bluuebunny/crossref_metadata_2025 huggingface dataset:
Self-excited mono-ion oscillator
Authors: Dehmelt Hans , Nagourney Warren , Sandberg Jon
Published: May 2006

We propose self-excitation as a potentially more sensitive technique for studying a mono-ion oscillator of frequency

v
z

≈ 0.1-100 MHz. This technique also makes only low demands on the harmonicity of the ion oscillation. It should therefore work with inexpensive, easily constructed rf traps. In our analysis, the bound ion between the trap electrodes is represented by an effective circuit resembling that of a piezoelectric quartz crystal. The feedback circuit developed, when operated below self-excitation threshold, may also make efficient electronic cooling of the ion possible, particularly in conjunction with a heterodyne feedback scheme. In the super-regenerative mode, the apparatus might function as a powerful atomic amplifier of the ion oscillation for an energy as low as a fraction of

hv
z

. These techniques may prove especially useful in conjunction with attempts to synthesize in an rf trap a loosely bound anti-hydrogen atom from a positron and antiproton.
Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid.
Authors: Saltiel A R , Cuatrecasas P
Published: May 2006

Insulin binding to plasma membrane receptors results in the generation of substances that acutely mimic the actions of the hormone on certain target enzymes. Two such substances, which modulate the activity of the high-affinity cAMP phosphodiesterase (EC 3.1.4.17), have been purified from hepatic plasma membranes. The two have similar properties and activities but can be resolved by ion-exchange chromatography and high-voltage electrophoresis. They exhibit a net negative charge, even at pH 1.9, and an apparent molecular weight of approximately 1400. The generation of these substances from membranes by insulin can be reproduced by addition of a phosphatidylinositol-specific phospholipase C purified from Staphylococcus aureus. This enzyme is known to selectively hydrolyze phosphatidylinositol and release from membranes several proteins that are covalently linked to phosphatidylinositol by a glycan anchor. Both enzyme-modulating substances appear to be generated by the phosphodiesterase cleavage of a phosphatidylinositol-containing glycolipid precursor that has been characterized by thin-layer chromatography. Some of the chemical properties of these substances have been examined. They appear to be related complex carbohydrate-phosphate substances containing glucosamine and inositol. These findings suggest that insulin may activate a selective phospholipase activity that hydrolyzes a membrane phospholipid, releasing a carbohydrate-containing molecule that regulates cAMP phosphodiesterase and perhaps other insulin-sensitive enzymes.
Molecular basis of hemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor.
Authors: Diuguid D L , Rabiet M J , Furie B C , Liebman H A , Furie B
Published: May 2006

A mutant factor IX, designated factor IXCambridge, was isolated from a patient with hemophilia B. This protein includes an 18-residue propeptide attached to the NH2 terminus of factor IX. A point mutation at residue -1, from an arginine to a serine, precludes cleavage of the propeptide by a processing protease and interferes with gamma-carboxylation of the factor IX, indicating the importance of the leader sequence in substrate recognition by the vitamin K-dependent carboxylase. This represents an example of an enzyme defect due to the presence of a point mutation in a precursor protein (preproenzyme) that is the cause of a human hereditary disease. This defect will serve as a prototype for understanding the molecular basis of some forms of hemophilia and other hereditary enzyme deficiencies.
Importance of the loop at residues 230-245 in the allosteric interactions of Escherichia coli aspartate carbamoyltransferase.
Authors: Middleton S A , Kantrowitz E R
Published: May 2006

Site-directed mutagenesis has been used to replace tyrosine-240 with phenylalanine in each of the catalytic chains of aspartate carbamoyltransferase. Tyrosine-240 is part of a loop in the structure of the enzyme, between residues 230 and 245, which undergoes a substantial conformation change as the enzyme becomes ligated [Krause, K. L., Volz, K. W. & Lipscomb, W. N. (1985) Proc. Natl. Acad. Sci. USA 82, 1643-1647]. The mutant enzyme with phenylalanine at position 240 has substantially reduced homotropic interactions and an increased affinity for the substrate aspartate but displays no alteration in maximal observed specific activity. The Hill coefficient decreases from 2.4 for the wild-type enzyme to 1.8 for the mutant, and the aspartate concentration at half the maximal observed velocity decreases from 11.9 mM to 4.7 mM at pH 8.3. Heterotropic interactions of the mutant enzyme are altered to a lesser extent. The catalytic subunit derived from the mutant enzyme exhibits kinetics identical to that of the wild-type catalytic subunit. Reactivity of the mutant enzyme with p-hydroxymercuribenzoate suggests that the unligated enzyme exists in an altered conformation. The properties of the mutant enzyme are explained in terms of the structure of the wild-type enzyme, and a model is proposed to account for the allosteric interactions of the wild-type enzyme in terms of specific interactions involving the 230-245 loop of the enzyme.
Processing of phage T4 td-encoded RNA is analogous to the eukaryotic group I splicing pathway.
Authors: Ehrenman K , Pedersen-Lane J , West D , Herman R , Maley F , Belfort M
Published: May 2006

