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PubMed1999

Selective sensitivity of early postmitotic retinal cells to apoptosis induced by inhibition of protein synthesis.

In previous work we showed that apoptosis in retinal tissue from developing rats can be induced by inhibition of protein synthesis (Rehen et al. 1996, Development, 122, 1439-1448). Here we show that recent postmitotic cells are the cells sensitive to apoptosis triggered by blockade of protein synthesis. To label all proliferating cells in the retina, a series of injections of the nucleotide analogue, bromo-deoxy-uridine (BrdU, 60 mg/kg b.w.), was given in rat pups. Then, explants of the retina were incubated in vitro with the inhibitor of protein synthesis anisomycin (1.0-3.2 microg/mL) for 1 day to induce apoptosis. Detection of apoptotic bodies under differential interference contrast microscopy was combined with immunocytochemistry for BrdU, proliferating cell nuclear antigen (PCNA) or for various markers of retinal cell differentiation. Despite the large number of BrdU- and PCNA-labelled cells in the tissue, the vast majority of the cells that underwent apoptosis were postmitotic cells which have left the mitotic cycle 3-4 days before. However, these cells were not labelled with antibodies to calretinin, calbindin, rhodopsin or to a Muller glial cell marker, suggesting that these are early postmitotic neurons. We suggest that during migration and initial differentiation, the apoptotic machinery is blocked by suppressor proteins, thus allowing recent postmitotic cells to find their final positions and differentiate while protected from apoptosis.

Animals↗

PubMed1996

Evaluation of asymptomatic microscopic haematuria--influence and clinical relevance of osmolality and pH on urinary erythrocyte morphology.

OBJECTIVE: To determine whether the morphological distinction between 'dysmorphic' and 'eumorphic' erythrocytes in urinary sediment differentiates microscopic haematuria (MH) from reno-parenchymal and post-renal bleeding. MATERIALS AND METHODS: The erythrocyte morphology of 2145 urinary sediments from 1391 patients with MH was evaluated by interference-contrast microscopy and compared with the osmolality, pH and specific gravity of the urine samples. RESULTS: Compared with more concentrated urine specimens, samples of < 700 mOsmol/kg showed significantly lower percentages of dysmorphic erythrocytes; there was a similar reduction in this percentage at a pH > or = 7. In addition, erythrocytes lysed in diluted or alkaline urine and therefore, under these conditions, no diagnosis could be made. CONCLUSION: The assessment of erythrocytes in urinary sediment should be performed only under 'standard conditions', i.e. in concentrated and acidic urine, > or = 700 mOsmol/kg and a pH < 7. The presence of > or = 90% dysmorphic erythrocytes in patients with asymptomatic MH, the absence of proteinuria, a normal blood pressure and normal radiological examination indicates 'reno-parenchymal MH', requiring a long-term follow-up with a routine evaluation twice a year, but no immediate treatment in most cases. In contrast, the presence of > or = 90% eumorphic erythrocytes or even 'mixed' results (10-90% eumorphic erythrocytes) indicates 'post-renal MH', requiring a complete urological evaluation.

Decision Trees↗

PubMed2001

A degeneration-based hypothesis for interpreting fibrillar changes in the osteoarthritic cartilage matrix.

The collagen fibrillar architectures in the general matrix of cartilage slices removed from both normal and osteoarthritic femoral heads were examined by both differential interference light microscopy and scanning electron microscopy. Whereas the normal general matrix contained a finely differentiated pseudo-random weave of fibrils developed from an interconnected array of radial elements, the osteoarthritic general matrix was characterised by the presence of structurally distinct regions consisting of strongly aligned radial bundles of fibrils and associated intense tangles or 'knotted' features. Simple structural models were developed to explore possible transformation structures based on two different types of interconnectivity in the three-dimensional fibrillar network. These models support the hypothesis that the distinctive ultrastructural features of the osteoarthritic general matrix can develop as a consequence of largely passive degradative changes occurring in the fibrillar weave originally present in the normal matrix. This could, in principle, occur independently of any new structure that might develop as a consequence of any upregulation of collagen associated with the osteoarthritic process.

