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Results for “Morphological Characterization”

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PubMed1993

Receptor-mediated endocytosis of transferrin at the blood-brain barrier.

Rat brains were perfuse with a transferrin-peroxidase conjugate (Tf-HRP) to characterize morphologically the endocytic pathway of transferrin in blood-brain barrier endothelial cells. Electron microscopic evaluation of rat brains perfused with Tf-HRP at 4 degrees C and subsequently warmed to 37 degrees C for brief periods of time (2 minutes) showed sequestration of Tf-HRP in clathrin coated pits and vesicles on the luminal membrane of the brain endothelium. After 5 minutes of warming, diaminobenzidine (DAB) reaction product was present in vesicular structures 250-500 nm in diameter and in associated tubules morphologically identified as large or sorting endosomes. Recycling endosomes were also heavily labelled at this time point. Almost no DAB reaction product remained in the cerebral endothelial cells when the warming period was longer than 15 minutes. Other rat brains were perfused with Tf-HRP at 30 degrees C for 15 minutes prior to fixation and DAB cytochemistry. In these studies, brain endothelial cells contained large amounts of DAB reaction product, mostly localized in 50-100 nm vesicles and tubules, often in the Golgi region of the cells. Coated pits and vesicles and large endosomes were also heavily labelled. Transcytosis of Tf-HRP was not identified in either perfusion protocol. Ultrastructural, indirect immunocytochemical localization of transferrin receptors showed that the transferrin receptor is highly polarized at the blood-brain barrier and is localized only on the apical membrane, in contrast to other polarized epithelial cells, like hepatocytes, in which the receptor is present on the basolateral membrane. The evidence supports an iron transport model in which iron-loaded transferrin is taken up by receptor-mediated endocytosis at the luminal membrane of brain capillaries. The iron then dissociates from transferrin in endosomal compartments and is transcytosed by unknown mechanisms, while the transferrin is retroendocytosed.

Animals↗

PubMed2001

Differential effect of GalNAcalpha-O-bn on intracellular trafficking in enterocytic HT-29 and Caco-2 cells: correlation with the glycosyltransferase expression pattern.

Our previous work has shown that long-term treatment of mucus-secreting HT-29 cells with 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha-O-bn), a competitive inhibitor of O-glycosylation, induced several phenotypic changes, in particular a blockade in the secretion of mucins, which are extensively O-glycosylated glycoproteins. Here, we have analyzed the effects of GalNAcalpha-O-bn upon the intracellular trafficking of basolateral and apical membrane glycoproteins at the cellular and biochemical levels in two types of cells, HT-29 G(-) and Caco-2, differentiated into an enterocyte-like phenotype. In HT-29 G(-) cells, but not in Caco-2 cells, DPP-IV and CD44 failed to be targeted to the apical or basolateral membrane, respectively, and accumulated inside intracytoplasmic vesicles together with GalNAcalpha-O-bn metabolites. We observed a strong inhibition of alpha2,3-sialylation of glycoproteins in HT-29 G(-) cells correlated to the high expression of alpha2,3-sialyltransferases ST3Gal I and ST3Gal IV. In these cells, DPP-IV and CD44 lost the sialic acid residue substituting the O-linked core 1 structure Galbeta1-3GalNAc (T-antigen). In contrast, sialylation was not modified in Caco-2 cells, but a decrease of alpha1,2-fucosylation was observed, in correlation with the high expression of alpha1,2-fucosyltransferases Fuc-TI and Fuc-TII. In conclusion, in HT-29 G(-) cells, GalNAcalpha-O-bn induces a specific cellular phenotype, which is morphologically characterized by the formation of numerous intracellular vesicles, in which are accumulated defectively sialylated O-glycosylproteins originally targeted to basolateral or apical membranes, and GalNAcalpha-O-bn metabolites.

Caco-2 Cells↗

PubMed1988

A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin.

