Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure

Nuclear organelles, unlike many cytoplasmic organelles, lack investing membranes and are thus in direct contact with the surrounding nucleoplasm. Because the properties of the nucleoplasm and nuclear organelles influence the exchange of molecules from one compartment to another, it is important to understand their physical structure. We studied the density of the nucleoplasm and the density and permeability of nucleoli, Cajal bodies (CBs), and speckles in the Xenopus oocyte nucleus or germinal vesicle (GV). Refractive indices were measured by interferometry within intact GVs isolated in oil. The refractive indices were used to estimate protein concentrations for nucleoplasm (0.106 g/cm3), CBs (0.136 g/cm3), speckles (0.162 g/cm3), and the dense fibrillar region of nucleoli (0.215 g/cm3). We determined similar protein concentrations for nuclear organelles isolated in aqueous media, where they are no longer surrounded by nucleoplasm. To examine the permeability of nuclear organelles, we injected fluorescent dextrans of various molecular masses (3-2000 kDa) into the cytoplasm or directly into the GV and measured the extent to which they penetrated the organelles. Together, the interferometry and dextran penetration data show that organelles in the Xenopus GV have a low-density, sponge-like structure that provides access to macromolecules from the nucleoplasm.     

Synaptonemal complexes,telomeric nucleoli and the karyosphere in achiasmatic oocyte meiosis of the carob moth

Synaptonemal complexes and telomeric nucleoli are involved in the spatial organization and regular distribution of homologous chromosomes in meiosis of the achiasmatic female carob moth. The bivalents are held together from zygotene to metaphase by the Synaptonemal complexes. These are attached to telomeric nucleoli which appear during early meiotic prophase and are unique to the oocyte. The telomeric nucleoli fuse during prophase and the chromosomes concentrate into a small karyosphere before prometaphase. During the final stages of prophase elements of the Synaptonemal complex are found in the periphery of the fibrillar region of the telomeric nucleoli.     

Use of bacteria for rapid,pH-neutral,hydrolysis of the model hydrophobic carboxylic acid ester p-nitrophenyl picolinate

Abstract

Caulobacter crescentus, Escherichia coli and Bacillus subtilis cultures promote the hydrolysis of the model ester p-nitrophenyl picolinate (PNPP) at neutral pH with high efficiency. Hydrolysis is related to cell concentration, while the interaction of PNPP with both bacterial cells and their extracellular molecules is required for a maximum rate of PNPP hydrolysis in C. crescentus cultures. Furthermore, C. crescentus cultures hydrolyse PNPP at concentrations useful in synthetic chemistry.     

Asymmetric distribution of metals in the Xenopus laevis oocyte: a synchrotron X-ray fluorescence microprobe study.

The asymmetric distribution of many components of the Xenopus oocyte, including RNA, proteins, and pigment, provides a framework for cellular specialization during development. During maturation, Xenopus oocytes also acquire metals needed for development, but apart from zinc, little is known about their distribution. Synchrotron X-ray fluorescence microprobe was used to map iron, copper, and zinc and the metalloid selenium in a whole oocyte. Iron, zinc, and copper were asymmetrically distributed in the cytoplasm, while selenium and copper were more abundant in the nucleus. A zone of high copper and zinc was seen in the animal pole cytoplasm. Iron was also concentrated in the animal pole but did not colocalize with zinc, copper, or pigment accumulations. This asymmetry of metal deposition may be important for normal development. Synchrotron X-ray fluorescence microprobe will be a useful tool to examine how metals accumulate and redistribute during fertilization and embryonic development.     

Separate groups in the oocyte population of cows, their cytogenetic analysis and capacity for in vitro maturation

Heterogenic population of the follicular oocytes of bovine ovaries has been divided into 5 groups on the basis of morphological characteristics. Chromatin structure, frequency of degenerative changes and the ability to maturation in vitro of different oocyte groups have been studied. The correlation of morphological characteristics of native oocytes and their ability to mature in vitro have been shown. The bovine follicular oocyte classification allows to pick out more homogeneous oocyte groups in comparison with the population as a whole and the oocyte group which is the best for maturation in vitro.     

