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Results for “Microinjection”

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PubMed1983

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin.

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.

Animals↗

PubMed1984

Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes.

Nuclei of amphibian oocytes contain large amounts of actin, mostly in unpolymerized or short-polymer form. When antibodies to actin or actin-binding proteins (fragmin and the actin modulator from mammalian smooth muscle) are injected into nuclei of living oocytes of Pleurodeles waltlii, transcription of the lampbrush chromosomes, but not of the rRNA genes, is inhibited. When transcription is repressed by drugs or RNA is digested by microinjection of RNAase into oocyte nuclei, an extensive meshwork of actin filament bundles is seen in association with the isolated lampbrush chromosomes. These observations indicate a close relationship between the state of nuclear actin and transcriptional activity and suggest that nuclear actin may be involved in transcriptional events concerning protein-coding genes.

Actins↗

PubMed1988

Microinjection of synthetic Xhox-1A homeobox mRNA disrupts somite formation in developing Xenopus embryos.

The structural similarity between Drosophila and vertebrate homeobox genes begs the question of whether the vertebrate gene products affect cell fate and pattern formation. To study the function of the Xenopus homeobox protein, Xhox-1A, we microinjected fertilized Xenopus eggs with an excess of synthetic Xhox-RNA and assayed for effects on development. The predominant phenotype is a disturbance in somite formation. When embryos are injected with Xhox-1A mRNA, but not with control mRNAs, morphogenesis of somites occurs chaotically and individual segments are lost. Histological staining, in situ hybridization, and immunohistochemistry indicate that the disorganized somitic tissue has differentiated into muscle cells. Overall, these results suggest that correct regulation of the Xhox-1A gene may be important for the normal development of the segmented somite pattern in early embryos. Moreover, the inferred role of Xhox-1A in somite formation indicates that there may be molecular parallels between mechanisms of segmentation in flies and vertebrates.

Animals↗

PubMed1988

Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies.

Early events in ligand-induced endocytosis of the EGF receptor have been examined. A mutant EGF receptor devoid of intrinsic protein-tyrosine kinase activity bound EGF and dimerized normally yet failed to undergo ligand-induced internalization. Immunofluorescence microscopy revealed that receptors lacking kinase activity failed to undergo the ligand-induced internalization characteristic of receptors with kinase activity. Monoclonal anti-phosphotyrosine antibodies effectively inhibited phosphorylation of exogenous substrates in vitro and, when microinjected into cells containing active EGF receptors, prevented internalization of the receptor when cells were subsequently challenged with EGF. These results point to a crucial role for the kinase activity of the EGF receptor in the process of ligand-induced endocytosis of receptors, and imply that a phosphorylated substrate(s) is required.

Antibodies, Monoclonal↗

PubMed1989

Microinjection of antibodies to a 62 kd mitotic apparatus protein arrests mitosis in dividing sea urchin embryos.

Previously, we described a 62 kd protein that is a component of the isolated sea urchin mitotic apparatus. This protein is a substrate for an endogenous, calcium/calmodulin-dependent protein kinase also associated with the mitotic apparatus. Phosphorylation of the 62 kd protein directly correlates with the depolymerization of microtubules in isolated mitotic apparatuses. Here we report a test of the function of the 62 kd protein in vivo. Double labeling studies using a monoclonal antibody to tubulin and an affinity purified antibody specific for the 62 kd protein reveal that the 62 kd protein co-localizes with mitotic apparatus microtubules. When affinity purified antibodies to the 62 kd protein were microinjected into dividing sea urchin embryos, mitosis was blocked in a stage-specific manner. The results are discussed with respect to the role of the 62 kd protein in the metaphase-anaphase transition.

Animals↗

PubMed1990

Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts.

We have examined the effects of elevating the intracellular levels of p34cdc2 kinase by microinjection into living mammalian cells. These studies reveal rapid and dramatic changes in cell shape with cells becoming round and losing the bulk of their cell-substratum contact. Such effects were induced at all times in the cell cycle except at S phase and were fully reversible at S phase or mitosis. Similar results were obtained with the homogeneous catalytic subunit of p34cdc2 kinase or p34cdc2 kinase associated with cyclin B. These alterations were accompanied by a marked reduction in interphase microtubules without the spindle formation, actin microfilament redistribution, and premature chromatin condensation. Although these changes closely mimic the events occurring during early phases of mitosis, p34cdc2 kinase-injected cells were not induced to pass further into division. These data provide detailed evidence that p34cdc2 kinase plays a major prerequisite role in the rearrangement of cellular structures associated with mammalian cell mitosis.

Actin Cytoskeleton↗

PubMed1986

The use of DNA-intercalating dye to monitor cell fusion and microinjection.