We describe a method for permeabilizing and extracting cells that preserves both structure and function whilst allowing the cell derivatives to be handled freely. Cells are encapsulated in microbeads of agarose; the coat of agarose, which is freely permeable to small molecules, forms a protective layer around fragile cell constituents. Cells are then permeabilized by the non-ionic detergent Triton X-100 or antibody and complement in a buffer whose ionic composition mimics that of the cytoplasm. The resulting structures have been characterized morphologically (by immunofluorescence and electron microscopy) and biochemically. Lysis with Triton removes both cell and nuclear membranes, and extracts most of the cytoplasm to leave chromatin surrounded by cytoskeleton; nucleus and cytoplasm then become accessible to triphosphates, enzymes and antibodies. Lysis with complement permeabilizes the cell membrane but leaves the nuclear membrane intact; triphosphates and restriction enzymes, but not antibodies, can then enter both nucleus and cytoplasm. Both types of lysis yield preparations whose chromatin template remains essentially intact, and which is replicated and transcribed at rates close to, or greater than, those found in vivo. Treatment of complement-lysed cells with Triton reduces the very efficient DNA synthesis, implying that the nuclear membrane is involved, directly or indirectly, in replication.

Cell Nucleus↗

PubMed1993

Sensory and neurosecretory innervation of leech nephridia is accomplished by a single neurone containing FMRFamide.

The neural control of the excretory system of the medicinal leech Hirudo medicinalis has been characterized morphologically and chemically using light and electron microscopy, immunocytochemistry and biochemistry. Immunoreactivity against RFamide-like peptides revealed elaborate neuronal aborizations of a neurone in the nephridium, around the urinary bladder sphincter and in the central nervous system. The processes arose from the nephridial nerve cell (NNC), a previously identified receptor neurone. Using a combination of reverse-phase high pressure liquid chromatography, radioimmunoassay and subsequent Edman degradation and mass spectrometry, authentic FMRFamide has been identified as the major peptide of the NNC. Sensory and neurosecretory innervation of the nephridia is thus accomplished by a single neurone, which is thought to modulate nephridial performance.

Amino Acid Sequence↗

PubMed1998

Cell lines established from fetal brains of p53-deficient mice.

Brains from 7 p53-deficient mice on fetal day 13 were divided into 4 regions: cerebral cortex, cerebellum, upper spinal cord, and the rest of brain. They were trypsinized and cultured in medium containing 10% fetal calf serum. By dilution culture, 138 clonal lines were established from every regions. The lines were characterized morphologically and immunocytochemically as neuronal, glial, myogenic, or unidentified. They were cultured for more than a year, indicating that the lines are immortalized. p53-deficiency alone is sufficient for establishing clonal cell lines of the central nervous system.

Animals↗

PubMed2002

Endocytic pathway in mouse cardiac cells.

Primary cultures of heart muscle cells provide powerful tools for cardiac cell biological research that permits both physiological and biochemical approaches. In the present study we analyzed the endocytosis of cardiac cells and presented morphological characterization of the endocytic machinery using markers, which enabled us to follow the fluid-phase, receptor-mediated endocytosis and the internalization of large particles. Our results demonstrated the route of the internalized cargo to early endosomes followed or not by its discharge in the late compartments. We also confirmed the ability of cardiac muscle cells to ingest large particles such as the mannosylated ligand zymosan A, and even internalize whole eukaryotic cells such as the protozoan parasite Trypanosoma cruzi. Since endocytosis is involved in many important cellular functions, the present work contributes to the knowledge of possible additional roles played by cardiac muscle cells besides their well known ability to act as physically energetic cells in the body, constantly contracting without tiring.

Animals↗

PubMed2004

Short term Caco-2/TC7 cell culture: comparison between conventional 21-d and a commercially available 3-d system.

The Caco-2 cell model is a valuable tool for studying intestinal biotransformation of xenobiotics and to evaluate the potential of human intestinal absorption of new compounds. These properties were evaluated with Caco-2/TC7 cells in accelerated conditions to reduce maturation lag time from 21-d to 3-d in order to increase time and labor efficiency. Transmission electron and fluorescent microscopy were used for morphological characterization. Alkaline phosphatase and lactate dehydrogenase activities were assessed within time. Cytochrome P450 expression was studied by RT-PCR. Apparent permeabilities of a set of passively absorbed molecules across Caco-2/TC7 cell monolayers were determined to evaluate potential of both systems for prediction of human intestinal absorption. Microscopic images revealed that cells under both conditions differentiated as enterocyte-like cells but did so heterogeneously in the 3-d model. TEER values have shown that the 3-d model is a leakier cell system with higher mannitol Papp (cm/s). Biochemical characterization (hydrolase activities, CYP450 expression) suggested that the 3-d model was at a lower maturation level than the 21-d model. Carrier-mediated uptake of L-Phe was lower in the 3-d model suggesting that this model has limited application for mechanistic studies. Reasonable correlation was obtained between the two models (r2=0.88, p>0.01) for 11 passively absorbed compounds with high potential of rank ordering of compounds. Although results suggested that the 3-d cells are under-differentiated, they could be usable to estimate the oral absorption of passively absorbed compounds.

