bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b.
Authors: Parkos C A , Dinauer M C , Walker L E , Allen R A , Jesaitis A J , Orkin S H
Published: May 2006

Cytochrome b comprising 91-kDa and 22-kDa subunits is a critical component of the membrane-bound oxidase of phagocytes that generates superoxide. This important microbicidal system is impaired in inherited disorders known as chronic granulomatous disease (CGD). Previously we determined the sequence of the larger subunit from the cDNA of the CGD gene, the X chromosome locus affected in "X-linked" CGD. To complete the primary structure of the cytochrome b and to assess expression of the smaller subunit, we isolated cDNA clones for the 22-kDa polypeptide by immunoscreening and confirmed their authenticity by direct N-terminal protein sequencing. Although the deduced amino acid sequence of the 22-kDa subunit is not overtly similar to other known cytochromes, we observed a 31-amino acid stretch of 39% identity with polypeptide I of mitochondrial cytochrome c oxidase centered on a potential heme-coordinating histidine. Similarities in the hydropathy profiles and spacing of histidines of the 22-kDa protein and myoglobin suggest structural motifs in common with other heme-containing proteins that are not readily revealed by primary amino acid sequences. Although RNA for the larger subunit has been found only in cells of the phagocytic lineage, stable RNA encoding the 22-kDa subunit was observed in all cell types. However, the stable 22-kDa protein was detected only in phagocytic cells that were expressing the larger subunit RNA. This observation suggests that the large subunit may play a role in regulating the assembly of the heterodimeric cytochrome b.
Carboxyl-terminal amino acid sequences of two variant forms of the gamma chain of human plasma fibrinogen.
Authors: Francis C W , Müller E , Henschen A , Simpson P J , Marder V J
Published: May 2006

The gamma chain of human fibrinogen has been shown to be heterogeneous; three forms of various sizes are normally present in plasma. To further characterize the two less prevalent elongated variants, we have purified the three forms of the gamma chain, isolated unique tryptic fragments, and sequenced the variant portions of each chain. The intermediate-sized form (gamma 55) has a sequence identical to the longest variant (gamma 57.5) from residue Val-408 to Pro-423, which is the C terminus of the gamma 55 chain. The gamma 57.5 chain extends an additional four amino acids. The C-terminal amino acid sequence and corresponding nucleotide sequence of the gamma 55 chain show marked similarity with an elongated gamma-chain variant of rat fibrinogen. In addition, a remarkable similarity in both amino acid and nucleotide sequence was noted between the human gamma 55 chain and the C-terminal extension in human and bovine A alpha chain of fibrinogen, which are encoded by the cDNA but are posttranslationally cleaved and are not found in plasma. The findings suggest an evolutionary conservation in the C-terminal amino acid sequence found in both the fibrinogen A alpha and gamma chains in several species.
Interaction of a common cellular transcription factor, ATF, with regulatory elements in both E1a- and cyclic AMP-inducible promoters.
Authors: Lin Y S , Green M R
Published: May 2006

We have previously identified a cellular transcription factor, ATF, which is involved in the expression of multiple adenovirus E1a-inducible genes. Here we show that ATF also binds to regulatory elements in cellular cAMP-inducible promoters. In these cellular promoters, ATF binds to a consensus sequence that has been previously shown to be necessary and in some instances sufficient for cAMP-inducible transcription. Furthermore, we show that binding of ATF to these promoter elements is required for the constitutive in vitro transcriptional activity of the cAMP-inducible somatostatin gene. Taken together, our results suggest that a common cellular transcription factor, ATF, can be regulated in vivo by two apparently unrelated inducing agents: the adenovirus E1a protein and cAMP.
Constitutive synthesis of activator protein 1 transcription factor after viral transformation of mouse fibroblasts.
Authors: Piette J , Hirai S , Yaniv M
Published: May 2006

Transcription factor activator protein 1 (AP1) interacts with the promoter region of a number of genes that are stimulated by growth factors present in serum or by agents such as phorbol 12-myristate 13-acetate (PMA) that partially mimic their action. To investigate the possible role of AP1 in the control of cellular growth and in transformation of mammalian cells, we monitored its activity by binding to a specific DNA probe in normal or transformed mouse NIH 3T3 cells. A 3- to 4-fold increase in the DNA-binding activity was found after serum stimulation of quiescent NIH 3T3 cells. A 2- to 3-fold activation was found after treatment with PMA or dibutyryl-cAMP, suggesting that different signal-transducing pathways could activate AP1 factor in these cells. PMA stimulation was dependent on new protein synthesis. In contrast to normal cells, a high serum-independent AP1 DNA-binding activity was found in NIH 3T3 cells transformed by simian virus 40. These results suggest that constitutive AP1 synthesis may be a crucial step in cellular transformation.
ATP-dependent association of nuclear proteins with isolated rat liver nuclei.
Authors: Imamoto-Sonobe N , Yoneda Y , Iwamoto R , Sugawa H , Uchida T
Published: May 2006