Adult↗

PubMed2006

Radixin is required to maintain apical canalicular membrane structure and function in rat hepatocytes.

BACKGROUND & AIMS: Ezrin-radixin-moesin proteins are cross-linkers between the plasma membrane and actin filaments. Radixin, the dominant ezrin-radixin-moesin protein in hepatocytes, has been reported to selectively tether multidrug-resistance-associated protein 2 to the apical canalicular membrane. However, it remains to be determined if this is its primary function. METHODS: An adenovirus-mediated short interfering RNA (siRNA) was used to down-regulate radixin expression in collagen sandwich-cultured rat hepatocytes and morphologic and functional changes were characterized quantitatively. RESULTS: In control cultures, an extensive bile canalicular network developed with properly localized apical and basolateral transporters that provided for functional excretion of fluorescent cholephiles into the bile canalicular lumina. siRNA-induced suppression of radixin was associated with a marked reduction in the canalicular membrane structure as observed by differential interference contrast microscopy and F-actin staining, in contrast to control cells exposed to adenovirus encoding scrambled siRNA. Indirect immunofluorescence showed that apical transporters (multidrug-resistance-associated protein 2, bile salt export pump, and multidrug-resistance protein 1) dissociated from their normal location at the apical membrane and were found largely associated with Rab11-containing endosomes. Localization of the basolateral membrane transporter, organic anion transporting polypeptide 2 (Oatp2), was not affected. Consistent with this dislocation of apical transporters, the biliary excretion of glutathione-methylfluorescein and cholylglycylamido-fluorescein was decreased significantly in the radixin-deficient cells, but not in the control siRNA cells. CONCLUSIONS: Radixin is essential for maintaining the polarized targeting and/or retaining of canalicular membrane transporters and is a critical determinant of the overall structure and function of the apical membrane of hepatocytes.

Animals↗

PubMed2002

[Application of Optical Coherence Tomography (OCT) in middle ear surgery].

BACKGROUND: Middle ear surgery has reached an advanced stage of development over the last few decades. The use of biocompatible materials offers new possibilities in ossiculoplasty. The exact calculation concerning the length of the implant to be used, however, still poses considerable difficulties and is an additional cause for a remaining air conduction difference or a further surgical intervention. METHODS: Optical coherence tomography (OCT) is a non-invasive imaging procedure for the imaging of tissue structures with a resolution accurate to micrometres. OCT can be operated touch-free and for this reason does not require any means of contact between applicator and sample. In the present study an optical coherence inferometre was coupled to an operating microscope and used in 5 stapedoplasties and 5 tympanoplasties type III in order to determine the length of the prosthesis to be used. RESULTS: The use of OCT implies an intraoperative measuring time of only a few seconds. The coupling in the optical path of an operating microscope allows trouble-free handling. The measurement of middle ear structures has an accuracy of 30 micrometer. The postoperative audiological results show a good auditory performance. CONCLUSIONS: Initial experience with OCT indicates that the technology will be extremely interesting for otosurgery. Improvements in functional pattern will enable routine intervention in connection with an operating microscope. Further OCT applications for tissue differentiation will have to follow.

Equipment Design↗

PubMed1999

[Diagnostic procedure in primary ciliary dyskinesia].

BACKGROUND: Primary ciliary dyskinesia (PCD) is usually diagnosed by ultrastructural investigations of nasal or bronchial mucosa. Less invasive techniques for quick and cost-effective diagnosis of PCD should be evaluated. METHODS: In 32 patients with suspected PCD, saccharin transport time, ciliary beating of nasal respiratory epithelium, and ultrastructure of nasal mucosa biopsies were investigated. RESULTS: In 13 patients, PCD was excluded by a normal saccharin transport time (< 20 min). In 19 patients, respiratory cells were obtained by nasal brushing. Frequency, coordination, and amplitude of ciliary beating were examined using interference contrast microscopy and scored. PCD was excluded in 10 of 19 patients, who revealed normal ciliary activity. In the remaining 9 patients, nasal mucosa specimens for ultrastructural investigation were obtained. Ultrastructural alterations indicating PCD were found 4 of 9 specimens, in 2 specimens no cilia were found and in 3 specimens alterations indicating secondary inflammatory alterations were found. CONCLUSIONS: PCD may be efficiently excluded in several cases using cost-effective diagnostic techniques. The definite diagnosis in the remaining cases requires ultrastructural investigations which should be performed in specialized centers. In the present study, PCD in a carefully preselected group had a surprisingly high prevalence.