Biological Transport↗

PubMed1996

Microcalcification clustering parameters in breast disease: a morphometric analysis of radiographs of excision specimens.

X-ray microradiography of surgically excised breast specimens offers the possibility of morphological characterization of calcifications. When combined with digital imaging techniques there exists added potential for obtaining valuable basic quantitative morphometric information regarding differences between microcalcifications in tissues exhibiting evidence of fibrocystic change, benign and malignant tumours. A total of 157 excised breast specimens from 84 patients were microradiographed using a Softex Super Soft X-ray unit and Kodak AA high resolution industrial film. A Quantimet 570C image analysis system was used to digitize and analyse the microradiographs. Of the 157 microradiographs, 51 (from 30 patients) revealed microcalcification clusters. The existence of significant differences between the three identified categories of tissue were indicated by clustering parameters. These included the number of particles per cluster, area of clusters, maximum distance to nearest neighbour, and geometric mean distance to nearest neighbour. The distribution pattern index (DPI), another of the clustering parameters used in this study, has been observed to be a particularly powerful discriminator. The value for fibrocystic change was found to be significantly smaller (0.514) than that for benign tumour (0.796) whilst that for benign tumour was observed to be significantly larger than that for malignant tumour (0.604) at a p-value of less than 0.05 (Kruskal-Wallis one-way analysis of variance).

Adolescent↗

PubMed2002

Feline vaccine-associated fibrosarcoma: morphologic distinctions.

Forty-four primary feline vaccine-associated fibrosarcomas and 16 recurrences were examined histologically for detailed morphologic characterization with emphasis on tumor grade, presence of neoplastic multinucleated giant cells, presence and proportion of T and B lymphocytes within the tumor, and thin and intermediate filament contents of neoplastic and stromal cells. The microvascularity and proliferation rates of central and peripheral areas of the tumors were also quantified by computerized image analysis. For primary fibrosarcomas, 11 of 44 (25%) were grade I, 21 of 44 (47.7%) were grade II, and 12 of 44 (27.3%) were grade III. The recurrences followed a similar pattern: 4 of 16 (25%) were grade I, 8 of 16 (50%) were grade II, and 4 of 16 (25%) were grade III. A positive correlation was found between the presence of neoplastic multinucleated giant cells and tumor grade. These cells were present in 9 of 12 (75%) of grade III and none of the grade I tumors. Prominent peritumoral lymphoid aggregates or follicles were present in 59% of the tumors, and many contained high proportions of T lymphocytes, varying from 19 to 87%. All fibrosarcomas were immunoreactive for vimentin and 28 of 44 (64%) were reactive for alpha-smooth muscle actin. The actin-positive cells were either part of the tumor or formed a capsule around tumor nodules. The peripheral vascularity was significantly higher than the central vascular density but no difference was found in tumor cell proliferation rates between the two areas. Centrally located, fluid-filled micro- or macrocavitations were frequently observed in the large vaccine sarcomas and probably formed secondary to rapid tumor growth and central necrosis.

Animals↗

PubMed2006

A novel fluorescent tracer for visualizing coupled cells in neural circuits of living tissue.

Gap junctions have diverse roles in a wide variety of tissues and have recently become a subject of intense investigation in neural circuits where synchrony and oscillations may play an important part. In circuits where gap junctions are present, the possibility arises of identifying intercommunicating cells via introduction of tracer into one cell and observing its spread into its coupled neighbors. Staining the coupled cells by this means opens the door to many vital techniques including paired-cell electrophysiology, RT-PCR, and morphological characterization of previously unknown coupled cells. Tracers commonly used at the present time are not generally suitable for these purposes in many tissues, including neurons. This paper describes how a fluorescent nuclear tracer, Po-pro-1, can be used to visualize coupled cells in several types of retinal neurons thought to be comprised of different connexin proteins including Cx36, Cx45, Cx50, and Cx57.