In vitro association of Xenopus nucleoplasmin and mammalian nonhistone chromosomal high mobility group 1 (HMG1) protein with nuclei isolated from rat liver was examined. Efficient association of nuclear proteins with isolated nuclei requires ATP, HCO3-, and Ca2+. Association occurred at 33 degrees C but not at 4 degrees C. ATP could be replaced by adenosine 5'-[alpha,beta-methylene]triphosphate (pp[CH2]pA), a nonhydrolyzable ATP analog. pp[CH2]pA associated with nuclei at 33 degrees C and nucleoplasmin and HMG1 rapidly associated with the pp[CH2]pA-bound nuclei at 4 degrees C. Competition studies showed that these associations at both 33 degrees C and 4 degrees C were specific. More than 80% of the bindings of nuclear proteins to the nuclear surface were blocked by wheat germ agglutinin.
Induction of vitellogenin in primary monolayer cultures of cockerel hepatocytes.
Authors: Boehm K D , Hood R L , Ilan J
Published: May 2006

A primary monolayer culture system from cockerel hepatocytes was established. The cultures synthesize and secrete proteins that comigrate with authentic serum proteins on polyacrylamide gels and are found in the same relative abundance. Addition of estradiol increased the synthesis of apoprotein B, found in very low density lipoprotein, under all culture conditions. Vitellogenin synthesis could not be induced directly by estradiol. However, when serum was obtained from cockerels injected with estradiol 4 days before blood collection and included in the culture medium, the cultures secreted a protein identified immunologically as vitellogenin by affinity chromatography. Furthermore, addition of growth hormone or prolactin to cultured cockerel hepatocyte monolayers resulted in the synthesis and secretion of a polypeptide that comigrates with authentic vitellogenin on polyacrylamide gels.
Transfer of retinol from parenchymal to stellate cells in liver is mediated by retinol-binding protein.
Authors: Blomhoff R , Berg T , Norum K R
Published: May 2006

Newly absorbed chylomicron remnant retinyl ester is endocytosed by parenchymal liver cells, and retinol is subsequently transferred to perisinusoidal stellate cells in liver. In the present study we have used several approaches to elucidate the mechanism for the paracrine transfer of retinol between liver parenchymal and stellate cells. In one series of experiments, chylomicrons labeled with [3H]retinyl palmitate or with retinyl [3H]palmitate were injected intravenously into rats. It was shown that the retinol as well as the palmitate moiety were initially taken up in parenchymal liver cells. However, only the retinol moiety was detected in stellate cells, indicating that the retinyl ester is hydrolyzed before retinol is transferred to stellate cells. It is well known that parenchymal liver cells secrete retinol bound to retinol-binding protein (RBP), and we have recently found that stellate cells do have RBP receptors. Here we report that antibodies against RBP completely block the transfer of retinol from parenchymal to stellate cells. These findings indicate that following uptake of chylomicron remnant retinyl ester in parenchymal cells, the retinyl ester is hydrolyzed, and retinol secreted from parenchymal cells on RBP is taken up by stellate cells by means of RBP receptors.
Gene sharing by delta-crystallin and argininosuccinate lyase.
Authors: Piatigorsky J , O'Brien W E , Norman B L , Kalumuck K , Wistow G J , Borras T , Nickerson J M , Wawrousek E F
Published: May 2006

The lens structural protein delta-crystallin and the metabolic enzyme argininosuccinate lyase (ASL; L-argininosuccinate arginine-lyase, EC 4.3.2.1) have striking sequence similarity. We have demonstrated that duck delta-crystallin has enormously high ASL activity, while chicken delta-crystallin has lower but significant activity. The lenses of these birds had much greater ASL activity than other tissues, suggesting that ASL is being expressed at unusually high levels as a structural component. In Southern blots of human genomic DNA, chicken delta 1-crystallin cDNA hybridized only to the human ASL gene; moreover, the two chicken delta-crystallin genes accounted for all the sequences in the chicken genome able to cross-hybridize with a human ASL cDNA, with preferential hybridization to the delta 2 gene. Correlations of enzymatic activity and recent data on mRNA levels in the chicken lens suggest that ASL activity depends on expression of the delta 2-crystallin gene. The data indicate that the same gene, at least in ducks, encodes two different functions, an enzyme (ASL) and a structural protein (delta-crystallin), although in chickens specialization and separation of functions may have occurred.
Toward an objective classification of cells in the immune system.
Authors: Lefkovits I , Kuhn L , Valiron O , Merle A , Kettman J
Published: May 2006

The relative abundance of individual proteins shared among clones of lymphocytes provides a meaningful basis for cellular classification. Twelve clones of T cells (obtained by limiting dilution) were analyzed by two-dimensional gel electrophoresis for polypeptide content and then evaluated by the computational technique known as principal component analysis. As a result, relatedness of the clones was established and expressed in terms of taxonomic distances. The data show that a comprehensive and objective classification of the cells involved in the immune system can be approached.
Molecular cloning of human T-cell lymphotrophic virus type I-like proviral genome from the peripheral lymphocyte DNA of a patient with chronic neurologic disorders.
Authors: Reddy E P , Mettus R V , DeFreitas E , Wroblewska Z , Cisco M , Koprowski H
Published: May 2006

