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At least 595 records · Page 33Linked to original sources

PubMed1989

Effect on vasopressin release of microinjection of angiotensin II into the paraventricular nucleus of conscious rats.

We have studied in conscious unrestrained rats the role of angiotensin II receptors in the paraventricular nucleus (PVN) in the control of vasopressin secretion under basal conditions and after 24 h of water deprivation. In euhydrated rats, the microinjection rostral to the PVN of 1 and 5 ng of angiotensin II produced a transient, dose-dependent increase in the plasma vasopressin concentration. There was also a transient, but not dose-dependent, rise in mean arterial blood pressure (MABP), without a change in heart rate. However, the microinjection of angiotensin II into the PVN and sites caudal to it was without effect on the measured variables. In dehydrated rats, on the other hand, microinjection of 5 ng angiotensin II, both into the PVN and rostral to it, resulted in a prolonged increase in the plasma vasopressin concentration. The response from the rostral site was more rapid and was associated with an increased MABP. These findings suggest that angiotensin II receptors rostral to the PVN may participate in the control of vasopressin release in both euhydrated and dehydrated states, whereas angiotensin II receptors in the PVN are effective only following dehydration.

Angiotensin II↗

PubMed1972

Tracer microinjection study of renal tubular phosphate reabsorption in the rat.

To determine the sites of tubular phosphate reabsorption in the nephron, microinjection studies were undertaken, utilizing isotonic electrolyte solutions, containing either 1.4 or 8.0 mM phosphate and radioactive PO(4)-(33)P and inulin-(3)H, in rats made mildly diuretic by infusion of mannitol. The injected sites were localized by the technique of latex dissection. The relation between proximal tubular length and per cent (33)P recovery for injections of 1.4 mM phosphate (physiological amounts) suggest that relatively little reabsorption of phosphate occurs in the distal 30% of the proximal tubule compared with the proximal portion of the tubule. The corresponding recoveries for proximal tubular microinjections of 8.0 mM phosphate fall along a smooth curve tending to plateau with essentially complete (33)P recovery (> 95%) beyond 50% of the tubule. Absolute reabsorption of injected phosphate for both concentrations (i.e., absolute efflux per unit tubular length in the proximal tubule) was independent of phosphate delivery, since the relationship between reabsorption and site of injection was no different for the two concentrations. Distal convoluted tubular microinjections for both phosphate concentrations showed complete recovery of (33)P from all injection sites. THE DATA INDICATE THAT: (a) no phosphate reabsorption occurs in the distal convoluted tubule or in the collecting duct, (b) phosphate efflux per unit tubular length is greater in the first one-third of the proximal tubule than in the remaining two-thirds, and (c) in the last two-thirds of the proximal tubule, absolute phosphate reabsorption is relatively small and might be limited by factors other than the amount or concentration of injected phosphate.

Animals↗

PubMed2003

Development of a vibratory microinjection method.

To reduce cellular damage by pronuclear microinjection and nuclear transfer, we have recently developed a vibratory microinjection method. A micropipette was fixed to a piezoelectric ceramic with a resonance frequency of 70 kHz. When this micropipette was vibrated, it easily entered a mouse-fertilized egg without any sharp depression of the cell body, whereas a sharp, deep depression at the insertion site was observed when the micropipette was not vibrated. A depression rate defined as a rate of a depth of depression over an original cell diameter was utilized as an index of cellular deformation. The depression rates with and without vibration were 11.1 +/- 5.2% (N = 24) and 40.4 +/- 8.8% (N = 16), respectively (P < 0.0001, Student's t-test). In conclusion, the vibratory microinjection method is a new, useful option for gene transfer because it resulted in much less cellular deformation, therefore implicating less cellular damage.

Animals↗

PubMed1989

Expression of microinjected hsp 70/CAT and hsp 30/CAT chimeric genes in developing Xenopus laevis embryos.

The expression of microinjected chimeric genes containing Drosophila hsp 70 and Xenopus hsp 70 and hsp 30 promoters linked to the reporter gene coding for bacterial chloramphenicol acetyltransferase (CAT) was examined during early development of Xenopus laevis. Heat-inducible expression of fusion genes containing either the Drosophila hsp 70 promoter (1100 bp) or the Xenopus hsp 70 promoter (750 bp) was first detectable after the midblastula stage of development. This coincides with the embryonic stage at which the endogenous hsp 70 gene is first heat-inducible. A Xenopus hsp 30/CAT fusion gene containing 350 bp of promoter sequences was also heat-inducible after the midblastula stage unlike the endogenous hsp 30 genes which were not heat-inducible until the early tailbud stage (stage 23-24). Sequences that are present within either the coding or 3' region of the hsp 30 clone do not cause the microinjected hsp 30 gene to be developmentally regulated in a normal manner. Additionally, microinjected hsp 30 gene sequences have no effect on the developmental regulation of endogenous hsp 30 genes which continue to be activated at the tailbud stage of development. Our data suggest, that an inhibitory system, which may control the expression of the endogenous hsp 30 gene during development, does not regulate the expression of the injected hsp 30 gene.

