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At least 1,567 records · Page 87Linked to original sources
PubMed1982

Enzymatic replacement in vitro of the first anticodon base of yeast tRNAAsp: application to the study of tRNA maturation in vivo, after microinjection into frog oocytes.

A combination of several enzymes, RNase-T1, nuclease S1, T4-polynucleotide kinase and T4-RNA ligase were used to prepare and modify different fragments of yeast tRNAAsp (normal anticodon G U C). This allowed us to reconstitute, in vitro, a chimeric tRNA that has any of the four bases G, A, U or C, as the first anticodon nucleotide, labelled with (32p) in its 3' position. Such reconstituted (32p) labelled yeast tRNAAsp were microinjected into the cytoplasm or the nucleus of the frog oocyte and checked for their stability as well as for their potential to work as a substrate for the maturation (modifying) enzymes under in vivo conditions. Our results indicate that the chimeric yeast tRNAsAsp were quite stable inside the frog oocyte. Also, the G34 was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q34; U34 into mcm5s2U and mcm5U. In contrast, C34 and A34 were not transformed at all neither in the cytoplasm nor in the nucleus of the frog oocyte. The above procedure constitutes a new approach in order to detect the presence of a given modifying enzyme inside the frog oocyte; also it provides informations about its cellular location and possibility about its specificity of interaction with foreign tRNA.

Animals↗
PubMed1983

Post-transcriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes.

An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not.

Animals↗
PubMed1982

Microinjection of interferon and 2',5'-oligoadenylate into mouse L cells and their effects on virus growth.

By means of a new type of microinjection apparatus, which has a micropipette located in a hole through the optical axis of the condenser lens, we injected interferon (IFN) or 2',5'-oligoadenylate (2-5A) into mouse L cells, and observed their antiviral effects on the multiplication of vesicular stomatitis virus (VSV). After injection, cells were infected with VSV, and labeled with [3H]uridine in the presence of actinomycin D. The proportion of cells infected with VSV which carried radioactive virus-RNA was determined by autoradiography. IFN introduced directly into L cells had no effect on the virus growth. This result supports the idea that IFN molecules exert their effect from outside the cell membrane without penetrating into the cytoplasm. 2-5A, on the other hand, was able to inhibit the growth of VSV effectively when injected into L cells. The antiviral effect was dependent on the dose of 2-5A injected, and moreover the effect was transient, since it disappeared completely after 24-h incubation.

Adenine Nucleotides↗
PubMed2003

Fate of postacrosomal perinuclear theca recognized by monoclonal antibody MN13 after sperm head microinjection and its role in oocyte activation in mice.

Monoclonal antibody (mAb) MN13 labels mouse sperm head postacrosomal perinuclear theca (PT), which is possibly involved in oocyte activation during fertilization. The antigenic site is expressed after mild sonication followed by treatment with dithiothreitol (DTT) or heat (45 degrees C), and is visible as a thick band in the postacrosomal region. The presence of protease inhibitors in the sonication medium suppresses the exposure of MN13 epitope (MN13p), suggesting the involvement of a proteolytic reaction in this process. Spermatozoa do not express MN13p after the induction of acrosome exocytosis by Ca(2+) ionophore, zona binding, or during zona penetration, a strategy that ensures safe delivery of postacrosomal PT proteins to oocytes after fusion. MN13 labeling was not detectable during fertilization by zona-free in vitro fertilization, suggesting that the antigenic site does not react with proteolytic enzymes during sperm-oocyte fusion and the antibody does not recognize the nascent epitope. Microinjection of sperm heads prepared by sonication and DTT treatment led to the activation of metaphase II oocytes. The oocyte activating function of such sperm heads was significantly diminished after labeling with MN13 prior to intracytoplasmic sperm injection (ICSI), but labeling with antiequatorin antibody MN9 activated oocytes with a frequency similar to that of unlabeled sperm heads. The sperm heads in inactive oocytes formed premature chromosome condensations (PCCs), which were invested by independent metaphase-like spindles. These observations indicate that the postacrosomal PT recognized by mAb MN13 is involved in oocyte activation. MN13p is dissociated from sperm heads during the early stages of decondensation after ICSI. In activated oocytes, MN13-labeled fine granules were redistributed in the midzone spindle region, whereas in inactive oocytes they formed a ring around the polar regions of the metaphase II and PCC spindles.

