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Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effect of colchicine on the redistribution of L-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes.

To define the role of cytoplasmic microtubules in the biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells, we studied the effect of colchicine on the incorporation of L-[1,5,6-3H]fucose into Golgi, lateral basal and microvillus membranes. Colchicine was administered intraperitoneally before or after injection of radioactive fucose. The incorporation of radioactivity into Golgi membranes was little affected by colchicine, which did not prevent the redistribution of most of the labelled glycoproteins from the Golgi complex into other parts of the villus cell. The incorporation of labelled glycoproteins into the microvillus membrane was greatly inhibited by colchicine given 2 h or 10 min before the radioactive fucose: all labelled glycoproteins present in this membrane were equally affected. In contrast, the administration of colchicine considerably increased the incorporation of radioactivity into the lateral basal part of the plasmalemma, and prevented the disappearance of most of the labelled glycoproteins from this membrane at late times after fucose injection. These results suggest that cytoplasmic microtubular structures are important for the polarization of the intestinal villus cell and the biogenesis of the microvillus membrane, although playing little or no role in the movement of membrane components from the Golgi complex to the lateral basal part of the plasmalemma.

Animals↗

Smooth muscle cell differentiation in mammary stromo-epithelial lesions with evidence of a dual origin: stromal myofibroblasts and myoepithelial cells.

AIMS: We investigated the origin of myoid cells in benign stromo-epithelial lesions of the breast in order to ascertain their myoepithelial or myofibroblastic origin. METHODS AND RESULTS: We selected 22 stromo-epithelial lesions of the breast and reviewed their morphological features at haematoxylin-eosin (H&E) level. The lesions were classified as fibrous stromo-epithelial lesions (without evidence of myoid differentiation at H&E level) (13 cases), type 1 myoid stromo-epithelial lesions (myoid cells directly merging with the myoepithelial layer) (three cases), type 2 myoid stromo-epithelial lesions (bundles of myoid cells unrelated to the glands) (six cases). All cases were studied immunohistochemically and myoid stromo-epithelial lesions were also studied with electron microscopy. The myoid component in two out of three cases of type 1 myoid lesions showed immunohistochemically co-expression of smooth muscle and myoepithelial markers. In contrast, the remainder showed immunohistochemical results identical to those found in type 2 myoid lesions (positivity with SMA, desmin, calponin, CD34 and bcl2 and negativity with cytokeratin 14 and p63). Ultrastructural study confirmed the presence of cells with myoepithelial features in type 1 myoid lesions and of cells with myofibroblastic features in type 2 myoid lesions. CONCLUSIONS: Myoid cell differentiation is common in stromo-epithelial lesions of the breast, and is evident in H&E sections in up to 40% of cases. In addition, the origin of myoid cells is myofibroblastic in most cases, but in some cases, cells present immunohistochemical and ultrastructural evidence of myoepithelial origin.

Adult↗

Renal epithelial neoplasms: the diagnostic implications of electron microscopic study in 55 cases.

Several unsettled histogenetic, nosologic and diagnostic considerations for renal epithelial tumors may have ultrastructural ramifications. Yet, a comprehensive electron microscopic study of renal epithelial neoplasms, in light of the recent classification, is not available. The ultrastructural findings from fifty-five renal epithelial neoplasms [31 clear cell renal cell carcinomas (RCC), 11 papillary RCC, 5 chromophobe RCC, 3 sarcomatoid RCC and 5 oncocytomas] were correlated with their light microscopic appearance. Clear cell RCC showed long microvilli similar to the brush border of the normal proximal tubules, with abundant cytoplasmic lipid and glycogen. Papillary RCC showed variably sized microvilli, and small amounts of cytoplasmic lipid, but no glycogen. Chromophobe RCC showed many cytoplasmic vesicles and abnormal mitochondria, with rare short and stubby microvilli. Renal oncocytoma showed many mitochondria with a few vesicles in the apical portion of the cytoplasm and rare short and stubby microvilli. The eosinophilic cell variants of clear cell RCC, papillary RCC and chromophobe RCC showed ultrastructural features similar to those of their respective prototypes, except for an increased numbers of mitochondria in the cytoplasm. One sarcomatoid clear cell RCC showed skeletal muscle differentiation. Two types of cytoplasmic inclusions, i.e. hyaline globules and granules similar to those in the Paneth cells (PC-like granules) were identified only in clear cell RCC, which displayed distinctive ultrastructural features. The current EM study demonstrates distinctive ultrastructural features of renal epithelial neoplasms. The findings lend additional support to the current classification of the pertinent tumor types, facilitate the differential diagnoses, and provide insights into the possible histogenesis of renal epithelial neoplasms.

