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PubMed1985

Ultrastructure of the olfactory neuron of the bullfrog: the dendrite and its microtubules.

The ciliated dendritic bulb of the olfactory neuron of the bullfrog was studied with the electron microscope, with emphasis on microtubular elements. Methods used included various fixation procedures with and without detergent extraction, serial sectioning, microtubule polarity assays, and an assay to demonstrate F-actin. Structural continuity exists, via microtubules, between the ciliary membrane and the perikaryon of the neuron. One type of structural link connects the distal end of the basal body to the plasma membrane and, in slightly oblique cross sections of the basal body, the link shows a highly characteristic tripartite profile resembling a claw hammer. The six to ten basal bodies of a dendritic bulb have a lateral foot that serves as an organizing center for microtubules, and these microtubules (totaling about 150) extend toward the perikaryon in the basal half of the epithelium. Polarity assays indicate that the attached or minus ends of dendritic microtubules are in the dendritic bulb, with their plus or fast-growing ends near or within the perikaryon of the neuron. It is shown that dendritic microtubules are depolymerized by direct osmium tetroxide fixation, in contrast to olfactory axonal microtubules, which persist after such fixation. F-actin appears to be abundantly present in the dendritic bulb of the neuron, and it is possible that this actin could play a role in shape changes of the dendrite. The various findings provide new information about the olfactory dendrite, its microtubule organizing centers, and the nature and relationships of its microtubules.

Actins↗

PubMedDate not supplied

Cytokeratin intermediate filaments of rat hepatocytes: different cytoskeletal domains and their three-dimensional structure.

A new method of visualizing the three-dimensional architecture of the cytokeratin filaments of the intact rat hepatocyte in situ has been achieved. Frozen sections of liver cut 10 micron thick were serially extracted to remove all elements of the cells except the intermediate filaments. Parallel sections were stained with monoclonal antibodies to the two main cytokeratins found in bile duct and liver cells. Immunofluorescent antibody and immunogold electron microscopy techniques were used to identify the proteins morphologically. Several new observations resulted from these studies. The pericanalicular sheath of intermediate filaments was visualized using steropairs as an uninterrupted branching tubular structure composed of cytokeratins located in the cell cortex of adjacent hepatocytes. Intermediate filaments in the cell cortex formed a distinct sheet of matted filaments which enveloped the entire hepatocyte. The cortical intermediate filaments were in continuity with the pericanalicular sheath and the filaments located within the cytoplasm. The intermediate filaments are attached to the centrioles and appeared to tent the nuclear lamina-pore complex at points of contact. Monoclonal antibodies to rat liver intermediate filament cytokeratins (CK49 and CK55) each stained intermediate filaments located in the cell cortex, within the cytoplasm and at the nucleus. By immunogold staining, some of the intermediate filament filaments were shown to contain both cytokeratins. Filaments which did not stain were thought to be either actin at the cell periphery or nuclear lamins around the nucleus. It is concluded that the cytokeratins form a specialized framework for the cell cortex, canaliculus, centrioles and the nucleus of hepatocytes. The filaments run continuously throughout the cytoplasm without terminating.

Animals↗

PubMed1991

Physico-chemical and immunological characteristics of pituitary prolactin from water buffaloes (Bubalus bubalis).

Prolactin (PRL) was purified from freshly frozen pituitary glands of water buffaloes (Bubalus bubalis) by a combination of existing procedures of Ellis and Jiang and Wilhelmi involving serial extraction of different pituitary proteins. The partially purified preparation was further fractionated on DEAE-Sephadex followed by Sephadex G-100 chromatography. This was finally purified on HPLC. This preparation was found to be homogeneous by SDS-PAGE and HPLC and had a single N-terminus amino acid (Threonine). The molecular size was estimated to be 24K +/- 0.5 by SDS-PAGE and approximately 25K by GPC-HPLC. The buffalo PRL gave a dose dependent inhibition curve in a rat liver based radio receptor assay with a potency of 30-35 I.U./mg and also in a partial homologous RIA using 125I-buffalo PRL and rabbit anti-oPRL serum giving a potency of 30 I.U./mg. Metabolic labelling studies using 35SO4(2-) with buffalo pituitary minces showed the incorporation of radioactive sulfate into immunoprecipitable PRL-like material. Physico-chemical characterization of the site of the linkage between sulfate and PRL revealed the presence of Tyr-O-SO4 in bu-PRL. A high affinity monoclonal antibody (MAB) with Ka of 10(10) L/M, belonging to IgG1 isotype, and capable of cross reacting with ovine and bovine PRL was generated. This MAB was conformation specific as reduced and carboxymethylated PRL did not react with it. A homologous RIA system using this MAB has been standardised.

Animals↗

PubMed1979

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Animals↗

PubMed1986

Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes.

