors: Irani M , Orosz L , Busby S , Taniguchi T , Adhya S
Published: May 2006

When the gal operator region is present in a multicopy plasmid it binds to all ("titrates") the gal repressor and "induces" the chromosomal gal operon. To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal genes from repression. Then with such an Oa plasmid operator revertants were isolated that had reacquired the ability to release repression. Both sets of mutations have been localized by DNA sequence analysis. When the Oa mutations were transferred from the plasmid to the chromosome by recombination these mutant operators were found to make gal expression constitutive (independent of repressor) but still dependent on cAMP, whereas the previously reported gal operator mutants (Oc) are constitutive both in the presence and in the absence of cAMP. The titration method of isolating mutants enables the isolation of strains with operator mutations that also affect normal promoter activity, and it provides an easy way to isolate revertants of operator mutations.
Transmission of naturally occurring lymphoma in macaque monkeys.
Authors: Hunt R D , Blake B J , Chalifoux L V , Sehgal P K , King N W , Letvin N L
Published: May 2006

Spontaneously occurring rhesus monkey lymphomas were transmitted into healthy rhesus monkeys by using tumor cell suspensions. The naturally arising tumors included an immunoblastic sarcoma and an undifferentiated lymphoma. Recipient animals developed undifferentiated lymphomas, poorly differentiated lymphomas, or parenchymal lymphoproliferative abnormalities suggestive of early lesions of lymphoma. Some of these animals developed such opportunistic infections as cytomegalovirus hepatitis and cryptosporidiosis. They also showed evidence of an abnormal circulating peripheral blood mononuclear cell. These findings, all characteristic of the acquired immune deficiency syndrome (AIDS) of macaques, suggest a link between these transmissible lymphomas and AIDS in macaque monkeys.
Characterization and location of myc homologous sequences in human cytomegalovirus DNA.
Authors: Gelmann E P , Clanton D J , Jariwalla R J , Rosenthal L J
Published: May 2006

Specific DNA fragments from human cytomegalovirus (HCMV) strains Towne and AD169 exhibited homology to myc DNA sequences under hybridization conditions corresponding to a 22-28% base mismatch. In a specific subset of hybridizing HCMV fragments, the homology was restricted to the 5' half of viral v-myc and the 5' half of human c-myc. No hybridization was observed between HCMV fragments and the 3' v-myc and 3' human c-myc probes. In Towne DNA, myc homologous sequences mapped in four regions within the long unique segment (0.070-0.094, 0.134-0.156, 0.454-0.470, and 0.591-0.605 map unit) and one region in each of the short terminal repeats (0.832-0.847 and 0.984-1.0 map unit). In strain AD169, myc homology mapped in three regions within the long unique segment (0.123-0.147, 0.174-0.198, and 0.583-0.606 map unit) and one region in each of the short terminal repeats (0.833-0.863 and 0.976-1.0 map unit). By utilizing probes specific for the 5' and 3' portions of v-myc and human c-myc, we established that the regions of homology in a specific subset of HCMV restriction fragments corresponded to the 5' half of myc and were not due to MC29 viral helper sequences, flanking cellular sequences, or binding of probe to G.C-rich DNA.
Carbachol can be released at a cholinergic ganglionic synapse as a false transmitter.
Authors: Baux G , Tauc L
Published: May 2006

Carbachol was injected into a presynaptic cholinergic neuron in the buccal ganglion of Aplysia and the quantal aspects of the Cl- -dependent postsynaptic response to a prolonged stimulation were analyzed by a statistical fluctuation method. The calculated amplitude of the miniature postsynaptic current was increased with respect to control. Statistical fluctuation analysis was also used to analyze the postsynaptic response obtained during ionophoretic application of acetylcholine and carbachol. The calculated unitary channel current was found to be greater for carbachol than for acetylcholine. This increase could explain the larger miniature postsynaptic current seen after intracellular injection of carbachol into the presynaptic neuron if carbachol was released at the synapse as a false transmitter. This conclusion was supported by the observation that it was possible to restore transmission at a synapse previously blocked by presynaptic intracellular injection of acetylcholinesterase with a presynaptic injection of carbachol.
Rational approximations to solutions of linear differential equations
Authors: Chudnovsky D. V. , Chudnovsky G. V.
Published: May 2006

Rational approximations of Padé and Padé type to solutions of differential equations are considered. One of the main results is a theorem stating that a simultaneous approximation to arbitrary solutions of linear differential equations over C(
x
) cannot be “better” than trivial ones implied by the Dirichlet box principle. This constitutes, in particular, the solution in the linear case of Kolchin's problem that the “Roth's theorem” holds for arbitrary solutions of algebraic differential equations. Complete effective proofs for several valuations are presented based on the Wronskian methods and graded subrings of Picard-Vessiot extensions.
Correlation of crystallographically determined and computationally predicted hydrogen-bonded pairing configurations of nucleic acid bases.
Authors: Ornstein R L , Fresco J R
Published: May 2006

