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PubMed2004

Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance.

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of "hemifusion" is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

Cell Surface Extensions↗
PubMed1995

Resting membrane potentials: a student test of alternate hypotheses.

The frog sartorius muscle is a model tissue for demonstrating to physiology students the principles underlying both membrane phenomena and hypothesis testing. Myocytes can be impaled with conventional glass microelectrodes to measure membrane voltage (Vm). Further, Vm is observed as extracellular K+ is altered and a K+ channel blocker is added. After the experiment, students examine the underlying assumptions of the Nernst equilibrium and the Goldman-Hodgkin-Katz equation. They ultimately determine which of the two algorithms best predicts the measured Vm. In addition, students learn micromanipulation and impalement techniques. This experiment facilitates the student's understanding of membrane permeability, ionic gradients, and membrane voltage.

Algorithms↗
PubMed2004

Cell stiffness and receptors: evidence for cytoskeletal subnetworks.

Viscoelastic models of cells often treat cells as homogeneous objects. However, studies have demonstrated that cellular properties are local and can change dramatically on the basis of the location probed. Because membrane receptors are linked in various ways to the intracellular space, with some receptors linking to the cytoskeleton and others diffusing freely without apparent linkages, the cellular physical response to mechanical stresses is expected to depend on the receptor engaged. In this study, we tested the hypothesis that cellular mechanical stiffness as measured via cytoskeletally linked receptors is greater than stiffness measured via receptors that are not cytoskeletally linked. We used a magnetic micromanipulator to apply linear stresses to magnetic beads attached to living cells via selected receptors. One of the receptor classes probed, the dystroglycan receptors, is linked to the cytoskeleton, while the other, the transferrin receptors, is not. Fibronectin-coated beads were used to test cellular mechanical properties of the cytoskeleton without membrane dependence by allowing the beads to endocytose. For epithelial cells, transferrin-dependent stiffness and endocytosed bead-dependent stiffness were similar, while dystroglycan-dependent stiffness was significantly lower. For smooth muscle cells, dystroglycan-dependent stiffness was similar to the endocytosed bead-dependent stiffness, while the transferrin-dependent stiffness was lower. The conclusion of this study is that the measured cellular stiffness is critically influenced by specific receptor linkage and by cell type and raises the intriguing possibility of the existence of separate cytoskeletal networks with distinct mechanical properties that link different classes of receptors.

Cell Line↗
PubMed1981

Fluorescence microlymphography.

Microneedles, 0.2 mm o.d., were connected to a microsyringe and mounted on a micromanipulator. Under microscopic control, 0.01 ml of a 25% solution of FITC-labeled dextran-40 or dextran-150 were injected into the subepidermis at the big toe near the nailfold or in the medial ankle region. Fluorescence intravital microscopy revealed a network of lymphatic microvessels. The comparison with recent anatomic studies reveals that the reticular network visualized by FITC-dextran corresponds to the network in the stratum papillare. In 20 healthy subjects lymphatic capillaries were detected in a restricted area on the lateral aspect of the big toe. In 10 patients with primary lymphedema, the dye expanded to almost the entire dorsal skin surface of the big toe. In two cases, enlarged and tortuous microvessels of pathologic shape were observed. Fluorescence microlymphography is a simple and nearly atraumatic approach for depicting the intravital anatomy of human skin lymphatic capillaries.

Adult↗
PubMed2001

Gene expression analysis of single neoplastic cells and the pathogenesis of Hodgkin's lymphoma.

