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PubMed2006

Trypanosoma cruzi: clones isolated from the Colombian strain, reproduce the parental strain characteristics, with ubiquitous histotropism.

Clonal histotropism and biological characters of five clones isolated during the early acute phase of the infection of Swiss mice with the Colombian strain of Trypanosoma cruzi (T. cruzi I), Biodeme Type III, were investigated. Clones were isolated from mice at the 10th and the 30th day of infection with the Colombian strain. Isolation was performed by micromanipulation and injection of one trypomatigote blood form into newborn mice, followed by passages into suckling mice for obtaining the inocula for the experimental groups. Mice infected with parental strain were also studied. All the clones have shown the basic characteristics of Biodeme Type III, with the same patterns of parasitemia, tissue tropism, morphological characters and isoenzymic profiles, such as the parental strain. Histotropism was most intense to myocardium and skeletal muscles, with intense lesions found in the advanced phase (20th to 30th day of infection). Both parental strain and the clones were seen to parasitize several organs and tissues; amastigote nests were identified in the cytoplasm of macrophages, adipose cells, smooth muscle of intestinal wall and Auerbach's neuronal plexus. The findings of the present study confirm the homology of the clones isolated from the Colombian strain, with predominance of a 'principal clone' and an ubiquitous distribution of parasites belonging to a same clone.

Animals↗
PubMed2004

Genetic segregation of natural Saccharomyces cerevisiae strains derived from spontaneous fermentation of Aglianico wine.

AIMS: Investigation of the meiotic segregation of karyotypes and physiological traits in indigenous Saccharomyces strains isolated from Aglianico (South Italy) red wine. METHODS AND RESULTS: Segregation was studied in F1 and F2 descendants. Tetrads were isolated from sporulating cultures by micromanipulation. The spore clones were subjected to karyotype analysis by pulse-field gel electrophoresis (Bio-Rad model CHEF-DR II) and to various physiological tests. Certain chromosomes of the isolates showed 2:2 segregation patterns in F1 but proved to be stable in F2. The ability of cells to utilize maltose also segregated in a 2 : 2 manner in F1 and did not segregate in F2. Resistance to CuSO4, SO2 tolerance, the fermentative power and the production of certain metabolites segregated in both F1 and F2 generations and showed patterns indicating the involvement of polygenic regulation. CONCLUSIONS: The analysis revealed a high degree of genetic instability and demonstrated that meiosis can improve chromosomal and genetic stability. SIGNIFICANCE AND IMPACT OF THE STUDY: Winemaking is critically dependent on the physiological properties and genetic stability of the fermenting Saccharomyces yeasts. Selection of clones from F2 or later generations can be a method of reduction of genetic instability.

Chromosomes, Fungal↗
PubMed2006

Thermostatic tissue platform for intravital microscopy: 'the hanging drop' model.

Intravital microscopy imposes the particular problem of the combined control of the body temperature of the animal and the local temperature of the observed organ or tissues. We constructed and tested, in the rat ileum microcirculation preparation, a new organ-support platform. The platform consisted of an organ bath filled with physiological solution, and contained a suction tube, a superfusion tube, an intestine-support hand that was attached to a micromanipulator and a thermometer probe. To cover the intestine we used a cover glass plate with a plastic ring glued on its upper surface. After a routine procedure (anaesthesia, monitoring and surgery), the intestine segment (2-3 cm long) was gently exteriorized and placed on the 'hand' of the organ support. A small part of the intestine formed a small 'island' in the bath that was filled with physiological salt solution. The cover glass was secured in place. The physiological salt solution from the superfusion tube, which was pointed to the lower surface of the cover glass, formed a 'hanging drop'. The objective of the microscope was then immersed into distilled water that was formed by the cover glass plastic ring. The 'hanging drop' technique prevented any tissue quenching, ensured undisturbed microcirculation, provided for stable temperature and humidity, and permitted a clear visual field.

Animals↗
PubMed2005

Nuclear fusion occurs during mating in Candida albicans and is dependent on the KAR3 gene.

