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PubMed2001

Microinjection and growth of bacteria in the cytosol of mammalian host cells.

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an "escape" step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfA-regulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.

Animals↗

PubMed1973

Microinjection of rabbit hemoglobin messenger RNA into amphibian oocytes and embryos.

After microinjection of hemoglobin mRNA from rabbit reticulocytes (9S mRNA) into Xenopus laevis oocytes, the cytological structure remains normal. The peroxidase reaction does not show an accumulation of synthesized hemoglobin in the germinal vesicle. Fertilized Pleurodeles Waltlii eggs synthesize rabbit hemoglobin after injection of 9S mRNA; hemoglobin synthesis decreases as development progresses. Micromeres and macromeres synthesize equal amounts of hemoglobin, despite the fact that the micromeres contain many more ribosomes. Descendants of injected blastomeres divide more slowly and do not differentiate; they ultimately undergo cytolysis.

Amphibians↗

PubMed1977

Microinjection analysis of envelope-glycoprotein messenger activities of avian leukosis viral RNAs.

Virion RNA from the avian leukosis virus Rous-associated virus 2 (RAV-2) and poly(A)-containing RNAs from RAV-2-infected chick embryo fibroblasts were microinjected into fibroblasts transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV), which is deficient in viral envelope glycoprotein. Production of infectious RSV following these injections depended upon the viral envelope-messenger activity of the injected RNA. This system constituted a sensitive and rigorous assay system for viral envelope-messenger RNA. It was found that 21S mRNA from RAV-2-infected cells expressed the highest activity, while 35S mRNA expressed comparatively little. In addition, RAV-2-virion RNA expressed little messenger activity. The rate of formation of infectious RSV following 21S mRNA injections reached a peak near 9 hr, which was followed by a rapid decline. Evidence has been obtained that a small fraction of both 35S virion RNA and 35S mRNA from virus-infected cells was encapsulated into virus particles following their injection into virus-producing cells.

Animals↗

PubMed1977

Frog oocytes synthesize and completely process the precursor polypeptide to virion structural proteins after microinjection of avian myeloblastosis virus RNA.

After microinjection of Xenopus laevis oocytes with RNA from avian myeloblastosis virus, viral structural proteins p27, p19, p15, and p12 are formed by a sequence of posttranslational cleavages of a high-molecular-weight precursor polypeptide. The 60-70S RNA aggregate or its 30-40S RNA subunits obtained by heat or formamide treatment possess the same ability to serve as template in X. laevis oocytes. The processing pattern of virus-specific precursor polypeptides is the same in X. laevis oocytes as in chick embryo fibroblasts infected with avian myeloblastosis virus, but the processing takes place at a much slower rate.

Animals↗

PubMed1979

Prevention of genetic anemias in mice by microinjection of normal hematopoietic stem cells into the fetal placenta.

Mice homozygous for mutant genes at the W locus have a marked macrocytic anemia that is fatal in some genotypes. The defect is believed to originate in the developmentally pluripotent hematopoietic stem cell population. Anemia is first grossly manifest on day 13 of gestation, when the liver is the chief hematopoietic organ. The known paucity of blood-forming foci in livers of homozygotes and the limited formation of their erythrocytes suggested that such fetuses-unlike normal ones-might have conditions favorable for in utero seeding of genetically normal hematopoietic tissue. If this were accomplished before day 13, the anemia might essentially be prevented, or at least substantially mitigated, and normalcy soon achieved by cell selection. This proved to be the case. Allogeneic normal fetal liver cells were microinjected into the blood vessels of the fetal placenta on day 11 of gestation. Of eight mutant homozygotes born from segregating matings, six (four W/W, two W(v)/W(v)) were successfully populated with donor cells. Strain-specific hemoglobin markers demonstrated replacement of the erythroid lineage with the normal type, the rate of substitution being more rapid in the W/W (ordinarily more anemic) recipients. Strain-specific isozyme differences revealed that white blood cells were also replaced. Thus, the initial selective pressure, hence the W-mutant phenotypic lesion, must have occurred at the pluripotent stem cell stage. The animals remained immunologically tolerant of the donor cells and no graft-versus-host reaction occurred. The early introduction of hematopoietic cells differing genetically from all the other tissues of the animal provides possibilities for tracing normal hematopoietic lineages in vivo, for analyzing cell and tissue interactions, such as those between lymphocytes and thymus, and for clarifying the etiology of other blood or immune insufficiencies or malignancies.

Anemia, Macrocytic↗

PubMed1979

In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts.

