Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Negative Staining”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,639 records · Page 91Linked to original sources

Evaluation of the range of areas of the fluorescein staining patterns of the tarsal conjunctiva in man.

Fluorescein dye is commonly used to highlight the tarsal conjunctiva in man to facilitate diagnosis of papillary conjunctivitis. A quantitative analysis of the fluorescein-highlighted features could be useful in both assignment of severity grades as well as for objective comparisons but no data is available on the distributions of sizes (areas) of these features. An exploratory, range-finding study was undertaken to provide data on feature areas that might be encountered. After application of fluorescein, 35 mm photographs were taken at a fixed distance with zone 1/zone 2 (Allansmith) of the tarsal plate aligned perpendicular to the camera. The photographs were projected, an overlay of 150 to 180 contiguous features outlined by fluorescein made and planimetry used to assess the areas and area distributions of the features on tarsal plates clinically graded with stages 1 to 4 papillary hypertrophy. For zone 1 of the tarsal plate in asymptomatic subjects, a fluorescein solution highlights the limits of features that enclose an area averaging 30,000 microns2 (range 8000 to 120,000; median area of close to 35,000 microns2). The areas of these features were generally normally distributed and there was little variation in feature areas across the zone. At different stages of clinical papillary conjunctivitis, the areas of the negative staining patterns are larger. Averages of 60,000 to 90,000 microns2, median values of close to 70,000 microns2 and individual feature areas up to 350,000 microns2 were seen in mildly symptomatic patients. These distributions were generally heterogeneous and showed a skewed distribution. Areas averaging up to 0.755 mm2 (range 0.6 to 3.2 mm2) were measured in moderate to severe papillary hypertrophy.

Cell Count↗

CynD, the cyanide dihydratase from Bacillus pumilus: gene cloning and structural studies.

The cyanide dihydratase in Bacillus pumilus was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in Escherichia coli. Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has K(m) and V(max) values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative Pseudomonas stutzeri strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of B. pumilus (strain 8A3) was cloned and expressed. This suggests that cynD, the gene coding for the cyanide dihydratase, is not unique to the C1 strain of B. pumilus and is not a reflection of its origin at a mining waste site.

Amino Acid Sequence↗

Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by Amplicor PCR.

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a PCR-based assay and culture techniques. Results of the PCR assay (Amplicor Mycobacterium tuberculosis Test) were compared with results of standard culture techniques with cultures held for 6 weeks. One thousand nine specimens were included: 301 retrospective specimens (frozen at -70 degrees C and later tested by PCR) and 708 prospective specimens (tested within 1 day of processing). One hundred sixty-two (16%) of the specimens were culture positive for M. tuberculosis; 83 (51%) of these were also fluorochrome stain positive. The sensitivity and specificity of the Amplicor PCR compared with those of culture were 83% (134 of 162 specimens) and 97% (800 of 827 specimens), respectively. The sensitivity for fluorochrome stain-positive specimens was 99%, and that for fluorochrome stain-negative specimens was 66%. The great majority of the 28 PCR-negative, culture-positive specimens were low positives; 27 were smear negative and 19 contained < 100 CFU of M. tuberculosis per ml. The 27 PCR-positive, culture-negative specimens included 24 that were positive by repeat testing by alternate primer PCR and were from patients with tuberculosis on antimicrobial therapy. With these considered culture misses, the final sensitivities of PCR and culture were 85, and 87%, respectively, while the specificities were 99.6 and 100%, respectively. After normal laboratory processing of sputum specimens, the Amplicor PCR assay can be completed in 8 h. Thus, it is possible to have results available within 10 h of specimen submission.

Fluorescent Dyes↗

Osteomyelitis/septic arthritis caused by Kingella kingae among day care attendees--Minnesota, 2003.