It is now well established that mating can occur between diploid a and alpha cells of Candida albicans. There is, however, controversy over when, and with what efficiency, nuclear fusion follows cell fusion to create stable tetraploid a/alpha cells. In this study, we have analysed the mating process between C. albicans strains using both cytological and genetic approaches. Using strains derived from SC5314, we used a number of techniques, including time-lapse microscopy, to demonstrate that efficient nuclear fusion occurs in the zygote before formation of the first daughter cell. Consistent with these observations, zygotes micromanipulated from mating mixes gave rise to mononuclear tetraploid cells, even when no selection for successful mating was applied to them. Mating between different clinical isolates of C. albicans revealed that while all isolates could undergo nuclear fusion, the efficiency of nuclear fusion varied in different crosses. We also show that nuclear fusion in C. albicans requires the Kar3 microtubule motor protein. Deletion of the CaKAR3 gene from both mating partners had little or no effect on zygote formation but reduced the formation of stable tetraploids more than 600-fold, as determined by quantitative mating assays. These findings demonstrate that nuclear fusion is an active process that can occur in C. albicans at high frequency to produce stable, mononucleate mating products.

Base Sequence↗
PubMed1979

Morphological study of autologous rosettes (A-RFC).

The morphology of autologous rosette forming cells (A-RFC) has been studied by light and electron microscopy. Autorosette forming cells were obtained from the spleen of adult thymectomized (ATx) or nude mice, and from neonatal thymus grafts during the regeneration phase. Among the autorosettes seen by light microscopy, 76% of them were formed by lymphoid cells which included a high proportion of large lymphocytes (48%). Electron microscopy study performed after isolation of A-RFC by micromanipulation demonstrated that 74% were formed by large lymphoid cells with an immature nucleus. The finding of such large-sized lymphoid cells with immature nuclei is in agreement with our previous results indicating that A-RFC might belong to an immature T cell class.

Animals↗
PubMed1984

Failure to distinguish ultrastructurally between T4+ (helper) and T8+ (suppressor/cytotoxic) T-cell subsets.

Human peripheral T-cell subpopulations revealed by monoclonal antibodies by means of a rosetting method were isolated by micromanipulation and submitted to electron microscopic analysis. The T3+ subset (total T cells) displayed a high degree of heterogeneity, including multiple transitional forms, from cells with a high nuclear to cytoplasmic ratio and rare organelles to cells with a low nuclear to cytoplasmic ratio and a complex system of cytoplasmic organelles. T4+ (inducer/helper) and T8+ (suppressor/cytotoxic) cell subpopulations were shown to have no evident distinguishing characteristics. They both displayed the same morphological variation mentioned for T3+ lymphocytes. On morphometric analysis, these two cell subsets were very similar, with only slight differences for cell surface roughness, volume of mitochondria, extent of nuclear indentation, and surface area of the rough endoplasmic reticulum. The significance of these minor morphological differences is discussed.

Animals↗
PubMed1987

Embryonic kidney in organ culture.

Organotypic cultures can be used for cultivating embryonic kidney rudiments either intact or dissociated into two interacting tissue components. This allows micromanipulations that would hardly be possible in vivo. Especially beneficial is the transfilter technique in which the interacting components are cultured on the opposite sides of a porous filter membrane. This model system allows precise timing of inductive interaction and temporal correlation of various developmental events. On the other hand, the manipulations and the in vitro conditions seem to cause metabolic changes, and long-term cultivation will result in damage to the tissues due to insufficient nutrition. Induced mesenchymal cells cultivated in monolayer cultures behave differently from those in three-dimensional organotypic explants: in the former, differentiation is largely impaired whereas in the latter, development closely resembles that in vivo. Although an organ culture system can never fully mimic embryogenesis in vivo, it offers advantages over cell cultures.

Animals↗
PubMed1984

Assay of ATP synthesis using single giant mitochondria.

Two assays of the capacity of single giant mitochondria or mitoplasts to phosphorylate ATP from Pi and ADP have been developed. One depends on the placement of a single mitochondrion next to a glycerinated myofibril, by micromanipulation. With the appropriate controls, contraction of the myofibril serves as an indication of ATP synthesis. The other assay similarly requires the isolation of one single mitochondrion but the assay of ATP synthesized uses the luciferin-luciferase reaction with a conventional photometric system. With the latter, we found that either impalement with microelectrodes or electrophoretic microinjection of two dyes, Lucifer Yellow CH or pyranine , into the inner space have no effect on the phosphorylative capacity of mitochondria or mitoplasts. The electric potential across the mitochondrial membrane was monitored during the assay and found to be small and generally positive inside.

Adenosine Triphosphate↗
PubMed1985

Protonmotive force and photophosphorylation in single swollen thylakoid vesicles.

