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At least 1,315 records · Page 73Linked to original sources
PubMed1997

Mutants in the Saccharomyces cerevisiae RAS2 gene influence life span, cytoskeleton, and regulation of mitosis.

We investigated the phenotypic consequences in Saccharomyces cerevisiae of a disruption allele (ras2::LEU2) and of a dominant mutant form (RAS2ala18,val19) of RAS2. In addition to the phenotypes described earlier for these mutants, we observed a small increase in the life span for the disruption allele and a drastic decrease of life span for the dominant mutant form, as compared with the isogenic wild type. This was found by analyzing these alleles in two different genetic backgrounds with nearly the same results. Life spans were determined by micromanipulating mother cells and counting generations until no further cell division occurred. A morphological analysis of the terminal phenotypes of very old mother cells was performed showing enlarged or rounded cells and in some cases elongated buds, some of which were difficult to separate from the mother cell. This was observed in wild-type cells, as well as mutant cells. However, the dominant RAS2 mutant (but not the wild-type or ras2::LEU2 mutant cells) after 2 days on complex media displayed phenotypes similar to the terminal phenotype of old mothers. A substantial fraction of the cells were enlarged and generated elongated buds, they lost Calcofluor staining of the bud scars, the cell surface appeared folded, the actin cytoskeleton was aberrant, and the mitotic spindle and the cytoplasmic microtubles were defective in their proper orientation, resulting in aberrant mitoses and empty buds. These phenotypic characteristics of the RAS2ala18,val19 mutation could be causative for the previously observed rapid loss of viability of these cells in stationary phase.

Actins↗
PubMedDate not supplied

Microinjection into crane-fly spermatocytes.

We were successful in microinjecting fluorescently labelled material into crane-fly spermatocytes. In our experiments, we obtained four results. (i) In most attempts, the membrane stretched around the micropipette and prevented entry of fluorescent material, even when the micropipette appeared to be pushed completely through the cell. This confirms suppositions from earlier micromanipulation experiments that the elastic membrane prevents the micropipette needle from entering the cell. (ii) In some attempts, cells lysed upon contact with the micropipette. (iii) In other attempts, we successfully injected fluorescent material into cells. (iv) Fluorescent material left the cells after injection, often passing into adjacent cells. Although our success rate is low, microinjection into crane-fly spermatocytes is indeed possible.

Animals↗
PubMed2006

Pulling the chromatin.

Nucleosome is the basic subunit of the chromatin, which organizes the genomic DNA within the cell nucleus. It was understood in the last decade that beside the DNA compaction it plays an important role in the regulation of the gene expression. In its intact form, the nucleosome represents an important mechanical barrier and, among others, it prevents access to the DNA and blocks the transcription elongation. Therefore, it has become important to know the forces and energies necessary to destabilize the nucleosome in order to understand the DNA-related processes. Stretching the chromatin fibre using micromanipulation techniques (e.g. optical tweezers) is an ideal approach to study the nucleosomal stability and the parameters that can modify it. In this short review we will discuss the existing data and potential difficulties that this state-of-the-art technique still has to overcome.

Chromatin↗
PubMed1998

The origin of Hodgkin and Reed/Sternberg cells in Hodgkin's disease.

One of the characteristic features of Hodgkin's disease (HD) is the presence of a small population of often bizarre-looking large mono- or multinucleated Hodgkin and Reed-Sternberg (HRS) cells within the affected tissue. Recent cytogenetic investigations, studies of Epstein-Barr virus (EBV) genomes present in HRS cells, and analyses of Ig gene rearrangements amplified from single, micromanipulated HRS cells show that these cells largely represent clonal populations. The finding of Ig gene rearrangements in HRS cells in most cases of HD identifies B cells as the precursors of HRS cells in most if not all cases. Furthermore, the presence and pattern of somatic mutations within the rearranged Ig genes show that HRS cells in classical (i.e. nodular sclerosis, mixed cellularity, and lymphocyte depletion HD) as well as lymphocyte predominant (LP) HD originate from germinal center (GC) B cells. Ongoing somatic mutation and evidence for selection link HRS cells from LP HD to a mutating, antigen-selected GC B cell. In classical HD, the finding of "crippling" mutations and lack of stringent selection for antigen receptor expression suggests that in this case HRS cells are derived from a compartment of GC B cells that were destined to die but escaped apoptosis by some transforming event. One candidate for the latter is EBV infection.

