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PubMed2005

Identification of "Candidatus Thioturbo danicus," a microaerophilic bacterium that builds conspicuous veils on sulfidic sediments.

Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the epsilon-subdivision of the Proteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name "Candidatus Thioturbo danicus."

Bacteria, Aerobic↗
PubMed1967

Diploid hybridization in a heterothallic haploid yeast, Saccharomyces rouxii.

By crossing of a heterothallic haploid yeast, Saccharomyces rouxii, we have succeeded in obtaining diploid hybrids. This paper shows one possible method of breeding heterothallic haploid yeasts for industrial application. S. rouxii is highly salt-tolerant and plays an important role in shoyu and miso fermentation. Therefore, genetic improvements of the properties are of commercial importance. Since newly isolated S. rouxii could neither conjugate nor sporulate on sporulation media commonly used, a suitable medium for conjugation and sporulation of S. rouxii was firstly investigated. A 5% NaCl Shoyu-koji extract agar was found to be most efficient. Next, we tried to get diploid strains by mass culture of two mating types on the conjugation medium, but several phenomena made this difficult: (i) zygotes quickly sporulated before budding; (ii) several zygotes showed terminal budding, but the buds could not grow into diploid cells, suggesting they would be heterocaryon; and (iii) a few zygotes lost their viability. After trying to isolate and cultivate a large number of zygotes in various combinations of crossing by micromanipulation, we fortunately recognized that large cells arose from some combinations. The analysis of ploidy suggested that the large cells would be diploid. Also, they showed sporulation of typical Saccharomyces, i.e., two to four spores in an unconjugated ascus. The diploid strains thus obtained were highly salt-tolerant and stable in liquid medium. Therefore, the procedure presented here would be effective for breeding salt-tolerant S. rouxii.

Conjugation, Genetic↗
PubMed2002

Small variant STEVOR antigen is uniquely located within Maurer's clefts in Plasmodium falciparum-infected red blood cells.

Malaria parasite antigens encoded by multigene families are important factors in virulence and in disease pathology. In Plasmodium falciparum, the virulence factor PfEMP-1 is encoded by the var multigene family and is exposed at the infected erythrocyte surface. PfEMP-1 is clonally variant, allowing the parasite to evade host immunity. The recently identified P. falciparum stevor multigene family and its products also have the potential to be involved in similar important aspects of host-parasite interactions. Here, we show tightly regulated stage-specific transcription of stevor occurring over just a few hours of the asexual parasite life cycle. Only a subset of stevor genes are transcribed in parasite populations maintained in cultures and in single micromanipulated parasites. Antibodies against STEVOR recognize proteins of the expected size (approximately 37 kDa) and localize STEVOR in Maurer's clefts, unique membranous structures located in the cytoplasm of infected erythrocytes. The fact that the timing of stevor expression and the location of STEVOR are clearly distinct from those of other parasite variant antigens suggests that this gene family may have a novel role in P. falciparum biology.

Animals↗
PubMed2003

Termite gut symbiotic archaezoa are becoming living metabolic fossils.

Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.

Amino Acid Sequence↗
PubMed2005

Cell biology of conidial anastomosis tubes in Neurospora crassa.

Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.

Cell Nucleus↗
PubMed1975

Morphology and viability of large bodies of streptococcal L-forms.

A procedure is described for the massive formation and isolation of large bodies of group A and D streptococcal L-forms. Up to 90% individual bodies of 20 to 100 mum in diameter can be produced in pour plates of nutrient gelatin and subsequently harvested by simple micromanipulation. The growth of these giant bodies was followed by light microscopy and their ultrastructure and internal architecture was examined by transmission and scanning electron microscopy. Large bodies had a honeycomb structure of vacuoles. Numerous small units could be observed inside, protruding into, and between vacuoles. Intact large bodies (30 to 60 mum), when placed in small amounts of broth and incubated at 35 C, initiated turbid cultures, but when set on agar, they ruptured, releasing internal granules and producing typical L-form colonies. The number of internal colony-forming units (CFU) was correlated with the size of large bodies. Up to 200 CFU were detected in bodies of 40 to 60 mum in diameter, whereas corpuscles of 20 to 30 mum averaged only three CFU. The inefficiency of replication was apparent with the determination by light and electron microscopy that at least 100 times as many granules and elementary corpuscles as CFU were produced inside large bodies.