Crystals of pairs of H-bonded nucleic acid bases are generally grown from nonaqueous solutions. We have been able to predict the H-bonded configuration of most of the base pairs in such crystals by using an empirical-potential function we recently developed for calculating the energetics of such interactions in chloroform solution. The following configurations were computationally predicted to predominate and are those observed in crystal structures: the Watson-Crick G.C configuration instead of two competing configurations; the Hoogsteen-type configurations for A.T, A.U, and A.br5U instead of Watson-Crick-type configurations; the Watson-Crick-type configurations for 2-aminopurine.br5U instead of the purine N3-type configuration; the Watson-Crick-type configurations for 8-bromo-2,6-diaminopurine.T instead of the Hoogsteen or purine-N3-type configurations; the syn-anti configuration for br8A.br8I instead of the anti-anti configuration; the Watson-Crick-type configurations for br8A.br5U instead of the Hoogsteen-type configurations; and the Hoogsteen-type configurations for me8A.T instead of the Watson-Crick configurations. In addition, the H-bonded base triplet br5U.2,6-diaminopurine.br5U was calculated to have Hoogsteen and Watson-Crick-type configurations but not the purine N3-type configuration. Apparently, lattice forces and chance nucleation of a minor base pairing configuration are not significant when the stability difference between the preferred and alternative configurations exceeds a relatively small value. In one case, in order to correctly predict the base pairing configuration in the crystal, it was necessary to include a contribution due to a C--H...O bond, suggesting that this type of H bond can make a significant contribution to base pair stability.
5-methylcytosine is localized in nucleosomes that contain histone H1.
Authors: Ball D J , Gross D S , Garrard W T
Published: May 2006

We have developed a procedure for purifying highly specific polyclonal antibodies against 5-methylcytosine. These antibodies were used to probe the distribution of 5-methylcytosine among fractionated nucleosomes of mouse cell chromatin. Our results demonstrate that at least 80% of the 5-methylcytosine is localized in nucleosomes that contain histone H1. Native nucleosomes that lack histone H1 or possess high mobility group proteins package DNA that is 1.6- to 2.3-fold undermethylated. We suggest that the preferential association of methylated sequences with histone H1 has functional significance because DNA methylation has been linked to gene inactivation and histone H1 is known to promote chromatin condensation.
Recombination between sequences in nonhomologous positions.
Authors: Sugawara N , Szostak J W
Published: May 2006

Crossing-over between the dispersed repeated sequences found in eukaryotic genomes would generate chromosomal rearrangements. The stability of the yeast genome suggests the existence of some constraint on the ability of these sequences to interact by recombination. We have constructed strains with two alleles of the his3 gene located on different chromosomes. Gene conversion accounts for the majority of the recombination events between these genes, but about 10% of the events are crossovers that result in a reciprocal translocation. When one of the alleles is on an autonomously replicating centromere plasmid, recombination is 5- to 10-fold more frequent than when both alleles are chromosomal, suggesting that higher-order chromosome structure may play a role in restricting interchromosomal recombination. We have also used the translocation to deduce the orientation of the his3 and rRNA genes relative to their centromeres.
Cycling of peripheral blood and marrow lymphocytes in cyclic neutropenia.
Authors: Engelhard D , Landreth K S , Kapoor N , Kincade P W , De Bault L E , Theodore A , Good R A
Published: May 2006

A 16-month-old male patient with cyclic neutropenia was found to have cyclic fluctuations of monocytes, lymphocytes, platelets, and eosinophils in the peripheral blood. Changes in lymphocyte counts were not obviously related to B, T, or natural killer cells. All classes of immunoglobulins were elevated throughout the cycle. Studies of the marrow morphology revealed remarkable cyclic oscillations of lymphoid as well as myeloid lineage cells. Granulocyte-macrophage progenitors (CFU-c) cycled and were virtually absent 1 wk prior to the neutropenic nadir. The cyclic changes in marrow lymphoid cell numbers were primarily due to changes in numbers of surface immunoglobulin negative (sIg-), cytoplasmic Ig+ (cIg+) pre-B cells. Pre-B cell numbers cycled from normal to extraordinarily elevated values with the same periodicity but reciprocal to the neutrophil cycle. We propose that the primary defect in cyclic neutropenia may either be a periodic failure of an early myeloid differentiation factor or a blunted response of early myeloid precursors to a common hemopoietic growth factor. This may lead to periodic fluctuations in the production or delivery of growth factors (or factor) that influence early stages of differentiation of other hemopoietic cells, including pre-B cells. The essential periodic deficiency is consequently reflected in deficient production of CFU-c accompanied by excessive production or accumulation of pre-B cells (and probably other hemopoietic precursors) in the marrow.
Reexamination of diversifying selection of polymorphic allozyme genes by using population cages in
Drosophila melanogaster
Authors: Yamazaki Tsuneyuki , Kusakabe S. , Tachida H. , Ichinose M. , Yoshimaru H. , Matsuo Y. , Mukai Terumi
Published: May 2006

