bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and functional organization of the colicin E1 operon.
Authors: Waleh N S , Johnson P H
Published: May 2006

We analyzed the structural and functional relationships among independently cloned segments of the plasmid ColE1 region that regulates and codes for colicin E1 (cea), immunity (imm), and the mitomycin C-induced lethality function (lys). On the basis of physiological properties, restriction endonuclease mapping, and DNA sequence analysis, the following recombinant plasmids were determined to represent three major functional classes: pNP12 (cea+, imm+, lys+), pNP4 (cea+, imm+, lys-), and pNP6 (cea+, imm-, lys-). Our results have established the order, boundaries, and relative orientation of the three structural genes, the location of the promoter region for imm gene transcription, and the predicted amino acid sequences of the imm and lys gene products. Hydropathicity analysis suggests that both proteins have hydrophobic amino acid segments characteristic of membrane-associated proteins. A model for the structure and expression of the colicin E1 operon is proposed in which the cea and lys genes are expressed from a single inducible promoter that is controlled by the lexA repressor in response to the SOS system of Escherichia coli. The imm gene lies between the cea and lys genes and is expressed by transcription in the opposite direction from a promoter located within the lys gene. This arrangement of structural genes indicates that the transcriptional units for all three genes overlap. We suggest that the formation of anti-sense RNA may be an important element in the coordinate regulation of gene expression in this system.
The 2-A resolution structure of a thermostable ribonuclease A chemically cross-linked between lysine residues 7 and 41.
Authors: Weber P C , Sheriff S , Ohlendorf D H , Finzel B C , Salemme F R
Published: May 2006

The crystal structure of Lys-7-(dinitrophenylene)-Lys-41-cross-linked ribonuclease A has been determined by molecular replacement and refined by restrained least-squares methods to an R factor of 0.18 at 2.0-A resolution. Diffraction intensity data were collected by using a conventional diffractometer and an x-ray area detector. Comparison of the thermostable cross-linked protein and the native enzyme shows them to be structurally similar, with a rms difference in backbone and side-chain atoms of 0.52 and 1.34 A, respectively. Native and modified proteins additionally show 35 common bound solvent sites and similar overall temperature factor behavior, despite localized differences resulting from cross-link introduction, altered crystal pH, or lattice interactions with neighboring molecules. These results are discussed in the context of proposals on the origins of thermostability in the cross-linked enzyme.
Can villin be used to identify malignant and undifferentiated normal digestive epithelial cells?
Authors: Robine S , Huet C , Moll R , Sahuquillo-Merino C , Coudrier E , Zweibaum A , Louvard D
Published: May 2006

We have investigated the presence of villin (a Ca2+-regulated actin binding protein) in various tissues (normal or malignant) and in established cell lines by using sensitive immunochemical techniques on cell extracts and immunofluorescence analysis on frozen sections. Our results show that villin is a marker that can be used to distinguish normal differentiated epithelial cells from the simple epithelia lining the gastrointestinal tract and renal tubules. Villin is found in the absorptive cells of the small and large intestines, in the duct cells of pancreas and biliary system, and in the cells of kidney proximal tubules. Furthermore, undifferentiated normal and tumoral cells of intestinal origin in vivo and in cell culture express villin. Therefore, expression of villin is seen in cells that do not necessarily display the morphological features characteristic of their terminally differentiated state, such as the microvilli-lined brush border. We suggest the possible clinical implications of using villin as a marker in the diagnosis of metastatic adenocarcinomas.
Size and behavior in ants: Constraints on complexity
Authors: Cole Blaine J.
Published: May 2006

In this paper I investigate the behavioral complexity of ants in relation to brain size. The volume of the corpora pedunculata, involved in the selection of motor programs, and the antennal lobes, involved in processing olfactory information, are directly related to the volume of the brain of an ant. The volume of the optic lobe, involved in visual processing, is not related to total brain volume. Brain volume is allometrically related to head width in ants. Behavioral complexity, as assayed by the size of the behavioral repertoire within the nest, is directly related to the cube of head width. Behavioral complexity is thus related to the 3/2 power of brain volume. The size of the behavioral repertoire does not appear to correlate with any ecological characteristics of the species but may be a passive consequence of body size. Determination of body size by numerous ecological factors may place constraints on the complexity of behavior that an ant can achieve.
Induction and upregulation by interleukin 2 of high-affinity interleukin 2 receptors on thymocytes and T cells.
Authors: Reem G H , Yeh N H , Urdal D L , Kilian P L , Farrar J J
Published: May 2006

We show that purified recombinant interleukin 2 (rIL-2) alone induces the expression of high- and low-affinity interleukin 2 (IL-2) receptors in vitro on human T cells and thymocytes that have not been activated previously by lectins or other inducing agents. IL-2 receptors are expressed after 24 hr, as determined by the binding of 125I-labeled monoclonal anti-IL-2 receptor antibody 2A3, which binds equally to high- and low-affinity receptors. High-affinity receptors were distinguished from low-affinity receptors by the binding of 125I-labeled IL-2 to T cells and by the proliferative response of thymocytes to IL-2, in concentrations that selectively interact with the high-affinity class of IL-2 receptors. The IL-2-induced proliferation of thymocytes in vitro induced by IL-2 alone is dependent upon the concentration of IL-2 and is inhibited by monoclonal anti-Tac antibody, indicating that the proliferative response is mediated by the binding of IL-2 to the receptors. In addition, we demonstrate that IL-2 augments the number of high-affinity receptors on concanavalin A-activated thymocytes. These results document that IL-2 acts as a hormone that induces the activation of thymocytes and T cells, as evidenced by the de novo induction of biologically active, high-affinity IL-2 receptors. IL-2 also upregulates the expression of high-affinity IL-2 receptors on activated thymocytes. These observations illustrate the biologic importance of the regulatory role of IL-2 in the immune response.
Thyrotropin upregulates alpha 1-adrenergic receptors in rat FRTL-5 thyroid cells.
Authors: Corda D , Kohn L D
Published: May 2006