Swollen vesicles generally 40 micron in diameter were prepared from spinach chloroplasts. These vesicles appear to originate from thylakoids. The present study reports results obtained with individual vesicles using micromanipulative procedures. The electric potential across the membrane was measured with microelectrodes and the pH of the internal space was calculated from the fluorescence of the pH indicator pyranine. The individual vesicles photophosphorylate as measured with luciferin-luciferase. Impalement with microelectrodes did not affect the ability of individual vesicles to photophosphorylate. However, there was no significant membrane potential either with continuous illumination or light flashes. In contrast, we found a delta pH of 3.7 under photophosphorylative conditions and the incubation with the appropriate buffers blocked photophosphorylation presumably by preventing formation of a pH gradient. We propose that, in these vesicles, the membrane potential plays no role in photophosphorylation, whereas a pH gradient is obligatory.

Adenosine Triphosphate↗
PubMedDate not supplied

Direct measurement of the tail beat frequency of human sperm by flash light synchronization.

Based on the synchronization between a periodic vibration and flashing light, we designed a device to determine the flagellar beating frequency of human sperm. The head of a spermatozoon was either held by the tip of a micropipette operated with a micromanipulator or adhered by itself on the surface of a glass slide when the sperm swam out of the micropipette into the fresh Ham's F-10 medium. The beat frequency of the flagellum was measured by synchronization of the frequency-adjustable flash light built on an inverted microscope. The light frequency synchronizer included a controller, a pulse generator, a signal counter, and a flash illuminator. During each measurement, the spermatozoon was transferred to the center of the observing field and the frequency generator created a series of signals which developed flashing signals onto the sample plane. When the vibration of the flagellum was observed as a constant two-step-like movement after frequency adjustment, the beating frequency was read from the signal counter and the count was twice that of the beat frequency of the sperm tail. As the flash signal was decreased to half of this frequency, an apparently immobilized sperm flagellum was observed and the exact beating frequency could then be determined. This device was then used to measure the effect of pentoxifylline on sperm motility. The results showed that the increase of tail beat frequency as measured by this device is well correlated with the increase of beat cross frequency as detected by a computer-assisted semen analyzer. These findings suggest that this flash light synchronization device is a reliable and useful system for the assessment of sperm motility.

Humans↗
PubMed2005

Frequent genetic recombination in natural populations of the marine cyanobacterium Microcoleus chthonoplastes.

A culture-independent method for multilocus sequence typing of Microcoleus chthonoplastes was developed based on mechanical separation of individual cyanobacterial filaments from natural microbial mat populations through micromanipulation, subsequent polymerase chain reaction (PCR) amplification and sequence analysis of three genetic loci (kaiC, petB/D, rDNA-ITS). Among 81 individuals sampled from intertidal sand flats of the North Sea and Baltic Sea, we found 8-14 different sequences (alleles) per genetic locus, resulting in 36 distinct genotypes with unique allele profiles. Non-congruent phylogenetic gene trees for the three loci analysed and split decomposition analysis indicated the occurrence of horizontal genetic exchange. The index of association determined for the entire population was 0.096, indicating that recombination occurs frequently enough to cause almost random association (linkage equilibrium) among alleles. Analysing individuals from three different locations in the North Sea and Baltic Sea, we did not find evidence for geographic subdivisions between populations.

Alleles↗
PubMed2005

DNA identification of fetal cells isolated from cervical mucus: potential for early non-invasive prenatal diagnosis.

OBJECTIVES: To develop a reliable method to isolate fetal cells for genetic diagnosis. DESIGN: Aspiration of cervical mucus from pregnant women in the first trimester. SETTING: Pregnant women were recruited before an elective termination of pregnancy. POPULATION: Sixty pregnant women (7-10 weeks of gestation). METHODS: Fetal cells were isolated from aspirated cervical mucus of pregnant women using a combination of enzymatic digestion, fluorescent immunohistochemistry, micromanipulation and single-cell DNA allelic profiling. MAIN OUTCOME MEASURES: The isolation and identification of fetal cells. RESULTS: The transformation of the tenacious cervical mucus into a single-cell suspension enabled the isolation and identification of fetal cells by fluorescent immunohistochemistry. Confirmation of fetal origin was accomplished by single-cell DNA allelic profiling alongside known maternal cells. CONCLUSIONS: This novel non-invasive method is rapid and efficient with results attainable within 24 hours as early as seven weeks of gestation. The technique would offer earlier reassurance and the option of first trimester therapeutic abortions to both high and low risk pregnant women.