Several investigators have reported increased heterozygosity for allozyme markers in laboratory experiments simulating a variable environment and have concluded that there is diversifying selection for allozyme variants. We found no change in heterozygosity in 28 large population cages where temperature, food, and light were each set at three levels, two constant and one variable, in a factorial design. Specifically, there was no change in heterozygosity when 0, 1, 2, or 3 factors were variable. The discrepancy between our negative results and earlier positive ones is that our experiments were started from a large caged population of flies which had been maintained for 6 years—long enough to be very near to linkage equilibrium. We suggest that the earlier results may have been the result of linkage between neutral, or nearly neutral, allozyme markers and selected loci or in some cases, inversions.
DNA fragments in Friend erythroleukemia cells induced by dimethyl sulfoxide.
Authors: Pulito V L , Miller D L , Sassa S , Yamane T
Published: May 2006

The idea of the correlation between DNA damage caused by dimethyl sulfoxide (Me2SO) and the induction of erythrodifferentiation in murine erythroleukemia (MEL) cells needs reevaluation as a result of the hydroxyapatite chromatographic analysis of DNA from Me2SO-treated and untreated cells as a function of the time in culture. Because Me2SO causes an arrest in G1 phase and consequent delay in DNA synthesis, the valid comparison would be of the DNA samples taken from control and induced cells at the same stage of growth rather than at the same time in culture. At the same stage of growth, there is no evidence of the induction of DNA damage by Me2SO. Presumably, so-called degradation reported in the past stemmed from the fragments of DNA replication.
Mismatch repair in Streptococcus pneumoniae: relationship between base mismatches and transformation efficiencies.
Authors: Claverys J P , Méjean V , Gasc A M , Sicard A M
Published: May 2006

Genetic transformation in Streptococcus pneumoniae involves the insertion of single-stranded pieces of donor DNA into a recipient genome. Efficiencies of transformation strongly depend on the mutations (markers) carried by donor DNA. Markers are classified according to their transforming efficiencies into very high, high, intermediate, and low efficiency. The last is approximately 1/20th as efficient as the first. This marker effect is under the control of the Hex system, which is thought to correct mismatches at the donor-recipient heteroduplex stage in transformation. To investigate this effect, wild type, mutant, and revertant DNA sequences at five genetic sites within the amiA locus were determined. The results show that low-efficiency markers arise from transitional changes A . T to G . C. The transversion A . T to T . A corresponds to an intermediate-efficiency marker. Transversions G . C to T . A and G . C to C . G lead to high-efficiency markers. Among the eight possible mismatches that could exist transiently at the heteroduplex stage in transformation, only two--namely, A/G and C/C--are not corrected by the Hex system. It is noteworthy that the four possible base pairs (A . T, T . A, G . C, and C . G) have been encountered at the very same site (amiA6 site), which constitutes a good illustration of the marker effect. DNA sequence analysis also reveals that short deletions (33 or 34 bases long) are integrated with very high efficiencies. These results confirm that the Hex system corrects point mismatches harbored in donor-recipient heteroduplexes thousands of bases long. The correction pattern of the Hex system toward multiple-base mismatches has also been investigated. Its behavior toward double-base mismatches is complex, suggesting that neighboring sequences may affect the detection of mispaired bases.
Temperature-compensated circadian clock in the pineal of Anolis.
Authors: Menaker M , Wisner S
Published: May 2006

The pineal organ of the lizard Anolis carolinensis can be maintained for up to 10 days in superfused organ culture. During this time it synthesizes and releases melatonin into the medium flowing slowly over it. Collection of timed aliquots of medium and subsequent analysis for melatonin by radioimmunoassay reveal circadian rhythms of melatonin output by the isolated pineal. These rhythms persist for many cycles in constant darkness and at several constant ambient temperatures ranging from 22 to 37 degrees C. The period of the rhythm is temperature compensated (Q10 approximately equal to 1.14) and the rhythm is synchronized by light-dark cycles imposed on the cultured gland. This isolated vertebrate organ displays the three major properties of circadian systems and must therefore contain one or more circadian oscillators.
Effects of sodium pentobarbital anesthesia and neurotrophic factor on the maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.
Authors: Koelle G B , Ruch G A , Uchida E
Published: May 2006

