bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of a T-lymphotropic retrovirus from naturally infected sooty mangabey monkeys (Cercocebus atys).
Authors: Fultz P N , McClure H M , Anderson D C , Swenson R B , Anand R , Srinivasan A
Published: May 2006

Healthy mangabey monkeys in a colony at the Yerkes Regional Primate Research Center were found to be infected with a retrovirus related to human immunodeficiency virus (HIV). Virus was isolated from peripheral blood cells of 14 of 15 randomly selected mangabeys. All virus-positive animals had antibodies to the mangabey virus at the time of virus isolation and, in a retrospective study, 82% of mangabey serum samples obtained in 1981 had antibodies to the virus. The newly isolated retrovirus is (i) morphologically identical to HIV by electron microscopy; (ii) serologically related to the human virus by enzyme immunoassay, immunoblotting experiments, radioimmunoprecipitation, and neutralization; and (iii) cytopathic for human OKT4+ cells. The mangabey virus also shares these properties with the simian T-lymphotropic virus type III (STLV-III) recently isolated from diseased macaques and from healthy African green monkeys (STLV-IIIAGM). However, the mangabey virus, like STLV-IIIAGM, differs from both HIV and STLV-III in that it apparently does not cause clinical immunodeficiency or disease following natural infection of the host from which it was isolated. Comparison of the virus-host interactions of these isolates may be valuable in defining determinants of pathogenicity for cytopathic retroviruses.
ras p21 deletion mutants and monoclonal antibodies as tools for localization of regions relevant to p21 function.
Authors: Lacal J C , Aaronson S A
Published: May 2006

Deletion mutants of the viral Harvey ras oncogene were generated by removing different lengths of the gene from either the amino or the carboxyl terminus. The deletion mutants, ras p21 expressed in Escherichia coli, yielded proteins of approximately 8, 10, 12, 14, 17, 18, 19, and 20 kDa. These proteins were utilized to identify epitopes recognized by a series of recently generated monoclonal antibodies as well as some previously reported monoclonal antibodies. Monoclonal antibodies that inhibited GTP binding, a major biochemical activity of the p21 protein, recognized two major regions of the protein. These regions were localized from amino acids 5 to 69 and 107 to 164, respectively, and were separated by another stretch from residues 70 to 106, whose antigenic determinants were not directly involved in GTP binding. Thus, the mapping of epitopes within the p21 molecule recognized by monoclonal antibodies has made it possible to localize important functional regions within the ras p21 molecule.
Human leukemia cells synthesize and secrete proteins related to platelet-derived growth factor.
Authors: Pantazis P , Lanfrancone L , Pelicci P G , Dalla-Favera R , Antoniades H N
Published: May 2006

Human leukemia cells in culture (HL-60) synthesize and secrete proteins that are recognized by antiserum to human platelet-derived growth factor (PDGF). The molecular mass of the intracellular proteins immunoprecipitated by PDGF antiserum ranged from 34 kDa to 240 kDa. PDGF-related proteins were also identified in the conditioned medium of the cells. Several of these immunoprecipitated proteins were glycosylated. A single protein of 46 kDa was immunoprecipitated from the cell-free translation products of mRNA obtained from the leukemia cells. Antiserum to the C but not to the N terminus of the predicted amino acid sequence of the transforming protein p28sis/PDGF-2 also immunoprecipitated proteins secreted by the HL-60 cells. These findings provide a direct demonstration for the synthesis and secretion of PDGF-like proteins by leukemia cells in culture. These proteins do not appear to be coded by the known c-sis/PDGF-2 locus since no sis mRNA was detectable in the HL-60 cells.
Somatic hybridization in the Gramineae:
Saccharum officinarum
L. (sugarcane) and
Pennisetum americanum
(L.) K. Schum. (pearl millet)
Authors: Tabaeizadeh Zohreh , Ferl Robert J. , Vasil Indra K.
Published: May 2006

Somatic hybrid cell lines with embryogenic capacity were obtained by fusion of protoplasts isolated from an embryogenic cell line of sugarcane with inactivated protoplasts of an
S
-(2-aminoethyl)-L-cysteine (Aet-Cys)-resistant cell line of pearl millet. Initial selection for putative hybrids was performed by plating protoplast-derived microcolonies onto medium containing 300 μg of Aet-Cys per ml. Calli growing in the presence of Aet-Cys were screened with respect to their electrophoretic pattern of phosphogluconate dehydrogenase (6-phospho-D-gluconate; EC 1.1.1.44), a dimeric enzyme. The somatic hybrid calli showed both parental homodimer bands plus an intermediate heterodimer band. Somatic hybrid lines were characterized further by electrophoretic patterns of shikimate dehydrogenase (shikimate; EC 1.1.1.25) and arylesterase (aryl-ester hydrolase; EC 3.1.1.2). All cell lines expressed the nuclear genomes of both parents. Use of a maize ribosomal DNA probe allowed the identification of diagnostic ribosomal DNA fragments in
Xba
I digests of total DNA in both parents and the somatic hybrids. The somatic hybrid lines carried both sugarcane and pearl millet ribosomal DNA fragments. The somatic hybrid calli formed somatic embryos. The banding pattern of phosphogluconate dehydrogenase from the somatic embryos was similar to that of the somatic hybrid calli.
K channels are expressed early in human T-cell development.
Authors: Schlichter L , Sidell N , Hagiwara S
Published: May 2006