FRTL-5 rat thyroid cells grown and maintained in a medium containing 0.05 nM thyrotropin have a 10-fold higher number of alpha 1-adrenergic receptors on their cell surface than FRTL-5 cells maintained in the absence of thyrotropin in their medium. The increased number of alpha 1-adrenergic receptors, measured as increased specific [3H]prazosin binding per microgram of DNA, is not associated with any changes in Kd values for prazosin. Thyrotropin increases the number of alpha 1-adrenergic receptors by inducing their biosynthesis, as evidence by the inhibitory effects of cycloheximide or actinomycin D; the effect on biosynthesis is cAMP-mediated, since 8-bromoadenosine 3',5'-cyclic monophosphate, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine can mimic the thyrotropin effect in both extent and time course. The alpha 1-adrenergic receptors on FRTL-5 thyroid cells have been functionally linked to iodide efflux into the follicular lumen and to the iodination of thyroglobulin--i.e., to the formation of thyroid hormones; the alpha 1-adrenergic receptor signal is mediated by Ca2+ rather than by cAMP and involves arachidonic acid intermediates. The present data thus describe a unique upregulation phenomenon wherein the sequential expression of two receptors (thyrotropin and alpha 1-adrenergic) and two distinct signal systems (cAMP and Ca2+) are apparently a necessary prelude to thyroid hormone homoeostasis.
Activation of tracheal smooth muscle contraction: synergism between Ca2+ and activators of protein kinase C.
Authors: Park S , Rasmussen H
Published: May 2006

The effects of divalent ionophores (A23187 and ionomycin), Ca2+ channel agonist (BAY K 8644), and protein kinase C (C-kinase) activators [phorbol 12-myristate 13-acetate (PMA), mezerein] on bovine tracheal smooth muscle contraction were investigated. A23187 (5 microM) and ionomycin (0.5 microM) produced a prompt but transient contraction. C-kinase activators either produced no effect--e.g., PMA at 200 nM--or produced a rise in tension that was slow in onset but then gradually increased--e.g., mezerein at 400 nM. In contrast, ionophores and C-kinase activators, in combination, acted synergistically to produce a prompt and sustained contractile response that is reminiscent of that observed in response to carbachol, a cholinergic agonist. In addition, BAY K 8644 (20 nM), which has a minimal effect on tension by itself, could significantly enhance contraction induced by C-kinase activators. The contraction induced by all of these agents was quickly reversed either by removal of extracellular Ca2+ or upon addition of forskolin, an activator of adenylate cyclase. A similar reversal of carbachol-induced contraction by forskolin was observed with carbachol-induced contraction. These findings strongly suggest that C-kinase plays an important role in mediating tracheal smooth muscle contraction.
Complete nucleotide sequence of the genome of bovine leukemia virus: its evolutionary relationship to other retroviruses.
Authors: Sagata N , Yasunaga T , Tsuzuku-Kawamura J , Ohishi K , Ogawa Y , Ikawa Y
Published: May 2006

We report the complete 8714-nucleotide sequence of the integrated bovine leukemia virus genome and deduce the following genomic organization: 5' LTR-gag-pol-env-pXBL-3' LTR, where LTR represents a long terminal repeat and pXBL represents a region containing unidentified open reading frames. This genomic structure is similar to that of human T-cell leukemia virus. The LTR contains a putative splice donor site in the R region. The gag gene encodes a precursor protein with the form NH2-p15-p24-p12-COOH. The NH2- and COOH-terminal regions of the pol product show stronger homologies with those of avian, rather than murine, type C retrovirus, and its structure is identical to that of avian virus. The env gene encodes a surface glycoprotein (gp51) and a transmembrane protein (gp30). In contrast to the pol product, the gp30 shows stronger sequence homology with a murine, rather than avian homologue, indicating the chimeric nature of the bovine leukemia virus genome. Comparisons of the best conserved pol sequences and overall genomic organizations between several major oncoviruses allow us to propose that bovine leukemia and human T-cell leukemia viruses constitute a group, designated as type "E," of Oncovirinae.
The structure of Marek disease virus DNA: the presence of unique expansion in nonpathogenic viral DNA.
Authors: Fukuchi K , Tanaka A , Schierman L W , Witter R L , Nonoyama M
Published: May 2006

DNA of Marek disease virus (MDV) consists of two unique regions UL and US flanked by long inverted repeat regions TRL and IRL, and short inverted repeat regions TRS and IRS, respectively, similar to herpes simplex virus DNA. Comparison of restriction patterns between pathogenic and nonpathogenic MDV DNA was made to identify a region of viral DNA different between these two types of MDV, as it may be responsible for the tumorigenicity of MDV in chickens. The results indicated that BamHI-D and -H, located at the long inverted repeat regions TRL and IRL, were specifically expanded in nonpathogenic viral DNA. The location of the expanded region has been determined within 1.5 kilobase pairs of the Bgl I/Pst I fragment of BamHI-D and -H, close to the junction between the inverted repeat and the unique region. The possibility that a gene responsible for tumor induction may be disrupted by such expansion has been discussed.
Intracellular free calcium levels are reduced in mitotic Pt K2 epithelial cells.
Authors: Keith C H , Maxfield F R , Shelanski M L
Published: May 2006