Several features of the split td gene of phage T4 suggest an RNA processing mechanism analogous to that of the self-splicing rRNA of Tetrahymena and other group I eukaryotic introns. Previous work has revealed conserved sequence elements and the ability of td-encoded RNA to self-splice in vitro. We show here that a noncoded guanosine residue is covalently joined to the 5' end of the intron during processing. Further, we demonstrate the existence of linear and circular intron forms in RNA extracted from T4-infected cells and from uninfected Escherichia coli expressing the cloned td gene. Sequence analysis of the intron cyclization junction indicates that the noncoded guanosine and one additional nucleotide are lost from the 5' end of the intron upon cyclization. This analysis places a uridine residue upstream of the cyclization site, in analogy to three other group I cyclization junctions. These striking similarities to the splicing intermediates of eukaryotic group I introns point not only to an analogous processing pathway and conserved features of cyclization site recognition but also to a common ancestry between this prokaryotic intervening sequence and the group I eukaryotic introns.
Affinity chromatography with an immobilized RNA enzyme.
Authors: Vioque A , Altman S
Published: May 2006

M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid.
The major histocompatibility complex class I heavy chain as a structural subunit of the human cell membrane insulin receptor: implications for the range of biological functions of histocompatibility antigens.
Authors: Due C , Simonsen M , Olsson L
Published: May 2006

Monoclonal antibodies against some of the monomorphic determinants of major histocompatibility complex (MHC) class I molecules reduce insulin binding and precipitate 125I-labeled insulin receptor preparations. A monoclonal antibody with specificity for the insulin binding site on the cell membrane insulin receptor of human cells was used to precipitate insulin receptors from human cell lines and resulted in distinct bands of Mr approximately 130,000, 90,000, and 45,000. The Mr 45,000 molecules thus precipitated were subjected to NaDodSO4/PAGE, eluted from the gels, and found to react with monoclonal antibodies against monomorphic and a polymorphic MHC class I determinant known to be expressed on the cell line used as receptor source. Moreover, a murine thymoma line (RI) with MHC class I expression bound significant amounts of insulin, whereas a MHC class I-negative variant had low insulin binding capacity. Reduction in the density on human cells of the MHC class I heavy chain was obtained by capping with antibodies to beta 2-microglobulin or to the MHC class I heavy chain and resulted in decreased insulin binding, whereas down-regulation of insulin receptors induced increased density of MHC class I molecules. It is concluded that the MHC class I heavy chain and the tetrameric insulin receptor are structurally associated in the cell membrane and suggested that this association may occur by displacement of beta 2-microglobulin by the insulin receptor.
Differences in sequences encoding the carboxyl-terminal domain of the epidermal growth factor receptor correlate with differences in the disease potential of viral erbB genes.
Authors: Gamett D C , Tracy S E , Robinson H L
Published: May 2006

Eleven recently isolated erbB-transducing viruses as well as avian erythroblastosis virus (AEV)-R (ES4) and AEV-H have been characterized for the type of disease they cause, their ability to transform fibroblasts in culture, their ability to cause disease in pedigrees of chicken that differ in susceptibility to erbB-induced erythroblastosis, and the structure of their erbB genes. Differences in each of the biological parameters correlated with differences in erbB sequences encoding the C-terminal domain of the epidermal growth factor receptor (EGFR). Seven viruses were strain restricted in their ability to induce erythroblastosis and did not transform fibroblasts. These seven viruses contained v-erbB genes encoding the complete C terminus of the EGFR. AEV-R and AEV-H were not pedigree restricted in their ability to induce erythroblastosis and could transform fibroblasts. These viruses contain v-erbB genes that lack codons for the immediate C terminus of the EGFR. Three viruses caused angiosarcoma and one caused fibrosarcoma. The angiosarcoma and fibrosarcoma-inducing viruses were not strain restricted and did not cause erythroblastosis. The v-erbB genes of each of these viruses contained extensive internal deletions or 3' truncations in sequences encoding the C-terminal domain of the EGFR.
Control by cations of opioid binding in guinea pig brain membranes.
Authors: Paterson S J , Robson L E , Kosterlitz H W
Published: May 2006

In membrane suspensions from guinea pig brain or cerebellum, NaCl, LiCl, NH4Cl, and KCl inhibit the equilibrium binding at 25 degrees C of the selective mu-agonist [3H][2-D-alanine,4-methylphenylalanine,5-glycinol]enkephalin ([D-Ala2,MePhe4,Gly-ol5]EK), the selective delta-agonist [3H][2-D-penicillamine,5-D-penicillamine]enkephalin ([D-Pen2,D-Pen5]-EK), and the selective kappa-agonist [3H]dynorphin A-(1-9). Choline chloride inhibits mu- and kappa-binding but not delta-binding. The relative activities of these monovalent salts and the slopes of the dose-response curves are site-dependent. Binding at the kappa-binding site is also inhibited by CaCl2, MnCl2, and MgCl2. On the other hand, these divalent salts potentiate delta-binding, and MnCl2 and MgCl2 have both potentiating and inhibitory effects on mu-binding; CaCl2 inhibits but does not potentiate mu-binding. Thus, the mechanisms by which monovalent cations inhibit opioid binding differ from those of divalent cations, and the mechanisms of action of both monovalent and divalent cations may differ at each site. When the antagonist [3H]naloxone, rather than the agonist [3H][D-Ala2,MePhe4,Gly-ol5]EK, is used to label the mu-binding site, the main effect of NaCl is to potentiate binding; a 22-fold higher concentration of LiCl is required to inhibit binding. The effects of NH4Cl, KCl, MnCl2, MgCl2, CaCl2, and choline chloride are little changed when [3H]naloxone is the ligand.
In vitro transcription of human mitochondrial DNA: accurate termination requires a region of DNA sequence that can function bidirectionally.
Authors: Christianson T W , Clayton D A
Published: May 2006

