Changes in development and ultrastructure of Aspergillus niger mycelium, strain with increased tolerance to toxic substances from beet molasses.

Effects of a defoamer and toxic molasses compounds on development and ultrastructure of A. niger mycelium, strain Z, characterized by high tolerance to these substances and producing citric acid in surface fermentation on proper molasses media with 70% yield were presented. Spumol BJ in concentration of 5 microliters/100 cm3 as well as toxic molasses compounds stimulated the process of swelling and germinating of conidia. Moreover, giant conidia, unable to germinate, appeared. Developing mycelium with dispersed hyphae became mucilaginous after 17-20 h culture, which indicated the process of sinking but after 24 h some part of the mycelium developed normally. Electron microscopic observations of mycelium developing in the presence of the toxic substances showed along with electron-transparent cytoplasm in a consequence of decrease in ribosome number, changes in ultrastructure of mitochondria. It may be assumed that one of the reasons of the above described abnormalities in development and ultrastructure of mycelium was a disturbance of respiration processes. The appearance of deposits of electron-dense material in mitochondria suggested the existence of a defence mechanism, eliminating toxic substances.     

Changes in development and ultrastructure of Aspergillus niger mycelium in the presence of toxic substances from molasses in citric acid fermentation medium.

Effects of a surface-active agent (Spumol BJ) and toxic molasses compounds on development and ultrastructure of Aspergillus niger strain R-16 mycelium producing citric acid in surface fermentation on molasses medium with 80% yield are presented. Microscopic observations showed that Spumol BJ in concentration of 5 microliters/100 cm3 as well as toxic molasses compounds stimulated the process of swelling and germinating of conidia. Giant conidia unable to germinate, appeared along with typical ones. Delicate mycelium outgrowing from germinating conidia however, sank after 20-24 h culture. Observations of hyphae in electron microscope showed changes in the ultrastructure and dimensions of mitochondria followed by successive degeneration of protoplast. The data obtained indicate that one of the reasons disqualifying raw molasses for biotechnological processes might be the excessive amount of surface-active antifoaming additives.     

Toxicity of L-Tryptophan photoproduct on recombinationless (rec) mutants of Salmonella typhimurium

l-Tryptophan after exposure to black light becomes toxic for recombinationless (rec) mutants of Salmonella typhimurium. Fifty-six radiation-sensitive mutants were screened for sensitivity to the tryptophan photoproduct; the rec and exr (X-ray sensitive) mutants are sensitive, whereas the uvr, hcr, and wild-type strains are resistant. A number of catabolic products of tryptophan and compounds related to tryptophan were screened for toxicity to rec strain; these are nontoxic or far less toxic for rec strains than irradiated l-tryptophan. The toxic photoproduct is relatively stable to drying and basic hydrolysis at 90 C, indicating that it is a stable organic compound, eliminating peroxide as the toxic component. It was also observed that the toxic product is a photooxidation product since it is formed only when l-tryptophan is irradiated in the presence of air.     

甘蓝热胁迫叶片细胞的超微结构研究

高温胁迫引起植物体损伤是很常见的,人们已从多方面探讨高温胁迫对植物的影响,主要工作集中在生理生化、膜结构、热激蛋白及作物产量等方面［１—４］,超微结构的变化也有少量报道［５—７］。但目前要建立一个高温对植物体影响的模式还比较困难。本文探讨了甘蓝不同耐热品种叶片细胞超微结构在热胁迫后的差异,目的是建立植物耐热程度的细胞学指标,为育种工作者选育耐热性品种提供细胞学依据。材料和方法试验材料为甘蓝（Ｂｒａｓｓｉｃａｏｌｅｒａｃｅａｖａｒ．ｃａｐｉｔａｔａＬ．）耐热品种“夏光甘蓝”和感热品种“京丰甘蓝”。…     

Ultrastructural Distribution of DNA within Plant Meristematic Cell Nucleoli during Activation and the Subsequent Inactivation by a Cold Stress

