Effect of environmental factors on Fonsecaea pedrosoi morphogenesis with emphasis on sclerotic cells induced by propranolol

The influence of growth conditions, as well as of propranolol on Fonsecaea pedrosoi morphogenesis was established using the chemically defined media of Czapeck-Dox (CD) and Butterfield (BF). Mycelial growth of F. pedrosoi in both media was obtained at room temperature (25 °C) for 14 days, without shaking, whereas conidia formed at 37 °C, for 4 days, in shaken cultures and could be isolated free from the mycelium by filtration in gauze. At low pH (2.5–3.0), there appeared sclerotic cells attached to normal hyphae. When propranolol was added to the CD medium moniliform hyphae were observed, whereas this drug in the BF medium induced formation of sclerotic cells. Ultrastructural examination revealed that the propranolol-induced sclerotic cells were very similar to those observed in infected tissues.     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

Screening of Bacterial Cellulose-producing Acetobacter Strains Suitable for Agitated Culture

Extensive screening for cellulose-producing Acetobacter strains suitable for agitated culture was done by developing the screening conditions. A total of 2096 strains were isolated; isolation from fruits was particularly efficient. The cellulose productivities of 412 isolates were estimated by culturing in two different media under both shaken and static conditions. No correlation between the amounts of cellulose accumulated in shaken and static cultures was observed. Higher cellulose accumulation was obtained in the shaken cultures using a corn steep liquor/fructose-based medium than a conventional yeast extract/peptone/glucose-based one. Many isolates showed higher cellulose accumulation than well-known cellulose-producing strains. The producer that yielded the highest cellulose accumulation in shaken culture was selected and named Acetobacter sp. BPR 2001. Using this strain, cellulose was produced in a jar fermentor.     

The growth of isolated barley embryos cultivated under different conditions

Isolated barley embryos were cultivated aseptically in three different complete media. The growth of the primary root of each embryo was measured during four days of cultivation. Embryos were cultivated on three different consistencies of media: on agar plates, on cellulose tissue moistened with the medium and in liquid shaken cultures. The last way of cultivation yielded the highest degree of the growth of roots. Optimum combination of conditions in shaken liquid medium was selected on the basis of systematic study. The growth of roots of isolated embryos under appropriate conditions approaches that of roots of seedlings cultived on moistened blotting paper.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose. Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose.
Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.     

Mellein and 4-Hydroxymellein Production by Aspergillus ochraceus Wilhelm

Mellein and 4-hydroxymellein are isocoumarin compounds produced by Aspergillus ochraceus Wilhelm. They are structurally similar to the dihydroisocoumarin moiety of ochratoxin A, a toxic metabolite of the same fungus, and they possibly have similar biological properties. Production of mellein and 4-hydroxymellein on synthetic media and natural solid substrates was determined. Several carbon and nitrogen sources supported production of these metabolites in stationary culture. Additional zinc and molybdenum increased production of both metabolites in stationary culture, but were not required for maximum production in shaken culture. Mellein and 4-hydroxymellein were produced on yellow corn, but neither was produced on wheat, peanuts, or soybeans.     

Water activity as a key parameter of synthesis reactions: The example of lipase in biphasic (liquid/solid) media

Ester synthesis catalyzed by Candida cylindracea lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was investigated in solid/liquid biphasic media containing the enzyme preparation and reactants without addition of organic solvents not participating in the reaction. Although the effects of water on enzyme kinetics have been abundantly studied in nearly anhydrous media, reactions in which water is produced have not been investigated. The effect of water produced by the reaction itself on the enzymatic activity was studied. The dispersion of water in a shaken, nearly anhydrous medium was shown to be responsible for the lack of activity of the enzyme. In contrast, when slowly shaken, the enzyme was fully activated by the water furnished as a product of the reaction. However, when experiments were performed in a two-phase aqueous/organic system with previously solubilized enzyme in water, the enzyme activity was increased by shaking and was of the same order of magnitude as in nearly anhydrous media. Under low water activity conditions, a powerful agitation can lead to slower reaction rate, because water, a product of esterification, is not retained in the microenvironment of the enzyme to activate it. The activation effect of water produced by the reaction was clearly shown using enzyme preparations shaken in an anhydrous medium and previously equilibrated at low water activities (a_w = 0.13 and 0.69). This activation did not occur for an enzyme preparation equilibrated at high a_w (0.89) or for a preparation gently shaken in a water-saturated medium. The lag time preceding activation of the enzyme increased with the extent of enzyme dehydration. The mass of the enzyme preparation was shown to be a parameter affecting the capacity of the lipase to produce enough water in its immediate environment. The lack of activity observed for a small quantity of enzyme was eliminated by addition of heat-denaturated lipase.     

