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At least 685 records · Page 38Linked to original sources

PubMed1993

Microinjection of dynorphin into the supraoptic and paraventricular nuclei produces antidiuretic effects through vasopressin release.

The mechanisms for the antidiuretic effects of dynorphin (DYN), an endogenous kappa-agonist, microinjected into the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei were investigated. DYN decreased the urine outflow rate dose-dependently from 5 to 20 nmol in the SON and PVN, and it increased vasopressin release. Microinjection of des-Tyr-DYN (a non-opioid peptide) into the SON produced antidiuretic effects with similar potency to that of the DYN-induced effects. However, in the PVN, the effects of des-Tyr-DYN were very markedly weaker than those of DYN. The DYN-induced antidiureses in the SON were partially inhibited by phenoxybenzamine, timolol and atropine, but not by naloxone. Those in the PVN were partially inhibited by naloxone, timolol and atropine, but not by phenoxybenzamine. Synthetic specific kappa-agonists, U50, 488H and Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro- Arg-Leu-Arg-Gly 5-aminopentylamide (DAKLI), microinjected into the PVN also produced antidiuretic effects in a dose-dependent manner. The order of antidiuretic potency was DAKLI > DYN > U50,488H, which was the same as that of kappa-receptor binding affinity. The DAKLI-induced antidiureses in the PVN were not inhibited by naloxone. These results suggested that DYN caused antidiureses by vasopressin release, through adrenergic and cholinergic mechanisms in the SON and PVN. Only the DYN-induced effects in the PVN were mediated, at least partially, through opioid receptors, perhaps the kappa-subtype.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed2000

FSH beta-subunit gene expression in long-term ovariectomized rat after pulsatile intracerebroventricular microinjections of GnRH.

OBJECTIVES: The purpose of this study was to examine whether in long-term ovariectomized rats direct pulsatile intracerebroventricular microinfusions of GnRH would result in frequency dependent biosynthesis of pituitary FSH beta-subunit mRNA. METHODS: Stainless steel cannula was stereotaxically implanted into the third ventricle of ovariectomized rats. 1 nM of GnRH microinjections were given at frequency 1, 2, 4 pulses/hour during 5 hours. Pituitary FSH beta-subunit mRNA level was determined by Northern-blot and serum FSH concentration was examined by RIA. RESULTS: Exogenous GnRH (1nM) induced a significant increase of pituitary content of FSH beta-subunit mRNA when administered 30 or 60 min intervals over 5 hours to ovariectomized rats. RESULTS: GnRH microinjections given at frequency of 1 pulse/hour were mostly effective resulting both in 85% increase of FSH beta-subunit mRNA level as compared to control as well as a significant (78%) stimulation of FSH release. CONCLUSIONS: our data show that in a long term ovariectomized rats (without steroid and gonadal peptides supplementation) a direct pulsatile intracerebroventricular microinjections of GnRH induce frequency-dependent pituitary FSH beta-subunit mRNA biosynthesis in the same mode as it was reported for gonadectomized and steroid replaced rats. It seems therefore that this method would represent an interesting alternative to the use of classical agents to disconnect, in vivo, the pituitary from hypothalamic GnRH influence. It may also provide a physiological data concerning regulatory aspects of gonadotropin subunits biosynthesis.

Journal Article↗

PubMed2002

Microinjection of adrenomedullin into rostral ventrolateral medulla increases blood pressure, heart rate and renal sympathetic nerve activity in rats.

The present study was undertaken to examine the effects of microinjection of adrenomedullin (AM) into rostral ventrolateral medulla (RVLM) on mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in 34 anesthetized Sprague-Dawley rats. The results obtained are as follows. (1) Following microinjection of AM (10 micromol/L, 200 nl) into the RVLM, MAP, HR and RSNA were significantly increased from 99.09+/-3.32 mmHg, 370.78+/-7.84 bpm and 100+/-0% to 113.57+/-3.64 mmHg (P>0.001), 383.28+/-7.38 bpm (P>0.001) and 123.72+/-2.74% (P>0.001), respectively. (2) Pretreatment with microinjection of calcitonin gene-related peptide receptor antagonist CGRP8-37 (100 micromol/L, 200 nl) did not change the effects of AM. (3) L-arginine (100 mg/kg, 0.2 ml, i.v.), an NO precursor, abolished the effects of AM. This study demonstrates that AM acting at the rostral ventrolateral medulla may produce significant cardiovascular responses, the effects are not mediated by CGRP receptor but may be abolished by NO.