Using a fluorescence ratio method, we have studied the intracellular free calcium levels in individual quin-2-loaded mitotic cells under the microscope. We have found that intracellular free calcium concentrations in Pt K2 epithelial cells drop by approximately 50% as they pass through mitosis. Calcium levels in interphase cells were 53 +/- 7 nM. During prophase, free cytoplasmic calcium begins to decrease, reaching 28 +/- 3 nM in prometaphase. Calcium levels remain low until the nuclear envelope is re-formed in late telophase, when they increase again to interphase levels. This decrease in overall free calcium in mitosis suggests that the mitotic cell has mechanisms for the general sequestration, and perhaps local release, of calcium ions.
Interleukin 2 regulates its own receptors.
Authors: Smith K A , Cantrell D A
Published: May 2006

The cell surface density of high-affinity membrane receptors for the T-lymphocytotrophic hormone interleukin 2 (IL-2) determines the rate of T-cell-cycle progression. Since 10-fold greater numbers of IL-2 receptor molecules were found by using a radiolabeled monoclonal antibody reactive with IL-2 receptors (anti-Tac) compared with binding of IL-2, the functional relationship of the binding sites recognized by both of these ligands was assessed. In the presence of cycloheximide, IL-2 binding sites declined with a half-time (t1/2) of 2.6 hr, whereas the decay of anti-Tac binding sites was much slower (t 1/2 = 6.4 hr). Moreover, after limited membrane proteolysis, the half-time for the reappearance of IL-2 binding sites was remarkably similar to its decay (t 1/2 = 2.2 hr), while Tac antigen reappearance was markedly retarded, returning to only 20% of original levels within 5 hr after proteolysis. Addition of homogeneous immunoaffinity-purified IL-2 to cell populations that expressed equivalent IL-2 and anti-Tac binding sites resulted in a time- and temperature-dependent 8- to 10-fold enhancement of Tac epitope expression and, simultaneously, a 20-30% diminishment of detectable high-affinity IL-2 binding sites. As the magnitude of the IL-2-dependent proliferative response correlated with the density of high-affinity IL-2 binding sites, rather than Tac antigen levels, quantitation of Tac epitope density does not provide a reliable indication of IL-2-responsiveness among activated T-cell populations. Instead, IL-2-receptor interactions actually promote the loss of IL-2 responsiveness by diminishing the density of high-affinity binding sites at the time that Tac antigen levels are increased.
The liver excretes large amounts of heme into bile when heme oxygenase is inhibited competitively by Sn-protoporphyrin.
Authors: Kappas A , Simionatto C S , Drummond G S , Sassa S , Anderson K E
Published: May 2006

TinIV-protoporphyrin IX (Sn-protoporphyrin) potently inhibits heme degradation to bilirubin in vitro and in vivo, and it completely suppresses neonatal hyperbilirubinemia in experimental animals, including primates. It also reduces plasma bilirubin levels in certain naturally occurring or induced forms of jaundice in animals and man. We have examined in this study the fate of that fraction of heme whose degradation to bile pigment is inhibited in vivo by administration of this heme oxygenase (EC 1.14.99.3) inhibitor. In bile-duct-cannulated rats, infused exogenous heme is rapidly converted to biliary bilirubin; a small amount of the infused heme is excreted into bile as well. Sn-protoporphyrin, administered with the exogenous heme, markedly increased (3- to 4-fold) the amount of heme excreted into bile and greatly diminished biliary output of bilirubin. The increase in biliary heme output exceeded the decrease in bilirubin excretion elicited by the inhibitor metalloporphyrin. In the same experimental model, Sn-protoporphyrin substantially decreased the conversion of heme, derived from heat-damaged erythrocytes, to biliary bilirubin. This decrease in biliary bilirubin output was accounted for entirely by a prompt and marked increase in biliary excretion of unmetabolized heme. The enhanced biliary excretion of unmetabolized heme following administration of Sn-protoporphyrin is a newly defined and biologically important response associated with use of this synthetic heme analogue. The features of the action of this compound in vivo--suppression of formation of the potentially neurotoxic metabolite, bilirubin; enhancement of disposal of the untransformed substrate (heme) of the enzyme that it inhibits; and its own elimination without metabolic alteration--define some of the characteristics of a therapeutically useful chemical.
Two alkaline phosphatase genes positioned in tandem in Bacillus licheniformis MC14 require different RNA polymerase holoenzymes for transcription.
Authors: Hulett F M , Wang P Z , Sussman M , Lee J W
Published: May 2006