A conventional method for microinjection, using erythrocyte ghosts as the injection vector, has been modified to provide a protocol for the highly efficient delivery of small quantities of material into the cytoplasm of target cells. The technique is applicable for use with a variety of proteins, sugars, nucleotides and dyes. When the intercalating dye propidium iodide is included within the sealed ghosts their subsequent fusion with target cells can be continuously monitored by fluorescence spectroscopy, providing a convenient and sensitive parameter of cell-cell fusion. The protocol can be adapted for use with both adherent and non-adherent target cells, and can be used to monitor the relative effectiveness of a variety of fusogenic agents.

Cell Fusion↗

PubMed1988

Limitations of the technique of pressure microinjection of excitatory amino acids for evoking responses from localized regions of the CNS.

The aim of this study, performed on anaesthetized cats and rabbits, was to test the assumption that pressure microinjections of excitatory amino acids cause long-lasting excitation of neurones located close to the injection site. Unitary action potentials or antidromic field potentials were recorded from respiratory or 'reticular' neurones in the medulla oblongata and from phrenic motoneurones at different distances from the injection site. Injection of 10-150 nl (5-150 nmol) of L-glutamate or DL-homocysteic acid into these areas resulted in complex and widespread neuronal events. Generally, more distant neurones (500-1300 microns) were excited for variable periods of time (3-15 min), while neurones in the vicinity of the injection site (0-500 microns) showed, after a brief period of excitation time, a long-lasting (up to 30 min) decrease in excitability or silencing of discharge, probably due to a depolarizing block and disturbances in the ionic composition of the extracellular space. These findings show that interpretation of physiological responses following such injections should not be based on an assumption of local neuronal excitation. Some recommendations regarding the use of this technique are made.

Action Potentials↗

PubMed1995

Studies on the 3-dimensional architecture of dendritic spines and varicosities in human cortex by confocal laser scanning microscopy and Lucifer yellow microinjections.

A method for 3-dimensional (3-D) visualization of dendritic spines and varicosities in human cortical neurons is described. Intracellular microinjection of Lucifer Yellow was used to display the morphology of dendrites on pyramidal and non-pyramidal neurons. Confocal laser scanning microscopy was used for imaging, and 3-D reconstructions and analysis of spines and varicosities were performed. The frontal, temporal, parietal and occipital cortices, and hippocampus in normal and pathological human brains were studied. Using this technique spines can be visualized from both sides of dendrites, which are 'hidden' in 2-D representations, and therefore not usually included in the extimation of dendritic spine density/total spine numbers. In patients with Rett's syndrome and some epilepsy patients, a regional loss of dendritic spines ('naked' dendrites) was found. These results will be included in the Human Brain Mapping Project.

Adolescent↗

PubMed1994

Dual channel confocal laser scanning microscopy of lucifer yellow-microinjected human brain cells combined with Texas red immunofluorescence.

A method for visualization of individual human brain cells and their dendritic extensions in combination with immunofluorescence is described. Microinjection of Lucifer Yellow was used to reveal the dendritic morphology of cortical brain cells. Indirect immunofluorescence with Texas Red as label was used to investigate the distribution of 3 different groups of immunogens: enzymes (monoamine oxidase A and B), receptors (beta-adrenoceptor protein), and synaptic vesicle proteins (synapsin I and synaptophysin) in each cortical slice. A dual-channel confocal laser scanning microscope with an argon/krypton laser was used for imaging these double-stained fluorescent specimens. Lucifer Yellow and Texas Red were recorded simultaneously or separately, taking advantage of the different activating lines (488 lambda and 568 lambda) of the laser and using the two filter blocks (K1 and K2) supplied with the instrument (BioRad MRC-600) for recording the emission of either fluorophore. Using this technique we have demonstrated the localization of immunoreactive material in relation to the dendritic morphology of cortical cells.

Brain↗

PubMed1987

Projections of supraspinal structures to the phrenic motor nucleus in rats studied by a horseradish peroxidase microinjection method.

Autonomic nervous functions are considered to be influenced by the system controlling respiratory function, and vice versa. Recently, it became apparent that there is an interaction between the nervous systems controlling circulatory and respiratory functions, but anatomical knowledge about these control systems is still scanty. To compensate this lack of anatomical knowledge about the location of respiratory neurons, supraspinal structures projecting to the phrenic motor nucleus were studied using rats for purpose of comparison with our previous findings in the cat. After the microinjection of a small amount of horseradish peroxidase (HRP) into the phrenic motor nucleus, which was physiologically identified, some 2151 HRP-labeled cells were identified in the 5 selected experiments. These cells were distributed in the medulla (88.7%) and pons (11.3%). The majority were concentrated in and around the two nuclei involved in the cardiovascular control system, i.e. the nucleus para-ambiguus (55.5%) which is known as a pressor-tachycardia-apnea response zone and nucleus retrofacialis (10.3%) which is known as a depressor-bradycardia-apnea response zone. This distribution was similar to that in the cat except that there were many labeled cells in the nucleus tractus solitarii of the cat (21.5%) but only a few in the rat (1.0%). The insignificant solitario-phrenic projection in the rat suggests a different organization of respiratory nuclei in this species.

