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[Analysis of transfer of microinjected zygotes in production of transgenic mice].

The data on transfer of mouse eggs microinjected with DNA during production of transgenic mice were analyzed. The transfer of mouse eggs into both oviducts did not lead to a reliably higher birth rate. It did not affect the frequency of recipients' pregnancy and, although somewhat increased the frequency of multiple birth, led, finally, to unjustified loss of the major part of viable DNA-injected eggs. We recommend transferring no less than 15 microinjected eggs only in one oviduct of each recipient. The transfer into another oviduct is acceptable if the transfer into the first oviduct failed or its outcome is doubtful.

Animals↗

PubMed1991

[The effect of microinjections of vasopressin into the inhibitory centers of the brain stem on rat behavior].

Microinjection of the arginin-vasopressin into the medial parabrachial nucleus, n. cuneiformis, raphe median nucleus and raphe magnus nucleus suppressed spontaneous locomotor activity and orienting response to electrical stimulation of hypothalamus in rats. Microinjections of 5,6-dihydroxytriptamine in n. raphe magnus blocked the inhibition of behaviour elicited by vasopressin.

5,6-Dihydroxytryptamine↗

PubMed1990

Effect of microinjections of apamin into the A10 dopamine region of rats: a behavioral and neurochemical analysis.

Hyperpolarization of dopamine neurons by activation of D2 and gamma-aminobutyric acidB receptors involves an increased conductance of K+ ions. Apamin, a blocker of Ca2(+)-activated K+ channels, has been reported to increase activity of dopamine neurons. Increased activity of the mesolimbic dopamine system is associated with increased motor activity. Thus, we investigated the behavioral and neurochemical effects of acute and daily microinjections of apamin into the A10 region of the rat. Apamin increased motor activity in a dose-dependent manner, and this effect was blocked by pretreatment with 0.1 mg/kg haloperidol. In postmortem analysis, 6.0 pmol of apamin significantly increased the levels of dihydroxyphenylacetic acid in the A10 region and of dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens, and 2.0 pmol of apamin significantly increased the level of dopamine in the prefrontal cortex. In vivo dialysis in the nucleus accumbens of freely moving rats revealed that apamin elevated extracellular dopamine metabolites. Rats receiving daily microinjections of apamin into the A10 region did not exhibit an augmentation in motor activity, suggesting that rats did not become sensitized to chronic treatment. These data are discussed in terms of the role of apamin-sensitive dopamine mechanisms in motor behavior and sensitization of these motor behaviors.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2006

[Magnesium ions prevent development of hyperkinesis produced by picrotoxin intrastriatal microinjections into the rat neostriatum].

The effects of daily bilateral microinjections of GABA-A receptor antagonist picrotoxin (2 mcg) into rostral neostriatum of the rats in chronic experiments were investigated. The picrotoxin was injected in volume of 1 mcl of saline or in 1 mcl of 1.0 M or 1.5 M MgCl2 solution; 1 mcl of saline or 1.0 M MgCl2 solution were injected in control groups of animals. The impairment of the avoidance conditioning in shuttle box, changes in free locomotor activity and the stereotypic choreo-myoclonic limb jerks were registered in rats with picrotoxin with the saline microinjections. The motor deviation registered are similar with Huntington chorea hyperkinesis in humans. The addition of magnesium ions into injected solution prevented both the lyperkinesis and condition behaviour realization impairment. As the magnesium is a universal calcium channel blocker, the calcium homeostasis of striatal neurons impairment as one of principal elements of hyperkinesis pathogenesis is proposed.

Animals↗

PubMed1990

De novo formation of cytokeratin filaments in calf lens cells and cytoplasts after transfection with cDNAs or microinjection with mRNAs encoding human cytokeratins.

