T.P. van der Krift

Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta‐(L‐alpha‐aminoadipyl)‐L‐cysteinyl‐D‐valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N‐synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200–800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.

Automated high-throughput electron tomography by pre-calibration of image shifts

Electron tomography is a versatile method for obtaining three‐dimensional (3D) images with transmission electron microscopy. The technique is suitable to investigate cell organelles and tissue sections (100–500 nm thick) with 4–20 nm resolution. 3D reconstructions are obtained by processing a series of images acquired with the samples tilted over different angles. While tilting the sample, image shifts and defocus changes of several µm can occur. The current generation of automated acquisition software detects and corrects for these changes with a procedure that incorporates switching the electron optical magnification. We developed a novel method for data collection based on the measurement of shifts prior to data acquisition, which results in a five‐fold increase in speed, enabling the acquisition of 151 images in less than 20 min. The method will enhance the quality of a tilt series by minimizing the amount of required focus‐change compensation by aligning the optical axis to the tilt axis of the specimen stage. The alignment is achieved by invoking an amount of image shift as deduced from the mathematical model describing the effect of specimen tilt. As examples for application in biological and materials sciences 3D reconstructions of a mitochondrion and a zeolite crystal are presented.

Purification of nonspecific lipid transfer protein (sterol carrier protein 2) from human liver and its deficiency in livers from patients with cerebro-hepato-renal (Zellweger) syndrome

The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and found to be very similar to that of the nonspecific lipid transfer protein from bovine and rat liver with, as main feature, the absence of arginine, histidine and tyrosine. By way of a specific enzyme immunoassay using affinity-purified antibodies, the levels of nonspecific lipid transfer protein were determined in human livers. Levels varied from approximately 150 ng nonspecific lipid transfer protein per mg 105,000 X g supernatant protein for juvenile and adult humans to 40 ng per mg supernatant protein for a young infant. Levels of nonspecific lipid transfer protein in livers of infants with cerebro-hepato-renal (Zellweger) syndrome were extremely low (i.e., 2 ng per mg supernatant protein). Immunoblotting revealed the presence of crossreactive proteins of molecular masses of 40,000 and 58,000. The 40 kDa and 58 kDa proteins occurred in control livers, whereas only the 40 kDa protein was present in Zellweger livers. As in rat the 58 kDa protein could be demonstrated in a peroxisomal preparation isolated from an adult liver. A possible link between the occurrence of nonspecific lipid transfer protein and the presence of peroxisomes is discussed.

Structural differences between two types of basidiomycete septal pore caps

The septal pore cap (SPC) of Trichosporon sporotrichoides CBS 8245 is vesicular-tubular, connected with flat-tubular endoplasmic reticulum (ER), and stains densely with zinc/iodine/osmium tetroxide, as does the ER. The SPC of Schizophyllum commune CBS 340.81 is more complex, about 600 nm in diameter, with perforations of 80-120 nm diameter, and stains less densely with zinc/iodine/osmium tetroxide than the ER. In high-pressure frozen and freeze-substituted hyphae of T. sporotrichoides the ER is present parallel to the dolipore septa, and electron-dense material occurs opposite the septal pore channel; the SPC rarely showed smooth vesicular-tubular membranes, suggesting that this is an ephemeral function of the SPC. The SPC of S. commune has a smooth outer and inner membrane, which enclose a matrix with a palisade-like substructure. A thin layer of electron-dense material covers the inner surface of the SPC of S. commune, from which beaded filamentous structures connect the SPC and the pore-occluding material. These filamentous structures may maintain the intracellular position of the SPC and possibly play a role in plugging the septal pore channel. The septal pore swellings of T. sporotrichoides contain more 1,6-beta-glucan than the septum, and intracellular glucans are also present near the septal pore channel. This cytosolic 1,6-beta-glucan in T. sporotrichoides may serve as a matrix to keep the tubular membranous structures of the SPC together. In contrast, 1,6-beta-glucan is not observed in the SPC and in the pore-occluding material of S. commune, and hyphal septa of this species show less labelling of 1,6-beta-glucan than the septal swelling. The evolutionary transition from simple to more complex types of SPCs may have resulted in a requirement for different components to maintain the morphological integrity and cell biological function.

Field emission gun-scanning electron microscopy of septal pore caps of selected species in the Rhizoctonia s. l. complex

Techniques of freeze-fracturing and cyto- plasmic maceration were combined to reveal the sep- tal pore cap in some members of the Rhizoctonia s.l. complex by the use of field emission gun-scanning

The perforate septal pore cap of Basidiomycetes

The interactions between cells of lower fungi may occur through plasmodesmata-like structures, which have been visualized electron microscopically in fungal septa. Developing zygospores of the zygomycetous speciesRhizopus sexualis andGilbertella persicaria contain transcellular strands in septa which separate young gametangia from the suspensors. These simple, unbranched strands occur at intervals in the gametangial wall, and resemble plasmodesmata (Hawker et al.1966). The structures were found to be connected with the endoplasmic reticulum. Plasmodesmata-like structures have also been demonstrated in septa of hyphae of the ascomycetous yeastsGeotrichum candidum (Kirk and Sinclair1966) and inEndomycopsis fibuliger (Takada et al.1965). Septa ofGalactomyces geotrichum exhibit unbranched plasmodesmata-like structures, extending through the cell wall. The width of these channels is40-60 nm, with a desmotubule of10 nm diameter (Marchant1976) which connects endoplasmic reticulum on either side of the septum. Plasmodesmata-like structures occur in few fungi, while in the majority of fungi with septate hyphae the protoplasm of neighbouring cells is connected through the septal pore channel.

Structure and Function of the Nonspecific Lipid Transfer Protein (Sterol Carrier Protein 2)

Rat liver contains proteins that in vitro modulate microsomal enzymes of cholesterol biosynthesis (for a recent review, see reference 1). One of these cytosolic modulators is sterol carrier protein 2 (SCP2) which stimulates the microsomal conversion of intermediates between lanosterol and cholesterol (2–4). This protein also facilitates net mass transfer of cholesterol between membranes (5–7). Through this action SCP2 stimulates the formation of cholesterol esters by microsomal membranes (6,8,9) and the conversion of cholesterol into pregnenolone by adrenal mitochondria (1,10). However, a recent study with rat hepatocytes and Reuber H35 hepatoma cells failed to provide support for an important role of SCP2 in cholesterol biosynthesis and esterification in situ (11). Additional questions about its role in cellular cholesterol metabolism were raised by the observation that SCP2 may be secreted by Morris hepatoma cells (12).

Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells: EXTREMELY THIN LAYER PLASTIFICATION

Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μ m2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.