Mature human T lymphocytes proliferate in response to the mitogen phytohemagglutinin (PHA), but immature thymocytes lacking the T3 receptor (T3- thymocytes) do not. Because functioning K channels are required for proliferation of mature T cells, we asked whether immunoincompetent T3- thymocytes lack normal K channels. We report that T3- thymocytes have a K+ current similar to that of mature peripheral T cells--that is, similar voltage dependence, activation and inactivation kinetics, and pharmacology. Moreover, the maximal specific K+ conductance is the same for both cell types, implying a similar density of activable channels in each cell. In assessing the functional responses of the channels to PHA, we found that the K+ current of immature and mature cells responds similarly to the mitogen. Responses near the threshold voltage for activating the K+ current were variable; the K+ conductance and rate of activation were increased, decreased, or unchanged after PHA treatment. For several cells, the voltage dependence of the conductance and activation kinetics was shifted in opposite directions. At more positive voltages, PHA consistently caused a 10-20% suppression of conductance that was not due to the addition of an inward current, to changes in the time course of activation or inactivation, or to changes in the steady-state level of inactivation. The effects of PHA on the K+ current cannot be explained by a simple shift in surface potential, as has been hypothesized to be involved in its triggering of T-cell proliferation. Taken together, our findings show K channels are expressed very early in T-cell differentiation, possibly before thymic processing, differential responses of the K+ current to PHA do not account for the failure of T3- thymocytes to proliferate, and changes in surface potential are probably not a necessary early event in activation of T cells by PHA.
Genomic diversity of the acquired immunodeficiency syndrome retroviruses is reflected in alteration of its translational products.
Authors: Devare S G , Srinivasan A , Bohan C A , Spira T J , Curran J W , Kalyanaraman V S
Published: May 2006

We have isolated retroviruses from six acquired immunodeficiency syndrome (AIDS) and three lymphadenopathy syndrome (LAS) patients by cocultivation of patients' lymphocytes with phytohemagglutinin-stimulated normal T cells. In an effort to address the extent to which these viruses have identical genetic information or there is divergence in their genomic sequences, we have compared the nine retrovirus isolates by the following criteria: (i) antigenic cross-reactivity by highly specific and sensitive competition radioimmunoassay for the major internal antigen; (ii) restriction site mapping analysis; and (iii) immunoblot analysis and metabolic labeling of viral structural proteins and their analysis by polyacrylamide gel electrophoresis. The data indicate that individual retroviruses have significant restriction site polymorphism in their genome even though they were isolated from patients residing at one geographic location. Furthermore, this polymorphism is reflected in the variation of the apparent size of the gag and env gene-encoded structural proteins. The heterogeneity in AIDS retrovirus-encoded proteins may be due to either substitutions in the primary amino acid sequence of the protein or deletions or additions in the coding regions of proteins. The alterations in viral structural proteins among various AIDS retroviruses could have important implications in antigenic properties and/or pathogenicity in development of the disease, its detection, and ultimately its eradication.
Hexose transport control in a fibroblast metabolic mutant can be promoted more effectively by D-allose than by glucose.
Authors: Ullrey D B , Kalckar H M
Published: May 2006

By studying the energy-requiring control of the hexose transport system (the transport "curb") in a lung fibroblast mutant called the phosphoglucose isomerase mutant (because it is devoid of the enzyme phosphoglucose isomerase) the following features were noted. The aldohexose D-allose, if added over 20 hr to a culture of the mutant, promotes the development of an intense curb of the hexose transport system, greatly surpassing that brought about by incubation with glucose. The allose-mediated curb can be circumvented by various metabolic inhibitors as well as by the presence of other aldohexoses such as mannose.
Fibroblast growth factor treatment of Swiss 3T3 cells activates a subunit S6 kinase that phosphorylates a synthetic peptide substrate.
Authors: Pelech S L , Olwin B B , Krebs E G
Published: May 2006

Exposure of quiescent cultures of Swiss 3T3-D1 cells to bovine brain acidic fibroblast growth factor (FGF) enhanced phosphorylation of a 31-kDa protein tentatively identified as 40S ribosomal subunit S6 (S6). Soluble extracts from FGF-treated as compared with quiescent fibroblasts exhibited up to 3-fold higher kinase activity towards S6 in exogenously added rat liver 40S ribosomes and a synthetic peptide, RRLSSLRA. This peptide was patterned after a phosphorylation site sequence in S6 and was phosphorylated with an apparent Km corresponding to 0.18 mM. Optimal activation of the S6 kinase with pure mitogen at 10 ng/ml occurred within 15 to 20 min exposure to FGF. Half-maximal stimulation of the FGF-induced S6 kinase was attained with FGF at 0.4 ng/ml. The S6 kinase in crude extracts utilized both [gamma-32P]ATP (apparent Km congruent to 6-8 microM) and [gamma-32P]GTP (apparent Km congruent to 3 microM), but the ability to utilize GTP was lost after partial purification of the kinase. The FGF-stimulated kinase had an apparent Mr of about 95,000 as determined by chromatography on Sephacryl S300 but appeared to be retarded on TSK 400 HPLC columns, since it eluted with an apparent Mr of 29,000. Treatment of Swiss 3T3 cells with the tumor promoter phorbol 12-myristate 13-acetate (PMA) activated the FGF-stimulated S6 kinase. However, protein kinase C was not required to mediate the FGF activation of the S6 kinase, as FGF still evoked a two-fold activation of the S6 kinase in phorbol ester-pretreated, protein kinase C-depleted cells.
Loss of tumor-suppressive function during chemically induced neoplastic progression of Syrian hamster embryo cells.
Authors: Koi M , Barrett J C
Published: May 2006

