Biomedical subjects

J A Hobot

Publications and source records attributed to J A Hobot.

10 recordsLinked to original sources

PubMed1991

Intracellular location of the autolytic N-acetylmuramyl-L-alanine amidase in Bacillus subtilis 168 and in an autolysis-deficient mutant by immunoelectron microscopy.

Antisera against purified autolytic N-acetylmuramyl-L-alanine amidase from Bacillus subtilis 168 were prepared in rabbits. They neutralized the enzymatic action of the purified amidase acting on isolated sodium dodecyl sulfate (SDS)-treated walls from the same organism. They also inhibited the lysis of native walls, but only after the walls lysed partially. Amidase adsorbed to insoluble walls still combined with antibody. Antisera did not stop the lysis of whole cells. Lowicryl HM20 sections of both strain 168 and its autolytic mutant strain FJ6 were prepared by the progressive-lowering-of-temperature technique, immunolabeled with the antisera, and visualized with colloidal gold particles as markers. The highest concentration of gold particles seemed to be in the septa of dividing cells, followed by the side walls. There was some labeling of the cytoplasm. Adsorption of sera with SDS-treated walls reduced the overall labeling of sections considerably but did not alter the relative intracellular distribution of particles. The results for strains 168 and FJ6 were similar. Labeling of SDS-treated walls unexpectedly revealed the presence of a wall-bound amidase fraction.

Antibodies, Bacterial↗

PubMed1991

Strategies for improving the cytochemical and immunocytochemical sensitivity of ultrastructurally well-preserved, resin embedded biological tissue for light and electron microscopy.

Many techniques for processing tissue into resin are available, varying from conventional room temperature to low temperature procedures. The problem is to choose an appropriate method to suit the biological specimen under study. Room temperature approaches with aldehyde and osmium fixation do not give optimal retention of immunoreactivity. Osmium can be removed from sections, but recovery of immunosensitivity is reduced. Osmium post-fixation can be omitted, but heat polymerization of resins causes tissue extraction and loss of immunoreactivity. Alternative techniques rely on the use of milder polymerization methods and avoid osmium. However, while providing an improvement, this alone is not sufficient to maximize tissue reactivity. Fixation with high concentrations of glutaraldehyde (greater than 1%) and processing into resin at either room or low temperature results in retention of similar levels of immunoreactivity. Low concentration glutaraldehyde (less than 0.2%) fixation for short periods of time (less than 60 minutes) produces improved tissue immunoreactivity and allows low concentrations of antigen at secondary sites to be detected. However, the tissue is now only minimally stabilized and is prone to extraction and conformational damage during processing. It can be partially protected by employing one of two strategies: processing at room temperature with partial dehydration (upto 70% solvent) and rapid embedding in LR White or Lowicryl K4M at 0 degrees C, or processing at progressively lower temperatures (PLT) and embedding in Lowicryl at -35/-50 degrees C. In a third strategy, specimens sensitive to very low fixative concentrations are cryo-immobilized, then resin embedded after substitution or freeze-drying (this latter method awaiting evaluation for inclusion in our strategical approach).

Acrylic Resins↗

PubMed1989

Cell wall assembly in Bacillus subtilis: visualization of old and new wall material by electron microscopic examination of samples stained selectively for teichoic acid and teichuronic acid.

Uranyl acetate staining of thin sections allowed a distinction to be made between cell wall material that contains teichoic acid and that which contains teichuronic acid. The stain was used to study the pattern of wall assembly in Bacillus subtilis undergoing transitions between growth conditions leading to incorporation of the different anionic polymers. The results showed that new material is incorporated along the inner surface of the cylindrical region of the wall confirming, by a more direct method, results obtained earlier with teichoic acid specific phages. New material appears to be evenly distributed along the inner surface and no evidence was obtained for the presence of specific zones of incorporation.

Bacillus subtilis↗

PubMed1989

Cell wall assembly in Bacillus subtilis: partial conservation of polar wall material and the effect of growth conditions on the pattern of incorporation of new material at the polar caps.

The use of phage SP50 as marker for cell wall containing teichoic acid in Bacillus subtilis showed clear differences in the rates at which new wall material becomes exposed at polar and cylindrical regions of the wall, though the poles were not completely conserved. Following transition from phosphate limitation to conditions that permitted synthesis of teichoic acid, old polar caps fairly rapidly incorporated enough teichoic acid to permit phage binding. Electron microscopy suggested that the new receptor material spread towards the tip of the pole from cylindrical wall so that phages bound to an increasing proportion of the pole area until only the tip lacked receptor. Eventually, receptor was present over the whole polar surface. Direct electron microscopic staining of bacteria collected during transitions between magnesium and phosphorus limitations showed that new material was incorporated at the inner surface of polar wall and later became exposed at the outer surface by removal of overlying older wall. The apparent partial conservation of the pole reflected a slower degradation of the overlying outer wall at the pole than at the cylindrical surface, the rate being graded towards the tip of the pole. The relative proportions of the new wall material incorporated into polar and cylindrical regions differed in bacteria undergoing transitions that were accompanied by upshift or downshift in growth rate. These differences can be explained on the basis that growth rate affected the rate of synthesis of cylindrical but not septal wall.