Adrenomedullin↗

PubMed2003

[Sodium chloride step gradients centrifuge to fractionate foreign DNA fragment for microinjection].

DNA microinjection is the most popular and reliable method of producing transgenic animals. The purity of foreign DNA plays an important role for the success of microinjection. In this study, we introduced the use of sodium chloride step gradients in fractionating foreign DNA fragment for microinjection. The data demonstrated that,compared with the conventional agarose gel extraction method, NaCl purification scheme of toreign DNA could improve the treated embryo survival and foreign DNA intergration rate markedly.

English Abstract↗

PubMed1982

Differential effects of microinjections of d-amphetamine into the nucleus accumbens or the caudate putamen on the rat's ability to ignore an irrelevant stimulus.

Latent inhibition is an attentional process by which animals learn to ignore an irrelevant stimulus. Rats received either 0 or 30 preexposures to a tone which was later used as a conditioned stimulus (CS) in a two-way avoidance task. Tone preexposure resulted in retarded conditioning (i.e., latent inhibition) in animals which received microinjections of saline or amphetamine in the caudate-putamen and for those which received microinjections of saline in the nucleus accumbens. This latent inhibition effect, however, was not present in animals which received d-amphetamine microinjections in the nucleus accumbens. The failure of CS preexposure to retard conditioning in these animals was not due to drug-induced changes in either tone or shock sensitivity. The results are discussed in terms of the role of the mesolimbic dopamine system in learning to ignore an irrelevant stimulus and the use of LI as a possible animal model of the attentional deficit that seems to characterize some subpopulations of schizophrenic humans.

Animals↗

PubMed2026

A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.

Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.

Animals↗

PubMed2000

Transfer of foreign gene to giant freshwater prawn (Macrobrachium rosenbergii) by spermatophore-microinjection.

We developed a spermatophore-microinjection (SMI) technique that allows exogenous DNA fragments to be transferred easily into the giant freshwater prawn (Macrobrachium rosenbergii), an important aquacultural shellfish and aquatic invertebrate model. From 28 to 1, 000 ng of the circular plasmid pGL, in a total volume of 1 microl, were directly microinjected into spermatophores. Fertilization and hatching of prawns created with SMI were completed in vivo. Fertilization and hatching rates in the SMI treatments did not differ from those of the untreated control group. The genomes of free swimming, SMI-created larvae (21 days after fertilization) were analyzed using PCR and Southern blot analyses. A product with a molecular mass of 680 bp was amplified. It corresponded to amplifications of pGL, and Southern blot analysis revealed that the amplified band was positive. The gene transfer rate was primarily dependent on the concentration of DNA during SMI. The higher the concentration of pGL, the higher the rate of gene transfer. PCR and Southern blot analyses detected the existence of foreign DNA in 16 of 23 samples (70%) of genomic DNA isolated from hatched larvae in the 750 ng pGL SMI treatment. SMI, described here for the first time, is the simplest and most efficient method for mass producing transgenic giant freshwater prawns.

Animals↗

PubMed2001

Investigation of nucleo-cytoplasmic transport using UV-guided microinjection.

Active nucleo-cytoplasmic transport is mediated by dynamic signal-mediated pathways. We investigated the effects of transcription inhibitors or fluorescent lectins on nuclear import mediated by nuclear localization signals (NLSs). Therefore, a novel experimental approach that allows the controlled sequential introduction of fluorescent substances into living cells was established. A microinjection system equipped with an UV-source enabled us to identify fluorescent-labeled cells for the subsequent introduction of additional fluorescent compounds, in order to study their interactions in vivo. Cells were initially labeled either by expression of autofluorescent proteins or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited import mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipartite NLS revealed that the bipartite signal was more active in import. Sequential injection of differentially labeled nuclear import and export substrates allowed monitoring of import and export simultaneously in the same living cell. The introduced experimental approach will also be useful to analyze a variety of biological processes in living mammalian cells.

Amino Acid Sequence↗

PubMed1984

Expression of mRNAs microinjected into somatic cells.

The microinjection of macromolecules into living somatic cells has been used to assess the biological activity of a wide variety of macromolecules. This article describes and evaluates the methodology of microinjection with micropipettes and reviews information on the expression of globin mRNAs injected into normal and transformed cultured cells. A brief account is also given of other mRNAs that have been successfully translated in cultured cells.