Xenopus laevis oocyte as a model for the study of the cytoskeleton

At the beginning of diplotene, the oocyte of Xenopus laevis is a cell of about 10–20 microns destined to increase 10,000-fold its size when the oocyte becomes filled with yolk platelets and has accumulated a great number of pigment granules in a half of its periphery. Its internal architecture is gradually accomplished during growth because of several factors, especially because of cytoskeletal changes. In the fully-grown oocyte, the cytoskeleton appears to sustain the eccentrically located germinal vesicle through arms radiating from the cortex to the germinal vesicle, a unique organization not to be found in other Amphibians. In this report, we summarized and analysed steps of cytoskeletal proteins and related mRNAs organization and function throughout diplotene stage, highlighting our studies in this animal model. The cytoskeletal proteins appear to exploit their activity with respect to ribosomal 60S subunit maturation and during translation. Most importantly, the polarity of the oocyte is achieved through a sophisticated and highly organized localization of mRNAs and cytoskeletal proteins in one side of the cell. This asymmetry will start the construction of the oocyte polarity that is instrumental for determining the characteristic of this cell, which will become an embryo. Moreover, in the same time membrane composition, conditioned by the underlying cytoskeletal organization, will acquire the prerequisites for sperm binding and fusion.     

Use of a lectin affinity selector in the search for unusual glycosylation in proteomics

The purpose of the work described in this paper was to develop a new approach to the identification of glycoprotein with particular types of glycosylation. The paper demonstrates N-glycosylation sites in a glycoproteins can be identified by (1) proteolysis with trypsin, (2) lectin affinity selection, (3) enzymatic deglycosylation with peptide-N-glycosidase F (PNGase F) in buffer containing 95% H(2)(18)O, which generates deglycosylated peptide pairs separated by 2 or 4 amu, (4) reversed-phase separation of the peptide mixture and MALDI mass analysis, (5) MS-MS sequencing of the ion pairs, and (6) identification of the parent protein through a database search. This process has been tested on the selection of glycopeptides from lactoferrin and mammaglobin, and the identification of the ion pairs of fetuin glycopeptides. Glycosylation sites were identified through PNGase hydrolysis in H(2)(18)O. During the process of hydrolyzing the conjugate, Asn is converted to an aspartate residue with the incorporation of (18)O. However, PNGase F was observed to incorporate two (18)O into the beta-carboxyl groups of the Asp residue. This suggests that the hydrolysis is at least partially reversible.     

Use of nile red as a fluorescent probe for the study of the hydrophobic properties of protein-sodium dodecyl sulfate complexes in solution.

Our results show that the noncovalent dye 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) can be used as a fluorescent probe to study the hydrophobic properties of proteins associated with the anionic detergent sodium dodecyl sulfate (SDS). Nile red can interact with both SDS micelles and protein-SDS complexes. The enhancement of Nile red fluorescence observed with diverse types of proteins occurs at SDS concentrations lower than the critical micelle concentration of this detergent. This is also observed using the covalent fluorophore rhodamine B isothiocyanate. Additional results obtained in studies in solution show that the fluorescence intensity and the spectral characteristics of Nile red associated with different proteins complexed with SDS are very similar. These spectroscopic similarities are probably related to the equivalent synchrotron X-ray scattering results found for various protein-SDS complexes in solution. The scattering results suggest that SDS induces the formation of complexes in which the basic structural properties are independent of the different initial structures of native proteins. We speculate that Nile red is bound to regions with equivalent hydrophobic characteristics located in the uniform structures produced by the association of SDS with proteins.     

Fluorescamine, a fluorescence probe for amino groups in histochemical studies of plant cells and the effect of mercury fixation

Synopsis When fixed in mercuric chloride solutions and stained with Fluorescamine, histological plant specimens emit a strong fluorescence. The fluorophore distribution is topologically identical to the staining pattern revealed by visible light methods for nucleoproteins, but the fluorescence mode of viewing preparations gave greater sensitivity and contrast than transmitted light absorption methods. The parameters that influence the formation of the fluorescent image in plant cells are discussed. The results obtained indicate that the mercury-Fluorescamine reaction is an ideal histochemical procedure for collecting qualitative and analytical information on plant nuclei and on the changes of nucleolar architecture that occur during the cellular developmental cycle.     

