bluuebunny/crossref_metadata_2025 huggingface dataset:
Benzodiazepine/gamma-aminobutyric acid receptor deficit in the midbrain of the seizure-susceptible gerbil.
Authors: Olsen R W , Wamsley J K , McCabe R T , Lee R J , Lomax P
Published: May 2006

The density of benzodiazepine/gamma-aminobutyric acid receptor binding sites was lower in the midbrain of seizure-susceptible gerbils compared to control seizure-resistant gerbils. Binding of [3H]diazepam to high-affinity brain-specific sites in membrane homogenates of gerbil brain showed a 20-30% lower binding in midbrain (but not other regions) in adult seizure-susceptible gerbils than in controls. This binding deficit was localized by tissue slice autoradiography with [3H]flunitrazepam to the substantia nigra and mesencephalic periaqueductal gray regions, while higher binding was observed in the interpeduncular nucleus. These differences were also seen in animals sacrificed immediately after a seizure. A parallel deficit of [3H]bicuculline methochloride binding to low-affinity gamma-aminobutyric acid receptors also was seen in the same midbrain regions. Scatchard plot analysis showed that the benzodiazepine binding deficit in the nigra was due to a lower number of binding sites with not significant difference in affinity. Lower [3H]flunitrazepam binding was likewise seen in younger animals (29% lower at 30 days of age, 38% at 60 days, and 21% at 90 days), indicating that the midbrain receptor deficit is present in the seizure-susceptible gerbil prior to the age of onset of seizures at 50-100 days. Therefore, these changes are not likely to result from seizures but reflect genetically determined biochemical differences that could play a role in the expression of seizure susceptibility. The deficit in midbrain benzodiazepine/gamma-aminobutyric acid receptors in the seizure-susceptible gerbil would be consistent with the hypothesis that a deficit of gamma-aminobutyric acid-mediated inhibition might contribute to some kinds of epilepsy.
Sequential changes in DNA methylation patterns of the rat phosphoenolpyruvate carboxykinase gene during development.
Authors: Benvenisty N , Mencher D , Meyuhas O , Razin A , Reshef L
Published: May 2006

The cytosolic phosphenolpyruvate carboxykinase [PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] gene was isolated from a rat genomic library, and a map of the methylatable sites C-C-G-G and G-C-G-C has been constructed. The extent of methylation of 18 sites in the PEPCK gene in adult liver, kidney, spleen, and heart muscle and in fetal liver has been analyzed using the 5-methylcytosine sensitive enzymes Hpa II and Hha I. This analysis revealed extensive undermethylation of the PEPCK gene in the adult liver and kidney (PEPCK-expressing tissue), whereas the gene in adult spleen and heart muscle as well as in fetal liver (PEPCK-nonexpressing tissues) was heavily methylated. However, unlike the gene in the adult nonexpressing tissues, a region in the middle of the gene was found to be partially hypomethylated in fetal liver. This hypomethylation correlates with the competence of the fetal liver gene to be expressed. Treatment of fetuses by in utero injection of 5-azacytidine causes a hypomethylation-associated activation of the PEPCK gene. Taken together, the present findings suggest a sequential loss of methyl groups during development. When related to PEPCK gene expression, the sequential loss of methyl groups demonstrates an early stage prior to transcription characterized by hypomethylation of discrete sites and a later developmental hypomethylation of all sites associated with the mature active PEPCK gene around the time of birth.
Mechanism of inhibition of DNA gyrase by analogues of nalidixic acid: the target of the drugs is DNA.
Authors: Shen L L , Pernet A G
Published: May 2006

Norfloxacin is a nalidixic acid analogue and one of the most potent DNA gyrase inhibitors. To study the mechanism of this important class of inhibitors, the binding of [3H]norfloxacin to gyrase and substrate DNA was measured. We found that, contrary to prior belief, norfloxacin does not bind to gyrase but instead binds to DNA. This was demonstrated by both equilibrium dialysis and membrane filtration techniques. Binding to ColE1 and pBR322 plasmids showed a primary process that is saturated at a norfloxacin concentration about equal to its supercoiling Ki (1.8 X 10(-6) M) and is followed by weaker secondary binding. The apparent Kd values are 1 X 10(-6) M for both plasmids. The molar binding ratio at this initial saturation point is extremely low: only 4 X 10(-4) norfloxacin per nucleotide for both plasmids. The binding of norfloxacin to DNA plasmids is nonintercalative, as shown by the fact that the drug binds preferentially to single-stranded DNA rather than to double-stranded DNA. The binding is reduced at high salt concentration, has a pH optimum between 4.5 and 6.5, and does not require divalent ions. The binding affinities of other nalidixic acid analogues were estimated by an indirect competition method. The calculated apparent Kd values of these analogues correlate well with their Ki values, providing strong evidence that the binding affinity of the drug to DNA determines biological potency.
Cloning determinants of pathogenesis from
Pseudomonas syringae
pathovar
syringae
Authors: Niepold Frank , Anderson Dirk , Mills Dallice
Published: May 2006