Cell Division↗
PubMed1978

Spore location patterns in sporulating doublets of Bacillus cereus and Bacillus megaterium, derived from single doublet isolates with differing sporulation geometry.

The distribution of spore loci in pairs of Bacillus cereus and B. megaterium showed different degrees of polarization from random location towards location at the old end of the sporangium. When individual doublets containing both spores at either the old or new ends of the sporangia were isolated by micromanipulation, subsequent culture showed the same spore location patterns.

Bacillus megaterium↗
PubMed1989

Evidence for the involvement of a cytoplasmic factor in the aging of the yeast Saccharomyces cerevisiae.

The life spans of individual Saccharomyces cerevisiae cells were determined microscopically by counting the number of buds produced by each cell to provide a measure of the number of cell generations (age) before death. As the cells aged, their generation times increased five- to sixfold. The generation times of daughter cells were virtually identical to those of their mothers throughout the life spans of the mothers. However, within two to three cell divisions after the daughters were detached from their mothers by micromanipulation, their generation times reverted to that characteristic of their own age. Recovery from the mother cell effect was also observed when the daughters were left attached to their mothers. The results suggest that senescence, as manifested by the increase in generation time, is a phenotypically dominant feature in yeast cells and that it is determined by a diffusible cytoplasmic factor(s) that undergoes turnover. This factor(s) appeared to be transmitted by a cell not only to its daughter, but also indirectly to its granddaughter. In separate studies, it was determined that the induced deposition of chitin, the major component of the bud scar, in the yeast cell wall had no appreciable effect on life span. We raise the possibility that the cytoplasmic factor(s) that appears to mediate the "senescent phenotype" is a major determinant of yeast life span. This factor(s) may be the product of age-specific gene expression.

Cell Division↗
PubMed1965

HEMOLYSIN OF MYCOPLASMA PNEUMONIAE.

Somerson, Norman L. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Robert H. Purcell, David Taylor-Robinson, and Robert M. Chanock. Hemolysin of Mycoplasma pneumoniae. J. Bacteriol. 89:813-818. 1965.-Discrete colonies of Mycoplasma pneumoniae or M. laidlawii on agar produced complete (beta) lysis of guinea pig erythrocytes. Crowding of colonies on agar plates, omission of yeast extract from the medium, and incubation under reduced oxygen tension inhibited hemolysin production. Colonies which were not viable after ultraviolet radiation or heating at 56 C did not produce hemolysin. In addition, hemolysis was suppressed in old cultures in which viability was diminished, and was eliminated when colonies were removed from the agar surface by micromanipulation. The hemolysin passed through a viscose dialysis membrane. These findings suggest that the hemolysin is labile, continuously released by the colonies, of low molecular weight, and probably nonprotein in nature.

Agar↗
PubMed1968

Spontaneous lethal sectoring, a further feature of Escherichia coli strains deficient in the function of rec and uvr genes.

Eight recombination-deficient (Rec(-)) mutants of Escherichia coli were studied. Progeny lines were obtained on solid media, by means of micromanipulation, and the colony-forming ability of individual cells was analyzed. Cells of all eight strains gave rise to colony-forming as well as non-colony-forming descendants ("lethal sectoring"). Lethal sectors, i.e., groups of non-colony-forming cells which originate from a common ancestor, appeared with frequencies per generation ranging between 4 and 20% in Rec(-) strains, whereas lethal sectors were rare in Rec(+) strains (less than 1%). A strain carrying a mutation (uvrA6) in one of the genes involved in pyrimidine dimer excision from deoxyribonucleic acid (DNA) showed twice as many lethal sectors per generation as a strain with the genotype uvrA(+). Similarly, a double mutant (AB2480, uvrA6, recA13) showed twice as much spontaneous lethal sectoring as the corresponding Rec(-) strain (uvrA(+), recA13). The kinetics of growth curves obtained in nutrient broth and the frequency of non-colony-forming units in stationary-phase broth cultures indicate clearly that lethal sectors occur in liquid cultures too. The causes for spontaneous lethal sectoring are unknown at present. It seems reasonable to assume that gene uvrA and the rec genes are somehow involved in the repair of spontaneously occurring DNA lesions, since a deficiency in this type of repair may cause lethal sectors. The extent to which spontaneous lethal sectoring (observed in all Rec(-) strains of E. coli studied) may contribute indirectly to the failure to form recombinants is discussed.