A medium for commercial production of the halophilic Micrococcus nuclease.

A simple synthetic medium (glutamate-sucrose medium) was devised for production, during growth in shaken flasks, of extracellular halophilic nuclease (nuclease H) by a moderate halophile, Micrococcus varians subsp. halophilus. A simple medium consisting of 0.7% ammonium sulfate, 1.0% glucose, minerals, three vitamins, and 2 M NaCl gave good growth and excellent production of nuclease H in a jar fermentor when the pH was adjusted to 7.5 to 8.0 during cultivation.     

A new ecological adaptation to high sulfide by a Hydrogenobacter sp. growing on sulfur compounds but not on hydrogen

Thermophilic bacteria were isolated from a sulfide-rich, neutral hot spring in Iceland on gelrite minimal medium with 16 mM thiosulfate. The isolates were aerobic, obligate chemolithoautotrophs and used thiosulfate and sulfur as electron donors, producing sulfate from both substrates. No growth was observed with hydrogen as the sole electron donor, and no hydrogenase activity was detected. The cells were gram-negative and usually single, 4-5 microm long and 0.7 microm in diameter and formed sulfur globules after a few days of incubation. By SSU rRNA sequence comparisons, the bacterium was placed in the genus Hydrogenobacter with the closest relative to be Calderobacterium hydrogenophilum with 98.3% sequence similarity. This novel bacterium shows an ecological adaptation to high sulfide springs and is differentiated from its closest known relatives by lack of H2 oxidation, deposition of sulfur and lower growth temperature.     

Growth and Development of Pelargonium Pith Cells in vitro

Pelargonium pith callus cultured in media containing auxin and cytokinin will not differentiate formed organs if monthly subcultures are made into fresh medium. Left on semisolid medium for prolonged periods, such callus gives rise to nodules containing lignified cells. Such nodules can be stimulated to produce shoots and roots if cultured for one month in shaken liquid medium or for longer times on semisolid mediium lacking both auxin and cytokinin. The subsequent development of organs is best on semisolid medium. Entire plants have heen produced in this way from initial pith explants containing several hundred cells.     

Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.     

Reduction of nitro-BT by some components of the chloroform-methanol extract of the grey matter of the rat brain.

An attempt was made to extract the substance present in the nerve fibres of the cerebral cortex and basal ganglia which reduced Nitro-BT in alkaline medium pH 9-5 [4,5]. The investigations were performed on 40 white Wistar rats of both sexes and ca. 200 g of body weight. The brains taken for studies were fixed in Baker's formalin for 1,5 hr, thereafter grinded and homogenized in chloroform-methanol solution supplemented up to 100 ml of final volume after homogenisation. Thus prepared solution was left for 24 hr at 4 degrees C temperature and than centrifuged 2000 g/min. The extract was evaporated and the sediment further investigated. The following fraction were received: 1. fraction soluble in aceton, 2. fraction soluble in ethylalcohol, 3. fraction soluble in ether, 4. fraction soluble in water and 5. the sediment. Each fraction was incubated with a standard medium containing Nitro-BT. After incubation the amount of reduced Nitro-BT in each of the incubation medium was spectrophotometrically measured. The most intense reduction was found in the incubation medium with the water fraction. The spectrophotometrically determined reduction of Nitro-BT of the water fraction compared with that of the gangliosides solution were similar. The addition into the incubation media biogenic amines and N,N-diethyl-p-phenylenediamine enhances the reduction of Nitro-BT significantly. According to the results obtained an interference of gangliosides and of the investigated amines in the reduction of Nitro-BT is suggested.     

TIP47 associates with lipid droplets

Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose differentiation-related protein (ADRP; also called adipophilin), which is found in a wide array of cell types, and the perilipins, which are restricted to adipocytes and steroidogenic cells. TIP47 was initially identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose 6-phosphate receptor, yet its sequence is highly similar to the lipid droplet protein, ADRP, and more distantly related to perilipins. Hence, we hypothesized that TIP47 might be associated with lipid droplets. In HeLa cells grown in standard low lipid-containing culture media, immunofluorescence microscopy revealed that the cells had few lipid droplets; however, TIP47 and ADRP were found on the surfaces of the small lipid droplets present. When the cells were grown in media supplemented with physiological levels of fatty acids, the amount of neutral lipid stored in lipid droplets increased dramatically, as did the staining of TIP47 and ADRP surrounding these droplets. TIP47 was found primarily in the cytosolic fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-containing culture medium, while ADRP was undetectable in these fractionated cell homogenates. When HeLa and MA10 Leydig cells were lipid-loaded, significant levels of ADRP were found in the floating lipid droplet fractions and TIP47 levels remained constant, but the distribution of a significant portion of TIP47 shifted from the cytosolic fractions to the lipid droplet fractions. Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein.     

Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos?

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, > 38 years; younger, < or = 38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean +/- SD: 24.1 +/- 2.5 microm vs 23.1 +/- 3.3 microm; p = 0.01) but ZP density was equal (2.8 +/- 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 +/- 2.2 microm vs 21.7 +/- 1.6 microm; p = 0.28) and density (2.9 +/- 0.7 nm vs 2.8 +/- 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 +/- 2.7 microm vs 19.1 +/- 5.0 microm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 +/- 0.5 nm vs 3.3 +/- 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 +/- 0.5 nm vs 2.9 +/- 0.7 nm, p < 0.001; younger: 3.3 +/- 1.0 nm vs 2.8 +/- 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.     

Effect of the aeration conditions on the biosynthesis of oxytetracycline and the formation of organic acids by a culture of Actinomyces rimosus]

The effect of the aeration conditions on oxytetracycline biosynthesis and production of organic acids by Act. rimosus was studied. Intensive biosynthesis of oxytetracycline in shaken flasks with concentrated complex media was observed at the rate of oxygen dissolution in the liquid ranging from 14 to 25 mg/1/min. Lower rates of the oxygen dissolution up to 7 mg/1/min resulted in decreased rates of the culture growth and the medium component consumption, decreased antibiotic levels, production of significant amounts of pyruvic and acetic acids.     

Extracellular proteinase fromEnterococcus faecalis subsp.liquefaciens

Growth and extracellular proteinase production byEnterococcus faecalis subsp.liquefaciens was studied on several culture media and under different incubation conditions. The organisms grew well and developed extracellular proteinase activity on proteinaceous media, but when it grew on Collins basal medium (lacking of protein), growth was poor and proteinase activity was not detected. The activation energy for growth was estimated to be 116 kJ/mol, the optimum being at 37°C. Proteinase production was not affected by temperature in the range studied (7–45°C). Growth rate was not affected by aeration although a higher amount of microorganisms was observed on shaking the culture during incubation. Likewise, extracellular proteolytic activity was about twice higher in cultures shaken at 2.3 or 3.3 Hz than in those shaken at 0 or 1.3 Hz.     

Formation of nano-hydroxyapatite in gelatin droplets and the resulting porous composite microspheres

A one-pot strategy was first presented in this paper to synthesize gelatin/hydroxyapatite (HAP) composite microspheres in a water-in-oil (W/O) emulsion. Using gelatin droplets as microreactors and colloid protective medium, needle-like nano-HAP crystals (5 nm x 60-100 nm) in form of clusters were homogeneously and orderly precipitated within gelatin matrix. The results of scanning electron microscopy (SEM) revealed that the as-prepared microspheres with an average diameter of 7.5 microm displayed a narrow particle size distribution, a high dispersity and a naturally porous structure. This microsphere material is expected to have a great potential for both controlled drug release and faster bone in-growth in bone tissue engineering.     

Physiological status and change of cytoplasmic lipid droplets in the pheromone-producing cells of the silkmoth, Bombyx mori (Lepidoptera, Bombycidae)

Changes in size and number of cytoplasmic lipid droplets were quantified in the pheromone gland (PG) of Bombyx mori before and after adult eclosion. Two days before eclosion, size and number of droplets are small (diameter is 2-7 microm) and few. The formation and significant proliferation of larger droplets (5-12 microm) take place between 2 days and 1 day before eclosion. From the day of emergence until day 3 a fluctuation in size and number of lipid droplets during the photophase (4h intervals) is observed. The changes are more characteristic and dramatic on the day of emergence and first day, while attenuation of these changes can be observed from the second day and seems to disappear by day 4. Bombykol content, at each respective time, is in good correlation with the observed fluctuation in lipid droplet parameters. Highest bombykol production daily is observed towards the early evening, when lipid droplets are the smallest (2-4 microm) and most numerous. By day 4, however, this regularity also ceases. In 24h old mated females PG cell structure is quite similar to newly emerged ones. In glands of 72 h old decapitated females the formation of 'extra' large lipid droplets is remarkable. In vivo pheromone biosynthesis activating neuropeptide (PBAN) treatment, however, induced the formation of many small droplets, although numerous larger ones also remained. The morphological changes in lipid droplets and cellular dynamics associated with the external signal of PBAN in the PG suggest a storage-pool function of the lipid droplets.