Cell hybrids between normal, early-passage Syrian hamster embryo cells and a highly tumorigenic, chemically transformed hamster cell line, BP6T, were formed, selected, and analyzed. Tumorigenicity and anchorage-independent growth were suppressed in the hybrid cells compared to the tumorigenic BP6T cells. These two phenotypes segregated coordinately in these cells. To determine at what stage in the neoplastic process this tumor-suppressive function was lost, two chemically induced immortal cell lines were examined at different passages for the ability to suppress the tumorigenic phenotype of BP6T cells following hybridization. Hybrids of BP6T cells with the immortal, nontumorigenic cell lines at early passages were suppressed for tumorigenicity and anchorage-independent growth. This tumor-suppressive ability was reduced in the same cells at later passages and in some cases nearly completely lost, prior to the neoplastic transformation of the immortal cell lines. Subclones of the cell lines were heterogeneous in their ability to suppress tumorigenicity in cell hybrids; some clones retained the tumor-suppressive ability and others lost this function. The susceptibility to neoplastic transformation of these cells following DNA transfection with the viral ras oncogene or BP6T DNA inversely correlated with the tumor-suppressive ability of the cells. These results suggest that chemically induced neoplastic progression of Syrian hamster embryo cells involves at least three steps: induction of immortality, activation of a transforming oncogene, and loss of a tumor-suppressive function.
Analysis of sugar-binding sites in mammalian cell nuclei by quantitative flow microfluorometry.
Authors: Sève A P , Hubert J , Bouvier D , Bourgeois C , Midoux P , Roche A C , Monsigny M
Published: May 2006

Quantitative flow microfluorometry of neoglycoprotein (bovine serum albumin coupled to sugar and to fluorescein) binding demonstrated the existence of sugar-binding sites (i.e., lectin-like molecules) in isolated BHK cell nuclei. The very similar labeling intensities obtained with nuclei isolated by cell lysis and with permeabilized karyoplasts obtained by enucleation strengthened the idea that the binding sites are borne by actual nuclear structures and not by cytoplasmic or membrane-derived contaminants. With both nuclei-isolation procedures, neoglycoproteins (containing similar numbers of sugar residues) used as markers can be similarly classified. Fluorescence microscopy further indicated that in both nuclear preparations, the neoglycoprotein binding sites were associated with the nucleoli as well as with nucleoplasmic ribonucleoprotein elements. Nuclei from exponentially growing cells bound much greater amounts of neoglycoprotein than did nuclei from contact-inhibited cells.
Immunologic relationship between platelet membrane glycoprotein GPIIb/IIIa and cell surface molecules expressed by a variety of cells.
Authors: Plow E F , Loftus J C , Levin E G , Fair D S , Dixon D , Forsyth J , Ginsberg M H
Published: May 2006

A polyclonal antiserum to platelet membrane glycoprotein GPIIb/IIIa was used to detect antigenically related molecules on a diverse panel of human cells. Umbilical vein endothelial cells, erythroleukemic HEL cells, and diploid fetal lung GM1380 fibroblasts expressed GPIIb/IIIa-related molecules, as judged by immunofluorescence and immunoprecipitation of surface-labeled proteins. The GPIIb and GPIIIa subunits were both present and were of similar molecular weight in these cell types. These molecules were synthetic products of the cells, as shown by immunoprecipitation of intrinsically labeled proteins. Promyeloid U937 cells could be induced by 4 beta-phorbol 12-myristate 13-acetate to synthesize and express GPIIb/IIIa-related molecules on their cell surface. The GPIIb/IIIa-related molecules were not precisely identical in the various cell types, based on slight differences in electrophoretic mobility and their failure to react with monoclonal antibodies specific for each subunit of platelet GPIIb/IIIa. These results suggest the existence of a widely distributed family of GPIIb/IIIa-related molecules. This family of "cytoadhesins" may share a common function in cellular adhesive reactions.
Spermidine synthase of Escherichia coli: localization of the speE gene.
Authors: Tabor C W , Tabor H , Xie Q W
Published: May 2006

We have obtained Escherichia coli mutants lacking spermidine synthase (putrescine aminopropyltransferase) and have found that the mutated gene (speE) is located immediately upstream from the gene coding for S-adenosylmethionine decarboxylase (speD); these genes are located at 2.7 minutes on the E. coli chromosome. Both genes are present in a 1795-base-pair fragment of E. coli DNA that was cloned into pBR322. Deletion of 105 bases upstream of speE caused a coordinate loss of both activities, indicating that speE and speD constitute a single operon. speE and speD have also been cloned separately in a high-expression vector; strains carrying these plasmids overproduce the respective enzymes.
Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons.
Authors: Bizub D , Wood A W , Skalka A M
Published: May 2006