Kingella kingae is a fastidious gram-negative coccobacillus that colonizes the respiratory and oropharyngeal tract in children. K. kingae occasionally causes invasive disease, primarily osteomyelitis/septic arthritis in young children, bacteremia in infants, and endocarditis in school-aged children and adults. Although diagnosis of this organism frequently is missed, invasive disease is uncommon. Only sporadic, non-epidemiologically linked cases have been reported previously. In October 2003, the Minnesota Department of Health (MDH) investigated a cluster of two confirmed cases and one probable case of osteomyelitis/septic arthritis caused by K. kingae among children aged 17-21 months attending the same toddler classroom in a day care center. All reported within the same week with onset of fever, preceding or concurrent upper respiratory illness (URI), and refusal to bear weight on the affected limb. This report summarizes these cases and describes the epidemiologic investigation of the day care center. The findings underscore the need for clinicians and laboratorians to consider K. kingae infection in young children with Gram stain--negative or culture-negative skeletal infections.

Arthritis, Infectious↗

The isolation and properties of the protein calliphorin.

1. A procedure for the isolation of the protein calliphorin from larvae and pupae of the blowfly Calliphora erythrocephala is described. 2. The calliphorin preparation shows a single component in the ultracentrifuge at pH6.3 and gives a single band when stained for protein after agar-gel or starch-gel electrophoresis at pH6.3 or 8.6. Immunoelectrophoresis yields only one arc, associated with the stained spot, to a rabbit antiserum known to react with 13 other soluble components of Calliphora pupae. 3. Calliphorin has s(0) (20,w) 19.4S, D(0) (20,w) 3.25x10(-7)cm(2).s(-1) and f/f(0) 1.22, indicating a molecular weight of 528000 and a compact symmetrical structure. The molecular weight determined by the meniscus-depletion sedimentation-equilibrium method is 529000. 4. In 6.2m-guanidine hydrochloride calliphorin dissociates into six components each with a molecular weight of about 87000. Calliphorin reversibly dissociates into components with sedimentation coefficients of about 7S as the pH is raised progressively above pH6.5. 5. Calliphorin has an unusually high tyrosine and phenylalanine content (442 and 400mol/mol of protein respectively), a relatively high methionine content (162mol/mol of protein) and very little cystine or cysteine (18mol/mol of protein). The E(280)/E(250) ratio is 3.2. The pure protein contains 0.4-0.5% carbohydrate. 6. When examined in the electron microscope by the negative staining technique the protein is seen to consist of particles which are right prisms, being 105A wide and 65A high, rectangular in side view and curvilinear equilateral triangles in surface view.

Amino Acids↗

Cryptococcal infection in sarcoidosis.

A 48-year-old man with a history of sarcoidosis was transferred to the Mayo Clinic for evaluation and management of progressive neurologic decline. Two years before admission, he was admitted to a local hospital with mental status changes accompanied by ataxia and severe headache. A diagnosis of pulmonary and central nervous system sarcoidosis was made based on computed tomography of the head, lumbar puncture, and chest radiography. A mediastinoscopy with lymph node biopsy exhibited noncaseating granulomas and negative stains for microorganisms. Prednisone therapy was initiated at 80 mg/day. Clinical improvement was apparent for 13 months during steroid therapy until the slow taper reached a dosage of 20 mg/day. At that time, the patient was readmitted to the local hospital with severe confusion and skin lesions. When intravenous methylprednisolone therapy for presumed central nervous system sarcoidosis did not improve the patient's mental status, he was transferred to the Mayo Clinic. Physical examination of the thighs revealed large, well-marginated, indurated, irregularly bordered, violaceous plaques and rare, umbilicated, satellite papules with central hemorrhagic crusts (Fig. 1A). Superficially ulcerated plaques with a similar appearance to the thigh lesions were coalescing around the lower legs (Fig. 1B). A skin biopsy specimen of the thigh demonstrated abundant numbers of encapsulated organisms and minimal inflammatory response (Fig. 2). Skin, blood, and cerebrospinal fluid cultures confirmed the presence of Cryptococcus neoformans. Amphotericin and flucytosine combination therapy was initiated, and steroid dosages were gradually tapered. A test for human immunodeficiency virus was negative. The patient was dismissed from hospital after a complicated 2-month course resulting in improved mental status but progression of the lower extremity ulcerations as a result of polymicrobial infection.

Amphotericin B↗

Immunocytochemical detection and phenotypic characterization of micrometastatic tumour cells in bone marrow of patients with prostate cancer.