In continuation of a previously reported study, the superior cervical ganglia of cats were preganglionically denervated bilaterally under sodium pentobarbital anesthesia. The following day cats were reanesthetized and infused via the common carotid artery with an aqueous extract of cat brain, spinal cord, and sciatic nerves for periods of 24, 12, 6, and 3 hr, without ligation of the external carotid or lingual arteries as was done previously. Values for acetylcholinesterase and butyrylcholinesterase of superior cervical ganglia at 48 hr postdenervation were all considerably above those of denervated controls. However, values for cats infused with 0.9% NaCl solution and for noninfused cats in which sodium pentobarbital anesthesia was maintained during the 24- to 48-hr postdenervation period were similarly elevated, to approximately twice the values in denervated controls. Ligation of the external carotid and lingual arteries at 24 hr postdenervation was found to oppose the preservation of acetylcholinesterase and butyrylcholinesterase contents of the ganglia induced by barbiturate anesthesia. When arterially ligated cats were infused with extract for periods of 12, 6, or 3 hr, beginning 24 hr postdenervation, acetylcholinesterase contents of superior cervical ganglia were elevated significantly above those of reanesthetized, arterially ligated controls after 12 and 6 hr but not after 3 hr of infusion, at 48 hr postdenervation.
RNA-dependent RNA polymerases of plants
Authors: Fraenkel-Conrat H.
Published: May 2006

The existence of RNA-dependent RNA polymerases (EC 2.7.7.48) in plants has been definitely proven by their isolation in pure form from cucumber and tobacco in our laboratory and from cowpea at Wageningen. These enzymes are single-chain proteins of 100-130 kilodaltons. They show clear physical and biochemical differences characteristic for a given plant species, even when their amounts in the plants were greatly increased prior to isolation by infection with the same virus. The role of these enzymes in plant physiology remains unknown.
Relationship between number of spleen colonies and 125IdUrd incorporation into spleen and femur.
Authors: Inoue T , Bullis J E , Cronkite E P , Hübner G E
Published: May 2006

Graded numbers of bone marrow (BM) cells were injected into fatally irradiated mice. Eight days later the mice were given 3.0 microCi (1 Ci = 3.7 X 10(10) Bq) of 125IdUrd to label proliferating cells in the spleen and BM. On day 9 the mice were killed and the spleens and femurs were removed for splenic colony assay and measurement of radioactivity in the spleen and femurs. The number of splenic colonies shows a linear relationship with dose of marrow cells injected from 10(4) to 10(5) cells. The slope of the curve of spleen colonies versus number of cells injected is less than 1, implying that the fraction seeded in spleen decreases with number of cells injected. Above 10(5) and below 10(4) there is a striking departure from the simple linearity. Below 2 X 10(3) cells injected, the logarithm of the observed colony yield is linear with logarithm of the number of cells injected. Poisson calculation of the average number of pluripotent stem cells that should be present with numbers of marrow cells injected below 2 X 10(3) followed closely the actual observations. The data show that there is no detectible proliferation in the BM until the dose of marrow cells exceeds 3.5 X 10(4) cells. Induction of cells into cycle increases the seeding into the BM, and thymidine cytocide drastically reduces seeding in the BM, leading us to conclude that the BM is repopulated almost exclusively by stem cells in DNA synthesis.
Cloned measles virus-specific T lymphocytes from a twin with multiple sclerosis.
Authors: Richert J R , McFarland H F , McFarlin D E , Bellini W J , Lake P
Published: May 2006

A measles virus-specific T lymphocyte clone derived from a measles-responder twin with multiple sclerosis has been produced. The cloned cells proliferate in response to measles virus but not mumps, vaccinia, or canine distemper viruses. The clone recognizes an antigen that is distributed in both the membrane and cytoplasmic fractions of cells infected with measles virus. Antigen is presented to the clone equally well by irradiated mononuclear cells obtained from the autologous subject and from her healthy, measles-nonresponder, HLA-identical twin. Studies with a panel of HLA-typed irradiated mononuclear cells indicate that antigen is presented to the clone in the context of a gene product linked to, but not identical with, HLA-DRw2. The clone produces interleukin 2 and has surface determinants recognized by OKT-3, OKT-4, and anti-HLA-DR monoclonal antibodies, but not by OKT-8. It thus appears to belong to the helper/inducer subpopulation of T lymphocytes.
Initiation of DNA replication in vitro by a DNA-membrane complex extracted from Bacillus subtilis.
Authors: Benjamin P , Firshein W
Published: May 2006

Initiation of DNA replication has been observed in vitro with a DNA-membrane complex extracted from Bacillus subtilis. Antibiotics known to interfere with various aspects of initiation inhibited DNA synthesis significantly in vitro, whereas a mutant resistant to one inhibitor failed to respond to its presence. The inhibitory effects occurred primarily when the immediate RNA precursors (ribonucleoside triphosphates) were present in the assay solution but not significantly when the precursors were omitted. Complexes extracted from a temperature-sensitive initiation mutant were almost incapable of synthesizing DNA at the restrictive temperature but displayed extensive synthesis at the permissive temperature. A strong indication of semiconservative DNA synthesis was obtained in vitro after density-shift experiments involving incubation of the complex with a heavy-density DNA precursor, followed by neutral and alkaline CsCl density gradient centrifugation. A significant amount of chain elongation or repair (or both) was also observed.
Nucleotide sequence of v-rel: the oncogene of reticuloendotheliosis virus.
Authors: Stephens R M , Rice N R , Hiebsch R R , Bose H R , Gilden R V
Published: May 2006