Adolescent↗

PubMed1994

Effect of culture in vitro and organ culture on the dry mass of preimplantation mouse embryos.

The dry mass of mouse embryos cultured in vitro in medium alone or in an organ culture system were measured by means of the Vickers M86 scanning microinterferometer. The data were compared with previous data on the dry mass of preimplantation embryos in vivo. The metabolism of embryos cultured in vitro differs from that of fresh embryos. In cultured embryos, dry mass decreases throughout the 2-cell stage whereas the dry mass is increasing at this stage in vivo. Embryos in an organ culture system regain a dry mass profile, similar to that observed in vivo at the late cleavage stage. These results support the view that conditions for embryo metabolism are suboptimal in vitro and that, although the oviduct may confer some advantage on developing embryos in vitro, it is unable fully to support the pattern of metabolism, as assessed by dry mass, observed in vivo.

Animals↗

PubMed2004

Biological glass fibers: correlation between optical and structural properties.

Biological systems have, through the course of time, evolved unique solutions for complex optical problems. These solutions are often achieved through a sophisticated control of fine structural features. Here we present a detailed study of the optical properties of basalia spicules from the glass sponge Euplectella aspergillum and reconcile them with structural characteristics. We show these biosilica fibers to have a distinctive layered design with specific compositional variations in the glass/organic composite and a corresponding nonuniform refractive index profile with a high-index core and a low-index cladding. The spicules can function as single-mode, few-mode, or multimode fibers, with spines serving as illumination points along the spicule shaft. The presence of a lens-like structure at the end of the fiber increases its light-collecting efficiency. Although free-space coupling experiments emphasize the similarity of these spicules to commercial optical fibers, the absence of any birefringence, the presence of technologically inaccessible dopants in the fibers, and their improved mechanical properties highlight the advantages of the low-temperature synthesis used by biology to construct these remarkable structures.

Animals↗

PubMed2006

A physical model of axonal damage due to oxidative stress.

Oxidative damage is implicated in the pathogenesis of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases, and in normal aging. Here, we model oxidative stress in neurons using photogenerated radicals in a simplified membrane-encapsulated microtubule system. Using fluorescence and differential interference contrast microscopies, we monitor photochemically induced microtubule breakdown on the supported region of membrane in encapsulating synthetic liposomes as a function of lipid composition and environment. Degradation of vesicle-encapsulated microtubules is caused by attack from free radicals formed upon UV excitation of the lipid-soluble fluorescent probe, 6-(9-anthroyloxy)stearic acid. Probe concentration was typically limited to a regime in which microtubule degradation was slow, and microtubule degradation was monitored by changes in the observed protrusion of the membrane surface. The kinetics of microtubule degradation are influenced by lipid saturation level, fluorescent probe concentration, and the presence of free-radical scavengers. This system is sufficient to reproduce some degenerative morphologies found in vivo.

Axons↗

PubMed2006

Spatiotemporal control of spindle midzone formation by PRC1 in human cells.

We have examined the role of PRC1, a midzone-associated, microtubule bundling, Cdk substrate protein, in regulating the spatiotemporal formation of the midzone in HeLa cells. Cdk-mediated phosphorylation of PRC1 in early mitosis holds PRC1 in an inactive monomeric state. During the metaphase-to-anaphase transition, PRC1 is dephosphorylated, promoting PRC1 oligomerization. Using time-lapse video microscopy, RNA interference, 3D immunofluorescence reconstruction imaging, and rescue experiments, we demonstrate that the dephosphorylated form of PRC1 is essential for bundling antiparallel, nonkinetochore, interdigitating microtubules to establish the midzone that is necessary for cytokinesis. Our results thus indicate that PRC1 is an essential factor in controlling the spatiotemporal formation of the midzone in human cells.

