bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet-derived growth factor synthesis in mesangial cells: induction by multiple peptide mitogens.
Authors: Silver B J , Jaffer F E , Abboud H E
Published: May 2006

Platelet-derived growth factor (PDGF) has been implicated in several nonmalignant pathophysiological processes, including proliferative diseases of the kidney. Glomerular mesangial cells secrete a PDGF-like factor and express the PDGF A-chain and c-sis (or B-chain) mRNAs. We report here that both mRNAs are induced by serum and this effect can be mimicked by recombinant PDGF, which also markedly stimulates DNA synthesis. Other growth factors, such as epidermal growth factor (EGF), transforming growth factor type alpha, basic fibroblast growth factor (bFGF), and tumor necrosis factor type alpha (TNF-alpha) also are mitogenic for human mesangial cells and induce expression of the PDGF mRNAs. EGF, TNF-alpha, and bFGF also stimulate these cells to secrete a PDGF-like factor. Furthermore, anti-PDGF antibody partially abrogates the mitogenic effect of EGF, suggesting that mitogen-stimulated PDGF synthesis in mesangial cells is at least partly responsible for cell growth induced by other growth factors. In contrast to these results, transforming growth factor type beta (TGF-beta), while inducing both mRNAs, is not mitogenic, indicating that its effect on message levels can be dissociated from DNA synthesis. These data suggest that several peptide growth factors regulate the growth of mesangial cells and that PDGF may be an effector molecule that plays a role in the mitogenic response to many of these growth stimuli.
Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides.
Authors: Kräusslich H G , Ingraham R H , Skoog M T , Wimmer E , Pallai P V , Carter C A
Published: May 2006

Retroviral capsid proteins and replication enzymes are synthesized as polyproteins that are proteolytically processed to the mature products by a virus-encoded proteinase. We have purified the proteinase of human immunodeficiency virus (HIV), expressed in Escherichia coli, to approximately 90% purity. The purified enzyme at a concentration of approximately 20 nM gave rapid, efficient, and specific cleavage of an in vitro synthesized gag precursor protein. Purified HIV proteinase also induced specific cleavage of five decapeptide substrates whose amino acid sequences corresponded to cleavage sites in the HIV polyprotein but not of a peptide corresponding to a cleavage site in another retrovirus. Competition experiments with different peptides allowed a ranking of cleavage sites. Inhibition studies indicated that the HIV proteinase was inhibited by pepstatin A with an IC50 of 0.7 microM.
Nucleotide substitution at major histocompatibility complex class II loci: evidence for overdominant selection.
Authors: Hughes A L , Nei M
Published: May 2006

To study the mechanism of maintenance of polymorphism at major histocompatibility complex (MHC) loci, synonymous and nonsynonymous (amino acid-altering) nucleotide substitutions in the putative antigen-recognition site (included in the first domain of the MHC molecule) and other regions of human and mouse class II genes were examined. In the putative antigen-recognition site, the rate of nonsynonymous substitution was found to exceed that of synonymous substitution, whereas in the second domain the former was significantly lower than the latter. In light of a previous theoretical study and parallel findings in class I MHC loci, we conclude that the unusually high degree of polymorphism at class II MHC loci is caused mainly by overdominant selection (heterozygote advantage) operating in the antigen-recognition site.
Regulation of the low density lipoprotein receptor and hydroxymethylglutaryl coenzyme A reductase genes by protein kinase C and a putative negative regulatory protein.
Authors: Auwerx J H , Chait A , Deeb S S
Published: May 2006

Transcription of the low density lipoprotein receptor (LDL-R) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase genes was rapidly and transiently induced (8.5- and 2.3-fold, respectively) early during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of the human monocytic leukemia cell line THP-1. The levels of mRNA coding for LDL-R and HMG-CoA reductase increased soon after induction, reached a maximum (12- and 7-fold increase, respectively) in 2-3 hr, and then rapidly returned to the low constitutive levels observed before induction. The stability of LDL-R mRNA did not change significantly during differentiation, whereas that of HMG-CoA reductase mRNA decreased by about 5-fold 6 hr after the addition of PMA. Transcriptional induction of both LDL-R and HMG-CoA reductase genes (5.6- and 2-fold, respectively) was also observed when undifferentiated cells were treated with cycloheximide (CHX), resulting in a transient increase in steady-state mRNA (7- and 3-fold, respectively). These results suggest that expression of the two genes is maintained at low constitutive levels in uninduced THP-1 cells by a protein with a short half-life. Superinduction of both genes occurred when PMA and CHX were added simultaneously. The induction of LDL-R and HMG-CoA reductase mRNAs during early macrophage differentiation is mediated by protein kinase C. It is hypothesized that protein kinase C acts directly or indirectly to inactivate the labile negative regulatory protein. Induction of LDL-R mRNA was also observed when the human hepatocarcinoma cell line Hep G2 was treated with PMA and CHX, suggesting that this mechanism of regulation may exist in several cell types.
Expression of haptoglobin-related protein and its potential role as a tumor antigen.
Authors: Kuhajda F P , Katumuluwa A I , Pasternack G R
Published: May 2006