We have investigated the precise location of DNA within the meristematic cell nucleolus ofZea maysroot cells andPisum sativumcotyledonary buds, in the course of their activation and induced inactivation following a subsequent treatment at low temperature. For this purpose, we combined the acetylation method, providing an excellent distinction between the various nucleolar components, with thein situterminal deoxynucleotidyl transferase-immunogold technique, a highly sensitive method for detecting DNA at the ultrastructural level. In addition to the presence of DNA in the condensed chromatin associated with the nucleolus, we demonstrated that a significant label was detected in the nucleolus of quiescent cells in both plant models. Evident labels were also found in the dense fibrillar component of actived nucleoli. Whereas in inactivated nucleoli no significant label was observed within the dense fibrillar component, an intense label was seen over the large heterogeneous fibrillar centres only during inactivation. The granular component was never significantly labelled. These results appear to indicate that the DNA present in the dense fibrillar component of activated nucleoli withdraws from this structure during its inactivation and becomes incorporated in the large fibrillar centres. These observations suggest that in plant cells inactivation of rRNA genes is clearly accompanied by changes in the conformation of ribosomal chromatin.     

Comparative studies on cell stimulatory, permeabilizing and toxic effects induced in sensitive and multidrug resistant fungal strains by amphotericin B (AMB) and N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl-amphotericin B methyl ester (MFAME) is a new derivative of amphotericin B, which is characterised by low toxicity to mammalian cells and good solubility in water of its salts. The antifungal activity and effects of MFAME towards Candida albicans and Saccharomyces cerevisiae multidrug resistant MDR(+) and sensitive MDR(-) strains was compared with those of parent compound. The results obtained indicate that MDR(+) S. cerevisiae was sensitive to MFAME as well as to AMB. MFAME exhibited the same effects on fungal cells studied as parent antibiotic. The two antibiotics, depending on the dose applied induced cell stimulation, K+ efflux, and/or had a toxic effect.     

LIGNIFICATION DURING SECONDARY WALL FORMATION IN COLEUS: AN ELECTRON MICROSCOPIC STUDY

The fine structure of lignin deposition was examined in developing secondary walls of wound vessel members in Coleus. KMnO₄, which was used as the fixative, selectively reacts with the lignin component of the cell wall and thus can be used as a highly sensitive electron stain to follow the course of lignification during secondary wall deposition. Lignin was first detected as conspicuuos electron-opaque granules in the primary wall in the region where the secondary wall thickening arises and as fine granular striations extending into the very young secondary wall. As the secondary wall develops lignification becomes progressively more extensive. In cross sections the lignified secondary wall appears as concentric, fine granular striations; in tangent al or oblique sections it is seen as delicate, beaded fibrils paralleling the long axis of the thickening. High magnification of tangential or oblique sections shows that the fibrillar appearance is due to the presence of alternating light and dark layers each approximately 25-35 A wide. It is assumed that the light layers are the cellulose microfibrils and the dark regions contain lignin which fills the space between the microfibrils. KMnO₄, by selectively reacting with lignin, thus negatively stains the cellulose microfibrils revealing their orientation and dimensions.     

Distribution of fibrillar centers and silver-stained components in the nucleolus of human Sertoli cells

The nucleolus of the human Sertoli cell consistently showed three distinct, spontaneously segregated parts: 1. one or two large, silver-positive fibrillar centers; 2. strands of dense fibrillar component continuous with the dense cords surrounding the fibrillar center. These components were also silver-positive; 3. a granular, silver-negative mass. These observations show that in the human Sertoli cell the number of fibrillar centers is far lower than the diploid number of NORs. They also suggest that the fibrillar center might contain several NORs in this cell type.     