Animals↗

PubMed1991

Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture.

In human skin fibroblasts microinjected with purified human immunodeficiency virus type 1 protease (HIV-1 PR), stress fibers were lost and alterations in nuclear morphology and condensation of nuclear chromatin were observed. Thereafter, the vimentin intermediate filament (IF) network collapsed. No effect was seen on the microtubules. While complicated by loss of affected cells from the substratum, a minimum estimate of the proportion of cells demonstrating these effects is 50%. Observation of single cells demonstrated that these effects were largely irreversible and were steps leading to the death of the HIV-1 PR-injected cells. After microinjection of various dilutions of the HIV-1 PR, it was observed that the changes in nuclear morphology and chromatin condensation were detectable under conditions where little or no effect was observed on both stress fibers and the IF network. Proteins of cells labelled with [35S]methionine and microinjected with either HIV-1 PR or BSA were subjected to two-dimensional gel electrophoresis. The major differences in the gel patterns were a diminution in the amount of vimentin and the appearance of novel products comigrating with cleavage products obtained after treatment of vimentin with HIV-1 PR in vitro. Thus, the HIV-1 PR is capable not only of cleaving IF subunit proteins in vivo, but also can catalyze alterations in other cellular structures.

Bisbenzimidazole↗

PubMed1992

Microinjection of antibodies against talin inhibits the spreading and migration of fibroblasts.

Talin is believed to be one of the key proteins involved in linking actin filaments to extracellular matrix receptors in focal adhesions. Our strategy for studying the function of talin has been to inactivate talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, well-spread morphology, but failed to migrate effectively. Our experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum.

Animals↗

PubMed1995

Import of microinjected proteins bearing the SKL peroxisomal targeting sequence into the peroxisomes of a human fibroblast cell line: evidence that virtually all peroxisomes are import-competent.

Peroxisomes import virtually all of their membrane and matrix proteins post-translationally. It is presently unknown whether, in mammalian cells, their exists a pool of mature peroxisomes which have received their complement of proteins and are import-incompetent. Previous work has shown that fibroblasts are capable of importing microinjected peroxisomal proteins into peroxisomes. This report describes the import of a hybrid peroxisomal protein into virtually all peroxisomes of the microinjected cell. The peroxisomal import was uniform in both short and long incubations. Pretreatment of the cells with cycloheximide did not affect the import of the peroxisomal protein, nor was there any difference in the distribution of the imported protein. Sequential microinjection experiments demonstrated that peroxisomes that had imported luciferase were capable of importing another peroxisomal protein injected 24 hours later. These results suggest that, in fibroblasts, all peroxisomes have associated protein-import machinery; this evidence does not support the hypothesis that there exists a pool of import-incompetent peroxisomes.

Cell Line↗

PubMed1986

Antidiuretic effects of methionine-enkephalin and 2-D-alanine-5-methionine-enkephalinamide microinjected into the hypothalamic supraoptic and paraventricular nuclei in a water-loaded and ethanol-anesthetized rat.

Effects of methionine-enkephalin (ME) and 2-D-alanine-5-methionine-enkephalinamide (DAMEA) microinjected into the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, which contain neurons synthesizing and releasing antidiuretic hormone, upon the outflow and the osmotic pressure of urine and the other visceral functions were studied in a rat which was loaded with water and anesthetized with ethanol. These opioid peptides when microinjected into the SON or PVN induced potent antidiuretic effects in dose-dependent and time-dependent manners with no significant effects on the other visceral functions. The approx. ED50 values for DAMEA were 1.3 (in the SON) and 0.7 (in the PVN) nmol, and the values for ME were 110 (in the SON) and 60 (in the PVN) nmol. The antidiuretic effects showed slow onset and long duration, with a minimal urine outflow at approx. 0.5 hr after microinjection and an approx. 2 hr-duration. The effects induced by the opioid peptides were inhibited by pretreatment with naloxone or atropine, without effects of pretreatment with alpha- or beta-adrenoceptor antagonists, suggesting that the antidiuretic effects were mediated through an opioid receptor having low sensitivity to naloxone and also possibly mediated through a muscarinic receptor which was stimulated probably by the ACh released by the opioid peptides.

