Analysis of the expression of p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus (Linnaeus, 1758).

Ontogenesis comprises a series of events including cell proliferation and apoptosis and resulting in the normal development of the embryo. Protein p53 has been described as being involved in the development of several animal species. The aim of this study was to analyze the expression of protein p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus and to correlate it with the histogenesis of structures present in this tissue. We used 24 embryos (at 12-20 days of incubation) and the thymus of two chickens. Immunohistochemical analysis was performed with the ABC indirect method. The expression of p53 in the gastroesophageal mucosa increased during the formation of the organ, mainly at the stages during which tissue remodeling and cell differentiation began. In the esophagus at stages 42 and 45, we observed immunoreactive (IR) cells in the surface epithelium and in early esophageal glands. In the proventriculus at stages 39-45, IR cells were present in the epithelial mucosa and rarely in the proventricular glands. In the gizzard after stage 42, we found IR cells mainly in the medial and basal epithelial layers of the mucosa and especially within the intercellular spaces that appeared at this phase and formed the tubular gland ducts. Thus, protein p53 occurs at key stages of development: in the esophagus during the remodeling of esophageal glands, in the proventriculus during the differentiation of the epithelium of the mucosa and in the gizzard during the formation of tubular glands.

Adenoviral gizzard erosion in broiler chickens in Germany.

Avian adenovirus infections cause important disease complexes in chickens, but many of the viruses also infect chickens without resulting in overt disease. Previously several outbreaks of gizzard erosions caused by a fowl adenovirus A serotype-1 (FAdV-1) were reported from Japan. Here we report an outbreak of gizzard erosions in 12 broiler flocks in Germany in 2011. Chickens had a reduced daily weight gain and a higher total mortality rate of up to 8%. The birds showed a severe detachment of the koilin layer and ulcerative to necrotizing lesions of the underlying mucosa. Histopathologically, necrotizing ventriculitis with basophilic, intranuclear inclusion bodies in epithelial cells was diagnosed. Immunohistochemistry, egg culture, and electron microscopic examination revealed adenovirus-like particles in the samples. No concurrent infectious agent could be identified. The virus was genotyped as FAdV-1 by PCR and subsequent sequencing. Phylogenetic analysis of the hexon loop L1 gene yielded 100% sequence identity to the chicken embryo lethal orphan strain. These findings suggest that outbreaks of adenoviral gizzard erosion can lead to significant economic losses in Germany and may be caused by an unusual virulent FAdV-1 strain.

Gastric gross and microscopic lesions caused by the UNAM-97 variant strain of infectious bronchitis virus after the eighth passage in specific pathogen-free chicken embryos.

Herein we report a description of gross and microscopic lesions found in specific pathogen-free chicken embryos caused by UNAM-97 infectious bronchitis virus (IBV) variant strain after the eighth passage. Embryos were divided into three groups and were inoculated in the chorioallantoic sac with 0.2 mL of UNAM-97, Mass 41 IBV (positive control), or sterile PBS (negative control). Forty-eight hours later the allatoic fluid was taken and used to start a cycle of eight passages through 9-d-old embryos. Seven days after the last passage, embryos were harvested and macroscopic lesions in all organs were recorded. Proventriculus and gizzard samples were obtained from all embryos and routinely processed for microscopic and ultrastructural examinations. The UNAM-97 IBV variant strain caused two macroscopic lesions uncommon for Mexican strains: thin-walled proventriculus and gizzard, as well as urate accumulation within an extra-embryonic peritoneal sac, leaving the body through the umbilical duct and accompanied by the yolk sac. At microscopic level, two relevant findings were observed to be produced by this variant. In the proventriculus, there was a decrease in the gland papillary branching, while the gizzard showed a significant reduction in mucosa thickness and tubular-to-proliferative-cell ratio, as well as an absence of hyaline secretion in the lumen. Electrodense material scattered in proventricular and gizzard cells was observed, with a structure consistent with that of coronaviruses. These pathological chicken embryo findings have not been reported as being caused by other IBV strains in Mexico.

Caldesmon exhibits a clustered distribution along individual chicken gizzard native thin filaments.