Animals↗

PubMed1988

Inhibition of baroreflex following microinjection of GABA or morphine into the nucleus tractus solitarii in rabbits.

The possibility that putative transmitters may influence the aortic nerve stimulation-produced bradycardia and depressor responses was examined in urethane- and chloralose-anesthetized, paralyzed and artificially ventilated rabbits. The ipsilateral microinjection of gamma-aminobutyric acid (GABA, 2-4 micrograms) or morphine hydrochloride (2-4 micrograms) into the nucleus tractus solitarii (NTS) area could partially block the evoked bradycardia and depressor responses produced by stimulation of the aortic nerve without influencing the basal blood pressure and heart rate. This blocking effect of either GABA or morphine was dose-related. Pretreatment with GABA receptor antagonist bicuculline methiodide (0.15-0.20 micrograms) and opiate receptor antagonist naloxone hydrochloride (1-2 micrograms) into the same medullary area completely abolished the effect of GABA and morphine, respectively. Application of bicuculline also greatly antagonized the effect of morphine, but the blocking effect of GABA on the evoked bradycardia and depressor responses still existed following the pretreatment of naloxone. These results indicate that GABAergic and opiate systems present at the NTS exert an inhibitory influence on the evoked baroreflexes and inhibitory effect of the latter may be related to the activation of GABAergic receptor in this nucleus.

Animals↗

PubMed1990

Complementation of N-hydroxyethyl-N-chloroethylnitrosourea hypersensitivity of Mex- cells by microinjection of mRNA from Mex+ cells.

Chinese hamster ovary (CHO-9) and HeLa MR cells lack detectable amounts of O6-alkylguanine DNA alkyltransferase (phenotypically Mex-) and are hypersensitive to the toxic effect of N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU), as compared to Mex+ derivatives. Microinjection of size-fractionated polyA+ mRNA extracted from HeLa S3 (Mex+) into CHO-9 and HeLa MR cells, or from ataxia telangiectasia (Mex+) into HeLa MR cells, gave rise to an increase in survival following treatment with toxic doses of HeCNU. Transient complementation of the Mex- phenotype was achieved with an RNA population 0.8-1 kb in size.

Animals↗

PubMed1986

Influence of atropine microinjection into nucleus reticularis thalami on activity of lateral geniculate nucleus neurones in freely moving rats.

The influence of the microinjection of atropine into the visual part of the nucleus reticularis thalami (TR) on flash-evoked unit responses of the dorsal lateral geniculate nucleus (dLGN) was investigated in freely moving rats. Atropine induced a significant prolongation and accentuation of the postexcitatory inhibitory phases in water-deprived trained rats, to which light flashes acted as conditioned stimuli and initiated a drinking behaviour. In satiated relaxed rats atropine did not change the unit activity significantly. Acetylcholine injected into TR caused a facilitation of the dLGN transmission. Since the decrease of flash-evoked excitatory response phases in thirsty, trained rats as compared to satiated ones could not be blocked by atropine, not all behaviourally dependent changes are mediated by a disinhibitory action of cholinergic fibres on TR.

Animals↗

PubMed1996

Bicuculline microinjections into the ventral tegmental area of the rat: alteration of self-stimulation thresholds and of cytochrome oxidase activity in the brain.

Abuse of drugs that potentiate GABAergic neurotransmission, namely benzodiazepines, is difficult to understand because this potentiation should elicit, among other effects, a decrease in activity within the mesolimbic system. Abuse of benzodiazepines is difficult to understand since the opposite, namely an increase in mesolimbic activity, has been implicated in drug abuse as well as in the rewarding effect of direct mesolimbic stimulation. In order to evaluate how the activity of the mesolimbic system depends on mesolimbic GABAergic influence, a GABAA receptor antagonist, bicuculline methiodide, was unilaterally injected into the ventral tegmental area and its effect on self-stimulation thresholds derived from stimulations applied to the same area was evaluated. Microinjection of 15, 20 and 30 ng increased the stimulation threshold. This decrease in stimulation efficiency lasted no more than 15 min after which baseline levels were obtained. Such a decrease is paradoxical considering that the manipulation should have released the ventral tegmentum from a tonic inhibitory influence. The metabolic consequences of repeated injections of 30 ng bicuculline were furthermore evaluated by cytochrome oxidase histochemistry. The staining was found to be weak around the injection site and dense in the ipsilateral nucleus accumbens. Release of a tonic GABAergic inhibition added to some cytotoxic damage probably resulted in an increased metabolic activity of this system. The presently reported paradoxical response of the ventral tegmentum and mesolimbic system to a GABAergic challenge may account for the paradoxical relationship between some behavioral properties of the mesolimbic system and GABAergic drugs.