Monoclonal antibodies (mAbs) specific for cytokeratins are potent probes for the identification of disseminated individual epithelial tumour cells in mesenchymal organs such as bone marrow. We have used a monoclonal antibody (mAB) against cytokeratin 18 (CK18) for the detection of individual metastatic tumour cells in bone marrow aspirates from 84 patients with carcinoma of the prostate. CK18+ cells were detected in a sensitivity of 1 per 8 x 10(5) marrow cells using the alkaline phosphatase anti-alkaline phosphatase (APAAP) system for staining. We were able to detect CK18+ tumour cells in the marrow of 33% of patients with stage N0M0 prostate cancers. The incidence of CK18+ cells showed a significant correlation with established risk factors, such as local tumour extent, distant metastases and tumour differentiation. For further characterization of such cells in patients with prostate cancer, we developed an immunocytochemical procedure for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate-specific antigen (PSA). In a first step, cells were incubated with a murine mAb against PSA, followed by gold-conjugated goat anti-mouse antibodies. In a second step, a biotinylated mAb to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. CK18+ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 14 patients with carcinomas of the prostate. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hyperplasia (BPH).(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow Diseases↗

Utilization of antihepatocyte clone OCH1E5 (Hep Par 1) in histological evaluation of liver tumors.

Diagnosis of hepatocellular carcinoma (HCC) is not always easy on simple hematoxylin and eosin (H&E) stain. The diagnostic problems arise when tumor shows pseudoglandular, pleomorphic or clear cell differentiation. Various tumors markers have been described with varying sensitivity and specificity. Monoclonal antibody Hep Par 1 (OCH1E5) which is specific for hepatocytes offers great help in separation of these tumors. The aim of the present study was to determine utility of Hep Par 1 (OCH1E5) in differentiating HCC from metastatic tumors and cholangiocarcinoma. Total of 62 cases of liver tumors obtained from biopsies, resected or autopsy specimens were included in the study. Slides having representative sections were subjected to immunohistochemistry with monoclonal antibody Hep Par 1 (Dako Corp) using avidin biotin technique with primary antibody dilution of 1:40. Adjacent nontumorous hepatocytes were taken as positive control. Slides were examined by experienced pathologist without any information of clinical or H&E diagnosis. Cases were considered positive for Hep Par 1 if tumor cells showed cytoplasmic brown colored granules. The intensity and distribution (diffuse/ focal) of immunoreactivity was noted. Subsequently immunohistochemistry results were correlated with histology and clinical diagnosis. Hep Par 1 antibody was positive in 26 (42 %) and negative in 36 (58 %) liver tumors. On correlating with H&E sections, out of 26 positive cases, 25 (89.2%) were HCC and one was the case of metastasis of mucin secreting adenocarcinoma. From 36 tumors with negative staining 3 were cases of HCC, 27 metastatic adenocarcinomas and 6 cholangiocarcinomas. Only one case of liver metastasis of mucin secreting adenocarcinoma showed positivity. None of the cases of cholangiocarcinoma showed positivity for Hep Par 1. The three HCCs which did not take up staining for Hep Par 1 were 2 cases of moderately differentiated HCC having pseudoglandular pattern and a case of well differentiated HCC with trabecular arrangement. In 11(44%) cases staining was diffuse while in 14 (56%) it was focal but intense. Hep Par 1 is a useful marker in differentiating HCC from metastaic tumors and cholangiocarcinoma with sensitivity and specificity of 89 % and 97 % respectively and positive predictive value of 96 %. However one should be aware of limitations of immunohistochemistry.

Adult↗

Automated cytochemistry in non-Hodgkin's lymphoma: a new method for determination of cells from lymph node biopsy.

Cell suspension prepared from the lymph node biopsy of patients with non-Hodgkin's lymphoma (NHL), metastatic carcinoma (MC) and non-neoplastic lymphadenopathies (NL) were analyzed by the Hemalog D, automated differential counter. The preparation of lymph node cells is described first in this study. The results show that the percentage of large cells (diameter greater than 13.5 micron) stained negatively with peroxidase (LUC, large unstained cells) was remarkably increased in patients with NHL (mean +/- SEM = 18.6 +/- 3.1%) and was particularly increased in the subgroup, diffuse histiocytic type (31.1 +/- 5.3%). Patients with MC had a raised percentage of nonspecific esterase-positive cells (9.2 +/- 1.4%) compared to patients in the NHL and NL groups. Patients in the NL group had low percentages of both LUC (5.3 +/- 0.7%) and nonspecific esterase-positive cells (1.8 +/- 0.2%). Quantitation of cells in the lymph node by using the Hemalog D may assist in the diagnosis of lymph node diseases.