Mammalian mitochondrial genomes have a presumptive transcription termination site at the 16S rRNA-tRNALeu gene boundary. We have developed an in vitro system from human KB cells that terminates transcription at this gene boundary. By S1 nuclease protection, the 3' ends of terminated transcripts were mapped 3 and 4 base pairs upstream of the 16S rRNA-tRNALeu gene boundary, in agreement with in vivo data. By high-resolution sizing of transcripts, the 3' end was mapped 7 +/- 1 base pairs downstream from the gene boundary. Termination occurs with equal efficacy from transcriptional initiation at the heavy- or light-strand promoter. All template nucleotide sequence information necessary for termination appears to be located near the termination site itself. An unexpected observation is that the termination region functions bidirectionally.
Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.
Authors: Murakami Y , Eki T , Yamada M , Prives C , Hurwitz J
Published: May 2006

In vitro replication of DNA containing the polyoma (Py) virus origin of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase alpha in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo.
Inositol cyclic triphosphate [inositol 1,2-(cyclic)-4,5-triphosphate] is formed upon thrombin stimulation of human platelets.
Authors: Ishii H , Connolly T M , Bross T E , Majerus P W
Published: May 2006

Cleavage of polyphosphoinositides in vitro by phospholipase C results in formation of both cyclic and noncyclic inositol phosphates. We have now isolated the cyclic product of phosphatidylinositol 4,5-bisphosphate cleavage, inositol 1,2(cyclic)-4,5-triphosphate [cIns(1:2,4,5)P3], from thrombin-treated platelets. We found 0.2-0.4 nmol of cIns-(1:2,4,5)P3 per 10(9) platelets at 10 sec after thrombin; none was found in unstimulated platelets or in platelets 10 min after thrombin addition. We conclude that cIns(1:2,4,5)P3 is a major product of polyphosphoinositide metabolism in thrombin-stimulated platelets.
Gene-for-gene interactions of five cloned avirulence genes from
Xanthomonas campestris
pv.
malvacearum
with specific resistance genes in cotton
Authors: Gabriel Dean W. , Burges Adrienne , Lazo Gerard R.
Published: May 2006

A total DNA clone bank of a strain of
Xanthomonas campestris
pv.
malvacearum
(Xcm) was constructed in the cosmid vector pSa747 and transfected into
Escherichia coli
. The Xcm strain carries at least nine identifiable avirulence (
A
) genes. Clones in
E. coli
were mated individually into a recombination-proficient Xcm isolate carrying no known
A
genes. Screening was for incompatibility on congenic cotton host lines that differ by single specific resistance (
R
) genes. Ten different cosmid clones conferring race-specific avirulence were recovered. In most cases, the same
A
gene clone was recovered independently several times. Using the congenic host lines and the merodiploid transconjugant pathogen strains, five of the
A
genes were shown to specifically interact, gene-for-gene, with individual
R
genes in the congenic cotton lines. Some
A
/
R
gene interactions appeared qualitatively different from others, suggesting that the physiological mechanism(s) of gene-for-gene specified incompatibility may be unique to the interactive gene pair. All
A
genes appeared to be chromosomally determined, three were found linked on a single 32-kilobase clone, and the rest were spaced more than 31 kilobases apart. Colinearity of the cosmid inserts with the Xcm recipient (carrying no known
A
genes) chromosome was demonstrated in two of the three tested. This and other evidence suggests that at least some
A
genes in bacteria may have the equivalent of virulence (
a
) alleles. The genetics of race specificity in this phytopathogenic bacterium appeared in all respects to be identical to that found in phytopathogenic fungi.
Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening
Authors: DellaPenna Dean , Alexander Danny C. , Bennett Alan B.
Published: May 2006

The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-α-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of
in vitro
translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)
+
RNA isolated from ripe tomato fruit. The cDNA library was inserted into a λ-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa
in vitro
translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.
Functional domains of the yeast regulatory protein GAL4.
Authors: Johnston S A , Zavortink M J , Debouck C , Hopper J E
Published: May 2006