Genotypic variation of the responses to chromium toxicity in four oilseed rape cultivars

Heavy metal toxicity in soils has been considered as major constraints for oilseed rape (Brassica napus L.) production. In the present study, toxic effects of chromium (Cr) were studied in 6-d-old seedlings of four different cultivars of B. napus (ZS 758, Zheda 619, ZY 50, and Zheda 622). The elevated content of Cr inhibited seedling growth, decreased the content of photosynthetic pigments, and activities of antioxidant enzymes, as well as increased the content of malondialdehyde and reactive oxygen species in all the cultivars. The Cr content in different parts of plants was higher in Zheda 622 than in other cultivars. The electron microscopic study showed changes in ultrastructure of leaf mesophyll and root tip cells at 400 μmol Cr, and these changes were more prominent in Zheda 622. An increased size and number of starch grains and number of plastoglobuli, damaged thylakoid membranes, and immature nucleoli and mitochondria were observed in leaves. In roots, enlarged vacuoles, disrupted cell walls and cell membranes, an increased number of mitochondria and a size of nucleolus, as well as plasmolysis (in Zheda 622) were observed. On the basis of these findings, it can be concluded that cv. Zheda 622 was more sensitive to Cr as compared to other three cultivars.     

Localization and Solubilization of Colicin Receptors

Envelope fractions isolated from Escherichia coli K-12 C600 and from colicin-resistant and colicin-tolerant (Tol II) mutants derived from this strain were separated on sucrose gradients into cell wall-enriched and cytoplasmic membrane-enriched fractions. These fractions were tested for their ability to neutralize colicins of the E and K groups. Neutralization activity was found in the cell wall-enriched fraction from the parent and the Tol II mutant but was absent from all fractions from the resistant mutant. This was also tested with several other E. coli strains. In all cases, sensitive strains contained the neutralization activity, whereas resistant strains did not. The neutralization activity was solubilized from cell walls or cell envelopes of sensitive or Tol II strains by extraction at room temperature with Triton X-100 plus ethylenediaminetetraacetic acid. The solubilized activity was precipitated by 20% ammonium sulfate, 70% ethanol, or 10% trichloroacetic acid. The activity was destroyed by treatment of the solubilized preparation with trypsin or periodate. These results suggest that this colicin-neutralization activity is due to the presence of specific receptors localized in the cell wall and that intact protein and a carbohydrate are required for this receptor to bind colicin.     

Repair of functional and ultrastructural alterations after thermal injury of Physarum polycephalum

Repair of thermal injury of Physarum polycephalum Schw. plasmodia has been studied by light and electron microscopy. As a result of heating the plasmodia for 10 min at 42°C both the unordered and shuttle protoplasmic streaming were arrested; the outer plasmodial membrane showed alterations at sites of contact with water; the onset of the next mitosis was considerably delayed. The plasmodial ultrastructure was markedly disturbed, including disappearance of the granular component of the nucleoili and a compact, almost fibrillar structure of the latter. The mitochondria became distorted and their intracristal spaces enlarged while the outer and inner membranes appeared in some places to be separated. Glycogen particles disappeared from the cytoplasm. Recovery of both types of protoplasmic streaming of the motility of the plasmodium, of the resistance of its membrane to contact with water, and of the ability of the organism to go through the cell cycle went all hand in hand with the normalization of the structure of nucleoli, mitochondria and cytoplasm. All of the functional and structural characteristics are normalized within ca. 9 h following heating.     

Tinopal AN as a selective agent for the differentiation of phytopathogenic and saprophytic Pseudomonas species

