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At least 289 records · Page 16Linked to original sources

PubMed1997

Cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats.

In this study we determined the cardiovascular effects produced by microinjection of angiotensin peptides [Angiotensin-(1-7) and Angiotensin II] and angiotensin antagonists (losartan, L-158,809, CGP 42112A. Sar1-Thr8-Ang II, A-779) into the rostral ventrolateral medulla of freely moving rats. Microinjection of angiotensins (12.5-50 pmol) produced pressor responses associated to variable changes in heart rate, usually tachycardia. Unexpectedly, microinjection of both AT1 and AT2 ligands produced pressor effects at doses that did not change blood pressure in anesthetized rats. Conversely, microinjection of Sar1-Thr8-Ang II and the selective Ang-(1-7) antagonist, A-779, produced a small but significant decrease in MAP an HR. These findings suggest that angiotensins can influence the tonic activity of vasomotor neurons at the RVLM. As previously observed in anesthetized rats, our results further suggest a role for endogenous Ang-(1-7) at the RVLM. The pressor activity of the ligands for AT1 and AT2 angiotensin receptor subtypes at the RVLM, remains to be clarified.

Angiotensin I↗

PubMed1999

Microinjections of carbachol into the phrenic motor nucleus inhibit phrenic nerve activity in the rat.

Microinjections (50 nl) of carbachol (2.5-500 microM) into the phrenic motor nucleus (PMN) of anesthetized rats caused a dose-dependent decrease in the phrenic nerve (PN) burst-amplitude. Prior microinjections of pirenzepine and methoctramine (1 mM, each) into the PMN, in separate experiments, significantly attenuated the carbachol-induced inhibition of PN activity. These results suggest that inhibition of PN activity induced by microinjections of carbachol into the PMN is mediated via M(1) and M(2) receptors. Since pirenzepine and methoctramine microinjections into the PMN did not alter the control PN activity, it was concluded that in anesthetized rats cholinergic inputs to the PMN, if any, are not tonically active. It is possible that muscarinic receptors in the PMN come into play only under specific conditions such as activation of a reflex mechanism which alters PN activity. These hypotheses remain to be tested.

Animals↗

PubMed1989

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels.

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1998

Behavioural topography in the striatum: differential effects of quinpirole and D-amphetamine microinjections.

Behavioural evidence has accumulated that supports the hypothesis that specific territories of the striatum contribute differentially to the control of motor behaviours. The present experiments compare the behavioural effects of microinjections of amphetamine (20 microg/0.5 microl) with those elicited by the D2-class dopamine receptor agonist quinpirole (3 microg/0.5 microl) following direct microinjection into three anatomically distinct sectors of the striatum: the nucleus accumbens, the ventrolateral striatum and the anterodorsal striatum. Our findings demonstrate that site-specific behavioural responses are induced by microinjections of amphetamine, but not of quinpirole, into the striatum. Our results suggest that widespread areas of the striatum are implicated in the induction of a syndrome of sedation, yawning and motor inhibition, observed readily following microinjections of quinpirole into the striatum. This evidence supports both homogeneity and segregation of function in the striatum at the behavioural level. Further, the results suggest that the elicitation of site-specific action sequences at the level of the striatum seems to require cooperative interactions between D1-class and D2-class dopamine receptors.

Amphetamine↗

PubMed1997

Microinjected cDNA encoding JAK2 protein-tyrosine kinase induces DNA synthesis in NIH 3T3 cells.

Microinjection of expression plasmids encoding either JAK2 or hyperactive (Ndelta661)rJAK2 into serum-starved NIH 3T3 cells resulted in 20-30-fold induction of DNA synthesis. Control microinjections of buffer or parental pcDNA3 vector resulted in only 3-5-fold induction of DNA synthesis. Induction of DNA synthesis was blocked when plasmid encoding JAK2 was microinjected in the presence of the JAK2-selective inhibitor AG-490, whereas AG-490 did not block DNA synthesis induced by microinjected plasmid encoding (Ndelta661)rJAK2. The ability of JAK2 to initiate the G(o)/S cell cycle transition is comparable to that of other proto-oncogenes, and supports a mechanistic role for overexpressed Janus kinases in carcinogenesis.

