Identification of a CArG Box-Dependent Enhancer within the Cysteine-Rich Protein 1 Gene That Directs Expression in Arterial but Not Venous or Visceral Smooth Muscle Cells

Smooth muscle cells (SMCs) are heterogeneous with respect to their contractile, synthetic, and proliferative properties, though the regulatory factors responsible for their phenotypic diversity remain largely unknown. To further our understanding of smooth muscle gene regulation, we characterized the cis-regulatory elements of the murine cysteine-rich protein 1 gene (CRP1/Csrp1). CRP1 is expressed in all muscle cell types during embryogenesis and predominates in vascular and visceral SMCs in the adult. We identified a 5-kb enhancer within the CRP1 gene that is sufficient to drive expression in arterial but not venous or visceral SMCs in transgenic mice. This enhancer also exhibits region-specific activity in the outflow tract of the heart and the somites. Within the 5-kb CRP1 enhancer, we found a single CArG box that binds serum response factor (SRF), and by mutational analysis, demonstrate that the activity of the enhancer is dependent on this CArG element. Our findings provide further evidence for the existence of distinct regulatory programs within SMCs and suggest a role for SRF in the activation of the CRP1 gene.     

Engrailed expression and body segmentation in the honeybee Apis mellifera

Summary Honeybee embryos were stained with a monoclonal antibody raised against the Drosophila engrailed protein. The antibody was found to label rows of nuclei in the transverse grooves that form the earliest external sign of metameric germ band organization. These grooves demarcate metameric units about seven cell rows wide, of which about three rows with reduced apical cell surfaces account for the grooves. The en stripes appear in the grooves as soon as these form and grow from one to about four cells in width and thus completely overlap the groove. During the rudimentary germ band retraction, the grooves shift slightly backwards relative to both the en stripes and the trachdeal pits. The spatio-temporal pattern by which the series of grooves and stripes arises is quite striking. Both become visible first in the gnathal and thoracic regions, then in the pregnathal parts of the head and in the abdomen. The stripes arise essentially in an antero-posterior sequence. In addition, the earliest stripes to form display a pattern of alternating intensities whereas the later stripes, those in the abdomen, arise with approximately equal strength. The latter trait was earlier observed in the grasshopper, while the former is known from Drosophila where, however, the strong stripes correspond to the weak stripes in the honeybee.     

bcd: A mutation affecting the width of organelle domains in the cortex of Tetrahymena thermophila

Summary A single-gene recessive mutation, bcd (broadened cortical domains), of Tetrahymena thermophila is characterized by a variable broadening of the spatial domains within which cortical organelles, including both the contractile vacuole pores (CVP) and oral apparatus (OA), are formed. The phenotype is not temperature-sensitive. During the development of the organelles of the mutant prior to cell division, extra CVPs and extra oral primordia (OP) appear near ciliary rows adjacent to the rows at which these structures normally form. In the later stages of development, some, but not all, of these extra structures are resorbed, or in the case of the oral domain, multiple adjacent OPs may be completely or partially integrated into a single enlarged OA. When multiple OAs persist, one or more of these may display a reversed orientation reminiscent of those encountered in janus mutants. However, unlike janus, bcd cells do not express any sign of a mirror-image global organization.Our results can best be accounted for by postulating that the bcd mutation affects some common determinant of the widths of both CVP and OA domains. Studies are in progress which explore the relationship between this width-determining mechanism(s) and the mechanism(s) determining the location of cortical organelles around the cell circumference.     

Bristle patterns and clones along a compartment border in the anterior wing margin ofDrosophila hydei

Summary The bristle pattern along the first longitudinal vein of the wing ofD. hydei differs from that ofD. melanogaster. Instead of a triple row,D. hydei and some allied species show a pattern of five parallel bristle rows of which the medial row (MR) is comparable to the medial triple row (MTR) ofD. melanogaster. Cells of the MR can be made homozygousyellow (y) by induction of mitotic recombination in heterozygousy/y ⁺ females. Until 70 h after egg laying (AEL), the MR clones inD. hydei overlap with one or more of the accompanying dorsal and ventral bristle rows. Between 70 and 120 h AEL the MR clones only overlap with dorsal bristle rows. Some time later they also become separated from both dorsal rows. The resulting MR clone pattern fits with the overall longitudinal clone pattern in the wing blade ofD. melanogaster described by Bryant (1970) and others. The MR clones inD. hydei, however, often show a fragmented appearance with many indentations of the surroundingy ⁺ tissue even when induced after fixation of the DV compartment boundary. This result contrasts with the commonly held notion, derived from work withD. melanogaster, that compartment boundaries are smooth lines.     

