Thrombasthenic mice generated by replacement of the integrin αIIb gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment

Abstract To analyze the transcriptional activity of the gene encoding the α subunit of the platelet integrin αIIbβ3during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore theknock-in of the tk gene into the αIIb locus resulted in the knock-out of the αIIb gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the αIIbglycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from αIIb-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet α-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia.

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Thrombasthenic mice generated by replacement of the integrin αIIb gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment

Blood ◽

10.1182/blood.v96.4.1399.h8001399_1399_1408 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1399-1408 ◽

Cited By ~ 2

Author(s):

Diana Tronik-Le Roux ◽

Valérie Roullot ◽

Christel Poujol ◽

Thierry Kortulewski ◽

Paquita Nurden ◽

...

Keyword(s):

Transcriptional Activation ◽

Fibrin Clot ◽

Lineage Commitment ◽

Gene Encoding ◽

Α Subunit ◽

Knock Out ◽

Toxic Gene ◽

Specific Locus ◽

Deficient Mice

To analyze the transcriptional activity of the gene encoding the α subunit of the platelet integrin αIIbβ3during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore theknock-in of the tk gene into the αIIb locus resulted in the knock-out of the αIIb gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the αIIbglycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from αIIb-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet α-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia.

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The role of EP2 receptors in mediating the ultra-long-lasting intraocular pressure reduction by JV-GL1

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2020-317762 ◽

2020 ◽

pp. bjophthalmol-2020-317762

Author(s):

Jacques A Bertrand ◽

David F Woodward ◽

Joseph M Sherwood ◽

Jenny W Wang ◽

Darryl R Overby

Keyword(s):

Intraocular Pressure ◽

Ex Vivo ◽

Hypotensive Activity ◽

Long Term Effects ◽

Gene Encoding ◽

Knock Out ◽

Pharmacological Mechanism ◽

Knock Out Mice

BackgroundA single application of JV-GL1 substantially lowers non-human primate intraocular pressure (IOP) for about a week, independent of dose. This highly protracted effect does not correlate with its ocular biodisposition or correlate with the once-daily dosing regimen for other prostanoid EP2 receptor agonists such as trapenepag or omidenepag. The underlying pharmacological mechanism for the multiday extended activity of JV-GL1 is highly intriguing. The present studies were intended to determine EP2 receptor involvement in mediating the long-term ocular hypotensive activity of JV-GL1 by using mice genetically deficient in EP2 receptors.MethodsThe protracted IOP reduction produced by JV-GL1 was investigated in C57BL/6J and EP2 receptor knock-out mice (B6.129-Ptger2tm1Brey/J; EP2KO). Both ocular normotensive and steroid-induced ocular hypertensive (SI-OHT) mice were studied. IOP was measured tonometrically under general anaesthesia. Aqueous humour outflow facility was measured ex vivo using iPerfusion in normotensive C57BL/6J mouse eyes perfused with 100 nM de-esterified JV-GL1 and in SI-OHT C57BL/6J mouse eyes that had received topical JV-GL1 (0.01%) 3 days prior.ResultsBoth the initial 1-day and the protracted multiday effects of JV-GL1 in the SI-OHT model for glaucoma were abolished by deletion of the gene encoding the EP2 receptor. Thus, JV-GL1 did not lower IOP in SI-OHT EP2KO mice, but in littermate SI-OHT EP2WT control mice, JV-GL1 statistically significantly lowered IOP for 4–6 days.ConclusionsBoth the 1-day and the long-term effects of JV-GL1 on IOP are entirely EP2 receptor dependent.

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Reduced autophagy in livers of fasted, fat-depleted, ghrelin-deficient mice: Reversal by growth hormone

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423643112 ◽

2015 ◽

Vol 112 (4) ◽

pp. 1226-1231 ◽

Cited By ~ 43

Author(s):

Yuanyuan Zhang ◽

Fei Fang ◽

Joseph L. Goldstein ◽

Michael S. Brown ◽

Tong-Jin Zhao

Keyword(s):

Growth Hormone ◽

Blood Glucose ◽

Calorie Restriction ◽

Gene Encoding ◽

Glucose Levels ◽

Blood Glucose Levels ◽

Increase Blood Glucose ◽

Plasma Gh ◽

Deficient Mice

Plasma growth hormone (GH) and hepatic autophagy each have been reported to protect against hypoglycemia in the fasted state, but previous data have not linked the two. Here we demonstrate a connection using a mouse model of fasting in a fat-depleted state. Mice were subjected to 1 wk of 60% calorie restriction, causing them to lose nearly all body fat. They were then fasted for 23 h. During fasting, WT mice developed massive increases in plasma GH and a concomitant increase in hepatic autophagy, allowing them to maintain viable levels of blood glucose. In contrast, lethal hypoglycemia occurred in mice deficient in the GH secretagogue ghrelin as a result of knockout of the gene encoding ghrelin O-acyltransferase (GOAT), which catalyzes a required acylation of the peptide. Fasting fat-depleted Goat−/− mice showed a blunted increase in GH and a marked decrease in hepatic autophagy. Restoration of GH by infusion during the week of calorie restriction maintained autophagy in the Goat−/− mice and prevented lethal hypoglycemia. Acute injections of GH after 7 d of calorie restriction also restored hepatic autophagy, but failed to increase blood glucose, perhaps owing to ATP deficiency in the liver. These data indicate that GH stimulation of autophagy is necessary over the long term, but not sufficient over the short term to maintain blood glucose levels in fasted, fat-depleted mice.

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Regulation of spermatogenesis in McCune–Albright syndrome: lessons from a 15-year follow-up.