Transposon mutagenesis and a cosmid genomic library of DNA from the bean pathogen
Pseudomonas syringae
pathovar
syringae
were used to identify and isolate sequences essential for pathogenesis. Strain PS9021, derived by Tn5 mutagenesis, was determined to be nonpathogenic on
Phaseolus vulgaris
cultivar Red Mexican and incapable of inducing a hypersensitive response in
Nicotiana tabacum
. This mutant also produced fluidal rather than firm colonies on selected agar media. A Tn5-containing
Eco
RI fragment from PS9021 was cloned and used to probe 1500 members of a genomic library constructed with DNA from the pathogenic parent strain and the wide host range cosmid pVK102. One member that hybridized to the probe contained a cosmid with a 30-kilobase-pair insert (pOSU3101) that complemented the mutant phenotypes when mobilized into PS9021. A restriction endonuclease cleavage site map of pOSU3101 was constructed and sequences essential for pathogenesis were determined by subcloning. Approximately 8.5 kilobase pairs of the insert were essential for restoration by complementation of pathogenesis and hypersensitive response and wild-type colony morphology in strain PS9021.
Endonuclease III (nth) mutants of Escherichia coli.
Authors: Cunningham R P , Weiss B
Published: May 2006

Two strains that overproduce endonuclease III were found in a colony bank containing hybrid ColE1-Escherichia coli plasmids. The enzyme was identified in crude extracts by the degradation of partially depyrimidinated DNA in the presence of EDTA, by its sedimentation velocity, and by its associated thymine glycol-DNA glycosylase activity. An insertion mutation was produced by cloning the kanamycin-resistance gene of Tn5 into the plasmid copy of the nth gene. The mutation was then transferred to the chromosome in the following steps: (i) selection for chromosomal integration of the plasmid at 42 degrees C in a temperature-sensitive polA strain, (ii) curing via temperature shifts, and (iii) phage P1-mediated transduction of a new host. The insertion mutant, as well as a separately isolated deletion mutant, had no measurable glycosylase activity for DNA containing thymine glycol. Although such residues are common lesions in oxidized or irradiated DNA, the mutants were not unusually sensitive to H2O2 or gamma-rays. The insertion mutation had a mutator effect (4- to 22-fold enhancement) on one tested allele.
Rearrangement and transcription of a T-cell receptor beta-chain gene in different T-cell subsets.
Authors: Hedrick S M , Germain R N , Bevan M J , Dorf M , Engel I , Fink P , Gascoigne N , Heber-Katz E , Kapp J , Kaufmann Y
Published: May 2006

The functionally defined sets of T lymphocytes--helper T cells, cytotoxic T cells, and suppressor T cells--were examined for the possible involvement of a recently identified T-cell receptor beta gene locus in receptor formation. Since gene rearrangements are required for functional gene expression, cloned T-cell lines from each of the groups were surveyed for the expression of unique gene rearrangements. In addition, cell lines that showed gene rearrangements were further tested for the expression of the mature 1.2- to 1.3-kilobase mRNA transcribed from a productive gene rearrangement. The results of such experiments show that helper and cytotoxic T cells may use a common beta chain of the receptor, whereas suppressor cells do so rarely, if at all.
Human major histocompatibility complex class I antigens: residues 61-83 of the HLA-B7 heavy chain specify an alloreactive site.
Authors: Walker L E , Ketler T A , Houghten R A , Schulz G , Chersi A , Reisfeld R A
Published: May 2006

A chemically synthesized peptide (sequence in text) homologous to residues 61-83 of the HLA-B7 heavy chain, induced antibodies that specifically recognized the HLA heavy chain-beta 2-microglobulin complex and the free heavy chain of the HLA-B7 antigen. These antibodies specifically immunoprecipitated the HLA-B7 beta 2-microglobulin complex solubilized from human lymphoblastoid cells by nonionic detergents and reacted with free HLA-B7 heavy chains in blots on nitrocellulose. These observations suggest that the antigenic conformation of this region of the HLA-B7 molecule is independent of the presence of beta 2-microglobulin and that amino acid residues 61-83 mimic an alloreactive site expressed by the HLA-B7 antigen.
Demonstration of inter- and intraspecies differences in serotonin binding sites by antibodies from an autistic child.
Authors: Todd R D , Ciaranello R D
Published: May 2006

Serotonin (5-HT) binding sites from bovine and rat cerebral cortex membranes share pharmacological properties that allow both to be subclassified by the same criteria. We show here that [3H]5-HT binding sites from human cortex also possess pharmacological properties that follow the same subclassification scheme as for bovine and rat cortex. In addition, we show that solubilized 5-HT1 and 5-HT3 sites from all three species have an s20,w value of 3.4. Despite these similar pharmacological and physical characteristics, we can demonstrate antigenic differences between receptor types and species. Human 5-HT1A sites can be distinguished from human 5-HT1B, 5-HT2, and 5-HT3 sites and from equivalent sites in rat and bovine cortex. The anti-human 5-HT1A antibodies were discovered in the blood of an autistic child and may have clinical or etiologic significance for this disorder.
Topological mapping of acetylcholine receptor: evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide.
Authors: Young E F , Ralston E , Blake J , Ramachandran J , Hall Z W , Stroud R M
Published: May 2006

Antibodies were raised against two synthetic peptides whose sequences correspond respectively to the COOH-terminal end (residues 501-516) of the protein encoded by the gene for the delta chain and to a proposed cytoplasmic region (residues 350-358) of the beta chain of the acetylcholine receptor from Torpedo californica. Binding of the COOH-terminal antibody to the acetylcholine receptor in intact, receptor-rich vesicles was tested by radioimmunoassay and by precipitation with immobilized protein A. In both cases, binding was detected only after treatment of the vesicles with detergent, suggesting that the segment of the receptor that is recognized by this antibody is on the cytoplasmic side of the membrane. Electron microscopy of tissue from Torpedo electric organ labeled with colloidal gold-conjugated second antibodies established that both anti-receptor antibodies bind to the cytoplasmic surface of the postsynaptic membrane. These experiments give ultrastructural evidence that the COOH-terminal segment of the delta chain as well as residues 350-358 of the beta chain are on the cytoplasmic surface. They strongly support a model in which each of the receptor subunits crosses the membrane five times in which one transmembrane segment of each chain contributes to the formation of a central ion channel.
Modification of chromium(VI)-induced DNA damage by glutathione and cytochromes P-450 in chicken embryo hepatocytes.
Authors: Cupo D Y , Wetterhahn K E
Published: May 2006