Our earlier immuno-gold electron microscopic study indicated that the distribution of caldesmon (CaD) on actin filaments is not uniform and is restricted to the vicinity of the myosin filaments (Mabuchi K, Li Y, Tao T, Wang CLA (1996) J Muscle Res Cell Motil 17: 243). This suggested that CaD could effectively inhibit muscle contraction, if those actin filaments in the vicinity of myosin filaments were saturated with CaD. In the present study we further examined the distribution of CaD along isolated, crude and purified native thin filaments (NTF). Individual CaD molecules on purified NTF were visualized with the aid of a chemical crosslinker, 5,5'-dithiobis(2-nitrobenzoic acid), which efficiently crosslinks CaD to actin (Graceffa P, Adam LP, Lehman W (1993) Biochem J294: 63), and of a monoclonal anti-CaD antibody. The results indicated that individual NTF had alternating CaD-rich and CaD-deficient regions. Moreover, we found that the N-termini of all CaD molecules in a given cluster appeared on the same side of an actin filament. Electron microscopic images of crude NTF immunoprecipitated by a polyclonal antibody clearly indicated that the spacing between the CaD clusters is wide enough for myosin heads to interact with actin subunits. Similar clustering of CaD was also observed in plastic embedded tissue sections. These observations raise the possibility that CaD is not acting as a simple on/off switch, but more likely as a modulator, of smooth muscle contraction.

Electron microscopic study of tropomyosin binding to actin filaments reveals tethered molecules representing weak binding.

The binding of tropomyosin (Tm) to actin filaments was studied using the rotary shadowing technique. Electron micrographs showed, in addition to free Tm molecules, some Tm molecules bound to actin filaments at only one end creating "tethered molecules." The ratio of free and bound Tm was determined by comparing densities of free Tm molecules in micrographs with those in Tm alone specimens. At a 1 to 7 ratio of 17 nM chicken gizzard Tm (gTm) and 120 nM actin filaments, the sum of the tethered (7%) and the free was only 29% of total Tm. The missing Tm molecules are likely to be those bound to actin filaments lengthwise and not visualized by the technique. At a lower ratio of Tm (3 nM) to actin (120 nM), the majority of bound Tm appeared to be tethered. At a lower actin concentration but a higher ratio of Tm (12 nM) to actin (24 nM), 57% of bound Tm appeared to be tethered. The populations of free and tethered molecules were also affected by different salt and glycerol concentrations or type of Tm used: more with rabbit skeletal (sTm) than with gTm. The tethering indicates that the interaction of Tm molecules to actin filaments is stronger at one end than at the rest of the molecule. The bundling of actin filaments was observed under certain conditions (e.g., approximately 2 microM actin and approximately 0.5 microM sTm), suggesting that both ends of Tm molecules can bind to actin filaments but one end binds much more weakly than the other. All of these observations support the fact that free and tethered molecules are under rapid equilibrium and that incidental encounter of the ends of two tethered Tm molecules which are at the right positions, or of one tethered and a free molecule, would strengthen the remaining actin binding site(s), resulting in tight binding, thus, the tethered molecules represent a weak form of binding.

Substructure of cytoplasmic dense bodies and changes in distribution of desmin and alpha-actinin in developing smooth muscle cells.

The substructure of assembling cytoplasmic dense bodies (CDBs) and changes in the distribution of desmin and alpha-actinin during development of smooth muscle were studied in gizzard samples from 10- and 16-day embryos and from 1- and 7-day post-hatch chickens. CDBs in these cells lack the density of CDBs in mature or adult smooth muscle cells and, thus, allow observations of the changes inside CDBs. The random filament orientation seen in younger embryonic cells is first modified to include relatively small patches of IFs that are somewhat straighter and are approaching a side-by-side arrangement. As development proceeds, the IFs in these arrays become straighter, are parallel over longer lengths of the IFs and later acquire the density characteristic of mature CDBs. Anti-desmin labeling in embryonic 10- and 16-day cells showed that desmin intermediate filaments (IFs) were located in the myofilament compartment but were concentrated in or near assembling CDBs. Anti-desmin labeling shifted to the perimeter of CDBs after hatching. Cross sections, longitudinal sections, and stereo pairs all show that IF profiles are present inside unlabeled assembling CDBs. Anti-alpha-actinin labeling was directly on CDBs and was often associated with the cross-connecting filaments (CCFs) (average diameter of 2-3nm) inside CDBs. We propose, based on these data, that desmin IFs, alpha-actinin-containing CCFs, and actin filaments are the principal components of the substructure of assembling CDBs. We also present a proposed model for CDB assembly.