Human T-cell lymphotropic virus type 1 (HTLV-I), the etiologic agent of human T-cell leukemia, has recently been shown to be associated with neurologic disorders such as tropical spastic paraparesis, HTLV-associated myelopathy, and possibly with multiple sclerosis. In this communication, we have examined one specific case of neurologic disorder that can be classified as multiple sclerosis or tropical spastic paraparesis. The patient suffering from chronic neurologic disorder was found to contain antibodies to HTLV-I envelope and gag proteins in his serum and cerebrospinal fluid. Lymphocytes from peripheral blood and cerebrospinal fluid of the patient were shown to express viral RNA sequences by in situ hybridization. Southern blot analysis of the patient lymphocyte DNA revealed the presence of HTLV-I-related sequences. Blot-hybridization analysis of the RNA from fresh peripheral lymphocytes stimulated with interleukin 2 revealed the presence of abundant amounts of genomic viral RNA with little or no subgenomic RNA. We have cloned the proviral genome from the DNA of the peripheral lymphocytes and determined its restriction map. This analysis shows that this proviral genome is very similar if not identical to that of the prototype HTLV-I genome.
Inhibition of surface membrane maturation in schistosomula of Schistosoma mansoni.
Authors: Wiest P M , Tartakoff A M , Aikawa M , Mahmoud A A
Published: May 2006

The surface membrane of the multicellular parasite Schistosoma mansoni is radically reorganized during the transformation of cercariae into schistosomula. The current study investigates factors involved in maturation of the surface from a trilaminate to a multilaminate membrane. When maturation was induced in the presence of puromycin (900 microM), the acquisition of a multilaminate surface and stainability with fluorescein-conjugated Con A were similar to that of control parasites. Similarly, although organisms treated with monensin (0.1 microM) for 3 hr showed large vacuoles in the perinuclear cytoplasm of the subtegumental cells, the surface membrane became multilaminate. In contrast, microtubule-active drugs interfered with maturation: the surface remained largely trilaminate and the percentage of organisms binding Con A to their surface was significantly reduced. Furthermore, large accumulations of multilaminate bodies were found in the subtegumental cells of colchicine-treated parasites, whereas few were seen in the controls. Colchicine-treated schistosomula failed to mature to adult worms upon injection into mice and, like cercariae, they were water tolerant. We therefore conclude that the components that constitute the schistosomula surface preexist in cercariae and suggest that they are stored in multilaminate bodies before being transported to the surface with the help of microtubules. The acquisition of the multilaminate membrane may be essential for survival of the parasites in vivo and in vitro.
Mitogen and lymphokine stimulation of heat shock proteins in T lymphocytes.
Authors: Ferris D K , Harel-Bellan A , Morimoto R I , Welch W J , Farrar W L
Published: May 2006

We have examined the effects of phytohemagglutinin (PHA) and the polypeptide growth factor interleukin 2 (IL-2) on the synthesis of the 70- and 90-kDa heat shock proteins (HSP70 and HSP90, respectively) in human T lymphocytes. Resting T cells (G0) stimulated with PHA responded with a generalized increase in protein synthesis that included HSP70. Gel blot analysis indicated that steady-state levels of HSP70 mRNA were not specifically modulated by PHA. Synthesis of HSP90 protein, however, peaked very rapidly following PHA stimulation and decreased sharply after 1 hr. When IL-2-dependent human T cells, synchronized by IL-2 deprivation, were treated with IL-2, synthesis of HSP70 mRNA was increased as much as 15-fold. HSP70 and HSP90 protein synthesis increased significantly upon IL-2 stimulation of human T lymphocytes. Two distinct members of the ancient family of heat shock genes, HSP70 and HSP90, are shown to be stimulated at the activation and progression stages of lymphocyte mitogenesis, which suggests that genetic mechanisms evolved from primitive stress/adaptation responses may be conserved in mammalian cellular activation.
Developmental control of the excitability of muscle: transplantation experiments on a myotonic mouse mutant.
Authors: Füchtbauer E M , Reininghaus J , Jockusch H
Published: May 2006

Developmental aspects of an animal model of myotonia, the mouse mutant called "arrested development of righting response" (ADR phenotype), were studied. Adult ADR muscle is characterized by a low chloride conductance of the membrane, leading to hyperexcitability, and by a low parvalbumin content. The myotonic hyperexcitability (as measured by the extent of "aftercontractions") of ADR muscle increased steeply between postnatal days 9 and 18, by which time it had approached the adult level. To study the tissue autonomy of the myotonic phenotype, muscle grafts were performed in all four combinations between ADR and wildtype (WT phenotype) donors and hosts. In most experiments, the relative contributions of donor and host to the regenerated muscles were determined by an allelic marker (glucose phosphate isomerase). In WT and ADR hosts, ADR grafts showed myotonic responses that in WT nude mouse hosts were incomplete and similar to those of juvenile ADR muscle. In no case did grafts from WT donors show any myotonia. This shows that the myotonic ADR phenotype is based on an intrinsic muscle property most likely related to the plasma membrane. The parvalbumin contents of grafted muscles, when compared with those of untransplanted muscles, indicated graft-host interaction in the expression of this secondary phenotypic property.
Somatic diversification of the S107 (T15) VH11 germ-line gene that encodes the heavy-chain variable region of antibodies to double-stranded DNA in (NZB x NZW)F1 mice.
Authors: Behar S M , Scharff M D
Published: May 2006