Amacrine Cells↗

PubMed1983

How does lung structure affect gas exchange?

The lung is characterized morphologically by establishing a very large surface and an exceedingly thin barrier between air and blood. A model for relating these structural features to the lung's gas exchange function is first developed. It is then shown that DO2 estimated by morphometry is about two times larger than that estimated by physiology; there are possible reasons for this. Comparing animals of high activity (dog, horse) with corresponding species of lower activity (man, cow) reveals that DO2 is proportional to O2 needs. The mechanical properties of the lung are discussed which allow such a large surface with such a thin barrier to be maintained lifelong. Surfactant properties of the lining layer are important factors in stabilizing the alveolar surface. Repair processes are also essential and require metabolic activities of the cells lining the barrier. The case of adult respiratory distress syndrome is used to illustrate the consequences of severe damage to the cell linings of alveoli: the barrier is thickened, and a good part of the alveolar surface is flooded by edema fluid, so that gas exchange is severely impaired.

Animals↗

PubMed1998

Apoptosis, the heat shock response, hyperthermia, birth defects, disease and cancer. Where are the common links?

Many cells die during normal prenatal development. Throughout postnatal life, production of new cells is balanced by death of older cells to maintain the normal mass of organs and tissues. In these situations, cell death is usually in the form of apoptosis, characterized morphologically by shrinkage of cellular contents within their membranes, condensation and margination of chromatin against the nuclear membrane and phagocytic removal by macrophages or adjacent cells of the organ. It is initiated and controlled by a complex set of gene-directed activities. The process is tidy and avoids the inflammatory effects of degenerating cellular contents on other tissues. The capacity to undergo this form of cell death is lost in neoplastic cell lines. In embryos the normal process of apoptosis has been termed programmed cell death, and in prenatal and mature animals a number of toxic agents can also cause morphologically typical forms of apoptosis. The heat shock (HS) response occurs in a wide range of plants and animals as a basic reaction that assists survival and recovery from the effects of heat and other toxic agents. It appears to be an extension of the cellular mechanism by which newly synthesized proteins are received by other ('chaperone') proteins to be transported within cells and folded into their functional configurations. Chaperone proteins adhere to hydrophobic sites on newly synthesized proteins, preventing the formation of functionless aggregates by random adhesion to hydrophobic sites on other proteins. After disengagement, the new protein can assume its proper configuration. Chaperone proteins are normally present in embryos, the genes encoding for their synthesis becoming activated particularly at inductive and rapid growth stages of organ formation. Hyperthermia and some other toxic agents also activate a set of inducible heat shock genes to synthesize induced HS proteins that adhere to uncovered hydrophobic sites on the heat denatured proteins, preventing random associations and allowing reconstitution or assisting degradation of irreparably damaged proteins. Irreparable damage usually results in a morphologically typical form of apoptotic cell death. Knowledge of signals initiating the response is incomplete but includes prostaglandin release, amplification by kinase cascades of mitogen and stress-activated protein signals, binding of the HS factor to the HS element on the HS gene. Maternal hyperthermia is a proven teratogen in all species studied. The HS response is inducible in early embryonic life but it fails to protect embryos against damage at certain stages of development. An embryo must absorb a threshold 'dose' of heat if defects are to be caused, the dose being the product of the level and the duration of elevation above the normal maternal temperature. The lowest elevation causing damage is 2-2.5 degrees C. Low elevations require longer durations and as the elevation increases, the time required is reduced logarithmically. Heat-induced defects are most common in the central nervous system (CNS) and include open neural tube, microencephaly, microphthalmia and neurogenic contractures. Apoptotic cells are found in these organs soon after threshold doses of heat. The periods of high susceptibility are brief, occurring at the time of organ induction and, paradoxically, at this stage, chaperone protein synthesis is at high levels, presumably to protect this process. Susceptibility might be due to gene activity being concentrated into organ induction with chaperone proteins being unavailable for repair of heat-denatured proteins. With activation of the HS response, normal protein synthesis is suspended (perhaps including those controlling induction of organs) and protective HS proteins are produced which rescue the embryo, but survival is achieved at the expense of normal development.