Animals↗
PubMed2004

Biomimetic nanoscale reactors and networks.

Methods based on self-assembly, self-organization, and forced shape transformations to form synthetic or semisynthetic enclosed lipid bilayer structures with several properties similar to biological nanocompartments are reviewed. The procedures offer unconventional micro- and nanofabrication routes to yield complex soft-matter devices for a variety of applications for example, in physical chemistry and nanotechnology. In particular, we describe novel micromanipulation methods for producing fluid-state lipid bilayer networks of nanotubes and surface-immobilized vesicles with controlled geometry, topology, membrane composition, and interior contents. Mass transport in nanotubes and materials exchange, for example, between conjugated containers, can be controlled by creating a surface tension gradient that gives rise to a moving boundary or by induced shape transformations. The network devices can operate with extremely small volume elements and low mass, to the limit of single molecules and particles at a length scale where a continuum mechanics approximation may break down. Thus, we also describe some concepts of anomalous fluctuation-dominated kinetics and anomalous diffusive behaviours, including hindered transport, as they might become important in studying chemistry and transport phenomena in these confined systems. The networks are suitable for initiating and controlling chemical reactions in confined biomimetic compartments for rationalizing, for example, enzyme behaviors, as well as for applications in nanofluidics, bioanalytical devices, and to construct computational and complex sensor systems with operations building on chemical kinetics, coupled reactions and controlled mass transport.

Biological Transport↗
PubMed1986

O2 uptake in periportal and pericentral regions of liver lobule in perfused liver.

O2 uptake by the perfused liver decreased at O2 concentrations considerably higher than levels that caused NADH reduction when the input O2 concentration was varied. The maximal rate of O2 uptake was two- to threefold higher in periportal (137 +/- 8 mumol . g-1 . h-1; O2 concentration = 478 +/- 37 microM) than pericentral regions (59 +/- 5 mumol . g-1 . h-1; O2 concentration = 263 +/- 21 microM); however, the O2 concentration required for half-maximal O2 uptake was similar (approximately 20 microM) in the two areas. The infusion of atractyloside, antimycin A, or KCN inhibited O2 uptake in both zones by 50-85%, indicating that O2 uptake in both regions was largely dependent on mitochondrial electron transport. The content of ATP and ADP and ATP:ADP were similar in microdissected samples from periportal and pericentral areas. In contrast, when livers were perfused in the retrograde direction, O2 uptake was two- to threefold greater in pericentral than in periportal regions. Maximal rates of O2 uptake correlated with the local O2 concentration irrespective of the direction of flow when the electrode was moved across the liver lobule with a micromanipulator. Lower rates of O2 uptake in pericentral areas were not altered appreciably by infusion of agents known to uncouple oxidative phosphorylation (DNP), increase ADP supply (fructose), or increase the NADH redox state (ethanol or octanoate). These data are consistent with the hypothesis that maximal rates of O2 uptake are regulated, in part, in the perfused liver by O2 concentrations far above the Km of cytochrome oxidase for O2.

Adenosine Diphosphate↗
PubMed1977

Contractile stimuli in collecting lymph vessels.

Contractility was investigated in collecting lymph vessels of rat and guinea pig mesentery. Lymphatic diameter and micropressure were simultaneously recorded under normal conditions and during micromanipulation of intralymphatic pressure. Spontaneous activity involved characteristic changes in intralymphatic pressure coincident with opening and closure of upstream and downstream valves. Contractions were irregular but still predictable by noting the trend of intraluminal pressure as it approached a threshold level. Mechanical obstruction upstream of lymph vessels reduced lymph pressure and contraction frequency. Microinjection or withdrawal of fluid caused contraction frequency. Microinjection or withdrawal of fluid caused contraction frequency to rise and fall, respectively. Contraction rate was not affected by the level of general anesthesia. Lymphatic wall tension, as calculated from pressure-radius variables, correlated well with contraction frequency, suggesting a myogenic origin for the contractile mechanism. However, lymphatic smooth muscle may be inherently unstable since some contractility persists despite an absence of pressure stimuli.