These studies describe the detection of a haptoglobin species, its characterization as the HPR gene product, and its association with both pregnancy and neoplasia. Previous work showed that the early recurrence of human breast cancer correlated with immunohistochemical staining with a commercial antiserum ostensibly directed against pregnancy-associated plasma protein A (PAPP-A). Use of this antiserum to guide purification of the putative antigen led to the present identification and purification of a strongly immunoreactive protein species distinct from PAPP-A that was present in the plasma of pregnant women at term. Unlike PAPP-A, a homotetramer of 200-kDa polypeptides, the immunoreactive protein consists of a light (alpha) chain (16.5 kDa) and a heavy (beta) chain (40 kDa); protein microsequencing of the beta chain showed it to be a member of the haptoglobin family. The alpha chain of this haptoglobin species differs from ordinary haptoglobin 1 and 2 alpha chains both structurally and immunologically and represents the product of the HPR gene, haptoglobin-related protein (Hpr), since (i) the apparent molecular mass is the same as that predicted for Hpr alpha chain, (ii) the peptide map differs from that of haptoglobin 1 in a manner predicted by the HPR nucleotide sequence, (iii) monospecific antibodies that react with epitopes shared by the unique alpha chain and a synthetic peptide derived from the HPR nucleotide sequence do not detect these epitopes in either haptoglobin 1 or 2, and (iv) sequences of alpha-chain peptides were consistent with this identification, excluding haptoglobin 1 but not haptoglobin 2. The immunohistochemical reactivity of antibodies raised to the synthetic Hpr peptide is similar to that of anti-PAPP-A. Moreover, staining of neoplastic breast tissue is abolished by preincubation with purified Hpr.
Estrogen-responsive element of the human pS2 gene is an imperfectly palindromic sequence.
Authors: Berry M , Nunez A M , Chambon P
Published: May 2006

Using chimeric recombinants transfected into HeLa cells and a transient expression assay, we demonstrate that the 5'-flanking region of the pS2 gene from position -428 to position -324 exhibits both constitutive and estrogen-inducible enhancer activity. The estrogen-inducible activity, but not the constitutive activity, was inhibited by antiestrogens. ICI 164,384 behaved as a pure antagonist, whereas hydroxytamoxifen was a partial agonist-antagonist. The estrogen-responsive element of the pS2 gene has been narrowed down by site-directed deletion mutagenesis to a 13-base-pair (position -405 to position -393) imperfectly palindromic sequence, which in isolation can confer estrogen inducibility to the heterologous rabbit beta-globin gene promoter. On the other hand, the sequences responsible for the constitutive enhancer activity are spread over the entire region.
Transduction heats in retinal rods: tests of the role of cGMP by pyroelectric calorimetry.
Authors: Hagins W A , Ross P D , Tate R L , Yoshikami S
Published: May 2006

The sensory dark current of vertebrate retinal rods is believed to be controlled by light activation of a chain of coupled biochemical cycles that finally regulate the cationic conductance of the plasma membrane by hydrolytically reducing the level of cGMP in rod outer segment cytoplasm. The scheme has been tested by measuring heat production by live frog retinas when stimulated with sequences of light flashes of progressively increasing energy. Using pyroelectric poly(vinylidene 1,1-difluoride) detectors that simultaneously measure transretinal voltage and retinal temperature change, four heat effects assignable to known biochemical cycles in rods have been found. As the dark current shuts down after a flash causing 180-1800 rhodopsin photoisomerizations per rod, a heat burst, q1, raises the retinal temperature 1-2 microK. q1 is closely regulated in size and slightly precedes dark current shutdown. Isobutylmethylxanthine slows and enlarges q1, delaying the dark-current response. Increasing cytoplasmic Ca2+ stops the dark current without affecting q1. Although rod heat production is consistent with splitting of 1-3 microM of free cytoplasmic cGMP during transduction, the kinetics of the two processes do not match the predictions of current cGMP control models.
Chromosomes of older humans are more prone to aminopterine-induced breakage.
Authors: Esposito D , Fassina G , Szabo P , De Angelis P , Rodgers L , Weksler M , Siniscalco M
Published: May 2006

