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PubMed2001

[First attempts in preimplantation genetic diagnosis: blastomere biopsy].

There is an increasing expectation from couples that serious inherited diseases should be recognized at the earliest stages of embryonic development. A valuable tool for early prenatal diagnosis, preimplantation genetic diagnosis (PGD), involves the removal of 1 or 2 blastomeres from an in vitro fertilized embryo with micromanipulator (blastomere biopsy) without affecting the viability of the embryo. Genetic analysis of the removed blastomeres is performed to determine whether the embryo carries the genes responsible for the examined disease. Based on the results of the genetic analysis it is possible to transfer only unaffected embryos to the uterus. In this study, the authors performed blastomere biopsy on 35 embryos at the 6-10 cells stage. A total of 104 blastomeres were analyzed. On follow-up, 64% of biopsied embryos were cleaved and 43% developed to the morula or blastocyst stage. The introduction of this new procedure into the field of assisted reproduction can provide an alternative for couples who do not want to give birth to children affected by a genetic disease but would reject induced abortion after a positive prenatal diagnosis.

Abortion, Induced↗
PubMed1999

Successful pregnancy in a case of azoospermic infertility by using testicular sperm for intracytoplasmic injection into the oocyte.

Non-obstructive azoospermia used to be considered an untreatable cause of infertility. By the microinjection technique, however, sperm that has been surgically extracted from the testis can be injected into the oocyte cytoplasm. The injected eggs can be transferred to the uterus or fallopian tubes to initiate a pregnancy. A healthy baby boy conceived by using this method was delivered in November 1997. This micromanipulation technique offers couples in which the man has non-obstructive azoospermia the chance of having their own offspring. The methodology used and a brief discussion of its merits are presented.

Journal Article↗
PubMed2000

[Analysis of TCR-gamma gene rearrangement in single H/R-S cells of Hodgkin disease].

OBJECTIVE: To detect the rearrangement of T cell receptor gamma chain gene in single H/R-S cell as an indication of early T lymphocyte differentiation. METHODS: CD30 positive H/R-S cells were isolated with micromanipulator from the frozen sections and analyzed by single-cell PCR. TCR-gamma chain gene rearrangement was detected through semi-nested PCR. In the first round of PCR, a pair of TCR-gamma primers which amplify the segments between Vgamma and Jgamma region (including the N region) of TCR-gamma gene mixing with a pair of beta-globin primers were used. In the second round, only the products positive beta-globin were amplified with the primers for TCR-gamma gene. RESULTS: A 268 bp beta-globin gene specific product was obtained in 24 out of 56 H/R-S cells from 6 cases of Hodgkin's disease. After the first round of PCR, no rearrangement of the TCR-gamma gene was noticed at a single cell level. CONCLUSION: H/R-S cells from the selected cases were not derived from the T-cell lineage but possibly from the B-cell lineage.

Adolescent↗
PubMed2001

Cloned murine fetuses produced by nuclear transfer using metaphase-arrested embryonic stem cells.

We examined whether metaphase nuclei could be used as nuclear donors in nuclear transfer in mice. The reconstructed embryos were developed to fetuses in both the metaphase-nuclear transfer and the G1-nuclear transfer. We also performed enucleation of oocytes following nuclear injection (injection-enucleation method) using microinjection method with a piezo-driven micromanipulator in order to produce the cloned murine fetuses. We found that this method could shorten time for manipulation in comparison with the conventional method performing nuclear injection following enucleation of oocytes (enucleation-injection method). We produced successfully cloned fetuses by the injection-enucleation method. Furthermore, there was no difference of developmental efficiency in reconstructed embryos from between B6D2F1 and ddY strain as oocyte donor.

Animals↗
PubMed2002

[Factors affecting the frequency of concealed heterokaryosis in Saccharomyces cerevisiae].