The skeletal muscle transverse tubular Mg-ATPase: identity with Mg-ATPases of smooth muscle and brain.

Purified chicken skeletal muscle transverse tubule (T-tubule, TT) membrane preparations contain a very active Ca- or Mg-ATPase (EC 3.6.1.3) previously thought to be a T-system-specific marker enzyme. The function of the Mg-ATPase has not yet been determined although its prominent activity and concentration in junctional complexes supports a possible role in the excitation-contraction cycle. An essential component of the Mg-ATPase has been identified as a M(r) 85,000 glycoprotein (85k-GP). Polyclonal antibodies raised against the TT 85k-GP were specific and exhibited no cross-reactivity with other skeletal muscle proteins on immunoblots. Using this anti-85k-glycoprotein IgG, we have explored other chicken tissues to determine the tissue distribution of the 85k-GP. Antibody reactive polypeptides of M(r) 85,000 were found in gizzard smooth muscle, brain, heart, spleen, and lung tissue. The brain and smooth muscle membrane proteins were further purified and characterized for 85k-GP-associated Mg-ATPase activity. The brain and smooth muscle enzymes exhibited properties indistinguishable from the skeletal muscle TT-specific Mg-ATPase with regard to a series of activators and inhibitors, amino terminal amino acid sequences, and the effects of deglycosylation. The enzyme in all three tissues was inhibited by the diacylglycerol kinase inhibitor R 59022. Identification of the TT Mg-ATPase in gizzard smooth muscle has allowed the investigation of the Mg-ATPase membrane topology using isolated whole smooth muscle cells. The data support an ecto-orientation for the smooth muscle cell enzyme. Although the orientations of the brain and skeletal muscle enzymes have not been conclusively determined, the nearly identical properties of all three enzymes argues for an ecto-orientation of the active sites of these enzymes as well. The responsiveness of the three enzymes to regulatory lipids suggests that the ecto-Mg-ATPase may serve as a master switch controlling extracellular ATP concentrations and ligand accessibility to P1- and P2-purinoceptors. It is also proposed that the ecto-MgATPase may regulate ATP accessibility to ectoprotein kinases in a variety of tissues, and, in brain, the ecto-MgATPase may modulate the neurotransmitter role of ATP.

Scanning transmission electron microscopic mass determination of in vitro self-assembled smooth muscle myosin filaments.

We have measured the mass per unit length of in vitro self-assembled smooth muscle myosin filaments, using scanning transmission electron microscope darkfield images of freeze-dried samples. The measured values were integral multiples, usually 1, 2, 3 or 4, of 75 kDa per nm. The data corroborate an earlier proposal, that these filaments are built from monolayer sheets of molecules, each sheet having two antiparallel myosin molecules per 14.3 nm of its length.

Down-regulation of the chicken alpha 5 beta 1 integrin fibronectin receptor during development.

We have characterized the diversity of the chicken beta 1 integrin family and studied the expression of individual receptors during development. The diversity of the beta 1 integrin family was investigated by affinity purifying the beta 1 integrins from a variety of adult and embryonic tissues. These purifications reveal the relative levels of expression and also the differential expression of the alpha subunits in those tissues. Monoclonal antibodies were generated against the prominent 'band 1' of the embryonic chicken integrins and used to characterize the expression of this alpha subunit in embryonic and adult tissues. This alpha subunit is shown to be the chicken homologue of human alpha 5 fibronectin receptor. The chicken alpha 5 beta 1 integrin is the most prominent beta 1 integrin in the embryo and is expressed on the majority of cell types through the day 17 stage. The distribution of this receptor in the embryo closely parallels the distribution of its ligand, fibronectin. In adult tissues, expression of this receptor is greatly diminished relative to the expression of other alpha subunits. The cell type distribution is highly restricted: limited primarily to the vasculature and to connective tissue regions. These studies reveal a prominent role for the alpha 5 beta 1 integrin in embryonic cell types and a down-regulation of this receptor on many cell types during development.

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon.

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.

Conformational change of turkey-gizzard caldesmon induced by specific chemical modification with carbodiimide.

Water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to internally cross-link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86,974 Da of chicken-gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook. R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13,873-13,879). Comparative circular dichroism analysis indicated a decrease of the alpha-helix content from 43% to 36% resulting from the chemical modification. The 1H-NMR spectra of the native and modified caldesmon showed that the covalent cross-linking affected mainly the central and N-terminal parts of the molecule. The C-terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90-kDa proteolytic N-terminal fragment to give an internally cross-linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F-actin bundles. These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F-actin bundles or weaker interactions between actin filaments.

Calcium adenosine triphosphatase and secretion of koilin membrane in the gizzard of the fowl.

The localization of calcium adenosine triphosphatase (Ca(2+)-ATPase) was determined histo- and ultracytochemically in the gizzard gland cells of the adult domestic fowl. Surface and chief gland cells exhibited faint and inconstant basolateral activity in contrast to basal cells, whose basolateral cell membrane constantly showed deposition on the external side. Intracellular enzyme activity was localized on the luminal aspect of Golgi membranes in all types of gland cells. Lysosomes also reacted positive for Ca(2+)-ATPase. Neither membranes of secretory vesicles nor cortical cytoplasm of the secretory pole exhibited enzyme activity. From these results it is speculated that calcium is not essentially involved in the secretion of the koilin membrane in terms of storage of the secretory material, transport to the secretory surface and release into the lumen. Ca(2+)-ATPase activity rather seems to be related to differentiation and maturation processes and to intracellular storage of Ca2+.

Supercontracted state of vertebrate smooth muscle cell fragments reveals myofilament lengths.

Isolated cell preparations from chicken gizzard smooth muscle typically contain a mixture of cell fragments and whole cells. Both species are spontaneously permeable and may be preloaded with externally applied phalloidin and antibodies and then induced to contract with Mg ATP. Labeling with antibodies revealed that the cell fragments specifically lacked certain cytoskeletal proteins (vinculin, filamin) and were depleted to various degrees in others (desmin, alpha-actinin). The cell fragments showed a unique mode of supercontraction that involved the protrusion of actin filaments through the cell surface during the terminal phase of shortening. In the presence of dextran, to minimize protein loss, the supercontracted products were star-like in form, comprising long actin bundles radiating in all directions from a central core containing myosin, desmin, and alpha-actinin. It is concluded that supercontraction is facilitated by an effective uncoupling of the contractile apparatus from the cytoskeleton, due to partial degradation of the latter, which allows unhindered sliding of actin over myosin. Homogenization of the cell fragments before or after supercontraction produced linear bipolar dimer structures composed of two oppositely polarized bundles of actin flanking a central bundle of myosin filaments. Actin filaments were shown to extend the whole length of the bundles and their length averaged integral to 4.5 microns. Myosin filaments in the supercontracted dimers averaged 1.6 microns in length. The results, showing for the first time the high actin to myosin filament length ratio in smooth muscle are readily consistent with the slow speed of shortening of this tissue. Other implications of the results are also discussed.

The cytoskeletal and contractile apparatus of smooth muscle: contraction bands and segmentation of the contractile elements.

Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin-containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.

Differential expression of tenascin splicing variants in the chick gizzard and in cell cultures.

Tenascin is a large disulfide-linked hexameric extracellular matrix glycoprotein. It is a multidomain protein containing many repeated structural units such as heptad-, EGF-like-, and fibronectin type III repeats, as well as a homology to the globular domains of beta- and gamma-fibrinogen. In the chick embryo three major tenascin variants exist. They arise from one gene by alternative splicing of three of its 11 fibronectin type III repeats. Monoclonal antibodies against the alternatively spliced domains allowed us to study the expression of tenascin variants in tissue sections and in cell cultures. In the gizzard, the largest tenascin variant was only detected in the smooth muscle layer and the connective tissue below the epithelium of the villi, whereas the shortest tenascin variant was predominant in the tendons and the intramuscular connective tissue. Differential expression of tenascin variants was also obtained in cell cultures of chick embryo fibroblasts. Fetal calf serum equally stimulated the accumulation of all three tenascin variants, whereas after transformation with polyomavirus middle-T only the secretion of the largest tenascin variant was greatly enhanced.

Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria.

In avian smooth muscle cells, desmin-containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, including the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti-desmin and protein-A-colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.

Development of smooth muscle: ultrastructural study of the chick embryo gizzard.

The growth and differentiation of smooth muscle in the chicken gizzard were studied by electron microscopy from the 10th day in ovo to 6 months after hatching; during this period the organ grows 1000-fold in weight. At the earliest stage studied, smooth muscle cells, interstitial cells, and fibroblasts are immature but can already be clearly distinguished. The structural components of muscle cells develop in a characteristic sequence. Mitochondria are more abundant in immature muscle cells (8% in 14 days embryos and 7% in 19 days embryos) than in the adult (5%). Caveolae are virtually absent in the 11 day embryo; they become more common at the end of embryonic life, but continue to increase in relative frequency after hatching. Gap junctions appear around the 16th day in ovo as minute aggregates of connexons, which then grow in size, probably by addition of new connexons. In the earliest stages studied, myofilaments occupy 25% of the cell profile and are assembled into bundles accompanied by dense bodies and surrounded by loosely arranged intermediate filaments. By contrast, membrane-bound dense bands are scarce until the latter part of embryonic life, an observation suggesting that myofilament formation and alignment is not a process initiated near the cell membrane or directed by the cell membrane, and that only late in development bundles of myofilaments become extensively anchored to dense bands over the entire cell surface: at that time myofilaments occupy more than 75% of the cell volume. The muscle cells increase about four-fold in volume over the period studied; the 1000-fold increase in muscle volume is mainly accounted for by an increase in muscle cell number. Mitoses are found in the gizzard musculature at all embryonic ages with a peak at 17-19 days; they occur in muscle cells with a high degree of differentiation. These cells divide at a stage when they are packed with myofilaments and form junctions with neighbouring cells: the mitotic process affects the middle portion of the cell, which takes up an ovoid shape and eventually divides, whereas the remaining portions of the cell do not differ in appearance from the surrounding muscle cells. At all stages of development the population of muscle cells has a uniform appearance (apart from the cells in mitosis), and the growth and differentiation seem to proceed at the same pace in all the cells. There are no undifferentiated cells left behind in the tissue for later development.

Vinculin interaction with permeabilized cells: disruption and reconstitution of a binding site.

Fluorescently labeled vinculin binds to focal contact areas in permeabilized cells independent of actin (Avnur, Z., J. V. Small, and B. Geiger, 1983, J. Cell Biol., 96:1622-1630), but the nature of the binding site is unknown. In this study we have examined the interaction of vinculin with these sites in permeabilized L6 myoblasts to define conditions that perturb the binding and subsequently to reconstitute it. Mild treatment with low concentrations of protease prevents vinculin incorporation without gross changes in the cytoskeleton or extensive protein breakdown. Exposure to buffers of moderate ionic strength also reduces subsequent vinculin binding without large morphological effects. These extraction conditions were used to obtain a fraction from gizzard which was able to restore the vinculin localization. Talin, actin, and vinculin itself were able to alter the binding of labeled vinculin to permeabilized cells and each also interacted with vinculin in gel overlays; however, they were unable to reconstitute the binding site in treated permeabilized cells. The results show a requirement for an as yet unidentified protein to capacitate vinculin binding to focal contact sites and suggest that this protein is peripheral and interacts directly with the binding site.

Structure of the glandular layer and koilin membrane in the gizzard of the adult domestic fowl (Gallus gallus domesticus).

The koilin membrane is formed by the secretions of gland, crypt and surface epithelial cells. Glands form a continuous layer and are arranged in groups of 10-20. They are straight tubes about 500 microns long and 15 microns in diameter and produce rodlets of hard koilin. Hard koilin rodlets (5 microns diameter) form clusters of five or six as they pass through the crypts and enter the koilin membrane. Each rodlet hardens within its gland and maintains its individuality throughout its entire length. Rodlet clusters have previously been called 'rods'. Most of the softer koilin, which fills the spaces between the rodlet clusters, is produced by the surface epithelial cells. These cells form gentle arches between the cavities of adjacent crypts. Horizontal branches between rodlet clusters ('rods') do not exist. There is approximately twice as much surface koilin as rodlet koilin within the membrane. Abrasion of the koilin membrane is not uniform but occurs in a patchy fashion.