Southern transfer analysis of Bacillus licheniformis MC14 DNA, using as probe a DNA fragment from within the coding region of a previously cloned alkaline phosphatase (APase) gene, revealed a second area of hybridization adjacent to the cloned APase gene. A second APase gene (APase II) was subcloned from the same plasmid clone, pMH8, from which the first APase gene (APase I) had been subcloned. The two genes are arranged in tandem with several hundred base pairs separating them. Immunoblot analysis showed that both code for Mr 60,000 proteins that crossreact with anti-APase. Both proteins enzymatically cleave 5-bromo-4-chloro-3-indolyl phosphate. In vitro transcription showed that APase I and APase II are transcribed in the same direction but that the two genes require different forms of Bacillus RNA polymerase: sigma 55- and sigma 37-containing RNA polymerase holoenzymes, respectively.
Nitrogenase reductase: A functional multigene family in
Rhizobium phaseoli
Authors: Quinto Carmen , De La Vega Humberto , Flores Margarita , Leemans Jan , Cevallos Miguel Angel , Pardo Marco Aurelio , Azpiroz Ricardo , De Lourdes Girard Maria , Calva Edmundo , Palacios Rafael
Published: May 2006

The complete coding sequence of the nitrogenase reductase gene (
nifH
) is present in three different regions of a
Rhizobium phaseoli
symbiotic plasmid. Homology between two of the regions containing
nifH
coding sequences extends over 5 kilobases. These in turn share 1.3 kilobases of homology with the third region. The nucleotide sequences of the three nitrogenase reductase genes were found to be identical. Site-directed insertion mutagenesis indicated that none of the three genes is indispensable for nitrogen fixation during symbiosis with
Phaseolus vulgaris
. This implies that at least two of the reiterated genes can be functionally expressed.
Molecular cloning of a cDNA for human alpha-L-fucosidase.
Authors: Fukushima H , de Wet J R , O'Brien J S
Published: May 2006

A lambda gt11 human hepatoma cDNA expression library was screened with antibodies to human alpha-L-fucosidase, a lysosomal enzyme whose activity is deficient in the human autosomal recessive disease fucosidosis. Three positive clones were identified after screening 9 X 10(6) plaques. One of these was sequenced and found to be spurious, probably representing an out-of-frame cDNA that gave rise to amino acid sequences of unknown length that crossreacted with alpha-L-fucosidase. A second clone, lambda AF3, was isolated which, after establishment in Escherichia coli BNN103, gave rise to a fusion protein of Mr 154,000 containing a human fragment of Mr 40,000 that represented 80% of the mature processed enzyme (Mr 50,000). Southern blot analysis of mouse and human chromosomal DNA confirmed the human origin of insert AF3. The nucleotide sequence of AF3 was determined and colinearity was established between 270 nucleotides and 90 amino acids in alpha-L-fucosidase. AF3 was found to contain 1058 base pairs and to code for 347 amino acids of alpha-L-fucosidase. Four potential glycosylation sites were identified. The frequency of lambda AF3 in the hepatoma library was 0.0018%.
Cyclization of RNA 3'-terminal phosphate by cyclase from HeLa cells proceeds via formation of N(3')pp(5')A activated intermediate.
Authors: Filipowicz W , Strugala K , Konarska M , Shatkin A J
Published: May 2006

RNA 3'-terminal phosphate cyclase has been partially purified from HeLa cells. In the presence of ATP and Mg2+, cyclase preparations catalyze conversion of RNA 3'-terminal phosphate to the 2',3'-cyclic phosphodiester. The mechanism of 3'-phosphate cyclization was studied with oligoribonucleotides containing terminal 2'-deoxy- or 2'-O-methylribose. Incubation of these substrates with cyclase and ATP results in formation of the corresponding activated 3'-terminal structures, dN(3')pp(5')A and Nm(3')pp(5')A. It is proposed that an intermediate step in cyclization is transfer of the adenylyl group from ATP to the 3' phosphate of RNA. Rapid attack of the adjacent 2'-OH normally follows, resulting in elimination of AMP and formation of the cyclic phosphodiester. Cyclase preparations can be covalently labeled with [alpha-32P]ATP, suggesting that an earlier step in the cyclization reaction involves formation of an adenylylated enzyme intermediate.
Xenogeneic antiserum to soluble products from activated lymphoid cells inhibits interleukin 1-mediated functions in the helper pathway of cytolytic-effector-cell differentiation.
Authors: McMannis J D , Plate J M
Published: May 2006

We have produced an antiserum that inhibits interleukin 1-mediated functions in immune responses. Skin graft-induced helper factor-containing supernatant (SgHFS) was used an immunogen in rats. The resultant antiserum was immunosuppressive of T-cell functions both in vivo and in vitro. We have further studied the effects of this antiserum on cell surface molecules that are involved in the generation of cytolytic effector T cells. Rat anti-SgHFS inhibited the differentiation of precytolytic effector cells in mixed lymphocyte cultures by blocking the helper-cell pathway. Both the level and the kinetics of interleukin 2 production were affected as the duration of rat anti-SgHFS pretreatment was increased. Interleukin 1 production after 24 hr in culture was unaffected. Monokines, including partially purified interleukin 1, actively compete with the rat anti-SgHFS by activating helper cells and thus circumvent suppression. Rat anti-SgHFS inhibits interleukin-1-mediated functions by a time-dependent active process. Thus, the target cell surface molecule(s) affected by the rat anti-SgHFS are associated with interleukin 1 function and may be the interleukin 1 receptor.
Stimulation of Na+/H+ antiport is an early event in hypertrophy of renal proximal tubular cells.
Authors: Fine L G , Badie-Dezfooly B , Lowe A G , Hamzeh A , Wells J , Salehmoghaddam S
Published: May 2006