Clone Cells↗
PubMed2002

Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis.

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.

Animals↗
PubMed2005

Development of Microelectromechanical Systems (MEMS) forceps for intraocular surgery.

AIM: To develop silicon microforceps for intraocular surgery using Microelectromechanical Systems (MEMS) technology, the application of microchip fabrication techniques for the production of controllable three dimensional devices on the micrometre scale. METHODS: Prototype MEMS forceps were designed and manufactured for intraocular surgery. Scanning electron microscopy was used to evaluate device tip construction. Designs using both thermal expansion actuators and conventional mechanical activation were tested in human cadaver eyes and in vivo rabbit eyes to assess functionality in standard vitreoretinal surgery. RESULTS: MEMS forceps were constructed with various tip designs ranging from 100 mum to 2 mm in length. Scanning electron microscopy confirmed accurate construction of micro features such as forceps teeth as small as tens of micrometres. In surgical testing, the silicon forceps tips were effective in surgical manoeuvres, including grasping retinal membranes and excising tissue. The mechanical actuator design on a 20 gauge handle was more operational in the intraocular environment than the thermal expansion actuator design. While handheld operation was possible, the precision of the forceps was best exploited when mounted on a three axis micromanipulator. CONCLUSION: MEMS microforceps are feasible for conventional vitreoretinal surgery, and offer advances in terms of small scale, operating precision, and construction tolerance.

Animals↗
PubMed2002

Quantification of histological changes after calibrated crush of the intraorbital optic nerve in rats.

BACKGROUND: Traumatic optic nerve lesions (TONL) are probable but unpredictable consequence after severe midface or skull base trauma. Based on a previously described rat model, the authors developed a new model in order to simulate optic nerve crush during trauma on the optic canal. METHODS: To achieve a calibrated TONL, a microinjuring device was designed that made it possible to assess the correlation between a defined trauma and the neuronal degeneration in the rat retinal ganglion cell (RGC) layer. This device is based on a small dynamometer mounted onto a conventional micromanipulator. The supraorbital approach was chosen to expose the extracranial optic nerve. RESULTS: In this rat model (n=100, Wistar strain) the parameters of "force" and "time" could be precisely monitored during the experiment. The decrease in the mean number of retinal neurons (N) according to the pressure exerted (2-30 cN x mm(-2)) on the optic nerve was linear for 1, 6, and 15 minutes of injuring time; the decrease in N for varying injuring forces also appears to be nearly linear. CONCLUSION: The results show that this model provides a reliable method for studying quantitatively the anatomical effects of TONL on the RGC layer and the optic nerve itself, and may allow the design of treatment strategies following TONL.

Animals↗
PubMed1997

Prenatal detection of fetal aneuploidies using transcervical cell samples.

In the course of an investigation aimed at detecting the presence of trophoblastic cells in the endocervical canal of pregnant women between 7 and 17 weeks of gestation, several cases of aneuploidies were observed using a fluorescent in situ hybridisation (FISH) assay. The cases include fetal chromosome 21 and 18 trisomies, triploidy and sex chromosome aneuploidies. The results were confirmed by testing placental tissues obtained after termination of pregnancy (TOP). In two of these cases, clumps of cells with the morphology of trophoblasts were isolated from the transcervical cell (TCC) samples using micromanipulation. FISH and fluorescent polymerase chain reactions (PCR), performed on these clumps, showed them to be exclusively of fetal origin. These results show that prenatal diagnoses of major aneuploidies can be performed by FISH using whole TCC samples, or on isolated clumps of cells by FISH and PCR assays.

Adult↗
PubMed1998

A simple combined microdissection and aspiration device for the rapid procurement of single cells from clinical peripheral blood smears.