The importance of mutational activation of the Ha-ras protooncogene in polycyclic aromatic hydrocarbon-induced mouse skin tumors was investigated in a complete carcinogenesis model using repetitive applications of 7,12-dimethylbenz[a]anthracene (DMBA), or in an initiation-promotion model using a single application of dibenz[c,h]acridine (DB[c,h]ACR) or benzo[a]pyrene (B[a]BP) followed by chronic treatment with phorbol 12-myristate 13-acetate. DNA isolated from carcinomas induced by DMBA or DB[c,h]ACR, but not by B[a]P, efficiently transformed NIH 3T3 cells, and a high percentage of the transformed foci had an amplified Ha-ras gene. Restriction enzyme Southern blot analysis and DNA sequencing revealed that the amplified Ha-ras genes of the transformants had an A----T transversion in the second position of the 61st codon. The same mutation was also detected in primary tumor DNA in a high percentage of the DMBA- or DB[c,h]ACR-induced carcinomas. Identification of the mutation in NIH 3T3 cells transformed with DNA from DB[c,h]ACR-induced benign skin papillomas suggests that it is an early event in skin carcinogenesis. Thus, mutation of the 61st codon of the Ha-ras-1 gene appears to be a critical step in the formation of mouse skin tumors induced in both of the two models tested. Our analyses also delineate two other classes of hydrocarbon-induced carcinomas--namely, tumors whose DNAs efficiently transform 3T3 cells but do not contain mutated ras genes and tumors whose DNAs do not transform 3T3 cells.
Taste and salivary function.
Authors: Weiffenbach J M , Fox P C , Baum B J
Published: May 2006

Human taste perception was remarkably unimpaired in eight individuals with severe, chronic failure of all major and minor salivary gland function. Subjective reports of taste experience and objective measures of suprathreshold sensitivity were within normal limits for the overwhelming majority of these individuals. Impairments of threshold sensitivity for at least one quality was common, but normal thresholds for all four qualities were observed in one individual. These data demonstrated that the functional integrity of the taste system is not dependent upon the presence of normal saliva in the mouth. Thus, the suggestion that a salivary factor is responsible for the maintenance of taste receptor end organs can be rejected and attention directed toward other mechanisms, not dependent on saliva, that might account for the unusual resistance of these cells to environmental insult.
Atrial natriuretic factor stimulates Na/K/Cl cotransport in vascular smooth muscle cells.
Authors: O'Donnell M E , Owen N E
Published: May 2006

Atrial natriuretic factor (ANF) is a collective term used to describe a group of peptides isolated from mammalian atria which have vasorelaxant activity as well as diuretic and natriuretic activity. Recently, ANF peptides have been shown to bind to specific receptors on vascular smooth muscle cells (VSMC) and to cause an elevation in cGMP levels. We have previously demonstrated that VSMC possess a prominent, cyclic-nucleotide-sensitive Na/K/Cl cotransport system. In the present study, the effects of the ANF peptide rat atriopeptin III (rAP III) were measured on Na/K/Cl cotransport of VSMC by using primary cultures derived from rat thoracic aorta. It was found that rAP III caused a marked elevation of Na/K/Cl cotransport. Maximal stimulation occurred at 100 nM, and the dose of rAP III required for half-maximal potassium influx (K1/2) was 9 nM. We also investigated the effect of rAP III on cGMP levels in VSMC. It was found that rAP III increased cGMP in a dose-dependent manner, with a K1/2 value of 10 nM. Finally, we measured the effect of the permeable cGMP analog 8-bromo-cGMP on Na/K/Cl cotransport. It was found that 8-bromo-cGMP stimulated cotransport to the same extent as did a saturating dose of rAP III (K1/2 = 0.2 microM). Saturating doses of rAP III and 8-Br-cGMP in combination did not stimulate cotransport in an additive manner, suggesting that rAP III probably does not elevate cGMP via inhibition of phosphodiesterase. These findings suggest that activation of Na/K/Cl cotransport via elevations in cGMP may be associated with ANF-mediated vasorelaxation and/or ANF-mediated diuresis and natriuresis.
Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells.
Authors: Connor J A
Published: May 2006

Intracellular free calcium levels have been measured in cultured central nervous system (CNS) cells by using the fluorescent indicator fura-2 and digital imaging techniques. Cells were plated from rat embryo diencephalon (embryonic day 17 or 18), with nearly all of the cells surviving dissociation having undergone final mitosis within the previous 24 hr. The initially spherical cells were observed within the first 24 hr in culture when they were extending processes but had not established a network of fibers that would prevent the identification of the origin of a given fiber. Cells that were rapidly extending showed high Ca2+ levels in the regions of growth. Where processes had just emerged from the soma or where growth was proceeding from more than one pole, Ca2+ levels were uniform and estimated levels of 500 nM were commonly seen. In active growth cones distant from the soma, Ca2+ levels exceeded 200 nM, whereas the soma levels were in the 60-80 nM range. Nonextended and extended cells that had stalled had uniform Ca2+ levels in the range of 30-70 nM. The results show that high Ca2+ levels are at least a correlate of extension in CNS cells and that under some conditions the region of high calcium can be localized to a small part of the cell.
Interaction between proteins localized in membranes.
Authors: Grasberger B , Minton A P , DeLisi C , Metzger H
Published: May 2006

