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At least 1,567 records · Page 87Linked to original sources
PubMed1993

Genomic analysis of human hepatocellular carcinomas using Restriction Landmark Genomic Scanning.

Restriction Landmark Genomic Scanning (RLGS) was used to examine the multiple alterations of genomic DNAs that occur in association with transformation and development of malignancy in primary hepatocellular carcinoma (HCC). Genomic DNAs from HCC and its normal counterpart were cleaved by the restriction enzyme NotI, radiolabeled at the cleavage sites, and then size-fractionated by two-dimensional electrophoresis using HinfI as the second cleavage enzyme. About 2000 spots were recognized, whose position and intensity reflect the locus and the copy number of the corresponding restriction sites. Using this system in combination with micromanipulation of HCC to eliminate possible carry-over of nonmalignant cells, we detected six spots that were decreased in intensity in common to three different HCCs, along with five that were intensified spots. In addition, several spots showed changes that were nonoverlapping among different tumors.

Carcinoma, Hepatocellular↗
PubMed1993

Determination of adhesion force between single cell pairs generated by activated GpIIb-IIIa receptors.

A biophysical approach was used to directly determine the avidity of the junction between two Chinese hamster ovary (CHO) cells bearing recombinant GpIIb-IIIa in the presence and absence of fibrinogen. Micromanipulation was used to induce conjugation of the cell pairs with or without activating the GpIIb-IIIa molecules with monoclonal antibody (MoAb) 62. Activation of GpIIb-IIIa caused an increase in the force required to separate the conjugates. The molecular bonding force between cells bearing activated GpIIb-IIIa and fibrinogen molecules was found to be 2.1 x 10(-7) dyne, which is 3.7 times higher than that between nonactivated GpIIb-IIIa and fibrinogen (5.7 x 10(-8) dyne). The results provide a quantitative assessment of the molecular bonding force between fibrinogen and the GpIIb-IIIa expressed on cell surface. The findings indicate that the activation of GpIIb-IIIa leads to an increase in the adhesive force in CHO cell aggregation by increasing the strength of the GpIIb-IIIa-fibrinogen bonds rather than the number of these bonds.

Animals↗
PubMed1993

Monoclonal antibody J11d.2 recognizes cell cycle-dormant, primitive hematopoietic progenitors of mice.

It was reported that monoclonal antibody (MoAb) J11d.2 reacts with mature blood cells of mice but not with their progenitors. We tested in culture studies whether this antibody could be used for enrichment for primitive marrow progenitors. The majority of colony-forming cells including multipotential progenitors in the marrow cells from 5-fluorouracil (5-FU)-treated mice were J11d.2+, whereas most of the progenitors from normal mice were J11d.2-. In addition, formation of multilineage colonies from J11d.2+ in both 5-FU-treated and normal mice was augmented by interleukin 6. These observations indicated that MoAb J11d.2 recognizes cell cycle-dormant progenitors. We have recently described a simple method that provides 800-fold enrichment for the progenitors in post-5-FU marrow cells using MoAb D7 (anti-Ly-6A/E). When this method was modified to include sorting with MoAb J11d.2, D7+ J11d.2+ cells were 2,250-fold enriched for multipotential progenitors. Micromanipulation and culture of individual D7+ J11d.2+ cells showed that average plating efficiency of the cell population is approximately 70% and that about 30% of the progenitors are lymphohematopoietic in nature. These data demonstrate that J11d.2 is a useful MoAb for the isolation of primitive hematopoietic progenitors of mice.

Animals↗
PubMed1993

[Labeling of yeast protoplasts by neutral red and nile blue for fusion experiments].

Neutral red and Nile blue were found to be suitable for staining protoplasts from various strains of Saccharomyces cerevisiae. The absorption of stains by the protoplasts was dependent on the pH value, for both stains. At a pH of 7.9 the protoplasts of the fusion partners could be stained differently and therefore it was possible to observe the fusion between the various strains of yeast. Consequently the prerequisites were created for the following micromanipulation.

Fluorescent Dyes↗
PubMed1993

Inhibitory effects of dopamine on noradrenaline-induced constriction of arterioles in vivo in the striated cremaster muscle.

The effect of dopamine on the arterioles (50.8-95.2 microns) in the cremaster muscle was examined to determine its effect on microcirculation. Anesthetized rats were used under a light microscope connected to a videocamera. Drugs were applied using small round filter paper (370 microns in diameter) containing the drug and placed in the immediate vicinity of the arteriole on the cremaster with a micromanipulator. The dose of the drug applied was represented by concentration of the drug solution in which the filter paper was immersed. Dopamine (10(-6)-10(-4)M) induced neither constriction nor dilation of the arteriole in the cremaster. Papaverine (10(-2)M) did not dilate the arteriole. However, the arterioles were constricted by noradrenaline (10(-6)-10(-4)M) and vasopressin (10(-7)M) in a dose-dependent manner. Noradrenaline (10(-4)M)-induced constriction was blocked by concomitant application of dopamine (10(-4)M). This effect of dopamine was antagonized by SCH23390 (10(-3)M). However, isoproterenol (10(-3)M) did not affect the arteriole, nor inhibit noradrenaline (10(-4)M)-induced constriction of the arterioles. While forskolin (10(-2)M) alone did not produce constriction or dilation of the arterioles, it inhibited noradrenaline (10(-4)M)-induced constriction of the arteriole. These results suggest that dopamine prevents the constriction of the arteriole induced by noradrenaline, by activation of DA1 receptors, which activates adenylate cyclase.

Abdominal Muscles↗
PubMed1993

Stapedectomy surgery in residency: KTP-532 laser versus argon laser.

Stapedectomy for otosclerosis is one of the most demanding otologic procedures, especially for residents in training. The reported series in the literature, as well as our own experience with the traditional resident-performed complete stapedectomy, have been disappointing. The number of patients available to train residents in stapedectomy techniques has steadily declined. The challenge to the otologist involved in teaching residents and fellows is to find a method that will be safe and effective. Over the past 5 years, we have had an opportunity at the University of Texas Medical Branch at Galveston to compare two methods of performing a laser stapedotomy. The two methods involve using the KTP-532 laser with a micromanipulator and using the hand-held argon laser. Although the wavelengths of these two lasers are similar, the methods used in applying them are very different. Twenty-five consecutive cases in each group were reviewed with regards to technical difficulty, complication rate, operative time, and air-borne gap closure. The hand-held argon laser emerged as the preferred technique for teaching a routine stapedotomy to residents.

Adult↗
PubMed1995

Negative regulation of early T lymphopoiesis by interleukin-3 and interleukin-1 alpha.

We recently developed a two-step clonal cell culture system for murine lymphohematopoietic progenitors that are capable of producing myeloid and B-lymphoid progenies and characterized their cytokine requirements. We subsequently observed that addition of interleukin-3 (IL-3) or IL-1 alpha to permissive cytokine combinations in primary culture abrogates the B-lymphoid potential but not the myeloid potential of the lymphohematopoietic progenitors. We now describe a similar negative regulation of the T-cell potential of the lymphohematopoietic progenitors. Lin- Ly-6A/E+ marrow cells from 5-fluorouracil-treated mice were plated individually by micromanipulation in methylcellulose culture with steel factor (SF) and IL-11 for 8 days. The resulting colonies were tested for myeloid potential by reculturing part of each colony in secondary myeloid suspension culture. Remainders of individual primary colonies were injected intravenously into scid mice for determination of T- and B-lymphoid potentials. Approximately 10% of the progenitors that differentiated along myeloid lineages in culture reconstituted T- and B-cell compartments in scid mice. However, when scid mice were injected with colonies pooled from cultures containing steel factor, IL-11, and either IL-3 or IL-1 alpha, there was no reconstitution of thymocytes or spleen T cells. These results suggest negative regulatory roles for IL-3 and IL-1 alpha in the early stages of T lymphopoiesis.

Animals↗
PubMed1995

[Intra-embryonic hematopoiesis in mice].

In vertebrate embryos, all hemopoietic tissues that are successively active during ontogeny (fetal liver, thymus, spleen and bone marrow) are colonized by extrinsic hemopoietic stem cells (HSC). The exception to this rule is the yolk sac (YS) whose progenitors arise in situ. Moreover, the YS is the first hemopoietic site to appear in the embryo. In consequence, it was considered as the primary source of the HSC that transfer from one site to the other until the demonstration that, in birds, the source of definitive HSC was located in the aortic region of the embryo. To assess a possible contribution of intraembryonic sites to mouse hemopoietic development, we developed an in vitro approach that permits the detection of multipotent hemopoietic progenitors in the region surrounding the dorsal aorta. This new site, the paraaortic splanchnopleura (P-Sp), is active from the 10 somite stage to the stage of fetal liver colonization by HSC. The in vitro analysis of the hemopoietic potential of the P-Sp revealed that: 1) progenitors present in this region are multipotent since a single micromanipulated P-Sp cell can give rise to mature B and T lymphocytes and various myeloid cells, when cultivated in appropriate conditions; 2) the P-Sp is the only intraembryonic site endowed with this potential; 3) numbering progenitors present in the YS and P-Sp indicated that precursors appear in both locations at the 10-12 somite stage reaching 15 in each site at 9.5 dpc (25 somites), shortly before the beginning of fetal liver colonisation. During a cytological study of the 9-11 dpc embryo aimed to disclose the cellular basis for our experimental results, two potential hemopoietic sites were uncovered. Cells clusters that resemble avian intraaortic hemopoietic clusters were found in the arteries located close to the coelomic cavity (dorsal aorta, umbilical and omphalomesenteric arteries). Moreover, groups of cells strikingly similar to YS blood islands were uncovered in the mesentery. All these sites are located in areas formed from the P-Sp and are fully developed at the onset of fetal liver colonization. These hemogenic sites most probably develop from the hemopoietic precursors present in the P-Sp.

Animals↗
PubMed1996

Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin/Reed-Sternberg cells of a Hodgkin's lymphoma patient.

A novel cell line, L1236, was established from the peripheral blood of a patient with Hodgkin's disease (HD). Two Ig VH and one Vkappa gene rearrangements were amplified by polymerase chain reaction (PCR) from the DNA of this cell line, demonstrating its derivation from the B-cell lineage. To test the cell line for its clonal relationship to the Hodgkin/Reed-Sternberg (H-RS) cells in the patient, single H-RS cells were micromanipulated from tissue sections of a tumor-infiltrated bone marrow specimen from that patient and analyzed by PCR for Ig gene rearrangements. The same rearrangements detected in the cell line were also found repeatedly in H-RS cells of the biopsy material. Thus, L1236 is the first cell line established from a case of HD for which a derivation from the H-RS cells of the patient could be demonstrated. Furthermore, the selective isolation of identical V gene rearrangements from multiple single H-RS cells demonstrates that these cells represented a clonal population in the patient.

Adult↗
PubMed1996

[Hodgkin disease--pathogenesis and therapy].

Hodgkin's lymphoma exerts characteristics of a true hematopoietic neoplasia as well as of an atypic immune response. In affected lymphatic tissue a minority of malignant Hodgkin-Reed Sternberg [H-RS] cells is surrounded by a majority of reactive, nonmalignant cells. Clinical features and biological studies suggest a pronounced, but inefficient, T-cell response against a yet undefined [viralS/B] target antigen expressed on H-RS cells. The role of the Epstein-Barr virus [EBV] which can be detected in the H-RS cells in about 50% of cases remains to be defined. By micromanipulation of single H-RS cells from frozen sections and subsequent enzymatic amplification of nucleic acids [single cell PCR] for the first time a monoclonal B-cell origin of H-RS cells could be demonstrated in several cases of Hodgkin's disease. Therapeutic strategies in Hodgkin's disease, in contrast to non-Hodgkin's lymphoma, are not based on the histological subtype, but rather on precise determination of the stage of disease. Treatment of choice in limited disease stages without risk factors is radiation therapy. Intermediate stages are treated with combined chemo- and radiation therapy, advanced stages with intensive chemotherapy [subsequent radiation only on bulk tumors]. Thus, about 85% of all patients suffering from Hodgkin's disease, can be cured definitely. High-dose chemotherapy followed by autologous stem cell transplantation in relapsed Hodgkin's lymphoma is currently proofed in clinical studies.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice.

Recently, the ligand for the Mpl receptor (ML) was identified to be thrombopoietin, the principal regulator of megakaryocytopoiesis and thrombopoiesis. We examined the effects of ML, as a single factor or in combinations with early acting factors such as steel factor (SF), interleukin (IL)-3, IL-1, IL-6, and granulocyte colony-stimulating factor (G-CSF), on colony formation from primitive progenitors of mice. Cells enriched for cell cycle dormant primitive progenitors were isolated from bone marrow cells of 5-fluorouracil (5-FU)-treated mice by a combination of Nycodenz density gradient separation, immunomagnetic selection for lineage-negative cells, and fluorescence-activated cell sorter (FACS) sorting for Ly-6A/E+Kit+ cells. ML, in the presence of erythropoietin, could support the formation of only a few megakaryocyte colonies. However, ML acted synergistically with SF or IL-3 to support the formation of multiple types of hematopoietic colonies including multilineage colonies. Effects of the combination of ML and SF on multipotential progenitors were not mediated through other cells, as demonstrated by micromanipulation of individual progenitors. In suspension culture, the combination of ML and SF increased the number of multipotential progenitors. ML also acted synergistically with IL-11, IL-6, or G-CSF to support colony formation in serum-containing, but not in serum-free, cultures. However, the multilineage colony formation seen in serum-containing culture was completely abrogated by addition of ACK2, a neutralizing antibody to Kit protein. Serial observation (mapping studies) of colony development from multipotential progenitors suggested that ML triggers the cell division of dormant progenitors. Based on these observations, we propose that ML can function as an early acting cytokine and stimulate the proliferation of cell cycle dormant progenitors by shortening their G0 period.

Animals↗
PubMed1996

[Evaluation of sperm morphology using the "strict" method].

The asset of evaluation of the morphology of sperm cells for prediction of their fertilizing power is generally accepted. Advances in the sphere of extracorporeal fertilization and the onset of micromanipulation techniques (intracytoplasmic injection of a single sperm cell - IC-SI) called for a stricter evaluation which is the prerequisite for a high standard indication and selection of different methods of extracorporeal fertilization. We obtained the consent of Prof. Gruger and Prof. Menkveld from the Tygerberg University (South African Republic) who elaborated this evaluation, to publish their criteria in our journal.

Humans↗
PubMedDate not supplied

[Vascular microanastomosis with diode laser. Control study and morphological results].

Direct diode laser carotid (0.7-1.2 mm) end-to-end microanastomosis versus contro-lateral manual suture microanastomosis (CMA) were performed in 70 Wistar rats. The laser beam-wavelength 830 nm- was transmitted through a micromanipulator and provided a focused spot of 300 microns in diameter. After placement of three 10.0 Ethilon stitches for edge coaptation, the laser anastomosis was performed using laser shots (average 3) of 500 mW power, 4.5 s duration and 700 W/cm2 irradiance each. CMA was performed by six 10.0 stitches. Good vascular flow was confirmed by macroscopic controls, Doppler spectral analysis (n = 548) and digitalized angiography (n = 18) performed from day 0 to day 210. The patency rate (93%) as well as the complication rate (5/66) were similar in both series. Nevertheless light and scanning electron microscopy (n = 82) showed the morphological superiority of laser anastomosis: reendothelialization by day 3 and medial collagen restructuration by day 10 whilst after CMA, the arterial wall repair was delayed and the medial scar occupied by fibrotic tissue. The other advantages of the laser anastomosis were, shorter operating time minimizing organ ischemia (13 min versus 22 min), and slight endothelial trauma reducing thrombogenic risk. The technical advantages of diode laser were pointed out: miniaturization, no special maintenance and decreasing price of diodes.

Anastomosis, Surgical↗
PubMed1996

Molecular cloning of a rhodopsin gene from salamander rods.