A variety of studies suggest that members of the S107 (T15) heavy-chain variable-region gene family contribute to the autoimmune response of mice and humans to DNA. To identify the germ-line gene(s) involved and the degree of somatic diversification that occurs in such autoantibodies, we determined the mRNA sequence of the heavy and light chains of a group of monoclonal anti-DNA antibodies encoded by the S107 VH11 germ-line gene in (NZB x NZW)F1 mice. We also cloned and sequenced the VH11 germ-line gene of the NZB and NZW parental strains. The VH11 coding sequences of the two strains were identical. Comparison with this heavy-chain germ-line sequence showed that the variable regions of the monoclonal antibodies had undergone considerable somatic diversification.
Human monoclonal antibodies produced by primary in vitro immunization of peripheral blood lymphocytes.
Authors: Borrebaeck C A , Danielsson L , Möller S A
Published: May 2006

A general procedure is described for the production of human monoclonal antibodies from peripheral blood lymphocytes immunized in vitro against T-cell-dependent antigens. These lymphocytes immunized in culture were used to produce human-human or human-mouse hybridomas secreting monoclonal antibodies specific for digoxin, hemocyanin, a recombinant fragment of the gp120 envelope glycoprotein of human immunodeficiency virus (PB1), or a melanoma-associated antigen (p97). Depletion of a lysosome-rich cell population, containing large granular lymphocytes, monocytes, cytotoxic T cells, and a subset of CD8-positive T cells, was shown to be crucial before the cells could be immunized in vitro. This depletion was accomplished by treating the peripheral blood lymphocytes with the lysosomotropic agent L-leucine methyl ester. In addition, the in vitro immunization had to be supported by interleukin 2, gamma-interferon, and B-cell growth and differentiation factors, derived from irradiated, pokeweed-mitogen-stimulated human T cells. The production of human monoclonal antibodies from primary, antigen-specifically activated peripheral lymphocytes might obviate the need to immunize volunteers or patients.
Active transport of urea by mammalian kidney.
Authors: Beyer K H , Gelarden R T
Published: May 2006

With the aid of three basic organic compounds, 2-pyrazinoylguanidine, 3-amino-2-pyrazinoylguanidine, and 3,5-diamino-6-fluoro-2-pyrazinoylguanidine, urea was shown to be secreted by the renal tubules of the dog both from the lumen and into the lumen independent of urine flow.
Aging results in an unusual expression of Drosophila heat shock proteins.
Authors: Fleming J E , Walton J K , Dubitsky R , Bensch K G
Published: May 2006

We used high-resolution two-dimensional polyacrylamide gel electrophoresis to evaluate the effect of aging on the heat shock response in Drosophila melanogaster. Although the aging process is not well understood at the molecular level, recent observations suggest that quantitative changes in gene expression occur as these fruit flies approach senescence. Such genetic alterations are in accord with our present data, which clearly show marked differences in the synthesis of heat shock proteins between young and old fruit flies. In 10-day-old flies, a heat shock of 20 min results in the expression of 14 new proteins as detectable by two-dimensional electrophoresis of [35S]methionine-labeled polypeptides, whereas identical treatment of 45-day-old flies leads to the expression of at least 50 new or highly up-regulated proteins. In addition, there is also a concomitant increase in the rate of synthesis of a number of the normal proteins in the older animals. Microdensitometric determinations of the low molecular weight heat shock polypeptides on autoradiographs of five age groups revealed that their maximum expression occurs at 47 days for a population of flies with a mean life span of 33.7 days. Moreover, a heat shock effect similar to that observed in senescent flies occurs in young flies fed canavanine, an arginine analogue, before heat shock.
Putative structure and functions of a poly-beta-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes.
Authors: Reusch R N , Sadoff H L
Published: May 2006

A poly-beta-hydroxybutyrate complex extracted from the plasma membranes of genetically competent Escherichia coli contained polyhydroxybutyrate:polyphosphate:calcium in molar ratios approximating 1:1:0.5. The chain length of the polyhydroxybutyrate was estimated as 120-200 subunits, and that of the polyphosphate was estimated as 130-170 subunits. The extracted complex, when incorporated into liposomes, exhibited a lipid phase transition in the same temperature range as that of the membrane complex in whole cells as well as the same properties of irreversibility, lability, and sensitivity to chelating buffers. Space-filling molecular models and molecular energy minimization methods (Charmm) were used to develop and evaluate a plausible structure for the complex. It is proposed that the polyhydroxybutyrate forms an exolipophilic-endopolarophilic helix around an inner framework helix of calcium polyphosphate. The calcium ions link the two polymers by forming ionic bonds with phosphoryl oxygens of the polyphosphate and ion-dipole bonds with the ester carbonyl oxygens of the polyhydroxybutyrate. This symmetrical structure forms a channel through the membrane and may play a role in the transport of calcium, phosphate, and DNA.
Evidence for a cAMP-dependent nuclear factor capable of interacting with a specific region of a eukaryotic gene.
Authors: Lee C Q , Miller H A , Schlichter D , Dong J N , Wicks W D
Published: May 2006