Renal hypertrophy in vivo is achieved by an increase in protein content per cell and an increase in cell size with minimal hyperplasia. Hypertrophied renal tubular cells remain quiescent and demonstrate an increase in transcellular transport rates. This situation was simulated in vitro by exposing a confluent, quiescent primary culture of rabbit renal proximal tubular cells to either insulin, prostaglandin E1, or hypertonic NaCl for 24 or 48 hr. Protein per cell increased by 20-30% with little or no increase in [3H]thymidine incorporation into DNA. Mean cell volume was also increased in insulin- and hypertonic NaCl-treated but not in prostaglandin E1-treated cells. The lag period required to initiate DNA synthesis by a combination of insulin and hydrocortisone was the same in control and hypertrophied cells, indicating a quiescent state of the latter. Two hours of exposure to the growth stimuli increased amiloride-sensitive Na+ uptake, Na-dependent H+ efflux, and ouabain-sensitive Rb+ uptake, indicating that stimulation of Na+/H+ antiport (exchange) occurs as an early event in their action. Hypertrophied cells continued to demonstrate enhanced Na+/H+ antiport after the growth stimuli were removed for 3 hr, by which time their acute effects are reversed.
Isolation of morphine from toad skin.
Authors: Oka K , Kantrowitz J D , Spector S
Published: May 2006

A nonpeptide opioid was found in toad skin and purified to homogeneity by using HPLC with electrochemical detection. A nonpeptide opioid also was detected in bovine brain and adrenals as well as rabbit and rat skin, by reversed-phase HPLC following Sephadex G-15 column chromatography. The material in toad skin was identical to morphine by immunological, pharmacological, and physical chemical criteria.
Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits.
Authors: Criado M , Hochschwender S , Sarin V , Fox J L , Lindstrom J
Published: May 2006

Two monoclonal antibodies (mAbs 236 and 237) against a synthetic peptide composed of the same amino acid residues as the sequence 152-167 of the alpha subunit of the acetylcholine receptor were obtained, and their crossreaction with the synthetic peptide, alpha subunit, and solubilized receptor was demonstrated. Crossreaction with the synthetic peptide alpha 159-169 was less by a factor of 10(4), suggesting that the mAbs bind primarily to the sequence alpha 152-159. Cholinergic ligands did not inhibit mAb binding. No crossreaction was observed with the receptor in native membranes, but the mAbs could bind to receptor reconstituted into liposomes in which 50% of the receptors have their cytoplasmic surface oriented outside. When native membranes were permeabilized with saponin, mAbs directed against cytoplasmic determinants of the receptor could bind to them, but mAbs 236 and 237 could not. However, after treatments that removed peripheral proteins from the cytoplasmic surface, binding of both mAbs was observed. Further evidence for the cytoplasmic localization of this sequence was provided by observation of partial competition for binding between mAbs 236 and 237 and mAbs previously demonstrated to bind to the cytoplasmic surface of the receptor. To account for these findings, a model for the organization of the polypeptide chains in receptor subunits is proposed that has a total of seven transmembrane domains in each subunit, two of which are amphipathic and one of which is not alpha-helical.
Chicken triosephosphate isomerase complements an Escherichia coli deficiency.
Authors: Straus D , Gilbert W
Published: May 2006

We present the sequence of full-length chicken triosephosphate isomerase (D-glyceraldehyde 3-phosphate ketol-isomerase, EC 5.3.1.1) mRNA based on the analysis of cDNA and genomic clones. To isolate cDNA clones encoding the enzyme, we screened a muscle cDNA library with radioactively labeled cDNA made from RNA that had been enriched by immunoselection of polysomes. We blocked the signal caused by contaminating species in the probe with cloned DNA corresponding to the contaminants. Screening a chicken genomic library with cDNA coding for triosephosphate isomerase led to the isolation of phage containing the entire gene, which we used to map the transcriptional start. When placed downstream from a hybrid trp-lac promoter, the cDNA encoding the chicken enzyme programs the synthesis of functional protein, as judged by enzymatic criteria and by complementation of an Escherichia coli mutant that is deficient in bacterial triosephosphate isomerase.
Immunoglobulin VH gene structure and diversity in Heterodontus, a phylogenetically primitive shark.
Authors: Litman G W , Berger L , Murphy K , Litman R , Hinds K , Erickson B W
Published: May 2006

A mouse VH probe has been used to identify and isolate VH homologs in a DNA library of Heterodontus francisci (horned shark). The complete nucleotide sequence of one VH gene, HXIA, has been determined and found to exhibit striking organizational homology and nucleotide identity with mammalian prototype VH genes. Metric analysis of the complete sequence is consistent with the early phylogenetic diversification of framework and complementarity-determining regions (CDR). Both the predicted amino acid sequence and the specific hybridization of the CDR2-specific, synthetic oligodeoxynucleotide probe in spleen mRNA suggest that HXIA is functionally expressed. A probe consisting of the entire coding region of this gene hybridizes with multiple components in Southern blot analysis of Heterodontus genomic DNA and together with the identification of additional unique VH+-lambda clones indicates that considerable complexity is associated with the germline VH gene family in a contemporary species that represents an early stage in the phylogenetic development of the vertebrates.
Common haplotype dependency of high G gamma-globin gene expression and high Hb F levels in beta-thalassemia and sickle cell anemia patients.
Authors: Labie D , Pagnier J , Lapoumeroulie C , Rouabhi F , Dunda-Belkhodja O , Chardin P , Beldjord C , Wajcman H , Fabry M E , Nagel R L
Published: May 2006

