In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach

Many essential cellular processes are affected by transmembrane H(+) gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.     

Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets

BACKGROUND: Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V). METHODS: Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced. RESULTS: Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents. CONCLUSIONS: (1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.     

Netropsin suppresses the rise of activity of an intracellular proteolytic system in sporulatingBacillus megaterium

Intracellular catabolism of proteins labeled at the end of the exponential growth proceeded in two phases during sporulation. The first phase was induced by starvation and took place also in cells whose sporulation was inhibited by netropsin. The second phase of degradation, which was triggered at the onset of the irreversible sporulation phase, was inhibited by netropsin. Intracellular proteolytic activity determined in disintegrated cells, i.e., primarily the activity of the cytoplasmic Ca²⁺-dependent serine proteinase(s) at the first place, was increasing throughout the sporulation process and reached its maximum during the irreversible sporulation phase. Its increase was suppressed by netropsin. Fractionation of the cell sap by HPLC revealed a similar distribution of proteolytic activities in the extract from control and netropsin-inhibited cells. The antibiotic thus probably affected the activation, not the formation of the cytoplasmic serine proteinase(s). Netropsin also inhibited an increase of proteolytic activity in the membrane fraction, probably owing to the presence of two different proteolytic enzymes.     

The polarity of endogenous regulatory substances inBryophyllum crenatum leaves and stems

In isolated leaves ofBryophylluni crenatum the intensity of marginal bud formation decreases from the apex towards the blade base, which is associated with the decreasing content of enioganous gibberallins. As proved by Dostál (1930), the formation of marginal buds on transversely divided blade of the isolated leaf increases in comparison with the non-divided control leaf. The results of our experiments have revealed that the increase in the formation of marginal buds in the blade transversely divided into the apical, middle and basal parts is connected with the increasing level of endogenous gibberellins, especially in the apical part. This rising level appears as early as 7 days following blade division,i.e. at the time preceding the formation of marginal bud bases. InBryophyllum crenatum plants the level of endogenous cytokinins was estimated in apical, middle and basal leaves, as well as in adjacent internodia. Maximum content was found out in the leaves from the middle stem part, which is probably associated with the capacity of this part to form marginal buds spontaneously also in intact plants. However, prior to flowering the maximum of cytokinin activity is shifted to the apical stem part.     

Strahleninduzierte Mutationen bei Dermatophyten III. Untersuchungen über den Zusammenhang der Virulenz mit der Mikromorphologie und der keratinolytischen Aktivität

Zusammenfassung 1. Aus morphologisch normaler Kultur vonMicrosporon gypseum wurden die UV-Mutanten folgender acht Kategorien selektiert: 1. Kulturen, die Makrokonidien, Mikrokonidien und Chlamydosporen bilden, 2. Kulturen mit Makrokonidien und Mikrokonidien, 3. Kulturen mit Makrokonidien und Chlamydosporen, 4. Kulturen mit Makrokonidien und Chlamydosporen, 5. Kulturen nur mit Makrokonidien, 6. Kulturen nur mit Mikrokonidien, 7. Kulturen nur mit Chlamydosporen, 8. Kulturen vollständig ohne Sporen.2. Alle Kulturen dieser Mutantenkategorien zeigten in vitro die keratinolytische Aktivität und die Griseofulvinempfindlichkeit. Bei den Mutanten wurden nur quantitative Unterschiede in diesen Eigenschaften beobachtet. Manche mutierten Kulturen waren mehr als zehnmal griseofulvinsensitiver als die Ausgangskulturen.3. Aus den virulenten Kulturen vonM. gypseum wurden avirulente Kulturen isoliert.4. Die Kulturen, welche nur Chlamydosporen bildeten und solche, die überhaupt keine Sporenbildung aufwiesen, waren in allen Fällen avirulent. Die konidienbildenden (Makrokonidien, Mikrokonidien) mutierten Kulturen waren in den meisten Fällen virulent.5. Es wurde kein Zusammenhang zwischen der keratinolytischen Fähigkeit in vitro und der Virulenz der Kultur in vivo gefunden. Die in vitro erhöhte keratinolytische Aktivität des DermatophytenM. gypseum determiniert selbst nicht seine Virulenz.

---

Summary 1. From the morphologically normal strain ofMicrosporon gypseum eight groups of mutants were selected: 1. strains with production of macroconidia, microconidia and chlamydospores, 2. strains with macroconidia and microconidia, 3. strains with macroconidia and chlamydospores, 4. strains with microconidia and chlamydospores, 5. strains with macroconidia only, 6. strains with microconidia only, 7. strains with chlamydospores only, 8. mycelial strains without production of spores.2. All the strains of the eight group were keratinolytically active in vitro and sensitive to griseofulvin. Quantitative differences, in the features evaluated, were only found. Some mutants were more than 10 times more sensitive to griseofulvine in comparison with the wild strains.3. Avirulent strains were isolated from the virulent wild strainM. gypseum.4. The strains producing only chlamydospores and mycelial strains without sporulation were avirulent in all cases. The greater part of conidia producing strains remained virulent.5. No relationship between the degree of keratinolytic activity in vitro and virulence in vivo was found. The virulence itself cannot be determined only by an increase in keratinolytic activity in vitro.

---

---

Diese Mitteilung wurde auf dem III. Internationalen Dermatologischen Symposium in Bratislava (4.–6.X.1966) vorgetragen.     

Changes in organ growth ofChenopodium rubrum due to suboptimal and multiple photoperiodic cycles with and without flowering effect

The growth changes of cotyledons, leaves, hypocotyls and roots due to photoperiodic induction in short day plantChenopodium rubrum were investigated in relation to flowering. Six-day old plants were induced by photoperiods with a different number of dark hours. We found that the degree of inhibition which occurred during induction in the growth of leaves, cotyledons and roots similarly as the stimulation of hypocotyl is proportional to the length of dark period. The photoperiods with 12, 16 and 20 dark hours bring about marked inhibition of growth and at the same time induce flowering in terminal and axillary meristems. The inhibitory effect of critical period for flowering,i.e. 8 dark hours, is not apparent in all criteria used and even the flower differentiation is retarded. The photoperiods of 4 and 6 dark hours did not affect growth and were ineffective in inducing flowering even if their number has been increased. The experiments with inductive photoperiod interrupted by light break have clearly shown that growth pattern characteristic for induced plants can be evoked in purely vegetative ones. Such statement did not exclude the possible importance of growth inhibition as a modifying factor of flower differentiation. We demonstrated that the early events of flower bud differentiation are accompanied by stimulation of leaf growth. The evaluation of growth and development of axillary buds at different nodes of insertion enabled us to quantify the photoperiodic effect and to detect the effects due to differences in dark period length not exceeding 2 hours.     

Transport and localization of 6-(γ,γ-dimethylallylamino)-purine-8-14C cytokinin in apple trees

When cytokinin 6-(γ,γ-dimethylallylamino)-purine-8-¹⁴C was injected into the side root of a one-year-old apple graft, variety Cox's Orange Pippin, it was mainly transported acropetally, but a certain part of the radioactivity was also transported basipetally into the root below the place of injection. 6 days after application of the labelled cytokinin most of the radioactivity was detected in the xylem, 20–30 cm above the place of application. An appreciable quantity of radioactivity was transported toward the apical part of the plant as far as 100 cm from the point of application. Special analysis showed that 6 days after exposition about half of the remaining radioactivity was present in 6-(γ,γ-dimethylallylamino)-purine-8-C¹⁴ and the rest went to the other metabolites. The cytokinin was transported acropetally mainly in the transpiration stream.     

Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis

Colonies of Saccharomyces cerevisiae laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.