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PubMedDate not supplied

Noninvasive measurement of cell volume changes by negative staining.

To maintain the intracellular concentration of ions and small molecules on osmotic challenges, nature has developed highly sophisticated transport systems for regulating water and ion content. An ideal measurement technique for volume changes of cells during osmotic challenges has to fulfil two requirements: it has to be osmotically inert, and it should allow online monitoring of cell volume changes. Here, a simple fluorescence microscopy-based approach is presented. Using fluorescein as a negative stain, it is possible to monitor cell volume changes without affecting the functionality of cell membranes and cell osmolarity. Measurement of Madine-Darby canine kidney (MDCK) cells after hypo- and hyperosmotic challenges reveals the main advantages of this approach: besides providing precise and reproducible quantitative data on reversible cell volume changes, the viability of the cells can be assessed directly by the appearance of stain in the cytoplasm. This becomes evident especially after hypo-osmotic challenge of glutaraldehyde-treated cells, which become leaky after fixation, followed by a massive volume change. This new approach represents a very sensitive measurement technique for cell volume changes resulting from water or ion flux, and thus seems to be an ideal tool for studying cell volume regulatory processes.

Animals↗
PubMedDate not supplied

Computer image analysis of two-dimensional crystals of beef heart NADH: ubiquinone oxidoreductase fragments. I. Comparison of crystal structures in various negative stains.

We investigated the structure of two-dimensional crystals from bovine heart mitochondrial NADH: ubiquinone oxidoreductase. A detailed description of uranyl acetate-stained crystals demonstrated that they are composed of fragments in a spatial arrangement according to space group P4212 [J. Brink, S. Hovmöller, C.I. Ragan, M.W.J. Cleeter, E.J. Boekema and E.F.J. van Bruggen, European J. Biochem. 166 (1987) 287]. To gain more structural information on the crystal structure and to assess the effects of various negative stains on the structure preservation and appearance, we examined stained crystals by means of electron microscopy and image analysis. The space group P4212 appeared to be present for several stains tested, i.e. ammonium molybdate, uranyl acetate, uranyl nitrate and uranyl sulphate. Use of phosphotungstic acid and silicotungstate resulted in a reduction of symmetry to pseudo-P4212 or p4. Use of sodium tungstate led to a considerable loss of resolution to 3.8 nm at best, whereas otherwise 1.5 to 1.9 nm could be demonstrated. The lattice vectors were not affected by the stains; they were determined as a = b = 14.9 +/- 0.25 nm with gamma = 89.8 degrees +/- 0.6 degrees. Image analysis showed the presence of similar structures with the molybdate and uranyl compounds. Differences were observed in the case of the tungstate type of stains. Furthermore, the analysis revealed the complete absence of the four small pores of 2.0 nm diameter in the unit cell. This effect was observed irrespective of the type of stain and supporting film, and could be ascribed only to the glow-discharge treatment of the supporting film. The observed difference must be caused by changed interactions between the protein, stain and supporting film. Application of correspondence analysis and clustering algorithms to the various reconstructed images of the crystals showed that they could be separated into several clusters. Each of these clusters corresponded on the average to only one type of stain, whereas a further division according to the specific uranyl compounds was observed. This study therefore shows that under identical preparation conditions subtle differences between individual stains can be detected.

Animals↗
PubMedDate not supplied

The structure of dynein-c by negative stain electron microscopy.

Dynein ATPases contain six concatenated AAA modules within the motor region of their heavy chains. Additional regions of sequence are required to form a functional ATPase, which a previous study suggested forms seven or eight subdomains arranged in either a ring or hollow sphere. A more recent homology model of the six AAA modules suggests that these form a ring. Therefore both the number and arrangement of subdomains remain uncertain. We show two-dimensional projection images of dynein-c in negative stain which reveal new details of its structure. Initial electron cryomicroscopy shows a similar overall morphology. The molecule consists of three domains: stem, head, and stalk. In the absence of nucleotide the head has seven lobes of density forming an asymmetric ring. An eighth lobe protrudes from one side of this heptameric ring and appears to join the elongated cargo-binding stem. The proximal stem is flexible, as is the stalk, suggesting that they act as compliant elements within the motor. A new analysis of pre- and post-power stroke conformations shows the combined effect of their flexibility on the spatial distribution of the microtubule-binding domain and therefore the potential range of power stroke sizes. We present and compare two alternative models of the structure of dynein.

Animals↗
PubMed2004

Mismatch repair gene PMS2: disease-causing germline mutations are frequent in patients whose tumors stain negative for PMS2 protein, but paralogous genes obscure mutation detection and interpretation.