3T3 Cells↗

PubMed1996

Picrotoxin produces a "central" pain-like syndrome when microinjected into the somato-motor cortex of the rat.

In this study, we report the possibility of producing marked electrocorticographic changes and "pain-like" reactions, when the GABAA antagonist picrotoxin is microinjected unilateraly into the rat somato-motor Sml cortex in the region of the hind paw. After the microinjection, we observed continuous seizure isolated spikes, spikes-and-waves, bursts, and pain-like reactions, almost exclusively confined to the hind paw. These reactions considered of lifting off the floor, licking of the paw palm or digits, biting, paw tremors, and a peculiar paw position that we called "turn-in" paw. We also noted other behaviors, such as "limping," "neglected" paw, or rearing. The "pain-like" character of these manifestations was suggested by the fact that similar qualitative and quantitative data occurred consequent to the administration of 2.5% diluted formalin into the palm of the hind paw in different rats. Bringing together the electrocorticographic events and the behavioral reactions produced by Sml picrotoxin indicated that there was no obvious correlation between the phenomena, except that the tremor was always associated with the bursts. Sensory denervation of the hind paw, produced by sciatic and saphenous nerve transections, did not significantly modify either the ictal activity or the behavior. Finally, microinjection of naloxone prior to picrotoxin did not change the cortical events, but greatly diminished the "pain-like" reactions. All these results favor the cortical microinjection of a GABAA receptor antagonist as a good rat model for studying pain of "central" origin. They emphasize the possible role of the Sml cortex in such a phenomenon, and the deficit of cortical GABAergic processing, which can include an opioid link.

Animals↗

PubMed2003

Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy.

Oocytes collected by laparoscopic ovum pick-up (LOPU) were successfully used to produce transgenic goats by pronuclear microinjection of in vitro zygotes. Estrus cycles of 109 donor goats were synchronized using intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate and treatment with 70 mg NIH-FSH-P1 and 300 IU eCG to stimulate follicular development. Follicles were aspirated under laparoscopic observation. In vitro maturation (IVM) of oocytes was performed in M199 supplemented with hormones, kanamycin and 10% estrus goat serum. Following IVM, oocytes were cocultured with capacitated semen in TALP supplemented with 20% estrus goat serum for 15-20 h. The resulting zygotes were microinjected with a linear DNA fragment. In total, 3293 follicles were aspirated (15.7+/-9 follicles aspirated per donor) and 2823 oocytes were recovered (13.4+/-8 oocytes per donor). A total of 1366 zygotes were microinjected and transferred into 219 recipient goats by midventral laparotomy (average 6.2 embryos per recipient). A total of 150 kids were born, of which 9 (6 M: 3 F) were confirmed to be transgenic by PCR and Southern blotting analyses. These results demonstrate that acceptable transgenesis rates can be obtained in goats by DNA microinjection of in vitro produced zygotes.

Animals↗

PubMed1998

Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro.

Factors influencing the developmental potential of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n = 1053) were recovered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova). However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-free, growth factor-supplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively). Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes.

Animals↗

PubMed1996

Insulin microinjection into the nucleus tractus solitarii of the rat attenuates the baroreceptor reflex.

The presence of insulin and insulin binding sites in the central nervous system and the demonstration that central insulin has an effect on cardiovascular function has led to the hypothesis that insulin may play a role in mediating the baroreceptor reflex. To investigate this possibility, insulin was microinjected into depressor sites in the nucleus tractus solitarius (NTS), the first central synapse of the baroreceptor reflex, of urethane anesthetized rats. Microinjection of insulin into the NTS (110 nL of 1, 10 and 100 IU/mL) did not change mean arterial pressure (MAP) or heart rate (HR). However, insulin microinjection attenuated phenylephrine-elicited reflex bradycardia and depressor responses elicited by glutamate (GLU). The attenuation of GLU-elicited depressor responses was time-dependent for MAP changes and time and concentration-dependent for HR changes (p < 0.05). Insulin-like growth factor-1 microinjection into the NTS also attenuated GLU-elicited decreases in MAP (p < 0.05) but not HR. The effect of insulin on GLU-elicited responses was inhibited after peripheral adrenergic blockade by nadolol (1.0 mg/kg i.v.) but not after cholinergic blockade by methyl-atropine (2.0 mg/kg i.v.). These results demonstrate that insulin inhibits baroreceptor reflex responses in the NTS likely through an influence on the effects of excitatory amino acid neurotransmitters on the activity of NTS neurons involved in sympathetic control of the cardiovascular system.

Anesthetics, Intravenous↗

PubMed1998

Morphine analgesia in the formalin test: reversal by microinjection of quaternary naloxone into the posterior hypothalamic area or periaqueductal gray.

Bilateral microinjection of 5 nmol morphine into the posterior hypothalamic area (PHA), periaqueductal gray matter (PAG) or ventral tegmental area (VTA) elicits powerful suppression of nociceptive behaviors in the formalin test, an animal model of injury produced pain. The object of the present study was to determine whether analgesia in the formalin test (50 microl 2.5% formalin injected s.c. in one hindpaw) induced by systemically administered morphine requires opioid action at these sites, or other putative sites of opioid action. Morphine sulphate (6 mg/kg s.c.) produced almost complete analgesia in the second phase of the formalin test (30-50 min after formalin). Bilateral microinjection of the quaternary opioid antagonist naloxone methobromide (NxBr, 28 ng in 0.5 microl, 22 min after morphine) into the PHA completely abolished morphine analgesia, while NxBr into PAG partially reversed analgesia. Microinjection of NxBr into the VTA, central nucleus of the amygdala, habenula, striatum, nucleus accumbens or hypothalamic sites outside the PHA did not antagonize morphine analgesia, although microinjections into some of these sites appeared to reduce the cataleptogenic effects of morphine. The data indicate that the PHA and PAG are probably the primary sites of action of morphine in the formalin test.

Analgesics, Opioid↗

PubMed1998

Differential behavioral effects of angiotensin II microinjected unilaterally into the CA1 hippocampal area.

The behavioral responses of rats to unilateral microinjections of angiotensin II (ATII) into the left or right CA1 hippocampal area were studied. Unilateral (left or right) injections of ATII at a dose of 0.5 microg decreased locomotor activity but, at a dose of 1.0 microg, ATII increased it compared to the respective controls. The effect was more pronounced when ATII was microinjected into the left CAI hippocampal area. The elevated plus-maze experiments showed that ATII microinjections into the right CA1 hippocampal area at a dose of 0.5 microg decreased the ratio of the number of entries into the open arms to the total number of entries (into the open and closed arms). These findings suggest some asymmetric effects of ATII, depending on the dose, the behavioral test and the microinjected hemisphere.

Angiotensin II↗

PubMed1999

Development of pig embryos reconstructed by microinjection of cultured fetal fibroblast cells into in vitro matured oocytes.

Nuclear transfer as originally developed for use in amphibians involved microinjecting a nucleus directly into the cytoplasm of the oocyte. A major mammalian modification has been to use cell fusion to introduce the nucleus. Here we report using a microinjection method to introduce small and medium sized fibroblast cells into mature oocytes. Small cells were more likely to result in nuclear formation (30%) than larger cells (15%; P = 0.013). Small, confluent and serum starved cells resulted in nuclear formation more often (P < 0.048) than did cycling cells. The rate of nuclear formation was not dependent upon the media, (NCSU-23 or TL-Hepes without calcium) nor upon the duration of exposure to the media (1 h to 4 h) after microinjection but before activation. While such treatments did not have an effect on nuclear formation, treatment of parthenogenetically activated oocytes with calcium-free TL-Hepes reduced the percentage of blastocysts (P = 0.068. 11.2% vs. 18.3%) and increased the percentage of morula stage embryos (P = 0.007; 27.6% vs. 15.7%) as compared with culture in NCSU. Finally, small confluent cells were used for nuclear transfer and resulted in two presumptive blastocyst stage embryos [2/128 injected or 2/38 (5.3%) successful injections]. These results show that presumptive blastocyst stage embryos can result from microinjection of fibroblast cells to enucleated oocytes and thus may provide a method to create transgenic knockout animals.