Graessmann's microinjection technique was chosen to introduce fluorescently labeled muscle actin and other proteins into WI-38 human fibroblasts. The injected cells were examined during culture by fluorescence and reflection contrast microscopy. Within 30 min after injection, rhodamine-labeled actin was incorporated into a distinct network of fluorescent filaments, resembling the stress fibers observed by classical immunofluorescence microscopy. Cytochalasin b prevented the formation of these fibers, but colchicine did not. Neighboring fibers often converged into distinct focal points that appeared to be concentrated near the base of the cell. Examination of these fluorescent fibers and focal points by reflection contrast microscopy confirmed their close location to the substratum. After 6 hr of culture, fluorescent actin and the control proteins were segregated into granules located mainly near the cell nucleus. Thus, the injected actin both enters the intrinsic actin pool and participates in an assembly and disassembly of filamentous structures. Segregation into granules traces the natural turnover of this protein within the cell.

Actins↗

PubMed1979

Biological detection of specific mRNA molecules by microinjection.

We have used direct microinjection of messenger RNA into individual mouse and human cells to assay for specific translation products. We have been able to detect the synthesis of human fibroblast interferon, thymidine, kinase, hypoxanthine phosphoribosyltransferase, adenine phosphoribosyltransferase, and propionyl-CoA carboxylase in response to injected mRNA. Using the interferon system as a model, we have quantitated interferon synthesis and followed partial purification of interferon mRNA sequences on sucrose density gradients. The methods we have utilized should be applicable to other systems in which sensitive assays exist for gene products and should provide a screening procedure for isolating specific mRNA sequences.

Adenine Phosphoribosyltransferase↗

PubMed1980

Genetic transformation of mouse embryos by microinjection of purified DNA.

A recombinant plasmid composed of segments of herpes simplex virus and simian virus 40 viral DNA inserted into the bacterial plasmid pBR322 was microinjected into pronuclei of fertilized mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by the Southern blotting technique for the presence of DNA homologous to the injected plasmid. Two of 78 mice in one series of injections showed clear homology, though the injected sequences had been rearranged. Band intensities from the two positive mice were consistent with the presence of donor DNA in most or all of the cells of the newborns. These results demonstrate that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos. This technique affords the opportunity to study problems of gene regulation and cell differentiation in a mammalian system by application of recombinant DNA technology.

Animals↗

PubMed1980

Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture.

We have found that the calcium action potentials of bag cell neurons from the abdominal ganglion of Aplysia may be enhanced by intracellular microinjection of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The catalytic subunit was purified from bovine heart and shown to be effective in stimulating the phosphorylation of bag cell proteins in homogenates at concentrations of 10-50 nM. Intracellular injection into isolated bag cell neurons maintained in primary culture was through pressure applied to microelectrodes filled at the tip with catalytic subunit (5-22 muM). In 11 of 16 injected cells, both the slope of the rising phase and the height of the action potentials evoked by a constant depolarizing current were markedly enhanced relative to the pre-injection control (mean increases, 73% and 35%, respectively). This effect could occur with no change in resting potential or in the latency of the action potential from the onset of the depolarizing pulse. The effect was observed with enzyme dissolved in three different salt solutions (Na phosphate, K phosphate, or KCl). In two experiments, tetrodotoxin (50 muM) added to the extracellular medium had no effect on the enhanced action potentials. Subsequent addition of the calcium antagonist Co(2+), however, diminished or abolished the spikes. In more than half of the experiments, the injection of catalytic subunit was accompanied by an increase in the input resistance of the cells as measured by applying small hyperpolarizing current pulses. In three experiments, subthreshold oscillations in membrane potential resulted from the injections. Control injections (24 cells), carried out either with carrier medium alone or with heat-inactivated enzyme preparations, did not produce spike enhancement, increased input resistance, or oscillations. Our data suggest that the stimulation of intracellular protein phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase enhances the excitability of bag cell neurons by modifying calcium and potassium channels or currents.

Action Potentials↗

PubMed1982

Microinjection into an identified axon to study the mechanism of fast axonal transport.