Acrosome↗
PubMed1991

Thermostability of sperm nuclei assessed by microinjection into hamster oocytes.

Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronuclei. Mature, mammalian sperm nuclei, which are stabilized by protamine disulfide bonds, were moderately heat resistant. For example, they remained capable of pronucleus formation even after pretreatment for 30 min at 90 degrees C. Indeed, a temperature of 125 degrees C (steam) was required to inactivate hamster sperm nuclei completely. On the other hand, nuclei of rooster and tilapia spermatozoa and those of immature hamster and mouse spermatozoa, which are not stabilized by protamine disulfide bonds, were sensitive to heating; although some of them decondensed after exposure to 90 degrees C, none formed male pronuclei. Furthermore, nuclei of mature hamster sperm became heat labile when they were pretreated with dithiothreitol to reduce their protamine disulfide bonds. These observations suggest that the thermostability shown by the nuclei of mature spermatozoa of eutherian mammals is related to disulfide cross-linking of sperm protamines.

Animals↗
PubMed1993

Effect of maternal aging on transgene heritability in transgenic founder mice derived from zygotes microinjected with retroviral long terminal repeat-containing recombinant deoxyribonucleic acid.

To determine the stability of artificially introduced recombinant DNA in the mouse germline throughout the reproductive life, founder mice derived from fertilized eggs injected with retroviral long-terminal-repeat-containing recombinant DNAs were mated with congenic FVB/N mice. Tail DNA of all progeny were screened and restriction fragment patterns of the transgenes were examined. Litter size and percentage of transgene transmission at various reproductive age periods were analyzed. Microinjection of 1737 eggs with four different recombinant DNAs resulted in 12 female and 11 male transgenic mice; 2 males were sterile and the remaining 21 mice served as founders to produce 1087 F1 progeny. With increasing parental age, litter size decreased generally. The percentage of progeny inheriting the transgenes declined markedly with increasing aging of 4 female founders; this aging effect was not observed in male founders (p < 0.005). No apparent change in transgenes was detected in progeny from late reproductive stages.

Aging↗
PubMed1993

Inhibition of the tail flick reflex following microinjection of morphine into the amygdala.

Recent evidence indicates that the amygdala plays a critical role in the activation of brain stem antinociceptive systems during stress. In the present experiment, bilateral microinjection of morphine sulfate (10 micrograms) into the amygdala of pentobarbital-anesthetized rats resulted in a time-dependent elevation in latency of the tail flick reflex evoked by radiant heat. The most effective sites within the amygdala were in or immediately adjacent to the basolateral nucleus. The relative amplitude of the tail flick reflex did not differ as a function of repeated testing or morphine treatment. These results suggest that important forebrain inputs which normally activate endogenous antinociceptive systems in behaving animals may be manipulated and studied in detail using the anesthetized rat.

Amygdala↗
PubMed1994

Intracerebral microinjections of dermorphin: search for the epileptic induction thresholds.

To test the different sensitivity of several central structures to the epileptogenic action of mu opiate agonists, intracerebral microinjections of the selective mu agonist dermorphin were delivered into different areas of the rat dorsal and ventral hippocampus, septum, amygdala, entorhinal cortex, thalamus, striatum and neocortex. The dose of dermorphin (up to 6 nmol) necessary to trigger electrical epileptic events in each of these regions was studied. Epileptic discharges were triggered only in the ventral hippocampus (CA1 and CA3 areas), amygdala and entorhinal cortex. The epileptic induction threshold was the lowest in the CA1 area of the ventral hippocampus. The results suggest that when a mu opiate agonist is injected intraventricularly, the epileptic activity originates in the ventral hippocampus.