In this work, studies on the phenomenon of concealed heterokaryosis that we previously detected in the saccharomycetes yeast strains were continued. New approaches to high effectiveness of isolation of cytoductants carrying the concealed nucleus were implemented, and the composition of individual concealed heterokaryons, zygotic clones, and the first zygotic buds was analyzed by a micromanipulator. The relationship between a delay in the growth of the parental strain (a potential donor of the concealed nucleus) and a decline in the frequency of the appearance of concealed heterokaryons (HKC) was observed. It is assumed that different replication rates of two nuclei of the heterokaryon probably underlie the appearance of HKC. A drastically decreased level of replication of one of the parental nuclei may be connected with the fact that binuclear buds appear extremely rarely and give rise to the rapidly "purified" progeny consisting of cells carrying the second nucleus with normal replication. A lack of the phenotype allows rare binuclear cells to persist as concealed heterokaryons. HKC may be detected only when cells of either parental type are isolated on the corresponding selective media.

Cell Nucleus↗
PubMed1975

[Report on the experience in intracellular microelectrode studies of the external eye muscles with the stereotactic surgical device SESH-2].

The Soviet SESH-2 stereotactic surgical device for micro-electrode examinations offers technical advantages in the derivation of the intracellular muscle fibre membrane potential after resting and of the intracellular potential of the peripheric ocular muscles after irritation as well as in the study of the central irritation of the nuclei of the ocular muscles. The coupling of two hydraulic micromanipulators permits to perform in vivo various investigations of the ocular muscles by means of micro-electrodes. The numerous possibilities of variation and assembly according to the modular principle allows also simultaneous studies on ocular muscle mechanics.

Animals↗
PubMed2002

Clonal relation in a case of CLL, ALCL, and Hodgkin composite lymphoma.

Large cell lymphomas and Hodgkin disease may develop during the course of chronic lymphocytic leukemia (CLL). In some cases the transformed cells are Epstein-Barr virus (EBV)-positive and not clonally related to the CLL cells. In other cases the transformed cells have the same clonal rearrangements as the CLL cells. Here we describe a composite lymphoma in a patient with CLL that exhibits a combination of CLL/small lymphocytic lymphoma, large cell lymphoma with anaplastic morphology, and Hodgkin lymphoma (HL). Although the large cell lymphoma cells are CD45R0 and TIA-1-positive, suggesting a T- or 0-cell anaplastic large cell lymphoma (ALCL), the genetic analysis demonstrates immunoglobulin heavy chain (IgH) gene rearrangements for both alleles, carrying the same somatic mutations as observed in the CLL component. The Reed-Sternberg (R-S) cells in the Hodgkin component also strongly express TIA-1 but differ from the anaplastic large cells by the expression of CD15 and TARC and the presence of a prominent lymphocytic infiltrate. The ALCL and HL components both are EBV negative. Analysis of the IgH gene rearrangements in micromanipulated R-S cells revealed identical Ig gene rearrangements carrying the same somatic mutations as the CLL and the large cell components. The findings indicate transformation of the CLL cells into a large cell lymphoma with anaplastic morphology and a Hodgkin component.

Aged↗
PubMed2002

Cultivation of fetal erythroid precursors from maternal blood: isolation and characterization by PCR and FISH.

Cultivation of fetal progenitor cells from maternal blood offers the opportunity to produce sufficient fetal cells for prenatal molecular genetic and cytogenetic analysis. For in vitro cultivation, 10 ml of blood was collected from 22 women carrying a male fetus. After triple-density gradient centrifugation, the mononucleated cells were cultivated for 10 to 14 days in special hematopoietic growth medium. Red and white colored cell colonies were individually collected by micromanipulation. A representative sample from each colony was characterized by chromosome Y-specific polymerase chain reaction (PCR) systems. The remaining cells of the Y-positive colonies were used to perform chromosome preparations and fluorescence in situ hybridization (FISH) to detect XY-positive interphase nuclei and metaphases. Y-positive signals could be detected in 15 (68%) of the 22 analyzed blood samples. With SRY PCR 10.5% (40/379) of the collected red colonies were determined to be of fetal origin and 6.1% (32/522) of the colonies analyzed by amelogenin PCR were Y-positive. All collected white cell colonies were Y-negative. FISH analyses of PCR-positive colonies revealed that less than 30% of the cells within a colony are of fetal origin and reflect more precisely the actual situation within a single colony. Moreover the successful preparation of fetal metaphases in non-invasive prenatal diagnosis is presented.