The origin of the Reed-Sternberg cell of Hodgkin's disease remained clouded in mystery for almost a century after its discovery in 1898. The major obstacle to its understanding is that, unlike other cancers, the malignant cell of Hodgkin's disease is vastly outnumbered by surrounding non-neoplastic cells at approximately 1000:1. We have devised several strategies to isolate Reed-Sternberg T-cells to determine their origin, global gene expression and, ultimately, their pathogenesis. This has increased the number of genes known to be expressed in Reed-Sternberg cells by >100-fold to over 12,000. Approaches such as density gradients, microdissection, and cell sorting help to enrich Reed-Sternberg cells for genomic DNA analysis. However, single-cell micromanipulation of living Reed-Sternberg cells was required to determine the genome-wide gene expression profile of these cells. Combined analysis of single cells and cell lines revealed the expression of 2666 named genes. Further analysis with high-density gene expression microarrays has demonstrated the expression of approximately 12,000 genes by Reed-Sternberg cells. The gene expression profile is that of an aberrant germinal center B-lymphocyte that resists apoptosis through CD40 signaling and NFkappaB activation. Gene expression analysis of Hodgkin's disease is an extreme test case demonstrating the application of high-throughput gene expression studies even to individual cells from clinical samples.

B-Lymphocytes↗
PubMed1993

Adult phenotype in the mouse can be affected by epigenetic events in the early embryo.

Major epigenetic modifications apparently occur during early development in the mouse. The factors that induce such modifications are complex and may involve the various components of a zygote. We have started to explore whether changes in the nucleocytoplasmic composition brought about by micromanipulation can induce phenotypic effects through epigenetic modifications. Nucleocytoplasmic hybrids were therefore prepared by transplanting a female pronucleus into a recipient egg from a different genotype. As a result, the maternal genome was of a different genetic background as compared with the egg cytoplasm. Specifically, experimental zygotes had cytoplasm from the inbred strain C57BL/6, a maternal genome from DBA/2, and a paternal genome from C57BL/6 (termed BDB hybrids). The mirror-image combination, termed DBD, was also made. The reconstituted zygotes were transferred to recipients and allowed to develop to term. Mice born from manipulated zygotes showed transcriptional repression and DNA methylation of major urinary protein genes in their liver, as well as growth deficiency resulting in reduced adult body weight. No altered phenotype was observed in controls in which the maternal pronucleus was simply transplanted back into another zygote of the same genetic background. These results clearly demonstrate phenotypic as well as molecular effects on DNA methylation and expression of at least one gene. Phenotype was therefore no longer predicted by genotype as a result of epigenetic modifications in experimental embryos. What precisely triggers the phenotypic and epigenetic changes is unknown, but presumably, nucleocytoplasmic interactions in hybrid zygotes may be partly responsible.

Animals↗
PubMed2004

Microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes.

We systematically examined the impact of microtubules on distribution of actin filaments and positioning of cell cleavage using micromanipulation to progressively alter the symmetric distribution of spindle microtubules in grasshopper spermatocytes. The initial microtubule asymmetry was induced by placing a single chromosome at one spindle pole using a microneedle, which facilitates regional assembly of spindle microtubules. We augmented chromosome-induced microtubule asymmetry by further removing the aster from the achromosomal pole, producing unichromosome-bearing monopolar spindles. We created the highest spindle asymmetry by cutting early anaphase cells in two, each containing a full set of segregating chromosomes in a half-spindle. We demonstrate that the location of the spindle midzone, distribution of actin filaments, and position of cell cleavage depend on the amount of microtubule asymmetry generated, shifting up to 48.6+/-3.8% away from the spindle equator in cut cells. The positional shift is dynamic, changing incessantly as spindle microtubules reorganize during cytokinesis. These results suggest that microtubules continuously dictate the distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes.

Actin Cytoskeleton↗
PubMed1997

Time scale dependent viscoelastic and contractile regimes in fibroblasts probed by microplate manipulation.

Many essential phenomena in biology involve changes in cell shape. Cell deformation occurs in response to physical forces either coming from the external environment or intracellularly generated. In most tests of cell rheology, an external constraint is usually superimposed on an already mechanically active cell, thus the measurements may reflect both active motion and passive viscoelastic deformation. To show that active and passive processes could be distinguished on a time scale basis, we designed a novel piezo-controlled micromanipulation system to impose dynamic mechanical deformations on individual cells. Chick fibroblasts were seized between two glass microplates; one of the plates, more flexible, served as a sensor of the applied force. Controlled amounts of unidirectional compression and traction in the range of 10(-8)-10(-7) N were applied, using either step functions or sinusoidal signals at chosen frequencies. These tests allowed identification of three time scale dependent regimes. (1) A dominant elastic response, characterized by a linear stress-strain relationship, was especially apparent at short times (seconds); (2) A viscous behavior, characterized by force relaxation and irreversible cell deformation, was noticeable at intermediate times (minutes). Data from traction and oscillatory excitation tests were well fitted by a three-element Kelvin viscoelastic model, allowing the calculation of two elastic moduli in the range of 600-1,000 N/m2 and an apparent viscosity of about 10(4) Pa.s. (3) A contractile regime, in which actin-dependent traction forces were developed in response to uniaxial load was apparent at longer times (several tens of minutes). These forces were in the order of 4 x 10(-8) N above viscous relaxation. Thus we could distinguish, on a time scale basis, the specific contributions of passive viscoelasticity and active traction, and evaluate their mechanical characteristics within one experiment on a single cell.

Animals↗
PubMed1980

Locomotion and cell-substratum contacts of Xenopus epidermal cells in vitro and in situ.

Studies of tissue cell locomotion in culture have revealed much about cell motility, but whether behaviour in vitro resembles movement of the same cells in the animal is not clear. To investigate this, I compared the locomotion and cell-substratum contacts of epidermal cells from Xenopus tadpoles, migrating from explants on glass and plastic, with the same cells spreading in vivo during wound closure. Time-lapse cinemicrography showed that in both cases, cells spread by extending broad lamellipodia across the substratum, and did not form microspikes, filopodia, or blebs. The net rate of translocation was significantly slower in vitro, however, because cells both protruded lamellipodia slower and spent more time stationary or withdrawing, compared with cells in situ. The increased fluctuation seemed in part due to greater tension within the expanding sheet in vitro, since when tension was reduced, for example by wounding, the cells spread with less fluctuation and at a greater rate (6.5 micrometers/min compared with 0.77 micrometers/min). Micromanipulation showed that cells adhered to the substratum, both in situ and in vitro, by a broad contact where transmission electron microscopy (TEM) of sectioned material showed the cells to be less than 30 nm from the substratum. A similar separation was observed beneath cells in vitro when viewed in life with interference-reflexion optics (IRM). A few focal contacts (adhesion plaques) were also seen with IRM and TEM of cells in vitro, but were not seen with TEM of cells in situ. Submarginal as well as marginal basal cells of the advancing sheet adhere and spread on the substratum in both situations, whereas cells of the outer layer are passive. Hence, the overall pattern of migration of these cells is similar in vitro and in situ; the differences in rates of movement may be explained in part by the different degree of tension in the epithelium under the 2 conditions.

Animals↗
PubMed1980

Ultrastructure of polytene chromosomes of Drosophila isolated by microdissection.

Drosophila polytene chromosomes prepared by a new micromanipulative procedure, which avoids acid squashing, have been examined at the ultrastructural level in the electron microscope. Puffs at 2B, 68C, 74EF, 75B and 85EF, have been examined in some detail, along with the chromocentre and various interbands. The ultrastructure of these chromosomes, which have never been exposed to acid protein denaturants, compares favourably with that of classical acid-fixed specimens. Ribonucleoprotein particles in puffs are seen to be organized in linear arrays and evidence is adduced for looped transcription units. Particles with characteristic sizes and morphologies are observed near the chromocentre, in puffs and in interbands. In interbands RNP particles and 'superbead'-like chromatin particles may be distinguished. Drosophila polytene chromosomes isolated by micro-manipulation should prove useful for the localization of native chromosomal proteins at an ultrastructural level.

Animals↗
PubMed2005

Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR.

Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. However, there are no accurate, rapid and appropriate methods to determine the exact copy numbers of particular mRNAs in a single cell. We therefore developed a procedure for isolating a single, identifiable cell and determining the exact copy numbers of mRNAs within it. We first isolated the cerebral giant cell of the pond snail Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior brought about by associative learning of feeding behavior. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs). These transcription factors play an important role in memory formation across animal species. The protocol uses two techniques in concert with each other: a technique for isolating a single neuron with newly developed micromanipulators coupled to an assay of mRNAs by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The molecular assay determined the mRNA copy numbers, each of which was compared with a standard curve prepared from cDNA solutions corresponding to the serially diluted solutions of Lymnaea CREB mRNA. The standard curves were linear within a range of 10 to 10(5) copies, and the intra-assay variation was within 15%. Each neuron removed from the ganglia was punctured to extract the total RNA directly and was used for the assay without further purification. Using this two-step procedure, we found that the mRNA copy number of CREB repressor (CREB2) was 30-240 in a single cerebral giant cell, whereas that of CREB activator (CREB1) was below the detection limits of the assay (< 25). These results suggest that the CREB cascade is regulated by an excess amount of CREB2 in the cerebral giant cells. Our procedure is the only quantitative analysis for elucidation of the dynamics of gene transcription in a single cell.

Analysis of Variance↗
PubMed1980

The viability of early cleavage stages containing half the normal number of blastomeres in the sheep.

The developmental capacity of embryos produced by injection of single blastomeres of 2-cell embryos, pairs of blastomeres of 4-cell embryos or 4 blastomeres of 8-cell embryos into foreign zonae pellucidae was studied. The micromanipulated embryos were embedded in tiny agar cylinders, transferred to ligated oviducts of ewes on Days 2-8 of their oestrous cycle and recovered when the total age of the embryos was 5 1/2 to 6 1/2 days. Of 78 embryos transferred, 69 (88.5%) were recovereod, and of the latter 65 (94.2%) had developed at a normal rate. Thirty of these 'half' embryos were transferred to ewes on Day 6 of the oestrous cycle: 24 (80%) developed into lambs. There were no differences apparent between embryos derived from the 2-, 4- or 8-cell embryos with respect to developmental capacity. A further 12 'half' embryos were stored by deep-freezing for 1 or 2 months. After thawing, 9 of these were selected for transfer to ewes on Day 6: 3 developed into lambs, each of which was the monozygotic twin of a lamb resulting from the first series of transfers.

Animals↗
PubMed1988

Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse.

Zona cutting and zona drilling of the mouse oocyte significantly increased the fertilization rate (3.8-90%) at low sperm concentrations (less than 200,000/ml) compared with zona-intact controls (0-45%). More oocytes were fertilized after zona drilling. Zona cutting was associated with a low loss of oocytes (less than 1%), no increase in polyspermy and normal development in vitro and in vivo after fertilization. There was a 4% oocyte loss rate after zona drilling, mostly due to extrusion of the oocyte from the zona during the procedure. Hatching of blastocysts occurred about 12 h earlier for zona-drilled than for zona-cut and zona-intact control oocytes. Zona drilling was associated with a higher, but not statistically significant, rate of polyspermy at all sperm concentrations tested. The proportion of zygotes developing to the blastocyst stage was not different between the techniques (zona cut, 77%; zona drilled, 66%; control, 71%). Similarly, no difference was found in the percentage of embryos implanting after blastocyst transfer to the uterine horns of pseudopregnant female mice (zona cut, 67%; zona drilled, 68%; control, 77%). Transmission electron microscopy demonstrated the induced defects in the zona with no damage to the oocyte or oolemma. Parthenogenetic activation was not seen after either of the micromanipulative techniques. Both techniques have promise for application to the human.

Animals↗
PubMed1991

Development of enucleated mouse oocytes reconstituted with embryonic nuclei.

The chromosomes of mouse oocytes at telophase of the first meiotic division were removed using micromanipulation and differential interference microscopy. The enucleated oocytes were used as recipients for nuclear transplantation, after culture for 4-6 h. The newly synthesized proteins of the enucleated oocytes showed the same pattern as those of secondary oocytes matured in vivo. When the enucleated oocytes received a nucleus from late 2- and 8-cell embryos, or a cell from the inner cell mass (ICM) of blastocysts, 23, 4 and 10%, respectively, of reconstituted embryos developed to blastocysts. After transfer to recipient females, live young were produced from the reconstituted eggs that received a nucleus from late 2-cell embryos.

Anaphase↗
PubMed1994

[Production of transgenic mice from in vitro fertilized eggs cryopreserved by ultrarapid freezing].

In vitro fertilized mouse eggs (C57BL/6N), followed by ultrarapid freezing were used for production of transgenic mice by microinjection of the chicken beta-actin promoter-driven the firefly luciferase cDNA (beta act-Luc). Following micromanipulation, the survival rates of the cryopreserved eggs and of the fresh in vitro fertilized eggs (control) were 70.8% (131/185) and 71.9% (159/221), respectively. After transferring them into oviducts of psudopregnant recipients on Day 1, 13.6% (17/125) of the cryopreserved eggs developed to live offspring and 14.1% (21/149) of fresh eggs did so. It was confirmed by Southern blotting analysis that each two transgenic mice were produced from the cryopreserved eggs (12%, 2/17) and the fresh eggs (10%, 2/21). All of transgenic mice produced from both eggs showed the expression of the luciferase gene. These results indicate that the in vitro fertilized eggs cryopreserved by ultrarapid freezing, can be, easily and conveniently, used for generation of transgenic mice.

Animals↗
PubMed1998

Culture of human insulinoma cells: development of a neuroendocrine tumor cell- and human pancreatic islet cell-specific monoclonal antibody.

We report on the culture of human insulinoma cells derived from a 32-year-old male patient with hyperinsulinism due to an insulinoma of the pancreas. A single-cell suspension was made by passing insulinoma fragments through a fine-gauge stainless-steel mesh. Cluster-forming insulinoma cells resembling pancreatic islets grew in the presence of fibroblasts. The insulinoma cell clusters could be differentiated from fibroblasts by using in situ pan optic staining and specific immunocytochemical staining (anti-human insulin and anti-human insulinoma monoclonal antibody (mAb) D24). mAb D24 was generated using insulinoma cells as antigen for immunization of a Balb/C mouse and cell fusion by the hybridoma cell technique. The anti-insulinoma cell mAb recognized a 32 kDa protein on immunoblot analysis of neuroendocrine tumor cells. D24 mAb also reacted immunohistochemically with normal pancreatic beta-cells and tumors such as vipoma, gastrinoma and carcinoid. Insulinoma cell clusters separated from fibroblasts by micromanipulation and plated into multiwell culture dishes exhibited an insulin-secretion rate of approximately 30 U/100 cells per 24 h with no insulin-secretory response to elevated glucose concentration. Purified insulinoma cells incubated with 1 ng/ml human nerve growth factor expressed neurofilament and neurite extension. These findings together with earlier observations in animal models suggest that human pancreatic beta-cells share some properties with neurons and are related to other neuroendocrine cells in the gastrointestinal tract.

Adult↗
PubMed1995

Instructing the animal physiology graduate student in human assisted reproductive technology.

Animal physiology graduate students provide an excellent personnel resource for laboratories performing human assisted reproductive technology (ART) procedures. However, the basic training of these students falls short of what is required for this highly specialized field. We designed a course to enhance their education in this area via classroom and hands-on laboratory instruction in a hospital and university setting. Topics covered in the course included in vitro maturation, in vitro fertilization, embryo culture, embryo transfer, quality control, quality assurance, micromanipulation, and cryopreservation. These techniques were applied to a group project to evaluate the influence of spermatozoal quality and quantity on early embryonic development in cattle and humans. Student grades were based on 1) oral and written examinations; 2) demonstrated competency in laboratory techniques; 3) presentation of class project data at a state academy of science meeting; and 4) initiative, determination, and interest in the coursework. Three aspects of the course stood out as very positive. First, the team approach to accomplishing a class project was new to some of the graduate students. Second, a bond was formed between hospital- and university-based faculty that did and will continue to foster unique teaching and research opportunities between the two groups. Third, the opportunity for students to present research data in a formal setting was very rewarding. This course made the students keenly aware of the many aspects of ART and provided them with specialized skills that should make them more marketable in the field of reproductive technology.

Animal Husbandry↗
PubMedDate not supplied

Recent advances in clinical/molecular andrology.

During the last decade there were extensive investigations in clinical and molecular andrology with emphasis on assisted reproduction, micromanipulation techniques of gametes, sperm/egg interaction, male contraception, diabetes mellitus, varicocele, andropause versus menopause, sexual dysfunction, associated hypertension/stress, prostatic carcinoma and molecular parameters of male reproduction. Sperm hyperactivation is a required step in capacitation sequence. Sperm motility is measured by videotape to evaluate the Straight Line Velocity (microm/s) (VSLI). Fertilization/embryonic development results from single sperm transfer (S-MIST) and multiple sperm transfer. Fertilization/embryo development is achieved by injection of immotile sperm into the perivitelline space. To assess sperm viability, a supravital stain suitable for use in combination with immunofluorescent assay, Hoeschst 33258, is used. The dye fluoresces with an intense blue when bound to DNA. To assess sperm plasma membrane integrity, a hypo-osmotic swelling test (HOST) is performed, using fluoresceinated D-mannose enriched albumin (FITC-DMA). The ability of sperm to swell under hypo-osmotic conditions indicates an intact membrane. A human protein, C-peptide, thought to be a useless byproduct of insulin may protect against devastating heart and nerve damage that diabetes causes. Human diabetics may benefit from the substance. Over 15 million Americans have diabetes, in which blood sugar levels rise out of control. There are two types of diabetics: Type I diabetics produce no insulin, the hormone that regulates blood sugar. Type II diabetics are unable to use their insulin properly. Diabetics are at great risk of heart disease and nerve damage, as arteries throughout the body leak and nerve-cell impulses fail. C-peptide is a byproduct of insulin production; it can be produced by the body or synthetically. Production of this protein is not induced by insulin, so diabetics who take insulin do not get C-peptide with it. Varicocele occurs unilaterally on the left side in 78% to 93% of men. Typically the presence of a varicocele is associated with an abnormal semen analysis (sperm density and morphology) and a decreased testicular volume on the affected side. Impaired sperm motility occurs in 89.5% of all varicocele patients. Varicocele ligation improves semen parameters in two thirds of patients. A few studies on andropause included sexual dysfunction, hormonal changes, medical/psychological correlates of impotence, ostenopenia/osteoporosis and bone loss; indices of bone remodeling, testosterone supplementation, androgen, negative feedback and hypothalamo-pituitary-testicular axis. Prostatic cancer is the second leading cause of cancer death for men between the ages of 60 and 80. Early detection involves a simple blood test for prostate specific antigen (PSA). Regular screening and early detection are essential. This is an important test because a high antigen count can be the only symptom. Since no screening is 100% accurate, physicians recommend both a PSA blood test and a physical examination. Although heredity plays a major role in whether a man will develop prostate cancer, men who lead healthy lives can dramatically reduce their chances of cancer: low-fat diet, eating plenty of fruits and vegetables and not smoking. Recent advances in molecular andrology include peptide hormone binding proteins; gonadotropin-releasing hormone (GnRH) agonists/antagonists analog; gonadotropins/their receptors; growth factors/reproduction; peptides as intratesticular regulators; molecular cloning of reproductive proteins/peptides. Gene cloning is applied for characterization/expression of genes coding. The interaction of gp120 with CD4 receptor plays a role in syncytium formation, apoptosis and CD4 cell deletion in human immunodeficiency virus (HIV) infection. The recombinant V3 peptide of fragment 307-330 of HIV-1 can induce sperm head agglutination. The generation process of react

Contraception↗