In the yeast Saccharomyces cerevisiae regulation of the galactose/melibiose regulon rests on a dosage-dependent functional interplay between the positive regulator of transcription, the GAL4 protein, and the negative regulator, GAL80 protein. We have used this interplay to select in vitro generated fusions between the yeast ADH1 promoter and GAL4 coding sequence that overproduce GAL4 protein, allowing the identification of GAL4 protein in crude extracts from yeast. One type of these constructions produces a GAL4 protein that lacks its normal NH2 terminus. This protein is unable to complement a gal4 lesion but still retains a domain that functionally antagonizes the negative regulatory protein. One defect in this truncated protein is its inability to be concentrated in the nucleus. However, the nuclear localization defect is complemented by full-length GAL4 protein. The truncated protein also appears to effect changes in the relative transcriptional levels of the structural genes. These observations imply that GAL4 protein consists of several domains, including ones for nuclear localization, interaction with the negative regulatory protein, and, possibly, separable transcriptional activation domains for the structural genes.
Cis-acting sequences responsible for the transcriptional activation of human T-cell leukemia virus type I constitute a conditional enhancer.
Authors: Paskalis H , Felber B K , Pavlakis G N
Published: May 2006

Transcription from the long terminal repeat promoter of human T-cell leukemia virus type I is activated in the presence of a trans-activator protein, TA-I, encoded in the 3' part of the genome. A series of long terminal repeat mutants and hybrid promoter constructs have been studied in a transient expression assay for their ability to be activated in the presence of the trans-activator protein. The sequences responsible for trans-activation have properties similar to those of transcription enhancer elements. They act relatively independent of position and orientation and activate both the homologous as well as heterologous promoters only in the presence of the trans-activator protein. Therefore, the trans-activator protein of human T-cell leukemia virus type I acts via an inducible enhancement mechanism.
Correction of adenosine deaminase deficiency in cultured human T and B cells by retrovirus-mediated gene transfer.
Authors: Kantoff P W , Kohn D B , Mitsuya H , Armentano D , Sieberg M , Zwiebel J A , Eglitis M A , McLachlin J R , Wiginton D A , Hutton J J
Published: May 2006

A retroviral vector called SAX, containing the cloned human cDNA for adenosine deaminase (ADA), has been constructed and used to introduce the ADA gene into cultured T- and B-lymphocyte lines derived from patients with ADA deficiency. DNA analysis showed that the SAX vector was inserted intact into the T and B cells at approximately one copy per cell. The treated cells produced the characteristic isozymes of human ADA at a level similar to normal T and B lymphocytes. It is known that ADA-deficient lymphocytes are unusually sensitive to high levels of 2'-deoxyadenosine, and this is the mechanism thought to underlie the selective lymphocytotoxicity associated with ADA deficiency in vivo. Expression of the introduced ADA gene was sufficient to reverse the hypersensitivity of these genetically deficient lymphocytes to 2'-deoxyadenosine toxicity. These results support the suggestion that retroviral vector gene-delivery systems show promise for application to human gene therapy.
Identification and transmission of hepatitis B virus-related variants.
Authors: Wands J R , Fujita Y K , Isselbacher K J , Degott C , Schellekens H , Dazza M C , Thiers V , Tiollais P , Brechot C
Published: May 2006

We have identified long-incubation viral agents that share epitopes with hepatitis B virus (HBV). During chimpanzee infectivity studies, these agents may be recognized in the liver since they possess complementary nucleic acid sequences with HBV DNA; the genomic size was found to be 3.2 kilobases, identical to that of HBV. Liver injury was produced and there was antigen expression in hepatocytes. Chimpanzees were not protected by prior immunization with hepatitis B surface antigen; conversely, they were still susceptible to HBV after recovery from infection with such agents. These findings suggest that these hepatitis B virus-related variants appear to be immunologically distinct from HBV.
Cloning and DNA sequence analysis of the cDNA for the precursor of the beta chain of bovine follicle stimulating hormone.
Authors: Esch F S , Mason A J , Cooksey K , Mercado M , Shimasaki S
Published: May 2006

Follicle stimulating hormone (FSH) plays essential roles in the maintenance and development of oocytes and spermatozoa in normal reproductive physiology. FSH possesses two subunits, alpha and beta, the latter being responsible for FSH biological specificity. We have cloned and sequenced the cDNA encoding the FSH beta chain from a bovine anterior pituitary cDNA library. The mature molecule is 109 amino acids long and is preceded by a 20-amino acid putative signal peptide. RNA gel blot analysis of bovine pituitary RNA shows that the mRNA encoding beta chain of FSH is approximately 1.7 kilobases in length.
Molecular cloning of the breakpoint junction of a human chromosomal 8;14 translocation involving the T-cell receptor alpha-chain gene and sequences on the 3' side of MYC.
Authors: McKeithan T W , Shima E A , Le Beau M M , Minowada J , Rowley J D , Diaz M O
Published: May 2006