We present a conceptual framework for evaluating the effect on the self-association of proteins in membranes due to the presence of other proteins at high concentrations (excluded volume effect) and the high concentration and preoriented state of the reactive species. We have calculated the magnitude of such effects using plausible values for the concentrations of proteins in membranes, for the degree to which proteins may tilt and move vertically, and for their dimensions. Compared to the association of monomers tumbling freely in an experimentally realistic volume, we calculate that these factors alone can increase the likelihood of forming dimers 10(6)-fold and of forming trimers and higher oligomers many orders of magnitude greater. We discuss the implications of our calculations for experimental manipulations of membrane proteins, for biosynthetic assembly of multisubunit membrane proteins and formation of membrane lesions by assemblies of exogenous proteins, and for the activation of cellular events induced by the interaction of membrane receptors with themselves or with other membrane proteins.
Efficient reversion of simian sarcoma virus-transformation and inhibition of growth factor-induced mitogenesis by suramin.
Authors: Betsholtz C , Johnsson A , Heldin C H , Westermark B
Published: May 2006

Simian sarcoma virus, an acutely transforming primate retrovirus with capacity to induce gliomas and sarcomas in experimental animals, has acquired its transforming properties by transducing the cellular gene sequences that encode one of the constituent chains of platelet-derived growth factor. Suramin, a drug used in the treatment of trypanosomiasis and onchocerciasis, has previously been reported to inhibit the interaction of platelet-derived growth factor with its cell surface receptor. We show here that suramin efficiently reverts the simian sarcoma virus-induced transformed phenotype in human and rat fibroblasts and propose that this is due to neutralization of an externalized v-sis product. Moreover, we show that suramin inhibits the action of a broad spectrum of growth factors.
Induction of c-sis gene expression and synthesis of platelet-derived growth factor in human myeloid leukemia cells during monocytic differentiation.
Authors: Pantazis P , Sariban E , Kufe D , Antoniades H N
Published: May 2006

Phorbol esters induce the differentiation of human myeloid leukemia cells HL-60 and U-937 along the monocytic-macrophage lineage. This process has been associated with the induction of several cellular protooncogenes, including the c-fos and c-fms genes. We now report that phorbol ester-induced differentiation of the HL-60 and U-937 cells results in the induction of the expression of the c-sis platelet-derived growth factor 2 (PDGF-2) protooncogene. sis mRNA transcripts were not detectable in the uninduced cells but were detectable within 12 hr of phorbol ester induction. Concomitantly, the induced cells were shown to synthesize and secrete biologically active PDGF-like proteins, identified in the conditioned medium of the phorbol ester-treated cells by direct immunoprecipitation with PDGF antiserum. Addition of cycloheximide to phorbol ester-treated HL-60 cells superinduced sis mRNA transcripts. c-sis gene transcripts were also detected in freshly isolated human monocytes but not in human granulocytes or in HL-60 cells induced to differentiate along the granulocytic lineage. Activation of the c-sis/PDGF-2 gene in human hematopoietic cells during monocytic differentiation may serve in the mediation of physiologic functions of the differentiated cells by means of the secretion of potent PDGF-like mitogen.
Evasion of alternative complement pathway by Trypanosoma cruzi results from inefficient binding of factor B.
Authors: Joiner K , Sher A , Gaither T , Hammer C
Published: May 2006

During its differentiation in the insect vector to a stage infective for the mammalian host, Trypanosoma cruzi becomes resistant to lysis by the alternative pathway of complement. To elucidate the mechanism of complement evasion, we studied control of complement activation on the surface of the noninfective epimastigote and the infective culture-derived metacyclic trypomastigote stages (CMT) of T. cruzi. It was found that the predominant form of complement component C3 on epimastigotes is C3b, whereas the majority of C3 on CMT is in the form of the hemolytically inactive fragment iC3b, which cannot participate in C5 convertase formation or lead to deposition of the lytic C5b-9 complex. Our results also showed that C3 binds by a covalent ester linkage to surface molecules of different molecular weight in the epimastigote stage and CMT. Binding studies with purified complement components indicated that CMT do not support efficient formation of an alternative pathway C3 convertase. C3b on the parasite surface fails to bind the amplification component, factor B, rather than showing enhanced binding of the control component, factor H. These results identify the biochemical basis for evasion of complement-mediated killing in T. cruzi and reveal a mechanism for developmental regulation of complement activation.
Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger.
Authors: Besterman J M , Duronio V , Cuatrecasas P
Published: May 2006