The MutLalpha heterodimer formed by mismatch repair (MMR) proteins MLH1 and PMS2 is a major component of the MMR complex, yet mutations in the PMS2 gene are rare in the etiology of hereditary nonpolyposis colorectal cancer. Evidence from five published cases suggested that contrary to the Knudson principle, PMS2 mutations cause hereditary nonpolyposis colorectal cancer or Turcot syndrome only when they are biallelic in the germline or abnormally expressed. As candidates for PMS2 mutations, we selected seven patients whose colon tumors stained negative for PMS2 and positive for MLH1 by immunohistochemistry. After conversion to haploidy, truncating germline mutations of PMS2 were found in two patients (2192delTAACT and deletion of exon 8). These mutations abrogated PMS2 protein in germline cells by Western analysis. In two additional patients, PMS2 protein from one allele also was abrogated. Novel or previously described missense variants of PMS2 were detected, but their pathogenicity is undetermined. We detected and characterized a new transcript, PMS2CL, showing 98% sequence identity with exons 9 and 11-15 of PMS2 and emanating from a locus close to PMS2 in chromosome 7p. Its predicted protein product was not detected. Thus, in addition to several previously described PMS2-related genes resembling the 5' end of PMS2, at least one related gene resembles the 3' end of PMS2. In conclusion, both detectable and presently undefined germline mutations are deleterious and produce susceptibility to cancer by the two-hit mechanism. Paralogous genes interfere with mutation detection, resulting in underdiagnosis of PMS2 mutations. Mutation detection in PMS2 requires haploid DNA.

Adaptor Proteins, Signal Transducing↗
PubMed1982

Morphology of gel state phosphatidylethanolamine and phosphatidylcholine liposomes: a negative stain electron microscopic study.

The capture volumes (internal aqueous spaces) of liposomes prepared from a series of saturated phosphatidylcholines (PC) and saturated phosphatidylethanolamines (PE) had previously been found to be a function of lipid structure. PE vesicles have larger internal aqueous spaces than PC vesicles and for lipids with the same head group, capture volume increases with lengthening of the fatty acyl chains. Capture volume is determined by vesicle size, number of lamellae, and interlamellar distance. In this study, liposomes were found from a saturated PC or PE and their morphology studied In the gel state using the technique of negative staining transmission electron microscopy. The measured interlamellar distances were quite similar among these various lipids while the number of lamellae was found to decrease as the fatty acyl chain length increased. In general PEs form fewer lamellae than PCs and in particular mono- and di-methylated dipalmitoyl-PE form only unilamellar vesicles. The number of lamellae then appears to bear a relationship to the size of the capture volume in that liposomes with larger capture volumes have fewer lamellae.

Chemical Phenomena↗
PubMed2003

Capturing time-resolved changes in molecular structure by negative staining.

Imaging structural intermediates of biological processes is a key step in understanding biological function. Because intermediates are commonly short-lived, lasting only milliseconds, the main methods used to capture them have been conventional imaging of analog or inhibited states, having extended lifetimes, or rapid (millisecond timescale) freezing of intermediates with subsequent observation by cryo-EM. We have developed a simpler method that fixes structure on the millisecond timescale. The procedure consists of briefly (milliseconds) exposing the macromolecular structure of interest on an EM grid to conditions that initiate the structural change, then immediately fixing with uranyl acetate or tannic acid. Specimens are then observed by negative staining. The key finding that validates this approach is our demonstration that uranyl acetate, and in some cases tannic acid, fixes protein molecular structure on the millisecond timescale. This is demonstrated by our observation that exposure of actin and myosin filaments to these fixatives for as little as 10 ms is sufficient to fully preserve them against changes that normally induce rapid and major alteration in their molecular structure. Fixation appears to stabilize both ionic and hydrophobic bonds. This approach should be of general utility for studying transient molecular changes in many systems.

Actins↗
PubMed1978

Electron microscopy of metal-shadowed and negatively stained microtubule protein. Structure of the 30 S oligomer.

Microtubule protein purified from porcine brain was fixed at low protein concentration with glutaraldehyde under conditions which maximize the relative concentration of the ring-shaped 30 S oligomer. Fixed oligomer was separated from glutaraldehyde and other protein species by column chromatography. The fixed, isolated oligomer was deposited on electron microscopy grids, dehydrated, and then critical point-dried before shadow-coating with carbon/platinum alloy at a fixed angle. Analysis of the shadow lengths observed by electron microscopy revealed that the height of the 30 S oligomer is 15 nm. Microtubule protein deposited on electron microscope grids at high protein concentrations was examined by the negative stain technique and found to contain apparent stacks of oligomer from which the number of tubulin dimers per turn of the ring and the distance between turns could be determined. The number of subunits per turn was determined as 13.8. The distance between turns was found to be 7.4 nm, indicating that the 15 nm high, shadowed oligomers consisted of two turns. Additional information from the literature is considered and a model is presented for the oligomer. The model is a helix of 29 tubulin dimers and five high molecular weight protein molecules arranged so as to preserve intersubunit bonding patterns found in microtubules.

Animals↗
PubMedDate not supplied

Evaluation of three Mycobacterium leprae monoclonal antibodies in mucus and lymph samples from Ziehl-Neelsen stain negative leprosy patients and their household contacts in an Indian community.