Chromosomes, Human, Y↗
PubMed2002

Cerebrospinal fluid centesis at the cerebellomedullary cistern of kittens.

We needed an effective technique for obtaining cerebrospinal fluid (CSF) from young (2- to 18-week-old) kittens. Standard veterinary technique was not suitable, so we adapted a previously published technique for rats. We first established an effective isoflurane-only anesthetic protocol for young kittens. After inhalant anesthesia, the kittens were positioned on a supporting platform to gain flexion of the head and neck. A micromanipulator was used to hold and slowly advance the collection needle. At the time this report was written, we had collected a total of 33 samples from eight kittens without causing apparent neurologic deficits. Correct positioning of the animal and collection needle was critical for success. This procedure enabled the collection of approximately 0.5 ml CSF from kittens younger than 12 weeks and larger volumes from older kittens.

Age Factors↗
PubMed2002

Dispelling the "Nocardia amarae" myth: a phylogenetic and phenotypic study of mycolic acid-containing actinomycetes isolated from activated sludge foam.

Right-angle branched filaments and rods micromanipulated from activated sludge foam and mixed liquor were identified as putatively novel members of the genera Gordonia, Mycobacterium and Rhodococcus using a combination of chemical, molecular and morphological data. Pyrolysis mass spectrometric analyses of gordoniae isolated in both the present and a previous study revealed pyro-groups, distinct from validly described Gordonia species, which could be equated with those based on morphological properties and 16S rDNA data. Putative gordoniae assigned to one of these groups were found to be closely related to strains currently identified as "Rhodococcus australis". These strains were also found to have properties consistent with their classification in the genus Gordonia. The results of this study highlight the limitations of the microscopic approach to filament identification and cast further doubt on the view that foaming can be attributed to members of one or a few Nocardia species.

DNA, Bacterial↗
PubMed2002

Phylogenetic analysis and in situ identification of "Nostocoida limicola"-like filamentous bacteria in activated sludge from industrial wastewater treatment plants.

The diversity of filamentous bacteria present in industrial wastewater treatment plants was analysed by a combination of classical and molecular-biological approaches. Many unknown filamentous bacteria were observed in about 80 screened activated sludge samples from different industries with sometimes severe bulking sludge problems. A special focus was paid to filaments which resembled "Nostocoida limicola", a filamentous bacterium which was found to be present in many WWTPs. These filamentous bacteria are hardly cultivable and only one strain was obtained and maintained in co-culture with a yeast. The 16S rRNA sequences of several other "Nostocoida limicola"-like filamentous bacteria from different sludge samples were obtained by micromanipulation and different molecular-biological methods. The sequences were phylogenetically analyzed and specific molecular probes were developed and applied. The results clearly demonstrate that "Nostocoida limicola"-like filaments from industrial WWTPs are different from all other "Nostocoida limicola" types investigated so far. Our strains are affiliated to the alpha-subclass of Proteobacteria.

DNA Primers↗
PubMed1975

[K+, H+ and Ca++ concentrations in the perivascular fluid as a factor in the regulation of the lumina of the vessels of the pia mater].