PURPOSE: Salamander photoreceptor cells have been used widely as models in vision research. However, the salamander opsin genes had not been cloned. The purpose of this study was to clone a salamander rhodopsin and to determine its primary structure and cell type-specific expression. METHODS: Using salamander retina RNA as a template and Xenopus rhodopsin-specific oligonucleotides as primers, reverse transcription and polymerase chain reaction (RT-PCR) were used to amplify and clone a rhodopsin cDNA fragment. This fragment was used as a probe to isolate a full-length cDNA of the rhodopsin from a cDNA library of salamander retina. The dideoxynucleotide chain termination method was used to determine the nucleotide sequence. Single rod and cone cells were isolated by micromanipulation, and the absorbance spectra of the rod outer segments were measured with a photon-counting microspectrophotometer. Individual rod and cone cells were lysed for RT-PCR and Southern blot analysis to detect cell-specific expression of this gene. RESULTS: A 1.2 kb rhodopsin cDNA containing the full-length coding region of rhodopsin has been cloned and sequenced from the larval tiger salamander, Ambystoma tigrinum. This cDNA encodes 354 amino acids that, by hydropathy profile, could form seven transmembrane domains characteristic of other rhodopsins. Sequence identity was found with other amphibian rhodopsins at the nucleic acid (82% to 83%) and the amino acid (88% to 89%) levels. Key amino acids critical for structure and function of rhodopsin have been retained. The mRNA of this rhodopsin was identified in red rod cells (lambda max 506 nm). No expression of the gene was detected in cone cells. CONCLUSIONS: The cloned rhodopsin is a newly isolated member of the G protein-coupled receptor superfamily. This protein is expressed in rods but not in cones.

Ambystoma↗
PubMed1977

An ocular fixation device for freehand pars plana vitrectomy.

An adaptation of the stereotaxic micromanipulator is described which provides ocular support and stabilization during freehand vitrectomy. It consists of a baseplate for immobilization of the patient's head and an ocular fixation arm assembly which connects to the eye by grasping a special Flieringa ring sewed to the postlimbal sclera. The ocular fixation arm can be made alternately flexible or rigid, thereby providing needed ocular mobility or stability during the operation as is desired by the surgeon.

Ciliary Body↗
PubMed1996

The genetics of male infertility.

PURPOSE: We provide an up-to-date summary of the genetic aspects of male infertility. MATERIALS AND METHODS: The literature on male infertility was extensively reviewed. RESULTS: Genetic defects are associated with a variety of clinical presentations by the infertile man ranging from gonadotropin-releasing hormone deficiency to spermatogenic failure to obstructive azoospermia. Microsurgery and micromanipulation of gametes make it possible for many of these men to father children. However, with each successive breakthrough in treatment of male infertility there is an increased risk of transmitting genetic abnormalities to the progeny. CONCLUSIONS: Transmission of genetic defects through assisted reproductive techniques can have serious long-term implications. Assisted reproductive techniques should not be initiated in men with a possible or known genetic cause of infertility without prior genetic counseling and risk assessment. Clinicians and researchers involved in reproductive medicine must recognize that, although these techniques have revolutionalized the treatment of male infertility, they have the risk of passing genetic abnormalities to the progeny. Therefore, researchers must proceed cautiously with development and application of assisted reproductive technologies to avoid creating future generations of genetically abnormal individuals. The first step in accomplishing this goal is through an increased understanding of the genetic basis of male reproductive failure.

Chromosome Aberrations↗
PubMed1996

Soluble thrombopoietin receptor (Mpl) and granulocyte colony-stimulating factor receptor directly stimulate proliferation of primitive hematopoietic progenitors of mice in synergy with steel factor or the ligand for Flt3/Flk2.

In an effort to establish the specificity of the thrombopoietin (TPO) effects on murine multipotential progenitors, we tested the effects of soluble TPO receptor (sTPOR; sMpl) on multilineage colony formation that was supported by a combination of TPO and steel factor (SF). Surprisingly, sTPOR did not suppress colony formation from primitive progenitors. This led to the discovery that sTPOR synergizes with SF or Flt3/Flk2 ligand (FL) to support the formation of various types of hematopoietic colonies including multilineage colonies. The colonies supported by the combination of sTPOR and SF were capable of expressing both myeloid and B-lymphoid potentials. Studies using micromanipulation and serum-free culture showed that the effects of sTPOR and SF on the primitive progenitors are direct, not mediated by contaminating stromal cells, and not dependent on factors present in the serum. TPOR belongs to the cytokine receptor group that includes granulocyte colony-stimulating factor receptor (G-CSFR) and erythropoietin receptor (EPOR). Therefore, we tested the effects of sG-CSFR and sEPOR on primitive progenitors. sG-CSFR, but not sEPOR, was able to synergize with SF or FL in supporting the proliferation of primitive progenitors. The direct effects of the soluble receptors appear to be mediated through interactions with their respective membrane-bound receptors expressed on the primitive hematopoietic progenitors.

Animals↗