Animals↗
PubMed1986

Microvascular distribution of coronary vascular resistance in beating left ventricle.

To determine the distribution of resistance in the coronary vasculature, measurements of microvascular pressure and diameter were obtained with vasomotor tone intact and during coronary dilation produced by papaverine. We studied anesthetized, open-chest cats and used jet ventilation synchronized to the cardiac cycle to eliminate respiratory-induced cardiac motion. The system for measuring microvascular pressure compensated for cardiac motion with stroboscopic illumination of the microvessels and a computer-controlled electromechanical micromanipulator that moved a micropipette in synchrony with the heart. Pressures were measured with the servonull technique, and diameters were measured via a video system. Resistance was estimated from the pressure gradient from the aorta to a particular class (size) of coronary microvessel. During control conditions, with coronary vasomotor tone intact, myocardial perfusion was 139 +/- 9 ml X min-1 X 100 g-1 and was increased to 339 +/- 52 during papaverine infusion (P less than 0.05). During control conditions (mean arterial pressure 70-80 mmHg), approximately 25% of total coronary resistance was proximal to 200-microns diameter arterioles and approximately 20% was produced by arterioles between 100 and 200 microns diameter; there was approximately 20-mmHg pressure gradient from the aorta to 200-microns arterioles and a 30- to 35-mmHg gradient from the aorta to 100-microns arterioles. Present results also indicate that 55% of total coronary resistance is distal to the 100-microns arterioles under control conditions. Coronary vasodilation with papaverine (mean arterial pressure maintained at 70-75 mmHg with an aortic snare) produced a redistribution of resistance; only 10% of total resistance was proximal to the 200-microns arterioles, and the pressure gradient from the aorta to these vessels was only 5-10 mmHg. Thus, under these experimental conditions with vasomotor tone intact, a substantial portion (45%) of coronary resistance resides in relatively large (greater than 100-microns) coronary arterioles, and the distribution of resistance and microvascular pressures can be shifted with coronary vasodilation.

Animals↗
PubMed1994

Measuring activation patterns of the heart at a microscopic size scale with thin-film sensors.

To study the spread of excitation in ventricular heart preparations we have designed a fast, high-resolution recording and mapping system. Papillary muscles were dissected from the isolated guinea pig hearts. The preparation was fixed in a tissue bath and superfused with Tyrode solution. Linear and two-dimensional arrays of Ag/AgCl electrodes were made on glass with a thin-film technique. The transparent sensors with up to 24 electrodes (spaced 50, 90, or 180 microns apart) were positioned close to the surface of the preparation with a custom-designed three-dimensional micromanipulator. Extracellular signals were simultaneously recorded by a 24-channel data acquisition system with a 200 kHz per channel sample rate, with 12-bit amplitude resolution and a maximum data length of up to 3 MB. Digitized video images of the electrode array and the underlaying preparation were used to identify the locations of the recording sites. A UNIX-based computer system with a custom-designed data acquisition and database program was used to control the instruments and to manage the experimental data. This technique gave signals with excellent signal-to-noise ratios (up to 65 dB) and permitted accurate evaluation of the time and the site of the local activation with high resolution (to within 5 microseconds, 50 microns). We describe the spread of excitation within the area of a few cells and found a substantial dispersion of conduction velocities. Beat-to-beat comparison of activation patterns showed relatively small variations in the spread of excitation (a few microseconds).

Action Potentials↗
PubMed1999

Separation of cardiomyocytes and coronary endothelial cells for cell-specific RT-PCR.