Animals↗

PubMed2001

Neuroendocrine marker expression in thyroid epithelial tumors.

Tissue sections from 50 cases with thyroid tumors, composed of 11 follicular adenomas, 10 follicular carcinomas, 14 papillary carcinomas, 10 anaplastic carcinomas, and 5 medullary carcinomas, were immunohistochemically analyzed for representative neuroendocrine markers. Immunoexpression ratios of these neuroendocrine markers were as follows: Follicular adenomas, neuron-specific enolase (NSE)63.6%, synaptophysin (SynP) 45.5%, Leu7 27.3%, NCAM 45.5%, chromogranin A (CgA) 0%, SNAP25 0%; follicular carcinomas, NSE 90.0%, SynP 80.0%, Leu7 80.0%, NCAM 0%, CgA 0%, SNAP25 0%; papillary carcinomas, NSE 85.7%, SynP 78.6%, Leu7 100%, NCAM 7.0%, CgA 0%, SNAP25.0%; anaplastic carcinomas, NSE 10.0%, SynP 0%, Leu7 0%, NCAM 0%, CgA 0%, SNAP25 0%; medullary carcinomas, NSE 100%, SynP100%, Leu7 80.0%, NCAM 40.0%, CgA 100%, SNAP25 100%. The two follicular carcinomas, which were morphologically characterized by "insular" (or "alveolar") arrangements, showed distinct immunoexpression of NSE and SynP at the same time. By in situ hybridization (ISH), expression of mRNA for NSE was confirmed in cases with marked immunoexpression of NSE. Although no endocrine granules were found, our results suggested that a specific type of follicular carcinoma, i.e., insular variant, may be immaturely neuroendocrine-differentiated.

Adenocarcinoma↗

PubMed2002

Etiology and significance of the optically clear nucleus.

Papillary thyroid carcinoma (PTC) is diagnosed in both cytology and surgical pathology specimens on the basis of distinct nuclear morphology, characterized by nuclear elongation, chromatin clearing, intranuclear grooves, and inclusions. Although these nuclear features are specific to papillary carcinoma, they can be mimicked in some benign conditions. The majority of PTC cases do not pose diagnostic problems. However, a distinct subset of cases has generated controversy among experts. These cases are follicular patterned tumors that show minimal nuclear changes in PTC. Several investigators have explored the role of immunohistochemical markers in the histologic diagnosis of PTC. Somatic rearrangements of the RET protooncogene are the most frequent genetic abnormality found in PTC. The frequency of these rearrangements has varied according to the geographic region, radiation exposure, and methodologies used and histologic variant of PTC. Recent studies have suggested that RET/PTC may be the cause of this specific nuclear change in PTC; however, the role of RET/PTC in tumor progression still needs to be defined.

Adenocarcinoma, Follicular↗

PubMed2006

Expression of Dlx5 and Dlx6 during specification of the elbow joint.

The onset of elbow joint formation in the developing limb is characterized morphologically by the conversion of differentiated chondrocytes at the site of incipient joint formation into the densely packed flattened cells of the joint interzone. However, experimental studies have indicated that the elbow joint is specified well before joint interzone formation by a distinctive population of precursor cells located at the site in the developing limb bud at which the elbow joint will subsequently form. Here we show that during specification of the elbow joint in the chick limb bud, the homeodomain transcription factors Dlx5 and Dlx6 are highly expressed by a discrete group of cells that encompass the prospective elbow joint. The Dlx5- and Dlx6-expressing cells at the prospective elbow joint are located where the differentiating humerus branches into the radius and ulna. Thus, Dlx5 and Dlx6 are the earliest molecular markers of the presumptive elbow joint yet described. The onset of Dlx5 expression in the region of the presumptive elbow joint is shortly followed by the initiation of expression amongst the Dlx5-expressing cells of Gdf5, which encodes a secreted signaling molecule that is involved in regulating the onset of joint formation. These results suggest that Dlx genes may be involved in specification of the elbow joint and/or in providing positional information that specifies the site at which the elbow joint will form.

