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A region of the poliovirus genome coding for a cysteine protease was expressed in E. coli. Following separation in isoelectric focussing gels, four new proteins were identified and were shown to be products of the viral cDNA. Although a majority of the protein products is aggregated, the system offers the first practical basis for production of large quantities of a viral protease.
The voltage-activated Na channel (NaCh) is an integral membrane protein that is enriched at the neuromuscular end plate. Using loose-patch voltage-clamp and immunofluorescence, we have found that the aggregation of NaChs occurs late, during maturation of the neuromuscular junction. A decline in expression of embryonic NaCh mRNA and increase in adult NaCh mRNA precedes the onset of aggregation, and the appearance of functional adult NaChs coincides with NaCh aggregation. We tested the possibility that only the adult NaCh subtype could aggregate during development and found that both the embryonic and adult isoforms become concentrated at the synapse. The NaCh is the first postsynaptic membrane protein shown to become clustered postnatally, and the mechanism producing this aggregation appears to be different from the process producing aggregation of other synaptic proteins.
Gap junctional communication is important in many physiological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human beta 1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional beta 1 connexin was detected only in cells transfected with beta 1 connexin cDNA in the correct orientation (beta 1-BHK). Analysis of the cells by light microscopic immunocytochemistry indicated that beta 1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomerization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.
RNA interference (RNAi) is a novel phenomenon that can induce post-transcriptional gene silencing (PTGS) both in animals and plants. RNAi is effective in suppressing specific gene expression in the early mouse embryonic cells and in undifferentiated embryonic stem (ES) cells. In this study, we demonstrate that dsRNA is effective in inducing PTGS in differentiated ES cells: CD34+ embryoid body (EB) cells, as confirmed by western blot and immunocytochemical staining. PU.1 is a key transcription factor in myeloid differentiation. Undifferentiated ES cells do not express PU.1; however it is expressed when ES cells differentiate into EBs. PU.1 could be suppressed by the specific PU.1 dsRNA, but not non-specific Lamin A/C dsRNA, in the CD34+ EB cells when they were induced to myeloid differentiation in the presence of GM-CSF and IL-3. As a consequence, the level of expression of CD115 (M-CSF receptor), one of the downstream genes regulated by PU.1 is decreased in PU.1 dsRNA treated CD34+ EB cells, but not in Lamin A/C dsRNA treated cells. To explore this phenomenon in other myeloid gene, we also found that C/EBPalpha gene could be knocked down by C/EBPalpha dsRNA. Our finding demonstrates that RNAi is effective in inhibiting specific gene expression in differentiated ES cells. RNAi offers a new methodology for study of hematopoietic regulation using ES cell differentiation.
Mouse lymphoma cell line W7MG1 is stably infected with mouse mammary tumor virus and produces the viral envelope glycoprotein precursor Pr74, but the mature envelope proteins gp52 and gp33, which are derived from Pr74 by posttranslational processing, are produced only when the cells are cultured with a glucocorticoid agonist. The current study demonstrated that even when W7MG1 cells are grown with hormone, the conversion of Pr74 to gp52 and gp33 is an inefficient process in this cell line. At least 2 h of exposure to glucocorticoid were required to induce the appearance of gp52 and gp33; furthermore, Pr74 labeled in the absence of hormone was not converted to gp52 and gp33 upon subsequent addition of hormone. RNA synthesis inhibitors blocked the hormonal induction of gp52 and gp33, indicating that the hormone acts by promoting the expression of a new gene(s) required for the production of gp52 and gp33, rather than by inhibiting the expression of a gene(s) that prevents processing of Pr74. Subcellular fractionation studies demonstrated that Pr74 produced in either the presence or absence of hormone was associated primarily with the ER, whereas gp52 and gp33 were found in the Golgi and plasma membrane fractions. The Pr74 molecules from W7MG1 cells grown either with or without glucocorticoid coimmunoprecipitated with BiP/GRP78 and sedimented as aggregates of heterogeneous size. In contrast, Pr74 from virus-producing GR3A mouse mammary tumor cells, which process Pr74 more efficiently, sedimented as apparent monomers, dimers, and trimers.(ABSTRACT TRUNCATED AT 250 WORDS)
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Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.