We have studied the formation of new intermediate-sized filaments (IFs) by human cytokeratins (CKs) 8 and/or 19 in cultured bovine lens cells stably transfected with the corresponding cDNAs under SV40 promoter control. In the transfected cells, polypeptides of both type I and type II CKs were synthesized to near-equimolar amounts, formed heterotypic complexes and assembled into IFs with a peculiar tendency to accumulate into variously sized, often roundish aggregates in the juxtanuclear region, usually one per cell. Electron microscopy of these large CK IF aggregates revealed typical 7 to 12-nm IFs, tightly packed together in an apparently haphazard mode. By immunoelectron microscopy, the CK IFs could be readily distinguished from the vimentin IFs which were abundant in these cells. Electron microscopy also showed that many of the CK IF aggregates were located in the vicinity of the nucleus but did not have direct contact with the nuclear envelope; moreover, their location did not regularly correspond to those of the centrosomes and the Golgi apparatus. During enucleation of transfected cells in the presence of cytochalasin B, the CK aggregates were often retained in the cytoplast. After microinjection of CK 8 and 19 mRNAs, synthesized in vitro from cDNA molecules, into enucleated cytoplasts prepared from untransfected cells, CK IFs similar to those observed in microinjected whole cells were formed but often showed a wider cytoplasmic distribution. Our observations indicate that typical CK IFs can form, in vivo, in the absence of any nuclear structures. We discuss possible reasons for the tendency of the CK IFs to accumulate, in this cell line, into a juxtanuclear aggregate, in relation to similar CK-IF aggregates formed in certain normal cell types and upon toxic damages.

Animals↗

PubMed1992

Inhibition of DNA synthesis in living cells by microinjection of Gi2 antibodies.

Heterotrimeric guanine nucleotide binding proteins function in the coupling of a diverse span of cell surface receptors to a variety of intracellular signaling pathways, some of which stimulate cellular proliferation. With the recent discovery that mutated forms of G proteins are present in specific tumors, there has been an increased interest in the determination of the role of specific subtypes of G proteins in the regulation of cellular growth. We have attempted to determine which subtypes of G proteins are directly involved in serum-stimulated DNA synthesis through microinjection of inhibitory antibodies into living cells. Inhibitory rabbit polyclonal antibodies directed against specific Gi alpha subunits were introduced into living Balb/c 3T3 fibroblasts by microinjection, and the effect upon serum-stimulated DNA synthesis was examined. Results of these experiments indicate that Gi2 plays a direct role in serum-stimulated DNA synthesis in living cells and suggest that G proteins may function in a variety of mitogenic signaling pathways initiated by serum growth factors.

3T3 Cells↗

PubMed1991

Effect of human immunodeficiency virus type 1 protease on the intermediate filament subunit protein vimentin: cleavage, in vitro assembly and altered distribution of filaments in vivo following microinjection of protease.

The intermediate filament (IF) subunit protein vimentin is efficiently cleaved in vitro by purified human immunodeficiency virus type 1 (HIV-1) protease. Immunological data confirm that identical sites are cleaved when vimentin is polymerized into filaments or occurs as protofilaments. Preformed filaments require 10 times more protease to achieve the same extent of cleavage seen with protofilaments, suggesting that the cleavage sites are partially masked in IFs. The primary cleavage gives rise to molecule lacking most of the tail domain and which not only remains in preformed filaments, but also is capable of polymerizing into essentially normal 10 nm filaments. However, these filaments of the vimentin primary cleavage product show a propensity to form large lateral aggregates. The three secondary cleavage products of vimentin additionally lack portions of the head domain, are almost quantitatively released from preformed filaments and are not capable of forming filaments de novo. These results confirm and extend previous data obtained with desmin and provide a limit for that portion of the tail domain of type III IF subunit proteins that play a role in IF formation and stability. Microinjection of HIV-1 protease into cultured human skin fibroblasts resulted in a large increase in the percentage of cells with an altered and abnormal distribution of vimentin IFs. Most commonly, the IFs were observed to have collapsed into a clump with a juxtanuclear localization. The efficient cleavage of vimentin observed in vitro and the ability of microinjected HIV-1 protease to alter IF distribution in vivo suggest that IF proteins may serve as substrates within HIV-1 infected cells and may play a role in viral infection.

Amino Acid Sequence↗

PubMed1991

Microinjection of sulpiride into the nucleus accumbens increases ethanol drinking in alcohol-preferring (P) rats.

