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PubMed2000

Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus.

In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one.

Animals↗

PubMed1997

Diffusion of microinjected markers across the parasitophorous vacuole membrane in cells infected with Eimeria nieschulzi (Coccidia, Apicomplexa).

Cells infected with the intracellular parasite Eimeria nieschulzi were microinjected with lucifer yellow (457 Da), biocytin lucifer yellow (850 Da) and dextranrhodamine (10,000 Da). Immediately after injection of a mixture of the markers into the host cell cytoplasm, a differential diffusion pattern was observed in trophozoites and schizonts. Lucifer yellow and biotin lucifer yellow were seen to enter the parasitophorous vacuole, whereas the dextran was excluded. Since these markers cannot permeate cell membranes, this suggests that the Eimerian parasitophorous vacuole acts as a molecular sieve. This method allows the visualization and further characterization of the parasitophorous vacuole in living cells.

Animals↗

PubMed2003

Effect of IL-1beta microinjection into the posterior hypothalamic area on trigeminal nociception in the rat.

The hypothalamus has been implicated in the pathophysiology of the most disabling forms of primary headache, namely migraine and cluster headache. Interleukin-1beta (IL-1beta) is highly expressed in the hypothalamus. We recorded from the trigeminal nucleus caudalis of rats using extracellular electrophysiological methods from neurons responding to electrical stimulation of the peri-middle meningeal artery dura mater and having receptive fields in the ophthalmic division of the trigeminal nerve. Data were collected from fifteen clusters of wide-dynamic range neurons with stable baseline firing responses between 97 and 101% ( n=3 for each unit) to stimulation. Microinjection of IL-1beta into the posterior hypothalamus of 9 animals resulted in a modest inhibition of evoked trigeminal responses in three units, no effect in six and no overall effect for the entire cohort studied. The mean maximum response was a non-significant reduction in firing to 83+/-7% ( n=9) at 30 minutes post-injection of IL-1beta. There was some variation of effect dependent on site of injection with central posterior hypothalamus being the predominant area that resulted in inhibition. There was no inhibition in the six animals injected with vehicle (saline). If there is an important effect for IL-1beta in the posterior hypothalamus it is likely to be highly somatotopically restricted.

Animals↗

PubMed2006

Semi-quantitative expression and knockdown of a target gene in single-cell mouse embryonic stem cells by high performance microinjection.

Interactions of multiple genes and associated factors are involved in the differentiation and de-differentiation of embryonic stem (ES) cells. Quantitative analysis of these genes and factors is essential for the elucidation of their mechanism. To meet this requirement, we have investigated various experimental conditions for high performance microinjection into mouse ES cells. A speedy and rhythmic operation was found to be important and was accomplished robotically by using a single-cell manipulation technique and XY-address registrable culture dishes. Among many experimental parameters, the tip size of an injection capillary, the pressure condition, and the DNA concentration in the injection capillary were of critical significance. Their optimum values were 0.5-0.8 microm, 0.7 kgf/cm(2) for 30 ms, and 1-100 ng/microl, respectively. Under these conditions, semi-quantitative control of the EGFP gene expression in mouse ES cells and its knockdown was successfully demonstrated.

Animals↗

PubMed2006

Fluoro-Jade B staining following zymosan microinjection into the spinal cord white matter.

1. The fluorescein derivate Fluoro-Jade B (FJB), which primarily labels dead or dying neurons, was used to study the acute focal inflammation in the spinal cord white matter. Inflammation was induced by microinjection of the yeast particulate zymosan to evaluate the biological effects of intraspinal macrophages activation without the confounding effects of physical trauma. 2. A single bolus of zymosan (Sigma, 75 nL) was stereotaxically injected at the thoracic level into the lateral white matter of rat spinal cord. A standard Fluoro-Jade B staining protocol was applied to spinal cord sections at 6, 12, 24 h and 2, 4 days postinjection. Neutral Red, NADPH-diaphorase, Iba1-IR, and DAPI staining protocols accomplished examination of the cells participating in the acute inflammatory response. 3. Zymosan caused formation of clearly delineated inflammation lesions localized in the lateral white matter of the spinal cord. Fluoro-Jade B stained cells in the area of inflammation were not observed at 12 h postinjection while mild FJB staining appeared at 24 h and intense staining was observed at 2 and 4 days postinjection. 4. This study shows that the acute response to zymosan-induced inflammation in the rat spinal cord white matter causes a gradual appearance of phagocytic microglia/macrophages and delayed FJB staining of the inflammatory cells. 5. FJB, a reliable marker of dying neurons, is a more universal agent than formerly believed. One possible explanation for the gradual appearance of FJB-stained cells in the area of inflammation is that specific time is required for sufficient levels of proteins and/or myelin debris of axonal origin to appear in the cytoplasm of phagocytic microglia/macrophages.