Nuclear extracts prepared from the livers of rats treated with or without 8-bromo-cAMP were tested for their ability to bind to various fragments from the flanking region of the gene encoding phosphoenolpyruvate carboxykinase (GTP) [GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] known to contain the element that confers transcriptional regulation by cAMP. Using the nitrocellulose-filtration method, concentration-dependent, apparently saturable binding was seen that is both specific and cAMP dependent. Analysis of various fragments pinpointed the active binding region to positions within the -67 to -111 region, which coincides with the functional regulatory element as shown by recent transfection studies. Formation of an apparently single complex between a synthetic oligomer containing the region from -67 to -111 of the phosphoenolpyruvate carboxykinase gene and a factor in nuclear extracts from cAMP-treated rat liver was visualized by the gel-retardation method. Complex formation is both concentration and cAMP dependent and can be prevented by excess specific but not nonspecific competitor DNA. The congruity of the results with the two different methods suggests that the factor we have detected has properties consistent with a possible role as mediator of the transcriptional control exerted by cAMP in eukaryotic cells.
Malignant transformation by ras and other oncogenes produces common alterations in inositol phospholipid signaling pathways.
Authors: Alonso T , Morgan R O , Marvizon J C , Zarbl H , Santos E
Published: May 2006

The role of ras proteins in signal transduction was assessed by studying inositol phospholipid metabolism and inositol phospholipid-mediated cellular responsiveness to agonists in cells transformed by ras and other oncogenes. Specific alterations were observed in the inositol phospholipid cycle of ras-transformed fibroblasts, but similar changes were also produced by spontaneous transformation or transformation mediated by either membrane-associated oncogenes, such as src, met, or trk, or cytoplasmic oncogenes, mos and raf; the nuclear oncogenes fos and myc did not produce these changes. The alterations included (i) stimulation of phospholipase A2 activity as indicated by elevated levels of glycerophosphoinositol and nonesterified arachidonic acid and (ii) specific uncoupling between surface receptor-mediated stimulation by platelet-derived growth factor, bombesin, or serum and activation of intracellular phospholipase C. These findings suggest the existence of common biochemical pathways for transformation by cytoplasmic and membrane-associated oncogenes and are not consistent with the hypothesis that 21-kDa ras proteins (p21) are direct or distinct regulatory elements of phospholipase C or phospholipase A2 in inositol phospholipid signal transduction pathways.
Use of site-directed mutagenesis to elucidate the role of arginine-166 in the catalytic mechanism of alkaline phosphatase.
Authors: Butler-Ransohoff J E , Kendall D A , Kaiser E T
Published: May 2006

The guanidinium group of arginine-166 has been postulated to act as an electrophilic species during phosphorylation of alkaline phosphatase. Its role could be either to stabilize the developing negative charge on the oxygen of the leaving group or the pentacoordinate transition state or to help bind the -PO2-3 group. We have produced via site-directed mutagenesis two Escherichia coli alkaline phosphatase mutants (lysine-166 and glutamine-166) to test whether the guanidinium group plays a critical role in catalysis. Comparative kinetic characterization of the lysine-166 and glutamine-166 mutants indicates that the charge at residue 166 is not required for the hydrolysis of phosphate monoesters. Small decreases in kcat are observed for both the lysine and glutamine mutants, relative to the wild-type enzyme, but the value for the uncharged glutamine mutant is only one-third that of lysine. Thus, the stabilizing effect of the positively charged guanidinium group does not appear to play a major role in the rate-limiting step for substrate hydrolysis. A significant effect on the Km value is seen only for the glutamine mutant.
Regulation of the surface pressure of lipid monolayers and bilayers by the activity of water: derivation and application of an equation of state.
Authors: Wolfe D H , Brockman H L
Published: May 2006

A quasi-two-dimensional equation of state for liquid-type lipid monolayers has been derived and successfully applied to surface pressure-area isotherms obtained with a variety of lipids. For lipids with acyl moieties of similar length, the surface pressure and area at monolayer collapse can be accurately predicted from data obtained at lower surface pressure. Consideration of the rationalized activity coefficient as a linear scaler in an expression for surface pressure as a function of depth in the surface phase permits comparison of surface pressure-area data for monolayers with force-distance data for bilayers. This analysis shows the thermodynamic equivalence of monolayers at collapse and fully hydrated bilayers. It also supports the interpretation of the activity coefficient as a scaler and allows its determination solely from bilayer-derived data. Overall, the results show the common assumption that partial specific volume of water equals its bulk value to be inappropriate for the analysis of surface structure.
A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants
Authors: Delauney Ashton J. , Tabaeizadeh Zohreh , Verma Desh Pal S.
Published: May 2006

Tobacco plants expressing constitutive chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) activity were obtained by transformation with a chimeric CAT gene driven by the cauliflower mosaic virus 19S promoter. Plants expressing different levels of CAT activity were retransformed with vectors containing CAT sequences transcriptionally fused in the antisense orientation between the coding region of the hygromycin-resistance gene and the 3′ end of the nopaline synthase gene. Several plants regenerated on high concentrations of hygromycin exhibited a loss of CAT activity, whereas plants retransformed with a vector conferring hygromycin resistance but lacking the antisense CAT sequence showed no reduction in CAT activity. RNA blot analysis revealed a strong correlation between the degree of CAT gene inactivation and the levels of stable antisense transcripts accumulated. The possibility that CAT gene inactivation was due to transferred DNA instability was discounted since a kanamycin-resistance gene contiguous with the CAT gene was expressed normally, and DNA blot analysis indicated no loss or rearrangements of the transferred DNA fragments. Thus, the imposed selection pressure enabled the selection of plants expressing high levels of stable bifunctional antisense transcripts that inhibited the activity of the targeted gene.
Control of pollen hydration in
Brassica
requires continued protein synthesis, and glycosylation in necessary for intraspecific incompatibility
Authors: Sarker R. H. , Elleman C. J. , Dickinson H. G.
Published: May 2006