Acta Endocrinologica ◽

10.1530/eje-07-0494 ◽

2008 ◽

Vol 158 (6) ◽

pp. 921-927 ◽

Cited By ~ 14

Author(s):

Filippo De Luca ◽

Valérie Mitchell ◽

Malgorzata Wasniewska ◽

Teresa Arrigo ◽

Maria Francesca Messina ◽

...

Keyword(s):

Gene Encoding ◽

Α Subunit ◽

Mccune Albright Syndrome ◽

Sexual Precocity ◽

Albright Syndrome ◽

Histology And Immunohistochemistry ◽

Shed Light ◽

Mccune Albright

ContextMcCune–Albright syndrome (MAS) is a disorder caused by a post-zygotic gain-of-function mutation in the gene encoding the Gs-α protein. Sexual precocity, common in girls, has been reported in only 15% of boys, and little is known on the long-term evolution of MAS in males.ObjectiveIn a boy with MAS, we studied spermatogenesis, testis histology, and immunohistochemistry with the aim to shed light on seminiferous tubule activity.DesignA boy who presented at the age of 2.9 years with sexual precocity, monolateral macroorchidism, increased testosterone levels, and suppressed gonadotropins was followed up until the age of 18.ResultsThroughout follow-up testicular asymmetry persisted and gonadotropin and testosterone pattern did not change. At the age of 18, inhibin B was undetectable while α-immunoreactive inhibin was within normal range. Anti-Mullerian hormone level was slightly subnormal. Sperm cells were 3 900 000 per ejaculate. Histology of both testes showed spermatogonia, spermatocytes, and, in some tubes, matured spermatozoa. Sertoli cells were markedly stained with anti-inhibin α-subunit antibody in both the testes. There was no immunostaining of Sertoli, Leydig, or germ cells with anti-βA or anti-βB antibody. MAS R201H mutation was identified in both the testes.ConclusionThe 15-year follow-up in this boy with MAS demonstrated that autonomous testicular activation and gonadotropin suppression persisted over time. This provides an interesting model of active spermatogenesis despite long-term FSH suppression. It also suggests that FSH is needed for the full expression of the inhibin βB-subunit gene, an expression previously reported in the germ and Leydig cells of normal adult subjects.

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Faculty Opinions recommendation of Long-term control of Mycobacterium bovis BCG infection in the absence of Toll-like receptors (TLRs): investigation of TLR2-, TLR6-, or TLR2-TLR4-deficient mice.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1022880.264653 ◽

2004 ◽

Author(s):

Charles Czuprynski

Keyword(s):

Mycobacterium Bovis ◽

Toll Like Receptors ◽

Mycobacterium Bovis Bcg ◽

Deficient Mice

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Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants

Molecular Genetics and Genomics ◽

10.1007/s00438-007-0209-1 ◽

2007 ◽

Vol 277 (5) ◽

pp. 589-600 ◽

Cited By ~ 200

Author(s):

Chang-Sun Choi ◽

Hiroshi Sano

Keyword(s):

Abiotic Stress ◽

Transcriptional Activation ◽

Gene Encoding ◽

Tobacco Plants

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The G-Protein α-Subunit GasC Plays a Major Role in Germination in the Dimorphic FungusPenicillium marneffei

Genetics ◽

10.1093/genetics/164.2.487 ◽

2003 ◽

Vol 164 (2) ◽

pp. 487-499 ◽

Cited By ~ 2

Author(s):

Sophie Zuber ◽

Michael J Hynes ◽

Alex Andrianopoulos

Keyword(s):

G Protein ◽

Germination Rate ◽

Yeast Cells ◽

Class Iii ◽

Temperature Dependent ◽

Gene Encoding ◽

Α Subunit ◽

G Protein Α Subunit ◽

Opportunistic Human Pathogen

AbstractThe opportunistic human pathogen Penicillium marneffei exhibits a temperature-dependent dimorphic switch. At 25°, multinucleate, septate hyphae that can undergo differentiation to produce asexual spores (conidia) are produced. At 37° hyphae undergo arthroconidiation to produce uninucleate yeast cells that divide by fission. This work describes the cloning of the P. marneffei gasC gene encoding a G-protein α-subunit that shows high homology to members of the class III fungal Gα-subunits. Characterization of a ΔgasC mutant and strains carrying a dominant-activating gasCG45R or a dominant-interfering gasCG207R allele show that GasC is a crucial regulator of germination. A ΔgasC mutant is severely delayed in germination, whereas strains carrying a dominant-activating gasCG45R allele show a significantly accelerated germination rate. Additionally, GasC signaling positively affects the production of the red pigment by P. marneffei at 25° and negatively affects the onset of conidiation and the conidial yield, showing that GasC function overlaps with functions of the previously described Gα-subunit GasA. In contrast to the S. cerevisiae ortholog Gpa2, our data indicate that GasC is not involved in carbon or nitrogen source sensing and plays no major role in either hyphal or yeast growth or in the switch between these two forms.

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FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

Blood ◽

10.1182/blood.v92.11.3997 ◽

1998 ◽

Vol 92 (11) ◽

pp. 3997-4002 ◽

Cited By ~ 92

Author(s):

Dirk Meyer ◽

Carsten Schiller ◽

Jürgen Westermann ◽

Shozo Izui ◽

Wouter L. W. Hazenbos ◽

...

Keyword(s):

Hemolytic Anemia ◽

Autoimmune Hemolytic Anemia ◽

Knock Out ◽

Effector Cells ◽

Igg Isotypes ◽

Knock Out Mice ◽

Deficient Mice ◽

Experimental Autoimmune

Abstract In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

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