Immunoelectron microscopy of chemically fixed developing plant embryos.

The hydrophobic plant cell wall, large acidic central vacuole, diverse secondary compounds, intercellular airspaces, and rigid starch granules present obstacles to ultrastructure preservation and specimen sectioning. We describe modifications of fixation and embedding procedures successfully used with microbes, protists, and mammalian tissues that have overcome these obstacles. Vacuum infiltration is used to remove intercellular air rapidly replacing it with fixative and buffer preserving cellular ultrastructure while neutralizing the acidic vacuole. Vacuum infiltration of embedding resin ensures uniform embedding resin permeation allowing production of intact ultrathin sections that are stable under the electron beam and suitable for immunolabeling. The methodology described has been used for immunolocalization of non-specific lipid transfer proteins in the diverse cell types found in developing castor bean fruits but is suitable for all plant tissues.

Serial section immunoelectron microscopy of algal cells.

Electron microscopy when combined with immunogold labeling provides a 2D image of intracellular protein distribution. Cells are however 3D structures. We describe a method of serial section immunogold electron microscopy that allows a 3D cellular image to be reconstructed from a series of electron micrographs. Cells are fixed to preserve cellular ultrastructure and they are embedded in plastic allowing ultrathin sections to be obtained. The ribbon of ultrathin serial sections produced as the microtome sequentially cuts through the sample is labeled with a monospecific antibody to the protein of interest and then with protein-A gold making the antigen-antibody complex visible in the electron microscope. A common field of view from each serial section is photographed in the electron microscope. Using image analysis software, each digitized micrograph is sequentially aligned; immunolabel and cellular structures of interest are traced onto each micrograph; the micrographs are stacked; and the structures of interest are rendered as solid surfaces producing a 3D image of protein distribution within the cell.

Diversity of laccase among Cryptococcus neoformans serotypes.

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.

Intracellular protein localization by immunoelectron microscopy.

Eukaryotic cells are characterized by the presence of a number of membrane-bound organelles which have differing degrees of internal structure. After synthesis in the cytoplasm, proteins must be targeted to the appropriate organelle and localized to the correct suborganellular compartment. We describe a method for immunoelectron microscopy that can be used to localize a protein not only to the correct organelle but to the appropriate suborganellular compartment. Cells are fixed to preserve subcellular structures and ultrathin sections are labeled with a monospecific antibody to the protein of interest. Protein-A gold is used to visualize the antigen-antibody complex by transmission electron microscopy allowing the intracellular location of the antigen to be determined. The methodology described was developed to study protein localization in Euglena but it is applicable to most organisms.

Analysis of bacterial flora in dohyo soil.

OBJECTIVES: Sumo wrestling is one of the most popular sports in Japan. Injuries are not uncommon as this is a vigorous contact sport. Sumo wrestlers have little in the way of protective clothing; their main garb is the mawashi, making them prone to exposure to any microorganisms in the dohyo. The bacterial flora of the dohyo has received little attention. If the constituent flora is identified, then appropriate treatment or prevention of any bacterial lesions or infections incurred by the wrestlers is possible. METHODS: The Vitek AMS system used in this study was developed by McDonnell Douglas Corporation. In this system, the physiological and biochemical properties of Gram-positive and negative bacilli, Gram-positive and-negative cocci, and fungi isolated from clinical materials and environments are examined using test cards specifically for each microorganism group, and the results are automatically read by a computer and encoded. Obtained codes are compared with a built-in database, and bacterial species of test strains are identified. RESULTS: In this study, using the automatic identification kit VITEK or ATB, we describe the aerobic bacterial flora found in the dohyo over the four seasons of the year. We also investigated the effect of salt on the bacterial flora as sumo wrestlers toss salt on the dohyo before each match. We show the relationship between salinity changes and variations in the flora observed upon the addition of salt. Without salt, at the beginning of a match, Gram-negative bacteria predominate. When salt is added, there is a transient decrease in the incidence of flora followed by an increase in the incidence Grampositive cocci. CONCLUSIONS: Sixteen bacterial genera were identified using the bacterial identification systems in dohyo soil samples during the year. The number of identified bacterial species was 32. Even in the presence of salt, there is a measurable amount of bacterial flora in dohyo soil; salt does not act as an antibacterial agent.

Electron microscopic studies on bactericidal effects of electrolyzed acidic water on bacteria derived from kendo protective equipment.