Animals↗

PubMed1988

A quantitative evaluation of promoter activity by microinjection of chloramphenicol acetyltransferase hybrid genes into tissue culture cells.

The combination of DNA microinjection into tissue culture cells and the chloramphenicol acetyltransferase enzyme assay has been used to determine quantitatively the transcriptional activity of viral or eukaryotic promoters. As shown for a wide range of DNA concentrations and time periods between 2 and 16 h the assay allows the reliable and reproducible determination of promoter activities in defined cellular backgrounds. Only 50 cells are necessary for the test.

Acetyltransferases↗

PubMed1987

Enhanced degradation of oxidized glutamine synthetase in vitro and after microinjection into hepatoma cells.

Mixed-function oxidation of Escherichia coli glutamine synthetase has previously been suggested to mark the enzyme for intracellular degradation, and in vitro studies have demonstrated that oxidation renders the enzyme susceptible to proteolytic attack. In this study, the susceptibility of glutamine synthetase to degradation by purified proteases has been compared with the rate of degradation after microinjection into hepatoma cells. Upon exposure to an ascorbate mixed-function oxidation system the enzyme rapidly loses most of its activity, but further oxidation is required to cause susceptibility to extensive proteolytic attack either by a high-molecular-weight liver cysteine proteinase or by trypsin. The rate of degradation of biosynthetically 14C-labeled native and oxidized glutamine synthetase preparations after injection into hepatoma cells parallels their susceptibility to proteolysis in vitro. Native enzyme preparations and enzyme oxidatively inactivated, but not susceptible to extensive degradation by purified proteases, had similar intracellular half-lives; however, oxidized enzyme preparations that were susceptible to proteolytic breakdown in vitro were degraded almost ten times faster than the native enzyme within the growing hepatoma cells. These results suggest that the same features of the oxidized enzyme that render it susceptible to proteolysis in vitro are also recognized by the intracellular degradation system. In addition, they show that loss of enzyme activity does not necessarily imply decreased metabolic stability.

Animals↗

PubMed1990

Age-related change in the degradation rate of ovalbumin microinjected into mouse liver parenchymal cells.

Change in the degradation rate of ovalbumin (OVA) microinjected into liver parenchymal cells isolated from mice of various ages was studied. OVA was injected by osmotic lysis of pinosomes and the amount of OVA was determined by immunoblotting using purified antibody to OVA. Cellular activity as judged by rates of protein synthesis and degradation of pulse-labeled proteins was not affected by the injection. To localize injected OVA in the cells, cell extracts were fractionated by differential centrifugation and the amount of OVA and the activity of beta-D-galactosidase as a lysosomal marker enzyme of each fraction were determined. The ovalbumin was mostly found in the soluble fraction, while the beta-D-galactosidase activity was found in the particulate fraction, indicating that the ovalbumin was spread in the cytosol but was not present in the pinosomes or lysosomes. The average half-lives of OVA were 106, 113, and 164 h in the cells from young (3.5-6.5 months), middle-aged (13.5-20.0 months), and old (24.5-30.5 months) mice, respectively. Thus, the half-life of ovalbumin in the cells of senescent mice was about 50% longer than that in the cells of young or middle-aged mice. These results are in good agreement with those of our previous investigation, which showed that the half-life of inactivation of horseradish peroxidase was extended by about 50% in the hepatocytes from old mice (Ishigami and Goto, 1988, Mech. Ageing Dev., 46, 125-133).

Aging↗

PubMed1986

Use of reconstituted Sendai virus envelopes for fusion-mediated microinjection of double-stranded RNA: inhibition of protein synthesis in interferon-treated cells.

Poly(I).poly(C) molecules were trapped with reconstituted Sendai virus envelopes when added to the reconstitution system. A quantitative estimation indicated that about 10% of the added poly(I).poly(C) remained associated with the fusogenic viral envelopes. About 50% of the associated poly(I).poly(C) were found to be RNAase A resistant, enclosed within the viral envelopes. Incubation of loaded viral envelopes with HeLa or L-cells resulted in strong inhibition of protein synthesis, indicating fusion-mediated microinjection of the enclosed poly(I).poly(C). Introduction of poly(I).poly(C) into cultured cells by the use of reconstituted Sendai virus envelopes was as efficient as the introduction of these polynucleotides using the calcium phosphate coprecipitation technique. The inhibition of protein synthesis in L-cells but not in HeLa cells was dependent upon pretreatment with interferon. Incubation of poly(I).poly(C)-loaded viral envelopes with interferon-treated variant cells of the NIH 3T3 line, which possess a very low amount of RNAase L, resulted in only 25% inhibition of protein synthesis, compared to 85% inhibition observed in L-cells.