We have adopted a simplified version of the "cell hybrid cotransfer method" to test the hypothesis that human lymphocytes derived from elderly individuals have a higher chromosome instability. Peripheral blood lymphocytes from "old" male individuals and "young" controls were fused with a Chinese hamster cell line (CHO-YH21), yielding 10 HAT-resistant rodent-human clones from the old propositi and 22 from the young controls (HAT = hypoxanthine/aminopterin/thymidine). Both series of hybrid clones were analyzed with respect to the retention of the enzyme glucose-6-phosphate dehydrogenase and the surface antigen MIC2 identified by monoclonal antibody 12E7, two human X chromosome-linked markers located at opposite ends of the X chromosome. Cell hybrid clones with an X chromosome from a young control retained both markers in about 70% of the cells. In contrast, cell hybrid clones with an X chromosome from an old donor retained the MIC2 marker in only 30% of their cells. Slot-blot hybridization studies have established that the observed loss of the MIC2 marker is due to loss of the coding gene, not to suppression of its expression. Similar hybridization studies with molecular probes specific for other regions of the X chromosome suggest preferential chromosomal breakage sites. T lymphocytes from old donors were also found to have an LD50 for aminopterine significantly lower than the concentration of this drug in the HAT medium used to grow the hybrids, suggesting that the higher level of gene loss observed in the X chromosomes from old donors may be directly related to their increased sensitivity to the clastogenic effect of aminopterine. We speculate that the higher rate of chromosomal breakage and of marker loss observed along the "old-age" X chromosomes could be the result of "molecular scars" accumulated with aging at sites of constitutive chromosomal fragility.
Mature human eosinophils have the capacity to express HLA-DR.
Authors: Lucey D R , Nicholson-Weller A , Weller P F
Published: May 2006

Human eosinophils are known to lose Ia antigen expression as they mature, and, accordingly, eosinophils obtained from the blood of five eosinophilic donors and three of four normal donors failed to display the major histocompatibility complex class II antigen HLA-DR, as determined by flow cytometry. However, when eosinophils from these nine donors were maintained in culture with recombinant human granulocyte-macrophage colony-stimulating factor and murine 3T3 fibroblasts, HLA-DR consistently developed on the eosinophils. By days 4-6 of culture, 24-97% of eosinophils were HLA-DR+, and the eosinophils remained morphologically mature. In contrast, another class II antigen, HLA-DQ, was not detectable by flow cytometry on eosinophils from eight of nine donors. Cultured eosinophils were able to synthesize HLA-DR, as documented by the incorporation of [35S]methionine into immunoprecipitable HLA-DR heavy and light chains. These findings show that mature eosinophils can synthesize and express HLA-DR and provide a means whereby eosinophils may interact with CD4+ lymphocytes.
Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens.
Authors: Keydar I , Chou C S , Hareuveni M , Tsarfaty I , Sahar E , Selzer G , Chaitchik S , Hizi A
Published: May 2006

We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.
Purification of a putative brain somatostatin receptor.
Authors: He H T , Johnson K , Thermos K , Reisine T
Published: May 2006

The brain somatostatin (somatotropin release-inhibiting factor; SRIF) receptor was purified by affinity chromatographic techniques. A protein of 60 kDa could be purified from rat brain. The protein was eluted from a [D-Trp8]SRIF affinity column with either sodium acetate (pH 5.5) or free [D-Trp8]SRIF. The binding of the protein to the affinity column was prevented by free [D-Trp8]SRIF or the stable SRIF analogue SMS 201-996 but not by the inactive somatostatin 28-(1-14). The purified receptor could be covalently labeled by the 125I-labeled SRIF analogue CGP 23996. Excess [D-Trp8]SRIF blocked the binding of 125I-labeled CGP 23996 to the purified receptor, but somatostatin 28-(1-14) did not affect the binding. A 60-kDa protein was also purified from the anterior pituitary cell line AtT-20, which has a high expression of SRIF receptors. In contrast, no 60-kDa protein could be purified from CHO cells, which have no detectable SRIF receptors. These findings present evidence for the purification of the SRIF receptor.
Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225----serine) by its substrate CDP.
Authors: Mao S S , Johnston M I , Bollinger J M , Stubbe J
Published: May 2006

The B1 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1) has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson [Tabor, S. & Richardson, C. (1985) Proc. Natl. Acad. Sci. USA 82, 1074-1078]. This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, Cys-225 and Cys-759, have been changed to serines. Incubation of the [Ser225]B1 mutant with the B2 subunit, [U-14C]CDP, and the allosteric effector ATP results in production of cytosine, destruction of the tyrosyl radical in B2, radiolabeling of the protein, and cleavage of the B1 subunit into two pieces of 26 and 61.5 kDa. This process is independent of the identity of reductant. The [Ser759]B1 mutant reduces CDP in the presence of thioredoxin/thioredoxin reductase at 7.7% the rate of wild-type B1. When dithiothreitol is utilized as reductant, however, the rate of CDP reduction with [Ser759]B1 is identical to that observed with wild type.
rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA.
Authors: Felber B K , Hadzopoulou-Cladaras M , Cladaras C , Copeland T , Pavlakis G N
Published: May 2006