Anesthesia↗

PubMed1987

Enhancement of gastric acid output and mucosal blood flow by tripeptide thyrotropin releasing hormone microinjected into the dorsal motor nucleus of the vagus in rats.

Central effect of thyrotropin releasing hormone (TRH) on gastric acid output and mucosal blood flow (MBF) was examined in urethane anesthetized rats. TRH, microinjected into the dorsal vagal complex [the dorsal motor nucleus of the vagus (NDV), the nucleus tractus solitarius and area postrema] induced dose-dependent (0.5-50 pmole) increases in gastric acid output and MBF. In contrast, 5 pmole TRH microinjected into various hypothalamic regions had no effect on these gastric parameters. Administration of TRH intraventricularly (i.c.v.) also increased these gastric parameters; however, about a 10 times higher dose of TRH was required to obtain the same order of excitatory effects seen with microinjection to the dorsal vagal complex. Application of anti-TRH serum to the dorsal vagal complex inhibited the increases in gastric acid output and MBF induced by TRH (i.c.v.). The effect of TRH applied to the dorsal vagal complex and i.c.v. were not modified by a concomitant administration of atropine. These results suggests that NDV is probably the site of action of TRH in inducing gastric hyper-functions. The mode of action of TRH seems to be independent of cholinergic muscarinic mechanisms present in the NDV.

Animals↗

PubMed2003

Cough reflex induced by microinjection of citric acid into the larynx of guinea pigs: new coughing model.

We developed a new coughing model that evoked coughs by microinjection of citric acid into the larynx in unanesthetized unrestrained guinea pigs; additionally, we recorded synchronous sounds and waveforms of coughing utilizing built-in microphones and a whole body plethysmograph. The coughing model was able to distinguish a coughing response from other expiratory responses, such as an expiratory reflex or a sigh, by examining the waveform of the expiratory response and the existence of sound. It was not necessary to distinguish a cough from a sneeze, since the administration site was restricted to the larynx. Microinjection of 0.4 M citric acid, total of 20 microl (10 times, 2 microl at 30-s intervals), induced coughs (27.03 +/- 4.03 coughs in 10-min observation) that were stable and independent of the inhalation volume. In the inhalation studies, animals were exposed to citric acid only once, because the number of coughs remarkably decreased with repeated administration at intervals of 24 h (tachyphylaxis). However our coughing model was able to repeatedly challenge the microinjection of citric acid at an interval of 24 h. These results indicated that this coughing model was highly sensitive and correctly assessed the cough response.

Animals↗

PubMedDate not supplied

The metabolism of microinjected inositol trisphosphate in Xenopus oocytes.

Microinjection of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) into Xenopus oocytes evokes a complex physiological response composed of a transient and a slow depolarizing chloride current. We investigated the relationship between intracellular levels of Ins(1,4,5)P3 and the kinetics of the physiological response. Microinjected Ins(1,4,5)P3 was slowly degraded following first order kinetics of disappearance (t1/2 = 10 min). The degradation products were inositol bisphosphate (InsP2), inositol monophosphate (InsP) and inositol, as well as inositol tetrakisphosphate (InsP4). The rate of degradation of injected 3[H]-Ins(1,4)P2 was much greater (t1/2 = 3 min), indicating that the conversion of InsP3 to InsP2 may be the rate-limiting step in the degradation process. The slow degradation of 3[H]-Ins(1,4,5)P3 was not a result of its conversion to Ins(1,3,4)P3 since no accumulation of InsP3 was observed within 10 min of microinjection of 3[H]-Ins(1,3,4,5)P4. Activation of protein kinase C (PK-C) with a phorbol ester transiently increased the rate of conversion of 3[H]-Ins(1,4,5)P3 to InsP2. This, however, did not significantly affect the overall kinetics of 3[H]-Ins(1,4,5)P3 disappearance. Our results indicate that the kinetics of Ins(1,4,5)P3 degradation do not correlate well with the termination of both the rapid and the slow components of the physiological response. The termination of the slow component of the response, however, may be related to the decay of Ins(1,4,5)P3-induced 45Ca efflux, which lasted about 10 min.

Animals↗

PubMed2000

Generation of transgenic rats with YACs and BACs: preparation procedures and integrity of microinjected DNA.