Pollen hydration and self-incompatibility (SI) in
Brassica
have been studied by using a combination of
in vivo
video-microscopy and experiments with metabolic inhibitors. Experiments with cycloheximide confirm earlier observations that pollen hydration is regulated through protein synthesis. No protein or glycoprotein has positively been identified with this event; however, it is unlikely that the total pool of any particular glycoprotein is involved, but rather a newly synthesized or otherwise activated fraction. Micromanipulation of pollen on the stigmatic papillae suggests that access to this hydration regulation system is limited to members of the Brassicaceae: pollen grains of other species—even those possessing dry stigmas—fail to hydrate. It is proposed that an interaction between enzymes of the stigma surface and the superficial layer of the pollen grain coating creates continuity between the content of the papillar wall and the grain protoplast. Inhibition of protein synthesis also overcomes SI, and since the advent of regulated hydration and synthesis of the so-called
S
-gene glycoproteins coincide with the acquisition of the SI system, there is strong circumstantial evidence that the same molecular species is involved in both processes. Experiments with tunicamycin, which prevents glycosylation of glycoproteins, indicate that the glycosyl groups of the
S
-gene glycoprotein are required for the operation of the SI system but not for the regulation of hydration. Further experiments suggest that pollen is positively inhibited on incompatible papillae but that this inhibition is biostatic. Recovery from the effects of the SI system appears to involve the metabolism of an inhibitor by the pollen. SI in
Brassica
thus emerges as a sophisticated process under dynamic control in both the female and male partners. The evolutionary advantages of such a system are discussed.
Protective immunity and eosinophilia in IgE-deficient SJA/9 mice infected with Nippostrongylus brasiliensis and Trichinella spiralis.
Authors: Watanabe N , Katakura K , Kobayashi A , Okumura K , Ovary Z
Published: May 2006

To elucidate the roles of IgE antibody in host responses to helminth infection, selective IgE-deficient SJA/9 mice were infected with the nematode parasites Nippostrongylus brasiliensis, which elicits remarkable potentiated IgE production, and Trichinella spiralis, which induces strong anti-Trichinella IgE antibody production in normal mice. The kinetics of blood eosinophilia, worm burden after primary infection, and resistance to secondary infection in SJA/9 mice were the same in both infections as those in congenic SJL/J mice used as an IgE-producing control. These results indicate that the host responses examined here operate under IgE-independent mechanisms.
Differentiation of an interleukin 3-dependent precursor B-cell clone into immunoglobulin-producing cells in vitro.
Authors: Kinashi T , Inaba K , Tsubata T , Tashiro K , Palacios R , Honjo T
Published: May 2006

Precursors to B-cell lines with immunoglobulin genes in the germ-line context have been shown to be capable of generating mature B cells in vivo. We report here that an interleukin 3-dependent precursor B-cell line, LyD9, differentiated in vitro into mature B cells, producing IgM and IgG by coculture with bone marrow accessory (or stroma) cells or with dendritic cells and T cells. Up to 50% of IgM-positive cells, but no Thy-1-positive cells, appeared after the 7- to 10-day coculture. Induced LyD9 cells underwent heterogenous immunoglobulin gene rearrangement and synthesized mRNAs encoding immunoglobulin mu, gamma, and kappa chains. However, these cells did not show any rearrangement of genes encoding the alpha and beta chains of the T-cell receptor. The induction of differentiation by coculture with bone marrow stroma cells was blocked by anti-lymphocyte function-associated antigen 1 or anti-interleukin 4 antibody. These systems are useful for molecular biological studies on regulation of differentiation of bone marrow-derived cells into the B-cell lineage.
Beta 2-adrenergic mechanisms in experimental arthritis.
Authors: Levine J D , Coderre T J , Helms C , Basbaum A I
Published: May 2006

We have studied (i) the contribution of specific adrenergic receptors to the proinflammatory effects of the sympathetic nervous system in experimental arthritis and (ii) the phases of the disease during which the sympathetic nervous system influences joint injury. Severity of joint injury was measured radiographically 28 days after induction of adjuvant arthritis in control rats and in rats treated with a variety of sympatholytic agents at various times during the course of the disease. Rats treated with a nonspecific catecholamine depletor (reserpine) or a beta-adrenergic receptor antagonist (propranolol) had a delayed onset and significantly less severe joint injury than saline-treated controls when treatment began prior to injection of the adjuvant and continued to day 28 after the injection. When administered over the same treatment period, neither nonselective (phenoxybenzamine) nor selective [prazosin (alpha 1) and yohimbine (alpha 2)] alpha-adrenergic receptor antagonists affected the onset or severity of joint injury. Metoprolol, a beta 1 antagonist, was also without effect. In contrast, two beta 2 antagonists (butoxamine and ICI 118,551) significantly retarded disease onset and reduced the severity of joint injury. When reserpine or butoxamine treatment was initiated after the onset of clinically apparent arthritis, it was still possible to favorably influence the course of the disease. These data indicate an important contribution of the beta 2-adrenergic receptor to joint injury in experimental arthritis.
Sporadicity: Between periodic and chaotic dynamical behaviors
Authors: Gaspard P. , Wang X.-J.
Published: May 2006

