Biologically synthesized silver nanoparticles as potent antibacterial effective against multidrug-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most worrisome infectious bacteria due to its intrinsic and acquired resistance against several antibiotics and the recalcitrance of its infections; hence, the development of novel antimicrobials effective against multidrug-resistant P. aeruginosa is mandatory. In this work, silver nanoparticles obtained by green synthesis using a leaf extract and fungi were tested against a battery of clinical strains from cystic fibrosis, pneumonia and burnt patients, some of them with multidrug resistance. Both nanoparticles showed a potent antibacterial effect, causing severe damage to the cell wall, membrane and DNA, and inducing the production of reactive oxygen species. Moreover, the nanoparticles derived from fungi showed synergistic antibacterial effects with the antibiotics meropenem and levofloxacin for some clinical strains and both kinds of nanoparticles were nontoxic for larvae of the moth Galleria mellonella, encouraging further research for their implementation in the treatment of P. aeruginosa infections.

Cementum protein 1 transfection does not lead to ultrastructural changes in nucleolar organization of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Transfection of cementum protein 1 (CEMP1) into human gingival fibroblasts (HGFs) notably increases cell metabolism and results in overexpression of molecules related to biomineralization at transcriptional and protein levels. Therefore, HGF-CEMP1 cells are considered as putative cementoblasts. This represents a significant advance in periodontal research because cementum neoformation is a key event in periodontal regeneration. In addition, it is well known that important changes in cell metabolism and protein expression are related to nucleolar structure and the function of this organelle, which is implicated in ribosome biogenesis. The aim of this study was to determine the effect of transfecting CEMP1 gene in human HGF on the ultrastructure of the nucleolus. MATERIAL AND METHODS: Cells were processed using the conventional technique for transmission electron microscopy, fixed with glutaraldehyde, postfixed with osmium tetraoxide, and embedded in epoxy resin. Semi-thin sections were stained with Toluidine blue and observed by light microscopy. Thin sections were stained with uranyl acetate and lead citrate. For ribonucleoprotein detection, the staining method based on the regressive effect of EDTA was used. In addition, the osmium ammine technique was used for specific staining of DNA. RESULTS: The results obtained in this study suggest that transfection of CEMP1 into HGFs does not produce changes in the general nucleolar ultrastructure because the different components of the organelle are present as fibrillary centers, and dense fibrillar and granular components compared with the control. CONCLUSION: The transfection of CEMP1 into HGFs allows these cells to perform cementoblast-like functions without alteration of the ultrastructure of the nucleolus, evaluated by the presence of the different compartments of this organelle involved in ribosomal biogenesis.

Oocyte structure and ultrastructure in the Mexican silverside fish Chirostoma humboldtianum (Atheriniforme: Atherinopsidae).

The structural and ultrastructural features of gonads from endemic Mexican fish have received scarce attention. This study describes the histological and ultrastructural characteristics of oocyte from Chirostoma humboldtianum. The ovary is asynchronic, and as such, most phases of oocyte development are found in the same ovary. The complete process of oogenesis was divided in five stages: oogonium and folliculogenesis, primary growth, cortical alveoli and lipid inclusions, vitellogenesis, and maturation. The presence of big filaments, which appear at the end of primary growth, induces some common follicular adaptation. During primary growth, abundant ribosomes, the rough endoplasmic reticulum, and mitochondria are grouped in the cytoplasm. At the end of this stage, the Z1 layer of the chorion is developed, while microvilli start to be evident. In the cortical alveoli and lipid droplets phase, intense PAS positive vesicles, some of them containing nucleoid material, are observed in the peripheral cytoplasm and the lipid droplets take a more central position. In vitellogenesis, the proteic yolk accumulates in a centripetal way while the chorion is completely formed. During maturation, the germinal vesicle migrates to the animal pole, meiosis is restored, and there is nuclear breakdown. The oocyte increases its size and holds some oil droplets and a big fluid mass of yolk. On the outside, filaments completely surround the oocyte.

Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis.

