Expression of two cell wall proteins during the intracellular development ofEncephalitozoon cuniculi: an immunocytochemical andin situhybridization study with ultrathin frozen sections

Parasitology ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 815-825 ◽  
Author(s):  
V. TAUPIN ◽  
G. MÉTÉNIER ◽  
F. DELBAC ◽  
C. P. VIVARÈS ◽  
G. PRENSIER
Keyword(s):  
Cell Wall ◽  
Parasitophorous Vacuole ◽  
Spore Wall ◽  
Frozen Sections ◽  
Specific Expression ◽  
Developmental Patterns ◽  
Obligate Intracellular ◽  
A Cell ◽  
Ultrathin Frozen Sections

The microsporidianEncephalitozoon cuniculiis an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical andin situhybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts.swp1mRNA was preferentially synthesized in early sporogony whileenp1mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of theE. cuniculigenome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.

scholarly journals Tissue-Specific Expression of a Gene Encoding a Cell Wall-Localized Lipid Transfer Protein from Arabidopsis

1994 ◽  
Vol 105 (1) ◽  
pp. 35-45 ◽  
Author(s):  
S. Thoma ◽  
U. Hecht ◽  
A. Kippers ◽  
J. Botella ◽  
S. De Vries ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lipid Transfer Protein ◽  
Lipid Transfer ◽  
Gene Encoding ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Transfer Protein ◽  
A Cell

Estimation of the State of the Bacterial Cell Wall by Fluorescent In Situ Hybridization

1998 ◽  
Vol 64 (8) ◽  
pp. 3059-3062 ◽  
Author(s):  
Elena Bidnenko ◽  
Carine Mercier ◽  
Josselyne Tremblay ◽  
Patrick Tailliez ◽  
Saulius Kulakauskas
Keyword(s):  
Cell Wall ◽  
In Situ Hybridization ◽  
Cell Walls ◽  
Fluorescent In Situ Hybridization ◽  
Cell Level ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Identification Of Bacteria

ABSTRACT Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.

scholarly journals The false mussels (Mytilopsis leucophaeata) can be mechanical carriers of the shrimp microsporidian Enterocytozoon hepatopenaei (EHP)

2021 ◽  
Author(s):  
Natthinee Munkongwongsiri ◽  
Orawan Thepmanee ◽  
Kanokwan Lertsiri ◽  
Rapeepun Vanichviriyakit ◽  
Ornchuma Itsathitphaisarn ◽  
...  
Keyword(s):  
Spore Wall ◽  
Tissue Analysis ◽  
Asian Countries ◽  
Chain Reaction ◽  
Shrimp Ponds ◽  
Obligate Intracellular ◽  
Pcr Technology ◽  
Polymerase Chain ◽  
Enterocytozoon Hepatopenaei

Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasite causing hepatopancreatic microsporidiosis (HPM) in cultivated shrimp in Asian countries. One strategy to control EHP is to identify and eliminate biological reservoir(s) in shrimp ponds. Several marine and brackish-water organisms, including false mussels (Mytilopsis) have been reported to test positive for EHP using polymerase chain reaction (PCR) technology. Thus, we tested samples of commonly found Thai false mussel Mytilopsis leucophaeata from 6 EHP-infected shrimp ponds by PCR for the presence of EHP using the spore wall protein (SWP) gene primers. The mussel samples from all 6 ponds were positive. Subsequent bioassays carried out using naive mussels cohabitated with EHP-infected shrimp gave 100% SWP-PCR positive mussels at 20 days. One batch of such PCR-positive mussels was transferred for cohabitation with naive shrimp and gave 37.5% EHP-positive shrimp within 10 days. Tissue analysis of the EHP-PCR-positive mussels using light microscopy, in situ hybridization analysis for the SWP gene and electron microscopy did not confirm EHP infection. In summary, we obtained no evidence that Mytilopsis leucophaeata was itself infected with EHP. However, it was apparently capable carrying infectious spores for some period after ingestion and serving as a mechanical or passive carrier. The results support previous reports warning of the danger of feeding living or fresh bivalves to broodstock shrimp in hatcheries or shrimp in rearing ponds without prior heating or freezing.