The role of glutathione and cytochrome P-450 in the production of DNA damage by chromium(VI) was examined in chicken embryo hepatocytes by the alkaline elution technique. Cellular levels of glutathione and cytochrome P-450 were altered by treating the hepatocytes with N-acetyl-L-cysteine, buthionine sulfoximine, isopentanol, or beta-naphthoflavone. A dramatic increase in chromium(VI)-induced DNA strand breaks was observed after increasing glutathione levels in the cells. Chromium(VI)-induced DNA strand breaks were even more numerous when the level of cytochrome P-450 was also increased. Upon depletion of glutathione levels and induction of cytochrome P-450 or cytochrome P-448, little or no DNA strand breaks or DNA interstrand cross-links were observed after chromium(VI) treatment. Chromium(VI)-induced DNA-protein cross-links generally decreased after either increases or decreases in cellular levels of glutathione or cytochrome P-450 or P-448. These results suggest that glutathione enhances chromium(VI)-induced DNA damage through metabolic activation of chromium(VI). The possible production of reactive chromium species upon metabolism by glutathione and cytochrome P-450 or P-448 and their involvement in DNA damage is discussed.
Maize mitochondria synthesize organ-specific polypeptides.
Authors: Newton K J , Walbot V
Published: May 2006

We detected both quantitative and qualitative organ-specific differences in the total protein composition of mitochondria of maize. Labeling of isolated mitochondria from each organ demonstrated that a few protein differences are due to changes in the polypeptides synthesized by the organelle. The synthesis of developmental stage-specific mitochondrial polypeptides was found in the scutella of developing and germinating kernels. The approximately 13-kDa polypeptide synthesized by mitochondria from seedlings of the Texas (T) male-sterile cytoplasm was shown to be constitutively expressed in all organs of line B37T tested. Methomyl, an insecticide known to inhibit the growth of T sterile plants, was shown to be an effective inhibitor of protein synthesis in mitochondria from T plants.
Cell surface antigen of human neuroblastomas is related to nuclear antigen of normal cells.
Authors: Rettig W J , Chesa P G , Jennings M T , Spengler B A , Melamed M R , Oettgen H F , Biedler J L , Old L J
Published: May 2006

The localization of MC25, an antigen first detected on the surface of human neuroblastoma cells, was determined in cultured cells and tissues. Neuroblastoma cell lines (15/17) express the antigen on the surface and in the cytoplasm (scMC25+), whereas 156/160 cell lines derived from other normal and malignant human cell types are scMC25-. However, MC25 is found in the nucleus of scMC25- cells (nMC25+), presenting a discrete granular pattern. In scMC25+/nMC25- neuroblastoma lines, apparent antigen shifting from the cell surface/cytoplasm to the nucleus accompanies variant formation, which represents a transition in the neuronal differentiation program of these cells. Results of immunohistochemical studies with human tissues parallel the findings with cultured cells. Almost all cell types are scMC25-/nMC25+; basal cells of the epidermis are the only cells constitutively expressing cMC25; and a population of neurons are the only scMC25-/nMC25- cells. Alternative localization of MC25 to different cellular compartments and antigen shifting are reminiscent of the behavior of certain developmentally regulated antigens in Drosophila and Xenopus.
Use of pooled DNA samples to detect linkage disequilibrium of polymorphic restriction fragments and human disease: studies of the HLA class II loci.
Authors: Arnheim N , Strange C , Erlich H
Published: May 2006

A rapid method has been developed and used to search for restriction fragment length polymorphisms (RFLPs) that are in linkage disequilibrium with disease-associated loci. By using genomic blot-hybridization analysis with DQ beta-chain and DR beta-chain cDNA probes, we examined DNA polymorphisms within the HLA class II loci associated with susceptibility to insulin-dependent mellitus (IDDM). To facilitate the search for informative RFLPs, we compared pooled DNA samples from IDDM patients with pooled DNA samples from randomly selected control individuals, instead of using the conventional approach of examining DNA samples from individuals in two groups. (The conditions under which this approach is useful are treated theoretically in the Appendix.) Several specific polymorphic restriction fragments associated with IDDM were revealed by using this economical and rapid approach. The restriction enzymes and probes identified as informative in this screening were then used to analyze HLA-DR-typed IDDM families, homozygous typing cells, and unrelated individuals to determine the association of the specific restriction fragments with HLA-DR serological type and the frequency in control and IDDM populations. Some individual polymorphic fragments for which the IDDM population was enriched correlated strongly with HLA-DR3, whereas others correlated strongly with HLA-DR4. Some fragments (e.g., a 10-kilobase Taq I fragment detected with the DR beta probe) that were more prevalent in the IDDM population subdivided the serologically defined HLA-DR type and may be informative markers for IDDM susceptibility.
Additional products of the Tla locus of the mouse.
Authors: Chorney M J , Shen F W , Tung J S , Boyse E A
Published: May 2006

