A biphenyl type two-photon fluorescence probe for monitoring the mitochondrial membrane potential.

Here we describe the design and synthesis of a bifunctional two-photon fluorescence probe, N,N'-|dimethyl-4,4'-(biphenyl-2,1-ethenediyl)dipyridinium hexafluorophosphate (BP6). HeLa, Hek293, and Paramecium caudatum cells were stained with BP6. BP6 accumulated on the mitochondria of all three cell types when the mitochondrial membrane potential was high. As the mitochondrial membrane potential decreased following the addition of carbonyl cyanide m-chlorophenyl hydrazine, BP6 moved from the mitochondria to the nucleus in a reversible manner, depending on the mitochondrial membrane potential status. The maximum value of the two-photon absorption cross-section of BP6 is 250 GM (1 GM=1×10(-50) cm(4) s molecules(-1) photon(-1)). This value is 3 and 30 times larger, respectively, than that of the conventional mitochondria selective probes, rhodamine 123 and green fluorescence protein. These results suggest that BP6 should be useful for monitoring mitochondrial membrane potential by two-photon excitation.

Variability in secondary structure of 18S ribosomal RNA as topological marker for identification of Paramecium species.

Besides cytological and molecular applications, Paramecium is being used in water quality assessment and for determination of saprobic levels. An unambiguous identification of these unicellular eukaryotes is not only essential, but its ecological diversity must also be explored in the local environment. 18SrRNA genes of all the strains of Paramecium species isolated from waste water were amplified, cloned and sequenced. Phylogenetic comparison of the nucleotide sequences of these strains with 23 closely related Paramecium species from GenBank Database enabled identification of Paramecium multimicronucleatum and Paramecium jenningsi. Some isolates did not show significant close association with other Paramecium species, and because of their unique position in the phylogenetic tree, they were considered new to the field. In the present report, these isolates are being designated as Paramecium caudatum pakistanicus. In this article, secondary structure of 18SrRNA has also been analyzed as an additional and perhaps more reliable topological marker for species discrimination and for determining possible phylogenetic relationship between the ciliate species. On the basis of comparison of secondary structure of 18SrRNA of various isolated Paramacium strains, and among Paramecium caudatum pakistanicus, Tetrahymena thermophila, Drosophila melanogaster, and Homo sapiens, it can be deduced that variable regions are more helpful in differentiating the species at interspecific level rather than at intraspecific level. It was concluded that V3 was the least variable region in all the organisms, V2 and V7 were the longest expansion segments of D. melanogaster and there was continuous mutational bias towards G.C base pairing in H. sapiens.

Nanodiamond for intracellular imaging in the microorganisms in vivo.

Nanodiamond (ND) has great potential for bio labeling and drug delivery. In this work, the biocompatibility and bio labeling of ND are demonstrated via the interaction with cells and microorganisms, protists microorganisms Paramecium caudatum and Tetrahymena thermophile, in vitro and in vivo. We found the microorganism's living functions are not significantly affected by ND. The NDs were found entering the food vacuoles and later excreted by the microorganisms. The 5 nm ND was found more toxic compared to 100 nm ND, presumably due to the surface disordered carbons. Our results demonstrated nanodiamond can be used in bio imaging and matter delivery.

DNA polyfluorophores for real-time multicolor tracking of dynamic biological systems.

Dye-ing to live: Spectral limitations of common organic dyes make it difficult or impossible to visualize and follow multiple biological components in rapidly moving systems. The development of a multispectral set of improved DNA-scaffolded fluorophores is described. Their use in multicolor cellular imaging (see scheme) and in tracking of biological motions on the subsecond timescale is demonstrated.

New details indicated by different stainings during conjugation of ciliated protozoa Paramecium.

During conjugation of Paramecium caudatum, nuclear events occur in a scheduled program. Morphological studies on nuclear behavior during conjugation of P. caudatum have been performed since the end of the 19th century. Here we report on new details concerning the conjugation of P. caudatum through the staining of conjugating cells with protargol, carbol fuchsin solution, Hoechst 33342 and immunofluorescence labeling with monoclonal antibody of anti-α tubulin. 1) The crescent nucleus is a characteristic of the meiotic prophase of P. caudatum, has an unstained area. We stained this area with protargol, which was separated from the chromatin area and was not detected by the other stainings. 2) In regards to the four meiotic products, it has long been considered that only one product enters the paroral cone region (PC) and survives after meiosis. However, our protargol and immunofluorescence labeling results indicated that PC entrance of the meiotic product happened before the completion of meiosis instead of after. 3) In our previous study, protargol staining indicated the presence of a swollen structure around the central part of the "U" and "V" shaped spindles connecting the two types of prospective pronuclei. However, immunofluorescence labeling with anti-α tubulin antibodies gave a different image from protargol. All these observations form the basis for further studies of their molecular mechanisms.