Adenoma, Oxyphilic↗

African swine fever: Expression of interleukin-1 alpha and tumour necrosis factor-alpha by pulmonary intravascular macrophages.

To determine, in the acute form of African swine fever (ASF), the relationship between the appearance of pulmonary oedema and viral replication and expression of cytokines by pulmonary intravascular macrophages (PIMs), 14 pigs were inoculated intramuscularly with ASF virus (strain España'70) and killed in pairs on days 1-7 post-inoculation. Samples of lung were examined immunohistochemically and ultrastructurally. The immunohistochemical study was carried out with antibodies against interleukin-1 alpha (IL-1alpha), tumour necrosis factor-alpha (TNF-alpha), viral antigen of ASF (Vp73) and a myeloid marker (SWC3). Viral replication was observed mainly in PIMs, which at the same time showed intense activation, accompanied by the expression of IL-1alpha and TNF-alpha. The occurrence of interstitial oedema, neutrophil sequestration and fibrin microthrombi in septal capillaries coincided with high degrees of cytokine expression by infected PIMs. Alveolar macrophages did not show a significant change in cytokine expression as a result of ASF infection, and viral replication was detected in only a low percentage of these cells.

Acute Disease↗

Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy.

We report advances in quantitative fluorescent speckle microscopy to generate simultaneous maps of cytoskeleton flow and rates of net assembly and disassembly in living cells. We apply this tool to analyze the filamentous actin (F-actin) dynamics at the front of migrating cells. F-actin turnover and flow are both known to be factors of cell locomotion. However, how they are orchestrated to produce directed cell movements is poorly understood. Our approach to data analysis allows us to examine their interdependence. Our maps confirm the previously described organization of flow into a lamellipodium and a lamellum, both exhibiting retrograde flow; and a convergence zone, where lamellum retrograde flow meets with slow anterograde flow of cortical F-actin at the ventral side of the cell body. The turnover maps show the well known actin polymerization at the leading edge, but also indicate that approximately 90% of the polymer disassembles at the lamellipodium-lamellum junction. Strong depolymerization is also found in the convergence zone, where meshwork contraction is prominent. To determine whether contraction and depolymerization are coupled events, we have treated cells with calyculin A, which is known to promote myosin activity. Stimulated contraction was accompanied by accelerated retrograde flow and increased depolymerization throughout the lamellum, whereas disassembly at the lamellipodium-lamellum junction remained unaffected. There appear to be two distinct depolymerization mechanisms, of which one depends directly on meshwork contraction.

Actins↗

Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in the trophectoderm.

Secretions of the uterus support survival and growth of the conceptus (embryo/fetus and associated membranes) during pregnancy. Galectin-15, also known as OVGAL11 and a previously uncharacterized member of the galectin family of secreted beta-galactoside lectins containing a conserved carbohydrate recognition domain and a separate putative integrin binding domain, was discovered in the uterus of sheep. In endometria of cyclic and pregnant sheep, galectin-15 mRNA was expressed specifically in the endometrial luminal epithelium but not in the conceptus. In pregnant sheep, galectin-15 mRNA expression appeared in the epithelia between days 10 and 12 and increased between days 12 and 16. Progesterone induced and IFN-tau stimulated galectin-15 mRNA in the endometrial epithelium. Galectin-15 protein was concentrated near and on the apical surface of the endometrial luminal epithelia and localized within discrete cytoplasmic crystalline structures of conceptus trophectoderm (Tr). In the uterine lumen, secreted galectin-15 protein increased between days 14 and 16 of pregnancy. Galectin-15 protein was functional in binding lactose and mannose sugars and immunologically identical to the unnamed Mr 14,000 (14K) protein from the ovine uterus that forms crystalline inclusion bodies in endometrial epithelia and conceptus Tr. Based on the functional studies of other galectins, galectin-15 is hypothesized to function extracellularly to regulate Tr migration and adhesion to the endometrial epithelium and intracellularly to regulate Tr cell survival, growth, and differentiation. Galectins may be useful as cellular and molecular markers for endometrial function and receptivity, to enhance conceptus survival and development, and to evaluate and enhance fertility.