Animals↗

PubMedDate not supplied

The sarcoplasmic reticulum of smooth muscle fibers.

The ability of the sarcoplasmic (endoplasmic) reticulum (SR, ER) of smooth muscle cells to accumulate Ca was demonstrated by measuring the uptake of 45Ca in fibers which were chemically skinned with saponin, and by electron cytochemistry of the accumulated Ca. The Ca uptake was dependent on ATP and it was stimulated by oxalate, as it is the case in SR of striated muscle. Electron microscopy of the skinned smooth muscle preparations revealed the presence of calcium oxalate deposits in the reticulum. The SR vesicles were isolated from several smooth muscles. The purification was carried out by taking advantage of the density increase of the SR vesicles after loading with calcium in the presence of oxalate. Among the muscles investigated the smooth muscle of the pig stomach was found to be the most suitable and it was selected for further biochemical and morphological characterization of the SR vesicles. These vesicles, which contain calcium oxalate crystals, were able to accumulate an additional amount of Ca. The Ca uptake was supported by several energy yielding substrates. Their order of potency was ATP greater than dATP congruent to UTP greater than ITP greater than GTP congruent to CTP. The rate of Ca uptake was two orders of magnitude slower than that in SR of skeletal muscle. The measurement of the level of phosphorylated Ca transport intermediate showed that this difference is due to smaller number of calcium transport sites per vesicle. The distribution of intramembrane particles in freeze-fractured specimens is in agreement with this conclusion.

Animals↗

PubMedDate not supplied

Isolation and evaluation of tannin-degrading fungal strains from the Mexican desert.

Eleven fungal strains (4 Penicillium commune, 2 Aspergillus niger, 2 Aspergillus rugulosa, Aspergillus terricola, Aspergillus ornatus and Aspergillus fumigatus) were isolated, characterized morphologically and by their capacity to degrade tannins. Aspergillus niger Aa-20 was used as control strain. Several concentrations of hydrolysable tannin (tannic acid) were used as sole carbon source. All strains were able to degrade hydrolysable tannins. Aspergillus niger GH1 and PSH showed the highest tannin-degrading capacity (67 and 70%, respectively). Also, the fungal capacity to degrade condensed tannin (catechin) was tested. Aspergillus niger PSH and Penicillium commune EH2 degraded 79.33% and 76.35% of catechin. The results demonstrated the capacity of fungi to use hydrolysable and condensed tannins as carbon source.

Biodegradation, Environmental↗

PubMed2005

Quantal size is independent of the release probability at hippocampal excitatory synapses.

Short-term synaptic plasticity changes the reliability of transmission during repetitive activation and allows different neuronal ensembles to encode distinct features of action potential trains. Identifying the mechanisms and the locus of expression of such plasticity is essential for understanding neuronal information processing. To determine the quantal parameters and the locus of alterations during short-term plasticity of cortical glutamatergic synapses, EPSCs were evoked in hippocampal oriens-alveus interneurons by CA1 pyramidal cells. The robust short-term facilitation of this connection allowed us to examine the transmission under functionally relevant but widely different release probability (P(r)) conditions. Paired whole-cell recordings permitted the functional and post hoc morphological characterization of the synapses. To determine the quantal size (q), the P(r), and the number of functional release sites (N(F)), two independent quantal analysis methods were used. Light and electron microscopy were performed to identify the number of synaptic junctions (N(EM)) between the recorded cells. The mean number of functional release sites (N(F(f)) = 2.9 +/- 0.4; n = 8) as inferred from a simple binomial model with no quantal variance agreed well with the mean of N(EM) (2.8 +/- 0.8; n = 6), but N(F(f)) never matched N(EM) when they were compared in individual pairs; however, including quantal variance in the model improved the functional prediction of the structural data. Furthermore, an increased P(r) (4.8 +/- 0.8-fold) fully accounted for the marked short-term facilitation of EPSCs (5.0 +/- 0.7-fold), and q was independent of P(r). Our results are consistent with the "one-release site, one-vesicle" hypothesis.

Animals↗