Localization of stationary pronuclei during conjugation of Paramecium as indicated by immunofluorescence staining.

After the third prezygotic division during conjugation of Paramecium caudatum, migratory and stationary pronuclei are produced. The migratory pronuclei remain in the paroral region tightly against the conjugating boundaries; while the stationary pronuclei are located beside the migratory pronuclei. To date, however, it is not clear what causes this close side-by-side localization between migratory and stationary pronuclei. In the current study, immunofluorescence staining with monoclonal antibody of anti-α tubulin indicated that ''U'' or ''V'' shaped spindles connected the migratory and stationary pronuclei during the third prezygotic division. This observation accounts for the close localization between these two types of pronuclei.

Changes in Paramecium caudatum (protozoa) near a switched-on GSM telephone.

The protozoan Paramecium caudatum was examined under normal conditions versus aside a switched-on GSM telephone (900 MHz; 2 Watts). Exposed individuals moved more slowly and more sinuously than usual. Their physiology was affected: they became broader, their cytopharynx appeared broader, their pulse vesicles had difficult in expelling their content outside the cell, their cilia less efficiently moved, and trichocysts became more visible. All these effects might result from some bad functioning or damage of the cellular membrane. The first target of communication electromagnetic waves might thus be the cellular membrane.

Morphological apoptotic characteristics of the post-meiotic micronuclei in Paramecium caudatum.

In a previous study, the apoptotic degeneration of meiotic products outside the paroral region of Paramecium caudatum was indirectly demonstrated by means of "apofluor" staining. In this experiment, conjugating pairs and exconjugants of P. caudatum were stained with either "apofluor" or carbol fuchsin or both to find some direct evidence to demonstrate the apoptotic characteristics of this process. As a result, asynchronous meiotic nuclear degeneration was observed. Furthermore, a number of additional meiotic nuclei were found. Disintegrating/dividing meiotic nuclei outside the paroral region were observed, which might be the origin of these additional meiotic nuclei. Condensed chromatin and disintegrated chromatin attached to the nuclear membrane were also observed in degenerating nuclei, which are the typical morphological characteristics of apoptosis. Comparison of the cells stained by the above two methods indicated that "apofluor"-stained meiotic nuclei could not be detected by carbol fuchsin in some cells, which suggests a time lag between meiotic nuclear DNA degradation and their eventual disappearance. In this study, some direct evidence was found to show that the meiotic nuclear degeneration in P. caudatum is of apoptotic nature, which further confirmed our previous study (Yang et al. 2007) and indicated that morphological apoptotic characteristics discovered in multicellular organisms do exist in unicellular eukaryotic ciliate protozoa.

[Motor activity of infusoria: fundamental and applied aspects].

The article considers morpho-functional organization of the cilia, locomotor organelle of the infusoria, and demonstrates the complicity of locomotor behavior of these protista. The problem of control of locomotion of infusoria is whole organism in discussed; and conclusion is drawn that system of control of movements could be multilevel and include receptor, afferent, central, efferent and effector units. In this context the macronucleus, could act as a central integrator and coordinator of the locomotor behavior being closely connected with periphery by dynamic elements of cytoskeleton. The eradication of infusoria parasitizing in humans and animals by interrupting of locomotion of the protista is also discussed.

[Use of infusoria Paramecium caudatum for the evaluation of the toxicity of antigens of different causative quarantine agents].

The results of the evaluation of the toxicity of bacterial antigens obtained from the causative agents of plaque, glanders, melioidosis, cholera on infusoria of the species P. caudatum, as well as on cell lines L-929, CHO K-1 and peritoneal macrophages of BALB/c mice, are presented. As revealed in this study, the method of toxicity determination on infusoria is similar in its sensitivity to the methods of testing on. CHO K-1 and L-929 cells, but the former is simpler, more available and permits the determination of toxic doses producing disturbances in the vital activity of the infusoria, but not leading to their death.

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum.

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.