The selective toxicity of the respiratory inhibitor Tinopal AN (1,1-bis (3, N -5–dimethylbenzoxazol-2-yl) methine p -toluene sulphonate) towards phytopathogenic bacteria was investigated further and in general was confirmed using more than 160 additional strains of Pseudomonas spp. The mechanism(s) of the resistance shown by saprophytic fluorescent pseudomonads were studied to elucidate the differences between resistant saprophytic and sensitive phytopathogenic Pseudomonas species. Damage to, or partial removal of the cell wall of Tinopal AN-resistant Pseudomonas aeruginosa , resulted in a marked Tinopal AN-sensitivity, as judged by the ability of Tinopal AN to inhibit oxygen uptake. Furthermore, removal of part of the lipo-polysaccharide (LPS) component of the outer membrane also resulted in sensitivity. Mutants of Ps. aeruginosa with modified outer cell walls were tested for their reactions towards Tinopal AN, and two cell wall lipopolysaccharide (LPS) mutants of Escherichia coli (env Al) and Salmonella typhimurium (rfa) were, in contrast to the wild-type strains, found to be sensitive towards Tinopal AN. The results therefore suggest that the resistance of saprophytic pseudomonads towards Tinopal AN is due (at least in part) to the selectively permeable properties of the outer membrane of the cell wall. The usefulness of Tinopal AN for screening potentially phytopathogenic strains of Pseudomonas was confirmed.     

Silicon amelioration of aluminium toxicity and cell death in suspension cultures of Norway spruce (Picea abies (L.) Karst.)

A role for silicon (Si) in the amelioration of aluminium (Al) toxicity in gymnosperms is suggested by their codeposition in planta, including within needles. This study was designed to investigate Al/Si interactions at the cellular level using suspension cultures of Norway spruce. Toxic effects of Al were dependent on duration of Al exposure, concentration of Al, and pH. Toxicity was reduced when Si was present, and the effect was enhanced at pH 5.0 compared to pH 4.2. Study of the ultrastructure of Al-treated cells indicated that changes in cell wall thickening, degree of vacuolation, and the degeneration of mitochondria, Golgi bodies, ER and nucleus preceded cell death, and significant amelioration was noted when Si was also present. When the fluorescent dye Morin was employed to localise free Al, cells treated with Al and Si in combination showed less fluorescence than the cells treated with Al alone. Intensity of fluorescence depended on the concentration of Al, duration of treatment and pH. Notably, presence of Si reduced the concentration of free Al in the cell wall in parallel with amelioration of Al toxicity. We therefore propose that formation of aluminosilicate complexes in the wall and apoplasm provide a significant barrier to Al penetration and cell damage in Norway spruce.     

Ultrastructural Study in Leaf Cell of Brassica oleracea var. capitata Under Heat Stress

Ultrastructural changes of the leaf cells in response to heat stress in thermo-resistant cultivar and thermo-sensitive cultivar of Brassica oleracea var. capitata L. were investigated. No ultrastructural change was shown in mesophyll cell of the thermo-resistant cultivar. All membrane-bound structure remained intact. However, the cell ultrastructure of thermosensitive cultivar showed significant changes. One of the major effects of heat stress was the disruption of membrane structure. Chloroplasts and nucleus were extremely sensitive to heat stress. Chloroplasts rounded off in different extents and their outer membranes disappeared. Structural alteration of the thylakoid membrane were visualized. Large amount of plastoglobulis appeared within the chloroplast. Mitochondria were far more resistant to heat stress than chloroplasts. There was no distinct changes of mitochondria structure. Nucleus suffered from serious damage as heat caused disruption of nuclear envelope and condensation of nucleoplasm, showing, eventually, numerous fibrillar granulous material and irregularly shaped hollow spaces presented within the nucleus.     