We define the class of sporadic dynamical systems as the systems where the algorithmic complexity of Kolmogorov [Kolmogorov, A. N. (1983)
Russ. Math. Surv.
38, 29-40] and Chaitin [Chaitin, G. J. (1987)
Algorithmic Information Theory
(Cambridge Univ. Press, Cambridge, U.K.)] as well as the logarithm of separation of initially nearby trajectories grow as
n

v
0

(log
n
)

v
1

with 0 <
v
0
< 1 or
v
0
= 1 and
v
1
< 0 as time
n
→ ∞. These systems present a behavior intermediate between the multiperiodic (
v
0
= 0,
v
1
= 1) and the chaotic ones (
v
0
= 1,
v
1
= 0). We show that intermittent systems of Manneville [Manneville, P. (1980)
J. Phys. (Paris)
41, 1235-1243] as well as some countable Markov chains may be sporadic and, furthermore, that the dynamical fluctuations of these systems may be of Lévy's type rather than Gaussian.
One-dimensional diffusion of Escherichia coli DNA-dependent RNA polymerase: a mechanism to facilitate promoter location.
Authors: Ricchetti M , Metzger W , Heumann H
Published: May 2006

The mechanism of promoter location by DNA-dependent RNA polymerase of Escherichia coli was investigated. The occupancies of DNA fragments carrying the A1 promoter of bacteriophage T7 were analyzed as a function of the length of flanking sequences adjacent to the promoter. Competition between the promoters on different fragments showed qualitatively that DNA sequences downstream of the promoter enhanced promoter occupancy, whereas upstream flanking sequences had little or no influence on occupancy. This was studied quantitatively by using a set of DNA fragments with four identical A1 promoters (I-IV) equidistant from each other, but with different lengths of flanking sequences upstream from promoter I and downstream from promoter IV. The relative occupancies of these promoters showed that downstream DNA sequences of up to 250 base pairs increased the occupancy of the adjacent promoter, whereas upstream sequences longer than 70 base pairs had little or no effect on occupancy. Promoter occupancies measured as a function of the length of the downstream flanking DNA sequences were fit by a published theory that takes into account an enhancement of signal-sequence location by linear diffusion.
Transcriptional activation of HLA-DR alpha by interferon gamma requires a trans-acting protein.
Authors: Blanar M A , Boettger E C , Flavell R A
Published: May 2006

Stimulation of the human epithelial-like cell line, HeLa, with interferon gamma (IFN-gamma) induces steady-state levels of HLA-DR alpha mRNA. Using a sensitive RNase-mapping procedure, we detect induced HLA-DR alpha mRNA as early as 8 hr after IFN-gamma treatment; maximal accumulation occurs by 48 hr. Treatment with the protein synthesis inhibitor, cycloheximide, abolishes the IFN-gamma-induced accumulation of HLA-DR alpha mRNA, indicating that de novo synthesis of a trans-acting protein factor is required for induction of this major histocompatibility complex class II gene. Nuclear run-off transcription assays revealed that IFN-gamma acts by directly stimulating the transcription rate of HLA-DR alpha. Similarly, IFN-gamma increased the transcription rate of the class I HLA-A2-encoding gene as well as that of the human invariant chain gene. IFN-gamma-induced transcription of HLA-DR alpha and of the invariant chain gene was blocked by treatment with cycloheximide, but IFN-gamma-induced transcription of HLA-A2 was unaffected. Our findings show that transcriptional induction of HLA-DR alpha and the invariant chain gene by IFN-gamma requires the action of an unidentified trans-acting protein.
Standard energy metabolism of a desert harvester ant,
Pogonomyrmex rugosus
: Effects of temperature, body mass, group size, and humidity
Authors: Lighton J. R. B. , Bartholomew George A.
Published: May 2006

Pogonomyrmex rugosus
(Hymenoptera: Formicidae) is an important seed predator in the Mojave Desert of the southwestern United States. Its standard rate of O
2
consumption (
˙Vo
2
) varied significantly with temperature (
˙Vo
2
= 10

(-1.588 + 0.0315
T
)

, where
˙Vo
2
is ml·g
-1
·hr
-1
and
T
is body temperature in °C). The ratio of the
˙Vo
2
values at 10°C increments in body temperature,
Q
10
, also varied with temperature; methods of calculating
˙Vo
2
from temperature with a shifting
Q
10
are described.
˙Vo
2
also varied with body mass (
˙Vo
2
= 0.0462
M
0.669
, where
˙Vo
2
is ml·hr
-1
and
M
is body mass in g).
˙Vo
2
was inversely related to relative humidity and was independent of group size. The rise in
˙Vo
2
at low relative humidities was caused by increased activity and resulted in higher rates of net water loss. The primary metabolic adaptation to xeric conditions in
P. rugosus
appears to be a lower-than-predicted metabolic rate.
Identification and sequencing of cDNA clones encoding the granule-associated serine proteases granzymes D, E, and F of cytolytic T lymphocytes.
Authors: Jenne D , Rey C , Haefliger J A , Qiao B Y , Groscurth P , Tschopp J
Published: May 2006