The effects of dopamine antagonists microinjected into the nucleus accumbens (Acb) on alcohol-drinking behavior were studied in the selectively bred alcohol-preferring (P) line of rats. P female rats (N = 8) were given access to food and water ad lib, while availability of a 10% (v/v) ethanol solution was limited to 1 hr/day. After implantation of guide cannulas bilaterally into the Acb and recovery from surgery, the rats were microinjected with either the D1 antagonist SCH 23390 (0.1 to 2.0 micrograms/side), the D2 antagonist sulpiride (0.1 to 2.0 micrograms/side) or vehicle, and ethanol intake was monitored. The D1 antagonist SCH 23390 had little effect on alcohol intake although the 0.5 ug/side dose produced a small increase which was not statistically significant. The D2 antagonist sulpiride increased, in a dose dependent manner, the intake of ethanol to as high as 215% of control values (F(4,28) = 39.9; p < 0.001). Additional experiments indicated that the 2.0 micrograms/side dose of sulpiride did not alter the consumption of a 14% glucose nor a 0.25% saccharin solution. These data suggest that D2 receptors in the Acb are important in the regulation of alcohol drinking by the P line of rats.

Alcohol Drinking↗

PubMed1990

The different changes of phrenic nerve activity and frequency elicited by microinjection of L-glutamic acid into ventrolateral nucleus of the tractus solitarius in cats.

There is only limited information of the neurotransmitters in central respiratory control. L-glutamic acid has been proposed as the primary neurotransmitter in the nucleus of the tractus solitarius (NTS) in cats. To test whether there is a respiratory effect of glutamic acid in the respiratory center or not, we microinjected L-glutamic acid (1 M, 0.1 microliter) via 1 microliter Hamilton microsyringe into the ventrolateral nucleus of NTS, namely dorsal respiratory group (DRG) over two-second intervals with continuous monitoring of phrenic nerve activity (PNA), frequency (f), end tidal CO2, blood pressure and heart rate. Glutamate induced various respiratory changes including: increase in PNA and f decrease in PNA and f [corrected] increase in PNA but decrease in f and decrease in PNA but increase in f. In addition to regular changes of PNA or f elicited by glutamate microinjection were found. Other patterns of irregular rhythmic changes such as absence of PNA, continuous phrenic nerve discharge and irregular phrenic nerve discharge with reflex apnea, apneusis and irregular respiration respectively were also observed. Glutamic acid appears to significantly modulate respiratory drive in DRG. We suggested that L-glutamic acid may be a neurotransmitter in the respiratory center and be involved in central respiratory control.

Animals↗

PubMed1990

[Effects of microinjection of tutin into the nucleus tractus solitarius on the phrenic nerve discharge in rabbits].

Experiments were performed on 44 urethane anesthetized, paralyzed, bilaterally vagotomized and artificially ventilated rabbits. Unilateral microinjection of different concentrations of tutin (0.1 microgram/microliter, 1 microgram/microliter, 5 micrograms/microliters, 10 micrograms/microliters, n = 8 for each group) into the nucleus tractus solitarius (NTS) region could induce reversible increase in respiratory frequency in the phrenic neurogram due to the shortening of inspiratory and expiratory duration and a decrease of the peak amplitude of the integrated phrenic nerve activity. However, no significant effects on arterial blood pressure (BP), heart rate (HR) and electroencephalogram (EEG) were noted. Of the four concentration groups of tutin application, 1 microgram/microliter group already attained a maximal effect. Microinjection of 5 micrograms/microliters and 10 micrograms/microliters tutin into NTS region produced further a disturbance of respiratory rhythm exhibited as transient phrenic nerve discharge during expiratory phase or irregular prolongation of expiratory duration. Unilateral injection of 1 microliter normal saline into NTS region (n = 8) had no significant effects on phrenic activity, BP, HR and EEG. The results suggest that NTS area might be involved in the transition of respiratory phases in the rabbit, and that tutin has a direct stimulating action on the respiratory phase switching mechanism of the NTS region, thus facilitating the transition of respiratory phases with an increase of respiratory frequency.