As anucleate cell particles, platelets do not lend themselves to analysis by traditional cell and molecular biology techniques. Moreover, while valuable information may be gleaned from studies of messenger RNA in platelets, the rapid events in platelets are not governed by or dependent on alterations in gene expression. In contrast, proteomics, the study of the protein complement of a genome, will have a major impact on platelet biology. It offers the opportunity to comprehensively describe the proteins involved in discrete elements of platelet function, from the subsecond events following platelet activation and adhesion through to platelet aggregation and granule secretion. As the function of every protein is understood and as the mechanisms that regulate protein modifications are unravelled, we will discover a wealth of proteins that are themselves potential therapeutic agents or novel targets for the development of diagnostics and drugs. Here we review the current applications of proteomics to platelet research. We briefly describe various proteomic approaches to unravel platelet biology, including the documentation of platelet proteins, the investigation of thrombin-activated phosphotyrosine signaling networks and the analysis of the proteins that are secreted upon platelet activation. Proteomics is a young field and there are only a handful of published examples applying proteomics to platelet research. This number will increase over the next few years, as advances in analytical methods allow a more functional analysis of the platelet proteome.
Nucleolipids are hybrid molecules composed of a nucleobase, a nucleoside, a nucleotide or an oligonucleotide (either DNA or RNA), and a lipophilic moiety, which might be either simply a single- or double-chained alkyl (or alkenyl) moiety or a carbocyclic hydrocarbon such as cholesterol, a vitamin, or a bile acid. This review covers all aspects of nucleolipids, namely their natural occurrence, their synthesis, their molecular recognition, as well as aggregation behavior, either in aqueous or non-aqueous solution. Potential future aspects of nucleolipids in material sciences and for the elucidation of biochemical reactions in living cells are discussed.
In culture the human teratocarcinoma cell line HT-H generates both adherent monolayer and free-floating aggregates. Some populations of aggregated cells develop further to form cystic bodies. A previous study showed the morphological resemblance of the cystic bodies to cells of blastocyst of preimplantation embryo. In this study, HT-H adherent cells were further separated into two subpopulations, fast adhering and slow adhering cells. Fast adhering cells produce fibronectin, spread well onto substratum, and do not proliferate. In contrast, slow adhering cells do not produce fibronectin. Trophoblastic markers were examined in each morphological stage of HT-H cells and the following results were obtained. Only fast adhering cells produce progesterone. Human chorionic gonadotropin was secreted preferentially by fast adhering cells, about six times less by slow adhering cells, and was not secreted by aggregates or cystic bodies. All stages of HT-H cells express c-fos but only fast adhering cells express c-fms oncogene. Cytokeratin 18 was expressed in all stages of HT-H cells. The level of cytokeratin 18 is modestly decreased from adherent to aggregates further into cystic bodies. These results indicate that HT-H cells share properties with cells in trophoblast, placenta, and extraembryonic endoderm. Spontaneous differentiation of HT-H cultures results in the appearance of fast adhering cells which exhibit biochemical properties expected for syncytiotrophoblast.
P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.
OBJECTIVE: Extracellular calcium influences chondrocyte differentiation and synthesis of extracellular matrix. Previously, calcium concentrations ranging from 0.1 mM to 2 mM have been used in vitro and these studies indicated that low calcium concentrations were generally favorable for chondrocyte culture. Our objective was to extend these findings to yet lower calcium concentrations and to comprehensively examine effects on morphology and phenotype in two culture systems. METHODS: Serum-free media containing 1 mM, 50 microM or 15 microM of calcium and a serum-containing medium were used to culture chondrocytes in suspension and in monolayer, at high and low inoculation density. RESULTS: In monolayer, at low and high density, removing serum and decreasing calcium concentration decreased cell spreading and lowered collagen type I expression whereas collagen type II expression remained stable. In suspension, cells aggregated for all media tested; however, aggregates were smaller and looser in the absence of serum. CONCLUSION: The serum-free 50 microM and 1 mM calcium media provide good alternatives to classical media for monolayer culture since both growth and chondrocyte phenotype were maintained. In suspension culture, the serum-free 1mM calcium medium also possesses the beneficial properties of limiting aggregate size while maintaining growth and phenotype.
Slac2-a/melanophilin regulates melanosome transport in mammalian skin melanocytes by linking melanosome-bound Rab27A and an actin-based motor protein, myosin Va. Slac2-a consists of an N-terminal Slp homology domain (SHD), which has been identified as a specific GTP-Rab27-binding domain, a myosin Va-binding domain (MBD) in the middle region, and an actin-binding domain (ABD) at the C-terminus. Mutations in the slac2-a/mlph gene cause the abnormal pigmentation (i.e., perinuclear melanosome aggregation in melanocytes) in human Griscelli syndrome type III and in leaden mice because of the inability to form the tripartite protein complex consisting of Rab27A, Slac2-a, and myosin Va. In this chapter we describe the methods, including in vivo melanosome distribution assay combined with dominant-negative approaches and RNA interference technology, that have been used to analyze the function of Slac2-a in melanosome transport in melanocytes.
BACKGROUND/AIMS: Angiogenesis is essential in liver regeneration. However, only little is known about sinusoidal endothelial cell proliferation and the role of different angiogenic growth factors and their receptors during regeneration. METHODS: Seventy percent hepatectomy was carried out on male rats. Serial changes in endothelial cell proliferation were evaluated by immunohistochemistry. The mRNA expressions of angiogenic growth factors (vascular endothelial growth factor (VEGF) and angiopoietins 1 and 2) and their receptors (flt-1, flk-1, Tie-1 and Tie-2) in the whole liver were evaluated by semi-quantitative RT-PCR. RESULTS: Significant elevation of endothelial cell proliferation started at 48 h and peaked at 72 h after hepatectomy. The ratio of sinusoids to liver tissue area initially decreased at 72 h, and thereafter, significantly increased at 96 h. VEGF related factors had early peaks, which coincided with the endothelial proliferation. flt-1, flk-1 and VEGF expressions peaked at 24, 48 and 72 h, respectively. angiopoietin/Tie factors peaked at 96 h, except Ang-2, which gradually increased and peaked at 168 h. CONCLUSIONS: During liver regeneration, hepatocyte proliferation was followed by endothelial cell proliferation. The VEGF family and angiopoietin/Tie system may have distinct roles in angiogenesis, with an enhanced expression of the VEGF family in the early phase of regeneration followed by angiopoietin/Tie expression.
Congenital absence of platelet glycoproteins IIb and IIIa (GPIIb and GPIIIa) results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIb and GPIIIa are present as a heterodimeric, noncovalent complex in the platelet plasma membrane and function as the fibrinogen receptor. To characterize synthesis of these two proteins, RNA isolated from a human leukemia cell line that contains GPIIb and GPIIIa was translated in a wheat germ cell-free system. Polyclonal antibodies specific for each protein immunoprecipitated distinct [35S]methionine-labeled precursors, indicating that GPIIb and GPIIIa are translated from separate mRNAs. Moreover, using specific antibodies against either intact unreduced GPIIb or the beta subunit, we obtained evidence for synthesis of a common polypeptide precursor for GPIIb alpha and GPIIb beta. Based on experiments using microsomal membranes, it appears that GPIIb is integrated into the platelet membrane with little or no cytoplasmic component. These results suggest that precursors of GPIIb and GPIIIa may be encoded by separate genes and that each precursor is processed before delivery to the plasma membrane.