We have studied 42 homozygous beta-thalassemia patients from Algeria and 34 sickle cell anemia patients from Senegal and Benin, determining the relationship between haplotypes, Hb F, and G gamma-globin/A gamma-globin ratios. Populations selected have a high frequency of haplotype homozygotes because of consanguinity (Algeria) and geographic homogeneity (West Africa). We find in beta-thalassemia patients, that haplotype IX in haplotypic homozygotes and heterozygotes, haplotype III in heterozygotes, and the Senegal haplotype in sickle cell anemia patients are all linked to high G gamma-globin expression. In addition, haplotypes IX and Senegal, but not haplotype III, have high Hb F levels. All of these haplotype have a common subhaplotype (+- ) in the gamma-globin gene region. In addition, haplotypes IX, III, and Senegalese sickle cell anemia patients exhibit hematological amelioration of their disease. Conversely, haplotypes I, V, and A in thalassemia patients, which also have a common subhaplotype (-----), and the Benin subhaplotype (--++-) in sickle cell anemia patients are all associated with low G gamma-globin and low Hb F levels. Low G gamma-globin expression in the adult is associated with two haplotypes that are not common between thalassemia and sickle cell anemia patients. We conclude that the determinant for high G gamma-globin expression is haplotype-linked to common and genetically dominant subhaplotypes in the two diseases. The total Hb F level, unlike the high G gamma-globin expression, however, is linked to haplotypes but not to subhaplotypes, thus dissociating the two genetic effects.
Suppressor T-cell factor(s) display an altered pattern of Igh (immunoglobulin heavy chain locus) genetic restriction when developed in an Igh-congeneic host.
Authors: HayGlass K T , Naides S J , Benacerraf B , Sy M S
Published: May 2006

Suppressor T-cell factor(s) (TsF1) inhibit the in vivo priming of azobenzenearsonate-specific cytotoxic T-cell responses. The activity of TsF1 is restricted by genes linked to Igh-1 allotypic markers. TsF1 obtained from B6.Igh-1n mice was unable to suppress the immune response in B6.Igh-1b mice and vice versa. However, TsF1 prepared from B6.Igh-1n T cells "parked" in an Igh-congeneic B6.Igh-1b environment displays an additional restriction specificity of the host. Thus, TsF1 prepared from these Igh-chimeric mice suppressed immune responses in both B6.Igh-1n (donor) and B6.Igh-1b (recipient) mice but not in mice of the unrelated strain BALB/c.Igh-1a. The results indicate that the establishment of the suppressor T-cell repertoire is dependent not only upon the genetic background of the individual T cell but also upon the influence of Igh-linked determinants present when T-cell clones are selected during the response.
Padé approximations and diophantine geometry
Authors: Chudnovsky D. V. , Chudnovsky G. V.
Published: May 2006

Using methods of Padé approximations we prove a converse to Eisenstein's theorem on the boundedness of denominators of coefficients in the expansion of an algebraic function, for classes of functions, parametrized by meromorphic functions. This result is applied to the Tate conjecture on the effective description of isogenies for elliptic curves.
Lysosomotropic agents selectively potentiate thrombin-induced acid hydrolase secretion from platelets.
Authors: Van Oost B A , Smith J B , Holmsen H , Vladutiu G D
Published: May 2006

Thrombin induces partial secretion (up to 60%) of beta-N-acetyl-D-hexosaminidase (EC 3.2.1.52) from untreated platelets. Preincubation of platelets with 10 mM NH4Cl for up to 2 hr resulted in a time-dependent and marked stimulation of thrombin-induced secretion of both this enzyme and other acid glycosidases from platelets. The enhancement of the thrombin-induced secretion was not due to cell lysis, and NH4Cl alone did not cause leakage of lysosomal enzymes into the medium. The effect could be reversed by reincubating the platelets in NH4Cl-free medium. Stimulation of thrombin-induced secretion also was produced by a series of aliphatic primary amines from methylamine to butylamine, and by micromolar concentrations of chloroquine. The effect of weak bases on platelets appeared to be quite specific for enhancing lysosomal enzyme secretion. Thrombin-induced secretion of adenine nucleotides from dense granules and of beta-thromboglobulin from alpha granules was slightly enhanced by NH4Cl but was slightly inhibited by methylamine. The only direct effect of the weak bases on platelets was the displacement of serotonin from dense granules. Accumulation of weak bases in acidic pools in the platelets (e.g., lysosomes) might, therefore, be responsible for the enhanced secretion of lysosomal enzymes. By using controlled digitonin-induced platelet lysis, it was found that preincubation of platelets with NH4Cl lowered the digitonin concentration required for enzyme solubilization. We suggest that loading of lysosomes with weak bases dissociates already bound enzyme inside the lysosomes, resulting in a more effective discharge upon stimulation by thrombin.
Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells.
Authors: Sutherland B M , Bennett P V , Freeman A G , Moore S P , Strickland P T
Published: May 2006

Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.
Synthesis and secretion of proteins resembling platelet-derived growth factor by human glioblastoma and fibrosarcoma cells in culture.
Authors: Pantazis P , Pelicci P G , Dalla-Favera R , Antoniades H N
Published: May 2006

