Multiple molecular forms of endothelial cell factor VIII related antigen

Heterogeneous factor VIII related antigen isolated from endothelial cell postculture medium was characterized. On crossed immunoelectrophoresis, slow moving less anodal populations of factor VIII related antigen molecules were more prominent in endothelial cell postculture medium than in plasma. The protein synthesized by endothelial cells appeared as two discrete protein bands of different molecular weight in sodium dodecyl sulfate polyacrylamide gels. In contrast the factor VIII related antigen isolated from plasma moved as a single protein band in an identical gel system. The factor VIII related antigen from endothelial cell postculture medium was immunoisolated using monospecific rabbit antibody to normal human plasma factor VIII related antigen, electrophoresed on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with ¹²⁵I, trypsinized and subjected to peptide mapping using two-dimensional high voltage electrophoresis and thin-layer chromatography. The two forms of endothelial cell factor VIII antigen had virtually identical peptide maps. These studies suggest that the molecular heterogeneity of the factor VIII related antigen sytem reflects polymeric associations of identical subunits. Circulation in vivo may alter the ratio of polymer subsets.     

Identification of tumour-induced changes in endothelial cell surface protein expression: an in vitro model

The selective destruction of the supporting vasculature of tumours has been proposed as a means of therapy. Fundamental to this approach is the identification of suitable targets on tumour-endothelium. To detect proteins that may be up-regulated on the luminal (apical) surface of tumour-associated endothelium confluent endothelial cells were examined following incubation with tumour cell conditioned medium (TCM) from, or co-culture with, a range of breast carcinoma and small cell lung carcinoma (SCLC) cell lines. Exposed endothelial membrane proteins were labelled with sulpho-NHS-biotin and detected by enhanced chemiluminescence following two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blotting. TCM induced varying levels of proliferative activity in endothelial cells; generally breast TCM contained greater mitogenic activity than SCLC TCM. Exposure of human breast and lung microvascular, and umbilical vein endothelial cells to soluble tumour cell factors from several breast cancer and SCLC cells lines produced similar changes in luminal protein profiles: Breast cancer cells and in particular the MDA-MB-231 cell line induced the most pronounced changes. The expression of six proteins was altered consistently on endothelial cells stimulated with soluble tumour cell factors. However, similar changes were observed following incubation with ECGS suggesting that they were related to endothelial cell proliferation per se. As these proteins were altered in breast and lung microvascular, and umbilical vein endothelial cells stimulated by a variety of breast cancer and SCLC cell lines they support the potentially broad applicability of anti-vascular approaches targeted at the endothelium.     

Endothelial cell proliferation induced by HARP: implication of N or C terminal peptides

HARP (Heparin Affin Regulatory Peptide) is a 18-kDa secreted protein displaying high affinity for heparin. It has neurite outgrowth-promoting activity, while there are conflicting results regarding its mitogenic activity. In the present work, we studied the effect of human recombinant HARP expressed in bacterial cells as well as two peptides (HARP residues 1-21 and residues 121-139) on the proliferation of three endothelial cell types derived from human umbilical vein (HUVEC), rat adrenal medulla (RAME), and bovine brain capillaries (BBC) either added as a soluble form in the cell culture medium or coated onto the culture plate. HARP added in a soluble form in the culture medium had no effect on the proliferation of BBC, HUVEC, and RAME cells. However, when immobilized onto the cell culture plate, HARP had a concentration-dependent mitogenic effect on both BBC cells and HUVEC. The peptides presented as soluble factor induced a significant concentration-dependent mitogenic effect on BBC cells but only a small effect on HUVEC and RAME cells. When they were immobilized onto the cell culture plate, the mitogenic effect was much greater. The most responsive cells were BBC that expressed and secreted in the culture medium the higher amounts of HARP.     

Human adipose tissue endothelial cells promote preadipocyte proliferation

Adipogenesis is preceded by development of a microvascular network, and optimal functioning of adipose tissue as an energy store and endocrine organ is dependent on extensive vascularization. We have examined the role of endothelial cell-derived factors that influence the proliferation of human preadipocytes. Microvascular endothelial cells and preadipocytes were isolated from human omental and subcutaneous adipose tissue biopsies by use of a developed procedure of collagenase digest, immunoselection, and differential trypsinization. Conditioned medium from microvascular endothelial cell cultures promoted the proliferation of preadipocytes (P = <0.001) and (to a lesser extent) other cell types. No depot-specific differences in mitogenic capacity of microvascular endothelial cell medium or of preadipocyte response were observed. These results indicate that adipose tissue endothelial cells secrete soluble adipogenic factor(s).     

Release of an endothelial cell growth factor from cultured porcine thyroid follicles

It has been proposed from in vivo studies that thyroid angiogenesis during thyroid enlargement may be due to paracrine mitogenic factors released by epithelial thyroid cells. To study this paracrine growth regulating communication between thyroid cells and endothelial cells in vitro, culture medium from isolated porcine thyroid follicles was investigated for a growth promoting effect on porcine aortal endothelial cells. Serum-free conditioned medium (CM) from thyroid follicles in suspension culture contains a dose-related mitogenic activity which stimulates endothelial cell growth up to 197%. Stimulation of the thyroid follicles with TSH (1 mU/ml) significantly reduced the mitogenic activity for endothelial cells in CM to 131%. Thyroid hormones had no influence on mitogenic activity in CM. When follicles were treated with iodide (20 microM) during CM production, no proliferation of endothelial cells was observed by this CM. In contrast, CM from epidermal growth factor-treated thyroid follicles significantly enhanced the mitogenic activity for endothelial cells up to 235%. The mitogenic activity was precipitable by saturated ammonium sulfate, showed high affinity to heparin by chromatography on heparin-sepharose, and was abolished after treatment of CM with trypsin. On gel electrophoresis the heparin-binding fraction showed a double band with a mol wt of 15 and 15.5 k. These data show a paracrine mitogenic activity on endothelial cells released by thyroid follicles which is regulated by TSH, epidermal growth factor, and iodide in parallel with the direct effect of these substances on thyroid cell growth. The data suggest that the mitogenic factor is a polypeptide, which belongs to the heparin-binding growth factors.     

Suramin inhibits C6 glioma-induced angiogenesis in vitro

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-μm thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-μm pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently from networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiongenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.     

Identification of an endothelial cell growth-inhibitory activity produced by human monocytes

The control of endothelial cell proliferation is important in a variety of processes including wound healing and tumor-induced angiogenesis. We have observed that normal unstimulated human monocytes isolated from the blood can inhibit human endothelial cell proliferation. Monocyte-conditioned medium was fractionated by gel filtration chromatography, yielding a 175-fold enrichment of a growth inhibitory activity, designated monocyte-derived endothelial cell inhibitory factor (MECIF). MECIF was found to be protease sensitive, resistant to acid treatment, and heat labile. When conditioned medium was subjected to HPLC gel filtration, the inhibitory activity was eluted as a single peak with a molecular weight of 50-70 kDa. Several characteristics distinguish MECIF from previously described monocyte/macrophage-derived inhibitory factors. Unlike TGF-beta, MECIF is heat labile and does not induce a mitogenic response in growth-arrested normal rat kidney cells. In addition, polyclonal antibodies specific for TGF-beta or INF-gamma do not inhibit MECIF activity. MECIF preparations show low levels of TNF-alpha, insufficient to promote the observed growth inhibitory effect. MECIF activity on human endothelial cells was found to be dose dependent and reversible. MECIF also appeared to be target cell selective in that it did not significantly alter the growth of human smooth muscle cells or skin fibroblasts. These data suggest that monocyte-derived factors may play a key role in inhibiting endothelial cell proliferation.     

Protein synthesis and secretion in human decidua of early pregnancy

Proteins synthesized and secreted by first trimester decidua in primary culture were identified. Explants were cultured for 24 h, in RPMI-1640 or Dulbecco's Modified Eagles Medium containing the radioactive amino acid 35S-methionine or 3H-proline. Electron microscopy of explants before and after 24 h of culture demonstrated the relative purity of the decidua, maintenance of cell integrity, and ultrastructural features indicative of active protein synthesis and secretion. Proteins synthesized and secreted by the explants into the medium were analyzed by fluorography of one-dimensional polyacrylamide gels in the presence of sodium dodecyl sulfate. By comparison of radiolabeled proteins from four women, eight 35S-methionine-labeled bands of 78, 70, 60, 50, 43, 34, 25, and 23 kDa were identified as common decidual peptides. A comparison of autoradiographs of the medium from decidual cultures to decidual cell homogenates showed that seven of these peptides were enriched in the culture medium. When labeled peptides from fibroblast cultures were compared to labeled proteins from decidual cultures each of the common decidual peptides (except the 70 kDa protein) occurred only in the decidual culture medium. Comparison of 3H-proline and 35S-methionine-labeled decidual proteins revealed that the 78, 70, 60, 50, and 34 kDa proteins were of similar fluorographic intensity when labeled with the two different amino acids. The 43, 25, and 23 kDa proteins appeared to contain more methionine, and proteins at 36, 20, 13, and 12 kDa were proline-rich, but contained less methionine. The seven decidual explant-specific, 35S-methionine-labeled secreted proteins were concentrated and purified by preparative gel electrophoresis, and antisera were generated to four of the putative decidual secretory proteins.     

Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of progesterone on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium

Summary The pattern of proteins synthesized and secreted in response to progesterone by guinea-pig endometrial epithelial cell cultured with estradiol-17 was investigated. Glandular epithelial cells were maintained in culture for 3 days on growth medium, then washed three times with a steroid-free medium. After this period, 2 × 10^-8 M estradiol-17 or 2 × 10^-8 M estradiol-17 plus 5 × 10^-7 M progesterone were added to the medium for 48 h. To study biochemical changes, the proteins were labeled by a 6 h pulse of ³⁵S-methionine. The proteins in medium and in cells were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. The addition of progesterone to estradiol-17 in the culture medium caused a change in the patterns of cellular and secreted proteins: one-dimensional gel electrophoresis showed that variation of 8 cellular proteins and 12 secreted proteins was caused by progesterone. Induction of individual proteins by progesterone treatment was observed by two-dimensional gel electrophoresis: one cellular protein (Mr 49000; pI 5.90) and one secreted protein (Mr 14300; pI 4.80) were specifically induced and might serve as markers of progesterone action.     

Characterization of a Mr 20,000 basic fibroblast growth factor-like protein secreted by normal and transformed fetal bovine aortic endothelial cells

A mitogenic and plasminogen activator (PA)-inducing activity for endothelial cells has been identified in serum-free culture medium of normal AG 7680 and transformed tumorigenic GM 7373 fetal bovine aortic endothelial (FBAE) cells. The activity binds to heparin-Sepharose and it is quenched by polyclonal anti-human placental basic fibroblast growth factor (bFGF) antibodies. In the serum-free conditioned medium of FBAE cells, the anti-bFGF antiserum recognizes an immunorective Mr 20,000 molecule which co-purifies with the mitogenic and PA-inducing activity on a heparin-Sepharose column. The partially purified Mr 20,000 bFGF-like molecule competes with the typical Mr 18,000 125I-bFGF form for the binding to high-affinity bFGF receptors in intact GM 7373 cells. Immunoprecipitation of biosynthetically labeled GM 7373 cells with anti-bFGF antiserum confirms the presence of a Mr 20,000 bFGF-like molecule in the conditioned medium of these cells and identifies the typical Mr 16,000 and Mr 18,000 bFGF forms and two high-molecular-weight immunoreactive Mr 22,000 and Mr 25,000 bFGF forms in their cell extract. Immunoreactive Mr 20,000 bFGF is detectable also in the conditioned medium of transformed nontumorigenic FBAE GM 7372 cells and of adult bovine aortic endothelial cells, but not in the culture medium of nonendothelial cell types, including rat and mouse fibroblasts, human hepatoma, and human endometrial adenocarcinoma cells. The results indicate that bovine endothelial cells secrete a Mr 20,000 bFGF-like molecule which shares several biological, biochemical, and immunological characteristics with the typical cell-associated Mr 18,000 bFGF.     

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-β1

Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β₁. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β₁ to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β₁ with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Supported by a Post-Doctoral Fellowship from the Swiss National Science Foundation (OK) and grant HL-RO1-28373 (JAM) from the Department of Human Services, Public Health Service, Washington, D.C.     