We have prepared a monoclonal antibody, AE11, that recognizes specifically a 195-kD protein (pI 5.4) of human keratinocytes. This antigen constitutes approximately 0.01-0.1% of total protein in keratinocytes of skin, esophagus, and cornea, and is readily detectable in these cells by immunofluorescent staining and immunoblotting. However, it is barely detectable in MCF mammary carcinoma cells and HeLa cells, and is undetectable in nonepithelial cell types. Results from serial extraction experiments have shown that this protein exists in two distinct pools: a Tris-soluble, and a Tris-insoluble but urea- or SDS-soluble one. The distribution of the 195-kD protein between these two pools appears to be differentiation-related, since relatively undifferentiated cells selected by a low-calcium medium contain primarily the soluble form, while highly differentiated cells contain mainly the insoluble form. Data from immunofluorescent staining and trypsin-sensitivity experiments suggest that the soluble form is cytoplasmic, whereas the insoluble form is submembranously located at the cell periphery of upper, differentiated cells. The insoluble, cell peripheral form of the 195-kD antigen increases progressively during epidermal differentiation; its insolubility appears to be related to the formation of disulfide-bond(s). These results indicate that the 195-kD protein, which has recently been suggested to be involved in cornified envelope formation (Simon, M., and H. Green, 1985, Cell, 36:827-834), undergoes significant changes in its solubility characteristics and intracellular location during keratinocyte maturation.

Antibodies, Monoclonal↗

PubMed1976

Synthesis of elastin. A rapid formation of lysine-derived crosslinks by chick embryo aorta.

Aortas of 13-day-old chick embryo were labeled for 0.5 hr with [14C]lysine and subjected to a serial extraction after chase for 1-24 hr with [12C]lysine. Substantial radioactivity was found in insoluble elastin after 3 hr chase. The effect of beta-amino-propionitrile on labeling with [14C]lysine was also examined. Each fraction was hydrolyzed and applied to a short column on an amino acid analyzer. Radioactivity was found in desmosine and isodesmosine of insoluble elastin as early as 1 hr after the beginning of chase. The radioactivity increased rapidly at 2 hr and very slowly thereafter. A large count, which was separated into five peaks on a long column, was observed in other lysine derivatives at 2 hr and increased steadily up to 24 hr, while the lysine count decreased from 1 : 0.5 to 1 : 6 against lysine derivatives and from 1 : 0.04 to 1 : 0.9 against quarter-desmosine after 24 hr. The oxidation of lysine residues incorporated during the 0.5 hr pulse was almost completed during the first 1 hr of chase, and these oxidized residues were incorporated into crosslinks during the following 1 hr. It is suggested that poorly crosslinked elastin accumulated in the soluble fractions. The presence of crosslinking derived from lysine residues was also indicated in the microfibril fraction.

Amino Acids↗

PubMed1986

Description and characterization of a surface lectin from Giardia lamblia.

The mechanisms by which the human enteric pathogen Giardia lamblia colonizes the proximal small intestine are poorly understood. Although the parasite possesses an attachment organelle on its ventral surface, the "sucking" disk, we considered that like many bacteria and some protozoa, G. lamblia might also have a surface membrane-associated modality for adherence to its host. Using an erythrocyte mixed-agglutination model, we demonstrated a parasite surface lectin with specificities for D-glucosyl and D-mannosyl residues. This lectin is soluble in Triton X-100, is calcium dependent, and is maximally active at pH 5.5 to 6.0. Partial purification was achieved by serial extraction of parasites in Triton X-100 followed by Sephadex G-150 affinity chromatography. The lectin could not be surface radiolabeled with 125I-Bolton-Hunter reagent, but radiolabeling of the hapten eluate from an affinity column produced four bands of 57,000 to 78,000 Mr on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. The biological function of this lectin is unknown. The presence of mannosyl residues on the luminal surface of human small intestinal epithelial cells suggests that there are receptors for Giardia lectin at the site of colonization.

Animals↗

PubMed1986

Identification and characterization of a major early cytomegalovirus DNA-binding protein.

We characterized a DNA-binding protein with an approximate molecular weight of 129,000 (DB129) which is present in the nuclei of cytomegalovirus- (strain Colburn) infected cells, but not in virus particles. Results of two types of experiments demonstrated that DB129 is a member of the early class of herpesviral proteins. First, time course pulse-labeling experiments showed that its synthesis begins after that of the immediate-early protein IE94, but prior to the appearance of late viral proteins, and was reduced at late times. Second, in the presence of inhibitors of viral DNA replication, DB129 continued to be made and accumulated to elevated levels. A second set of experiments showed that DB129 bound to single-stranded DNA in vitro and was eluted by a NaCl gradient in two peaks, one at about 0.2 M and the second at about 0.6 M. A similar pattern of release was observed when infected-cell nuclei were serially extracted with increasing NaCl concentrations. In addition, treatment of nuclei with DNase I selectively released DB129, along with a small but significant fraction of another DNA-binding protein, DB51. These results suggest that DB129 is associated with DNA in vivo and that it interacts directly with single-stranded DNA. It was also shown that cells infected with human cytomegalovirus (strain Towne) contain a slightly larger counterpart to DB129, which was designated DB140. Similarities between these proteins and the major DNA-binding protein of herpes simplex virus are discussed.

Cell Nucleus↗

PubMed1991

Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay.

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.

Animals↗

PubMed1992

An unexpected growth pattern: considerations in management.