Biopsy↗

Expression of mutant p53, c-erbB-2 and the epidermal growth factor receptor in transitional cell carcinoma of the human urinary bladder.

Expression of the p53, the epidermal growth factor receptor (EGFr; c-erbB-1) and c-erbB-2 proteins was studied in 82 patients with primary transitional cell carcinoma of the bladder using an immuno-histochemical method. Strong or moderate staining was found in 18% of tumours for p53 with weaker staining in a further 36% giving a total of 54% of tumours stained for p53. Strong staining was found in 15% of tumours for c-erbB-2 and in 31% for the EGFr. Tumours invading the bladder muscle were significantly more likely to be strongly stained positively for p53 and/or EGFr compared with superficial tumours: only 15% of invasive tumours were stained negatively for both p53 and EGFr. No statistical association was found between p53 and EGFr expression. Weakly positive associations were found between the expression of c-erbB-2 and p53 and between muscle invasive tumours and increased expression of c-erbB-2. Alterations in the expression of p53, c-erbB-1 and c-erbB-2 were found frequently in human transitional cell carcinoma of the urinary bladder and may be of clinical use in defining patient sub-groups of differing prognosis.

Aged↗

Fine-structure evidence for cell membrane partitioning of the nucleoid and cytoplasm during bud formation in Hyphomonas species.

Hyphomonas spp. reproduce by budding from the tip of the prosthecum, distal to the main body of the reproductive cell; thus, the chromosome must travel through the prosthecum to enter the progeny, the swarm cell. When viewed by electron microscopy, negatively stained whole cells, ultrathin-sectioned cells, and freeze-etched and frozen hydrated cells all had marked swellings of the cytoplasmic membrane (CM) in the prosthecum which are termed pseudovesicles (PV). PV were separated by constrictions in the contiguous CM. In replicating cells, PV housed ribosomes and DNA, which was identified by its fibrillar appearance and by lactoferrin-gold labeling. The micrographs also revealed that the CM bifurcates at the origin of the prosthecum so that one branch partitions the main body of the reproductive cell from the prosthecum and swarm cell. The results of this fine-structure analysis suggest models explaining DNA segregation and the marked asymmetric polarity of the budding reproductive cell.

Cell Division↗

Evaluation of the Merifluor-Legionella immunofluorescent reagent for identifying and detecting 21 Legionella species.

We evaluated a 33-valent polyclonal indirect immunofluorescent-reagent kit (Merifluor-Legionella; Meridian Diagnostics Inc., Cincinnati, Ohio) made for the detection of Legionella species by testing bacterial isolates, seeded sputum, and negative sputum samples. Use of the reagent according to the directions of the manufacturer gave false-negative staining of homologous culture isolates due to a prozone phenomenon; this was solved by diluting test strain suspensions. After this change in testing protocol was made, the reagent gave bright fluorescent staining with 31 of the 33 Legionella strains with which it supposedly reacts. Strongly reacting Legionella strains included the type strains of L. pneumophila serogroups 1 to 10, L. longbeachae serogroups 1 and 2, and serogroup 1 of L. anisa, L. bozemanii, L. cherrii, L. dumoffii, L. gormanii, L. hackeliae, L. jamestowniensis, L. jordanis, L. maceachernii, L. micadedi, L. oakridgensis, L. rubrilucens, L. sainthelensi, L. spiritensis, L. steigerwaltii, and L. wadsworthii. Type strains of L. erythra and L. feeleii fluoresced only dimly with the reagent. Of 10 non-Legionella bacteria known to cross-stain with other polyvalent antisera, 5 also cross-reacted with the Merifluor reagent; these included 3 Bacteroides fragilis and 2 Pseudomonas fluorescens strains. The lower limit of detection of L. pneumophila serogroup 1 in seeded sputum was about 5 x 10(4) to 5 x 10(5) cells per ml. None of 21 randomly collected sputum specimens tested contained fluorescing legionellalike organisms, but 6 specimens did contain brightly fluorescing bacteria atypical in morphology for Legionella species. The Merifluor-Legionella kit appears to perform as well as other polyclonal immunofluorescent reagents used for detection of Legionella species. Because of the cross-reactions observed, which are common to all polyclonal reagents, utilization of this reagent for either bacterial identification or detection must be performed in combination with culture.