rev (trs/art) is an essential human immunodeficiency virus type 1 (HIV-1) regulatory protein. rev increases the levels of the gag- and env-producing mRNAs via a cis-acting element in the env region of HIV-1, named rev-responsive element. Our results show that rev increases the stability of the unspliced viral mRNA, while it does not affect the stability of the multiply spliced viral mRNAs that do not contain the rev-responsive element. The study of mutated proviral constructs producing mRNA that cannot be spliced revealed that the effect of rev on stability is independent of splicing. Our experiments also indicate that rev promotes the transport of the viral mRNA containing the rev-responsive element from the nucleus to the cytoplasm. The proposed functions of rev are consistent with its nuclear localization as shown by immunofluorescence. The selective effects of rev on the levels of the viral mRNA suggest a model for feedback regulation by rev leading to a steady state of viral expression.
Destabilization of an alpha-helix-bundle protein by helix dipoles.
Authors: Gilson M K , Honig B
Published: May 2006

The finite difference Poisson-Boltzmann method is used to calculate the electrostatic work of assembling the four alpha-helices of Themiste dyscritum hemerythrin to form the protein's observed antiparallel helical bundle. The calculations account for the interaction of each helix dipole with the high-dielectric solvent as well as for pairwise interactions of the dipoles with each other. We find that the electrostatic work of assembly is dominated by unfavorable changes in dipole-solvent interactions rather than by favorable interactions between antiparallel helices. Furthermore, the electrostatic energy difference between the observed arrangement of helices in hemerythrin and at least one other possible helical arrangement is less than 1 kT. These results suggest that the helix dipole actually destabilizes the helical bundle and that it plays little or no role in producing the observed bundle geometry.
A 4-kDa maize chloroplast polypeptide associated with the cytochrome b6-f complex: subunit 5, encoded by the chloroplast petE gene.
Authors: Haley J , Bogorad L
Published: May 2006

Four polypeptides, three of which are chloroplast-encoded, have been shown to be associated with the thylakoid membrane cytochrome b6-f complex. In this report, the gene for a fifth polypeptide, which copurifies with the b6-f complex, is identified through the use of an antibody generated against a synthetic decapeptide predicted from a maize chloroplast DNA sequence. The deduced 37-amino acid sequence of the immunoreactive 4-kDa polypeptide is 100% and 86% conserved in the respective similar open reading frames encoded by Nicotiana tabacum and Marchantia chloroplast DNA. The 4-kDa polypeptide is present in both etioplasts and chloroplasts of maize and is found as well in spinach, tobacco, pea, wheat, and rice thylakoids. Similar to the other subunits of the b6-f complex, it is intrinsic to the membrane, and its hydrophilic COOH terminus is located at the stromal thylakoid surface. We propose to call the 4-kDa polypeptide "subunit 5" and the chloroplast gene that encodes it the petE gene.
Positive and negative regulatory elements in the mouse albumin enhancer.
Authors: Herbst R S , Friedman N , Darnell J E , Babiss L E
Published: May 2006

Most enhancer elements so far described are comprised of multiple DNA binding sites for proteins that act to increase the rate of transcription. In this report we show that the far upstream mouse albumin enhancer element (-10.5 to -8.5 kilobase pairs) is a composite of at least three functional regions: a negative region that suppresses an otherwise positive, liver-specific enhancer element and a third region that by itself is inactive but in conjunction with the remaining elements overrides the effect of the negative region. The protein that binds to the overriding element is C/EBP, a liver-enriched transcription factor, whereas the newly identified protein that is responsible for full liver-specific enhancer activity is found in HeLa cell and spleen extracts as well as in extracts prepared from the liver. This latter protein may therefore be an example of a widely distributed protein that because of cell-specific modification or interaction has a tissue-specific, positive action on transcription.
Angiogenin stimulates endothelial cell prostacyclin secretion by activation of phospholipase A2.
Authors: Bicknell R , Vallee B L
Published: May 2006

Angiogenin stimulates capillary and umbilical vein endothelial cell prostacyclin secretion but not that of prostaglandins of the E series. The response was quantitated by radioimmunoassay and by [3H]arachidonate labeling followed by analysis of the secreted prostaglandins. The stimulated secretion lasts for several minutes and is optimal at 2-4 min. The dose-response (peak at 1-10 ng/ml) is similar to that previously observed for activation of endothelial cell phospholipase C. Stimulated secretion was blocked by pretreatment with the inhibitors of prostacyclin synthesis, indomethacin and tranylcypromine, and also the specific inhibitor of phospholipase A2, quinacrine, as well as pertussis toxin and the diglyceryl and monoglyceryl lipase inhibitor RHC 80267. Stimulated secretion was also abolished in cells that were either pretreated for 48 hr with phorbol ester to down-regulate protein kinase C or incubated with the protein kinase inhibitor H7. Hydrolysis of phosphatidylinositol by phospholipase A2 appears to be the source of angiogenin-mobilized arachidonate; angiogenin-induced hydrolysis of phosphatidylcholine was not detected. Activation of phospholipase A2 occurs in the absence of an angiogenin-induced calcium flux. The results are discussed in terms of mechanisms of agonist-induced intracellular arachidonate mobilization and relevance to angiogenesis.
An alternatively spliced region of the human hexabrachion contains a repeat of potential N-glycosylation sites.
Authors: Gulcher J R , Nies D E , Marton L S , Stefansson K
Published: May 2006