The nucleotide sequence of v-rel, the oncogene carried by reticuloendotheliosis virus (REV), has been determined. The defective transforming genome REV arose through the insertion of v-rel (1,415 nucleotides) into the env gene of the helper virus REV-A. The predicted rel protein (503 amino acids) employs the REV-A env initiator and terminates within the p20E region of env. Because there are no natural antisera that detect the REV transforming protein, this nucleotide sequence provides the first step toward its isolation and characterization. The predicted protein is clearly distinct from all other reported transforming proteins but may be very distantly related to members of the src family.
pH regulation of divalent/monovalent Ca/K cation transport selectivity by a macrocyclic carrier molecule.
Authors: Hriciga A , Lehn J M
Published: May 2006

The lipophilic dicarboxylic acid-dicarboxamide macrocycle 1 is an efficient carrier for calcium and potassium transport through a liquid membrane. The process involves competitive Ca2+/K+ symport coupled to proton antiport in a pH gradient. It presents a very pronounced phenomenon of pH regulation of transport selectivity from preferential K+ transport to preferential Ca2+ transport as the pH increases from 2 to 9 in the starting aqueous phase containing the metal ions. The results demonstrate how carrier design allows control of the rate and selectivity of divalent/monovalent M2+/M+ cation transport.
Somatocrinin receptor coupled with cAMP-dependent protein kinase on anterior pituitary granules.
Authors: Lewin M J , Reyl-Desmars F , Ling N
Published: May 2006

The molecular mechanism of growth hormone release by synthetic somatocrinin was investigated on purified hog anterior pituitary secretory granules; the granules were found to contain a cAMP-dependent protein kinase that catalyzed [gamma-32P]-ATP histone phosphorylation with maximal rates ranging from 1 to 5 nmol of Pi incorporated per mg of protein per 20 min. The activity of this enzyme was further stimulated by somatocrinin. Stimulation was observed at concentrations as low as 0.3 pM, and the half-maximal effect was obtained with 35 +/- 8 pM (n = 4). Michaelis-Menten analysis of phosphorylation kinetics suggested that the peptide did not change significantly the reaction's Vmax, but produced a dramatic increase in enzyme affinity for cAMP: the apparent Km for the nucleotide decreased from 400 X 10(-9) M under unstimulated conditions to 15 X 10(-9) M in the presence of 100 pM somatocrinin. Furthermore, a Hill plot of concentration-dependence curve indicated the existence of negative cooperativity. At the concentration of 35 pM, the less potent analogs of somatocrinin [designated hpGRF-44 to indicate source (human pancreas, hp), activity (growth hormone-releasing factor, GRF), and amino acid composition], hpGRF-(1-37) and [Phe1]hpGRF-(1-40) had 20% and 7%, respectively, of the effect of somatocrinin. The biologically inactive analog hpGRF-(2-40) had no evident effect at concentrations up to 0.1 microM. Therefore, we suggest that somatocrinin stimulation of growth hormone release involves activation of exocytosis through a phosphorylation mechanism mediated by a granular receptor coupled with a cAMP-dependent protein kinase.
Eukaryotic initiation factor 4A is the component that interacts with ATP in protein chain initiation.
Authors: Seal S N , Schmidt A , Marcus A
Published: May 2006

Protein synthesis in a resolved homogenate of wheat germ requires ATP and eight factors functioning at the level of protein chain initiation. To identify the component(s) interacting with ATP, the different factors were treated with the ATP affinity analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and tested for their function in protein synthesis. The activity of eukaryotic initiation factor 4A (eIF4A) was strongly curtailed, whereas all other factors were unaffected. At a concentration of 250 microM, AMP, ADP, and ATP protected eIF4A against FSBA inactivation, whereas at a concentration 50 microM, protection was afforded only by ATP. GTP did not protect at a concentration of 250 microM. In another approach, the substrate analogue 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) was found to inhibit protein synthesis in a manner, at least in part, competitive with ATP. Supplementing a TNP-ATP inhibited reaction with eIF4A substantially reversed the inhibition. Except for a small effect by factor C1, no reversal was obtained with any other component. Finally, a preincubation of ribosomes with ATP, mRNA, and eIF4A resulted in the formation of a complex capable of TNP-ATP-resistant amino acid incorporation. These data are interpreted to indicate that the primary interaction of ATP is with eIF4A. A model is proposed reconciling this conclusion with other observations relevant to the mRNA . ribosome attachment reaction.
Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes.
Authors: Harrison J J , Anisowicz A , Gadi I K , Raffeld M , Sager R
Published: May 2006