Cross Reactions↗

Helical flagellation in Centipeda periodontii, a gram-negative, anaerobic bacillus from periodontitis lesions.

The insertion pattern of flagellation of Centipeda periodontii was determined from electron micrographs of negatively stained cells treated with various detergents to remove their outer cell wall layer(s) selectively. The best results were obtained with 0.2% sodium dodecyl sulphate and a treatment time of 1-2 min. Detergent-treated cells displayed a unique helical pattern of flagellation which we designate as helicotrichous (Gk n. helix, helix + Gk n. thrix, hair). Flagella originated from an electron-dense region approximately 180 nm wide, which spiraled around the cell body. The length of the insertion path averaged 2.6 micron per 360 degrees turn; its periodicity was 2.5 micron. An average of 19 flagella were inserted per 1 micron length of the electron-dense region. The effects of cell division and branching on flagellation were also examined.

Bacteria, Anaerobic↗

Comparison of direct electron microscopy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children.

An approximate 10% suspension in water of the first available stool sample from 411 infants and young children with acute gastroenteritis was examined by electron microscopy (EM) after 2 min of negative staining. This procedure enabled the detection of 88% of the 199 rotavirus infections, all of the 22 adenovirus infections, and 47% of the 15 approximately 27-nm virus infections ultimately detected by a combination of techniques, including immune electron microscopy (IEM) and rotavirus enzyme-linked immunosorbent assay (ELISA). Of the 204 infections detected by direct EM of stools, 76% were detected within 2 min of viewing, and 94% were detected within 6 min of viewing. Type 1 and type 2 rotavirus particles were visualized with approximately equal efficiency, although type 2 rotavirus infections were more common. Rectal swab preparations were clearly inferior to stool preparations for the detection of virus infection by direct EM. IEM examination was required for efficient visualization of viruses in rectal swab specimens. ELISA was the most sensitive method for the detection of rotaviruses; with this method, all infections in which rotavirus particles were visualized by EM or IEM were detected. However, 73% of the 1,834 specimens which were presumptively positive for rotavirus by conventional indirect ELISA proved to be falsely positive on the basis of EM, IEM, blocking ELISA, confirmatory ELISA, or a combination of these methods. False-positive rotavirus ELISA reactions apparently were eliminated when fecal specimens were tested in a modified confirmatory ELISA with a lower dilution of rotavirus-negative (pre-immunization) than rotavirus-positive (post-immunization) capture antibody from the same animal.

Acute Disease↗

Characterization of a major envelope protein from the rumen anaerobe Selenomonas ruminantium OB268.

Cell envelopes from the Gram-negative staining but phylogenetically Gram-positive rumen anaerobe Selenomonas ruminantium OB268 contained a major 42 kDa heat modifiable protein. A similarly sized protein was present in the envelopes of Selenomonas ruminantium D1 and Selenomonas infelix. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of Triton X-100 extracted cell envelopes from S. ruminantium OB268 showed that they consisted primarily of the 42 kDa protein. Polyclonal antisera produced against these envelopes cross-reacted only with the 42 kDa major envelope proteins in both S. ruminantium D1 and S. infelix, indicating a conservation of antigenic structure among each of the major envelope proteins. The N-terminus of the 42 kDa S. ruminantium OB268 envelope protein shared significant homology with the S-layer (surface) protein from Thermus thermophilus, as well as additional envelope proteins containing the cell surface binding region known as a surface layer-like homologous (SLH) domain. Thin section analysis of Triton X-100 extracted envelopes demonstrated the presence of an outer bilayer over-laying the cell wall, and a regularly ordered array was visible following freeze-fracture etching through this bilayer. These findings suggest that the regularly ordered array may be composed of the 42 kDa major envelope protein. The 42 kDa protein has similarities with regularly ordered outer membrane proteins (rOMP) reported in certain Gram-negative and ancient eubacteria.

