Biomedical subjects

W Liou

Publications and source records attributed to W Liou.

11 recordsLinked to original sources

PubMed1998

Experimental System for X-ray Cone-Beam Microtomography

: A laboratory test of X-ray tomography employing a diverging beam of X-rays rather than the usual parallel X-ray beam is described. We chose to test and demonstrate the advantages of divergent beam tomography by imaging an extracted juvenile human premolar using an ordinary dental X-ray source and a cooled CCD camera. Experiments with a three-piece cover-glass sample and with the human tooth demonstrated that three-dimensional reconstruction can be achieved at 34 µm per pixel resolution employing an X-ray tube spot 800 µm in its smallest direction without requiring close contact with the fluorescent screen. Reconstruction of a 256 x 256 pixel single-plane image from 100 projection images took only 45 sec on a personal computer with a Pentium 166 MHz processor. We have also demonstrated a volume reconstruction of 256 x 256 x 256 voxels from the data. Successful extension of this work to submicrometer projection X-ray microscopy is predicted. Improved resolution of medical tomography is another possible application.

Journal Article↗

PubMed1997

The autophagic and endocytic pathways converge at the nascent autophagic vacuoles.

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV--initial (AVi), intermediate (AVi/d), and degradative (AVd)--were defined by morphological criteria and immunogold labeling characteristics of marker enzymes. The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15-45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

Animals↗

PubMed1996

Improving structural integrity of cryosections for immunogold labeling.

Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution of this approach. We found that the step during which sections are thawed and transferred from the cryochamber to the supporting film on an EM grid is most critical for structural preservation. Surface tension of the transfer medium, on which sections are spread during thawing, can easily damage their structure by overstretching. By substituting a mixture of methylcellulose and sucrose for the conventional sucrose transfer medium, we were able to alleviate the problem of overstretching, thus improving greatly the structural integrity of thin cryosections. Also, material extraction from the sections after thawing causes structural damage, particularly when cross-linking is deficient. Incorporation of uranyl acetate in the transfer medium can then further help to maintain the structural integrity of the sections during the immunolabeling procedure. Excellent ultrastructure was featured in sections picked up and dried directly in methylcellulose/uranyl acetate mixtures. Such preparations can provide new insight into subcellular details and is an efficient back-up for immunolabeled sections in respect of their morphology. Cryosections from fresh frozen tissue can be preserved for immunolabeling by using transfer media that contain fixatives. This approach may have advantages if chemical fixation of tissue is thought to induce morphological artifacts or antigen redistribution.

Animals↗

PubMed1995

Ultrastructural and immunocytochemical characterization of autophagic vacuoles in isolated hepatocytes: effects of vinblastine and asparagine on vacuole distributions.

The interactions between the autophagic and the endocytic degradation pathways were investigated by means of immunogold labeling of autophagic vacuoles (AVs) in ultrathin frozen sections from isolated rat hepatocytes. AVs were identified by their autophagocytosed contents of the degradation-resistant cytosolic enzyme CuZn-superoxide dismutase (SOD). Another cytosolic enzyme, carbonic anhydrase (CAIII), was rapidly degraded in the lysosomes, making the vacuolar CAIII/SOD ratio useful as a rough indicator of the progress of autophagic-lysosomal degradation. Lysosomes could be recognized by the presence of the lysosomal membrane glycoprotein lgp120, which was absent from hepatocytic endosomes. Endocytic inputs into the AVs were detected by the presence of gold-conjugated bovine serum albumin (BSA-gold), taken up by fluid-phase endocytosis. All vacuoles recognized morphologically as AVs were SOD-positive, as were essentially all of the lysosomes (96%). The majority (72%) of the lysosomes also labeled positively for BSA within 2 h of endocytosis. The data are thus compatible with the notion that all lysosomes can engage in both autophagic and endocytic degradation. Lgp120 appeared to distinguish well between lysosomes and nonlysosomal AVs: the lgp120-negative AVs (nonlysosomes) had a CAIII/SOD ratio identical to that of the cytosol, indicating that no degradation had occurred. In the lgp120-positive AVs (lysosomes), the ratio was only 43% of the cytosolic value, consistent with substantial CAIII degradation. Among the nonlysosomal AVs (about one-third of all AVs), one-half were BSA-positive, suggesting that early AVs (autophagosomes) and suggesting that early AVs (autophagosomes) and intermediary AVs (amphisomes) that had fused with endosomes were equally abundant. These morphological data thus support previous biochemical evidence for a prelysosomal meeting of the autophagic and endocytic pathways. The microtubule inhibitor vinblastine inhibited the autophagic influx to the lysosomes, causing an accumulation of autophagosomes and a reduction in average lysosomal size. Vinblastine also inhibited the endocytic flux, thereby precluding the formation of amphisomes and of BSA-positive lysosomes. High concentrations (20 mM) of asparagine induced swelling of amphisomes and of BSA-positive lysosomes, probably reflecting an acidotropic effect of ammonia generated by asparagine deamination. Asparagine also caused an accumulation of autophagosomes, amphisomes, and BSA-negative lysosomes, presumably as a result of impaired fusion with the swollen BSA-positive lysosomes. The two agents thus appear to perturb the autophagic-endocytic-lysosomal vacuole dynamics by different mechanisms, making them useful in the further study of these complex organelle interactions.

Animals↗

PubMed1994

Expression of functional growth hormone receptor in a mouse L cell line infected with recombinant vaccinia virus.

The growth hormone receptor is a member of a large family of receptors including the receptors for prolactin and interleukins. Upon binding to one molecule of growth hormone two growth hormone receptor polypeptides dimerize. We have expressed the rabbit growth hormone receptor DNA in transfected mouse L cells infected with polymerase T7-producing vaccinia virus. The growth hormone receptor was synthesized as a 85-kDa protein and transported to the cell surface. Western blotting and metabolic labeling combined with immunoprecipitation using a rabbit antibody probably directed against the cytosolic domain of the receptor showed that its expression was dependent on both transfection of the growth hormone receptor DNA and vaccinia infection. Binding studies with 125I-labeled growth hormone demonstrated specific binding sites at the cell surface 20 h after transfection. Permeabilization with saponin showed that the growth hormone receptor binding sites were almost exclusively present at the cell surface with little intracellularly. Chemical crosslinking of the 125I-growth hormone complex resulted in a 180-kDa complex which could specifically be immunoprecipitated with the antiserum. Electron microscopic immunocytochemistry confirmed the presence of growth hormone receptor at the cell surface. Furthermore, specific growth hormone receptor antigen was also associated with intracellular membranes. These results thus show that this transient transfection system will be useful for cell biological studies of growth hormone receptor regulation.

Animals↗

PubMed1994

Ultrastructure and glycoconjugate histochemistry of the lens capsule during lens regeneration from the iris in the Newt.

The lens capsule of the regenerating lens develops from the basal lamina of the iris epithelium. As the lens differentiates and grows in size, the lens capsule increases in thickness by the formation of successive layers of basal laminar material resulting in a structure composed of increasing numbers of parallel lamellae. This initial arrangement may be lost and a more homogeneous composition of fine granules and filaments may characterize some parts of the capsule in older lenses. The developing lens capsule stains with the PAS and PAPD methods but not with the PAPS, AB and HID methods. Therefore, it appears to contain only neutral, non-acidic glycoconjugates (probably glycoproteins) which may contain fucose, galactose or mannose. This conclusion is supported by the incorporation of 3H-fucose into the developing lens capsule of regenerating lenses beginning at a stage with initial lens fiber formation. Except for the apparent absence of acidic glycoconjugates in the regenerating lens capsule, these events are similar to those described for developing lenses in chick and mammalian embryos. The differentiation of lens fibers by elongation, loss of organelles and accumulation of fine, electron-dense particles in the cytoplasm also parallels embryonic, lens fiber differentiation.

Animals↗

PubMed1993

Distribution of CuZn superoxide dismutase in rat liver.

Morphological and cell fractionation approaches were used to establish unambiguously the distribution of CuZn superoxide dismutase (CuZn SOD) in rat hepatocytes. Immunocytochemical observations revealed a primarily cytoplasmic localization of the enzyme. While only trace amounts were found in cell organelles like mitochondria and peroxisomes, lysosomes were labelled stronger than the cytoplasm. The presence of CuZn SOD in lysosomes was also identified in cell fractions of normal and Triton WR-1339-treated rats. Microscopic studies showed that the distribution of CuZn SOD was not affected by Triton, but the Triton treatment induced an apparent increase in the number and size of lysosomes with electron lucent contents which corresponded with a shift of lysosomes to low buoyant density fractions. The majority of CuZn SOD originally present in the mixed mitochondrial-peroxisomal-lysosomal fractions of the normal liver comigrated with the main peak of the lysosomal marker, beta-hexosaminidase, after Triton treatment. The peroxisomal marker, catalase, did not migrate with the CuZn SOD/beta-hexosaminidase-rich fractions in livers from Triton-treated animals. These results confirm earlier observations in rat liver cells, showing that CuZn SOD, a primarily cytosolic enzyme, accumulates in lysosomes. It is not present in significant amounts in rat hepatocyte peroxisomes.

Animals↗

PubMed1992

Morphology and glycoconjugate histochemistry of the palpebral glands of the adult newt, Notophthalmus viridescens.

The eyelids of the newt were studied in 10 microns serial paraffin and 1-2 microns plastic sections using standard histological stains and special stains for glycoconjugates. The eyelids contain four different glands. Simple acinar serous and simple acinar mucous glands occur in the skin; unicellular mucous glands occur in the conjunctiva; and convoluted tubular seromucous glands are present in connective tissue beneath the conjunctiva. The first two are identical to cutaneous glands found elsewhere on the head and body. The simple acinar serous glands are surrounded by myoepithelial cells and release their secretion, which is composed largely of proteins with minimal glycoconjugate content, by a holocrine mechanism. The secretory product of the simple acinar mucous glands is composed of neutral glycoconjugates with a minor content of acidic glycoconjugates; the mucin exhibits strong PAS and PAPD staining and weak staining by AB and PAPS methods. The unicellular conjunctival mucous glands secrete both neutral and acidic glycoconjugates as shown by positive reactions with PAS, PAPD, PAPS, and AB methods. Convoluted tubular seromucous glands in the ventral eyelid synthesize both proteins and neutral glycoconjugates. The mucous secretions of the conjunctival glands probably provide lubrication and protection for the cornea.

Animals↗

PubMed1990

Whole-mount preparations of mouse lens epithelium for the fluorescent cytological study of actin.

A technique is given for the preparation of a sheet of epithelial cells from the capsule of the crystalline lens. A new method is described for fixation and staining with fluorescent phalloidin or actin antibody in order to localize the actin cytoskeleton in this tissue. Optical section of the preparation resolves such actin features as apical polygonal arrays, sequestered actin bundles, perinuclear actin aggregates, observed here for the first time, and filamentous networks in the basal region of the cell. This method is superior to previous ones in its ability to preserve actin-abundant sectors distinctively.

Actins↗

PubMed1988

Actin filament patterns in mouse lens epithelium: a study of the effects of aging, injury, and genetics.

Using mainly fluorescence microscopy after rhodamine-phalloidin staining, the F-actin distribution in the mouse lens epithelium was studied with regard to the effects of age, genetic strain, and mechanical injury. These studies have revealed that aside from its association with the plasma membrane the structural organization of F-actin in the mouse lens epithelium in situ is characterized by two major configurations: (1) a filamentous arrangement in such patterns as stress fibers, polygonal arrays (PAs), and meshworks, and (2) a highly concentrated structure called a sequestered actin bundle (SAB). The aging study indicated that the SAB is a consistent character in C57BL/6 mice from the age of 5 wk on, but not in CF1 mice. The size and shape of the SAB change gradually with age as inferred from two-dimensional measurements. The genetic study on the SAB character using hybrids and congenic strains showed that it is inherited as a Mendelian dominant, probably multigenic mode. Finally, the injury study revealed a structural modification in cells around the wound, including flattening of cells at the edge and extension of processes into the wound space. In the rest of the epithelium, injury amplified membrane infolding and fluorescence of polygonal arrays but diminished the size and fluorescence intensity of SABs. These changes are thought to be correlated with wound repair involving cell division and migration. These studies illustrate the variability in F-actin expression in situ in lens epithelial cells that can be induced by intrinsic and extrinsic factors.

Actin Cytoskeleton↗

PubMed1986

Polygonal arrays of actin filaments in human lens epithelial cells. An aging study.

In order to determine the importance of lens actin filament configuration to lens accommodation, the pattern of actin filaments in the epithelium was studied in human lenses from different decades of life spanning the accommodative and non-accommodative years. Polygonal arrays of microfilaments were demonstrated in whole mounts of epithelium from normal and cataractous lenses using rhodamine phalloidin, an actin-specific, fluorescent-labeled probe. Tangential section transmission electron microscopy (TEM) studies confirmed that these arrays consist of central vertices and interconnecting filament rays, which line the apical end of each epithelial cell and appear to attach to the lateral membrane. These polygonal arrays were present in human lenses ranging from 25-94 yr of age. Measurements of intervertex distance showed remarkable constancy throughout the ages studied. In view of these findings, it is proposed that a possible function of these polygonal arrays is to stabilize the lens epithelium during lens flattening.

Actins↗