Animals↗
PubMed1996

Prolonged enhancement of REM sleep produced by carbachol microinjection into the amygdala.

The effect on sleep organization of carbachol microinjected into different amygdaloid nuclei was analysed in 12 cats. Single carbachol doses of 8 micrograms in 0.50 microliter saline were delivered unilaterally or bilaterally into the central, basal, lateral or basolateral amygdaloid nucleus. Carbachol administration into the central nucleus induced a prolonged (5 days) enhancement of both REM sleep and its preceeding slow wave sleep episodes with PGO waves (sommeil phasique a ondes lentes, SPHOL), which was more pronounced following bilateral than unilateral carbachol administration. However, neither SPHOL nor REM sleep changes were produced by administration of carbachol into the other amygdaloid nuclei. We conclude that cholinergic activation of the central amygdaloid nucleus produces a long-term facilitation of REM sleep occurrence.

Amygdala↗
PubMed1999

Microinjection of catalase cDNA prevents hydrogen peroxide-induced motoneuron death.

Oxidative stress is believed to play a central role in the pathogenesis of amyotrophic lateral sclerosis (ALS). We investigated the protective effects of overexpression of catalase in primary cultures of rat spinal motoneurons against the oxidative stress of hydrogen peroxide. Using microinjection, catalase-encoding cDNA was transferred into the motoneurons. In another approach, motoneurons were injected with a catalase solution. Both procedures elevated the intracellular antioxidant status of the cultured motoneurons as evidenced by a significant protection against H2O2 toxicity. We conclude that modulating the expression of enzymes involved in cellular defense against oxidative stress can render cells more resistant to oxidant toxicity.

Animals↗
PubMed2000

Subthalamic nucleus microinjections of 5-HT2 receptor antagonists suppress stereotypy in rats.

The subthalamic nucleus (STN) is an important mediator of basal ganglia output. We studied the effects of STN microinjections of the serotonin-2 (5-HT2) antagonists clozapine, mesulergine and M100,907 on apomorphine-induced stereotypic activity in the rat. Each compound profoundly decreased the expression of stereotypic behavior, with particularly strong effects to reduce gnawing behavior. Because M100,907 does not have appreciable affinity for dopamine D1 and D2 receptors, and since all three agents are 5-HT2 antagonists, the current data suggest that basal ganglia output related to orofacial movements can be significantly modified by 5-HT2 receptors. The results suggest that antipsychotics with serotonergic properties may have direct actions on the STN that influence their potential to produce orofacial and other motor side effects.

Animals↗
PubMed2000

Microinjection of opiates into the periaqueductal gray matter attenuates neuropathic pain symptoms in rats.

We have previously demonstrated that electrical stimulation of the ventral periaqueductal gray matter (PAG) produced analgesia in neuropathic pain in rats. Opioids were also shown to be involved in analgesic effects. This study sought to determine whether opiates microinjected into the ventral PAG produce analgesia. Male Sprague-Dawley rats were chronically implanted with a guide cannula in the PAG under pentobarbital anesthesia and both the tibial and sural nerves were completely cut. Pain sensitivity was postoperatively measured with a von Frey filament and acetone applied to the sensitive area for 1 week. Opioids such as [D-Ala2,N-MePhe4,Gly(ol)5]-enkephalin (DAMGO) and [D-Pen ,D-Pen5]-enkephalin (DPDPE) were injected into the PAG. DAMGO, a mu-opioid agonist, and DPDPE, a delta-opioid agonist, were highly effective in reducing neuropathic pain. These effects were reversed by naloxone. These results suggest that the neurons in the ventral PAG are activated by opioids to produce analgesia and that specific opioid receptors are involved in the descending pain inhibition system from the PAG.