We have cloned and sequenced two cDNA molecules that code for parts of two forms of human hexabrachion. The smaller clone has a sequence that corresponds to the previously published sequence of a cDNA clone coding for a part of chicken hexabrachion [Jones, F. S., Burgoon, M. P., Hoffman, S., Crossin, K. L., Cunningham, B. A. & Edelman, G. M. (1988) Proc. Natl. Acad. Sci. USA 85, 2186-2190]. It has eight consecutive domains similar to the type III homology units from fibronectin, several epidermal growth factor repeats, and a domain similar to the beta and gamma chains of fibrinogen. The larger clone has 5' and 3' ends that are identical to the smaller clone but also has an alternatively spliced 1.9-kilobase internal segment. The unique segment contains remarkable repeats of potential glycosylation sites and an additional seven type III homology units.
tus, the trans-acting gene required for termination of DNA replication in Escherichia coli, encodes a DNA-binding protein.
Authors: Hill T M , Tecklenburg M L , Pelletier A J , Kuempel P L
Published: May 2006

The components for termination of DNA replication in Escherichia coli include the terminator signals T1 and T2 and the trans-acting gene tus. We have shown previously that tus maps in a 4-kilobase region of the chromosomal terminus near T2. Through the use of deletion and insertion mutants, the location of the tus gene has now been precisely identified. We sequenced 2416 nucleotides in this region and identified a 927-base-pair open reading frame which encodes Tus. Insertion of a kanamycin-resistance gene in this open reading frame abolished tus activity. We also demonstrated that crude extracts of tus+ cells contain a protein which binds to the T2 terminator sequence.
Cell cultures of neuroblasts from rat olfactory epithelium that show odorant responses.
Authors: Coon H G , Curcio F , Sakaguchi K , Brandi M L , Swerdlow R D
Published: May 2006

We have developed procedures that permit isolation and propagation of clonal cell cultures from the olfactory epithelium of the 5- to 7-day-old rat that appear to represent the neuroblasts that repopulate the sensory neurons in the olfactory epithelium throughout life. The cell lines we report here synthesize neuron-specific enolase, which is a neuron marker, 43-kDa growth-associated protein, a protein associated with neuronal growth cones, and carnosine, a possible olfactory neurotransmitter. In two of the cell lines we have found dose-dependent cAMP accumulation following exposure to submicromolar concentrations of chemical odorants in the medium. These two cell lines show different patterns of odorant specificity when tested against a panel of six chemicals commonly used as test odorants. We anticipate that these and similarly derived cell lines will prove valuable in studying aspects of neurogenesis and olfaction.
The glutamate analog 2-amino-4-phosphonobutyrate antagonizes synaptic transmission from cones to horizontal cells in the goldfish retina.
Authors: Nawy S , Sie A , Copenhagen D R
Published: May 2006

In the retina, the glutamate analog 2-amino-4-phosphonobutyrate (APB) distinguishes a class of glutamate receptors that is thought to be found only on depolarizing bipolar cells (DBCs). We now report that APB is a potent antagonist of cone-driven horizontal cells in the goldfish retina. APB hyperpolarized the membrane to the same potential as cobalt Ringer's and blocked the light responses. APB acted specifically on the cone pathway, as it had no effect on rod-driven horizontal cells. The lowest effective APB concentration for antagonistic action on the horizontal cells (approximately 2 microM) was similar to the concentration for agonist action on DBCs. APB was not able to block the actions of exogenous glutamate or kainate on horizontal cells. We propose that the action of APB on the cone-horizontal cell synapse is mediated at a site that is distinct from the glutamate and kainate binding site. Therefore, APB is most probably acting at a different locus on the synaptic glutamatergic receptors of the horizontal cells or at presynaptic receptors located on the cones themselves.
On the mechanism of action of ribonucleases: dinucleotide cleavage catalyzed by imidazole and Zn2+.
Authors: Breslow R , Huang D L , Anslyn E
Published: May 2006

Cyclization/cleavage of the 2-(p-nitrophenyl) phosphate ester of propylene glycol is catalyzed by imidazole and, much more effectively, by Zn2+ with imidazole. In the latter case, the mechanism involves simultaneous Lewis acid/base catalysis. Similar Zn2+ and imidazole catalysis of cyclization/cleavage is seen with the dinucleotide 3',5'-UpU (uridylyluridine). Again, the zinc system is much more effective than is catalysis by imidazole alone, and in this case simultaneous Lewis acid/base catalysis substitutes for the sequential proton acid/base catalysis seen with polynucleotides or dinucleotides and imidazole buffer catalysts. A mechanism is proposed for catalysis of RNA cleavage by the enzyme ribonuclease A, and the relationship of that mechanism to the action of the enzyme model systems is discussed.
Nerve growth factor stimulates the hydrolysis of glycosylphosphatidylinositol in PC-12 cells: a mechanism of protein kinase C regulation.
Authors: Chan B L , Chao M V , Saltiel A R
Published: May 2006