Animals↗

PubMed1990

Microinjection of D-2-amino-7-phosphonoheptanoate into the dorsal periaqueductal gray matter reduces the pressor response to glutamate injected at the same site.

The current study assesses the influence of the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-7-phosphonoheptanoate (AP7) on the pressor effect of glutamate microinjected into the dorsal periaqueductal gray matter (DPAG) of urethane-anesthetized rats. Glutamate (20, 40 and 80 nmol/site) caused dose-related reproducible increases in systolic and diastolic blood pressure. Microinjection of saline into the DPAG did not alter the pressor effects of glutamate (80 nmol/site). Similar pretreatment with AP7 (2 nmol/site) significantly (P less than 0.05) attenuated the pressor effects of glutamate from +26.5 +/- 7.0 to +3.4 +/- 3.3 mmHg (systolic blood pressure). We conclude that the pressor effect of glutamate in the DPAG is mediated largely by activation of NMDA receptors.

2-Amino-5-phosphonovalerate↗

PubMed1990

Ventricular arrhythmia evoked by microinjection of picrotoxin into brain areas in rabbits.

To explore the main areas of brain responsible for arrhythmia evoked by icv picrotoxin (Pic), a very small dose of Pic (3 micrograms) was injected into different areas of brain in anesthetized rabbits. The short latency of arrhythmia was found after microinjection of Pic into posterior hypothalamus (PH, 5.6 +/- 0.9 min) and the medial part of tuberal region (7.5 +/- 2.9 min). The ventricular arrhythmia was abolished by vagotomy, iv phentolamine or diazepam, or pretreatment with adrenalectomy or vagotomy. Microinjection of phentolamine or diphenhydramine into PH prior to Pic postponed the arrhythmia. The results indicate that PH and the medial part of tuberal region are the most effective areas for Pic to induce arrhythmia, activations of both sympathoadrenomedullary and parasympathetic systems are involved in the genesis of arrhythmia.

Adrenalectomy↗

PubMed1989

Different respiratory patterns elicited by microinjection of L-glutamic acid into the ventrolateral nucleus of the tractus solitarius in cats.

Only limited information relating neurotransmitters to central regulation of the respiratory system exists. L-glutamic acid has been proposed as the primary neurotransmitter in the nucleus of the tractus solitarius (NTS) of cats. To test whether or not glutamic acid has an effect on the respiratory center, we microinjected L-glutamic acid (1 M, 0.1 microliter) via a 1 microliter Hamilton microsyringe into the ventrolateral area of the NTS dorsal respiratory groups (DRG) of unanesthetized, decerebrated cats at two-second intervals with continuous monitoring of tidal volume (VT), respiratory rate (f), end tidal CO2, blood pressure and heart rate. The results showed that glutamate induced the following respiratory changes: VT and f increased; VT and f decreased; and VT decreased but f increased. In addition to VT or f changes elicited by glutamate microinjection, changes in other rhythmic patterns such as apnea, apneusis and irregular respiration were observed. Glutamic acid appears to play a significant role in the modulation of the respiratory drive in the DRG. We, therefore, suggest that the excitatory amino acid L-glutamic acid may be involved in central respiratory control.

Animals↗

PubMed1989

Examining protein translocation in cell-free systems and microinjected Xenopus oocytes.

A variety of assays have been developed which permit rapid and unambiguous determination of the membrane topology adopted by newly synthesized proteins. Cell-free systems and microinjected Xenopus oocytes are two of the most attractive approaches for characterizing the elements in both the nascent polypeptide and the membrane which together determine the final orientation of the protein in the membrane. Careful analysis of the mechanism of protein translocation using these methods has revealed a number of unusual topologies. The applications of a number of different assays for endoplasmic reticulum membrane translocation are described for the most commonly used cell-free systems (wheat germ and reticulocyte lysate), as well as for microinjected Xenopus oocytes.

Animals↗

PubMed1989

Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA when microinjected into Xenopus oocytes.