Actin filament-organized local cortical endoplasmic reticulum aggregations in developing stomatal complexes of grasses

Endoplasmic reticulum (ER) immunolabeling in developing stomatal complexes and in the intervening cells of the stomatal rows (ICSRs) of Zea mays revealed that the cortical-ER forms distinct aggregations lining locally expanding wall regions. The polarized subsidiary cell mother cells (SMCs), displayed a cortical-ER-patch lining the wall region shared with the inducing guard cell mother cell (GMC), which disorganized during mitosis. In dividing SMCs, ER persisted in the preprophase band region and was unequally distributed in the mitotic spindle poles. The subsidiary cells (SCs) formed initially an ER-patch lining the common wall with the GMC or the young guard cells and afterwards an ER-ring in the junction of the SC wall with the neighboring ones. Distinct ER aggregations lined the ICSR wall regions shared with the SCs. The cortical-ER aggregations in stomatal cells of Z. mays were co-localized with actin filament (AF) arrays but both were absent from the respective cells of Triticum turgidum, which follow a different morphogenetic pattern. Experimental evidence showed that the interphase ER aggregations are organized by the respective AF arrays, while the mitotic ER aggregations by microtubules. These results revealed that AF and ER demarcated “cortical cytoplasmic domains” are activated below the locally expanding stomatal cell wall regions, probably via a mechanosensing mechanism triggered by the locally stressed plasmalemma/cell wall continuum. The probable role(s) of the local ER aggregations are discussed.     

Impact of intercropping aphid-resistant wheat cultivars withoilseed rape on wheat aphid (Sitobion avenae) and its natural enemies

The effects of intercropping of wheat cultivars and oilseed rape on the densities of wheat aphid, Sitobion avenae, and their arthropod natural enemies were evaluated. Three winter wheat cultivars with different resistant levels to S. avenae were used: ‘KOK’ (high resistance), ‘Xiaobaidongmai’ (low resistance) and ‘Hongmanghong’ (susceptible). The results showed that the densities of S. avenae were significantly higher on the monoculture pattern than on either the 8-2 intercropping pattern (eight rows of wheat with two rows of oilseed rape) or the 8-4 intercropping pattern (eight rows of wheat with four rows of oilseed rape). The mean number of predators and the mummy rates of S. avenae were significantly higher in two intercropping patterns than those in the monoculture pattern. The densities of S. avenae, ladybeetles, and mummy rate of S. avenae were significantly different among different wheat cultivars. The highest densities of S. avenae and ladybeetles were found on wheat cultivar Hongmanghong. The lowest densities of S. avenae associated with high mummy rate of S. avenae were found on wheat cultivar Xiaobaidongmai. The results showed that wheat-oilseed rape intercropping conserved more predators and parasitoids than in wheat monoculture fields, and partial resistance of wheat cultivar Xiaobaidongmai had complementary or even synergistic effects on parasitoid of S. avenae.     

Freeze fracture studies on the annelid septate junction

Summary Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80–120 Å, the distance between two particles (centre to centre) is 150–250 Å. Additionally striking rows of large particles (long diameter 150–200 Å). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100–150 Å, centre to centre distance approx. 250 Å) whereas the particles of the extracellular face (EF, mean diameter 200–250 Å) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.     

Experimental studies on the function of the cortical cytoplasmic zone of the preprophase microtubule band

Summary Centrifugation of young seedlings ofTriticum durum andTriticum aestivum for 8–10 hours at 1,500–2,000 x g causes a serious disorder of the spatial organelle relationships in the interphase as well as the preprophase and mitotic subsidiary cell mother cells (SMCs). The nucleus, most organelles and cytoplasm are displaced to the centrifugal end of the cell, while the vacuoles lie at the other end. However, after centrifugation, the preprophase microtubule bands (PMBs) are nucleated and remain at the expected position close to the guard cell mother cells (GMCs). In some elongated SMCs the PMBs become completely separated from the nucleus. The mitotic spindle exhibits variable orientation and is usually formed at some distance from the PMB cortical zone.Cytokinesis in SMCs is spatially highly disturbed and the cell plate shows a variety of unpredictable dispositions, which seem to be determined by: 1. the position of the preprophase-prophase nucleus and the orientation of the mitotic spindle as well as their spatial relationships to the PMB cortical zone, and 2. the space available for cell plate growth. Many of the daughter cells exhibit a highly variable shape and size in different planes. Usually one edge of the cell plate partly or totally joins the anticlinal parent wall adjacent to the PMB cortical zone.In some SMCs ofZea mays andTriticum aestivum, the junction regions of the periclinal walls with the anticlinal ones, lined by the PMB cortical zone in normal SMCs, are detectably thickened after the arrest of mitosis and the prevention of interphase microtubule formation by a prolonged colchicine treatment. In a small number of protodermal cells of the same plants, participating in the development of stomatal complexes, irregular wall bodies or incomplete wall sheets were formed at wall regions lined by the PMB cortical zone.The presented observations are in line with the following hypotheses: 1. the PMB cortical zone interacts with the growing edges of the cell plate attracting it to fuse with the underlying parent wall when the latter approaches the former at a critical distance, and 2. in SMCs particular regions of the PMB cortical zone and/or the adjacent plasmalemma promote the local wall deposition in the absence of microtubules.     

Cell death in late embryogenesis of the leech Helobdella

Cell death was characterized during stages 8 and 9 in the leech Helobdella with a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Using confocal analysis, the positions of dying cells were compared to rows of cells expressing the leech engrailed protein ht-en and to fluorescently marked cell lineages. Dying cells were present in diverse tissues. Some dying cells were in no obvious pattern, and others were in segmentally iterated patterns. Particular attention was paid to the ectoderm and mesoderm, where most of the cells examined died over a period equivalent to 1–4 h at 25°C. Segmentally iterated rows of dying cells were observed in the mesoderm just beneath the nf-derived ht-en expressing cell rows at a time when ht-en expressing cells were beginning to disappear. The position of these dying cell rows was consistent with a role in the partial deterioration of the septum. Received: 12 October 1998 / Accepted: 8 February 1999     

Where may reaction–diffusion mechanisms be operating in metameric patterning of Drosophila embryos?

Two general features of metameric patterning in Drosophilaare considered: (1) maintenance of a constant number of metameres (segments or parasegments) in the face of variation in length of the embryo; (2) expression of pattern by on-off switchings of particular genes, with only three or four rows of cells to each element of pattern. For each of these features, the general strategic question is raised: could reaction-diffusion theory account for this? In both cases, it is answered affirmatively. For the second feature, this review contains some hitherto unpublished computer simulations by one of us (K. Y. T.), illustrating that a reaction-diffusion mechanism can be transformed into a patterned switching mechanism by nothing more than compartmenting of the diffusion region. For the scale of three compartments to one pattern repeat unit (representing three rows of cells to a segment) the switching pattern predicted by computation is two-off to one-on. This resembles the pattern of expression of the engrailed gene, posteriorly localized in each segment.     

Pattern as a function of cell number and cell size on the second-leg basitarsus ofDrosophila

Summary The bristle pattern of the second-leg basitarsus inDrosophila melanogaster was studied as a function of the number and size of the cells on this segment in well-fed and starved wild-type flies, in triploid flies, and in two mutants (dachs andfour-jointed) that have abnormally short basitarsi. The second-leg basitarsi of well-fed, wild-type flies from 22 otherDrosophila species were studied in a similar manner. There are typically 8 longitudinal rows of evenly-spaced bristles on the second-leg basitarsus, and in each row the number of bristles was consistently found to vary in proportion to the estimated number of cells along the segment, and the interval between bristles was found to vary in proportion to the average cell diameter on the segment. These correlations are interpreted to mean that the spacing of the bristles within each row is controlled developmentally, whereas the number of bristles is not. The interval between bristles is evidently measured either as a fixed number of cells or as a distance which indirectly depends upon cell diameter.     

下延叉蕨和芽胞叉蕨配子体发育的研究

利用光学显微镜详细观察了叉蕨属(Tectaria)下延叉蕨(Tectaria decurrens)和芽胞叉蕨(T.fauriei)的配子体发育过程,记录了配子体各发育阶段的特征。结果表明:(1)下延叉蕨和芽胞叉蕨的孢子均为单裂缝,具周壁,由周壁形成纹饰,孢子极面观椭圆形,赤道面观豆形或肾形。(2)孢子萌发方式为向心型。(3)原叶体发育方式为三叉蕨型。(4)成熟原叶体心脏形,两翼向斜上方扩展。(5)均具单细胞和多细胞毛状体,在丝状体或片状体阶段出现。研究认为,从配子体发育角度看,叉蕨属是较进化的陆生真蕨类;毛状体的类型、位置和出现时间等特征在叉蕨属种间存在差异,可作为该属种间分类的特征。     

Involvement of Delta and Nodal signals in the specification process of five types of secondary mesenchyme cells in embryo of the sea urchin, Hemicentrotus pulcherrimus

Secondary mesenchyme cells (SMCs) of the sea urchin embryo are composed of pigment cells, blastocoelar cells, spicule tip cells, coelomic pouch cells and muscle cells. To learn how and when these five types of SMCs are specified in the veg₂ descendants, Notch or Nodal signaling was blocked with γ‐secretase inhibitor or Nodal receptor inhibitor, respectively. All types of SMCs were decreased with DAPT, while sensitivity to this inhibitor varied among them. Pulse‐treatment revealed that five types of SMCs are divided into “early” (pigment cells and blastocoelar cells) and “late” (spicule tip cells, coelomic pouch cells and muscle cells) groups; the “early” group was sensitive to DAPT up to the hatching, and the “late” group was sensitive until the mesenchyme blastula stage. Judging from timing of the shift of Delta‐expressing regions, it was suggested that the “early” group and “late” groups are derived from the lower and the middle tier of veg₂ descendants, respectively. Interestingly, numbers of SMCs were also altered with SB431542; blastocoelar cells, coelomic pouch cells and circum‐esophageal muscles decreased, whereas pigment cells and spicule tip cells increased in number. Pulse‐treatment showed that the “early” group was sensitive up to the mesenchyme blastula stage, while the “late” group up to the onset of gastrulation. Thus, it became clear that precursor cells of the “early” and “late” groups, which are located in different regions in the vegetal plate, receive Delta and Nodal signals at different timings, resulting in the diversification of SMCs. Based on the obtained results, the specification processes of five types of SMCs are diagrammatically presented.     

c-Ha-rasEJ transfection of rat aortic smooth muscle cells induces epidermal growth factor responsiveness and characteristics of a malignant phenotype

Although the role of several protooncogenes, including sis, myc, and myb in the regulation of growth and differentiation of vascular cells has been examined in some detail, limited information is available on the contribution of ras genes to these processes. In the present studies the influence of oncogenic ras transfection on the phenotypic expression of rat aortic smooth muscle cells (SMCs) was examined. Cultured rat aortic SMCs during early passage (P₄) were transfected by lipofection with c-Ha-ras^EJ in a pSV2 neo vector or with pSV2 neo vector alone. Stable transfectants were selected in G418 over a 6-week period. Oncogene-transfected cells (ras-LF-1) exhibited differences in morphology and growth pattern relative to vector controls (neo-LF-1), or naive SMCs, including the development of prominent processes and the appearance of focal cellular arrangements giving rise to latticelike structures. Southern analysis revealed multiple integration of oncogenic ras in ras LF-1 cells. Transfection of c-Ha-ras^EJ was associated with a twofold increase in p21 levels relative to pSV2 vector controls demonstrating that exogenous ras was expressed in these cells. Overexpression of ras p21 afforded SMCs a lower serum requirement for growth compared to vector controls, anchor-age independent growth on soft agar, and acquisition of epidermal growth factor (EGF) responsiveness. Stimulation of serum-deprived SMCs with 5% fetal bovine serum (FBS) increased steady-state levels of c-Ha-ras mRNA in both ras-LF-1 and neo-LF-1 but ras induction was more pronounced in ras-transfected cells. α-smooth muscle (SM) actin gene expression was markedly reduced in ras-transfected cells relative to vector controls. These results show that transfection of c-Ha-ras^EJ into aortic SMCs induces an altered phenotypic state characterized by alterations in growth factor-related signal transduction and tumorigenic potential. © 1994 Wiley-Liss, Inc.     

PDGF-induced proliferation in human arterial and venous smooth muscle cells: molecular basis for differential effects of PDGF isoforms

Platelet‐derived growth factor (PDGF) has been implicated in the pathogenesis of arterial atherosclerosis and venous neointimal hyperplasia. We examined the effects of PDGF isoforms on smooth muscle cells (SMCs) from arterial and venous origins in order to further understand the differential responsiveness of these vasculatures to proliferative stimuli. Serum‐starved human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Whereas, proliferation of arterial SMCs was strongly stimulated by PDGF‐AA, venous SMCs showed no proliferative response to PDGF‐AA, but instead demonstrated a significantly greater proliferative response to PDGF‐BB than arterial SMCs. Part of this difference could be attributed to differences in PDGF receptors expression. There was a 2.5‐fold higher (P < 0.05) density of PDGF receptor‐α (PDGF‐Rα) and a 6.6‐fold lower (P < 0.05) density of PDGF‐Rβ expressed on arterial compared to venous SMCs. Concomitant with an increased proliferative response to PDGF‐AA in arterial SMCs was a marked PDGF‐Rα activation, enhanced phosphorylation of ERK1/2 and Akt, a transient activation of c‐Jun NH2‐terminal kinase (JNK), and a significant reduction in expression of the cell‐cycle inhibitor p27^kip1. This pattern of signaling pathway changes was not observed in venous SMCs. No phosphorylation of PDGF‐Rα was detected after venous SMC exposure to PDGF‐AA, but there was enhanced phosphorylation of ERK1/2 and Akt in venous SMCs, similar to that seen in the arterial SMCs. PDGF‐BB stimulation of venous SMC resulted in PDGF‐Rβ activation as well as transactivation of epidermal growth factor receptor (EGF‐R); transactivation of EGF‐R was not observed in arterial SMCs. These results may provide an explanation for the differential susceptibility to proliferative vascular diseases of arteries and veins. J. Cell. Biochem. 112: 289–298, 2011. © 2010 Wiley‐Liss, Inc.     

The Supramolecular Organization of Photosynthetic Membranes of the Thallus Stage of Porphyra leucosticta Thuret. (Bangiales,Rhodophyta) Visualized by Freeze-Fracture

The supramolecular organization of the thylakoid membranes of the thallus stage in the red alga Porphyra leucosticta is studied in replicas of rapidly frozen and fractured cells. Freeze-fractured thylakoid membranes exhibit only two types of fracture faces (EF and PF), because the lamellae in red algal chloroplasts are not stacked. The PF reveals numerous, tightly packed, but randomly distributed particles (density range from 2970 to 3550 particles/μm²). In contrast, the EF particles appear organized into parallel rows, the spacing of which is about 60–70 nm (about 8–9 particles occur along 100 nm of the line that is formed). Significant numbers of single EF particles are randomly distributed between the EF particle rows. The particles on both fracture faces (PF and EF) fall into two size classes: 10 to 11 nm (major size class) and 14 to 15 nm (minor size class).     

Ultrastructural Observations on the Spermatozoa of Two Temnocephalids (Platyhelminthes)

The spermatozoa of two Temnocephalidae collected in Uruguay, Temnocephala iheringi Haswell, 1893 (Host: Pomacea canaliculata) and Temnocephala axenos Monticelli, 1899 (Host: Parastacus varicosus), were studied with a transmission electron microscope. In both species the spermatozoon is made up of a long sperm body which bears at one extremity two free flagella of the 9+‘1’ flatworm pattern. The sperm body contains the nucleus, mitochondria, dense bodies and parallel, cortical, longitudinal singlet microtubules. Along a part of the sperm body the palissade of the microtubules displays a spiral pattern in transverse sections. A part of the perimeter of the cell is thus lined by two overlapping rows of microtubules. This spiral pattern of the singlets is considered as a synapomorphy of the family Temnocephalidae. The singlet microtubules are interconnected by two kinds of links: tangential links between neighbouring singlets in the same row and radial links between singlets belonging to two rows. The presence of these links suggests that this structure could be a motile system of singlets.     

Arrangement of bristles as a function of bristle number on a leg segment inDrosophila melanogaster

Summary The arrangement of bristles on a leg segment of the fruitflyDrosophila melanogaster was studied in various mutants that have abnormal numbers of bristles on this segment. Eighteen mutations at six different genetic loci were analyzed, plus five double or triple mutant combinations. Recessive mutations at theachaete-scute locus were found to affect distinct groups of bristles:achaete mutations remove mechanosensory bristles, whereasscute mutations remove mainly chemosensory bristles. Mechanosensory bristles remain uniformly spaced along the longitudinal axis unless their number decreases below a certain threshold, suggesting that spacing is controlled by cell interactions that cannot function when bristle cells are too far apart. Above a certain threshold, bristle spacing and alignment both become irregular, perhaps due to excessive force from these same interactions. Chemosensory bristles occupy definite positions that are virtually unaffected by removal of individual bristles from the array. Extra chemosensory bristles develop only near the six normal sites. At two of the six sites the multiple bristles tend to exhibit uniform longitudinal spacing — a property confined to mechanosensory bristles in wild-type flies. To explain the various mutant phenotypes the following scheme is proposed, with different mutations directly or indirectly affecting each step: (1) spots and stripes are demarcated within the pattern area, (2) one bristle cell normally arises within each spot, multiple bristle cells within each stripe, (3) incipient bristle cells inhibit neighboring cells from becoming bristle cells, and (4) the bristle cells within each stripe become aligned to form rows and then repel one another to generate uniform spacing.