Thymocytes and leukemia cells of some mouse strains yield TL proteins, precipitable by anti-TL antiserum and by anti-TL monoclonal antibodies, that include not only the familiar heavy (H) chain of 45-50 kDa but also products of higher molecular mass. Production of a 53-kDa TL form by Tlad thymocytes was studied in detail. A cross was made between B10.M (Tlad) mice, which produce the 53-kDa TL, and mice of the A strain (Tlaa), which make only the usual H chain. Hemi-expression of apparently unaltered 53-kDa TL was observed in thymocytes of the Tlad/Tlaa heterozygous F1 progeny. Thus, there was no indication of positive or negative trans interaction with respect to production of the 53-kDa TL form associated with Tlad. We conclude that production of 53-kDa TL is governed intrachromosomally. Two-dimensional chymotryptic peptide maps of the TL H chain and the 53-kDa TL of Tlad thymocytes differed only by added features found in the map of the 53-kDa TL. With the exception of Tlaa, all Tla alleles (Tlab-f) yielded TL products of higher molecular weight than the accompanying H chain, although in the case of Tlab this was evident only in TL+ leukemia cells because Tlab thymocytes are TL-. For H-2, representing other class I genes, no products other than the familiar H chain were demonstrable under similar conditions.
T-cell antigenic sites tend to be amphipathic structures.
Authors: DeLisi C , Berzofsky J A
Published: May 2006

We propose, on the basis of physical chemical and biological requirements for T-cell activation by antigen, that sites on a protein that can stimulate T lymphocytes will be capable of forming a stable amphipathic structure (i.e., one with separated hydrophobic and hydrophilic surfaces), displaying periodicity in hydrophobic residues. A spectral analysis of the 12 antigenic sites to which the method could be applied indicates that the amphipathic periodicity hypothesis is valid for 10 of them, generally with reliabilities that are well above 98%, with periodicities compatible with an alpha-helical structure. An 11th case manifests a different type of amphipathicity. The analyses require only a knowledge of amino acid sequence. The finding that T-cell antigenic sites show a high correlation with amphipathicity greatly simplifies the search for such sites and is potentially important for vaccine development.
Site-specific carcinogen binding to DNA in polytene chromosomes.
Authors: Kurth P D , Bustin M
Published: May 2006

Treatment of Chironomus polytene chromosomes with the ultimate carcinogen benzo[a]pyrene diol epoxide I or in vivo administration of the parent hydrocarbon to larvae indicates that the carcinogen interacts with the genome in a nonrandom manner. Visualization of the carcinogen-DNA binding sites by immunofluorescence reveals that, in vivo, some sites are preferentially modified. The combined effects of DNA sequence, chromatin structure, and gene localization may lead to selective targeting of carcinogens to specific genomic regions. In polytene chromosomes the targeting effect is amplified, thereby making these chromosomes a uniquely suitable system for visualizing and studying site-specific interactions of carcinogens with the genome.
Discrimination between fibrin and fibrinogen by a monoclonal antibody against a synthetic peptide.
Authors: Scheefers-Borchel U , Müller-Berghaus G , Fuhge P , Eberle R , Heimburger N
Published: May 2006

Circulating soluble fibrin, observed in the blood of patients with ongoing intravascular coagulation, is generated from the plasma protein fibrinogen by the limited proteolytic action of thrombin. We report the production of a monoclonal antibody that discriminates between fibrin and fibrinogen in blood. The synthetic hexapeptide Gly-Pro-Arg-Val-Val-Glu, representing the amino terminus of the alpha chain of human fibrin, was used as immunogen. This hexapeptide is located within the A alpha chain of fibrinogen but becomes the amino terminus of the fibrin alpha chain, after fibrinopeptide A is removed by the action of thrombin, and thus becomes accessible for antibody binding. The monoclonal antibody we have prepared can discriminate between fibrin and fibrinogen and thus can be used in assay systems to quantitate soluble fibrin or, potentially, to image fibrin-rich thrombi.
Immunological detection of hemoglobin in bones of ancient Roman times and of Iron and Eneolithic Ages.
Authors: Ascenzi A , Brunori M , Citro G , Zito R
Published: May 2006

Using an immunological method (immunoblot), we have established that hemoglobin (or hemoglobin fragments) can be quantitatively determined in old and ancient bones, some of them dating back 4500 years. It is shown that the total recovery decreases with time, but it is still effective in the older specimens. Thus, the immunological assay may prove useful to solve problems relevant to paleontology and paleopathology.
Genes for skeletal muscle myosin heavy chains are clustered and are not located on the same mouse chromosome as a cardiac myosin heavy chain gene.
Authors: Weydert A , Daubas P , Lazaridis I , Barton P , Garner I , Leader D P , Bonhomme F , Catalan J , Simon D , Guénet J L
Published: May 2006