Ricin, Shiga toxin, and Shiga-like toxin II (SLT-II, Vero toxin 2) exhibit an RNA N-glycosidase activity which specifically removes a single base near the 3' end of 28 S rRNA in isolated rat liver ribosomes and deproteinized 28 S rRNA (Endo Y., Mitsui, K., Motizuki, M., & Tsurugi, K. (1987) J. Biol. Chem. 262, 5908-5912; Endo Y. & Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130, Endo, Y., Tsurugi, K., Yutsudo, T., Takeda, Y., Ogasawara, K. & Igarashi, K. (1988) Eur. J. Biochem. 171, 45-50). These workers identified the single base removed, A-4324, by examining a 28 S rRNA degradation product which was generated by contaminating ribonucleases associated with the ribosomes. To determine whether this N-glycosidase activity applies in living cells, we microinjected ricin into Xenopus oocytes. We also microinjected Shiga toxin and a variant of Shiga-like toxin II (SLT-IIv). All three toxins specifically removed A-3732, located 378 nucleotides from the 3' end of 28 S rRNA. This base is analogous to the site observed in rat 28 S rRNA for ricin, Shiga toxin, and SLT-II. Purified, glycosylated, ricin A chain contains this RNA N-glycosidase activity in oocytes. We also demonstrated that the nonglycosylated A subunit of recombinant ricin exhibits this RNA N-glycosidase activity when injected into Xenopus oocytes. Ricin, Shiga toxin, and SLT-IIv also caused a rapid decline in oocyte protein synthesis for nonsecretory proteins.

Animals↗

PubMed1989

[New data on the type of structural changes in DNA of simian adenovirus SA7 microinjected into mouse zygotes].

The structure of the transgene has been analysed in a new series of experiments on the transfer of adenovirus SA7 DNA into the mice zygotes by microinjection technique. The previous data on SA7 DNA elimination from the genomes of different organs (sceletal muscles, heart, tail) have been confirmed and detailed for the F0 and F1 generations of transgenic animals. The left end of adenoviral genome has been shown to be predominantly transfered after microinjections of SA7 DNA into the mice zygotes.

Adenoviridae↗

PubMed1988

Renal action of a novel uricosuric diuretic, S-8666. I. Clearance and tubular microinjection studies in rats.

Clearance and tubular microinjection techniques were used to evaluate the effects of a novel uricosuric diuretic, S-8666, on renal function and tubular absorption of urate by the rat kidney. Tubular sites of diuretic action of S-8666 were determined indirectly using osmolar clearance techniques. The i.v. injection of S-8666 at a dose ranging from 0.3 to 3.0 mg caused a dose-dependent increase in urine flow and sodium excretion. Potassium excretion was increased significantly but the increase was not marked as compared with sodium excretion. Glomerular filtration rate was not changed by S-8666. The diuretic response reached a maximum within 5 min and was retained for 45 min with 1 mg of S-8666. The comparison with the effect of furosemide revealed that furosemide was 13 times more potent than S-8666. Both the free water reabsorption on hydropenia and free water clearance in hydrated animals decreased with administration of S-8666. The urinary excretion of urate increased significantly after the administration of S-8666. By contrast, furosemide did not increase urinary excretion of urate. Total urinary urate recovery after S-8666 administration was higher after the microinjection of [14C]urate into early proximal tubule sites. We conclude that S-8666 acts as a uricosuric diuretic agent with the major site of altered urate absorption being in the proximal convoluted tubule and the major site of diuretic action being in the cortical and medullary diluting segments.

Animals↗

PubMed1986

Microinjection of the ras oncogene protein into nonestablished rat embryo fibroblasts.

Early (twice) and late (eighth) passage nonestablished rat embryo fibroblasts exhibit dramatically different responses to microinjection of the oncogenic form (T24) of the ras protein. Late passage cells respond like established cultures in that they both synthesize DNA and divide in response to the oncogenic protein. However, early passage cells are shown to be much less responsive to the introduction of this protein in terms of the threshold concentration required to elicit a mitogenic response. Furthermore, conditioned medium from late passage cells can both potentiate the effect of ras in early passage cells and stimulate colony formation in soft agar. In addition, we show that human cells can respond to microinjection of the ras oncogene protein by synthesizing DNA.

Animals↗