Treatment of PC-12 pheochromocytoma cells with nerve growth factor (NGF) results in the differentiation of these cells into a sympathetic neuron-like phenotype. Although the initial intracellular signals elicited by NGF remain unknown, some of the cellular effects of NGF are similar to those of other growth factors, such as insulin. We have investigated the involvement of a newly identified inositol-containing glycolipid in signal transduction for the actions of NGF. NGF stimulates the rapid generation of a species of diacylglycerol that is labeled with [3H]myristate but not with [3H]arachidonate. NGF stimulates [3H]myristate- or [32P]phosphate-labeled phosphatidic acid production over the same time course. Although NGF alone has no effect on the turnover of inositol phospholipids, it does stimulate the hydrolysis of glycosylphosphatidylinositol. The NGF-dependent cleavage of this lipid is accompanied by an increase in the accumulation of its polar head group, an inositol phosphate glycan, which is generated within 30-60 sec of NGF treatment. In an unresponsive PC-12 mutant cell line, neither the diacylglycerol nor inositol phosphate glycan response is detected. A possible role for the NGF-stimulated diacylglycerol is suggested by the inhibition of NGF-dependent c-fos induction by staurosporin, a potent inhibitor of protein kinase C. These results suggest that, like insulin, some of the cellular effects of NGF may be mediated by the phospholipase C-catalyzed hydrolysis of glycosylphosphatidylinositol.
Multiple post-transcriptional regulatory mechanisms in ferritin gene expression.
Authors: Mattia E , den Blaauwen J , Ashwell G , van Renswoude J
Published: May 2006

We have investigated the mechanisms involved in the regulation of ferritin biosynthesis in K562 human erythroleukemia cells during prolonged exposure to iron. We show that, upon addition of hemin (an efficient iron donor) to the cell culture, the rate of ferritin biosynthesis reaches a maximum after a few hours and then decreases. During a 24-hr incubation with the iron donor the concentrations of total ferritin heavy (H) and light (L) subunit mRNAs rise 2- to 5-fold and 2- to 3-fold, respectively, over the control values, while the amount of the protein increases 10- to 30-fold. The hemin-induced increment in ferritin subunit mRNA is not prevented by deferoxamine, suggesting that it is not directly mediated by chelatable iron. In vitro nuclear transcription analyses performed on nuclei isolated from control cells and cells grown in the presence of hemin indicate that the rates of synthesis of H- and L-subunit mRNAs remain constant. We conclude that iron-induced ferritin biosynthesis is governed by multiple post-transcriptional regulatory mechanisms. We propose that exposure of cells to iron leads to stabilization of ferritin mRNAs, in addition to activation and translation of stored H- and L-subunit mRNAs.
Amino acid substitutions in albumin variants found in Brazil.
Authors: Arai K , Huss K , Madison J , Putnam F W , Salzano F M , Franco M H , Santos S E , Freitas M J
Published: May 2006

Conventional horizontal starch-gel electrophoresis in four buffer systems and structural studies were performed on four albumin variants, and the findings were compared with similar previous data. Albumins Coari I and Porto Alegre I have a previously unreported amino acid substitution (glutamic acid replaced by lysine at position 358, denoted 358 Glu----Lys). The alteration in albumin Porto Alegre II (501 Glu----Lys) is the same as that found for three alloalbumins of Asiatic origin, designated Vancouver, Birmingham, and Adana. Albumin Oriximiná I has the same exchange as albumin Maku (541 Lys----Glu). Some of these findings can be explained only by the occurrence of independent mutations at the same site in the albumin gene. They also point to a third cluster of mutations in that gene, indicating hypermutability in some of its segments.
Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease.
Authors: Meek T D , Dayton B D , Metcalf B W , Dreyer G B , Strickler J E , Gorniak J G , Rosenberg M , Moore M L , Magaard V W , Debouck C
Published: May 2006

Recombinant human immunodeficiency virus 1 (HIV-1) protease, purified from a bacterial expression system, processed a recombinant form of its natural substrate, Pr55gag, into protein fragments that possess molecular weights commensurate with those of the virion gag proteins. Molecular weights of the protease obtained under denaturing and nondenaturing conditions (11,000 and 22,000, respectively) and chemical crosslinking studies were consistent with a dimeric structure for the active enzyme. The protease appropriately cleaved the nonapeptide Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 between the tyrosine and proline residues. HIV-1 protease was sensitive to inactivators of the aspartic proteases. The aspartic protease inactivator 1,2-epoxy-3-(4-nitrophenoxy)propane produced irreversible, time-dependent inactivation of the protease. The pH-dependent kinetics of this inactivator were consistent with the requirement of an unprotonated carboxyl group in the active site of the enzyme, suggesting that HIV-1 protease is also an aspartic protease.
Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.
Authors: Houtz R L , Stults J T , Mulligan R M , Tolbert N E
Published: May 2006

Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.
Altered binding of human histone gene transcription factors during the shutdown of proliferation and onset of differentiation in HL-60 cells.
Authors: Stein G , Lian J , Stein J , Briggs R , Shalhoub V , Wright K , Pauli U , van Wijnen A
Published: May 2006

Two sites of protein-DNA interaction have been identified in vivo and in vitro in the proximal promoter regions of an H4 and an H3 human histone gene. In proliferating cells, these genes are transcribed throughout the cell cycle, and both the more distal site I and the proximal site II are occupied by promoter-binding factors. In this report we demonstrate that during the shutdown of proliferation and onset of differentiation of the human promyelocytic leukemia cell line HL-60 into cells that exhibit phenotypic properties of monocytes, histone gene expression is down-regulated at the level of transcription. In vivo occupancy of site I by promoter factors persists in the differentiated HL-60 cells, but protein-DNA interactions at site II are selectively lost. Furthermore, in vitro binding activity of the site II promoter factor HiNF-D is lost in differentiated cells, and nuclear extracts from differentiated cells do not support in vitro transcription of these histone genes. Our results suggest that the interaction of HiNF-D with proximal promoter site II sequences plays a primary role in rendering cell growth-regulated histone genes transcribable in proliferating cells. It appears that while cell-cycle control of histone gene expression is mediated by both transcription and mRNA stability, with the shutdown of proliferation and onset mRNA stability, with the shutdown of proliferation and onset of differentiation, histone gene expression is regulated at the transcriptional level.
Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species.
Authors: Gould S J , Subramani S , Scheffler I E
Published: May 2006

The DNA polymerase chain reaction was developed for in vitro amplification of specific DNA sequences, and it has been used for a wide variety of purposes in several fields. We have developed an application of the polymerase chain reaction that is useful for the isolation of partial cDNA or genomic clones of conserved genes. We used this technique to clone the gene encoding the iron protein subunit (27 kDa) of succinate dehydrogenase (EC 1.3.5.1) from several species, including human, rat, Drosophila melanogaster, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Mixed oligonucleotide primers corresponding to two conserved regions of the protein were used in conjunction with genomic and cDNA templates in the reaction. The primers contained all possible nucleotide combinations that could encode the corresponding peptide sequences. These oligonucleotide mixtures contained 262,144 (2(18] and 8192 (2(13] unique sequences, respectively. Use of the polymerase chain reaction for homology probing allows one to utilize more complex mixtures of oligonucleotides as probes than is possible with filter hybridization screening techniques. In addition, the polymerase chain reaction offers the advantage of synthesizing the DNA product directly, in some cases obviating the need to construct cDNA or genomic libraries. This application of the polymerase chain reaction should be useful not only for the identification of conserved genes in a variety of species but also for the isolation of previously unknown members of gene families.
A region of a cyanobacterial genome required for sulfate transport.
Authors: Green L S , Laudenbach D E , Grossman A R
Published: May 2006

Using the cysA locus of Salmonella typhimurium as a heterologous probe, we have cloned a region of the Anacystis nidulans R2 (Synechococcus PCC 7942) genome involved in sulfate assimilation. The 8.3-kilobase-pair region encodes at least five transcripts that cannot be detected unless the cells are deprived of sulfur. One of the genes in this region has been sequenced, and the protein that it encodes is homologous to a polypeptide component of other permease systems of Escherichia coli and Salmonella. Insertional inactivation of the putative sulfate permease gene, designated cysA, as well as of other genes within this region, results in cysteine auxotrophy, reduced sulfate uptake, and altered expression of soluble and cytoplasmic-membrane polypeptides associated with sulfur starvation.
Characterization of an amyloid beta precursor protein that binds heparin and contains tyrosine sulfate.
Authors: Schubert D , LaCorbiere M , Saitoh T , Cole G
Published: May 2006

A secreted form of the amyloid beta protein precursor was isolated from the growth conditioned medium of the PC12 sympathetic nerve-like cell line. This protein is recognized by an antiserum that detects a protein of 140 kDa and a less abundant species of 115 kDa on NaDodSO4/acrylamide gels. The amyloid precursor proteins contain O-linked sugars and tyrosine sulfate and bind to the glycosaminoglycan heparin. These results suggest a role for extracellular sulfated glycoproteins in the pathogenesis of Alzheimer disease.
Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation.
Authors: Sun X H , Baltimore D
Published: May 2006