Myosin heavy chain (MHC) genes are expressed as several distinct isoforms in a tissue- and stage-specific manner; three skeletal muscle MHC isoforms appear sequentially during development. We have isolated cDNA clones, identified by RNA blot hybridization and by nucleotide sequence determination as coding for portions of the embryonic (pMHC2.2), perinatal (pMHC16.2A), and alpha(V1) cardiac (pMHC141 and pMHC101) MHC isoforms. These four probes and the adult skeletal MHC probe (pMHC32) have been used on Southern blots of genomic DNA to detect restriction fragment length polymorphisms defining the alleles for these genes in mouse species Mus musculus and Mus spretus. In this way, we followed the segregation of skeletal and cardiac MHC genes in 42 offspring resulting from an interspecies backcross. We found that the embryonic, perinatal, and adult skeletal MHC genes are clustered on chromosome 11 near the locus nude, the skeletal and cardiac MHC genes do not cosegregate, and the alpha(V1) cardiac MHC gene is located on chromosome 14 close to Np-1. This result is in contrast to that for other contractile protein genes such as the alkali myosin light chain and the actin multigene families, which are dispersed in the genome.
Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid.
Authors: Ling N , Ying S Y , Ueno N , Esch F , Denoroy L , Guillemin R
Published: May 2006

A Mr 32,000 protein with inhibin activity was isolated from porcine follicular fluid by heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and four reversed-phase HPLC steps. The isolated molecule is composed of two chains having molecular weights of 18,000 and 14,000, respectively, and bound together by disulfide bonds. Amino acid sequence analysis revealed the 10 NH2-terminal residues of the Mr 18,000 chain to be Ser-Thr-Ala-Pro-Leu-Pro-Trp-Pro-Trp-Ser- and those of the Mr 14,000 chain to be Gly-Leu-Glu-Xaa-Asp-Gly-Arg-Thr-Asn-Leu-. This Mr 32,000 protein specifically inhibits the basal secretion of FSH, but not that of LH, in the rat anterior pituitary monolayer culture system, with a half-maximal effective dose of 450 pg/ml.
Expression of biologically active bovine luteinizing hormone in Chinese hamster ovary cells.
Authors: Kaetzel D M , Browne J K , Wondisford F , Nett T M , Thomason A R , Nilson J H
Published: May 2006

Biologically active bovine luteinizing hormone (LH) has been obtained through expression of the alpha- and LH beta-subunit genes in stably transformed clones of DUXB11, a Chinese hamster ovary cell line deficient in dihydrofolate reductase (DHFR). Expression of alpha-and LH beta-subunit mRNAs of the expected sizes (approximately 910 and 770 nucleotides, respectively) were revealed by blot analysis after electrophoresis of total cellular RNA. Furthermore, presence or absence of the gonadotropin mRNAs in several clonal lines was directly correlated with the appearance of one or both bovine LH subunits in the culture medium. Media from three clones secreting significant immunoreactive levels of both subunits also stimulated the release of progesterone in ovine luteal cells, suggesting that the secreted LH was assembled into a biologically active and glycosylated dimer. Immunoprecipitation and NaDodSO4/PAGE of [35S]methionine-labeled proteins secreted from one of the clones, CHODLH20, further confirmed the presence of an alpha/beta dimer with apparent subunit molecular weights of 20,500 and 16,000, only slightly higher than those of pituitary alpha and LH beta subunits.
Interaction between two transcriptional control sequences required for tumor-antigen-mediated simian virus 40 late gene expression.
Authors: Brady J , Loeken M R , Khoury G
Published: May 2006

Transcriptional control signals required for tumor (T)-antigen trans-activation of the simian virus 40 (SV40) late promoter include T-antigen binding sites I and II and the SV40 72-base-pair (bp) repeats. We have used in vivo competition studies to examine how these signals function in relationship to one another. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences [map position (mp) 5171-272] results in quantitative removal of limiting trans-acting factor(s) required for late gene expression in COS-1 cells. Deletion of either the T-antigen binding sites (mp 5171-5243) or the 72-bp tandem repeat (mp 128-272) from the competitor plasmid results in markedly less efficient binding of the trans-acting factor, as judged by the loss of competition. Cotransfection of two separate plasmids, one containing the T-antigen binding sites I and II and the other containing the 72-bp repeats, fails to compete for the trans-acting factors. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the enhancer sequences also dramatically reduces the efficiency of competition. These results suggest that efficient binding of trans-acting factors requires the presence, in cis, of at least two SV40 regulatory domains. Our studies further suggest that the distance separating these two transcriptional signals is important.
Induction of the proto-oncogene fos by nerve growth factor.
Authors: Kruijer W , Schubert D , Verma I M
Published: May 2006

Nerve growth factor (NGF) causes the differentiation of PC12 cells to sympathetic neuron-like cells and also induces a rapid but transient expression of fos mRNA and protein. fos mRNA transcripts can be detected 5 min after the addition of NGF, are maximally abundant after 30 min, and then their levels decrease. fos protein synthesis parallels the expression of fos mRNA, and the induced fos proteins are located in the nucleus. cAMP, epidermal growth factor, the phorbol ester phorbol 12-myristate 13-acetate, and K+ depolarization also induce the fos gene. Growth of PC12 cells in the presence of dexamethasone, which induces differentiation into chromaffin-like cells, is not accompanied by fos expression. We propose that while fos gene induction is associated with the differentiation of PC12 cells to sympathetic nerve, its enhanced expression is primarily involved in the anabolic responses induced by NGF and many growth factors.
One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair.
Authors: Hare J T , Taylor J H
Published: May 2006