The MOLT-16 cell line, which was established from the malignant cells of a patient with T-cell acute lymphoblastic leukemia, is characterized by a translocation involving chromosome 8 (band q24) and chromosome 14 (band q11) [t(8;14)(q24;q11)]. To determine the position of the gene encoding the alpha chain of the T-cell receptor and of the protooncogene MYC (formerly c-myc) in relation to the breakpoint junction and to evaluate their possible role in the pathogenesis of T-cell neoplasia, we applied the techniques of in situ chromosomal hybridization, Southern blot analysis, and molecular cloning to MOLT-16 cells. Our results indicate that the breakpoint on chromosome 14 at band q11 occurs close to a joining sequence of the gene encoding the alpha chain of the T-cell receptor. The constant region and part of the joining region of this gene are translocated to the 3' side of the MYC exons. The breakpoints on chromosomes 8 and 14 are close to, but distinct from, those found in SKW-3, another T-cell leukemia cell line, which has a t(8;14). The identification of a breakpoint to the 3' side of MYC suggests that this recurring translocation is analogous to the variant t(2;8) and t(8;22) translocations observed in the B-cell malignancies.
Genetic-molecular basis for a simple Drosophila melanogaster somatic system that detects environmental mutagens.
Authors: Green M M , Todo T , Ryo H , Fujikawa K
Published: May 2006

We have developed a simple, objectively scorable test for the mutagenicity of chemical compounds which can be fed Drosophila melanogaster. The test depends upon the somatic reversion of the X chromosome, recessive eye color mutation, white-ivory (wi) to wild type (w+). Reversions are scored as clones of w+ facets in the wi eyes of eclosing adults. To increase the sensitivity, a tandem quadruplication containing four wi mutations was synthesized. Thus, in homozygous females eight wi mutations are potentially revertible. Six mutagenic compounds, all alkylating agents, all gave positive results at several concentrations tested. Molecular analysis demonstrates that the induced reversions, germinal and somatic, are associated with the loss of 2.9-kilobase DNA duplicated in the wi mutation.
The effect of hexapeptides on attachment and outgrowth of mouse blastocysts cultured in vitro: evidence for the involvement of the cell recognition tripeptide Arg-Gly-Asp.
Authors: Armant D R , Kaplan H A , Mover H , Lennarz W J
Published: May 2006

Differentiation of the nonadherent trophectoderm cells of the mammalian embryo into attachment-competent trophoblast cells appears to be a prerequisite to invasion of the uterine stroma. To investigate the molecular basis of trophoblast differentiation free of maternal environmental constraints, we used a model system in which attachment and outgrowth of trophoblast cells occurs in vitro. Recently, it was found that either fibronectin or laminin, both of which are extracellular matrix glycoproteins of the uterine stroma, will support trophoblast outgrowth in vitro. In this study we report that the outgrowth of blastocysts on fibronectin-coated dishes is inhibited in a dose-dependent manner by the presence of a hexapeptide containing the sequence Arg-Gly-Asp, which has been shown previously to be recognized by the fibronectin receptor. This peptide had no effect on laminin-mediated trophoblast outgrowth, suggesting that the trophoblasts contain different cell surface receptors for fibronectin and laminin. Trophoblast attachment and limited outgrowth also could be obtained on dishes to which the hexapeptide Gly-Arg-Gly-Asp-Ser-Pro was coupled. Under these conditions, however, outward migration of the trophoblast cells appeared to be reduced. Vitronectin, another adhesion molecule that apparently binds to cells via a cell surface receptor that recognizes Arg-Gly-Asp sequences, also was capable of supporting trophoblast outgrowth. These findings suggest that differentiation of cells of the trophectoderm into trophoblast cells with an invasive phenotype may involve the production of cell surface receptors for fibronectin and possibly for other proteins that contain the Arg-Gly-Asp recognition sequence.
Ethylene-regulated gene expression: molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris.
Authors: Broglie K E , Gaynor J J , Broglie R M
Published: May 2006

A full-length copy of bean leaf chitinase mRNA has been cloned. The 1146-base-pair insert of pCH18 encodes the 27-residue amino-terminal signal peptide of the precursor and 301 residues of the mature protein. Utilizing pCH18 as a hybridization probe, we have shown that the increase in translatable chitinase mRNA seen upon ethylene treatment of bean seedlings is due to a 75- to 100-fold increase in steady-state mRNA levels. Southern blot analysis of bean genomic DNA revealed that chitinase is encoded by a small, multigene family consisting of approximately four members. From our nucleotide sequence analysis of five additional chitinase cDNA clones, it appears that at least two of these genes are expressed. Three of the bean chitinase genes have been isolated from a Sau3A genomic library and partially characterized.
Heat shock response in Escherichia coli influences cell division.
Authors: Tsuchido T , VanBogelen R A , Neidhardt F C
Published: May 2006