Analgesics, Opioid↗
PubMed2000

Endomorphin-2 but not Leu-enkephalin modulates spatial learning when microinjected in the CA3 region of the rat hippocampus.

The recently discovered endogenous mu-selective opioid peptide, endomorphin-2, and the endogenous delta-selective opioid peptide, Leu-enkephalin, were tested for their ability to affect spatial learning in the Morris water task. It was found that microinjection of 10 nmol endomorphin-2 into the CA3 region of the rat hippocampus significantly impaired spatial learning. However, the two lower doses tested, 3.3 and 1 nmol, had no effect in this test. Leu-enkephalin did not have any effect on spatial learning at the two doses tested (10 and 3.3 nmol). Neither peptide had any effect on motor performance as measured by swim speed. The results indicate that mu-receptors in the CA3 region of the rat hippocampus are more relevant than delta-receptors for spatial learning.

Animals↗
PubMed2003

Hypotensive effects of neuromedin U microinjected into the cardiovascular-related region of the rat nucleus tractus solitarius.

Neuromedin U (NMU) is a brain-gut peptide with potent contractile effects on the uterus and smooth muscle. Intracerebroventricular injection of NMU reportedly decreased food intake and body-weight gain in the rat. We evaluated the effects of NMU delivered by microinjection into the rat nucleus tractus solitarius (NTS) on cardiovascular responses. At the concentrations used (5, 10 or 50 pmol), the intra-NTS injection of NMU in artificial cerebrospinal fluid produced a significant reduction in both the mean arterial pressure and heart rate. The hypotensive responses were dose dependent. Our findings suggest that NMU may act as a neurotransmitter or neuromodulative substance that causes excitation of neurons in the NTS and that it may play a role in cardiovascular regulation in vivo.

Animals↗
PubMed1981

Transcription and translation of the herpes simplex virus type 1 thymidine kinase gene after microinjection into Xenopus laevis oocytes.

The hybrid plasmid pTK1 consists of the herpes simplex virus type 1 (HSV-1) BamHI p fragment, which contains the thymidine kinase (TK) gene, inserted into the vector pAT 153. When pTK1 DNA was microinjected into nuclei of Xenopus laevis oocytes, functional HSV-1-specific TK was produced, showing that transcription and translation of the gene occurred. Investigation of pTK1-specific RNA by "Southern' blot hybridization revealed that all regions of the hybrid plasmid were transcribed by RNA polymerase II, but sequences present in TK mRNA were most highly represented in stable transcripts.

Animals↗
PubMed1992

Induction of specific macronuclear developmental mutations by microinjection of a cloned telomeric gene in Paramecium primaurelia.

In Paramecium, the differentiation of a highly polyploid macronucleus from a diploid nucleus is accompanied by an extensive reorganization of the genome, involving reduction in chromosome size and formation of new telomeres at heterogeneous, but reproducible, positions. The results presented here, as well as work by others, indicate that telomere addition regions are not strictly determined by the micronuclear sequence, but are at least partially controlled by the old macronucleus. It is shown that microinjecting a high copy number of a plasmid containing the G surface antigen gene into the macronucleus of wild-type cells specifically modifies the processing of the G gene-bearing micronuclear chromosome at the following autogamy. Telomeric repeats are added upstream of the gene, rather than at their wild-type position 5 kb downstream of its 3' end, resulting in the deletion of the gene from the new macronucleus. This macronuclear mutation is unstable at the following autogamy, giving rise to many different telomere addition regions in different postautogamous clones. However, after several successive autogamies, cell lines can be obtained in which the telomeres reproducibly form in the same region. In crosses with wild-type cells, these macronuclear mutations show cytoplasmic inheritance; the micronuclei of the mutants are shown to be fully functional. The implications for the mechanism of choice of telomere addition sites are discussed.

Animals↗