Although the occurrence of 5-methylcytosine (m5C) in DNA is widespread, the function of this modified base remains unclear. At some specific sites it apparently has an effect in controlling gene expression, but many sites do not appear to be involved in this regulation. Balanced against its regulatory usefulness at some sites is the mutational risk it imposes upon the cell. Deamination of m5C can lead to its replacement by thymine (T). One possible role for excess methylation is strand discrimination in the repair of mismatches. We constructed the complementary hemimethylated single-base-pair mismatches, G T and A C, at a CG site in simian virus 40 DNA, transfected these into the host African green monkey kidney cells (CV-1), and examined DNA of the progeny for repair at this site. Hemimethylation at two Hha I sites (Gm5CGC) bracketing the mismatch directed repair to occur only on the unmethylated strand. Methylation at the multiple Cm5CATGG and Gm6ATC sites, a pattern normally seen in bacteria, also instructed repair to proceed on the unmethylated strand, although less efficiently. Hemimethylation at only one site, adjacent to the mispaired bases (Hpa II, Cm5CGG) produced repaired molecules in a ratio that may represent random repair of the A C mismatch and strand-directed repair in the complementary G T mismatch. The -mCG- -GT- mismatch could result from deamination of m5C in the most commonly methylated dinucleotide in vertebrates, CpG. Methylation may be able to compensate for the errors it causes by serving as a mechanism for strand discrimination in correcting those errors. In addition, single-strand nicks were also shown to direct repair.
Requirement of endogenous interferon-gamma production for resolution of Listeria monocytogenes infection.
Authors: Buchmeier N A , Schreiber R D
Published: May 2006

Peritoneal exudate cells and splenic cells of mice infected with Listeria monocytogenes show increased production of interferon-gamma (IFN-gamma) after antigen or mitogen stimulation. When an IFN-gamma-specific enzyme-linked immunosorbent assay was used, increased production was first observed 2 days after infection in peritoneal cells and 4 to 6 days after infection in splenic cells. The increased production of IFN-gamma correlated with the clearance of Listeria from the peritoneal cavity and spleen. Macrophages derived from mice at these times were activated as evidenced by expression of nonspecific tumoricidal activity against 111In-labeled P815 mastocytoma cells. Injection of neutralizing monoclonal anti-IFN-gamma into 1-day-infected mice completely inhibited the generation of activated macrophages. Normal hamster IgG had no effect. In vivo, the monoclonal antibody also abrogated clearance of bacteria from the spleen and peritoneal cavity. Six days after injection of a sublethal dose of Listeria, the peritoneal cavity of control mice treated with normal hamster IgG was devoid of bacteria and the spleen contained less than 10(3) colony-forming units. However, mice treated with anti-IFN-gamma carried more than 8 X 10(6) colony-forming units in either anatomical site at day 6 and exhibited a higher mortality rate. These results indicate that IFN-gamma production is required for the in vivo generation of activated macrophages and the clearance of bacteria during Listeria infection.
Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome 18.
Authors: Cleary M L , Sklar J
Published: May 2006

The t(14;18)(q32;21) chromosomal translocation characteristic of follicular lymphomas is the most common cytogenetic abnormality known to be associated with any specific type of hematolymphoid malignancy. A fragment of DNA containing the crossover point between chromosomes 14 and 18 was cloned from the tumor cells of a patient with a follicular lymphoma carrying this translocation. Nucleotide sequence analysis of the breakpoint DNA revealed that the break in chromosome 14 occurred in joining region 4(J4) of the nonfunctional immunoglobulin heavy chain allele. This finding and other structural similarities of the breakpoint with the functional diversity region-joining region (D-J) joint in this lymphoma suggest that D-J recombination enzymes played a role in the mechanism of the t(14;18) translocation. Hybridization analysis of DNA from 40 follicular lymphomas showed that the majority of t(14;18) translocations occur on chromosome 18 DNA within 4.2 kilobases of the cloned breakpoint. A DNA probe from this breakpoint-cluster region detects transcription products in the tumor cells from which it was cloned and in a B-lymphoma cell line containing a t(14;18) translocation.
Neurokinin B is a preferred agonist for a neuronal substance P receptor and its action is antagonized by enkephalin.
Authors: Laufer R , Wormser U , Friedman Z Y , Gilon C , Chorev M , Selinger Z
Published: May 2006

Receptor specificity of the substance P-related peptides neurokinin A and neurokinin B was studied in the isolated guinea pig ileum. Substance P and the recently discovered neurokinins elicit contraction of the ileum both directly through action on a muscle cell receptor and indirectly through stimulation of a neuronal receptor, leading to release of acetylcholine, which causes muscle contraction via muscarinic receptors. Two specific assay procedures for the function of the neuronal receptor were developed. The muscular receptor was inactivated either by desensitization with the selective agonist substance P methyl ester or by receptor blockade with the selective antagonist [Arg6, D-Trp7,9, Me-Phe8]substance P-(6-11) hexapeptide. Both procedures revealed that the neuronal receptor is clearly distinct from the muscular receptor, since it exhibits different agonist specificity and is insensitive to antagonists of the muscular receptor. Neurokinin B was found to be the most potent agonist (EC50 = 1 nM) for the neuronal receptor. Furthermore, [D-Ala2, Met5]enkephalinamide inhibited in a naloxone-sensitive manner the effect of neurokinin B mediated via the neuronal receptor. These results suggest that the different mammalian tachykinins can play specific physiological roles by virtue of their distinct receptor specificities.
Differences in the side-chain metabolism of vitamin D3 between chickens and rats.
Authors: Yamada S , Ino E , Takayama H , Horiuchi N , Shinki T , Suda T , Jones G , DeLuca H F
Published: May 2006