The classic pathway for agonist-induced generation of diacylglycerol is via activation of a phospholipase C-mediated hydrolysis of the "phosphoinositides." We now report findings from a variety of cell types, which indicate that tumor-promoting phorbol diesters, serum, and platelet-derived growth factor activate within seconds the hydrolysis of phosphatidylcholine, as detected by the formation of diacylglycerol and phosphocholine. It is known that phorbol diesters do not stimulate hydrolysis of the phosphoinositides. Yet, in cells prelabeled with either [14C]oleate or [32P]orthophosphate, addition of the tumor promoter phorbol dibutyrate (PBt2) resulted in the rapid generation of both diacylglycerol and phosphatidate in a time- and dose-dependent manner. The fatty acid composition of the phosphatidate most resembled the fatty acid profile of phosphatidylcholine from the same cell type. Taken together, these findings suggested a role for protein kinase C in the generation of diacylglycerol (and phosphatidate) from phosphatidylcholine. To define further the pathways involved, the metabolism of cellular phosphatidylcholine was studied. In cells prelabeled with [3H]choline, addition of PBt2, but not 4 alpha-phorbol, stimulated the formation of intracellular phosphocholine within 45 sec. Furthermore, addition of platelet-derived growth factor (PDGF) or serum to "serum-starved" cells prelabeled with [3H]choline resulted in increased levels of intracellular phosphocholine within 15-30 sec. Thus, the data suggest that agonists that stimulate protein kinase C either directly (e.g., PBt2) or indirectly via activation of phosphoinositide hydrolysis (e.g., PDGF and serum) may stimulate degradation of phosphatidylcholine by phospholipase C in intact cells. However, prior down-regulation of protein kinase C by prolonged pretreatment of cells with PBt2 almost totally abolished subsequent stimulation of phosphatidylcholine degradation by PBt2 but only partially attenuated subsequent stimulation by PDGF and serum. These observations suggest that PDGF and serum act, at least partially, through a protein kinase C-independent mechanism. Lastly, the size of the cellular choline and CDP-choline pools were shown to be small and relatively insensitive to agonist addition, as compared to the size and behavior of the phosphocholine pool. Thus, the rapidly increased levels of phosphocholine (and diacylglycerol) arising in response to agonist addition appear to be derived directly from phosphatidylcholine by a phospholipase C-mediated mechanism.
Changes in gene expression associated with induced differentiation of erythroleukemia: protooncogenes, globin genes, and cell division.
Authors: Ramsay R G , Ikeda K , Rifkind R A , Marks P A
Published: May 2006

Hexamethylenebisacetamide (HMBA)-induced differentiation of murine erythroleukemia cells (MELC) is a multistep process involving an early latent period during which a number of metabolic changes have been detected, but the cells are not yet committed irreversibly to differentiate. Commitment is defined as the capacity of MELC to go on to express the program of terminal cell division and gene expression (such as the accumulation of globin mRNA) upon removal of the HMBA from the culture. In the presence of HMBA, a small proportion of MELC are committed by 10-12 hr and greater than 90% by 48-60 hr. The present study shows that, during the initial 4 hr of culture, HMBA causes a marked decrease in c-myb and c-myc and an increase in c-fos mRNA levels. With continued culture, the decrease in c-myb and the increase in c-fos mRNA persists, while c-myc mRNA returns to control levels before the time that MELC begin to show irreversible differentiation. Dexamethasone, which blocks expression of HMBA-induced MELC differentiation, does not alter the early pattern of changes in protooncogene mRNA nor the sustained elevation of c-fos, but it does inhibit the continued suppression of c-myb allowing c-myb to return toward control levels. Hemin, which induces MELC to accumulate globins but does not initiate commitment to terminal cell division, does not alter these protooncogene mRNA levels. These studies suggest that, although the early decrease in c-myb and c-myc and increase in c-fos mRNAs may be involved in the multistep events leading to differentiation, the continued suppression of c-myb is critical for HMBA-induced MELC commitment to terminal cell division.
Chromosomal assignment of the human erythropoietin gene and its DNA polymorphism.
Authors: Law M L , Cai G Y , Lin F K , Wei Q , Huang S Z , Hartz J H , Morse H , Lin C H , Jones C , Kao F T
Published: May 2006

Erythropoietin (EPO), a glycoprotein hormone, is the major physiological regulator of erythrocyte production in mammals. A cDNA clone containing the entire human EPO-coding region was used for Southern blot analysis of a series of human-Chinese hamster somatic cell hybrids containing different combinations of human chromosomes. Synteny analysis revealed 100% concordance between the EPO gene and human chromosome 7. Further localization to the region q11-q22 was accomplished by in situ hybridization of 3H-labeled human EPO cDNA to metaphase chromosomes prepared from both human lymphocytes and the cell hybrid 879-2a that contained human chromosomes 5, 7, 9, 12, and 21. In addition, restriction fragment length polymorphisms were detected at a frequency of approximately 20% in a Chinese population using restriction enzymes either HindIII or HinfI. These polymorphisms were inherited in a Mendelian fashion. Thus, the EPO marker is reasonably polymorphic and should be useful in linkage analysis with other genetic markers on chromosome 7, including the locus for cystic fibrosis.
Analysis of mutations in the transmembrane region of the aspartate chemoreceptor in Escherichia coli.
Authors: Oosawa K , Simon M
Published: May 2006

Site-specific mutagenesis was used to replace an alanine with a lysine residue and to create a deletion of seven amino acids into the first transmembrane region (TMI region) of the aspartate chemoreceptor in Escherichia coli. The mutations resulted in the loss of aspartate chemotaxis on tryptone motility plates. However, both mutant proteins were able to associate with the membrane and to bind aspartate. They were both refractory to methylation or to modification of the C-terminal region of the protein by the cheB gene product. These results suggested that the integrity of the TMI domain of the protein was required to maintain the function of the cytoplasmic portion of the receptor. The Lys-19 mutant retained the ability to generate a repellent response. Analysis of suppressor mutations of the Lys-19 mutation suggested that formation of an ion pair or specific changes in a 40 amino acid stretch in the cytoplasmic region of the protein (from amino acid 264 to amino acid 303) could suppress the effects of the Lys-19 mutation. The TMI region of the protein may be involved in transmembrane transmission of signals from the periplasmic portion of the cell to the cytoplasmic portion of the Tar protein.
Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels.
Authors: Zalman L S , Wood L M , Müller-Eberhard H J
Published: May 2006