5-Azacytidine (azaC), a drug that induces decreased methylation of DNA in mammalian cells, was shown previously to induce differentiation of mesenchymal cell types in CHEF/18 cells (Chinese hamster embryo fibroblasts). This paper describes the effectiveness of azaC in inducing tumorigenicity in CHEF/18 cells, previously shown to be nontumorigenic stable diploids. A short exposure of growing cells to 3 microM azaC induced tumor-forming ability in CHEF/18 stem cells. Pre-adipocyte clones and subclones derived from CHEF/18 by prior treatment with azaC were also found to be tumorigenic. Pre-adipocytes previously induced by insulin in the absence of azaC were mostly nontumorigenic, but one clone produced tumors and gave rise to both tumorigenic and nontumorigenic subclones. Karyotype analysis of 41 clones and subclones from azaC-induced and insulin-induced pre-adipocytes revealed a complete correlation between tumor-forming ability and the presence of trisomy for chromosome 3q. In addition, the tumorigenic and tumor-derived lines were demethylated at specific C-C-G-G sites in the preproinsulin, Ha-ras, and Ki-ras genes as revealed by blot hybridization to Msp I- and Hpa II-digested DNAs, whereas the nontumorigenic lines resembled the CHEF/18 controls. This three-way correlation between tumorigenicity, trisomy for 3q, and specific demethylation suggests that decreased DNA methylation may be involved both in differentiation and in tumorigenicity, and that azaC may induce chromosomal aberrations as well as altering DNA methylation.
Serotonin alters the subcellular distribution of a Ca2+/calmodulin-binding protein in neurons of Aplysia.
Authors: Saitoh T , Schwartz J H
Published: May 2006

We purified and radioiodinated calmodulin from Aplysia nervous tissue. Using blot analysis, we detected 14 calmodulin-binding proteins in extracts of the abdominal ganglion after gel electrophoresis in NaDodSO4. All 14 components are present in extracts from each of the major central Aplysia ganglia, but their relative proportions differed characteristically from one ganglion to another. When neural components are extracted with nonionic detergents in 2 M glycerol, we obtain a cytoplasmic and a membrane-cytoskeleton fraction. Of the 14 calmodulin-binding proteins, 2 are cytoplasmic, 10 are loosely associated, and 2 remain bound to the membrane-cytoskeleton complex. Treatment of isolated ganglia with serotonin, which has been shown to increase the content of cAMP within neurons, or with dibutyryl cAMP causes the partial dissociation of one of the loosely associated proteins, which has an apparent Mr of 55,000. In extracts prepared in the presence of glycerol and cAMP, this component also is dissociated from the membrane-cytoskeleton complex, but only if ATP is also present. These results suggest that a cAMP-dependent protein phosphorylation controls the subcellular distribution of the Mr 55,000 calmodulin-binding protein, whose state of phosphorylation itself, however, does not appear to be increased by cAMP.
Identification of the probable site of synthesis of butyrylcholinesterase in the superior cervical and ciliary ganglia of the cat.
Authors: Uchida E , Koelle G B
Published: May 2006

The source of butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) in the ganglion cells of the cat superior cervical and ciliary ganglia has been elusive, inasmuch as the enzyme is present in high concentrations in the neuropil, where it is confined largely to the dendritic and perikaryonal plasma membranes, but appears to be absent from the perikarya. In the present study, ganglionic butyrylcholinesterase was near-totally inactivated by the injection of tetramonoisopropyl pyyrophosphoramide (6.0 mumol/kg of body weight) intravenously. During the ensuing 72 hr, the regenerating enzyme became detectable by the copper thiocholine histochemical method in the somata of essentially all ganglion cells and in the neuropil. Results were similar in preganglionically denervated superior cervical ganglia and in normal ciliary ganglia. These findings suggest (i) that butyrylcholinesterase indeed is synthesized in the ganglion cell perikarya (presumably, the rough endoplasmic reticulum) and transported extremely rapidly to more peripheral cellular sites and (ii) that the synthesis is largely independent of control by any neurotrophic factor provided by the preganglionic axonal terminals. Similar studies were conducted in the rat. In this species, in contrast to the cat, the somata of essentially all ganglion cells of the superior cervical ganglion contain various but at least moderate concentrations of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) and propionylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). After injection of tetramonoisopropyl pyrophosphoramide, propionylcholinesterase reappeared in the ganglion cell somata before its accumulation in the neuropil, as would be expected.
Stable low molecular weight DNA in xeroderma pigmentosum cells.
Authors: Hurt M M , Beaudet A L , Moses R E
Published: May 2006