Microinjection into an axon of an identified invertebrate neuron is shown to be a useful technique for analyzing the mechanisms of fast axonal transport. It permits direct assessment of the effect of agents that cannot permeate the plasma membrane on the translocation of material in the axon. The actin filament depolymerizer DNase I, when injected into the axon of the Aplysia neuron R2, caused a local block of fast transport of [3H]glycoprotein. Two agents that should interfere with the functioning of actin filaments without causing extensive depolymerization, tne N-ethylmaleimide-modified nuclease S1 fragment of myosin (injected) and dihydrocytochalasin B (applied externally). had no effect. Together these results suggest that actin plays a structural role in the axonal cytoskeleton rather than a role in transport force generation, the effect of DNase I being mediated by structural disordering of the axoplasm. Experiments were also done with inhibitors of dynein, the microtubule-associated ATPase. erythro-9-[3-(2-Hydroxynonyl)]adenine blocked transport but vanadate was ineffective.

Actins↗

PubMed1982

Biosynthesis and secretion of catalytically active acetylcholinesterase in Xenopus oocytes microinjected with mRNA from rat brain and from Torpedo electric organ.

A novel technique was developed for monitoring the level of the mRNA species that direct the synthesis of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7), using microinjected Xenopus oocytes as a translation system. When injected with poly(A)-containing RNA from whole rat brain or rat cerebellum and from electric organ of Torpedo ocellata, Xenopus oocytes synthesize and secrete catalytically active cholinesterase. The newly synthesized enzyme, which is mostly secreted into the oocytes incubation medium, appears to be primarily AcChoEase because it is inhibited by the specific inhibitor BW 284C51. The new enzymatic activity can be detected after injection of as little as 12.5 ng of poly(A)-containing RNA per oocyte, and there is a linear dependence of the oocytes' ability to form AcChoEase on the amount of injected RNA. The AcChoEase mRNA displays a tau 1/2 of about 10 +/- 3 hr in injected oocytes. The abundance of AcChoEase mRNA in the total nonfractionated mRNA injected was calculated to be ca. 1 x 10(-5), a value similar to the level of AcChoEase protein determined in rat brain. The combination of the high turnover number of AcChoEase, the efficiency of the oocyte system, and the sensitivity of the assay used thus permit the accurate monitoring of the scarce mRNA species that direct the synthesis of this enzyme.

Acetylcholinesterase↗

PubMed1984

Ca2+ causes active contraction of bile canaliculi: direct evidence from microinjection studies.

Cytoplasmic microinjection of Ca2+ triggers contraction of bile canaliculi in freshly isolated monolayer cultures of rat hepatocytes. Unseparated paired hepatocytes were used to demonstrate this motility-based phenomenon. Only one cell of the pair was injected, but fluorescein spread from the target cell to the opposite cell; also, the contractions were always uniform, equally involving both hepatocytes that form the canaliculus, indicating that communication exists between the cell pairs. Inhibitors of calmodulin and actin filaments, trifluoperazine and cytochalasin B, respectively, inhibited the Ca2+-induced contractions. Hence, the mechanism of contraction has features in common with actin-myosin based cytoplasmic motility behavior found in other non-muscle cells.

Animals↗

PubMed1988

Automatic microinjection system facilitates detection of growth inhibitory mRNA.

Naturally quiescent human lymphocytes, consisting predominantly of T cells, contain mRNA(s) that can inhibit DNA synthesis when injected into either human diploid fibroblasts (IMR-90) or transformed recipient cells (HeLa). By using an automated capillary microinjection system and a fluorescent coinjection marker (fluorescein isothiocyanate-dextran), individually injected cells can be retrieved and analyzed for DNA synthesis. mRNA isolated from resting T cells is able to block the cells from entering the S phase. The block is reversible and leads to a delay in DNA synthesis. The inhibitory effect is not observed if the injected mRNA is isolated from growth-activated T cells. The disappearance of the inhibition coincides with the approach of the G1/S boundary in both the donor T cells and the recipient human fibroblasts. The mRNA of resting T cells was size-fractionated and the peak inhibitory activity was recovered in a fraction approximately equal to 1.5 kilobases long.

Cell Division↗

PubMed1990

Microinjection of a protein-tyrosine-phosphatase inhibits insulin action in Xenopus oocytes.