In vitro metabolism of 25-hydroxy-24-oxovitamin D3 was studied in kidney homogenates from vitamin D-supplemented chickens and rats. In chicken homogenates, 25-hydroxy-24-oxovitamin D3 was converted predominantly to 23,25-dihydroxy-24-oxovitamin D3, 24,25-dihydroxyvitamin D3, and 23,24,25-trihydroxyvitamin D3. In rat homogenates, 25-hydroxy-24-oxovitamin D3 was not converted to either 24,25-dihydroxyvitamin D3 or 23,24,25-trihydroxyvitamin D3, but it was converted to 23,25-dihydroxy-24-oxovitamin D3 and 23-hydroxy-24,25,26,27-tetranorvitamin D3. The latter metabolite was not produced by the chicken preparations. The stereochemical configuration at C-24 of the 24,25-dihydroxyvitamin D3 produced by chicken homogenates was determined to be S. This contrasts with the R configuration of 24,25-dihydroxyvitamin D3 produced by 24-hydroxylation of 25-hydroxyvitamin D3. These results suggest that chickens have an enzyme that can reduce the 24-oxo group to 24S-hydroxyl group, whereas rats do not.
Purification of neutral lens endopeptidase: close similarity to a neutral proteinase in pituitary.
Authors: Ray K , Harris H
Published: May 2006

A neutral endopeptidase (EC 3.4.24.5) that degrades alpha- and beta-crystallins occurs in mammalian lens. A procedure for purification of this enzyme from bovine lens is described. The enzyme appears to have a high molecular weight (Mr approximately equal to 700,000) and under denaturing conditions dissociates into at least eight polypeptide subunits with Mrs ranging from 24,000 to 32,000. A neutral proteinase in bovine pituitary has been reported previously to have similar structural characteristics. We have found that this enzyme purified from bovine pituitary is indistinguishable in molecular weight and in subunit composition from bovine lens endopeptidase. In addition, antiserum raised in rabbit against the purified lens enzyme crossreacts with bovine pituitary enzyme. When examined side by side in Ouchterlony double-diffusion tests, the two enzymes give a continuous precipitin line with no spurring. It is concluded that lens neutral endopeptidase and pituitary neutral proteinase are structurally closely similar, if not identical. This is a surprising result because it had been thought previously that the lens endopeptidase was unique to lens, where its crystallin substrates comprise a large proportion of the total tissue protein. In other tissues, crystallin is either absent or occurs, at most, in trace amounts.
Inhibition of the Mg(II).ATP-dependent phosphoprotein phosphatase by the regulatory subunit of cAMP-dependent protein kinase.
Authors: Jurgensen S R , Chock P B , Taylor S , Vandenheede J R , Merlevede W
Published: May 2006

We report potent inhibition of the Mg(II).ATP-dependent protein phosphatase, Fc.M, by the regulatory subunit dimer of type II cAMP-dependent protein kinase, RII2. The protein kinase catalytic subunit has no effect on phosphatase activity and is unable to substitute for kinase FA in the kinase FA- and Mg(II).ATP-mediated phosphatase activation reaction. Phosphatase inhibition was investigated as a function of RII2 concentration. The results suggest that RII2 both inhibits the active phosphatase and inhibits phosphatase activation. The inhibition is shown to be noncompetitive with respect to substrate (phosphorylase a). The potential physiological significance of this inhibition is discussed in terms of phosphorylation/dephosphorylation cascade systems involving this kinase and phosphatase.
A method to detect and characterize point mutations in transcribed genes: amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells.
Authors: Winter E , Yamamoto F , Almoguera C , Perucho M
Published: May 2006

A significant percentage of human tumors contain activated ras oncogenes that have acquired oncogenic potential as a result of somatic point mutations at codon 12 or 61 of the encoded ras gene product. We report here a method to detect and characterize mutations in ras genes that is based on the ability of pancreatic ribonuclease (RNase A; EC 3.1.27.5) to cleave RNA heteroduplexes containing single-base mismatches. Using this method, we show that certain human tumor cells contain mutant c-Ki-ras genes, and we define the nature and position of these mutations. At the same time, we describe the presence and estimate the expression of both normal and mutant c-Ki-ras alleles in the same tumor cells. This method should be useful for the diagnostic detection and characterization of single point mutations in expressed genes.
Cap-dependent RNA splicing in a HeLa nuclear extract.
Authors: Edery I , Sonenberg N
Published: May 2006

We have studied the involvement of the 5' cap structure in the splicing of precursor mRNAs in a HeLa nuclear extract. We show that precursor mRNAs are spliced efficiently only when they possess a cap structure and that preincubation of a HeLa nuclear extract rendered the splicing reaction highly sensitive to inhibition by cap analogues. This sensitization was dependent on exogenous Mg2+ but not exogenous ATP or GTP. These results demonstrate that splicing in a nuclear extract is highly dependent on the cap structure, as was demonstrated for the splicing process in a HeLa whole-cell extract [Konarska, M. M., Padget, R. A. & Sharp, P. A. (1984) Cell 38, 731-736], and thus support the contention that cap recognition is an important feature of eukaryotic mRNA biogenesis.
DNase I-resistant nontranscribed spacer segments of mouse ribosomal DNA contain poly(dG-dT).poly(dA-dC).
Authors: Thomas J R , Bolla R I , Rumbyrt J S , Schlessinger D
Published: May 2006