Animals↗

PubMed1991

Inhibition of intracellular granule movement by microinjection of monoclonal antibodies against caldesmon.

Monoclonal antibodies, C2, C9, C18, and C21, against chicken gizzard caldesmon (called high molecular weight isoform) were shown to crossreact with a low molecular weight isoform of caldesmon in chicken embryo fibroblasts (CEF). These antibodies were used in a microinjection study to investigate the in vivo function of caldesmon in nonmuscle cell motility. Injected cells did not appear to change their morphology significantly; the cells displayed a flat appearance and were able to ruffle and locomote normally. However, in the C21 injected cells, saltatory movements of granules and organelles appeared to be greatly inhibited. This inhibition of granule movement was reversible, so that by 3 hr after injection, granules in injected cells had already recovered to normal speed. The inhibition of granule movement in cells injected with C2, C9, or C18 antibody, or with C21 antibody preabsorbed with caldesmon, were not significantly different from that in uninjected cells. In a previous epitope study, we demonstrated that, of the antibodies used in this study, only C21 antibody was able to compete with the binding of caldesmon to Ca++/calmodulin and to F-actin, although both C21 and C2 antibodies recognized the same carboxyl-terminal 10K fragment of gizzard caldesmon [Lin et al., 1991: Cell Motil. Cytoskeleton 20:95-108]. The caldesmon distribution in C21 injected cells changed from stress-fiber localization to a more diffuse appearance, when the injection was performed at 10-30 mg/ml of C21 antibody. We have previously shown that a monoclonal anti-tropomyosin antibody exhibited motility-dependent recognition of an epitope, and that microinjection of this antibody specifically inhibited intracellular granule movements of CEF cells [Hegmann et al., 1989: J. Cell Biol. 109:1141-1152]. Therefore, it is likely that tropomyosin and caldesmon may both function in intracellular granule movement by regulating the contractile system in response to [Ca++] change inside nonmuscle cells.

Actin Cytoskeleton↗

PubMed1987

Restriction enzyme evidence for Alu sequence-mediated dispersion of microinjected genes in transgenic mice.

A human bacteriophage clone containing adult beta-globin genes with four Alu sequences was microinjected to produce transgenic mice. Southern blot analysis on the spleen of a transgenic mouse revealed an unusual hybridization pattern that suggested extensive dispersion of human DNA throughout the mouse genome. This pattern was reproducible using several restriction enzymes, including a noncutting enzyme. The hybridization pattern was not observed in other tissues, and sequences were not detected in progeny using the bacteriophage probe. However, hybridization of spleen DNA of offspring against a human Alu probe revealed genetic transmission of human Alu sequences. The results suggest dispersion of microinjected Alu sequences throughout the genome.

Animals↗

PubMed1992

Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium.

Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA+ or cnrB+) of the donor 9-24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Single-cell microinjection of cytochrome c can result in gap junction-mediated apoptotic cell death of bystander cells in head and neck cancer.

BACKGROUND: Gap junction intercellular channels are required for metabolic cooperation between cells and regulate normal tissue homeostasis by means of the transfer of small molecules between contacting cells. Not surprisingly, the gap junction phenotype is frequently lost during carcinogenesis in human tissues (including those of the upper aerodigestive tract), freeing individual cancer cells from the growth control signals of normal surrounding tissues and less aggressive adjacent cancer cells. We hypothesized that gap junctional intercellular communication (GJIC) could mediate a bystander effect (apoptotic cell death) in squamous cell carcinoma of the head and neck (SCCHN) cells adjacent to individually targeted SCCHN cells. METHODS: Single-cell microinjection of cytochrome c was used to induce apoptosis in target SCCHN cells with endogenous GJIC activity and in an SCCHN cell line with exogenously introduced GJIC activity. Apoptosis was followed in target and surrounding bystander cells through light and time course microscopic characterization. All of the preceding experiments were carried out in the absence and presence of 18-beta-glycerretinic acid, a pharmacologic inhibitor of GJIC. RESULTS: When cytochrome c was introduced into SCCHN cells with endogenous GJIC activity through single-cell microinjection, bystander effects (apoptosis of nontarget cells) were observed. When GJIC activity was blocked with the specific pharmacologic inhibitor of gap junctions, 18-beta-glycerretinic acid, a bystander effect was never seen in GJIC active SCCHN cell lines. CONCLUSIONS: Gap junction intercellular channels can mediate a bystander effect in SCCHN. Inconsistencies in our data will be discussed in the context of recent advances in this field, as well as our future research directions.