Analysis of a mutant in fam, a pleiotropic gene affecting cell division in Escherichia coli, revealed that this gene is probably identical to the heat shock regulatory gene htpR. The fam-715 mutant and different htpR mutants were found to share the following three characteristics: temperature-sensitive growth, faulty cell division, and inability to induce the normal cellular heat shock response. These defects were all corrected in fam and htpR mutants by complementation with plasmids carrying intact htpR+ or by recombination between these mutant alleles and a plasmid carrying only a portion of htpR. These results implicate the E. coli heat shock system in the regulation of cell division and raise the question of a similar role in other organisms.
Putative diazepam binding inhibitor peptide: cDNA clones from rat.
Authors: Mocchetti I , Einstein R , Brosius J
Published: May 2006

cDNA clones corresponding to the polypeptide that has been shown to be an endogenous diazepam binding inhibitor and may act as a physiological ligand for the benzodiazepine/beta-carboline receptor have been isolated from bacteriophage lambda recombinant libraries from rat hypothalamus, total brain, and liver. The clones contain an open reading frame corresponding to 87 amino acids. A signal sequence is not present. In addition to high levels of mRNA in various brain regions, RNA blot analysis reveals an abundance of diazepam binding inhibitor mRNA in many peripheral organs (e.g., testes, kidney, liver, and heart) that are known to be rich in peripheral benzodiazepine recognition sites. The size of the mRNA in all tissue examined is approximately 0.7 kilobase. Southern blot analysis of genomic DNA suggests the presence of about six genes in the rat, some of which may be pseudogenes.
Induced differentiation of a transformed clone of Ly-1+ B cells by clonal T cells and antigen.
Authors: Bishop G A , Haughton G
Published: May 2006

The present study used cocultures of clonally derived B and T cells, together with an antigen reactive with the membrane Ig of the clonal B cells, to address the issue of B-cell differentiation requirements. The B cells were CH12.LX, an in vitro grown subclone of a murine B-cell lymphoma, which bears surface IgM reactive with sheep erythrocytes. The T cells were alloreactive T-helper-cell hybridomas. Very small numbers of T-helper cells could induce differentiation of cloned B cells without the presence of accessory cells, but such induction was dependent upon the presence of the antigen recognized by the B cell. Induced differentiation of the B cells did not depend on metabolic activity of the T cells, and metabolically active T cells could be replaced by fixed cells or by monoclonal antibody reactive with the class II molecule of the B cell to deliver an Ia-specific differentiative signal. T cells, or alloantibody that reacted with the I-E molecule, induced differentiation of the B cells; those that reacted with the I-A molecule did not. These results define the minimal requirements for major histocompatibility complex-restricted, T-cell-mediated induction of B-cell differentiation.
Identification of two calcium channel receptor sites for [3H]nitrendipine in mammalian cardiac and smooth muscle membrane.
Authors: Rogart R B , deBruyn Kops A , Dzau V J
Published: May 2006

Various Ca-channel blockers differ in cardiovascular action despite common effects at the Ca channel. Many investigators have reported only a single high-affinity receptor for binding of [3H]nitrendipine, a dihydropyridine Ca-channel blocker. Its equilibrium dissociation constant (Kd) does not match the concentration of nitrendipine needed for a physiological effect on the mammalian cardiac Ca channel. The purpose of these studies was to clarify the existing discrepancy between pharmacological properties of nitrendipine receptors and the physiological effects of the dihydropyridine blockers. Of particular importance in this regard was to provide a pharmacological correlate for electrophysiological studies demonstrating multiple voltage-dependent conformational states of the Ca channel, which show differing affinities for the dihydropyridine Ca-channel blockers. By use of an improved ligand binding assay, our studies demonstrate both "high-affinity" and "low-affinity" [3H]nitrendipine receptors with Kd values corresponding well with observed physiologically effective nitrendipine concentrations. We detected two distinct populations of nitrendipine receptors in rat heart and bovine aortic membrane. A high-affinity Kd value of 0.2-0.3 nM was found, which seems to correspond to the physiologically functional state of the Ca channel in smooth muscle, since the Kd value is similar to the concentration at which nitrendipine inhibits contraction. However, in contrast to numerous other studies, we observed that the predominant component of [3H]nitrendipine binding (95-99%) had a low-affinity Kd value (235 nM). This putative low-affinity [3H]nitrendipine receptor may correspond to the physiologically functional state of the Ca channel in cardiac muscle.
Monoclonal antibody specific for an activated RAS protein.
Authors: Carney W P , Petit D , Hamer P , Der C J , Finkel T , Cooper G M , Lefebvre M , Mobtaker H , Delellis R , Tischler A S
Published: May 2006

Activated RAS transforming genes that encode proteins (p21s) with amino acid substitutions at positions 12, 13, or 61 have been detected in 10-20% of human neoplasms. This report describes a monoclonal antibody (DWP) raised against a synthetic peptide corresponding to amino acids 5-16 of a mutated RAS gene encoding Val instead of Gly at position 12. DWP reacted in competition assays with peptides containing Val or Cys at position 12, but did not react with peptides containing Gly, Arg, Ser, Ala, Asp, or Glu at position 12. Immunoblot analysis of transformed NIH cells and human carcinoma cell lines showed that DWP reacts specifically with activated RAS proteins containing Val at position 12 and not with normal p21s or p21s activated by other amino acid substitutions at positions 12 and 61. Immunohistochemical studies showed that DWP-labeled transformed NIH cells and human carcinoma cells contained p21s with either Val or Cys at position 12 but not normal or other activated p21s. In contrast to the specificity seen with human carcinoma cell lines, analysis of formalin-fixed, primary carcinoma specimens indicated that positive immunoperoxidase staining with DWP did not necessarily correlate with immunoblot and transfection assays for the presence of activated RAS proteins. Immunohistochemical studies did show, however, that DWP preferentially binds human carcinoma cells.
Introduction of genes into preimplantation mouse embryos by use of a defective recombinant retrovirus.
Authors: Rubenstein J L , Nicolas J F , Jacob F
Published: May 2006