Amino Acid Sequence↗

Spliced adenovirus-associated virus RNA.

We describe the structure of cytoplasmic RNA species transcribed from the DNA of adenovirus-associated virus, a defective parvovirus. The RNA was hybridized with minus strand template DNA and visualized in the electron microscope. Alternatively, the DNA.RNA duplex molecules were digested with nuclease S1 or Escherichia coli exonuclease VII and analyzed by agarose gel electrophoresis. A set of RNA species was observed with 5' terminal at map positions 5, 13, 19, or 39 and a 3' terminus and poly(A) tail at position 96 (one map unit is equivalent to 1% of genome length). Most of these RNAs are spliced and lack sequences approximately between positions 40 and 49. Some RNA preparations also contained unspliced molecules with 5' and 3' terminal at positions similar to those in the spliced RNA.

Base Sequence↗

Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor.

The type 1 human immunodeficiency virus (HIV-1) encodes a 27-kDa protein termed Nef (negative factor). Nef has been reported to down-regulate viral gene transcription directed by the HIV-1 long terminal repeat. To assess the possible role of Nef in the initiation or maintenance of viral latency, we prepared two different nef expression vectors (pNEF from the HXB-3 proviral clone; pNEF-2/3 from HXB-2 and HXB-3) and a control vector containing a frameshift mutation in the HXB-3 nef coding sequence (pNEF-fs). Consistent with prior studies, the Nef proteins produced by pNEF and pNEF-2/3 were approximately 27 kDa in size, posttranslationally modified by myristoylation, and primarily associated with cytoplasmic membrane structures. However, in contrast to previous reports, these Nef proteins failed to inhibit transcriptional activity of the HIV-1 long terminal repeat in any of a variety of cell types, including primary human T lymphocytes, Jurkat or YT-1 leukemic T cells, U-937 promonocytic cells, and nonlymphoid COS cells. Furthermore, HXB-3 proviral clones of HIV-1 containing either a wild-type or mutated version of the nef gene replicated in an indistinguishable manner when transfected into COS cells. Our findings suggest that Nef is neither a transcriptional inhibitor nor a negative viral factor under these assay conditions. Rather, we suggest that the primary biological function of this conserved HIV-1 protein has yet to be defined, perhaps reflecting an intrinsic shortcoming in the in vitro experimental systems presently available for the study of HIV-1.

Animals↗

Exon skipping during splicing of albumin mRNA precursors in Nagase analbuminemic rats.

Based on the observation that albumin transcripts accumulate in the liver nuclear RNA fraction of Nagase analbuminemic rats (NAR), it was proposed [Esumi, H., Takahashi, Y., Sato, S., Nagase, S. & Sugimura, T. (1983) Proc. Natl. Acad. Sci. USA 80, 95-99] [corrected] that a 7-base-pair deletion at the splice donor site of intron H-I of the albumin gene in these animals leads to impaired processing of albumin pre-mRNA. To identify the specific splicing abnormality, we examined the primary structure of cytoplasmic albumin mRNA across the junctions of exons G-H-I by RNase protection mapping, Northern blot hybridization, Southern blot analysis of polymerase chain reaction-amplified cDNA, and DNA sequencing. The major albumin mRNA species in NAR showed precise deletion of exon H, suggesting that this exon was skipped during albumin pre-mRNA processing. Since the intron G-H splice donor and acceptor sites and exon H sequence are normal, the finding of exon H skipping in NAR has important implications regarding the mechanism of splice site selection. Moreover, the NAR model provides an excellent system to study splicing in vivo in a higher animal.