We have investigated the distribution of U3 snRNA and rRNA in HeLa cells and normal rat kidney cells during interphase and mitosis. U3 snRNA, known to be involved in pre-rRNA processing, was detected in nucleoli and coiled bodies during interphase, whereas rRNA was distributed in the nucleoli and throughout the cytoplasm. By comparison, ribosomal protein S6 was detected in nucleoli, coiled bodies, and in the cytoplasm. During nucleologenesis, pre-rRNA was observed in newly forming nucleoli during late telophase but not in prenucleolar bodies (PNBs), whereas U3 snRNA was detected in forming nucleoli and PNBs. Similar findings to those reported here for the localization of U3 snRNA have been reported previously for the U3 small nuclear ribonucleoprotein fibrillarin. These results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis. The nucleolus is formed at specific telophase domains (nucleolar organizing regions) and the PNBs, containing factors essential for pre-rRNA processing, are recruited to these sites of rRNA transcription and processing.

In maize, two distinct ribulose 1,5-bisphosphate carboxylase/oxygenase activase transcripts have different day/night patterns of expression.

Several cDNAs encoding ribulose-1,5-bisphosphate carboxylase/oxygenase activase (Rubisco activase, RCA) were isolated from a maize (Zea mays L.) leaf cDNA library. Although all the cDNAs encoded the same polypeptide, the RCA beta isoform, they showed two different downstream-like elements (DST-like) at their 3' untranslated regions (UTRs). The Zmrca1 cDNAs had the subdomain I, and II and the Zmrca2 cDNAs, besides these subdomains, showed two repeats of the subdomain III. The presence of at least two different rca genes in the maize genome was demonstrated by Southern, and by PCR analysis using primers specific for the two cDNAs. Northern analysis with probes specific for each gene showed that the Zmrca2 was expressed as a 1.8 kb transcript, the Zmrca1 corresponded to a 1.4 kb transcript, and a 1 kb band was a stable degradation product of one or both transcripts. Although both mRNAs showed cyclic variations during a day/night period, with their highest levels before dawn, the Zmrca2 transcript showed stronger changes than the Zmrca1 transcript, presenting a twofold larger highest to lowest RNA accumulation ratio than the Zmrca1 transcript, implying that they have different turnover rates. Our results suggest that post-transcriptional mechanisms, mediated by the DST-like element might be involved in the circadian expression of the maize rca transcripts.

Immunolocalization of a human cementoblastoma-conditioned medium-derived protein.

Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum.

Localization of intranuclear RNA by electron microscopy in situ hybridization using a genomic DNA probe.

BACKGROUND: The presence of RNA in the cell nucleus is well known. However, a high resolution in situ hybridization evidence for the presence of RNA in some nuclear particles is still lacking. The aim of this work is to localize RNA in subnuclear particles using a novel ultrastructural in situ hybridization procedure. In this study, biotinylated genomic mouse DNA as a probe to localize total RNA in the nuclei of mouse hepatocytes was used. METHODS: The procedure is based on paraformaldehyde fixation and embedding in lowicryl resin. Thin sections are mounted in formvar-coated gold grids. Hybridization is performed on non-denatured thin sections. DNA-RNA hybrids are detected with streptavidin-10 nm gold particles complex. By controlling the time of nick-translation during incorporation of biotin into the probe, labeling in the fibrillar portions of the nucleoplasm is obtained. More digested probes generate more labeling in the granular components. Nucleoli were similarly labeled. RESULTS: As expected, no label was observed in the compact chromatin clumps. These results indicate that granular components as perichromatin granules in the nucleus contain more processed RNA than fibrillar portions. As a comparison, viral DNA sequences on denatured RNase-treated thin sections of adenovirus-2 (Ad-2)-infected human cells were detected. As previously reported, at late stages DNA was observed in the viral particles and surrounding nucleoplasm, where Ad-2 DNA is synthesized. CONCLUSIONS: The present procedure allows the study of intranuclear RNA distribution and will be useful for the analysis of RNA processing in several types of cells.

Implications for evolution of nuclear structures of animals, plants, fungi and protoctists.