Cervix Mucus↗
PubMed1996

The opportunistic pathogen Nocardia farcinica is a foam-producing bacterium in activated sludge plants.

A Gram-positive unicellular coccal-diphtheroid rod causing foam in an activated sludge plant was successfully isolated by micromanipulation. Phenotypic characterization and 16S rDNA sequencing identified it as Nocardia farcinica. This is the first report that this opportunistic pathogen is a foam-causing bacterium in activated sludge, and the clinical implications of these observations are discussed.

DNA, Bacterial↗
PubMedDate not supplied

Analysis of coccidian oocyst populations by means of flow cytometry.

Flow cytometry was employed as a tool to analyze and characterize batches of oocysts from laboratory and field isolates of Eimeria spp. from chickens and to propagate sub-populations of batches of oocysts. Oocyst batches were cleaned of debris by a combination of salt flotation, washing and treatment with dilute sodium hypochlorite (1.5% aqueous). Oocyst size and shape were registered by forward-angle light scatter with the argon laser excitation set at 488 nm at 300 mW. Sub-populations of oocysts were collected by map gating and used for microscopy or for propagation. The profile of particle size was characteristic for each species. Propagation of sub-populations of oocysts of specified sizes resulted in cultures of coccidia that were pure species or nearly pure species. The small size of E. mitis caused difficulty in separation from the remaining fine debris. This technique was useful for studying the mixed isolates by bit-map gating had the same limitations as micromanipulation because of the overlapping size of Eimeria spp. Characterization is further limited by the lack of suitable size/shape standards for flow cytometry.

Animals↗
PubMed2002

Concealed nuclei in Saccharomyces strains.

We have found that cells derived from heterokaryons (HK) showing phenotypical traits, coded by the nucleus of one parental strain and by the cytoplasm of the other, may produce mitotic progeny in which the second nucleus is apparently present but not expressed. This 'concealed' nucleus can be forced to expression after growth of these cytoductants on proper selective media. Using a micromanipulator, the buds containing both parental nuclei were isolated in various crosses. Cloning these HK from a rich medium (YEPD) revealed that nearly all of them were composed of a mixture of hybrid cells and cells of one of the parents. Cells of the other parent were present in a very small proportion, if detectable at all. We showed that the percentage of concealed HK decreases when limiting the growth of the strain that serves as a donor of the concealed nucleus. Consequently, our explanation for the presence of concealed nuclei in HK is the low production rate of daughter cells containing both nuclei, which accounts for the lack of a visible phenotype in HK, together with the low replication rate (or fast nuclease degradation) of one of the nuclei. In homosexual crosses, selection allows us to switch the concealed nucleus to normal replication rate. Some abnormalities of meiosis due to hidden nuclei are shown.

Cell Nucleus↗
PubMed2000

Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists.

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.

Journal Article↗
PubMed2002

Phylogeny of the filamentous bacterium Eikelboom Type 1851, and design and application of a 16S rRNA targeted oligonucleotide probe for its fluorescence in situ identification in activated sludge.

Micromanipulation was used to obtain an isolate (BEN 52) of Eikelboom Type 1851 from a bulking activated sludge plant. Its 16S rDNA sequence reveals its closest relative is 'Roseiflexus castenholzii', a member of the phylum 'Chloroflexi', class 'Chloroflexi', previously called the green non-sulfur bacteria. The 16S rRNA targeted oligonucleotide probe designed for fluorescence in situ hybridisation against this sequence successfully identified filamentous bacteria with the morphological features of Type 1851 in activated sludge samples from plants in several countries and different operational configurations.

Australia↗
PubMed1976

Effect of pre- and postnatal protein-calorie undernutrition on RNA-content in rat motor neurons.

Changes in the RNA-content of isolated anterior horn motor neurons from the 7th cervical segment were studied during the postnatal development of pre- and postnatally undernourished, and normally nourished rats. The motor neurons of the anterior horn were isolated by micromanipulation from Carnoy-fixed sections. The neuronal RNA-content was determined by microchemical methods according to Edström (1964). The observation was made that moderate pre- and postnatal undernutrition does not alter the normal RNA-accumulation in anterior horn motor neurons during the preweaning period. After weaning the cellular RNA incorporation decreases but again reaches normal values at the age of 39 days. The present results are compared with those of a previous study (Haltia 1970) in which rats were severely undernourished postnatally.

Age Factors↗