Use of a fluorescence spectroscopy technique to study the adsorption of sodium dodecylsulfonate on liposomes

The fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS) was used to study the surface adsorption of sublytic concentrations of the anionic surfactant sodium dodecylsulfonate (C(12)-SO(3)) on phosphatidylcholine (PC) bilayers. The number of adsorbed molecules was quantified by determination of the electrostatic potential (psi(o)) of the bilayers. The abrupt decrease in the fluorescence intensity detected even 10 s after the surfactant addition and the slight fluorescence variations with time indicated that the surfactant adsorption was very fast and almost complete. For a given number of monomers adsorbed a linear dependence between the lipid and C12-SO3 concentrations was obtained, indicating similar adsorption mechanism regardless of the surfactant concentration. Hence, a monomeric adsorption is assumed even in systems with a C12-SO3 concentration above its CMC. In addition, this linear correlation allowed us to determine the surfactant/lipid molar ratios (Re) (inversely related to the C12-SO3 ability to be adsorbed on liposomes) and the bilayer/aqueous phase coefficients (K). The fact that the lowest values for Re were always reached after 10 s of incubation corroborates the rapid kinetics of the process. The decrease in the C12-SO3 partitioning (K) when the number of surfactant molecules exceeded 15000 was possibly due to the electrostatic repulsion between the free and the adsorbed monomers, which could hinder the incorporation of new monomers on the charged surface of liposomes.     

Synthesis of 3-(12-hydroxy-p-carboranyl)propionic acid, a hydrophobic, N-terminal tyrosine-mimetic for peptides

A new, p-carborane containing analog of tyrosine, namely 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran(12)-12-yl]propionic acid was prepared from p-carborane in five steps involving hydroxypropylation of O-protected 1-hydroxy-p-carborane as the key transformation. The simple tyrosine mimetic can function as a hydrophobic surrogate for an N-terminal tyrosine residue in insect and mammalian neuropeptides to enhance the lipophilicity, and therefore, the cuticle and/or tissue permeability properties of mimetic analogs.     

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO(3)(-)-N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO(3)(-)-N mg of MLVSS(-1) h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [(13)C]methanol to biomark the DNA of the denitrifiers. The extracted [(13)C]DNA and [(12)C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [(13)C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [(12)C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [(14)C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

Accessibilities of the sulfhydryl groups of native and photooxidized lens crystallins: a fluorescence lifetime and quenching study

Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq, values were 16.6 x 10(7), 26.9 x 10(7), and 32.7 x 10(7) M-1 s-1 for alpha-, beta H-, and gamma-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kq value for acrylamide quenching of the major decay component of alpha-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kq of alpha-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) label in irradiated alpha-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated alpha-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of beta H-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kq for acrylamide quenching of the major component of the decay of AEDANS bound to beta H- or gamma-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated alpha-crystallin and of untreated and irradiated beta H- and gamma-crystallins, but it had no detectable role in the case of untreated alpha-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.     

Cellular localization of tolyporphins,unusual tetrapyrroles,in a microbial photosynthetic community determined using hyperspectral confocal fluorescence microscopy

Photosynthesis Research - The cyanobacterial culture HT-58-2, composed of a filamentous cyanobacterium and accompanying community bacteria, produces chlorophyll a as well as the tetrapyrrole...     

Use of actinomycin D for the specific quenching of fluorescence of deoxyribonucleic acid in cells stained with acridine aminoderivatives.

Actinomycin D specifically quenches the fluorescence of acridine orange and quinacrine bound to deoxyribonucleic acid in cytologic preparations, but does not change the fluorescence of these fluorochromes bound to RNA. The following fluorescence-cytochemical applications of techniques based on these findings can be suggested: (a) distinction between deoxyribonucleic acid and ribonucleic acid; (b) detection of double-stranded virus ribonucleic acid; (c) approximate estimation of the lengths of A-T sequences in deoxyribonucleic acid molecules.