Amino Acid Sequence↗

FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima--quantitation, GTP hydrolysis, and assembly.

We have cloned the ftsZ genes from Thermotoga maritima and Azotobacter vinelandii and expressed the proteins (TmFtsZ and AzFtsZ) in Escherichia coli. We compared these proteins to E. coli FtsZ (EcFtsZ), and found that several remarkable features of their GTPase activities were similar for all three species, implying that these characteristics may be universal among FtsZs. Using a calibrated protein assay, we found that all three FtsZs bound 1 mole guanine nucleotide per mole FtsZ and hydrolyzed GTP at high rates (> 2 GTP per FtsZ per min). All three required magnesium and a monovalent cation for GTP hydrolysis. Previous reports showed that EcFtsZ (and some other species) required potassium. We confirmed this specificity for EcFtsZ but found that potassium and sodium both worked for Az- and TmFtsZ. Specific GTPase activity had a striking dependence on FtsZ concentration: activity (per FtsZ molecule) was absent or low below 50 microg/ml, rose steeply from 50 to 300 microg/ml and plateaued at a constant high value above 300 microg/ml. This finding suggests that the active state requires a polymer that is assembled cooperatively at 50-300 microg/ml. A good candidate for the active polymer was visualized by negative stain electron microscopy--straight protofilaments and protofilament pairs were seen under all conditions with active GTPase. We suggest that the GTP hydrolysis of FtsZ may be coupled to assembly, as it is for tubulin, with hydrolysis occurring shortly after an FtsZ monomer associates onto a protofilament end. As a part of this study, we determined the concentration of EcFtsZ and TmFtsZ by quantitative amino acid analysis and used this to standardize the bicinchonic acid colorimetric assay. This is the first accurate determination of FtsZ concentration. Using this standard and quantitative Western blotting, we determined that the average E. coli cell has 15,000 molecules of FtsZ, at a concentration of 400 microg/ml. This is just above the plateau for full GTPase activity in vitro.

Actin Cytoskeleton↗

The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. Evidence for a cooperatively coupled, negatively charged homotetramer.

The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca2+ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [3H]ryanodine binding was retained upon solubilization of the complex in Chaps but was lost in Zwittergent 3-14. The purified complex migrated as a single peak with an apparent sedimentation coefficient of approximately 30 and approximately 9 S upon density gradient centrifugation and with isoelectric points of 3.7 and 3.9 upon two-dimensional gel electrophoresis in Chaps and Zwittergent 3-14, respectively. Electron microscopy of negatively stained samples indicated that the distinct four-leaf clover structure of the ryanodine receptor observed in Chaps disappeared following Zwittergent treatment of the 30 S complex and instead showed smaller, round particles. Ferguson plot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial and fully cross-linked and incompletely denatured complexes suggested a stoichiometry of four Mr approximately 400,000 peptides/30 S ryanodine receptor oligomer. [3H]Ryanodine binding to the membrane-bound receptor in 50 microM--1 mM free Ca2+ revealed the presence of both high affinity (KD = 8 nM, Hill coefficient (nH) = 0.9) and low affinity (nH approximately 0.45) sites with a ratio of 1:3. Reduction in free Ca2+ to less than or equal to 0.1 microM or trypsin digestion of the membranes resulted in loss of high affinity but not low affinity ryanodine binding (Hill KD = 5,000 nM, nH = 0.9). Addition of 20 mM caffeine to the nanomolar Ca2+ medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low affinity sites altered the rate of [3H]ryanodine dissociation from the high affinity sites. Single channel recordings of the purified ryanodine receptor channel incorporated into planar lipid bilayers also indicated the existence of high and low affinity sites for ryanodine, occupation of which resulted in formation of a subconducting and completely closed state of the channel, respectively. These results are compatible with a subunit structural model of the 30 S ryanodine receptor-Ca2+ release channel complex which comprises a homotetramer of negatively charged and allosterically coupled polypeptides of Mr approximately 400,000.

Animals↗

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro.

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.

Animals↗