CDC2 Protein Kinase↗

PubMed2003

Mdm20 protein functions with Nat3 protein to acetylate Tpm1 protein and regulate tropomyosin-actin interactions in budding yeast.

The evolutionarily conserved Mdm20 protein (Mdm20p) plays an important role in tropomyosin-F-actin interactions that generate actin filaments and cables in budding yeast. However, Mdm20p is not a structural component of actin filaments or cables, and its exact function in cable stability has remained a mystery. Here, we show that cells lacking Mdm20p fail to N-terminally acetylate Tpm1p, an abundant form of tropomyosin that binds and stabilizes actin filaments and cables. The F-actin-binding activity of unacetylated Tpm1p is reduced severely relative to the acetylated form. These results are complemented by the recent report that Mdm20p copurifies with one of three acetyltransferases in yeast, the NatB complex. We present genetic evidence that Mdm20p functions cooperatively with Nat3p, the catalytic subunit of the NatB acetyltransferase. These combined results strongly suggest that Mdm20p-dependent, N-terminal acetylation of Tpm1p by the NatB complex is required for Tpm1p association with, and stabilization of, actin filaments and cables.

Acetylation↗

PubMed2000

Connexin expression in electrically coupled postnatal rat brain neurons.

Electrical coupling by gap junctions is an important form of cell-to-cell communication in early brain development. Whereas glial cells remain electrically coupled at postnatal stages, adult vertebrate neurons were thought to communicate mainly via chemical synapses. There is now accumulating evidence that in certain neuronal cell populations the capacity for electrical signaling by gap junction channels is still present in the adult. Here we identified electrically coupled pairs of neurons between postnatal days 12 and 18 in rat visual cortex, somatosensory cortex, and hippocampus. Notably, coupling was found both between pairs of inhibitory neurons and between inhibitory and excitatory neurons. Molecular analysis by single-cell reverse transcription-PCR revealed a differential expression pattern of connexins in these identified neurons.

Animals↗

PubMed2003

Coloration strategies in peacock feathers.

We report the mechanism of color production in peacock feathers. We find that the cortex in differently colored barbules, which contains a 2D photonic-crystal structure, is responsible for coloration. Simulations reveal that the photonic-crystal structure possesses a partial photonic bandgap along the direction normal to the cortex surface, for frequencies within which light is strongly reflected. Coloration strategies in peacock feathers are very ingenious and simple: controlling the lattice constant and the number of periods in the photonic-crystal structure. Varying the lattice constant produces diversified colors. The reduction of the number of periods brings additional colors, causing mixed coloration.

Animals↗

PubMed1971

Potassium cyanate as an inhibitor of the sickling of erythrocytes in vitro.

The recent use of urea as a treatment for the crisis phase of sickle-cell anemia has prompted us to investigate the possibility that cyanate, which is in equilibrium with urea in solution, might itself prevent the sickling of erythrocytes. We have found that in contrast to the high concentration of urea (1 M) needed to prevent reversibly the in vitro sickling of 80% of the cells, potassium cyanate (0.01-0.10 M) irreversibly inhibits sickling to the same extent. The prevention of sickling is a function of the amount of [(14)C]cyanate incorporated into acidprecipitable protein (0.1-1.0 mol of cyanate per mol of hemoglobin). Most of the radioactivity is accounted for by carbamylation of the NH(2)-terminal valine residues of hemoglobin; there is no detectable carbamylation of the lysine or cysteine residues. The reactive species, HN=C=O (isocyanic acid), may be an analog of O=C=O since both compounds bind to the same valine residues of hemoglobin. Deoxygenated sickled cells also incorporate [(14)C]-cyanate, but the sickling is not reversed. Oxygenation results in normal morphology in 75% of these cells. Upon subsequent deoxygenation, these cells remain normal. Potassium cyanate (5 mM) was also found to be an effective inhibitor of the gelling of deoxyhemoglobin S.

Anemia, Sickle Cell↗

PubMed1972

Structural and functional characterization of isolated pancreatic exocrine cells.