Mucus and lymph smears collected from leprosy patients (9) and their household contacts (44) in the Caño Mochuelo Indian Reservation, Casanare, Colombia, were examined with monoclonal antibodies (MoAb) against Mycobacterium leprae. The individuals studied were: 5 borderline leprosy (BB) patients, 4 with a lepromatous leprosy (LL), all of whom were undergoing epidemiological surveillance after treatment and 44 household contacts: 21 of the LL and 23 contacts of the BB patients. The MoAb were reactive with the following M. leprae antigens: 65 kd heat shock protein, A6; soluble antigen G7 and complete antigen, E11. All the samples were tested with each of the MoAb using the avidin-biotin-peroxidase technique and 3,3 diaminobenzidine as chromogen. The patients and household contacts studied were all recorded as Ziehl-Neelsen stain negative. The MoAb which showed optimal reaction was G7, this MoAb permitted good visualization of the bacilli. Five patients with BB diagnosis and one with LL were positive for G7; of the BB patients' household contacts, 9 were positive for G7; 7 of the LL patients' household contacts were positive for the same MoAb. MoAb G7 allowed the detection of bacillar Mycobacterium spp. compatible structures in both patients and household contacts. G7 permitted the visualization of the complete bacillus and could be used for early diagnosis and follow-up of the disease in patients.

Antibodies, Bacterial↗
PubMedDate not supplied

Computer image analysis of two-dimensional crystals of beef heart NADH: ubiquinone oxidoreductase fragments. II. Comparison of frozen hydrated and negatively stained specimens.

Two-dimensional crystals of bovine NADH: ubiquinone oxidoreductase fragments were studied by cryo-electron microscopy and computer image analysis at two focus settings. The lattice parameters of the crystals were determined as a = 14.5 +/- 0.2 nm, b = 14.8 +/- 0.3 nm with gamma = 87.8 degrees +/- 0.2 degrees. Translation and rotation alignment resulted in a final resolution of 1.38 (close-to-focus) or 1.9 nm (1.3 microns underfocus). When only translational alignment was employed 1.6 nm resolution resulted (close-to-focus case). The application of a further correction procedure surprisingly lowered the achieved resolution to 2.2 nm. A combination of the projected structures for the two focus settings in Fourier space resulted in a noise-free image displaying enhanced image contrast for reciprocal spacings from 1/5.0 to 1/1.4 nm-1. The structure in amorphous ice comprises four separate quasi-identical motifs, each resembling a distorted triangle. Two symmetry-related (p2) motifs reveal a pore-like feature. In negative stain the structure has p4mm symmetry. The absence of in-plane symmetry elements (i.e. twofold rotation and screw axes) and other differences might be accounted for (at least in part) if the crystal studied was slightly tilted.

Animals↗
PubMed1973

Low resistance junctions in crayfish. II. Structural details and further evidence for intercellular channels by freeze-fracture and negative staining.

The ultrastructure of low resistance junctions between segments of lateral giant fibers in crayfish is studied in sections from specimens fixed either by conventional methods or by glutaraldehyde-H(2)O(2) or by glutaraldehyde-lanthanum. Cross sections through junctions fixed by conventional glutaraldehyde display the usual trilaminar profile of two parallel membranes separated by a narrow gap. Most of the junctional regions appear covered by 500-800 A vesicles which lie on both sides of the junction in rows adjacent to the membranes. Gross sections through junctions fixed by glutaraldehyde-H(2)O(2) display, in regions containing vesicles, membranes with a beaded profile. The beads correspond to globules approximately 125 A in width and approximately 170 A in height arranged in a hexagonal pattern with a unit cell of approximately 200 A. The globules of one membrane match precisely with those of the adjacent membrane, and opposite globules seem to come in contact with each other at the center of the junction. The membrane of the vesicles also contains globules. Occasionally the globules of the vesicles seem to join with those of the junctional membranes, apparently forming intracellular junctions. Injunctions negatively stained by lanthanum the globules are seen organized into two arrangements. Areas containing globules in a hexagonal array with a unit cell of approximately 200 A (swollen pattern) are seen adjacent to areas in which the globules are more closely and disorderly packed (close packing), the minimum center-to-center distance between adjacent globules being approximately 125 A. At higher magnification each globule appears composed of six subunits arranged in a circle around a central region occupied by lanthanum (possibly a pit).

Aldehydes↗
PubMed1986

Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.

The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease-sensitive fibrils 91 and 72 nm in length, respectively. A third class of only very sparsely distributed short fibrils (63 nm) was observed on mutant HB-V51, which lacks both wall-associated AgB and AgC antigens. The identity of these fibrils and whether they are present on the wild type are not clear. The function of long, protease-resistant fibrils of 178 nm, which are also present on the wild-type strain, remains unknown.

Adhesiveness↗