A simple method for analyzing the differential gene expression of coronary endothelial cells and cardiac muscle cells was developed. Cells were isolated from guinea pig hearts by collagenase digestion. In the diluted cell suspension, single cardiomyocytes and capillary fragments containing 6-15 endothelial cells could be identified morphologically. A simple "cell picker" was constructed using a polyethylene pipette with a tip diameter of approximately 150 micrometers that was attached to a micromanipulator and connected to an electric miniature valve. Intermittent suction pulses (1- to 2-cm water column) were applied by opening the valve for 100-200 ms at 1-s intervals. Cardiomyocytes (800-1,000) or capillary fragments (150) were picked under visual control using an inverted microscope. The cells were transferred to a reaction tube for RNA extraction, reverse transcription (RT), and DNA amplification (RT-PCR) with gene-specific and intron-spanning primers. All PCR products were verified by sequencing. Troponin T and endothelin-1 were found to be specific markers for guinea-pig cardiac muscle cells and coronary endothelial cells, respectively.

Animals↗
PubMed2000

A method for measuring the oxygen consumption of intact cell monolayers.

This report describes an open-air method for measuring the O(2) consumption (QO(2)) of intact monolayers of cultured cells. This method is based on Fick's second law of diffusion. It requires only a micromanipulator and a miniature O(2) electrode to measure the PO(2) gradient in the culture medium in the well. It was compared with the conventional oxygraph chamber method. Both methods gave the same value for QO(2) in freshly isolated rat type II cells: 166 +/- 15.3 nmol. h(-1). 10(6) cells(-1) for the open-air method and 151 +/- 11.6 nmol. h(-1). 10(6) cells(-1) for the oxygraph chamber method (n = 11 experiments). But the open-air method gave significantly larger values for QO(2) in cells cultured for 2 days (236 +/- 8.8 nmol. h(-1). 10(6) cells(-1)) than the oxygraph method (71 +/- 15.2 nmol. h(-1). 10(6) cells(-1); P < 0.001; n = 12 experiments). This suggests that the way cells are detached from their substratum to be placed in the oxygraph chamber affects their QO(2). The open-air method may be useful for studies on the metabolic properties of monolayers because the cells do not risk being damaged.

Animals↗
PubMed2002

Autoregulation of renal medullary blood flow in rabbits.

We examined the extent of renal medullary blood flow (MBF) autoregulation in pentobarbital-anesthetized rabbits. Two methods for altering renal arterial pressure (RAP) were compared: the conventional method of graded suprarenal aortic occlusion and an extracorporeal circuit that allows RAP to be increased above systemic arterial pressure. Changes in MBF were estimated by laser-Doppler flowmetry, which appears to predominantly reflect erythrocyte velocity, rather than flow, in the kidney. We compared responses using a dual-fiber needle probe held in place by a micromanipulator, with responses from a single-fiber probe anchored to the renal capsule, to test whether RAP-induced changes in kidney volume confound medullary laser-Doppler flux (MLDF) measurements. MLDF responses were similar for both probe types and both methods for altering RAP. MLDF changed little as RAP was altered from 50 to >or=170 mmHg (24 +/- 22% change). Within the same RAP range, RBF increased by 296 +/- 48%. Urine flow and sodium excretion also increased with increasing RAP. Thus pressure diuresis/natriuresis proceeds in the absence of measurable increases in medullary erythrocyte velocity estimated by laser-Doppler flowmetry. These data do not, however, exclude the possibility that MBF is increased with increasing RAP in this model, because vasa recta recruitment may occur.

Anesthesia↗
PubMed1996

Changes in the apical surface of chloride cells following acclimation of lampreys to seawater.

Scanning electron microscopy (SEM) was used to study the changes that occur in the morphological relationships between chloride and pavement cells in the gills during acclimation of young adult lampreys to seawater. Because chloride cells are located predominantly between lamellae and are thus obscured from view, the lamellae were removed with the use of a micromanipulator installed in a SEM. In gills of animals maintained in river water, chloride cells could then be seen to be dislike and typically to form single rows between successive lamellae. After acclimation to seawater, the apical surfaces of chloride cells lose their microvilli and change in shape from small circles to rectangles that extend the full width between successive lamellae. These changes result in an increase in the length of the paracellular pathway between chloride cells. Previous work has shown that the number of strands of the zonulae occludentes sealing this pathway declines under these conditions. This presumably leads to an increase in paracellular permeability of the gill epithelium, thereby providing the low-resistance paracellular shunt required for the passive movement of sodium into the environment during osmoregulation in seawater. The above changes are reversed by transfer of lampreys downward to 10% seawater.