Erythrocytes are poorly lysed by homologous complement, whereas they are readily lysed by heterologous complement. This phenomenon had been attributed to an interference by the cell surface with the action of complement components C8 and C9. To isolate the responsible membrane constituent, detergent-solubilized human erythrocyte (EH) membranes were subjected to affinity chromatography by using human C9-Sepharose. The isolated protein had a mass of 38 kDa and, incorporated into liposomes, was highly effective in inhibiting complement-mediated channel expression, including the C5b-8, membrane attack complex, and tubular polymer of C9 channels. Antibody produced to the 38-kDa protein caused a 20-fold increase in reactive lysis of EH by isolated C5b6, C7, C8, and C9. The antibody did not enhance C5b-7 uptake, but it affected C9 binding to the target cell membrane. Antibody to human decay-accelerating factor, used as a control, had no effect on reactive lysis of EH. Anti-38-kDa protein did not enhance the action on EH of C8 and C9 from other species, indicating that the action of this regulatory protein is species specific. It was therefore termed homologous restriction factor (HRF). Blood cells other than erythrocytes, such as polymorphonuclear leukocytes, also exhibited cell-surface HRF activity. In immunoblots of freshly isolated EH membranes, anti-38-kDa HRF detected primarily a 65-kDa protein, suggesting that the 38-kDa protein constitutes an active fragment of membrane HRF. Because of the specific binding reaction observed between HRF and C8 or C9, HRF was tested with anti-human C8 and anti-human C9. A limited immunochemical relationship of HRF to C8 and C9 could be established and solid-phase anti-C9 proved an efficient tool for the isolation of HRF from solubilized EH membranes.
RETRACTED: Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.
Authors: Ramarli D , Fox D A , Milanese C , Reinherz E L
Published: May 2006

Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte-binding protein, T11, represents an essential cell surface component of a human T-cell lineage activation pathway. Furthermore, it is known that the human T3-Ti T-cell antigen/major histocompatibility complex receptor complex is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2-receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and must possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells.
Encapsulation of adriamycin in human erythrocytes.
Authors: De Flora A , Benatti U , Guida L , Zocchi E
Published: May 2006

Adriamycin (doxorubicin) was encapsulated in human erythrocytes by means of a dialysis technique involving transient hypotonic hemolysis followed by isotonic resealing. Up to 1.6 mg of the drug was entrapped per ml of packed erythrocytes, with the efficiency of encapsulation being 60-80%. In vitro incubation of the Adriamycin-loaded erythrocytes in autologous plasma was accompanied by progressive release of unaltered Adriamycin in the medium. The efflux was still evident after 50 hr. The metabolism of encapsulated Adriamycin was restricted to limited formation of the C-13 hydroxylated metabolite, adriamycinol, in the normal erythrocytes but not in erythrocytes from individuals deficient in glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) activity. Reductive bioactivation of encapsulated Adriamycin to yield the corresponding aglycones was not observed in a variety of conditions. However, when NADPH ferredoxin reductase and ferredoxin, both purified from spinach leaves, were co-entrapped within erythrocytes and allowed to catalyze electron transfer to Adriamycin intracellularly under N2, a quantitative conversion to 7-deoxyadriamycin aglycone was obtained. Adriamycin-loaded erythrocytes did not show any significant oxidative damage, except for a variable increase of methemoglobin, suggesting some redox cycling between native Adriamycin and its semiquinone radical. Encapsulation of Adriamycin in autologous human erythrocytes may represent a therapeutic strategy for the slow release in circulation of this antineoplastic drug in order to reduce or prevent its adverse effects and especially the delayed cardiotoxicity that limits its use in patients with neoplastic disease.
Abnormal response to DNA crosslinking agents of Fanconi anemia fibroblasts can be corrected by transfection with normal human DNA.
Authors: Diatloff-Zito C , Papadopoulo D , Averbeck D , Moustacchi E
Published: May 2006

Primary skin fibroblast cell lines from patients with Fanconi anemia were cotransfected with UV-irradiated pSV2neo plasmids and high molecular weight DNA from normal human cells. Restoration of a normal cellular resistance to mitomycin C (MMC) was observed provided that a Fanconi anemia cell line is selected for DNA-mediated transformation (neo gene) and that at least two successive rounds of transfection are performed. Cells were selected by taking advantage of the higher proliferation rate and plating efficiency of the MMC resistant transformants. As estimated from reconstruction experiments, the frequency of transfer of MMC resistance lies between 1 and 30 X 10(-7). The MMC resistance phenotype was maintained for at least 10 generations following transfection. Evidence for DNA-mediated transformation also includes the recovery of a normal pattern of DNA semiconservative synthesis after treatment with 8-methoxypsoralen and 365-nm UV irradiation, and the presence of exogenous pSV2neo DNA sequences was shown by Southern blot analysis. The acquired MMC resistance is probably due to the presence of DNA from normal cells. Indeed, sensitivity to MMC was maintained when Fanconi anemia cells were cotransfected with the UV-irradiated pSV2neo plasmid mixed with their own DNA or with yeast or salmon sperm DNA. These negative results also render unlikely the selection of spontaneous MMC resistant revertants in transfection of Fanconi anemia cells with normal DNA. These experiments establish the prerequisites for the isolation of the gene(s) involved in the response to DNA crosslinking lesions in human cells.
A metabolite-regulated potassium channel in rat pancreatic B cells.
Authors: Misler S , Falke L C , Gillis K , McDaniel M L
Published: May 2006