Animals↗

Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm.

The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm. When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment. This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm. A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.

Animals↗

How a G protein binds a membrane.

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Ggamma C terminus and Galpha N terminus, and another near the Galpha C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.

Animals↗

TSAP6 facilitates the secretion of translationally controlled tumor protein/histamine-releasing factor via a nonclassical pathway.

Translationally controlled tumor protein (TCTP) is cytoplasmic and structurally related to guanine-nucleotide free chaperones. TCTP (also called histamine-releasing factor) has been described previously as a secreted protein that participates in inflammatory responses by promoting the release of histamine. How TCTP is eventually exported out of the cell to promote such activities is unknown. Here we show that TCTP secretion was insensitive to either brefeldin A or monensin, suggesting that it proceeds via an endoplasmic reticulum/Golgi-independent or nonclassical pathway. Moreover, our analyses also suggest that secreted TCTP originates from pre-existing pools. TSAP6, a p53-inducible 5-6 transmembrane protein, was found to interact with TCTP in a yeast two-hybrid hunt. GST pull down assays confirmed their direct interaction, and immunofluorescence analysis revealed their partial co-distribution to vesicular-like structures at the plasma membrane and around the nucleus. Functionally, the overexpression of TSAP6 consistently leads to enhanced secretion of both endogenously and exogenously expressed TCTP. Finally, we found TCTP in preparations of small secreted vesicles called exosomes, which have been suggested as a possible pathway for nonclassical secretion. Overexpression of TSAP6 also increased TCTP levels in exosome preparations. Altogether, these data identify a novel role for TSAP6 in the export of TCTP and indicate that this multipass membrane protein could have a general role in the regulation of vesicular trafficking and secretion.

Biomarkers, Tumor↗

Vangl1 protein acts as a downstream effector of intestinal trefoil factor (ITF)/TFF3 signaling and regulates wound healing of intestinal epithelium.

The intestinal trefoil factor (ITF/TFF3) protects intestinal epithelia from a range of insults and contributes to mucosal repair. However, the signaling events that mediate healing responses are only partially understood. To identify ITF signaling pathways, proteins that were Ser/Thr phosphorylated in response to ITF stimulation were immunoprecipitated from human colon carcinoma cell lines and identified by mass spectrometry. We demonstrated that Van Gogh-like protein 1 (also designated Vang-like 1 or Vangl1), a protein with four transmembrane domains, was Ser/Thr phosphorylated in response to ITF stimulation. Vangl1 was present in normal human colon and all intestinal epithelial cell lines (IEC) tested. In transfected IEC, FLAG-Vangl1 was mostly present in the Nonidet P-40 soluble fraction as detected by Western blotting, corresponding to the localization of endogenous protein in cytoplasmic vesicular structures by confocal microscopy with rabbit polyclonal anti-human Vangl1 antibody (alpha-Vangl1). Vangl1 cell membrane association increased with differentiation, as demonstrated by co-localization with E-cadherin in differentiated IEC. Increased Vangl1 phosphorylation after stimulation with ITF corresponded to decreased cell membrane association with E-cadherin. Functionally, Vangl1 overexpression enhanced ITF unstimulated and stimulated wound closure of IEC, whereas siRNA directed against Vangl1 inhibited the migratory response to ITF. Vangl1 protein may serve as an effector mediating the ITF healing response of the intestinal mucosa.

Animals↗

Analysis of lipolytic protein trafficking and interactions in adipocytes.

This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗

Distribution and localization of CMP-N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes.

An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.

Animals↗

The influence of different cultivating conditions on polymorphonuclear leukocyte apoptotic processes in vitro, I: the morphological characteristics of PMN spontaneous apoptosis.