The evolutionary variations of nuclear structure of animals, plants, fungi and protoctists were studied with electron microscopy by using techniques preferentially staining ribonucleoprotein (RNP) particles and chromatin. A remarkable similarity in the general morphological features of the RNP particles and chromatin arrangement is found in animals, plants and fungi. Important variations of these features were found in protoctists. These observations suggest that major evolutionary changes in the nuclear structure predate the acquisition of plastids by the ancestors of green plants. Once evolved, the nuclear structural pattern is conserved in plants and animals. Among protoctists studied, Kinetoplastida, Cryptomonadida and Volvocida have RNP particles and chromatin arrangement resembling those of plants and animals. These similarities may indicate a common ancestor. Important differences in the nuclear structure among Euglenida, Amebida, Cryptomonadida, Volvocida and Kinetoplastida support the view that Sarcomastigophora is a polyphyletic taxon. For the same reason Kinetoplastida and Euglenida must not be grouped in a monophyletic taxon. We propose that the variations of RNP particles may be related to the initial evolution of post-transcriptional processing.

Kinetics and characterization of cellular responses in the peritoneal cavity of mice infected with Taenia crassiceps.

Changes in the leukocyte population of the peritoneal cavity ensue immediately after infection with Taenia crassiceps metacestodes. Basophils and neutrophils decrease, whereas macrophages, monocytes, and lymphocytes increase to reach only modest levels by 6 wk and then diminish to nearly disappear by 15 wk when the parasite begins rapid reproduction. Eosinophils also appear early in infection, but then abate to lower levels that persist. In late infections, when the mass of cysticerci equals that of the mouse, the cysticerci grow among surprisingly few inflammatory cells. Mingling with the peritoneal inflammatory cells is a number of odd-looking cells that could correspond to the metaplasic mesothelial cells of the host or be of parasite origin. These cells are multinucleated, they aggregate in varigerated clusters, and form cystic structures in vitro; they also bind specific anti-T. crassiceps antibodies and specific T. crassiceps DNA probes in their nuclei. When the peritoneal cell exudate is reinjected intraperitoneally into naive mice, the odd-looking cells subsist for months, inducing in the host the synthesis of specific anti-T. crassiceps antibodies and immune resistance to challenge but do not reassemble into cysticerci even after 6 mo of inoculation. The early appearance and the immunogenic and antigenic properties of these odd-looking cells suggest they are important protagonists in the early host-parasite confrontation when the outcome of infection is set.

In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism.

We describe the nuclear organization of pre-mRNA processing components in HeLa cells upon adenovirus 2 infection and their relationship to the localization of viral RNA sequences. We observe a redistribution of cellular splicing factors as well as RNA polymerase II and heterogeneous nuclear ribonucleoprotein particle proteins to sites of viral RNA transcription. Similar results were obtained in cells transiently transfected with a plasmid containing a portion of the beta-tropomyosin gene. Our findings demonstrate a very close association between RNA transcripts and transcription and pre-mRNA splicing factors, suggesting that these processes are both temporally and spatially linked in the cell nucleus. Furthermore, these data suggest a recruiting mechanism that regulates the localization of transcription and splicing factors in response to the initiation of active transcription.

New evidence that Lacandonia granules are ultrastructurally related to perichromatin and Balbiani ring granules.

Lacandonia granules are abundant non-typical extranucleolar ribonucleoprotein particles found in the nucleus of Lacandonia schismatica, a rare plant showing spatial inversion of sex organs. In the present study, changes in the number of Lacandonia granules during flower development, and the presence of SR proteins and poly(A)+ RNA in the nuclei of L. schismatica were analyzed by electron microscopy, immunoelectron microscopy and ultrastructural in situ hybridization. Our results show an important reduction in the number of Lacandonia granules in the nuclei of cells of opened (post-anthesis) in relation to unopened (pre-anthesis) flowers, where granules are very abundant. The SR family of splicing factors and poly(A)+ RNA are present in both perichromatin fibers and Lacandonia granules. The developmental behavior, the presence of SR proteins, recently involved in post-splicing events, poly(A)+ RNA and the reported absence of snRNPs splicing factors in Lacandonia granules, suggest that these particles are involved in postranscriptional events as storage and/or transport of mRNAs. A similar situation is present in other nuclear RNP as perichromatin granules present in mammals and Balbiani ring granules of salivary glands of Chironomus. Based on similarities in morphological, developmental behavior, immunocytochemistry and in situ hybridization results, we conclude that Lacandonia, perichromatin and Balbiani ring granules may be also functionally similar structures.

Atomic force microscopy of the cell nucleus.