Double-labeling and high precision comparison of complex protein patterns on two-dimensional polyacrylamide gels

Combination of double-labeling and two-dimensional polyacrylamide gel electrophoresis allows high precision comparison of proteins from two different cultured cell lines. Extracts of cell lines labeled with ¹⁴C and ³H, respectively, are mixed and analyzed on a single two-dimensional gel. Fluorography detects both isotopes, but autoradiography through carbon paper detects only ¹⁴C. Proteins can be classified into classes common to both cell lines and those specific to each cell line. The method should be useful for a wide range of biochemical studies.     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18–116 erg cm⁻². To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm⁻² for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm⁻². The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Y _s <50 erg cm⁻²), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (ΔF _adh) show that ΔF _adh decreases with increasingY _s , and equals zero around 45 erg cm⁻² for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

A method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin

Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.     

Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture

Removal of vascular smooth muscle cells (SMC) from their native environment alters the biochemical and mechanical signals responsible for maintaining normal cell function, causing a shift from a quiescent, contractile phenotype to a more proliferative, synthetic state. We examined the effect on SMC function of culture on two-dimensional (2D) substrates and in three-dimensional (3D) collagen Type I gels, including the effect of exogenous biochemical stimulation on gel compaction, cell proliferation, and expression of the contractile protein smooth muscle alpha-actin (SMA) in these systems. Embedding of SMC in 3D collagen matrices caused a marked decrease in both cell proliferation and expression of SMA. The presence of the extracellular matrix modulated cellular responses to platelet-derived growth factor BB, heparin, transforming growth factor-beta1, and endothelial cell-conditioned medium. Cell proliferation and SMA expression were shown to be inversely related, while gel compaction and SMA expression were not correlated. Taken together, these results show that SMC phenotype and function can be modulated using biochemical stimulation in vitro, but that the effects produced are dependent on the nature of the extracellular matrix. These findings have implications for the study of vascular biology in vitro, as well as for the development of engineered vascular tissues.     

Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells.

Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.     

Molecular biology of C4 photosynthesis in Zea mays: differential localization of proteins and mRNAs in the two leaf cell types

Summary We have investigated the molecular basis of differential localization of enzyme activities in mesophyll(M) and bundle-sheath (B) cells of maize leaves. M protoplasts and B strands were prepared by enzymatic digestions and mechanical treatment of secondary leaves. Soluble and thylakoid membrane proteins from the two cell types were compared by one- and two-dimensional gel electrophoresis and quantitative rocket immunoelectrophoresis. In addition, several thylakoid polypeptides were identified by crossed immunoelectrophoresis using monospecific antibodies. M and B thylakoids show quantitative and qualitative differences in their polypeptide compositions. While the M thylakoids contain the normal complement of polypeptides, the B thylakoids are deficient in ferredoxin-NADP⁺ reductase, photosystem II reaction center polypeptides, and the light-harvesting chlorophyll a/b-protein complex. Comparison of the soluble proteins by two-dimensional gel electrophoresis revealed marked differences between M and B cells. The major proteins of one cell type are clearly absent from the other. These differences are paralleled by differences in the in vitro translation products of poly A⁺ RNA isolated from the two cell types. Immunoprecipitation experiments showed that mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) is localized exclusively in B cells, whereas mRNA encoding phosphoenolpyruvate carboxylase is detected only in M cells. cDNA clones encoding the carboxylase rbcS and the chlorophyll a/b binding protein were used as probes in Northern blot analysis. M cells contain no detectable RNA encoding rbcS but have a higher steady state level of RNA encoding the chlorophyll a/b-binding polypeptide compared to B cells. Taken together, our results demonstrate that differential gene expression in the two leaf cell types is regulated at the level of translatable mRNA, and, for at least two proteins, at the level of steady-state RNA.     

Comparison of proteins synthesized by anterior and posterior regions of Dictyostelium discoideum pseudoplasmodia.

In migrating pseudoplasmodia of the cellular slime mold Dictyostelium discoideum, cells in approximately the anterior quarter of the structure give rise to stalk cells, while the remainder produce spore cells. Certain biochemical and structural components have been found to differ between cells occupying these two positions, indicating that some differentiation has already occurred by this stage. To evaluate the degree of this differentiation we have compared the proteins being synthesized in different regions of the pseudoplasmodia. Migrating pseudoplasmodia were labeled with [³⁵S]methionine and cut into five segments, and the labeled proteins were resolved by two-dimensional gel electrophoresis and visualized by autofluorography. Of 500 polypeptides detected, 57 showed regional variations in labeling. Nearly all of these differences in labeling occurred between the anterior fifth and posterior four-fifths of the pseudoplasmodia, indicating that by this stage a marked degree of differentiation has occurred between the two cell types.