In B cells from dispersed rat islet of Langerhans we have identified an inward rectifying voltage-independent K+ channel whose behavior parallels the metabolically regulated potassium permeability (PK) found in tracer flux and microelectrode recording studies. In cell-attached patches of membrane, the channel is closed when any one of several substrates (glucose, mannose, leucine, or glyceraldehyde) is added to the cell's bathing solution but is reopened on addition of an appropriate metabolic inhibitor, which prevents utilization of that substrate. In inside-out excised patches, a K+ channel with nearly identical kinetic features is closed by addition of micromolar concentrations of ATP to the "cytoplasmic" solution. The ATP sensitivity of channel activity is modified by addition of ADP, suggesting competition at a nucleotide binding site. These results suggest the presence of a metabolically regulated K+ channel gated by intracellular concentrations of ATP or the ratio of ATP/ADP concentrations.
An essential function for acyl carrier protein in the biosynthesis of membrane-derived oligosaccharides of Escherichia coli.
Authors: Therisod H , Weissborn A C , Kennedy E P
Published: May 2006

Membrane-derived oligosaccharides are branched, substituted beta-glucans localized in the periplasmic space of Escherichia coli and other Gram-negative bacteria. The biosynthesis of membrane-derived oligosaccharides and of analogous periplasmic oligosaccharides found in plant bacteria is of particular interest because it is subject to strict osmotic regulation [Miller, K.J., Kennedy, E.P., and Reinhold, V.N. (1986) Science 231, 48-51]. An enzyme system catalyzing the synthesis of the (beta 1-2)-linked glucan backbone of E. coli membrane-derived oligosaccharides from UDP-glucose requires both a membrane component and a cytosolic protein termed transglucosylation factor. The factor has now been purified to apparent homogeneity and has been found to be identical to acyl carrier protein (ACP), the phosphopantetheine-containing protein of low molecular weight that has long been known to be essential for fatty acid synthesis in E. coli and other organisms. Both are small, heat-stable, highly anionic proteins with identical chromatographic and electrophoretic behavior. ACP of the highest purity has an activity in the transglucosylation system indistinguishable from that of the protein independently purified as transglucosylation factor. Antibody raised against pure ACP completely inhibits transglucosylation activity; this inhibition is overcome by titration of the antibody with either ACP or transglucosylation factor. These findings provide evidence for an essential function of ACP unrelated to the biosynthesis of lipid.
Basic fibroblast growth factor induces angiogenesis in vitro.
Authors: Montesano R , Vassalli J D , Baird A , Guillemin R , Orci L
Published: May 2006

Fibroblast growth factors (FGFs) are potent mitogens for vascular and capillary endothelial cells in vitro and can stimulate the formation of blood capillaries (angiogenesis) in vivo. A crucial event in this process is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. Using a recently developed in vitro model of angiogenesis, we show here that highly purified basic pituitary FGF can induce capillary endothelial cells to invade a three-dimensional collagen matrix and to organize themselves to form characteristic tubules that resemble blood capillaries. We also show that basic FGF concomitantly stimulates endothelial cells to produce a urokinase-type plasminogen activator, a protease that has been implicated in the neovascular response. The results demonstrate that basic FGF can stimulate processes that are characteristic of angiogenesis in vivo, including endothelial cell migration, invasion, and production of plasminogen activator.
Translocation of protein kinase C in human leukemia cells susceptible or resistant to differentiation induced by phorbol 12-myristate 13-acetate.
Authors: Homma Y , Henning-Chubb C B , Huberman E
Published: May 2006

We investigated the possible relationship between the susceptibility of cells to differentiation induced by phorbol 12-myristate 13-acetate (PMA) and the subcellular translocation of calcium- and phospholipid-dependent protein kinase (protein kinase C) activity from the cytosol to the membrane. These two events were analyzed in a number of human leukemia cell lines, including four cell variants of the promyelocytic cell line HL-60 that exhibit different degrees of susceptibility to PMA-induced differentiation. The phenotype of the differentiated cells was characterized by increased reactivity with monoclonal antibodies against maturation-specific cell surface antigens, increased nonspecific esterase activity, and acquisition of morphological cell maturation. Analysis of the subcellular distribution of protein kinase C activity in each of these cell types revealed that 90% of the kinase activity was present in the cytosolic fraction, with the remaining activity in the membrane fraction. Treatment of the differentiation-susceptible cells with 160 nM PMA resulted, within 5 min after treatment, in a greater than 60% decrease in protein kinase C activity in the cytosolic fraction and a greater than 1500% increase in the activity in the membrane fraction. No such subcellular redistribution of protein kinase C activity was found after treatment of the differentiation-resistant cells. On the basis of these findings, we suggest that the process of subcellular translocation of protein kinase C activity, initiated after the binding of PMA to this kinase, is required for the induction of cell differentiation by this phorbol diester.