Bacillus subtilis↗

PubMed1988

Intracellular location of the histonelike protein HU in Escherichia coli.

Immunocytochemical labeling of thin sections of cryosubstituted, Lowicryl-embedded Escherichia coli cells with protein A-colloidal gold was used to study the structural organization of the bacterial nucleoid. We found that the histonelike protein HU was not associated with the bulk DNA in the nucleoid but was located in areas of the cell where metabolically active DNA is associated with ribosomes and where single-stranded DNA, RNA polymerase, and DNA topoisomerase I were also located. The resolution of the methods used did not allow us to decide whether HU was associated either with ribosomes or with transcriptionally active DNA, nor could we demonstrate interaction of HU with either.

Bacterial Proteins↗

PubMed1987

Use of on-section immunolabeling and cryosubstitution for studies of bacterial DNA distribution.

Escherichia coli cells were very rapidly frozen and substituted at a low temperature with 3% glutaraldehyde in acetone. Infiltration and embedding with Lowicryl K4M were carried out at -35 degrees C. This procedure resulted in good structural preservation of both the nucleoid morphology and its DNA plasm, such that immunolabeling with the protein-A gold technique could be carried out. With antibodies specific for either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), it was shown that dsDNA was present throughout the nucleoid but that ssDNA was located on the nucleoid periphery. Chloramphenicol-treated cells, in which protein synthesis but not DNA replication is stopped, produced a characteristic ringlike nucleoid shape and had both dsDNA and ssDNA present throughout the annular section of the DNA plasm. The relationship between metabolically active DNA and overall bacterial genome organization is discussed.

Antibodies, Monoclonal↗

PubMed1987

Modern acrylics for post-embedding immunostaining techniques.

We describe two methods for rapid processing of biological tissues into LR White acrylic plastic. Both methods make use of LR White's compatibility with small amounts of water, enabling non-osmicated tissue to be only partially dehydrated before infiltration with the plastic, a procedure that improves the sensitivity of post-embedding immunocytochemistry. In addition, both methods are designed to reduce the time for which tissue is exposed to the damaging influence of the plastic monomer, which can cause extraction and sudden shrinkage. The tissue example used in the first method is immersion-fixed, surgically removed human pituitary which, by virtue of its thorough fixation, can be processed quickly at 50 degrees C using catalytic polymerization at room temperature. The concentration of the catalyst is critically set to prevent the temperature rising above 60 degrees C in the tissue blocks. Penetration of immunoperoxidase reagents into 330-nm LR White sections is demonstrated and possible modes of action are discussed. When "lightly" fixed tissue is processed as above, serious polymerization artifacts can result from autocatalysis. A second method, based on the first but employing slower polymerization at 0 degrees C, has therefore been developed. The high level of fine structure that can be retained using this method is illustrated by the demonstration of the trans-tubular Golgi in perfusion-fixed kidney of rat. Biotinylated lectin is localized to cells of the kidney proximal tubule with streptavidin-colloidal gold, to illustrate tissue reactivity. In a second example, the structure of the bacterial cell envelope is shown to be similar in appearance after partial dehydration and LR White embedding to that seen after progressive lowering of temperature, dehydration, and Lowicryl embedding.

Acrylic Resins↗

PubMed1985

Low temperature embedding with Lowicryl resins: two new formulations and some applications.

Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze-substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.

Acrylic Resins↗

PubMed1985

Shape and fine structure of nucleoids observed on sections of ultrarapidly frozen and cryosubstituted bacteria.

Very rapidly frozen cells of Escherichia coli and Bacillus subtilis were substituted at low temperature into acetone with 1% OsO4 and embedded in Epon. They showed ribosome-free spaces filled with globular and fibrillar material of up to 15 nm. The sizes of structures seen do not exclude DNA superstructures such as supercoils, aggregates, and nucleosomes. With the Feulgen analog osmium-ammines stain, DNA was localized within the ribosome-free space. The bulk of DNA, the nucleoid, is therefore a major part of, or identical to, the main ribosome-free space. The ribosome-free space would correspond directly to the light microscopy phase-contrast image of nucleoids in living bacteria. The shape of the ribosome-free space does not reflect intracellular salt concentrations, nor do the Feulgen-positive areas. The previously observed dependency on the salt concentration of the growth medium seems to be due to permeabilization induced by the chemical fixative at room temperature. The ribosome-free space is more cleft in appearance than the nucleoid obtained by fixation with OsO4 but more confined than its very dispersed form found after aldehyde fixation.

Bacillus subtilis↗

PubMedDate not supplied

New aspects of bacterial ultrastructure as revealed by modern acrylics for electron microscopy.

Modern acrylics can be used over a wide temperature range (+60 degrees C to -80 degrees C) for infiltration, embedding, and polymerization. They can be used in procedures involving chemical fixation or rapid freezing. This flexibility allows for studies to be carried out upon the effects that different parameters involved in preparing biological tissue for microscopy have upon structure and retention of immunoreactivity. With most preparative methods contributions have been made to our knowledge on bacterial structure in gram-negative and gram-positive cells. The future should lie in integrating the advantages of the various methods for the purpose of advancing our understanding of bacterial structure/function.

Acrylic Resins↗