Animals↗

PubMed1992

Lack of effect on the sodium efflux of the microinjection of D-Ins(1,4,5)P3 into ouabain-poisoned barnacle muscle-fibers.

A study has been carried out using relatively intact mature muscle fibers from the barnacle Balanus nubilus to see whether D-Ins(1,4,5)P3 stimulates the ouabain-insensitive Na efflux following its microinjection and whether this is accompanied by a contraction of the fiber. Part of the impetus for a study of this type came from the on-going debate between Vergara, Rojas and co-workers and Lea and co-workers, the former group holding the view that skinned barnacle fibers and skeletal fibers in general are responsive to this isomer. The evidence brought forward indicates that the injection of D-Ins(1,4,5)P3 in concentrations in the range of 10(-2) M to 10(-6) M into cannulated unskinned fibers pretreated with ouabain fails to increase the residual efflux, and additionally fails to elicit a contraction. A similar picture emerges with the use of non-hydrolyzable DL-Ins(1,4,5)P3[S]3, analog following its injection in a concentration of 0.5 microM. Higher concentrations of the analog were unavailable for use. This work also involved verification of the idea that an effect might be obtainable in depolarized fibers. However, doubling or tripling K0+ and injection of the isomer or the analog simultaneously failed to support this idea. Since nothing is known as to whether D-Ins(1,4,5)P3 influences the behavior of the Na(+)-Ca2+ exchanger when operating in the reverse mode, experiments were done to check this possibility. ATPNa2 which is though to activate Na(+)-Ca2+ exchange was injected prior to the isomer or the analog but no significant results were obtained. A similar line of reasoning was followed, that of activating the L-type Ca2+ channel by injecting GTPNa2 which in addition is known to activate adenylate cyclase. Again, neither the isomer nor the analog were effective. Thus, the only conclusion possible is that in relatively intact, mature barnacle fibers there is no coupling between the phosphoinositide signalling pathway and two other key systems, viz. the Na(+)-Ca2+ exchanger when operating in the reverse mode and the L-type Ca2+ channel. Equally clear is that for some unknown reason the ouabain-insensitive Na efflux and the contractile apparatus are insensitive to D-Ins(1,4,5)P3[S]3.

Adenosine Triphosphate↗

PubMed1985

DNA replication of single-stranded DNA of phage fd microinjected into Xenopus laevis eggs.

Single-stranded circular DNA of phage fd was microinjected into Xenopus laevis eggs and DNA synthesis was analyzed after incubation. An efficient DNA synthesis occurred in the eggs: 2.7 ng of DNA was newly formed in an egg whose volume is approximately 900 nl during a 5-h incubation at 20 degrees C. The DNA synthesis was sensitive to aphidicolin, suggesting that DNA polymerase alpha participates in the synthesis. Agarose gel electrophoresis showed that full size of fd DNA is synthesized and that replicative form I of fd DNA is the major form of the newly produced DNAs. These results suggest that priming of DNA synthesis, elongation of DNA chain and ligation of DNA strands take place co-operatively in the egg with single-stranded DNA as a template.

Animals↗

PubMed1984

Identification of penicillin diffusion at the onset of epileptogenesis in the cat striate cortex following differential laminar microinjection.

Diffusion patterns of 14C-labeled penicillin, resulting from microinjections into different neocortical laminae, were compared with simultaneous electrophysiologic monitoring of epileptogenesis across these laminae. Injections into layer IV produced response alterations quickly in all recorded layers even with penicillin confined to this granular cell layer. Supra- and infragranular layer injections also initiated epileptic response abnormalities, though much more slowly and only after penicillin had diffused into layer IV. Injections where penicillin diffusion was shown to be limited to either of the non-granular layers did not induce epileptogenesis. These results indicate not only that layer IV is the most penicillin-sensitive neocortical layer, but that (1) it is sufficient for epileptogenesis when initially injected and, that due to this heightened sensitivity, (2) it serves as a natural trigger for epileptiform response development in other layers when they are injected first. Neurochemical and neuromorphologic correlates within striate cortex are discussed in an attempt to identify the reason for this granule cell layer sensitivity to penicillin-induced epileptogenesis.

Animals↗

PubMed1984

'Dystonia'-like postural asymmetry after microinjection of ACTH N-terminal fragments but not after ACTH1-39 in rat brainstem suggests role of neuropeptide mutation in genetic movement disorder.

N-terminal fragments of adrenocorticotropin (ACTH) was reported to exert potent 'dystonia'-like effects on posture and locomotion following a unilateral microinjection into the rat brainstem. The high reliability of this phenomenon provided a suitable animal model for the study of these actions. The present structure-activity study showed that ACTH1-39, in contrast to its N-terminal fragments, did not have any 'dystonic' actions, however transient or slight. Thus, the folded conformation of [1-39] in vivo may prevent its N-terminal region from interacting with those CNS sites that trigger 'dystonic' actions. These results suggest that genetically-linked human dystonia may have originated in part as a consequence of a mutation in the processing of the ACTH molecule, resulting in an aberrantly-folded conformation that allows its N-terminal region to trigger the dystonic syndrome.

