Phagocytosis induced by thyrotropin in cultured thyroid cells is associated with myosin light chain dephosphorylation and stress fiber disruption.

The actin/myosin II cytoskeleton and its role in phagocytosis were examined in primary cultures of dog thyroid cells. Two (19 and 21 kD) phosphorylated light chains of myosin (P-MLC) were identified by two-dimensional gel electrophoresis of antimyosin immunoprecipitates, and were associated with the Triton X-100 insoluble, F-actin cytoskeletal fraction. Analyses of Triton-insoluble and soluble 32PO4-prelabeled protein fractions indicated that TSH (via cAMP) or TPA treatment of intact cells decreases the MLC phosphorylation state. Phosphoamino acid and tryptic peptide analyses of 32P-MLCs from basal cells showed phosphorylation primarily at threonine and serine residues; most of the [32P] appeared associated with a peptide containing sites typically phosphorylated by MLC kinase. Even in the presence of the agents which induced dephosphorylation, the phosphatase inhibitor, calyculin A, caused a severalfold increase in MLC phosphorylation at several distinct serine and threonine sites which was also associated with actomyosin and cell contraction. Phosphorylation of cell homogenate proteins or the cytoskeletal fraction with [gamma-32P]ATP indicated that Ca2+, EGTA, or trifluoperazine (TFP) has little effect on the phosphorylation of MLC. Both fluorescent phalloidin and antimyosin staining of cells showed distinct dorsal and ventral stress fiber complexes which were disrupted within 30 min by TSH and cAMP; TPA appeared to cause disruption of dorsal, and rearrangement of ventral complexes. Concomitant with MLC dephosphorylation and stress fiber disruption, TSH/cAMP, but not TPA, induced dorsal phagocytosis of latex beads. While stimulation of either A or C-kinase disrupts dorsal stress fibers and rearranges actomyosin, another event(s) mediated by A-kinase appears necessary for phagocytic activity.

Overexpression of the vimentin gene in transgenic mice inhibits normal lens cell differentiation.

To investigate the role of the intermediate filament protein vimentin in the normal differentiation and morphogenesis of the eye lens fiber cells, we generated transgenic mice bearing multiple copies of the chicken vimentin gene. In most cases, the vimentin transgene was overexpressed in the lenses of these animals, reaching up to 10 times the endogenous levels. This high expression of vimentin interfered very strongly with the normal differentiation of the lens fibers. The normal fiber cell denucleation and elongation processes were impaired and the animals developed pronounced cataracts, followed by extensive lens degeneration. The age of appearance and extent of these abnormalities in the different transgenic lines were directly related to the vimentin level. Electron microscopic analysis revealed that the accumulated transgenic protein forms normal intermediate filaments.

Uptake of maternal immunoglobulins in the enterocytes of suckling piglets: improved detection with a streptavidin-biotin bridge gold technique.

In ungulates, intestinal absorption of maternal immunoglobulins from colostrum plays a vital role in the acquisition of passive immunity during early neonatal life. In the present study we used post-embedding colloidal gold labeling to examine the intracellular localization of IgG in the jejunal enterocytes of miniature piglets suckled for 2 hr. Quantitation of the immunolabeling revealed that the most sensitive technique for IgG detection was the streptavidin bridge-gold technique. In this method, the LR White-embedded sections were labeled sequentially with biotinylated anti-porcine IgG, streptavidin, and biotinylated BSA conjugated to 10-nm colloidal gold. With this approach, we found the following sequence of maternal IgG accumulation: passage of IgG from colostrum through the brush border; binding to the apical plasma membrane; uptake in noncoated pits and invaginations; transport in endocytotic vesicles; and accumulation in granules in the apical cytoplasm.

Oligoclonal bands and the IgG index in multiple sclerosis: uses and limitations.

The relationship between two tests commonly used in the investigation of multiple sclerosis (MS), the IgG index and oligoclonal bands, has been assessed. Using an immunoblotting technique specific for IgG, analysis of cerebrospinal fluid for oligoclonal bands was found to provide greater diagnostic sensitivity than the IgG index without any loss of specificity. In patients without oligoclonal bands the IgG index had no diagnostic value for MS and in the presence of bands the magnitude of the index was unrelated to the clinical certainty of the diagnosis. High values of the IgG index were invariably associated with the presence of oligoclonal bands and the IgG index appeared to have no clinical significance independent of this relationship. Even as a screening test the IgG index has serious limitations.

Effect of dietary fat and cholesterol level on growing pigs selected for three generations for high or low serum cholesterol at age 56 days.