In mammals and plants, the cell nucleus is organized in dynamic macromolecular domains involved in DNA and RNA metabolism. These domains can be visualized by light and electron microscopy and their composition analyzed by using several cytochemical approaches. They are composed of chromatin or ribonucleoprotein structures as interchromatin and perichromatin fibers and granules, coiled bodies, and nuclear bodies. In plants, DNA arrangement defines chromocentric and reticulated nuclei. We used atomic force microscopy to study the in situ structure of the plant cell nucleus. Samples of the plants Lacandonia schismatica and Ginkgo biloba were prepared as for electron microscopy and unstained semithin sections were mounted on glass slides. For comparison, we also examined entire normal rat kidney cells using the same approach. Samples were scanned with an atomic force microscope working in contact mode. Recognizable images of the nuclear envelope, pores, chromatin, and nucleolus were observed. Reticulated chromatin was observed in L. schismatica. Different textures in the nucleolus of G. biloba were also observed, suggesting the presence of nucleolar subcompartments. The observation of nuclear structure in situ with the atomic force microscope offers a new approach for the analysis of this organelle at high resolution.

Cellular source of collagenase and TIMP-1 in carrageenin-induced granuloma.

We have examined the occurrence and cellular localization of interstitial collagenase and TIMP-1 mRNAs in a model of granuloma induced by carrageenin in guinea pigs. Granulomas were studied at 4, 7, 10, and 14 days after carrageenin injury using a combined protocol for in situ hybridization and immunofluorescence. Anti-vimentin monoclonal antibody was used to identify fibroblasts. Avidin-FITC and Texas red horse antimouse IgG were employed for detection of probes and antibody, respectively. Our results showed that during the extracellular matrix deposit phase (4 and 7 days), interstitial collagenase and TIMP-1 mRNAs were expressed only by fibroblasts as demonstrated by the colocalization of mRNA and vimentin. By contrast, during the initiation of the resorptive phase (10 and 14 days), fibroblasts and vimentin-negative cells, probably macrophages, expressed collagenase and TIMP-1. This study suggests that fibroblasts are the cell type expressing interstitial collagenase and TIMP-1 mRNA during all phases of the evolution of carrageenin granuloma and that macrophages, by contrast, express the mRNA for the enzyme and the inhibitor exclusively in the degradative phase.

Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells.

We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2)-infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with alpha-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10-14 hours post-infection as fine dots in the nucleoplasm. By 18-24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.

Nucleologenesis: use of non-isotopic in situ hybridization and immunocytochemistry to compare the localization of rDNA and nucleolar proteins during mitosis.

Using in situ hybridization and immunocytochemistry during interphase and mitosis, we have compared the distribution of ribosomal DNA (rDNA) to that of the nucleolar proteins fibrillarin and RNA polymerase I. During interphase, nucleolar proteins were localized at sites throughout the nucleolus while the bulk of rDNA was localized in a single restricted nucleolar area. During metaphase and anaphase, all six NORs were detected by in situ hybridization, Ag-staining, or by the immunolocalization of RNA polymerase I. During telophase, rDNA and RNA polymerase I were found in a distinct subset of the prenucleolar bodies (PNBs) which obviously must contain the nucleolar organizers. Other numerous PNBs are smaller in size and do not contain detectable amounts of rDNA or RNA polymerase I. Therefore, reconstruction of the nucleolus originates in telophase-specific domains which contain both rDNA and RNA polymerase I.

Cellular organization of pre-mRNA splicing factors in several tissues. Changes in the uterus by hormone action.

In the mammalian cell nucleus, splicing factors are distributed in nuclear domains known as speckles or splicing factor compartments (SFCs). In cultured cells, these domains are dynamic and reflect transcriptional and splicing activities. We used immunofluorescence and confocal microscopy to monitor whether splicing factors in differentiated cells display similar features. Speckled patterns are observed in rat hepatocytes, beta-cells, bronchial and intestine epithelia and also in three cell types of the uterus. Moreover, the number, distribution and sizes of the speckles vary among them. In addition, we studied variations in the circular form (shape) of speckles in uterine cells that are transcriptionally modified by a hormone action. During proestrus of the estral cycle, speckles are irregular in shape while in diestrus I they are circular. Experimentally, in castrated rats luminal epithelial cells show a pattern where speckles are dramatically rounded, but they recover their irregular shape rapidly after an injection of estradiol. The same results were observed in muscle and gland epithelial cells of the uterus. We concluded that different speckled patterns are present in various cells types in differentiated tissues and that these patterns change in the uterus depending upon the presence or absence of hormones such as estradiol.