Animals↗

PubMed1998

Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos.

Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 degrees C, 15 min) and 4-6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.

Animals↗

PubMed2000

Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos.

The efficiency of transgenic animal production would increase if microinjected embryos with a successfully integrated transgene could be identified prior to transfer. It is possible to detect microinjected DNA in embryos. However, no reliable system is able to distinguish between transgenes merely present as extrachromosomal DNA and those that have been integrated into chromatin. The experiments reported here were designed to determine if the inclusion of matrix attachment regions (MARs) would enhance the efficiency of transgenic embryos identification using a selection scheme based on the expression of green fluorescent protein (GFP). Pronuclei of mouse embryos were microinjected with GFP reporter gene under the control of three different promoters and flanked or not by three different MAR elements. Transgene expression profiles were followed by direct visualization of GFP in cultured microinjected embryos. Embryos at different developmental stages were classified according to their GFP expression and groups with the same expression pattern were transferred into oviducts of pseudopregnant female mice. Fetuses were collected between days 12-15, and their genomic DNA was purified and analyzed to detect transgene integration. We did not find any statistically significant difference between the percentage of transgenic fetuses produced from GFP-positive or GFP-negative embryos transferred at 4-cell, morula, or blastocyst stage. However, when MAR elements were included in the construct, we found that GFP-positive embryos transferred at the 2-cell stage produced a significantly higher percentage of transgenic fetuses than GFP-negative embryos, but MAR sequences did not completely eliminate false positives.

Animals↗

PubMed1999

Dose-dependent increase of transcapillary diffusion of sodium fluorescein after histamine microinjections.

BACKGROUND: To study the dose-dependent effects of histamine on capillary permeability in human skin, using the microinjection technique. PATIENTS AND METHODS: Eight healthy volunteers (2 w, 6 m; mean age 33 years) were included in the study. On two separate occasions, glass microcannulas with a tip diameter of 7 to 9 microns were inserted into the subepidermal layer of the skin at the distal medial tibia surface of each lower limb with a micromanipulator. In each subject, 0.5 microliter of 3 different concentrations of histamine solution (0.1/1000, 0.01/1000 and 0.001/1000) were injected and compared to the solvent (0.9% NaCl). Transcapillary diffusion of intravenously administered Na-fluorescein was assessed simultaneously using two fluorescence videomicroscopy systems. Off-line video densitometry was performed in an area of 0.56 mm2 around the injection sites and fluorescence light intensities were measured in arbitrary units (AU) at 10, 30, 60, 120 and 600 s after dye appearance. RESULTS: Compared to the solvent histamine microinjections resulted in a dose-dependent increase of mean fluorescence light intensities (FLI). Whereas mean FLI for the 0.001/1000 histamine injection was only significantly elevated 10 min after dye appearance (p < 0.05) an increase of mean FLI was already observed 10 s after dye appearance following the 0.1/1000 histamine injection (p < 0.05), which was more pronounced at later time points (p < 0.001). Mean FLI's for the 0.01/1000 histamine solution were in between and resulted in significantly elevated values 1 min to 10 min after dye appearance (p < 0.05). CONCLUSIONS: We conclude that the microinjection technique together with fluorescence videomicroscopy described previously [6] is able to document a dose-dependent effect of histamine microinjections on skin capillary permeability. The technique may facilitate to determine appropriate dosages not only of histamine in order to test the effect of antagonists on human skin capillary permeability.

Adult↗

PubMed2006

Microinjection of naltrexone into the central, but not the basolateral, amygdala blocks the anxiolytic effects of diazepam in the plus maze.