Adrenocorticotropic Hormone↗

PubMed1985

Seizures induced by intraventricular microinjection of ionized cobalt in the rat--a new experimental model of epilepsy.

A new animal model for epilepsy was successfully produced by microinjection of cobaltous chloride into the lateral cerebral ventricle of the rat. The median convulsive dose (CD50) and the median lethal dose (LD50) of CoCl2 was 0.45 microM/10 microliters (0.27-0.77 microM/10 microliters) and 1.07 microM/10 microliters (0.73-1.57 microM/10 microliters), respectively. The behavioral changes, electrocorticogram (ECoG), and the action of 5 classical anticonvulsants were studied using this new model. Seizures induced by cobaltous chloride are clinically similar to those produced by systemic administration of kainic acid and amygdala kindling. These are characterized by staring spells, wet dog shakes, mild convulsive movements, and stereotyped convulsions. ECoG findings demonstrated a unique epileptic burst during the wet dog shakes. Generalized epileptiform discharges were seen during typical seizures. The burst of spikes first occurred in the opposite temporal and frontal regions; and then became generalized. Among the 5 anticonvulsants studied, phenobarbital (30 mg/kg) and nitrazepam (3 mg/kg) completely antagonized the seizures; carbamazepine showed a moderate effect; and phenytoin as well as sodium valproate showed little effect. It is postulated that the seizures induced by cobaltous chloride may originate in the limbic system; and that cobalt ions are responsible for the seizure-inducing action. The mechanism remains to be investigated.

Animals↗

PubMed1985

Effects on the caudate spindle in rats of taurine and gamma-aminobutyric acid (GABA) microinjected into the substantia nigra: involvement of the dopaminergic system.

Spindle bursts (caudate spindle) were recorded from the frontal cortex in rats. The bilateral intranigral microinjection of taurine suppressed the caudate spindle, whereas the same dose of gamma-aminobutyric acid (GABA) did not suppress the caudate spindle. These results indicate that suppression of the caudate spindle induced by taurine may relate to activation of the nigrostriatal dopaminergic neurons.

Animals↗

PubMed1986

Calcitonin: brainstem microinjection but not systemic administration inhibits spinal nociceptive transmission in the cat.

In anaesthetized cats, nociceptive responses of lumbar dorsal horn neurons were studied during administration of salmon calcitonin (sCT). Systemic sCT administration (4-95 IU/kg i.v.) produced no change in neuronal responses produced by noxious skin heating or by impulses evoked electrically in afferent C-fibres. Responses to skin heating were reduced during electrical stimulation in the brainstem, but the efficacy of this descending inhibition was not altered by systemic sCT administration. In contrast, noxious heat responses were clearly reduced by microinjection of sCT into the mesencephalic periaqueductal grey or the medullary raphe regions. These results suggest that calcitonin or a related peptide could act at specific brainstem sites to inhibit the spinal transmission of nociceptive information.

Animals↗

PubMed1987

Bilateral intranigral microinjection of morphine and opioid peptides produces antinociception in rats.

Bilateral intranigral microinjection of morphine produced dose-related and naloxone-reversible antinociceptive effects on the tail-flick and hot-plate tests. Intranigral injection of enkephalin had antinociceptive effects on both tests, and dynorphin had an antinociceptive effect on the hot-plate test. This is the first report of evidence that nigral opiate receptors may mediate antinociception.

Animals↗

PubMed1987

Release of endogenous monoamines into spinal cord superfusates following the microinjection of phentolamine into the nucleus raphe magnus.

Previous studies have suggested that raphe-spinal neurons located in the nucleus raphe magnus (NRM) are tonically inhibited by noradrenergic neurons. Furthermore, blockade of the inhibitory noradrenergic input to the NRM induces antinociception which appears to be mediated by the release of both serotonin and norepinephrine in the spinal cord. The present experiments were designed to directly measure the release of endogenous serotonin and norepinephrine into spinal cord superfusates before and after the microinjection of the alpha-adrenergic antagonist phentolamine into the NRM. High-performance liquid chromatography with electrochemical detection was used to quantitate the monoamines. The injection of phentolamine into the NRM induced a significant increase in the amount of both norepinephrine and serotonin released in the spinal cord. This enhanced release was not observed following either the injection of phentolamine into sites outside the NRM or the injection of saline vehicle into the NRM. These results support the proposal that the antinociception induced by the blockade of the inhibitory noradrenergic input to the NRM is mediated by the activation of spinally-projecting serotonergic and noradrenergic neurons.

Animals↗