Animals↗

PubMed1985

Translation of Schistosoma mansoni antigens in Xenopus oocytes microinjected with mRNA from adult worms.

Oocytes from Xenopus laevis microinjected with RNA isolated from Schistosoma mansoni adult worms translated antigens recognized by sera from infected rats, humans, and from immunized rabbits. The pattern of immunoprecipitated proteins analysed by SDS-polyacrylamide gel electrophoresis was species specific in rats. Serum from infected Fischer rats recognized antigens of 20, 27 and several bands in the 50-60 kDa range whereas serum from infected Brown Norway rats also immunoprecipitated major bands at 29, 43 and 100 kDa. Human infection sera gave a very variable pattern of immunoprecipitation not apparently dependent on the patients' age. At least 20 different antigenic species could be identified ranging from 14 to 150 kDa. Some S. mansoni antigenic proteins could be isolated from the membrane fraction of the oocytes whereas notably the 29 kDa band was present mainly in the soluble fraction. N-Glycosylation of S. mansoni antigens occurred as evidenced by the effects of tunicamycin treatment and concanavalin A binding. A multiple series of bands between 50 and 60 kDa, present in the membrane fraction, were glycosylated and secreted from the oocytes. Monoclonal antibodies to larval stage surface antigens failed to immunoprecipitate oocyte translation products, but sera absorbed with live schistosomula identified at least three putative surface antigens of 100, 43 and 29 kDa. However, the 29 kDa molecule was neither synthesized into membranes, nor secreted from oocytes.

Animals↗

PubMed1988

Microinjections of growth hormone-releasing factor into the medial preoptic area/suprachiasmatic nucleus region of the hypothalamus stimulate food intake in rats.

The role of the suprachiasmatic nucleus/medial preoptic area region of the hypothalamus in the expression of rat hypothalamic growth hormone-releasing factor-induced feeding in the rat was examined. Rats were tested for their 90-min food intake following microinjections of growth hormone-releasing factor (0.0, 0.01, 0.1 or 1.0 pmol) aimed at the suprachiasmatic nucleus/medial preoptic area region. It was found that growth hormone-releasing factor dose-dependently stimulated food intake with the 1.0 pmol dose being the most effective, increasing food intake by approximately 200%. Injections outside the suprachiasmatic nucleus/medial preoptic area region were ineffective. These data are taken to suggest that the suprachiasmatic nucleus/medial preoptic area region of the hypothalamus is important for the central stimulatory effects of growth hormone-releasing factor on feeding.

Animals↗

PubMed1990

Guanosine modifications in runoff transcripts of synthetic transfer RNA-Phe genes microinjected into Xenopus oocytes.

We have investigated whether unmodified yeast phenylalanine transfer RNA as well as one of its precursors containing an intron of nineteen nucleotides in the anticodon (pre-tRNA-Phe) can become substrates for selected tRNA modification enzymes present in a eukaryotic cell. This study was done by microinjecting into the cytoplasm of Xenopus laevis oocytes transcripts completely deprived of the naturally occurring modified nucleotides; these were obtained in vitro from appropriate synthetic genes under the control of bacteriophage T7 promoter. During the in vitro transcription, 32P labels were introduced with the guanosine triphosphate thus allowing easy detection of guanosine modifications in tRNA by two-dimensional chromatography after complete digestion into 5'-mononucleotides by nuclease P1. Results indicate that modifications occur on five guanosines (at positions 10, 26, 34, 37 and 46) in yeast tRNA-Phe and only on three guanosines (at 10, 26 and 46) in yeast precursor tRNA-Phe. These are the modifications expected from the known nucleotide sequences of naturally occurring Xenopus and yeast tRNA-Phe, i.e. N2-methyl-G10, N2,N2-dimethyl-G26, 2'-O-methyl-G34, N1-methyl-G37 or Y nucleoside-37 and N7-methyl-G46. The rates of modifications occurring in the two kinds of tRNA-Phe are faster in the intron-less tRNA-Phe than in the intron-containing tRNA-Phe. However quantitative modifications are only observed after as long as 75 h incubation in the oocytes.

Animals↗