Fine Structure of Listeria monocytogenes in Relation to Protoplast Formation

Among the eight strains of Listeria monocytogenes tested for lysozyme sensitivity, two were resistant to lysozyme but became sensitive after lipase pretreatment. Among the other six, one was very sensitive to lipase and another one was extremely susceptible to lysozyme. Stable protoplasts were formed from the lysozyme-resistant strain (42) by lipase and lysozyme treatment, which completely digested the cell wall. The cell wall (uranyl acetate-lead stained) was of a thick triple-layered profile, with the intermediate layer of low density. Lipase treatment for a short time (60 min) did not cause any alteration in structure, but prolonged treatment (180 min) caused extensive digestion of the plasma membrane and the cell wall, liberating cytoplasmic material. When the cells were treated with either lipase or lysozyme, a small number of protoplasts were extruded through the partly digested or weakened transverse cell wall, leaving an almost intact cell wall ghost. There were small vesicular structures in the interspace between cell wall and plasma membrane. Mesosomes of varied organization were prominent in electron micrographs, both in sections and in negatively stained preparations. These were largely everted during protoplasting in the form of tubules and as small peripheral buds; a few small vesicles also remained as intrusive structures, some of which were very unusual because they appeared to be enclosed by the inner layer of plasma membrane alone. Lysis of the protoplasts by dilution of the sucrose, while maintaining a constant ionic environment, liberated many small vesicular structures and fibrillar nuclear material.     

Distribution of nucleolar proteins B23 and nucleolin during mouse spermatogenesis

The intracellular distribution of nucleolar phosphoproteins B23 and nucleolin was studied during mouse spermatogenesis, a process that is characterized by a progressive reduction of nucleolar activity. Biochemical analyses of isolated germ cell fractions were performed in parallel with the in situ ultrastructural immunolocalization of these two proteins by means of specific antibodies and colloidal gold markers, and by silver staining. RNA blot experiments showed that mRNA for nucleolin progressively decreased during spermatogenesis whereas mRNA for B23 increased in amount during early spermatogenic stages. Immunoblotting confirmed that both proteins were present during early spermatogenesis up to the round spermatid stage and absent from mature sperm. Immunoelectron microscopy revealed that in spermatogonia, leptotene and pachtyene spermatocytes, and in Golgi phase spermatids, B23 and nucleolin were localized in the dense fibrillar component and granular component of the nucleolus but not in the fibrillar centers. In the dense fibrillar residue of the cap phase spermatids, labeling with anti-nucleolin but not with anti-B23 was observed. During nucleolar inactivation, neither of the two polypeptides was dispersed to the nucleoplasm. Silver salts stained the fibrillar centers and dense fibrillar component but not the granular component of the nucleolus. Our results suggest that there is no direct relationship between nucleolar activity and the occurrence of B23 and nucleolin or silver staining. Moreover, we confirm that silver staining and the presence of B23 or nucleolin are not directly related to each other.by M. Trendelenburg     

Effect of antibiotic action on the submicroscopic structure of Salmonella. The action of monomycin on S. typhi]

The effect of subinhibitory concentrations of monomycin on the submicroscopic structures of 2 strains of S. typhi, 5 and 799 was studied. It was shown that formation of filamentoue forms, separation of the cell wall, thinning out of the cytoplasm granular component, increasing of the size of the matter with low electron optic density of fine granular structure were common in the cell response of both strains. Formation of vacuoli containing the thinned out granular component, filterable elements and complex membrane structures followed by their liberation into the medium and formation of the forms devoid of the cell walls was a characteristic peculiar property of the cell response in strain 799. The cells of strain 5 were characterized by formation of large granular osmiefilic matters and their excretion from the cells.     

New data concerning the functional organization of the mammalian cell nucleolus: detection of RNA and rRNA by in situ molecular immunocytochemistry.

We have investigated the fine spatial distribution of RNA and rRNA within the Ehrlich tumor cell nucleolus by in situ hybridization with a biotin-labeled probe and by two new strategies, the polyadenylate nucleotidyl transferase-immunogold technique and immuno-labeling with anti-RNA antibodies. Besides the presence, as expected, of RNA and rRNA in the granular component and the dense fibrillar component, we show, for the first time, significant label over all the fibrillar centers of the nucleoli. When RNA and DNA were detected simultaneously on the same sections, only the fibrillar centers were positive for both. These results throw light on the controversial subject of the precise location of transcribing rRNA genes within the nucleolus. The fibrillar centers, and not the dense fibrillar component, should thus be the site of rRNA synthesis.     

Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface

Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology.