Fig. 5. Generation of headless phenotype depends on functional paired domain and transactivation domain in ectopic Gsb. The ability of mutated Gsb proteins, which are encoded by the transgenes listed in the left column and whose structure is shown schematically in the middle column, to generate class I-IV headless phenotypes is indicated in the right column. For a detailed explanation, see text.

Figure 7. Schematic representation of the relevant components of the Fas-signaling pathways in relation to the cytoskeletal networks, the Golgi-sorting compartment, and the surface membrane. In simple epithelial cells like hepatocytes K8/K18, IFs and fibrillar actin (FA) are largely localized underneath the surface membrane, whereas microtubules (MT) extend throughout the cytoplasm. The Golgi compartment (GC), next to the nucleus (N) and the endoplasmic reticulum (not shown), is involved in the sorting out of the newly synthesized receptors, such as Fas and EGF Receptor (EGFR), before their targeting to appropriate membrane portions, via a microtubule-dependent process. A look at the steps of Fas-mediated apoptosis indicates that, once the receptor has properly reached the surface membrane, the intensity of the Fas activation becomes dependent on the density of the receptor and on its degree of clustering, an actin-dependent process. Stimulation by FasL leads to Fas trimerization and death-inducing signaling complex formation, which in turn can activate two distinct caspase-signaling pathways classified as type I and type II, respectively. Hepatocytes correspond to type II cells, and accordingly, a large part of the death signaling occurs via the release of cytochrome C (Cyto C) and subsequently the activation of procaspase-9, and so on. EGF provides protection against Fas-mediated apoptosis via an activation of the Akt pathway that leads to the inhibition of caspase-9. c-FLIP, a labile protective protein homologous to caspase but exhibiting an inactive catalytic site, regulates death receptor–mediated apoptosis. Significantly, the results reported here suggest that K8/K18 largely modulate the Fas density at the hepatocyte surface, in a manner that depends on the participation of microtubules. This dynamic interplay may involve a contribution of plectin, a known integrator of the cytoskeletal networks in many cell types.

Fig. 7.   A proposed model for involvement of ATM or ATR in activation of the apoptotic pathway. UVA or UVC activates ATM or ATR, respectively. In the UVA response, p53 and JNKs link ATM to activation of apoptotic pathways. ATR is linked by p53 and JNKs to the cellular apoptotic response induced by UVC. Sphingomyelinase or protein kinase C signaling to JNKs is also suggested to mediate activation of apoptosis by UVA or UVC, respectively. The arrows indicate direct (thick arrow) or indirect activation (thin arrow).

Flavonoid biosynthesis pathway. PAL: Phenylalanine ammonia-lyase; C4H: cinnamate-4-hydroxylase; 4CL: 4-coumaroyl:CoA-ligase; CHS: chalcone synthase; CHI: chalcone isomerase; FSI: flavone synthase; FSII: cytochrome P450 flavone synthase; IFS: cytochrome P450 isoflavone synthase; FHT: flavanone 3β-hydroxylase; DFR: dihydroflavonol 4-reductase; LAR: leucoanthocyanidin synthase; ANS: anthocyanidin synthase; 3GT: UDPG-flavonoid 3-O-glucosyl transferase. Red text indicates cytochrome P450 enzymes.

Fig. 4. erg6 strain treated for 5 minutes with BFA. (a) A tubular Golgi network with small dilations at the intersections of polygonal meshes (G) is seen next to a curved sheet of unperforated ER (er). At the left, a tubular network (white arrow) is seen in profile and is continuous with a portion of subplasmalemmal ER at the top-left. A small vacuole-like structure (V) is located next to the nucleus (N). Magnification x39,700. (b) Several small vacuole-like structures of variable staining densities (V) are in close contact with the nuclear envelope. At the bottom-left, one of these structures is continuous with an ER sheet (ER). A large intensely stained vacuole (*) is seen proximal to the nucleus (N). Magnification x38,500. (c) Next to the nucleus (N) and connected to a subplasmalemmal ER sheet (ER), there is a large tubular network (arrow) forming an ovoid mass. Light and dense vacuoles are also labeled (V). Magnification x40,100. (d) A tubular Golgi network with small dilations at the intersections of narrow, irregular meshes (G) is continuous at one side with a wide-meshed tubular network (arrow) and is connected on the right with a parallel array of anastomosed tubules (white arrow). N, nucleus. Magnification x43,350. (e) A parallel array of saccules (white arrow) is seen in proximity to an ER sheet (ER). A large vacuole is labeled (V). Magnification x82,100.

Fig. 1. General scheme illustrating the organization of autonomic parasympathetic control of airway functions. Central nervous system (CNS) cell groups (level 4) regulate the activity of airway-related vagal preganglionic neurons (AVPNs; level 3). Axons of the AVPNs, as the final common pathway out of the medulla oblongata, synapse on intrinsic ganglionic neurons within airway walls (level 2). These ganglia give rise to postganglionic fibers that control the function of specific effector targets (i.e., airway smooth muscle, mucous glands, and blood vessels). Sensory feedback for these systems occurs via sensory fibers originating from sensory ganglia (nodose and jugular ganglionic neurons). These fibers innervate sensory receptors and transmit information from the airways to the CNS. They modulate the activity of AVPNs through central multisynaptic pathways and they may affect function of effector organs via 2 ill-defined local networks that include axon reflex responses (level 1) and sensory innervation of intrinsic airway ganglia (level 2).

Fig. 1. CD induces PAI-1 expression in growth-arrested R22 cells as a function of concentration and time of exposure. CD (in final concentrations of 2, 5 or 10 µM) or DMSO vehicle alone (0 µM) was added to serum-deprived R22 cell cultures. Total RNA was isolated 4 hours later and northern blots hybridized with 32P-labeled PAI-1 and A50 cDNAs (A). PAI-1 mRNA levels increased as a function of CD concentration; maximal expression was attained with 10 µM CD (Hawks and Higgins, 1998{Go}). The histogram in (B) represents a quantitative analysis of PAI-1 mRNA abundance (mean±s.d.) from three different experiments normalized to the A50 signal. Immunoblot detection of PAI-1 protein on transfers of electrophoretic separations of R22 cell extracts (20 µg protein/lane) at various times after CD treatment (C). PAI-1 increased as early as 1 hour post-stimulation and reached maximal levels by approximately 4 hours.

Fig. 2. In situ distribution of CD-induced PAI-1 protein in quiescent R22 cells. The fractions of PAI-1 protein-expressing cells and the relative intensity of immunoreactive PAI-1 staining were assessed by fluorescence microscopy; both increased as a function of the time of CD exposure. Nuclei were visualized by DAPI staining. Although microfilament structure was progressively disrupted in 0.1 and 1.0 µM CD-treated cells, PAI-1 expression was only evident in 10 µM CD-stimulated cultures. Induction was more closely associated with changes in cell shape (Fig. 3) than actin skeleton disorganization. The apparent nuclear region accumulation of PAI-1 is probably the collective result of CD-induced cellular arborization and Golgi collapse around the nucleus.

Fig. 3. Inverse correlation between extent of morphological response of quiescent R22 cells to cytoskeletal disruption and PAI-1 transcript abundance. Cells maintained under serum-free culture conditions for 3 days were generally well spread with little membrane ruffling (A). A 4 hour exposure to 10 µM CD resulted in an arborized phenotype (B), although the cellular periphery remained well demarcated and easily visible for footprint imaging. Colchicine exposure (10 µM, 4 hours) (C) did not produce the same arborized effect as CD, although the cellular footprint area was similarly reduced (by 35%) in CD- and colchicine-treated cultures. Computerized imaging was done on 75 randomly selected individual cells per culture condition in three sets of 25 cells each. The footprint area was significantly reduced by treatment with either drug at the maximal PAI-1 transcript-inducing concentration of 10 µM (Fig. 4). Comparison between DMSO and CD-treated cells indicated an approximate reciprocal relationship between cellular footprint area and PAI-1 expression (D). The histogram in (D) illustrates the mean±s.d. (in arbitrary units) for triplicate determinations of footprint area and PAI-1 levels.