The author separed the vascular vessels of pia mater with a size of 20-150 microns in cats under penthobarbital narcosis. The perivascular area of the vessels was perfused by an artificial fluid with varying concentrations of potassium, hydrogen and calcium. The local contraction of the smooth vascular musculature was induced by stimulation with rectangular impulses by means of electrodes introduced by micromanipulators in immediate vicinity of a vessel. The changes were recorded by means of microphotographic images of the vessels. It was established that the contraction of the vessels was observed when there was a lack of potassium or at concentration of 100 meq/l. There was no change at 2,5-3,0 and at 50-60 meq/l of potassium. Vasodilatation was found at 10-20 meq/l. Electrical stimulation caused stronger contraction of the vessel, when there was a lack of potassium in comparison with the norm (2,7 meq/l). The increase of potassium caused a reduction of the dimensions of the effect due to electrical stimulation. The vascular dilatation, induced by 10-20 meq/l of potassium was not affected by the increase of the calcium concentration (6-9 meq/l) in artificial fluid. The vasodilatation, induced by an increase of hydrogen, was abolished by raising the concentration of calcium in the artificial fluid. The obtained results were discussed.

Animals↗
PubMed2002

Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane.

Calmegin is a putative testis-specific molecular chaperone required for the heterodimerization of fertilin alpha/beta and the appearance of fertilin beta on the sperm surface. Calmegin-deficient mice are almost completely sterile. The cause of the sterility initially was considered to be impaired abilities in sperm/zona pellucida (ZP) and sperm/egg plasma membrane (EPM) binding, and in the ascension of sperm to the oviduct, phenotypes similar to those seen in sperm from fertilin beta-deficient animals. We have developed a new method in which eggs were prepared without any detectable ZP3 on their surfaces by using a piezo-driven micromanipulator. Using these eggs and sperm containing the green fluorescent protein in their acrosomes, which can distinguish acrosome-intact from acrosome-reacted sperm, the binding and fusing abilities of calmegin-deficient sperm were reexamined. Under these conditions, acrosome-reacted sperm retained their ability to bind to and fuse with the EPM. The reduction in EPM binding of sperm from the calmegin(-/-) animals was apparently due to the artifactual binding of large numbers of acrosome-intact sperm from calmegin(+/-) mice to ZP remnants remaining on the EPM prepared with acidic Tyrode's solution. Thus, the sperm defect in calmegin-null animals is not at the level of sperm-EPM binding but rather may involve either sperm-ZP binding and/or sperm transit to the oviduct. Because fertilin beta is absent from calmegin-deficient mice, these results also suggest that the role of fertilin beta in sperm-EPM interaction needs to be reevaluated.

ADAM Proteins↗
PubMed2003

[Gene expression analysis in liver tissue at a single cell level by nested polymerase chain reaction and laser microdissection].

OBJECTIVE: To investigate the measurements of gene expressing at a single hepatocyte level. METHODS: Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR). RESULTS: Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells. CONCLUSION: Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.

Dissection↗
PubMed1999

[Microdissection of M chromosome in Vicia faba and its library construction].

Microdissection and microcloning technique was employed to construct the library of M chromosome in Vicia faba. The M chromosomes were microdissected with a micromanipulator and were put into a 0.5 ml Eppendorf tube, then digested with Sau3A. Sau3A linker adaptors were ligated to the end of chromosome DNA fragments, and two rounds of PCR were carried out with one chain of linker adaptor as the primer. The PCR products ranged in size from 300 base pair (bp) to 3000 bp with predominant fragments from 500 bp to 1500 bp. Southern hybridization analysis confirmed that PCR products originated from Vicia faba genome. The second round PCR products were cloned and about 102,000 recombinants were obtained. 118 recombinants were selected randomly for analysis. The inserts ranged in size from 150 bp to 3000 bp with an average of 690 bp. Dot blot was carried out for 100 clones with DIG labeled Vicia faba genome DNA as probes. The result revealed that 51% were low and unique copy sequences, 49% were repetitive sequences. M chromosome DNA library has not been reported before.

Chromosome Mapping↗
PubMed2002

[Characteristics of tenocyte adhesion to biologically-modified surface of polymer].