We examined two sets of genes expressed early in the developmental cycle of Dictyostelium discoideum that appear to be regulated by cyclic AMP (cAMP). The transcripts of both sets of genes were not detectable in vegetative cells. During normal development on filter pads, RNA complementary to these genes could be detected at about 2 h, peaked around 6 to 8 h, and decreased gradually thereafter. Expression of these genes upon starvation in shaking culture was stimulated by pulsing the cells with nanomolar levels of cAMP, a condition that mimics the in vivo pulsing during normal aggregation. Expression was inhibited by caffeine or by continuous levels of cAMP, a condition found later in development when in vivo expression of these genes decreased. The inhibition of caffeine could be overcome by pulsing cells with cAMP. These results suggest that the expression is mediated via the cell surface cAMP receptor, but does not require a rise in intracellular cAMP. mRNA from a gene of the second class was induced upon starvation, peaked by 2.5 h of development, and then declined. In contrast to the other genes, its expression was maintained by continuous levels of cAMP and repressed by cAMP pulses. These and other results on a number of classes of developmentally regulated genes indicates that changing levels of cAMP, acting via the cell surface cAMP receptor, are involved in controlling these groups of genes. We also examined the structure and partial sequence of the cAMP pulse-induced genes. The two tandemly duplicated M3 genes were almost continuously homologous over the sequenced portion of the protein-coding region except for a region near the N-terminal end. The two M3 genes had regions of homology in the 5' flanking sequence and showed slight homology to the same regions in gene D2, another cAMP pulse-induced gene. D2 showed extremely significant homology over its entire sequenced length to an acetylcholinesterase. The results presented here and by others suggest that expression of many early genes in D. discoideum is regulated via the cell surface cAMP receptor. We expect that many of these genes may play essential roles in early Dictyostelium development and could code for elements of the cAMP signal transduction pathway involved in aggregation.
The cadherin gene family encodes calcium-dependent adhesion molecules that promote homophilic interactions among cells. During embryogenesis, differential expression of cadherins can drive morphogenesis by stimulating cell aggregation, defining boundaries between groups of cells and promoting cell migration. In this report, the expression patterns of cadherins were examined by immunocytochemistry and in situ hybridization in the embryonic kidney, during the time when mesenchymal cells are phenotypically converted to epithelium and the pattern of the developing nephrons is established. At the time of mesenchymal induction, cadherin-11 is expressed in the mesenchyme but not in the ureteric bud epithelium, which expresses E-cadherin. The newly formed epithelium of the renal vesicle expresses E-cadherin near the ureteric bud tips and cadherin-6 more distally, suggesting that this primitive epithelium is already patterned with respect to progenitor cell types. In the s-shaped body, the cadherin expression patterns reflect the developmental fate of each region. The proximal tubule progenitors express cadherin-6, the distal tubule cells express E-cadherin, whereas the glomeruli express P-cadherin. Ultimately, cadherin-6 is down-regulated whereas E-cadherin expression remains in most, if not all, of the tubular epithelium. Antibodies generated against the extracellular domain of cadherin-6 inhibit aggregation of induced mesenchyme and the formation of mesenchyme-derived epithelium but do not disrupt ureteric bud branching in vitro. These data suggest that cadherin-6 function is required for the early aggregation of induced mesenchymal cells and their subsequent conversion to epithelium.
Two of the most potent cytokines regulating anterior pituitary cell function are leukemia inhibitory factor (LIF) and interleukin (IL)-6, which belong to the cytokine family using the common gp130 signal transducer. Recently, the expression and action of two other members of this family, IL-11 and ciliary neurotrophic factor (CNTF), on different cell lines has also been demonstrated. We studied the expression of the specific receptor subunits for CNTF in mammotropic, non-functioning and somatotropic tumors and the action of CNTF and IL-11 in the regulation of hormone secretion in these and normal pituitary cells. The mRNA for the alpha chain specific for the CNTF receptor was detected by Northern blot in tumors secreting prolactin (PRL) and GH and in non-functioning tumors. We found that both IL-11 and CNTF exerted a similar stimulatory effect on GH mRNA expression in somatotropic monolayer cell cultures from acromegalic tumors, but these cytokines had no significant influence on GH secretion. CNTF stimulates prolactin secretion in lactotropic monolayer cell cultures from patients with prolactinoma. In monolayer cell cultures from normal rat anterior pituitary, IL-11 and CNTF had no significant effect on the release of either GH or PRL, or on GH mRNA. However, when the cells were cultured in aggregate cultures, in which the three-dimensional structure of the cells is reconstituted, both cytokines, in doses at which they had no effect on monolayer cultures, significantly stimulated both PRL and GH secretion. These data show that IL-11 and CNTF may act as regulatory factors in anterior pituitary cells, in which the three-dimensional structure of the gland is of critical importance.