Xeroderma pigmentosum (XP) cells from several complementation groups contained more low molecular weight DNA upon alkaline sucrose gradient centrifugation than did other human cells examined. Under conditions in which only 5% of the DNA in normal cells sedimented at 16 S or less, 20% of the DNA in XP cells from complementation group A sedimented at 16 S or less. Because cells were layered directly onto the gradients for lysing of cells and denaturing of the DNA, it appears that this low molecular weight material is due to naturally existing gaps or alkali-sensitive sites, or both, in the cellular DNA. The increase in low molecular weight DNA seen in XP complementation group A cells also is seen in complementation groups C, D, and E. When prelabeled cells were incubated for increasing times after removal of the radioactive label, the amount of low molecular weight material remained constant over a 3-hr period. The introduction of the DNA-damaging agent, bleomycin, to prelabeled XP cells produced a surprising effect. The normal response of human cells to bleomycin is an increase in low molecular weight DNA, dependent on the dose of the drug and time of treatment. In XP cells the reverse was observed. That is, the low molecular weight DNA observed in untreated XP cells disappeared upon addition of the drug. The process responsible for the unusual response of XP cells to bleomycin is unknown, but these results are compatible with an inducible repair process.
Classes of unitarizable derived functor modules
Authors: Enright Thomas J. , Parthasarathy Rajagopalan , Wallach Nolan R. , Wolf Joseph A.
Published: May 2006

For a real semisimple Lie group
G
, the description of the unitary dual remains an elusive question. One of the difficulties has been the lack of technique for constructing unitary representations. Unitary induction from parabolic subgroups of
G
yields unitary representations by the very definition of these representations. However, not all unitary irreducible representations of
G
are obtained by this type of induction. In addition, we need derived functor parabolic induction [ef. Vogan, D. (1981)
Representations of Real Reductive Lie Groups
(Birkhäuser, Boston)] to describe all irreducible representations of
G
. For this second type of induction, the obvious analogues from parabolic subgroup induction regarding unitarity are false. In this announcement, we describe a setting where derived functor parabolic induction yields unitary representations of
G
. These results include proofs of unitarity for some of the representations conjectured to be unitary by Vogan and Zuckerman [(1983)
Invent. Math.
, in press] and also proofs of unitarity for some which lie outside the domain described in those conjectures.
An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast.
Authors: Emr S D , Schekman R , Flessel M C , Thorner J
Published: May 2006

The peptide mating pheromone alpha-factor and the hydrolytic enzyme invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae). An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and prepro-leader portion of the coding sequence of the alpha-factor gene (MF alpha 1) to a large fragment of the invertase gene (SUC2) lacking its 5'-flanking region and the coding information for the first four amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the alpha-factor precursor have been retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-alpha-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the alpha-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF alpha 1, the MF alpha 1-SUC2 fusion is expressed in alpha haploids at levels 65-75 times higher than in a haploids or in a/alpha diploids; also, high-level expression is eliminated in mat alpha 1 mutants but not in mat alpha 2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer alpha cell-specific expression to the hybrid gene.
Two Epstein-Barr viral nuclear neoantigens distinguished by gene transfer, serology, and chromosome binding.
Authors: Grogan E A , Summers W P , Dowling S , Shedd D , Gradoville L , Miller G
Published: May 2006

We recently identified, by means of cotransformation of LTK- cells, a region of the Epstein-Barr virus (EBV) genome (the BamHI K fragment) that encodes or induces an EBV nuclear neoantigen (EBNA) serologically related to the EBNA found in lymphoid cells carrying the entire EBV genome. We now find that a second EBV DNA fragment, BamHI M, is also able to give rise to cotransformed LTK- cells with stable expression of a nuclear antigen. The BamHI K and M fragments have no apparent DNA homology. Many human sera that are reactive to EBNA in Raji cells detect both antigens; however, certain anti-EBNA-positive human sera are discordant and react only with the BamHI M or only with the BamHI K nuclear antigen. Every Raji cell appears to express both "M" and "K" antigens; D98 Raji cells, a somatic cell hybrid, express only "K" antigen. The K antigen is found on metaphase chromosomes of LTK cells and Raji cells. The M-induced antigen is not located on chromosomes when the cells are in metaphase but is present as granules within the nucleus.
Degradation and resynthesis of gap junction protein in plasma membranes of regenerating liver after partial hepatectomy or cholestasis
Authors: Traub Otto , Drüge Petra Maria , Willecke Klaus
Published: May 2006