A case study spanning 16 years is presented. Routine treatment with serial extraction and an edgewise appliance for a Class I crowded occlusion began when the patient was 8 years old. By the time the patient was 17, his dentition had evolved, because of unanticipated growth, into a Class III malocclusion with complete-arch crossbite. The question of whether it is most appropriate to treat this patient in one or two surgical procedures is discussed, and some controversial aspects and concerns are presented.

Adolescent↗

PubMed1979

Isolation and partial characterization of prolactin from equine pituitary gland (hypophysis).

Highly purified equine prolactin was prepared from equine pituitary glands (hypophysis) by serial extractions with water at pH 5.5, 0.1 M (NH4)2SO4 at pH 4.0, and 0.25 M (NH4)2SO4 at pH 5.5 to remove other hormones, and then finally with 70% ethanol at pH 9.3 to 10.0 to extract prolactin. Preliminary purification of the extract involved salting out other substances with 0.1% NaCl at pH 9.0. Prolactin was precipitated out by adding three times the volume of 95% ethanol at 4 C. This prolactin preparation had a biological potency of 24 IU/mg. Further purification by isoelectric focusing on a pH gradient of 5 to 7 gave three prolactin components with the following characteristics: isoelectric point 5.8, 5.7, and 5.25; biological potencies (IU/mg) 35.6, 19.6, and 11.3. The major component had a molecular weight of 25,000, an isoelectric point of 5.8, and a biological potency of 35.6 IU/mg. Antiserum produced against this component did not cross-react with equine follicular stimulating hormone, luteinizing hormone, and growth hormone, but did cross-react with ovine and bovine prolactin. Human and murine prolactin had little cross-reactivity with the equine prolactin antiserum.

Animals↗

PubMed1990

Stabilisation of refraction following extracapsular cataract extraction.

Regular serial refraction was used to determine the rate of stabilisation of refraction following routine extracapsular cataract surgery in 85 eyes. Patients were divided into four groups depending on wound closure technique: limbal section closed by interrupted 8-0 virgin silk, 9-0 polydioxanone or 10-0 nylon sutures, and corneal section closed by continuous 10-0 nylon suture. Stabilisation of refraction, sufficient to prescribe satisfactory glasses, occurred at three months except in the interrupted 10-0 nylon group, when stabilisation was delayed to between four and five months. The factors affecting stabilisation and the causes of the differences observed are discussed.

Aged↗

PubMed1987

Microcomputer-aided reconstruction: a system designed for the study of 3-D microstructure in histology and histopathology.

We have designed a microcomputer system to reproduce 3-D tissue structures graphically from serial microscopic sections. The system, based on a Hewlett-Packard model 310 desktop computer, comprises a floppy disc drive, a hard-disc that extends the available user memory by adding 40 Mbytes, a colour memory-mapped graphics display, a graphics tablet and a graphics printer. A set of serial 2-D images, manually extracted from serial sections using a microprojector, are digitized on the tablet by tracing the contours of the structure of interest; up to ten different structures can be inputted and reproduced using a 'hidden line' effect. The software, written mainly in HP-BASIC 4.0, produces 3-D pictures of a tissue structure either as a 'stack of slices' with or without being 'tiled' with triangular patches, or in 'wire framing'. Any angle of rotation around the x, y and z axes is assigned for the image to be reconstructed, allowing the operator to obtain the best perspective. The system also discloses the internal connectivity of an object by reducing the structure to a network diagram; this is especially useful in analysing the topological properties of tissue structure. It is shown how, in some examples, the system contributes to a better understanding of tissue microstructures and their morbid changes and how, as an effective tool in morphology, it will aid future studies of histology and histopathology.

Adenocarcinoma↗

PubMed1988

Reproductive responses in male rats fed extracts of fescue seed infected by the endophytic fungus Acremonium coenophialum.

This study was undertaken to determine whether a methanol extract of endophyte-infected tall fescue seed contained the factor(s) that were responsible for the decrease in male reproductive potential when rats were fed toxic tall fescue seed. Twenty-four Sprague-Dawley male rats (ca. 225 g.) were randomly allocated to one of three dietary treatments containing 50% (w/w) rodent chow and 50%: (1) noninfected tall fescue seed; (2) noninfected tall fescue seed treated with concentrated methanol effluent from serial methanol extraction of infected seed; and (3) the extracted seed residue of seed that was greater than 95% infected by Acremonium coenophialum. Parameters assessed were testicular weight, epidydimal weight, parenchymal weight, progressive sperm motility, sperm motility, sperm count and daily sperm production potential. Significant reduction (P less than 0.05) for feed intake, average daily gain, testicular weight, epidydimal weight and daily sperm production potential were observed for treatment 2. These data suggest that the serial methanol extract contains the factor(s) responsible for the lowered reproductive potential in male rats. The extract contained 15,578 micrograms/ml of pyrrolizidine alkaloids and only trace amounts of ergopeptides. These results suggest that N-formyl and N-acetyl loline may play an important role in the fescue toxicity noted in the exposed rats.

Acremonium↗

PubMed1971