A protein-tyrosine-phosphatase (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), specific for phosphotyrosyl residues, was microinjected into Xenopus oocytes. This resulted in a 3- to 5-fold increase in PTPase activity over endogenous levels. The PTPase blocked the insulin-stimulated phosphorylation of tyrosyl residues on endogenous proteins, including a protein having a molecular mass in the same range as the beta subunit of the insulin or insulin-like growth factor I receptor. PTPase 1B also blocked the activation of an S6 peptide kinase--i.e., an enzyme recognizing a peptide having the sequence RRLSSLRA found in a segment of ribosomal protein S6 and known to be activated early in response to insulin. On the other hand, the insulin stimulation of an S6 kinase, detected by using 40S ribosomes as substrate, was unaffected even though PTPase 1B partially prevented the phosphorylation of ribosomal protein S6 in vivo. Mono Q chromatography of insulin-treated oocyte extracts revealed two main peaks of S6 kinase activity. Fractions from the first peak displayed S6 peptide kinase activity that was essentially abolished in profiles from PTPase 1B-injected oocytes. Material from the second peak, which was best revealed by using 40S ribosomes as substrate and had comparatively little S6 peptide kinase activity, was minimally affected by PTPase 1B. These observations suggest that at least two distinct "S6 kinases" are involved in ribosomal protein S6 phosphorylation in vivo and that the activation pathways for these enzymes differ in their sensitivity to PTPase 1B.

Animals↗

PubMed1991

Intracellular distribution of microinjected antisense oligonucleotides.

Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinjected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCl as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.

Animals↗

PubMed1995

Evaluation of calcium influx factors from stimulated Jurkat T-lymphocytes by microinjection into Xenopus oocytes.

Acid extracts of thapsigargin-activated Jurkat cells have been shown to have intracellular activity in inducing a dose-dependent rapid chloride current upon microinjection in Xenopus laevis oocytes. The extracts act by elevation of calcium through calcium entry. The factor(s) responsible for this activity have been termed calcium influx factor (CIF) and have been found to be small, relatively polar molecules (< 1000 daltons) whose activity is abolished by alkaline phosphatase treatment and potentiated by co-injection of okadaic acid (a protein phosphatase inhibitor). CIF is produced in a time-dependent manner following thapsigargin treatment of Jurkat cells, being first elevated above basal levels by 2 min. Intracellular CIF activity is completely absent from NG115-401L neuronal cells, which lack capacitative entry. On this basis, it appears that Jurkat cells, activated by stimuli that deplete internal calcium stores, produce one or more CIF activities acting intracellularly, and Xenopus oocytes may be a powerful tool to purify and characterize CIFs.

Animals↗

PubMed1999

Formation of peroxisomes from peroxisomal ghosts in a peroxisome-deficient mammalian cell mutant upon complementation by protein microinjection.

Most mammalian cell strains genetically deficient in peroxisome biogenesis have abnormal membrane structures called ghosts, containing integral peroxisomal membrane protein, PMP70, but lacking the peroxisomal matrix proteins. Upon genetic complementation, these mutants regain the ability of peroxisome biogenesis. It is postulated that, in this process, the ghosts act as the precursors of peroxisomes, but there has been no evidence to support this. In the present study, we investigated this issue by protein microinjection to a mutant Chinese hamster ovary cell line defective of PEX5, encoding a peroxisome-targeting signal receptor. When recombinant Pex5p and green fluorescent protein (GFP) carrying a peroxisome-targeting signal were co-injected into the mutant cells, the GFP fluorescence gathered over time to particulate structures where PMP70 was co-localized. This process was dependent on both Pex5p and the targeting signal, and, most importantly, occurred even in the presence of cycloheximide, a protein synthesis inhibitor. These findings suggest that the ghosts act as acceptors of matrix proteins in the peroxisome recovery process at least in the PEX5 mutant, and support the view that peroxisomes can grow by incorporating newly synthesized matrix proteins.

ATP-Binding Cassette Transporters↗

PubMedDate not supplied

Cutaneous infection with Mycobacterium fortuitum after localized microinjections (mesotherapy) treated successfully with a triple drug regimen.

Mesotherapy is a treatment method devised for controlling pain syndromes or diseases by subcutaneous microinjections given at or around the involved areas at short intervals of time. Different adverse effects have been described due to this modality of treatment. This report describes 3 patients with cutaneous infection caused by Mycobacterium fortuitum after mesotherapy. Three women, aged 24, 27 and 44 years, presented with similar clinical features, consisting of painful nodules located at the points where mesotherapy had been applied. A smear from a skin biopsy revealed the presence of acid-fast bacilli in all 3 cases. The specimen was cultured and eventually identified as M. fortuitum. A multidrug long-term regimen (combinations of 3 drugs from the following: ciprofloxacin, cotrimoxazole, clarithromycin and amoxicillin-clavulanic acid) was needed to achieve resolution of the lesions. After 15, 25 and 26 months of follow-up, no patient relapsed. Mycobacterium fortuitum is a rapidly growing mycobacterium that can lead to cutaneous infection after minor surgical procedures when aseptic measures are not adequate. Multiple drugs for several months are usually needed to treat this disease successfully.

Adult↗