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was cotransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.
Binding of a soluble factor of Escherichia coli to preproteins does not require ATP and appears to be the first step in protein export.
Authors: Watanabe M , Blobel G
Published: May 2006

We have constructed a mutant form of the maltose binding protein precursor, termed preMBP*, that, unlike its wild-type counterpart preMBP, retains translocation competence after synthesis. In a homologous Escherichia coli translation/translocation system, preMBP* was translocated either co- or posttranslationally with virtually 100% efficiency into inverted vesicles (INV) derived from the E. coli plasma membrane. Translation of preMBP* mRNA in a wheat germ system and subsequent incubation with INV yielded no translocation. However, addition of increasing amounts of an E. coli postribosomal supernatant (PRS) to the wheat germ extract stimulated preMBP* translocation with virtually 100% efficiency occurring at 100 micrograms of PRS per 50 microliters of incubation mixture. The activity in the E. coli PRS appears to be identical to the previously described "export" factor. The soluble activity can bind to preMBP* posttranslationally and in the absence of ATP. Subsequent targeting to INV and/or translocation, however, requires ATP. Binding of the soluble activity to preMBP* thus appears to be the first step in a multistep translocation reaction.
Bilaterians of the Precambrian—Cambrian transition and the annelid—arthropod relationship
Authors: Valentine James W.
Published: May 2006

The Late Proterozoic fossil record contains the remains of animals that may represent a grade of organization not found among living metazoans. These forms were segmented and large enough to require a hemocoel, yet evidently were not capable of forming penetrating burrows, which are essentially absent from contemporaneous sediments containing locally common but chiefly horizontal trace fossils. As has been noted, there is no evidence that Late Proterozoic invertebrates possessed a coelom suited for peristaltic burrowing. Therefore, the annelidan body plan had probably not appeared. It is not implausible, however, that coelomic spaces in the form of ducts or organ sacs were present in Late Proterozoic segmented forms. Uniramians, some of which employ the hemocoel hydrostatically in lobopodal locomotion, may be allied to segmented hemocoelic forms not unlike sprigginids. Coelomic spaces may have been exploited in some protoarthropod lineages to enhance pedal-wave locomotion, but probably there are no eucoelomic forms in arthropodan ancestry. Annelids may represent an early divergent branch of seriated worms, perhaps rather nemertine-like at first, that developed eucoelomic compartments only in Cambrian time. The extinct grade is most likely to have arisen from flatworm-like ancestors. Of all the proposed phylogenies examined, only that of Manton closely anticipates these interpretations of the early metazoan fossil record.
Light-chain gene expression before heavy-chain gene rearrangement in pre-B cells transformed by Epstein-Barr virus.
Authors: Kubagawa H , Cooper M D , Carroll A J , Burrows P D
Published: May 2006

Epstein-Barr virus transformation of B-cell-depleted bone marrow cells from human fetuses allowed us to identify novel cell types characterized by the expression of immunoglobulin kappa or lambda light chain without heavy chains. Four kappa-only clones with normal karyotype were obtained and examined for their immunoglobulin gene configurations and expression. All four clones had kappa-chain gene rearrangements at either one or both alleles, but the heavy-chain gene loci in these clones either were in germ-line context or had undergone only D-JH rearrangements (D and JH represent diversity and joining gene segments). All clones contained kappa mRNA of normal size at levels consistent with the protein level, except for one clone that no longer produced kappa protein. No mu mRNA or immunoglobulin heavy-chain molecules were detected in any of the kappa+ clones. The results suggest that the mu heavy-chain protein is not an obligatory prerequisite for light-chain gene rearrangements.
Interleukin 1 as an autocrine growth factor for acute myeloid leukemia cells.
Authors: Cozzolino F , Rubartelli A , Aldinucci D , Sitia R , Torcia M , Shaw A , Di Guglielmo R
Published: May 2006

Production of interleukin 1 (IL-1) by leukemic cells was studied in 13 cases of acute myeloid leukemia. Intracytoplasmic immunofluorescence studies showed that the cells invariably contained the cytokine. Endogenous labeling studies demonstrated that acute myeloid leukemia cells produced either only the 33-kDa propeptide or both the propeptide and the 17-kDa mature form of IL-1 beta. The 33-kDa propeptide IL-1 alpha was always produced but was less frequently released. Involvement of IL-1 in leukemic cell growth was investigated using two antibodies specific for IL-1 subtypes, which inhibited spontaneous cell proliferation in the six cases studied. After acid treatment of the cells, a surface receptor for IL-1 could be demonstrated, which mediated 125I-labeled IL-1-specific uptake by leukemic cells. Furthermore, recombinant IL-1 alpha or IL-1 beta induced significant cell proliferation in 10 of 12 cases. The above findings were uncorrelated with the cytologic type (French-American-British classification) of leukemia. Our studies suggest that IL-1 may act as an autocrine growth factor in most cases of acute myeloid leukemia.