Molecular analysis of cells from cytology specimens can help to establish a diagnosis in ambiguous cases. However, mutations in heterogeneous samples might not be detected because of the diluting effect of DNA from normal background cells. Even if a mutation were detected, it could not be traced back to a specific cell type. Molecular analysis of single cells circumvents this problem. Both mechanical and laser assisted methods have been described for the selective procurement of cells from histology slides; however, they have the drawback of either being technically demanding or expensive. Furthermore, it is nuclear whether they can be applied to cytology specimens. Finally, few of these techniques are able to procure single cells. Therefore, we developed a simplified combined microdissection and aspiration device for the rapid procurement of single cells from clinical cytology specimens. The principle of this device, called the cytopicker, is the combination of the microdissection tool, a steel cannula, with the aspiration tool, a glass capillary connected to a vacuum, into one device. Steel cannulae are optimal for microdissection of cells from the hard matrix of cytology specimens but aspirate poorly. On the other hand, glass capillaries are suboptimal for dissecting but aspirate very well. Combining both tools into one by inserting the capillary into the cannula allows optimal dissection using the cannula (with the glass capillary with-drawn and thus protected), followed by optimal aspiration using the capillary (after being advanced through the cannula). All movements of the device are controlled by just one micromanipulator, making the cytopicker inexpensive to manufacture. The cytopicker can rapidly and simply procure single cells, such as lymphoblasts, from cytology specimens, such as peripheral blood smears. DNA from these cells can be amplified by PCR. However, precautions have to be taken to avoid contamination. Once improved further, the cytopicker might facilitate molecular analysis in the routine cytology laboratory.

Blood Specimen Collection↗
PubMed1981

Attempts to produce monozygotic quadruplets in cattle by blastomere separation.

The blastomeres of eight-cell cow embryos were separated by micromanipulation into four pairs, inserted in foreign zonae pellucidae, embedded in agar and cultured for approximately four days in ligated sheep oviducts. Of 44 "quarter" embryos (11 monozygotic groups) transferred to sheep, 91 per cent had continued to develop at a normal rate and 77 per cent had formed small blastocysts with a single inner cell mass. Twenty-six blastocysts freed from the agar were transferred to heifers, each heifer receiving two monozygotic embryos, one to the tip of each uterine horn. Nine recipients were diagnosed pregnant by rectal palpation on day 50, six carrying twins. Thus 15 of the embryos had continued to develop including two sets of monozygotic quadruplets and one set of monozygotic triplets. Eight fetuses developed to full term, one set of monozygotic triplets, two sets of monozygotic twins (one set born dead) and one single.

Animals↗
PubMed1985

Multiple embryo-transplant offspring produced from quartering a bovine embryo at the morula stage.

A non-surgical embryo collection was completed on a day 7 superovulated Chianina donor cow. Because all but two of the ova from the collection were unfertilised and a surplus of potential recipients was available, one embryo (an excellent quality late morula) was dissected into four equal portioned 'quarter' embryos using a simplified micromanipulation procedure. Each quarter embryo was then placed in a 0.25 ml French straw and non-surgically transplanted to four different crossbred beef recipient females. The remaining embryo was similarly transplanted to a herd mate recipient as an intact embryo. One recipient returned to oestrus, one recipient had an extended post transfer cycle and the two remaining recipients produced a live quarter embryo transplant calf each within 24 hours of the other. The intact embryo placed in a herd mate recipient did not produce a transplant calf. To the authors' knowledge, these transplant offspring are the first live births reported from a non-surgically collected later-stage bovine morula (day 7), which had been dissected into quarters and then individually transplanted non-surgically to recipient females. The procedure was relatively simple to perform and was completed in less than one hour.

Animals↗
PubMed1998

B-chromosome origin in the endemic New Zealand frog Leiopelma hochstetteri through sex chromosome devolution.

The endemic New Zealand frog Leiopelma hochstetteri has variable numbers of mitotically stable B chromosomes. To assess whether the B chromosomes were derived from the autosome complement, they were isolated by micromanipulation and their DNA amplified by degenerate oligonucleotide primed PCR. Southern hybridizations of B chromosome DNA probes to genomic DNA from males and females characterized by differing numbers of B chromosomes demonstrated that the B chromosomes were derived from the univalent W sex chromosome characteristic of North Island populations. The presence of homologous B chromosome specific sequences from geographically distinct populations indicates a single origin of the B chromosomes. Furthermore, a primitive homology shared by B chromosomes and the W sex chromosome from an ancestral WZ/ZZ karyotype, which is still present in frogs from Great Barrier Island, shows that the B chromosomes originated soon after the univalent W sex chromosome had originated. Sequence analysis revealed that B chromosome DNA is composed of repeat sequences and has the potential to form stable hairpin structures. The molecular dynamics of these structures may reflect an inherent propensity to undergo rapid change in nucleotide sequence and chromosome structure.

Animals↗