Thirty-six female pigs selected for three generations for high (HS, n = 18) and low (LS, n = 18) serum cholesterol at 56 d of age were used to test the hypothesis that the two populations would respond differently to a high-fat, high-cholesterol diet (HD) and a low-fat, low-cholesterol diet (LD). The animals were four-way crosses (Chester White x Landrace x Large White x Yorkshire). All pigs were fed a standard corn-soybean meal starter diet from weaning (at 4 wk) to 8 wk of age and a grower diet from 8 to 12 wk of age. Initial serum total cholesterol concentration at 12 wk of age was higher (P less than .05) in HS than in LS pigs (94.6 vs 76.9 mg/dL, respectively). The effect of genetic background persisted throughout the 13-wk experiment (25 wk of age); there was no interaction between diet and genetic background in serum total cholesterol (final concentrations were 114.3 mg/dL in HS-HD; 107.0 mg/dL in HS-LD; 105.9 mg/dL in LS-HD; and 89.7 mg/dL LS-LD). Trends over time revealed significant effects of diet (P less than .01) and genetic background (P less than .01) on serum total cholesterol. There was no effect of genetic background on high-density lipoprotein-cholesterol concentration; high-density lipoprotein-cholesterol as a percentage of serum total cholesterol was similar for all groups: 43% for HS-HD, 48% for HS-LD, 42% for LS-HD, and 45% for LS-LD.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic compartment syndrome, an important cause of work-related upper limb disorder.

OBJECTIVES: Work-related upper limb disorders (WRULD) are common and disabling complaints in industry, but a tissue diagnosis can be difficult where the pain is diffuse and variable, and this prevents effective treatment. Diffuse arm pain is frequently found in those doing rapid or strenuous repetitive work, such as factory assembly workers or keyboard operators. Similar symptoms occur in the legs in athletes, where chronic compartment syndrome (CCS) is a recognized entity, so we investigated the possibility that this might also be caused by prolonged repetitive work. METHODS: Patients were selected if they were unable to continue with work because of chronic forearm pain. They were divided into three groups: 42 patients with symptoms consistent with CCS as defined in the text, 15 volunteers and patients with other arm complaints, and 10 asymptomatic arms of patients with unilateral CCS. We measured the pressure inside the extensor muscle compartment of the forearm at rest and after a 2 min repetitive gripping exercise using an electronic pressure-sensitive probe. RESULTS: The results show that CCS is a common and disabling forearm complaint associated with prolonged repetitive work. Fifteen patients have now had decompressive surgery on the extensor muscle compartment with good relief of symptoms. CONCLUSION: CCS is responsible for chronic peripheral neurological dysfunction in addition to muscle pain, and awareness of this diagnosis allows early identification and treatment of a currently unrecognized disorder with potential resolution of a long-lasting arm disability.

Behaviour and pathogenicity of Trichomonas vaginalis in epithelial cell cultures: a study by light and scanning electron microscopy.

The behaviour and pathogenic effects of Trichomonas vaginalis in mammalian cell cultures were studied using light microscopy and scanning electron microscopy. Six hours after inoculation of the parasites into the cell cultures about 10% of the epithelial monolayer was destroyed. The parasites adhered to the epithelial cells, developed an amoeboid morphology, and crawled over and under the monolayer of cells. These observations suggest that the adhesiveness, amoeboid morphology, and motility of T vaginalis may be important mechanisms in the injury caused to the vaginal epithelium.

Epithelial cell migration in the intestine.

Despite significant advances in our understanding of the roles of the cytoskeleton and matrix receptors in cell locomotion, derived largely from in vitro studies on the movement of epithelial cell sheets and isolated cells, the mechanism of epithelial cell migration in the adult intestine remains an enigma. The primary function of the epithelial cell cytoskeleton seems to be in the maintenance of the apical region of the epithelium facing the gut lumen. There we find the brush border, with its associated enzymes, and the intercellular adhesion complexes that give the epithelium its cohesiveness and its barrier function. Curiously, there is little in the way of an organized cytoskeleton in the basal region of the epithelium adjacent to the basement membrane on which the epithelium is presumed to migrate. In this short review, I focus on what is known about epithelial migration from our understanding of the structure of the epithelium and from studies on wound healing, and indicate some avenues for future study.

Behaviour and structure of the leading lamella in moving fibroblasts. I. Occurrence and centripetal movement of arc-shaped microfilament bundles beneath the dorsal cell surface.

Arc-shaped bundles of microfilaments are frequently found beneath the dorsal surface of the leading lamella of chick embryo fibroblasts. These structures, called arcs, form parallel to, and 2-10 micron from the leading edge of the lamella and they then move centripetally through the lamella and disappear in front of the cell nucleus. Arcs move centripetally at a mean speed of 1.33 (+/- 0.08 S.E.) micron min-1 relative to the substratum. Arcs are specifically associated with cells that are actively protrusive. They are common in fanshaped fibroblasts and in those cells that are newly spreading on a substratum. Arcs are absent from fibroblasts that have only small lamellae or that are polygonal. The correlation between are formation and protrusive activity is also clearly shown by the locomotory behaviour of fan-shaped cells. When protrusion of the lamella is increased by tail detachment, fibroblasts often develop numerous arcs, which chase each other backwards through the lamella. Conversely, arc formation ceases during contact inhibition of locomotion. In cells with large convex-edged lamellae there is a dorsal submembraneous sheath of microfilaments. All of the filaments comprising the sheath are oriented parallel to the margin of the lamella. Arcs are regions of the sheath where the microfilaments are more densely packed and hence visible by phase-contrast microscopy. There is no obvious relationship between the dorsally situated arcs and microfilament sheath, and the stress fibres that are associated with the ventral cell surface. The similarities between the movement and behaviour of arcs and the centripetal transport of particles on the surface of the lamella suggest a role for the microfilament sheath in the movement of particles.

Direct evidence for microfilament-mediated capping of surface receptors on crawling fibroblasts.

On moving fibroblasts, cell-surface receptors cross-linked by antibodies or lectins are cleared centripetally from regions of lamellar cytoplasm and collect as a cap over the perinuclear region. Current theories of the mechanism of receptor redistribution on cultured cells variously implicate membrane flow, lipid flow, surface waves and linkage to the cytoskeleton. The last, the anchorage model, is based on observations that ligand-induced clusters of receptors on a variety of cell types either attach to actin or align over structures containing actin, myosin and alpha-actinin. I show here that the capping of antibody receptors on crawling chick embryo fibroblasts is highly coordinated with the apparent centripetal movements of arcs, which are part of a dorsal cortical actin-microfilament sheath (DCMS). This phenomenon can be directly observed in living cells. The data support the anchorage model of membrane receptor mobility and suggest that there is a continuous flow of actin associated with fibroblast locomotion.

Morphology of fibroblasts in collagen gels: a study using 400 keV electron microscopy and computer graphics.

We have used 400 kilovolt intermediate voltage electron microscopy (IVEM) to examine thick sections of fibroblasts cultured in collagen gels. In these 3D collagen lattices, the long, narrow pseudopodial extensions that extend out and make contact with the collagen matrix exhibit a complex topography not seen in the processes put out by cells moving on planar substrata. For this reason, sections 1 to 2 microns thick that enclose a whole cell process are more informative of the overall morphology of the interaction between cells and the collagen than are thin sections. To aid the discrimination of topography of cell processes in stereo views of micrographs, some cells were labeled with antibodies and protein A-colloidal gold conjugates. The gold particles provided clear 3D reference points for computer-aided reconstructions of membrane topography from tilt series of IVEM images. Our results confirm that cells that move through collagen lattices lack the well-spread morphology of their counterparts moving on glass. They are generally rather spindly with several long branching anterior pseudopodia. We found that the cell bodies and major pseudopodial processes were cylindrical, as one might expect of cells in a 3D environment, but at the leading edge of advancing pseudopodia there are small flat extensions similar to those seen in cells on glass. This similarity suggests that the lamellipodium is a basic type of protrusive structure used by fibroblasts during locomotion on all types of substratum. The flattened shape of lamellipodia may be part of the mechanism by which cells sense the orientation of fibrillar extracellular matrices during embryonic morphogenesis.

Locomotion and cell surface movements of fibroblasts in fibrillar collagen gels.

We have used light microscopy and scanning, scanning transmission, transmission, and high voltage electron microscopy to study the shape and cell surface movements of chick embryo fibroblasts cultured in Vitrogen collagen gels. Cells in gels are less well spread than their counterparts on glass but they retain a dorso-ventral polarity, and move by extending long, narrow lamellar processes. We show here for the first time that cells moving in 3D substrata form perinuclear caps of cross-linked cell surface receptors.

Cell locomotion: new research tests old ideas on membrane and cytoskeletal flow.

Recent studies on the mobility of membrane markers on crawling cells indicate that there is no long-range centripetal flow of membrane proteins or lipids during cell locomotion. In this article we reflect on the history of ideas about membrane flow in cells, and we discuss how these new findings will shift the focus of research in cell locomotion away from the cell surface to the molecular interactions and dynamics of the actin cytoskeleton.

Reconstruction from stereo and multiple tilt electron microscope images of thick sections of embedded biological specimens using computer graphic methods.

The extraction and display of 3-D information using modern computer graphic equipment in the electron microscope is presented. Thick specimens were imaged at 400 kV. Modelling of vectors and the tilting of surfaces is discussed as is the accuracy of reconstructions.

On the mechanisms of cortical actin flow and its role in cytoskeletal organisation of fibroblasts.

In this review we discuss the organization of F-actin in motile fibroblasts in relation to the phenomenon of cortical actin flow; we review some of the mechanisms proposed to drive this process and then relate some of our new findings on the interactions of cortical flow and substratum adhesions in fibroblasts. It is our thesis that the centripetal flow of F-actin through the lamellipodium and leading lamella is the major determining factor organising the polarized stress fiber system and associated cell-substratum adhesions in crawling fibroblastic cells. The broad flattened region of cytoplasm anterior to the nucleus, called the leading lamella, is fringed with much thinner structures, the lamellipodia, which are the primary protrusive organelles of motile cells. Lamellipodia are filled with a criss-crossed network of actin filaments interspersed with small bundles or ribs. In the leading lamella, the actin cytoskeleton is largely confined to two cortical layers beneath the dorsal and ventral cell surfaces. The ventral cortex is engaged in cell adhesion; the dorsal cortex is made up of a circumferentially orientated sheet of actin filaments and bundles which we team the dorsal cortical microfilament sheath (DCMS). Stress fibers insert into the ventral adhesions and pass back through the lamella rising up to meet the DCMS. Cortical flow appears to be a constitutive process in most types of cells when they become motile. In fibroblasts a continuous centripetal flux of structure is seen flowing through the lamellipodium and into the more central regions of the lamella at approximately 0.1 micron per second. Some of the structures engaged in the flux pass back and merge into the DCMS which is also moving rearward at a slower rate of 1 to 5 microns per minute. We find that the formation of a stress fibre is a direct consequence of cortical flow. Initially, stress fibre formation involves the establishment of a focal adhesion between an F-actin bundle in the lamellipodium and the substratum. Subsequently, a fibre grows centripetally from the adhesion and elongates coordinately with the rearward flowing cortex. Cortical flow is restrained locally at the distal end of the nascent fibre leading to indentation and folding of the sheet of filaments. This fold develops into an arc. We demonstrate that mechanical linkages exist between the lamellipodium and the DCMS. Cytochalasin induces a sudden and massive centripetal collapse of the DCMS which drags the lamellipodia with it.(ABSTRACT TRUNCATED AT 400 WORDS)

Abnormalities of autonomic function in the Lambert Eaton myasthenic syndrome.

Two cases of Lambert Eaton syndrome unassociated with an underlying malignancy are described. Both had mild autonomic symptoms but markedly abnormal autonomic function tests. These results are suggestive of a widespread defect in cholinergic transmission in addition to that at the skeletal neuromuscular junction.

Lenten cell: ultrastructure, absorptive properties, and enzyme expression of a novel type of cell in the newborn and suckling pig intestinal epithelium.

BACKGROUND: The small intestinal epithelium is made up of columnar absorptive enterocytes and a smaller number of specialized non-absorptive cells, including goblet cells, enteroendocrine cells, M cells, cup cells, and tuft cells. During a study on milk protein absorption in newborn pigs, we identified an enterocyte that showed no uptake of milk proteins and that could be found only in the jejunum and ileum of pigs during the first 2 weeks of life. We call this previously undescribed enterocyte the lenten cell. METHODS: We used light microscopy and transmission electron microscopy in conjunction with immunolabelling and cytochemical techniques to determine the occurrence, ultrastructure, absorptive properties, and brush border hydrolase expression of lenten cells. RESULTS: Lenten cells constituted approximately 1-2% of the villous epithelium. They were seen in newborn and suckling pigs 1-9 days of age, but were not found in weaned pigs. Morphologically, lenten cells were spindle- or wineglass-shaped, with a ventrally sited nucleus and an electron-dense cytoplasm with numerous cytokeratin filaments. Lenten cells had a normal brush border with microvilli that were slightly thicker than those of absorptive enterocytes, but they did not express the brush border hydrolases lactase, aminopeptidase N, and alkaline phosphatase. Lenten cells did not endocytose milk proteins or horseradish peroxidase, but contained some endocytic or secretory vacuoles and a few dense granules. CONCLUSIONS: No role for lenten cells has been identified in this study, but presence of these cells during the neonatal period, when growth and differentiation of the gastrointestinal tract is at a peak, clearly suggests that lenten cells may play a role in this process.

Distribution of beta-adrenergic binding in fractionated porcine adipocytes.

1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes. 2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200-300 fmol dihydroalprenolol-bound receptors/mg protein. 3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient. 4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity. 5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.

Recurrent lobar pneumonia associated with idiopathic Eaton-Lambert syndrome.

A patient with idiopathic Eaton-Lambert syndrome presenting with recurrent pneumonia as a consequence of the underlying muscle weakness is described.