Two-cell and four-cell preimplantation mouse embryos were cocultured in vitro with fibroblasts producing the recombinant retrovirus M-MuLV-neo. No wildtype helper virus was detected in these cultures. Of the embryos that survived the in vitro cultivation and the reimplantation into foster mothers, 2 of 15 that were tested contained the proviral genome. The provirus integrated as a single copy at a unique site. We estimate that approximately equal to 20% of the cells in each of the two transgenic fetuses contained the provirus.
Gene control in broad host range plasmid RK2: expression, polypeptide product, and multiple regulatory functions of korB.
Authors: Bechhofer D H , Kornacki J A , Firshein W , Figurski D H
Published: May 2006

The korB gene of broad host-range plasmid RK2 prevents host-cell lethality by kilB and negatively controls RK2 replication. We precisely mapped the limits of korB to a region near korA, an autoregulated gene involved in control of several RK2 genes. The following results show that korA and korB are cotranscribed from the korA promoter: Mutants deleted for the korA promoter fail to express korB, even with korA function supplied in trans; the korA promoter is nonessential to korB if a heterologous promoter is present; and RNA produced in vivo has both korA- and korB-specific sequences. Analysis of polypeptides synthesized from wild-type and mutant korB plasmids in maxicells revealed that korB encodes a 52-kDa polypeptide, whose activity is extremely sensitive to changes in its carboxyl terminus but relatively unaffected by replacement of its amino terminus. The minimal korB-encoding region allowed us to identify two new regulatory functions, both of which duplicate previously known functions of korA. First, korB alone was found to control the kilB1 component of kilB, thus resolving the paradox of korA-independent control of kilB. Second, analysis of polypeptides from the korA-korB region in the presence and absence of korB, and studies with the korA promoter fused to the chloramphenicol acetyltransferase structural gene (cat) showed that korB, like korA, autoregulates expression of the korA-korB operon. We suggest that korA and korB gene products act as co-repressors in the control of certain RK2 genes.
Potassium channels mediate killing by human natural killer cells.
Authors: Schlichter L , Sidell N , Hagiwara S
Published: May 2006

Human natural killer (NK) cells in peripheral blood spontaneously recognize and kill a wide variety of target cells. It has been suggested that ion channels are involved in the killing process because there is a Ca-dependent stage and because killings by presensitized cytotoxic T lymphocytes, which in many respects resembles NK killing, is associated with changes in K and Na transport in the target cell. However, no direct evidence exists for ion channels in NK cells or in their target cells. Using the whole-cell variation of the patch-clamp technique, we found a voltage-dependent potassium (K+) current in NK cells. The K+ current was reduced in a dose-dependent manner by the K-channel blockers 4-aminopyridine and quinidine and by the traditional Ca-channel blockers verapamil and Cd2+. We tested the effects of ion-channel blockers on killing of two commonly used target cell lines: K562, which is derived from a human myeloid leukemia, and U937, which is derived from a human histiocytic leukemia. Killing of K562 target cells, determined in a standard 51Cr-release assay, was inhibited in a dose-dependent manner by verapamil, quinidine, Cd2+, and 4-aminopyridine at concentrations comparable to those that blocked the K+ current in NK cells. In K562 target cells only a voltage-dependent Na+ current was found and it was blocked by concentrations of tetrodotoxin that had no effect on killing. Killing of U937 target cells was also inhibited by the two ion-channel blockers tested, quinidine and verapamil. In this cell line only a small K+ current was found that was similar to the one in NK cells. We could not find any evidence of a Ca2+ current in target cells or in NK cells; therefore, our results cannot explain the Ca dependence of killing. Our findings show that there are K channels in NK cells and that these channels play a necessary role in the killing process. In contrast, the endogenous channel type in the target cell is probably not a factor in determining target cell sensitivity to natural killing.
Developmental and tissue-specific regulation of the Q10 class I gene by DNA methylation.
Authors: Tanaka K , Barra Y , Isselbacher K J , Khoury G , Jay G
Published: May 2006

The H-2 class I genes encode cell-surface glycoproteins that play a critical role in the immune presentation of aberrant cells. The Q10 class I gene, however, encodes a secreted glycoprotein that is highly homologous to the membrane-bound molecules. While the H-2 genes are activated in all tissue types, the expression of the Q10 gene is restricted to only the liver. Analysis of DNA from different tissues revealed a unique methylation profile for the Q10 gene in liver. Developmental activation of this gene in newborn mice is also reflected by a coordinated temporal change in DNA methylation. By comparing the methylation profiles between congenic mice, which differed in their levels of expression of the Q10 gene, it is observed that methylation at the 3'-flanking region correlates with expression. Methylations were at both CG and CC sequences. Since treatment of newborns with 5-azacytidine, which led to inhibition of methylation, resulted in the suppression of Q10, we conclude that hypermethylation in the 3'-flanking region is responsible, at least in part if not in full, for the activation of the Q10 gene in the liver.
Cell type-specific negative regulatory element in the control region of the rat alpha-fetoprotein gene.
Authors: Muglia L , Rothman-Denes L B
Published: May 2006