The amygdala is involved in behavioral and physiological responses to fear, and the anxiolytic properties of several drugs are localized to this region. Activation of endogenous opioid systems is known to occur in response to stress and a growing body of literature suggests that opioid systems regulate the properties of anxiolytic drugs. These experiments sought to elucidate the role of opioid receptors in the central (CeA) and basolateral (BLA) nuclei of the amygdala in regulating the anxiolytic properties of ethanol and diazepam. Male rats fitted with cannula received bilateral microinjections of the nonselective opioid receptor antagonist naltrexone (NAL) immediately followed by systemic delivery of either ethanol (1 g/kg) or diazepam (2 mg/kg) in the elevated plus maze. Both diazepam and ethanol decreased anxiety-like behavior. Delivery of NAL into the CeA blocked the anxiolytic properties of diazepam. Delivery of NAL into the BLA slightly increased open arm avoidance, but had no effect on the anxiolytic properties of diazepam. Microinjection of NAL into either nucleus failed to block the effects of ethanol. These results were specific to the anxiolytic properties of diazepam, since baseline behaviors were unaffected by microinjection of NAL. Microinjection of lidocaine produced results distinct from NAL and failed to block the anxiolytic actions of diazepam. These studies indicate distinct roles for opioid receptor systems in the CeA and BLA in regulating the anxiolytic properties of diazepam in the elevated plus maze. Further, opioid receptor systems in the CeA and BLA do not regulate the anxiolytic properties of ethanol in this test.

Acetylcholinesterase↗

PubMed1985

Degradation of erythrocyte-microinjected and scrape-loaded homologous cytosolic proteins by 3T3-L1 cells.

Homologous cytosol was introduced into 3T3-L1 cells by two different methods. Erythrocytes loaded with radiolabelled cytosolic proteins extracted from 3T3-L1 cells were fused with the aid of Sendai virus to 3T3-L1 cells, which were then seeded to confluent and non-confluent cultures. Cytosolic proteins were also introduced into cells by the technique of scrape-loading. In confluent cells, injected cytosolic proteins were recovered largely (54-93%) in a sedimentable (6 X 10(6) gav.-min) fraction from recipient cells irrespective of the method of introduction or of radiolabelling of the injected proteins [( 125I]iodination, reductive methylation with NaB3H4 and backbone labelling with L-[4,5-3H]leucine). The degradation of microinjected cytosolic proteins was in all cases inhibited by the lysosomotropic agent NH4Cl to a greater extent (32-75%) than that observed for endogenous cytosolic (less than or equal to 19%) proteins (labelled with L-[4,5-3H]leucine). In growing cells both endogenous total cell proteins and microinjected proteins were degraded at a slower rate than in confluent cell monolayers. The inhibition by NH4Cl of the degradation of both the endogenous and microinjected proteins is decreased compared with the inhibition observed in confluent monolayers. The results are discussed in terms of the cytoplasmic capacity to segregate microinjected homologous proteins before protein degradation can take place.

Ammonium Chloride↗

PubMed1993

Microinjection of Rap2B protein or RNA induces rearrangement of pigment granules in Xenopus oocytes.

Rap2B, a member of the ras superfamily of low-molecular-mass GTP-binding proteins, induced a characteristic rearrangement of the pigment granules in Xenopus oocytes following its microinjection, resulting in numerous unpigmented spots on the animal hemisphere. This phenomenon, termed 'mottling', was also induced by microinjection of in vitro-transcribed Rap2B RNA or of purified recombinant Rap2A. Following the microinjection of Rap2B, more than 90% of the oocytes showed signs of mottling within 10 h. The time course of mottling paralleled the association of the recombinant Rap2B with an oocyte membrane fraction. Like other members of the ras superfamily, Rap2B possesses a C-terminal CAAX motif that serves as a signal for post-translational processing. Mutation of the cysteine residue in the CAAX motif to serine prevents the association of Rap2B with oocyte membranes, and also prevents mottling. This result suggests that post-translational processing of Rap2B is required for the observed effect. Mottling was blocked by boiling Rap2B prior to its microinjection or by co-injection of the cytoskeletal reagent phalloidin.

Animals↗