Acclimatization↗
PubMed1994

Osmotic barrier of the parietal peritoneum.

Fluid movement into the peritoneal cavity results after instillation of a hypertonic solution. Some investigators have assumed that the peritoneum is a significant barrier to small solutes and have predicted that fluid would be drawn by an osmotic gradient into the cavity from the tissue surrounding the peritoneal cavity, resulting in tissue hydrostatic pressures well below atmospheric pressure. Contrary to this, we have previously shown that protein and fluid cross the peritoneum and enter the tissue at the same rate during either isotonic or hypertonic dialysis. To investigate the nature of the osmotic barrier of the peritoneum, the hydrostatic pressure profiles were measured in the abdominal wall of the rat during conditions of either isotonicity or hypertonicity in the peritoneal cavity and constant intraperitoneal hydrostatic pressure (Pip). Measurements were made with a micropipette mounted on a micromanipulator and connected to a servo-null pressure measurement system. No interstitial pressures below atmospheric pressure were observed with either type of solution in the peritoneal cavity. For the three Pip values tested, there were few significant differences between the corresponding pressure profiles of isotonic or hypertonic solutions. It is concluded that the parietal peritoneum is not a functional barrier to small solutes, which are often used to raise the osmolality of intraperitoneal solutions. This finding also implies that the tissue interstitium underlying the parietal peritoneum is not the source of water flow into the cavity, which is observed during hypertonic dialysis.

Abdominal Muscles↗
PubMed1994

Determinants of intrarenal oxygenation. I. Effects of diuretics.

To study renal cortical and medullary oxygen tensions, we used sensitive Clark-type O2 microelectrodes, inserted by micromanipulators into the cortex and medulla of kidneys of anesthetized rats. As previously reported, under basal conditions, medullary PO2 was significantly lower than cortical PO2. Furosemide, which inhibits reabsorptive transport in the medullary thick ascending limb, increased medullary PO2 from 16 +/- 4 to 35 +/- 4 mmHg (P < 0.0005) without altering cortical PO2. This effect, reproduced by ethacrynic acid and bumetanide, was selective for loop diuretics and was directly due to decreased tubular O2 consumption, since medullary blood flow was remarkably reduced by furosemide (-28 +/- 6% from baseline, P < 0.0001, as measured by a laser-Doppler probe). By contrast, acetazolamide, which decreases proximal tubule metabolism, selectively increased cortical PO2. These data are, in general, consistent with tubular metabolism as a major determinant of intrarenal oxygenation and suggest, in particular, that medullary reabsorptive work is at least in part responsible for renal medullary hypoxia.

Absorption↗
PubMed1995

Measurement of the EMG-force relationship in a human upper airway muscle.

The upper airway muscles play an important role in breathing, swallowing, and speaking, but little is known about the electromyogram (EMG)-force relationship of these muscles. We have measured the peak integrated EMG activity (iEMG) and force of human nasal dilator muscles (NDM) with a custom-designed headpiece that was attached via the forehead and upper lip. The headpiece contains a micromanipulator that holds a rod with a load cell mounted on its tip. The reproducibility of the force measurements was examined by measuring the lateral or "flaring" force of the NDM in multiple trials on two separate occasions in 13 subjects. For these studies the subjects were instructed to perform maximal voluntary contractions (MVCs). Test-retest reproducibility averaged 8.3% (coefficient of variation) for within-day comparisons and 13.7% between days. We also measured iEMG and NDM force during an incremental exercise test in nine of the subjects; they were instructed to breathe nasally throughout one 30-s epoch at rest and at each workload. The iEMG and force during peak exercise (175-275 W) averaged 81 +/- 26% (SD) MVC and 235 +/- 127 mN (approximately 75% MVC), respectively. The iEMG during incremental exercise was linearly related to the peak force (r = 0.90, P < 0.001). Contractile properties were measured in seven of the subjects by application of single supramaximal shocks (0.1-ms pulse) to the facial nerve. Twitch force averaged 9 +/- 6% MVC, and the time to peak force was 62 +/- 13 ms, which is considerably faster than that in human diaphragm or elbow flexors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Modulation of extracellular pH by glutamate and GABA in rat hippocampal slices.