Cytochemical and immunocytochemical study of nuclear structures of Lacandonia schismatica.

The interphase nucleus of the cells of several tissues of Lacandonia schismatica was studied using electron microscopy cytochemical and immunocytochemical methods. The EDTA staining procedure, preferential for RNP, contrasted the Lacandonia granules and perichromatin fibrils. These granules were found to be relatively resistant to RNAse hydrolysis, but they were easily digested if RNAse treatment was carried out after a short hydrolysis with pronase. Bismuth oxynitrate stained granular structures about 17 nm in diameter and the periphery of a few Lacandonia granules. The anti-snURNPs bound to RNP-containing fibrils in the perichromatin and interchromatin space and also to the periphery of some Lacandonia granules. Immunolabeling of DNA demonstrated numerous filaments of extended chromatin in the perichromatin and interchromatin spaces which were closely related to Lacandonia granules. These observations suggested that Lacandonia granules are equivalent to Balbiani ring granules of nuclei with polytene chromosomes and to perichromatin granules of other plant and animal nuclei. The small number of Lacandonia granules labeled in their periphery by anti-snURNP mAb were interpreted as being immature granules in the process of formation. The external or annular part of the ring-shaped structures is heavily labeled by anti-URNP mAbs but scarcely stained by the EDTA procedure. These features indicate that this region contains abundant proteins associated with snURNAs but probably little snURNAs. The synaptonemal-like complexes previously found in the interphase nuclei of Lacandonia are formed by two parallel masses of compact chromatin, which react with anti-DNA, and a central clear space crossed by fiber.(ABSTRACT TRUNCATED AT 250 WORDS)

Relations between nucleolar morphometric parameters and pre-rRNA synthesis in animal and plant cells.

In order to study if there are differences between cells of the same tissue with one and two nucleoli, nuclear and nucleolar volume, density of tritiated uridine incorporation, amount of DNA per nucleus and intensity of cytoplasmic basophilia were measured in mononucleolated and binucleolated rat epithelial endometrial cells, in onion root meristematic cells and in chick embryo matrix cells of the central nervous system, neuroblasts and neurons. No significant differences in nuclear volume, density of tritiated uridine incorporation and amount of DNA per nucleus were found between cells of the same type with diverse numbers of nucleoli. Binucleolated endometrial cells, matrix cells, and root meristematic cells have biphasic distributions of nucleolar volumes. One peak of this distribution roughly coincides with the nucleolar volume of mononucleolated cells, the other peak corresponds almost to double the volume. As the density of uridine incorporation is the same irrespective of the nucleolar number and volume, the cells with larger nucleolar volumes have higher pre-rRNA synthesis. These cells also have higher amounts of ribosomes in the cytoplasm, as revealed by the photometric study of basophilia. It is concluded that in this population of cells the ribosomal production is regulated to a higher steady equilibrium than in the general population. This difference is not due to polyploidism or to the increased DNA content of G2 phase cells. Binucleolated neuroblasts and neurons have nucleolar volumes similar to those of mononucleolated ones.

Changes in nuclear ribonucleoprotein constituents and chromatin disposition during neuronal differentiation and maturation.

Morphological and quantitative changes of ribonucleoproteic (RNP) structures and chromatin are studied in the nuclei of undifferentiated cells, neuroblasts and neurons in several degrees of maturation, in order to relate them to the drastic modifications in transcription and/or RNA processing taking place during cell differentiation. Undifferentiated (matrix) cells of 2-day embryos differ from that of 4-day embryos in their nucleolar volume and in the amount of compact chromatin. These differences are interpreted as the earliest signs of neuroblast differentiation. All over the process of matrix cell-bipolar-multipolar neuroblast differentiation there is a large spreading of compact chromatin well before any important change in RNP structures or in nuclear volume. The most remarkable increase in nuclear volume and in the amount of RNP particles occurs during the differentiation of multipolar neuroblasts to immature neurons, which is characterized by large synaptogenic activity. The interpretation of these changes is discussed in connection, on one hand with the metabolic effects of synapses, and on the other hand with the variations of gene expression taking place in cell differentiation and under other natural and experimental situations.