Polymorphonuclear leukocyte (PMN) populations incubated in vitro with normal human serum are save-regulated systems of spontaneous apoptosis. Light microscopy (LM), transmission (TEM), and scanning (SEM) electron microscopes were used for the evalution of PMN apoptopic alteration. Twelve-hour PMN populations were represented by optimal number of normal and different apoptotic forms. Their ultrastructural analysis showed that on this background, 3 apoptotic cell lines (code named "first," "second," and "third") were predominated. The following characteristics were featured: "first"--vacuolization of same organelles, release of their content outside, increase of general cytoplasmic density, nuclear filling with condensed chromatin, and formation of PMNs mainly into small, round, dense forms; "second"--involvement of micronuclei or nuclei in apoptosis, their displacement to the cytoplasmic membrane and separation from the cells, and cytoplasm had numerous intact granules almost until the completion of apoptosis; "third"--synchronous apoptotic process of the nuclei and cytoplasm, moderate electronic density of cytoplasm, and granular translocation to the cell surface. Secondary necrosis was completed mainly in the apoptotic process of the "second" and "third" lines. SEM surfaces confirmed the results of TEM. This research showed that neutrophil spontaneous apoptosis is a complicated process. The 3 apoptotic cell lines reflect different pathways characteristic for the studied systems under certain conditions of cultivation.

Apoptosis↗

Intracellular liposomes and cholesterol deposits in chronic cholecystitis and biliary sludge.

Ultrastructural study of a group of selected specimens of chronic cholecystitic gallbladders reveals cholecystocyte changes characterized by abraded and altered microvilli accompanied by mitochondrial damages in the apical regions as well as mucus accumulation with aggregated, angulated lysosomes and heterogeneous liposomes. These liposomes contain needle-like crystals, probably rich in cholesterol. Many fragments of cholecystocystes and damaged organelles or contents can be found in the biliary sludge. These data support previous reports suggesting that there is an association between cholecystitis and the presence of cholelithiasis, subsequent to the production of altered bile. The present data suggest that disintegrating, sloughed cholecystocyte contents also contribute to the bile sludge, a complex milieu enriched by lipids, cholesterol deposits, altered mucus due in part to changes in expression of apomucins. The instability of prolonged storage of such modified bile, caused and/or accompanied by other associated metabolic defects, including gallbladder sluggishness, would favor the nucleation and the enlargement of gallstones. Based on the aforementioned data, a comprehensive sequence for cholecystocyte ultrastructural alterations and pathologies is proposed, as a result of chronic cholecystitis.

Adult↗

Similarities between giant cell tumor of bone, giant cell tumor of tendon sheath, and pigmented villonodular synovitis concerning ultrastructural cytochemical features of multinucleated giant cells and mononuclear stromal cells.

The authors investigated ultrastructural cytochemical features of multinucleated and mononuclear stromal cells in giant cell tumor of bone (GCTB), giant cell tumor of tendon sheath (GCTTS), and pigmented villonodular synovitis (PVNS). Specimens of each tumor, respectively numbering 4, 4, and 3, were stained for tartrate-resistant acid phosphatase (TRAP) reactions and examined with an electron microscope. In GCTB and GCTTS, multinucleated cells, including some relatively small giant cells, showed TRAP activity and cytoplasmic features characteristic of osteoclasts, and also sometimes abundant rough endoplasmic reticulum and siderosomes. A few giant cells with macrophage-like features and slight TRAP activity were demonstrated in GCCTS and PVNS. In each tumor type, mononuclear cells showing TRAP activity shared cytoplasmic features with osteoclast-like multinucleated giant cells, while some others had macrophage-like features, and still others were poorly differentiated; a few mononuclear cells showed cell-to-cell contact. Ultrastructural similarities of TRAP-positive mononuclear cells in the three tumor types, and those between TRAP-positive multinucleated cells in GCTB and GCTTS, suggest a common cell lineage capable of multinucleated giant cell formation in the 3 tumors, despite differing histogenesis.

Acid Phosphatase↗