The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats from the standpoint of transgenic production efficiency and integrity of the introduced gene. In yeast artificial chromosome (YAC) transgenesis, three methods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the amplification element (AMP/RE), and no amplification of the YAC in yeast (AMP-). Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP-. Based on these results, we favor the AMP method in spite of the thymidine kinase gene-induced male sterility. In bacterial artificial chromosome (BAC) transgenesis, linear DNA fragments for microinjection prepared by three kinds of purification procedures were compared: Not I digestion and CsCl gradient ultra-centrifugation (Prep. 1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and beta-agarase digestion (Prep. 2), and CsCl gradient ultra-centrifugation, Not I digestion, pulse field gel electrophoresis, and beta-agarase digestion (Prep. 3). Although the efficiency of producing transgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep. 3 procedure. We therefore favor the Prep. 3 method for preparing BAC DNA fragments. These results indicate that the method used to prepare a large DNA fragment such as YAC and BAC DNAs is important in order to produce transgenic rats with an intact transgene.

Animals↗

PubMed1998

Respiratory effects of kynurenic acid microinjected into the ventromedullary surface of the rat.

Several studies demonstrate that, within the ventral medullary surface (VMS), excitatory amino acids are necessary components of the neural circuits involved in the tonic and reflex control of respiration and circulation. In the present study we investigated the cardiorespiratory effects of unilateral microinjections of the broad spectrum glutamate antagonist kynurenic acid (2 nmol/200 nl) along the VMS of urethane-anesthetized rats. Within the VMS only one region was responsive to this drug. This area includes most of the intermediate respiratory area, partially overlapping the rostral ventrolateral medulla (IA/RVL). When microinjected into the IA/RVL, kynurenic acid produced a respiratory depression, without changes in mean arterial pressure or heart rate. The respiratory depression observed was characterized by a decrease in ventilation, tidal volume and mean inspiratory flow and an increase in respiratory frequency. Therefore, the observed respiratory depression was entirely due to a reduction in the inspiratory drive. Microinjections of vehicle (200 nl of saline) into this area produced no significant changes in breathing pattern, blood pressure or heart rate. Respiratory depression in response to the blockade of glutamatergic receptors inside the rostral VMS suggests that neurons at this site have an endogenous glutamatergic input controlling the respiratory cycle duration and the inspiratory drive transmission.

Animals↗

PubMed1989

Subunit assembly and secretion of transthyretin: studies in a cell-free translation system and in microinjected Xenopus oocytes.

Transthyretin (TTR), or thyroid-binding prealbumin, is a protein of 55 kDa, composed of four identical subunits, which is synthesized by the liver and choroid plexus epithelium. In order to study the subunit assembly and secretion of TTR, cRNA encoding TTR was translated in a rabbit reticulocyte lysate or microinjected into Xenopus oocytes, and radiolabelled biosynthetic products were immunoprecipitated with an antibody against TTR and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. In the cell-free translation system in the absence of dog pancreatic microsomes, a single protein of Mr 17,000 was synthesized. In the presence of dog pancreatic microsomes, two proteins of Mr 15,000 and 37,000 were observed. The Mr 17,000 protein was identified as pre-TTR and the Mr 15,000 and 37,000 proteins as monomeric and dimeric forms of TTR. When the mRNA was microinjected into Xenopus oocytes both Mr 15,000 and 37,000 proteins were secreted into the media. It was shown that, under the SDS-PAGE conditions used in this study, the TTR tetramer dissociated to the dimeric form (Mr 37,000), but that there was no, or at least very little, further breakdown to the monomer. Therefore, to determine whether tetrameric or dimeric forms of TTR were secreted from the oocytes, the media from microinjected oocytes were subjected to gel permeation chromatography under non-dissociating conditions, and the eluted fractions analysed by SDS-PAGE. TTR eluted from the column as a dimer; there was no tetramer or monomer. The dimer, however, was completely dissociated to the monomer when analysed by SDS-PAGE, which suggested that incomplete or incorrect subunit assembly had occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Cardiovascular effects of microinjection of corticoids and antagonists into the rostral ventrolateral medulla in rats.

Experiments were performed on Wistar or Sprague-Dawley rats of both sexes. Microinjection of corticosterone (10 or 40 ng/0.1 microliter/site) into the rostral ventrolateral medulla (rVLM) caused an increase in systolic blood pressure (SBP), heart rate (HR) and pressor response induced by stimulation of the dorsal periaqueductal grey (dPAG) in the midbrain. Microinjection of aldosterone (10 or 40 ng/0.1 microliter/site) into the rVLM had similar effects showing a higher level and longer period than that of corticosterone. All these effects were dose-dependent. Microinjection of glucocorticoid antagonist RU 38486 (40 ng/0.1 microliter/site) or mineralocorticoid antagonist spironolactone (40 ng/0.1 microliter/site) caused a decrease in SBP, HR and the pressor response induced by stimulation of the dPAG. The inhibitory effects of spironolactone were more apparent. These results suggest that both corticoids could exert central modulatory effects on the resting cardiovascular activities and facilitate the pressor response during a defense reaction, and the rVLM is an essential area for the location of the central modulation. These effects may play an important role in the incidence and development of hypertension induced by stress.