Cytoplasmic granules of cytolytic T lymphocytes contain at least six related serine esterases (granzymes) that are released together with perforin, a pore-forming protein related to complement component C9, during target-cell lysis. Polyclonal antibodies were used to isolate a large number of cDNA clones from an expression library derived from cytolytic-T-cell mRNA. Three distinct full-length cDNA clones coding for granzymes D, E, and F were identified by restriction site mapping and nucleotide sequencing. The three deduced amino acid sequences are highly similar to one another (between 72% and 90% amino acid identities) and to the sequences of granzymes B and C, cathepsin G, and rat mast-cell proteases I and II (between 43% and 57% amino acid identities). Cysteine residues capable of forming intramolecular disulfide bonds are conserved, as are the catalytic-site residues characteristic of serine proteases. Comparison of the cDNA-derived protein sequences with the amino termini of the isolated granzymes provides evidence that they are stored in a fully processed, activated form after removal of the signal peptide and two additional residues (propeptide) at the amino terminus. Immunoelectron microscopic studies demonstrated that granzymes D, E, and F are present in the same morphologically distinct cytoplasmic granules in which perforin has been found previously.
Circulating complexes containing IgA and fibronectin in patients with primary IgA nephropathy.
Authors: Cederholm B , Wieslander J , Bygren P , Heinegård D
Published: May 2006

IgA antibodies from patients with primary IgA nephropathy bind to collagens I, II, and IV. Here we show that this binding is mediated by the collagen-binding site of fibronectin, which occurs in the circulation in complex with IgA. No antibodies binding directly to collagen were identified. The complexes were isolated by affinity chromatography on gelatin-Sepharose and heparin-Sepharose, both with affinity for fibronectin, followed by adsorption to anti-human IgA immobilized on agarose gel. The presence of fibronectin and IgA antibodies in the isolated complexes is shown by enzyme-linked immunosorbent assay, gel electrophoresis, and electrophoretic transfer followed by immunostaining. The presence of an IgA-fibronectin complex in serum and the binding of this complex to collagen demonstrate the necessity of removing fibronectin from serum prior to identifying anti-collagen antibodies.
Purification and subunit structure of a putative K+-channel protein identified by its binding properties for dendrotoxin I.
Authors: Rehm H , Lazdunski M
Published: May 2006

The binding protein for the K+-channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3800- to 4600-fold enrichment and a recovery of 8-16%. The high affinity (Kd, 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO4/polyacrylamide gel revealed three bands of Mr 76,000-80,000, 38,000, and 35,000. Interestingly, the binding site for 125I-labeled mast cell degranulating peptide, another putative K+-channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol.
Electrostatic complementarity within the substrate-binding pocket of trypsin.
Authors: Gráf L , Jancsó A , Szilágyi L , Hegyi G , Pintér K , Náray-Szabó G , Hepp J , Medzihradszky K , Rutter W J
Published: May 2006

The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methyl-coumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII greater than EPP greater than EPA greater than EIP approximately equal to EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.
Villin sequence and peptide map identify six homologous domains.
Authors: Bazari W L , Matsudaira P , Wallek M , Smeal T , Jakes R , Ahmed Y
Published: May 2006

Site-specific proteases and antisera to the amino terminus of villin have been used to show that villin is organized into seven protease-resistant domains. Six are contained in the amino-terminal Mr 87,000 villin core, a Ca2+-regulated actin-severing fragment, whereas the carboxyl-terminal domain includes the villin "headpiece," a fragment involved in bundling of actin filaments. Ca2+ inhibits proteolytic cleavage between domains in the amino-terminal half of villin. The protein sequence of villin deduced from a single cDNA clone contains a conserved sequence that is repeated six times and is found in each domain of the villin core. The conserved repeats are found in other actin-severing proteins but not in the villin headpiece. Our results suggest that actin-severing proteins are organized around a common Mr 14,000-17,000 domain.
c-src gene product in developing rat brain is enriched in nerve growth cone membranes.
Authors: Maness P F , Aubry M , Shores C G , Frame L , Pfenninger K H
Published: May 2006

Differentiating rat neurons express high levels of the protooncogene product pp60c-src, a 60-kDa tyrosine kinase of unknown function encoded by c-src. pp60c-src was found to be concentrated at least 9-fold in membranes from a subcellular fraction of nerve growth cones, the motile tips of outgrowing neuronal processes. Indirect immunofluorescence staining of cultured chick retinal explants showed pp60c-src in neuronal growth cones and processes, with the antigen particularly concentrated in growth cones of long neurites. pp60c-src in growth cone membranes was an active tyrosine-specific protein kinase with elevated tyrosine-specific protein kinase activity and reduced electrophoretic mobility characteristic of the form of pp60c-src in central nervous system neurons. pp60c-src was present at lower levels in subcellular fractions from mature rat brain but synaptosomal membranes were not enriched. Preferential localization of an active form of pp60c-src in nerve growth cone membranes and persistence of pp60c-src in mature neurons suggest that this tyrosine kinase is important in growth cone-mediated neurite extension and synaptic plasticity.