Changes in the total amount of the gap junction protein (
M
r
26,000) after partial hepatectomy or bile duct ligation and recanalization were investigated in rat liver membranes by quantitative immunoblot with rabbit antiserum to the
M
r
26,000 protein. The loss and reappearance of the
M
r
26,000 protein roughly paralleled loss and reappearance of gap junction plaques analyzed previously under similar physiological conditions by freeze-fracture of hepatocyte surfaces. The total amount of the hepatic
M
r
26,000 protein in liver plasma membranes and the total area of the hepatocyte surface occupied by gap junction plaques appeared to be proportional under these conditions. However, at the minimum, 28-35 hr after partial hepatectomy we still find about 15% of the
M
r
26,000 protein, in contrast to <1% of gap junction plaques, determined by morphometric analysis. This discrepancy is probably due to the fact that very small gap junction plaques, single connexons, and free
M
r
26,000 gap junction subunits are missed by the morphometric analysis. At the times of the minimal amount of the
M
r
26,000 protein in hepatic plasma membranes after partial hepatectomy or bile duct ligation we found that crude hepatic lysosomal membranes of these rats contained less
M
r
26,000 protein than lysosomal membranes of nonoperated control animals. Thus, we conclude that the decrease and increase of the total amount of the
M
r
26,000 protein cannot be explained only by dispersal and reuse of gap junction subunits but are largely due to degradation and resynthesis of the
M
r
26,000 protein. No significant change in the amount of the
M
r
21,000 protein that had been isolated with gap junction plaques was observed in liver plasma membranes after partial hepatectomy. This confirms our previous conclusion that the
M
r
26,000 and
M
r
21,000 proteins are independent of each other.
DNase I sensitivity of transcriptionally active genes in intact nuclei and isolated chromatin of plants.
Authors: Spiker S , Murray M G , Thompson W F
Published: May 2006

We have investigated the DNase I sensitivity of transcriptionally active DNA sequences in intact nuclei and isolated chromatin from embryos of wheat (Triticum aestivum L.). Nuclei or isolated chromatin was incubated with DNase I, and the extent of DNA digestion was monitored as percentage acid solubility. The resistant DNA and DNA from sham-digested controls were used to drive reassociation reactions with cDNA populations corresponding to either total poly(A)+RNA from unimbibed wheat embryos or polysomal poly(A)+RNA from embryos that had imbibed for 3 hr. Sequences complementary to either probe were depleted in DNase I-resistant DNA from nuclei and from chromatin isolated under low-ionic-strength conditions. This indicates that transcriptionally active sequences are preferentially DNase I sensitive in plants. In chromatin isolated at higher ionic strength, cDNA complementary sequences were not preferentially depleted by DNase I treatment. Therefore, the chromatin structure that confers preferential DNase I sensitivity to transcriptionally active genes appears to be lost when the higher-ionic-strength method of preparation is used. Treatment of wheat nuclei with DNase I causes the release of four prominent nonhistone chromosomal proteins that comigrate with wheat high mobility group proteins on NaDodSO4 gels.
Interferon-mediated inhibition of virus penetration.
Authors: Whitaker-Dowling P A , Wilcox D K , Widnell C C , Youngner J S
Published: May 2006

Pretreatment of mouse L cells with mouse interferon (IFN) inhibits the penetration of vesicular stomatitis virus without affecting viral adsorption. The inhibition of virus uptake by IFN is dose dependent and, at the highest dose tested (1,000 units/ml), reaches 65%; 24 hr of treatment with IFN are required for maximal effect. A similar inhibition of uptake of virus occurs in human diploid fibroblasts and primary chicken embryo fibroblasts treated with homologous IFN. No significant inhibition occurs when cells are treated with heterologous IFN. These results document a previously unrecognized antiviral effect of IFN--namely, inhibition at the level of viral uptake.
In vitro complementation as an assay for purification of adenovirus DNA replication proteins.
Authors: Ostrove J M , Rosenfeld P , Williams J , Kelly T J
Published: May 2006

As an approach to the purification of adenovirus-encoded DNA replication proteins, we have developed in vitro complementation assays that make use of viral mutants defective in DNA replication in vivo. Nuclear extracts prepared from cells infected with H5ts36 or H5ts125, two such mutants belonging to different complementation groups, were found to be defective in viral DNA replication in vitro. However, replication activity could be restored by mixing the two extracts. Replication activity in either extract also could be restored by addition of appropriate replication-deficient fractions purified from cells infected with wild-type adenovirus. By using such assays, H5ts36- and H5ts125-complementing activities were extensively purified. As expected, purified H5ts125-complementing activity consisted of a single major polypeptide, the 72-kilodalton (kDal) adenovirus DNA binding protein. The purified H5ts36-complementing activity consisted of the 80-kDal adenovirus terminal protein precursor and two other major polypeptides with apparent molecular masses of 140 and 65 kDal. Formation of the 80-kDal terminal protein-dCMP complexes, the proposed initial step in adenovirus DNA replication, required components in the purified H5ts36-complementing fraction and a cellular factor(s) but did not require the adenovirus DNA binding protein. The complete in vitro adenovirus DNA replication reaction was reconstituted from the purified H5ts36-complementing activity, the adenovirus DNA binding protein, and an extract from uninfected cells.