In this study we examined the in vitro characteristics of tenocyte adhesion to biologically-modified surface of polymer. Polylactic-co-glycolic acid (PLGA) 85/15 films were prepared by a solvent-casting technique. Each film was adhered onto the bottom of a chamber. The film was precoated with poly-D-lysine (PDL), and then coated with serum-free F12 medium containing various concentrations of fibronectin (FN), type I collagen (CN I), and insulin-like growth factor1 (IGF-1). The monoclonal antibodies (to FN and to CN I) with various dilutions were used to inhibit attachment of tenocytes to surface precoated with FN or CN I. Human embryonic tendon cells (HETCs) and transformed human embryonic tendon cells (THETCs) were used as the seeding cells. The system used for the measurement of adhesion force was the micropipette aspiration experiment system. The micropipette was manipulated to aspirate a small portion of the tenocyte body by using a small aspiration pressure. Then the pipette was pulled away from the adhesion area by micromanipulation. The minimum force required to detach the tenocyte from the substrate was defined as the adhesion force. The results showed that modification of FN or CN I by precoating significantly enhanced attachment of tenocytes to surface of polymer (P < 0.05). As antibodies to FN or CN I were added to a polymer film precoated with FN or CN I, the adhesion force decreased significantly (P < 0.05). We concluded that the specific adhesion forces of tenocytes to extracellular matrix adhesion proteins (FN and CN I) had coordinated action and showed good dependence on their precoating concentrations, and were inhibited by the antibodies to these adhesion proteins. Films precoated with IGF-1 strongly accelerated the adhesion of tenocytes to polymer. These results indicate that the specific adhesion of tenocytes to polymer can be promoted by coating extracellular matrix adhesive proteins and insulin-like growth factor1. It is of great importance to construct tissue-engineered tendon.

Biocompatible Materials↗
PubMed2003

Analysis of the presence of Trypanosoma cruzi in the heart tissue of three patients with chronic Chagas' heart disease.

It is still unclear to what extent myocarditis-associated, chronic Chagas' heart disease is due to persisting Trypanosoma cruzi. In the present study, we have analyzed tissue samples from the hearts of three patients with this disease. In situ hybridization provided little evidence for the presence of intact T. cruzi even at sites of strong inflammation. Nevertheless, micromanipulation techniques detected remnants of both T. cruzi kinetoplast DNA and nuclear DNA. Trypanosoma cruzi DNA was also detected in single macrophages dissected directly from frozen heart tissue sections. Thus, this analysis demonstrates that T. cruzi kinetoplast DNA and nuclear DNA are widely dispersed in the heart tissue, although in low amounts. Since we rarely detected intact T. cruzi parasites during the chronic phase of Chagas' heart disease, we can exclude heart tissue as a major parasite reservoir.

Animals↗
PubMed2003

Allelic losses on chromosome 6q25 in Hodgkin and Reed Sternberg cells.

We established a molecular cytogenetic approach to identify consistent genetic aberrations in classical Hodgkin lymphoma. Single laser-micromanipulated Hodgkin and Reed Sternberg (H-RS) cells and the respective germ line tissue were PCR-amplified using highly polymorphic microsatellite probes. Loss of heterozygosity and genomic imbalances of the fluorochrome-labeled microsatellites were determined by fragment length analysis. Eleven cases of in classical Hodgkin lymphoma (cHL) were initially screened with 21 microsatellite markers scattered over the entire genome. Loss of heterozygosity was detected in >40% of informative loci in most cases indicating a deletion of a substantial part of the genome of H-RS cells. Allelic losses and imbalances on chromosome 6q were detected in most of these cases. A deletion mapping of 6q was performed in 16 cases of cHL. This detailed analysis of 6q led to the identification of a 3.3-Mbp region around D6S311 flanked by D6S978 and D6S1564 that was altered in 11 of 14 cases of cHL analyzed. In conclusion, allelotyping of single H-RS cells revealed monoallelic chromosomal deletions and genomic imbalances on 6q that might affect genes critically involved in the pathogenesis of H-RS cells.

Alleles↗