Immunoprecipitation of proteins extracted from metabolically labeled human glioblastoma and fibrosarcoma cells with antiserum to platelet-derived growth factor (PDGF) showed that these cells express and secrete proteins that are recognized specifically by the antiserum. The molecular masses of immunoprecipitated proteins in the lysates of the malignant cells ranged from 16 kDa to 140 kDa. Both cell lines secreted a 31-kDa polypeptide with structural, immunological, and biological properties similar to those of human PDGF. These cell lines were shown to synthesize a 4.4-kb mRNA that contained sequences from all the six currently identified exons of the human c-sis gene. These data suggest that the PDGF-like proteins in the two mesenchyme-derived transformed cells are encoded at least in part by the c-sis locus.
Mouse major histocompatibility class I gene expression begins at midsomite stage and is inducible in earlier-stage embryos by interferon.
Authors: Ozato K , Wan Y J , Orrison B M
Published: May 2006

To determine the timing of major histocompatibility complex class I gene expression during embryonic development, binding of anti-class I antibodies and appearance of class I gene transcripts were examined in mouse embryos from the egg-cylinder stage through day 16 of gestation. By using two series of monoclonal antibodies reactive with monomorphic and polymorphic determinants of class I antigens, it was found that cell-surface expression of the antigens becomes detectable at a low level only after midsomite stage on gestation day 10, at a time when embryos are developed beyond primordial organogenesis and have partial blood circulation. In agreement with the above finding, a low level of class I mRNA became detectable in day 9 and older embryos in blot hybridization. The level of class I transcripts in embryos at least to day 13 remained less than 1/50th that in adult spleen cells. Cells from head-fold stage embryos (gestation day 8), which otherwise do not have an appreciable amount of class I mRNA or surface antigens, begin to express a high level of antigens upon treatment with mouse alpha/beta or gamma interferon. This induction of class I antigen expression appears to be stage specific in that embryos in an earlier egg-cylinder stage (day 6) failed to express the antigens after interferon treatment. A possible role of interferons in activating class I genes during in vivo embryonic development is suggested.
Regulation of cellular function by products of lysosomal enzyme activity: elimination of human natural killer cells by a dipeptide methyl ester generated from L-leucine methyl ester by monocytes or polymorphonuclear leukocytes.
Authors: Thiele D L , Lipsky P E
Published: May 2006

L-Leucine methyl ester (Leu-OMe) is a lysosomotropic compound that irreversibly removes natural killer cell (NK) function from human peripheral blood mononuclear cells. This effect was dependent on the presence of mononuclear phagocytes (M phi) or polymorphonuclear leukocytes (PMN) and was prevented by lysosomal inhibitors such as chloroquine or NH4Cl. When M phi or PMN were incubated with Leu-OMe, a product was formed that eliminated all NK function from mixed lymphocyte populations. This effect did not require the presence of M phi or PMN and was not prevented by lysosomal enzyme inhibitors. Thin-layer chromatography and mass spectral analysis revealed that this NK-toxic product was L-leucyl-L-leucine methyl ester (Leu-Leu-OMe). When human lymphocytes were exposed to greater than 50 microM Leu-Leu-OMe for 15 min, all NK function was irreversibly eliminated. Other dipeptide methyl esters containing nonpolar L amino acids caused similar effects, but substitution with amino acids containing polar or charged side chains or with D stereoisomers produced compounds that had no toxic effect on NK. These findings indicate that M phi and PMN can regulate NK function by releasing the dipeptide condensation product Leu-Leu-OMe generated from Leu-OMe via a lysosomally mediated mechanism. The data show that specific products of lysosomal enzyme activity may have potent effects on the function of adjacent cells.
Direct measurement of the electrogenic activity of o-type cytochrome oxidase from Escherichia coli reconstituted into planar lipid bilayers.
Authors: Hamamoto T , Carrasco N , Matsushita K , Kaback H R , Montal M
Published: May 2006

Turnover of o-type cytochrome oxidase purified from Escherichia coli and reconstituted into proteoliposomes leads to the generation of a transmembrane electrical potential (interior negative) by means of vectorial electron flow. In the experiments reported here, purified oxidase is reconstituted in planar lipid bilayers formed at the tip of patch pipets, and open-circuit membrane potentials generated by electron transfer are measured directly. Potentials of up to 4 mV (substrate side positive) are generated in the presence of reduced phenazine methosulfate or ubiquinol-1, and with both substrates, electrogenic activity is inhibited by cyanide. Furthermore, the membrane potential generated during oxidase turnover is inhibited progressively with applied voltages (substrate side positive), decreasing almost to zero at an applied voltage of 150 mV.
gamma-Fluoromethotrexate: synthesis and biological activity of a potent inhibitor of dihydrofolate reductase with greatly diminished ability to form poly-gamma-glutamates.
Authors: Galivan J , Inglese J , McGuire J J , Nimec Z , Coward J K
Published: May 2006