1. Alkaline extracellular pH transients evoked by afferent stimulation, and local pressure ejection of glutamate and gamma-aminobutyric acid (GABA), were studied in the CA1 region of rat hippocampal slices. Amino acid-evoked responses were obtained by use of a dual micromanipulator, with the tip of a double-barreled pH-sensitive microelectrode positioned 50 microns from a pressure ejection pipette. 2. At 31 degrees C, in Ringer solutions buffered with 26 mM HCO3- and 5% CO2, mean extracellular pH in submerged 300-microns slices was 7.15 +/- 0.12 (n = 27 slices), at a tissue depth of approximately 150 microns. In Ringer buffered with 35 mM HCO3- and 5% CO2, extracellular pH was 7.29 +/- 0.10 (n = 19 slices). 3. Repetitive stimulation of the Schaffer collaterals caused an extracellular alkaline shift in stratum oriens, pyramidale, and radiatum, averaging 0.05 +/- 0.03 pH units among all regions (n = 138), with a maximum response of 0.16 pH units. Alkaline transients of similar appearance were obtained by local ejection of glutamate (0.01-0.12 pH units, n = 110) and GABA (0.01-0.18 pH units, n = 137). Control ejection of these amino acids into dilute agar caused only small acid shifts. 4. Superfusion of 100 microM picrotoxin abolished the GABA-evoked alkaline shift but failed to inhibit the Schaffer collateral- and glutamate-evoked alkalinizations. 5. Superfusion of 10(-5)-10(-3) M acetazolamide acidified the baseline by 0.05-0.10 pH units and amplified the Schaffer collateral- and glutamate-evoked alkaline shifts.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetazolamide↗
PubMed2000

Zona hardening, zona drilling and assisted hatching: new achievements in assisted reproduction.

Prior to fertilization, the zona pellucida surrounding the mammalian oocyte acts as a species-specific sperm barrier and is involved in sperm binding. After fertilization, the zona plays a role in blocking polyspermic fertilization, it protects the integrity of the preimplantation embryo during early embryonic development, and also helps its oviductal transport. Zona hardening occurs naturally after fertilization in order to ensure this threefold function. A combination of lysins produced by the cleaving embryo or the uterus and physical expansion then reduces the zona thickness in preparation for hatching. Zona hardening, although not readily quantifiable, may also be induced by in vitro culture and by in vivo aging. Indeed, prolonged exposure of human oocytes and embryos to artificial culture conditions seems to impair their ability to implant. Implantation rates are also inversely correlated with advanced female age. Recently, failure of the embryonic zona pellucida to rupture following blastocyst expansion has been put forward as a possible contributing factor in implantation failure. In order to help embryos escape from their zonae during blastocyst expansion, different types of assisted hatching have been developed. Zona drilling involves the creation of an opening in the zona with acidified medium, whereas zona slitting is carried out in the same manner as partial zona dissection. In zona thinning, the zona is just made thinner over a certain area without a hole or a slit being created. More recently, laser-assisted hatching has been introduced. In vitro studies with both mouse and human embryos have indicated that an artificial gap in the zona pellucida significantly improves the hatching ability of blastocysts grown in vitro as compared to non-micromanipulated embryos. However, the clinical relevance of assisted hatching within an assisted reproduction program remains controversial and elusive. Very few randomized studies are available. Most reports are of retrospective analyses which report either no differences in implantation and pregnancy rates between assisted hatching and control embryos or better results after assisted hatching. Five randomized controlled studies suggest that assisted hatching - of no benefit to the overall patient population - might be of value in increasing embryo implantation rates only in selected cases. No further evidence exists for an age-related benefit from assisted hatching in patients with advanced maternal age.

Animals↗