Immunoferritin localization of intracellular antigens in positively stained frozen sections

1978 ◽  
Vol 36 (2) ◽  
pp. 168-169
Author(s):  
K. T. Tokuyasu
Keyword(s):  
Surface Tension ◽  
Water Surface ◽  
Thin Layers ◽  
The Other ◽  
Positive Staining ◽  
Frozen Sections ◽  
The Past ◽  
Ultrathin Frozen Sections ◽  
Definition Of

During the past investigations of immunoferritin localization of intracellular antigens in ultrathin frozen sections, we found that the degree of negative staining required to delineate u1trastructural details was often too dense for the recognition of ferritin particles. The quality of positive staining of ultrathin frozen sections, on the other hand, has generally been far inferior to that attainable in conventional plastic embedded sections, particularly in the definition of membranes. As we discussed before, a main cause of this difficulty seemed to be the vulnerability of frozen sections to the damaging effects of air-water surface tension at the time of drying of the sections.Indeed, we found that the quality of positive staining is greatly improved when positively stained frozen sections are protected against the effects of surface tension by embedding them in thin layers of mechanically stable materials at the time of drying (unpublished).

Localization of pancreatic enzymes in ultrathin frozen sections stained with metal labeled antibody

1978 ◽  
Vol 36 (2) ◽  
pp. 90-91
Author(s):  
Kenjiro Yasuda
Keyword(s):  
Fluorescent Antibody ◽  
Pancreatic Enzymes ◽  
Tissue Preparation ◽  
Zymogen Granules ◽  
Frozen Sections ◽  
En Bloc ◽  
Antibody Method ◽  
Ultrathin Frozen Sections ◽  
Fluorescent Antibody Method

Localization of amylase,chymotrypsinogen and trypsinogen in pancreas was demonstrated by Yasuda and Coons (1966), by using fluorescent antibody method. These enzymes were naturally found in the zymogen granules. Among them, amylase showed a diffuse localization around the nucleus, in addition to the zymogen granules. Using ferritin antibody method, scattered ferritin granules were also found around the Golgi area (Yasuda et al.,1967). The recent advance in the tissue preparation enables the antigen to be localized in the ultrathin frozen sections, by applying the labeled antibodies onto the sections instead of staining the tissue en bloc.The present study deals with the comparison of the localization of amylase and lipase demonstrated by applying the bismuth-labeled, peroxidase-labeled and ferritin-labeled antibody methods on the ultrathin frozen sections of pancreas, and on the blocks of the same tissue.

The Application of Ultracryotomy to Immunocytochemistry

1973 ◽  
Vol 31 ◽  
pp. 704-709
Author(s):  
R. G. Painter ◽  
K. T. Tokuyasu ◽  
S. J. Singer
Keyword(s):  
Frozen Sections ◽  
Protein Antigens ◽  
Carbon Coated ◽  
Antibody Conjugates ◽  
Ultrathin Frozen Sections

A technique for localizing intracellular antigens with immunoferritin conjugates directly on ultrathin frozen sections of glutaraldehyde-fixed tissues has been developed. This method overcomes some of the limitations of previously described procedures, since it avoids drastic fixation, dehydration and embedding procedures which could denature many protein antigens.Briefly cells or tissues were fixed with glutaraldehyde (0.5 to 2% for 1 hr), and ultrathin frozen sections were cut and mounted on grids covered with carbon-coated Formvar film by the procedure described previously. Such sections were stained with ferritin-antibody conjugates by methods described elsewhere.

Ultrathin frozen sections of yeast without aldehyde prefixation

1978 ◽  
Vol 36 (2) ◽  
pp. 58-59
Author(s):  
K. J. Böhm ◽  
a. E. Unger
Keyword(s):  
Aqueous Solutions ◽  
Biological Material ◽  
Yeast Cells ◽  
Frozen Sections ◽  
Good Preservation ◽  
Ultrathin Frozen Sections

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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