Viable isolated exocrine cells have been obtained from guinea-pig pancreas by a tissue dissociation procedure using crude collagenase (EC 3.4.4.19) and hyaluronidase (EC 3.2.1.35), chelation of divalent cations, and mild shearing forces. Cell yields are 50-60%, based on recovered DNA, and about 90% of the population consists of exocrine cells which, although rounded up, retain their in situ polarity with regard to regional distribution of zymogen granules and specialization of the former luminal plasmalemma. The isolated cells incorporate labeled amino acids into proteins at linear rates for at least 4 hr at levels comparable to pancreatic slices in vitro; more than 95% of the exocrine cell population is active in this process, as shown by autoradiography. In response to secretogogues at optimal doses (100 muM carbamylcholine; 0.1 muM pancreozymin, or 0.01 muM caerulein), the cells discharge up to 30% of their content of pulse-labeled secretory proteins to the medium over a 3-hr period; in the same time, the controls release about 5% of their content. The results indicate that isolated exocrine cells are capable of synthesizing the processing secretory proteins, and of responding directly to cholinergic and peptidic secretogogues.

Animals↗

PubMed1973

Affinity binding of intact fat cells and their ghosts to immobilized insulin.

Immobilized insulin, prepared by coupling insulin directly to agarose or through hydrocarbon "connecting arms," was demonstrated to be capable of firmly binding intact adipocytes and their ghosts. Various lines of evidence indicate that the insulin receptor on the plasma membrane, in addition to the insulin coupled to the agarose, was responsible for the observed binding. This evidence includes: (a) the finding that increasing the "arm" length increased the binding capacities of insulin-Sepharose affinity chromatographic columns, (b) specific inhibition and reversal by insulin and antiserum to insulin of the binding, as compared to lesser effects by other peptide hormones, (c) the indication that only the plasma membrane sacs, not the other cellular contaminants in the crude ghosts, are capable of binding, and (d) the impairment and restoration of trypsin-sensitive membrane binding sites that are also required for insulin biosensitivity. These findings support the idea that the insulin receptor is the trypsin-sensitive site. By use of the differential buoyant densities of the various cell-bead complexes that resulted from the interaction of adipocytes with insulin-Sepharose, a new procedure was developed to demonstrate and study the binding. These complexes could also be demonstrated by interference contrast microscopy. Binding readily occurred under conditions favorable for insulin stimulation of the cells. By coupling tracer amounts of [(125)I]insulin to Sepharose or insulin-Sepharose, the effects of anti-insulin antisera, free insulin, and other peptide hormones and supplemental factors on the buoyant-density distribution of the complexes could be measured, as well as the effects of other ligands coupled to Sepharose.

Adipose Tissue↗

PubMed1975

Donnan potential of rabbit skeletal muscle myofibrils I: electrofluorochromometric detection of potential.

The fluorescence of the dye CC-6 [(3-hexyl-2-(3-hexyl-2-benzoxazolinylidene)-1-propenyl)-benzoxazolium iodide] has been shown to indicate Donnan potentials in rabbit skeletal muscle myofibrils. These results are in agreement with previously published work in which the potentials were measured with microelectrodes on glycerol-extraced muscle fibers. The magnitude of the Donnan potential of the myofibrils has been shown to be dependent on the state (rigor or relaxed) of the system.

Adenosine Triphosphate↗

PubMed1980

Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus.

It was recently shown by combined immunofluorescence and interference reflection microscopy that a protein named vinculin, along with alpha-actinin, is concentrated at focal adhesion plaques inside cultured normal fibroblasts [Geiger, B. (1979) Cell 18, 193-205]. These plaques are the discrete, isolated sites of strong adhesions formed between the ventral surfaces of the cells and the substrata on which they are grown. We show that after transformation of fibroblasts by Rous sarcoma virus a majority of the cells have many fewer focal adhesion plaques and now exhibit a cluster of small patches that are immunolabelled for both vinculin and alpha-actinin. Such a cluster (rosette) is located near the ventral surface of the cell, usually partly under the nucleus. The significance that these altered distributions of vinculin and alpha-actinin may have for the rounding up and loss of adherence of transformed cells is discussed.

Actinin↗