OBJECTIVES: Kendo protective equipment is used without washing for a long time.Staphylococcus saprophyticus, Micrococcus luteus, andBacillus sphaericus are frequently isolated from the mask ('men' in Japanese) of kendo protective equipment during one year. To investigate the bactericidal effects of electrolyzed acidic water on these three bacteria, we observed their cellular structures by electron microscopy after treatment with such water. METHODS: Each bacterium isolated from 'men' was treated with electrolyzed acidic water and then observed under scanning and transmission electron microscopes. RESULTS: WhenS. saprophyticus was treated with electrolyzed acidic water and its cellular structures were observed under a transmission electron microscope, ghost cytoplasm was observed, in which no ribosomal granules or fibrous DNA structures were present, and the cell wall inner layer was detached from the outer layer. Under a scanning electron microscope, the structure of the cell wall surface layer was wrinkled, and round pores were partially formed, indicating that the cytoplasmic structures were flushed out of the cells treated with electrolyzed acidic water through the pores formed in the cell wall. InM. luteus, the destruction of ribosomal granules and that of DNA fibers were observed to be similar to those ofS. saprophyticus. ForB. sphaericus, the effect of electrolyzed acidic water was investigated using vegetative cells. A dissociation between the cytoplasm and cell wall wrinkled the cell surface layer. CONCLUSION: On the basis of above findings, electrolyzed acidic water was found to destroy the cellular structures of the three bacterial species frequently isolated from kendo men within a short time. Electrolyzed acidic water may be useful for disinfecting of kendo equipment.

Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena.

Euglena complex chloroplasts evolved through secondary endosymbiosis between a phagotrophic trypanosome host and eukaryotic algal endosymbiont. Cytoplasmically synthesized chloroplast proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the Euglena chloroplast. Euglena chloroplast preprotein pre-sequences contain a functional N-terminal ER-targeting signal peptide and a domain having characteristics of a higher plant chloroplast targeting transit peptide, which contains a hydrophobic stop-transfer membrane anchor sequence that anchors the precursor in the vesicle membrane. Pulse-chase subcellular fractionation studies showed that (35)S-labeled precursor to the light harvesting chlorophyll a/b binding protein accumulated in the Golgi apparatus of Euglena incubated at 15 degrees C and transport to the chloroplast resumed after transfer to 26 degrees C. Transport of the (35)S-labeled precursor to the chlorophyll a/b binding protein from Euglena Golgi membranes to Euglena chloroplasts and import into chloroplasts was reconstituted using Golgi membranes isolated from 15 degrees C cells returned to 26 degrees C. Transport was dependent upon extra- and intrachloroplast ATP and GTP hydrolysis. Golgi to chloroplast transport was not inhibited by N-ethylmaleimide indicating that fusion of Golgi vesicles to the chloroplast envelope does not require N-ethylmaleimide-sensitive factor (NSF). This suggests that N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are not utilized in the targeting fusion reaction. The Euglena precursor to the chloroplast-localized small subunit of ribulose-1,5-bisphosphate carboxylase was not imported into isolated pea chloroplasts. A precursor with the N-terminal signal peptide deleted was imported, indicating that the Euglena pre-sequence has a transit peptide that functions in pea chloroplasts. A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with the hydrophobic membrane anchor and the pre-sequence region C-terminal to the hydrophobic membrane anchor deleted was imported localizing the functional transit peptide to the Euglena pre-sequence region between the signal peptidase cleavage site and the hydrophobic membrane anchor. The Euglena precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase and the deletion constructs were not post-translationally imported into isolated Euglena chloroplasts indicating that vesicular transport is the obligate import mechanism. Taken together, these studies suggest that protein import into complex Euglena chloroplasts evolved by developing a novel vesicle fusion targeting system to link the host secretory system to the transit peptide-dependent chloroplast protein import system of the endosymbiont.

Presence of glyoxylate cycle enzymes in the mitochondria of Euglena gracilis.

Isocitrate lyase and malate synthase are specific enzymes of the glyoxylate cycle, used here as glyoxysomal markers. Both enzymes were found in the mitochondrial fraction after organelle fractionation by isopycnic centrifugation. Electron microscopy of this fraction indicated that mitochondria were the only recognizable organelles. Using an immunogold labeling method with anti-(malate synthase) antiserum, the only organelles stained in cells were the mitochondria. These results show that the glyoxylate cycle is present in mitochondria in Euglena.