Albumin and alpha-fetoprotein are evolutionarily related genes that show differential and complex patterns of regulation during development. We investigated the role of the sequences flanking the transcription initiation site of the rat alpha-fetoprotein gene in transient transfection assays of hepatic and nonhepatic cell lines. Chimeric flanking regions and deletion analysis have defined the following three functionally different regions: a cell type-specific enhancer(s), encompassing 3 kilobases, located between -7 kilobase pairs and -4 kilobase pairs; a cell type-specific promoter, inactive in the absence of an enhancer and comprising at most the 180 base pairs upstream from the site of transcription initiation; and a 550-base-pair region, located between the promoter and the enhancer (at -3.5 kilobases), down-regulates transcription initiation in a cell type-specific manner and is also capable of repressing heterologous promoters. The implications of these findings with respect to the complex pattern of AFP regulation are discussed.
Biosynthesis of zeatin from
N
6
-(Δ
2
-isopentenyl)adenine in
Actinidia
: Sites and seasonal changes in activity
Authors: Einset John W.
Published: May 2006

In
Actinidia arguta
(hardy kiwifruit) plants, the potential to accumulate the cytokinin zeatin (io
6
Ade) during feeding with a precursor,
N
6
-(Δ
2
-isopentenyl)adenine (i
6
Ade), varies depending on the tissue. This can be demonstrated by incubating explants for 24 hr on a basal nutrient medium supplemented with 30 μM i
6
Ade and then extracting the tissues and analyzing cytokinin contents using HPLC methodology. Under these conditions, the potential for io
6
Ade accumulation in tissue slices from growing roots was 93 nmol/g at the root tip, >200 nmol/g immediately behind the tip (1.0-mm diameter), ≈100 nmol/g in 2-mm diameter root, and progressively lower in older tissues. A similar gradient of io
6
Ade accumulation was detected in growing stems, with relatively low activity (40 nmol/g) in the terminal 0.5 cm, ≈170 nmol/g in the 5- to 15-cm interval, and about 25 nmol/g in stem tissues taken 15-100 cm from the tip. Growing leaves accumulated little io
6
Ade (7-26 nmol/g) during feeding, as did fruits (0-8 nmol/g) at various stages of development and maturation. Seasonally, root activity was detected as early as March 1, whereas stem activity did not appear until March 15. Thus, the activation of i
6
Ade metabolism in both of these organs preceded sap flow (March 29) and bud break (April 16) by several weeks. The results suggest that root and stem tissues may be sites of cytokinin biosynthesis in growing
Actinidia
plants and that cytokinin production may not be the critical factor controlling the beginning of shoot growth in the spring.
Absence of antibodies to the human immunodeficiency virus in sera from Africa prior to 1975.
Authors: Levy J A , Pan L Z , Beth-Giraldo E , Kaminsky L S , Henle G , Henle W , Giraldo G
Published: May 2006

Three different assays for detection of antibodies to the human immunodeficiency virus (HIV) were conducted on 677 sera obtained from 1964 to 1975 from male and female children and adults in Uganda and other countries in Africa. Several sera were collected from individuals with Kaposi sarcoma. No evidence of antibodies to the virus was noted up to 1975. These results strongly suggest that the emergence of HIV in Africa occurred relatively recently. Further studies are required to determine the geographic origin of the acquired immunodeficiency syndrome virus.
Mevinolin, an inhibitor of cholesterol biosynthesis, drastically depresses Ca2+ channel activity and uncouples excitation from contraction in cardiac cells in culture.
Authors: Renaud J F , Schmid A , Romey G , Nano J L , Lazdunski M
Published: May 2006

Mevinolin (MK803), a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) (Ki, 30 X 10(-9) M), depressed de novo synthesis of cholesterol in 11-day chicken embryonic cardiac cells cultured in lipoprotein-deficient serum (LPDS). Cardiac cells exposed to different concentrations of mevinolin for 1-3 days presented different electrophysiological and mechanical properties: The resting membrane potential, the rate of increase, and the shape of the action potential and contractile properties were changed at concentrations as low as 0.1 microM mevinolin. At a concentration of 1 microM mevinolin, the cardiac cells became quiescent and electrical stimulation induced action potentials of short duration without contraction. Isoproterenol and Bay K8644 were unable to restore excitability and contraction. Although the number of receptors for the tritiated Ca2+ channel blocker nitrendipine was the same in control and in mevinolin-treated cells, voltage-clamp data on isolated cardiac cells and 45Ca2+ flux experiments on monolayers showed that most of the slow Ca2+ channel activity was lost in mevinolin-treated cells. These results suggest that the disappearance of Ca2+ channel activity is most probably at the origin of the loss of cardiac contractility.