Apoptosis↗

PubMed1983

Microinjection of human cell extracts corrects xeroderma pigmentosum defect.

Cultured fibroblasts of patients with the DNA repair syndrome xeroderma pigmentosum (XP) were injected with crude cell extracts from various human cells. Injected fibroblasts were then assayed for unscheduled DNA synthesis (UDS) to see whether the injected extract could complement their deficiency in the removal of u.v.-induced thymidine dimers from their DNA. Microinjection of extracts from repair-proficient cells (such as HeLa, placenta) and from cells belonging to XP complementation group C resulted in a temporary correction of the DNA repair defect in XP-A cells but not in cells from complementation groups C, D or F. Extracts prepared from XP-A cells were unable to correct the XP-A repair defect. The UDS of phenotypically corrected XP-A cells is u.v.-specific and can reach the level of normal cells. The XP-A correcting factor was found to be sensitive to the action of proteinase K, suggesting that it is a protein. It is present in normal cells in high amounts, it is stable on storage and can still be detected in the injected cells 8 h after injection. The microinjection assay described in this paper provides a useful tool for the purification of the XP-A (and possibly other) factor(s) involved in DNA repair.

Cell Extracts↗

PubMed1985

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression.

Adenine Nucleotides↗

PubMed2003

Sensitivities of benzodiazepine receptor binding and muscarinic acetylcholine receptor binding for the detection of neural cell death caused by sodium nitroprusside microinjection in rat brain.

Sodium nitroprusside (SNP) was microinjected into rat cerebral cortex and changes in muscarinic acetylcholine receptor (mAChR) binding and benzodiazepine receptor (BZR) binding were followed for 24 h after the infusion using [(3)H]-N-methyl-4-piperidyl benzilate ([(3)H]-NMPB) and [(3)H]-flumazenil, respectively, as a radioligand. The microinjection of SNP dose-dependently caused significant neural cell death 3 h after infusion, with the area of cell death becoming extensive 24 h after infusion. Neither SIN-1 nor NOC-18, other types of NO donors, caused neural cell death. Together with the result that deferoxamine, an iron-chelating agent, protected SNP-induced brain injury indicated important roles of iron-related radicals in SNP cytotoxicity in rat brain. In vitro [(3)H]-NMPB binding was significantly reduced in parallel with the time course of neural cell death detected by TTC staining and Nissl staining. In contrast, [(3)H]-flumazenil binding was essentially unaltered during the 24-h period after the SNP infusion. Similar results were observed in in vivo binding experiments. In vivo [(3)H]-NMPB binding was found to be much more sensitive at detecting cell death caused by SNP. On the other hand, [(3)H]-flumazenil binding in vivo was relatively insensitive to SNP-induced cell death. These results indicate that mAChR binding may be superior to BZR binding for detecting cell death in brain tissue, in contrast to what was previously thought.

Animals↗

PubMed1993

Locomotor activity following intra-accumbens microinjections of dopamine D1 agonist SK&F 38393 in rats.

The present study examined the effects of the dopamine D1 receptor subtypes agonist SK&F 38393 on locomotor activities after bilateral microinjection (0.00, 0.01, 0.1, 1.0, 10.0 micrograms) into the nucleus accumbens (Acb). The dose of 0.1 microgram elicited the highest response rate across measures of locomotion, rearing and stereotypy behavior. On the other hand, the largest dose of 10.0 micrograms was associated with significant increase in center time behaviors. The data were supportive of the hypothesis that dose-related locomotor activities elicited by microinjections of SK&F 38393 into the Acb are independently mediated by D1 receptors.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1991

Microinjection of cultured rat embryos: studies with retinol, 13-cis- and all-trans-retinoic acid.

Retinol, all-trans-retinoic acid or 13-cis-retinoic acid were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships of the retinoids showed that the dysmorphogenic effects were qualitatively similar for all three, but were elicited by a low concentration of all-trans-retinoic acid (250 ng/ml), a 6- to 7-fold higher concentration of 13-cis-retinoic acid and an approximately 16-fold higher concentration of retinol. After microinjection of 2,000 ng/ml of retinol, no dysmorphogenesis was observed but instead an increase in all growth parameters as compared to the controls.

Amnion↗