A methotrexate (MTX) analog containing fluorine at the gamma-carbon of the glutamate moiety, gamma-fluoromethotrexate (FMTX), has been synthesized and evaluated for its biochemical and pharmacological properties. FMTX inhibition of dihydrofolate reductase from several sources is nearly equivalent to that shown by MTX. Most important, FMTX is an exceedingly poor substrate for folylpoly (gamma-glutamate) synthetase, the enzyme that catalyzes the biosynthesis of the highly-retained, cytotoxic MTX polyglutamates. Uptake experiments in H35 hepatoma cells show that FMTX accumulates to approximately the same extent as MTX at steady state. The rapid efflux of both derivatives is also very similar. The major difference detected in cells between the two compounds is the meager glutamylation of FMTX, due to the electronegative properties of the fluorine adjacent to the potential amide-forming carboxyl group. Exposure of dividing cells to 50 microM MTX for 2 and 6 hr results in the formation of 55 and 130 nmol, respectively, of the polyglutamates (more than two glutamate residues)/g of cell protein. With FMTX these values were reduced by 98% and 93%, respectively. Growth inhibition studies show that MTX is only 12-fold more toxic than FMTX when the cells are exposed to each derivative continuously for 72 hr. When the exposure time is reduced, a greater disparity between the inhibitory effects is observed; with a 2-hr pulse, MTX is 2300-fold more effective than FMTX. These data correlate with the effects of pulses of FMTX and MIX on de novo thymidylate biosynthesis in intact cells. The results indicate that of the parameters examined, the vastly reduced toxicity of FMTX after its removal from the culture medium is best correlated with impaired glutamylation. The data strongly suggest that prolonged toxicity of MTX is a result of metabolic conversion to MTX polyglutamates and that these effects are far more dramatic in short-term than in long-term exposure to the antifolates.
Modification of pK values caused by change in H-bond geometry.
Authors: Scheiner S , Hillenbrand E A
Published: May 2006

The competition between various groups for a proton is studied by ab initio molecular orbital methods. It is found that reorientations of the two groups involved in a H-bond can reverse the equilibrium position of the proton shared between them. Specifically, the carbonyl and hydroxyl groups were modeled by H2CO and HOH. In the H-bond between these two groups, association of the proton with the carbonyl (H2COH...OH2)+ is favored over the hydroxyl (H2CO...HOH2)+ when the latter group is situated along a lone pair of the carbonyl oxygen. However, displacement of the water to the C = O axis between the two carbonyl lone pairs reverses the situation and (H2CO...HOH2)+ is more stable. A similar reversal of stability is observed in the H-bond involving a Schiff base (modeled by CH2NH) and amine (NH3). In one arrangement where the lone pairs of the two groups point toward one another, the proton prefers the Schiff base to the amine--i.e., (H2CHNH...NH3)+ is more stable than (H2CHN...HNH3)+. On the other hand, rotation of the lone pair of the amine away from the Schiff base nitrogen results in proton transfer across to the amine. These shifts in stability correspond to reversal of relative pK of the groups involved. A fundamental principle emerging from the calculations is that ion-dipole electrostatic interactions favor transfer of a proton to the group that is positioned as closely as possible to the negative end of the dipole moment vector of the other. The ideas developed here suggest a number of means by which conformational changes may be utilized to shift protons from residue to residue within a protein molecule such as an enzyme or bacteriorhodopsin.
Mutations in the phosphoglucose isomerase gene can lead to marked alterations in cellular ATP levels in cultured fibroblasts exposed to simple nutrient shifts.
Authors: Plesner P , Ullrey D B , Kalckar H M
Published: May 2006

The generation of ATP in a hamster fibroblast mutant devoid of the enzyme phosphoglucose isomerase (PGI) has been studied and compared with that in the parental line, which is PGI+. Both cell lines could be maintained for 24 hr in hexose media devoid of L-glutamine. Under these conditions in mannose medium, both the parental line and the PGI mutant maintained high intracellular ATP levels. With glucose under the same conditions, the parental line was able to keep the ATP level high. In contrast, the mutant line lost most of its ATP pool after incubation with glucose; the ATP/ADP ratio fell about 80% after incubation in glucose medium. Addition of pyruvate, with or without glucose, preserved the ATP pool at high levels even in the mutant, as did the presence of L-glutamine. When the PGI mutant was maintained for 3-4 days in growth medium, containing 4 mM L-glutamine and 10% dialyzed calf serum, in which glucose was replaced by mannose, the UDP-glucose pool dwindled and mediated control of the hexose uptake system did not ensue, in contrast to results of the same exposure to glucose-containing medium.
α-Amanitin resistance is developmentally regulated in carrot
Authors: Pitto L. , Schiavo F. Lo , Terzi M.
Published: May 2006

Carrot cells are capable of inactivating α-amanitin only in embryogenic conditions (regenerating cells and embryoids). Instead, the mutant line a3 is capable of inactivating the drug also in nonembryogenic conditions (vegetative growth). The mutation is dominant in somatic hybrids and is pleiotropic, allowing expression during vegetative growth of other embryonal functions. The inactivation of α-amanitin is due to the oxidative activity of tyrosinase.
Morphogenetic rearrangement of injected collagen in developing chicken limb buds.
Authors: Stopak D , Wessells N K , Harris A K
Published: May 2006

A fundamental question concerning the development of the extracellular matrix is what factors control the arrangement of collagen fibrils within a tissue and at the same time allow for the great diversity of geometric forms exhibited by collagen. In this report, we test the possibility that physical forces within the embryo serve to organize collagen fibers into regular patterns. In particular, we test the prediction that patterns of stress having this morphogenetic function are generated by cell traction, the contractile force exerted by cells to propel themselves. To study the effects of these mechanical forces on the extracellular matrix, type I collagen was fluorescently labeled and injected into developing chicken wing buds. When the injected limbs were allowed to develop and then examined histologically, the exogenous collagen was found incorporated within normal connective tissues of the wing. The labeled collagen became arranged according to its site of injection, forming parts of tendons, perichondria, cartilages, perineuria, and blood vessels. Since the injected collagen formed a gel within minutes of its injection, the subsequent incorporation of this performed collagen within organized structures cannot be explained in terms of molecular self-assembly or other mechanisms occurring during collagen deposition. These results demonstrate that, within developing tissues, patterns of forces exist that are capable of physically rearranging collagen and determining its long-range order.