Aldosterone↗

PubMed1998

Microinjection of protein tyrosine phosphatases into fibroblasts disrupts focal adhesions and stress fibers.

Microinjection and scrape-loading have been used to load cells in culture with soluble protein tyrosine phosphatases (PTPs). The introduction of protein tyrosine phosphatases into cells caused a rapid (within 5 minutes) decrease in tyrosine phosphorylation of major tyrosine phosphorylated substrates, including the focal adhesion kinase and paxillin. This decrease was detected both by blotting whole cell lysates with anti-phosphotyrosine antibodies and visualizing the phosphotyrosine in focal adhesions by immunofluorescence microscopy. After 30 minutes, many of the cells injected with tyrosine phosphatases revealed disruption of focal adhesions and stress fibers. To determine whether this disruption was due to the dephosphorylation of FAK and its substrates in focal adhesions, we have compared the effects of protein tyrosine phosphatase microinjection with the effects of displacing FAK from focal adhesions by microinjection of a dominant negative FAK construct. Although both procedures resulted in a marked decrease in the level of phosphotyrosine in focal adhesions, disruption of focal adhesions and stress fibers only occurred in cells loaded with exogenous protein tyrosine phosphatases. These results lead us to conclude that although tyrosine phosphorylation regulates focal adhesion and stress fiber stability, this does not involve FAK nor does it appear to involve tyrosine-phosphorylated proteins within focal adhesions. The critical tyrosine phosphorylation event is upstream of focal adhesions, a likely target being in the Rho pathway that regulates the formation of stress fibers and focal adhesions.

Animals↗

PubMed1989

Effect of microinjected catalytic fragment of protein kinase C on morphological change in Swiss 3T3 cells.

Although phorbol esters can enhance formation of an active, catalytic domain of protein kinase C (PKC) in intact cells, little is known about the actual importance of the proteolytic pathway in mediating cellular responses to the phorbol esters or other PKC activators. To explore this issue, we examined the effect of microinjected catalytic fragment of PKC on Swiss 3T3 cell morphology. In contrast to the dramatic, rapid response upon phorbol ester treatment, catalytic fragment microinjected in the presence of bovine serum albumin or normal goat immunoglobulin G as carrier protein had no effect. A morphological response similar but not identical to the effect of phorbol ester treatment was obtained, however, if catalytic fragment was microinjected in the presence of normal rabbit immunoglobulin G rather than the usual carrier proteins. The normal rabbit immunoglobulin by itself was inactive. Although the mechanism remains undefined, normal rabbit immunoglobulin but not other carrier proteins modulated PKC activity in vitro. We conclude that the generation of free catalytic fragment of PKC cannot account for the morphological response of Swiss 3T3 cells to the phorbol esters; secondary factors may, however, potentiate its action.

Animals↗

PubMed1999

Effects of microinjection of the D2 dopamine antagonist raclopride into the ventral tegmental area on ethanol and sucrose self-administration.

From previous microinjection studies, a reciprocal feedback between the nucleus accumbens and the ventral tegmental area (VTA) has been implicated in the reinforcing stimulus actions of ethanol and sucrose. In these studies, the effects of self administration of ethanol or sucrose solutions on maintained responding were similar when a dopamine antagonist was injected in the nucleus accumbens or a dopamine agonist was injected into the VTA. Our study was performed to determine if the effects on responding that had been observed when a dopamine agonist was injected into the nucleus accumbens would occur after an injection of a dopamine antagonist into the VTA. Male, Long-Evans rats were initially trained to lever press using either 10% ethanol or 75% sucrose solutions as the reinforcers. Bilateral guide cannulae were implanted to allow microinjection into the VTA of differing doses of the dopamine D2 antagonist, raclopride. Only at the highest dose tested (10 microg) was any effect observed on responding maintained by either reinforcer. The effect was minimal and different from that observed after the microinjection of a dopamine agonist into the nucleus accumbens. This suggests that either the actions of the nucleus accumbens agonist manipulation involved other processes or that the level of enhanced dopamine release in the nucleus accumbens from the VTA antagonist injection was not sufficient to mimic the effect of the nucleus accumbens agonist injections.

Alcohol Drinking↗