The nontranscribed spacer regions (NTS) that adjoin the coding portion of mouse ribosomal DNA are protected in nucleoli against exhaustive DNase I digestion. Since these sequences are degraded by the enzyme after they are extracted by phenol, the protection is suggested to result from the binding of specific proteins. The nucleolar structure would thus be organized to protect NTS sequences and expose the coding sequences for transcription. We show here that these protected sequences include tracts of poly(dG-dT).poly(dA-dC). We also report that these sequences are localized in regions flanking the rRNA transcription unit. These sequences can potentially form Z-DNA. The organized DNase I-resistant NTS structure in which they participate could be involved in structuring the nucleolus or in regulating transcription because poly(dG-dT).poly(dA-dC) sequences and portions of spacer rDNA can serve as transcriptional enhancer elements.
Transient fluorescence in synchronously dividing Escherichia coli.
Authors: Layne S P , Bigio I J , Scott A C , Lomdahl P S
Published: May 2006

Using a spectrometer equipped with an optical multichannel analyzer as the detector, we observed the Stokes laser-Raman spectra of metabolically synchronous Escherichia coli from 100 to 2100 cm-1. After more than 400 separate recordings, at cell concentrations of 10(7)-10(8) per ml, no Raman lines attributable to the metabolic process nor to the cells themselves were found. However, we did find that synchronous E. coli cultures become more fluorescent during a limited phase of the division cycle. This transient increase in fluorescence may be ascribed to a variation in the redox state of a chemical species within the bacteria or to a variation of the intracellular optical field. The effect is reproducible in synchronous cultures and it is not seen in asynchronous ones. The results suggest that spectral features seen in previous laser-Raman spectra of synchronous bacteria (taken with scanning monochromators) are due to a time-dependent variation in bacterial fluorescence.
High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells.
Authors: Zachary I , Rozengurt E
Published: May 2006

Gastrin-releasing peptide (GRP) labeled with 125I at tyrosine-15 (125I-GRP) binds to intact quiescent Swiss 3T3 cells in a specific and saturable manner. Scatchard analysis indicates the presence of a single class of high-affinity binding sites of Kd = 0.5 X 10(-9) M and a value for the number of sites per cell of about 100,000. 125I-GRP binding was not inhibited by other mitogens for these cells, and cell lines that are mitogenically unresponsive to GRP do not exhibit specific GRP binding. Structure-activity relationships show a close parallel between the ability of a range of GRP-related peptides to both inhibit GRP binding and to stimulate mitogenesis. Further, GRP binding is selectively blocked in a competitive fashion by a novel bombesin antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P. In addition, this compound selectively inhibits GRP and bombesin-induced mitogenesis. These results demonstrate that the mitogenic response of Swiss 3T3 cells to peptides of the bombesin family is mediated by a class of receptors distinct from those of other mitogens for these cells.
Rapid change in mutation rate in a local population of Drosophila melanogaster.
Authors: Mukai T , Baba M , Akiyama M , Uowaki N , Kusakabe S , Tajima F
Published: May 2006

The lethal and detrimental loads per second chromosome rapidly increased from 1968 to 1970 in a local population of Drosophila melanogaster in Japan (lethal load, from about 0.16 to 0.38; detrimental load, from 0.125 to 0.231 [Watanabe, T. K., Watanabe, T. & Oshima, C. (1976) Evolution 30, 109-118]). When the homozygous loads were measured in 1983, the lethal load had decreased to approximately the original amount (0.19) but the detrimental load had stayed high (0.241). The rise and fall of the lethal load can be accounted for by a P-type element that invaded a population with M cytotype, producing a high mutation rate. The mutation rate fell back to the earlier value after the cytotype became P. That the detrimental load did not decrease can be explained by assuming a longer persistence for detrimental mutations in the population. Evidence for a P-type mutator factor is that the mutation rate of the wild-type chromosomes differs between the different cytoplasmic and chromosomal backgrounds, being lower in the background from which the chromosomes were taken.
Primary structure of phage mu transposase: homology to mu repressor.
Authors: Harshey R M , Getzoff E D , Baldwin D L , Miller J L , Chaconas G
Published: May 2006

The phage Mu transposase is essential for integration, replication-transposition, and excision of Mu DNA. We present the complete nucleotide and derived amino acid sequence of the transposase and analyze implications for transposase/DNA interaction. The NH2 terminus of the Mu transposase has considerable sequence homology with the Mu repressor and with the NH2 terminus of the transposase of the Mu-like phage D108. These three proteins are known to share binding sites on DNA. The protein sequence and predicted secondary structural similarities at the NH2 termini of the three proteins suggest a common DNA-binding region similar to the regions found in proteins of known structure. An internal sequence in the Mu A protein also shares these features. We anticipate that these regions will be involved in DNA recognition during transposition.
Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine.
Authors: Yang H Y , Fratta W , Majane E A , Costa E
Published: May 2006

Two peptides that crossreact with an antiserum raised against Phe-Met-Arg-Phe-NH2 were purified from bovine brain extract. Their structures were determined to be Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 and Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2. The sequences were determined by gas-phase sequencing, except for the COOH-terminal phenylalaninamides. These were assigned on the basis of the reactivity of the peptides with the anti-Phe-Met-Arg-Phe-NH2 antiserum, which appears to recognize the determinant -Arg-Phe-NH2. Both peptides were synthesized, and the synthetic peptides were found to have the same HPLC retention times as the endogenous Phe-Met-Arg-Phe-NH2-immunoreactive peptides, thus confirming the assignment of phenylalaninamide to the COOH-terminal positions. Both of the synthetic peptides were found to decrease tail-flick latency in rats, and the octapeptide was more active than the octadecapeptide. The octapeptide was found also to attenuate the prolongation of the tail-flick latency induced by morphine.