Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis

Mpl ligand (thrombopoietin [TPO]) is the physiological regulator of platelet production. In mice, mRNA encoding the Mpl ligand (Mpl-L) is predominantly found by Northern blot analysis in the liver and kidney. To investigate the mode of regulation of the Mpl-L gene, we have developed several experimental models of severe thrombocytopenia differing in their kinetics and an opposite model of chronic thrombocytosis. Northern analysis performed at various times after induction of a thrombocytopenic state demonstrates that, whatever the number of circulating platelets, no change in Mpl-L mRNA level occurs in liver and kidney. By ribonuclease protection assays, we analyzed the ratios between mRNAs coding for the wild-type Mpl-L form and various splice variants encoding inactive or nonsecreted Mpl-L proteins. No modification in levels of these various isoforms was detected confirming the data of a previous report. Because the highest level of Mpl-L bioactivity in sera was observed only in mice with drastically reduced numbers of both platelets and megakaryocytes, these results further suggest that not only platelets, but also megakaryocytes, must be involved in the regulation of the level of circulating Mpl-L. In addition, we show that no downregulation of wild-type Mpl-L mRNA and no change in the ratio of Mpl-L mRNA isoforms were detected in mice in which a chronic thrombocytosis was induced. Together, these different models extend and further confirm that the regulation of Mpl-L does not occur at a transcriptional level or by a modulation in the ratios of Mpl-L mRNA isoforms.

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LacI-Family Transcriptional Regulator DagR Acts as a Repressor of the Agarolytic Pathway Genes in Streptomyces coelicolor A3(2)

Frontiers in Microbiology ◽

10.3389/fmicb.2021.658657 ◽

2021 ◽

Vol 12 ◽

Author(s):

Maral Tsevelkhoroloo ◽

So Heon Shim ◽

Chang-Ro Lee ◽

Soon-Kwang Hong ◽

Young-Soo Hong

Keyword(s):

Gene Expression ◽

Type Strain ◽

Wild Type Strain ◽

Degradation Products ◽

Mrna Level ◽

Regulation Of Gene Expression ◽

Hydrolytic Enzymes ◽

Upstream Region ◽

Transcriptional Level ◽

Wild Type

Actinobacteria utilize various polysaccharides in the soil as carbon source by degrading them via extracellular hydrolytic enzymes. Agarose, a marine algal polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG), is one of the carbon sources used by S. coelicolor A3(2). However, little is known about agar hydrolysis in S. coelicolor A3(2), except that the regulation of agar hydrolysis metabolism is strongly inhibited by glucose as in the catabolic pathways of other polysaccharides. In this study, we elucidated the role of DagR in regulating the expression of three agarase genes (dagA, dagB, and dagC) in S. coelicolor A3(2) by developing a dagR-deletion mutant (Δsco3485). We observed that the Δsco3485 mutant had increased mRNA level of the agarolytic pathway genes and 1.3-folds higher agarase production than the wild type strain, indicating that the dagR gene encodes a cluster-suited repressor. Electrophoretic mobility shift assay revealed that DagR bound to the upstream regions of the three agarase genes. DNase 1 footprinting analysis demonstrated that a palindromic sequence present in the upstream region of the three agarase genes was essential for DagR-binding. Uniquely, the DNA-binding activity of DagR was inhibited by AHG, one of the final degradation products of agarose. AHG-induced agarase production was not observed in the Δsco3485 mutant, as opposed to that in the wild type strain. Therefore, DagR acts as a repressor that binds to the promoter region of the agarase genes, inhibits gene expression at the transcriptional level, and is derepressed by AHG. This is the first report on the regulation of gene expression regarding agar metabolism in S. coelicolor A3(2).

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Thioredoxin Is Involved in Oxygen-Regulated Formation of the Photosynthetic Apparatus of Rhodobacter sphaeroides

Journal of Bacteriology ◽

10.1128/jb.181.1.100-106.1999 ◽

1999 ◽

Vol 181 (1) ◽

pp. 100-106 ◽

Cited By ~ 20

Author(s):

Cecile Pasternak ◽

Kerstin Haberzettl ◽

Gabriele Klug

Keyword(s):

Mutant Strain ◽

Oxygen Tension ◽

Rhodobacter Sphaeroides ◽

Aminolevulinic Acid ◽

Mrna Level ◽

Photosynthetic Apparatus ◽

Transcriptional Level ◽

Wild Type ◽

Low Oxygen ◽

Low Oxygen Tension

ABSTRACT Thioredoxin, a redox active protein, has been previously demonstrated to be essential for growth of the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides. In the present study, the involvement of thioredoxin in the formation of the photosynthetic apparatus of R. sphaeroides WS8 was investigated by construction and analysis of a mutant strain disrupted for the chromosomal trxA copy and carrying a plasmid-borne copy of trxA under the control of the hybrid p trc promoter inducible by IPTG (isopropyl-β-d-thiogalactopyranoside). This strain was viable in the absence of IPTG but was affected in pigmentation. When shifted from high to low oxygen tension conditions, thetrxA mutant showed a reduced bacteriochlorophyll content in comparison to that of the wild type. Although thioredoxin is able to regulate aminolevulinic acid (ALA) synthase (the first enzyme of the tetrapyrrole biosynthetic pathway) activity by a dithiol-disulfide exchange, our mutant strain exhibited a level of ALA synthase activity identical to that of the wild type, suggesting that thioredoxin is involved in other steps to regulate the synthesis of the photosynthetic apparatus. Accordingly, we showed that the trxA mutation affects the oxygen-regulated expression of the puf operon encoding the pigment-binding proteins of the light-harvesting and reaction center complexes. Upon transition from aerobic to semiaerobic growth conditions, the maximal puf mRNA level was found to be 40 to 50% lower in the mutant strain than in the wild type. The stability of the puf transcripts was identical in both strains grown under low oxygen tension, indicating that the role of thioredoxin in regulating puf expression occurs at the transcriptional level.

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Homology-Dependent Silencing of the SC3 Gene in Schizophyllum commune

Genetics ◽

10.1093/genetics/147.2.589 ◽

1997 ◽

Vol 147 (2) ◽

pp. 589-596 ◽

Cited By ~ 1

Author(s):

Theo A Schuurs ◽

Eveline A M Schaeffer ◽

Joseph G H Wessels

Keyword(s):

Gene Silencing ◽

Genomic Dna ◽

Type Strain ◽

Cytosine Methylation ◽

Wild Type Strain ◽

Schizophyllum Commune ◽

Southern Analysis ◽

Transcriptional Level ◽

Wild Type

After introduction of extra copies of the SC3 hydrophobin gene into a wild-type strain of Schizophyllum commune, gene silencing was observed acting on both endogenous and introduced SC3 genes in primary vegetative transformants. Nuclear run-on experiments indicated that silencing acted at the transcriptional level. Southern analysis revealed that cytosine methylation of genomic DNA occurred. Moreover, SC3 silencing was suppressed by exposure to 5-azacytidine during growth. After growth of SC3-suppressed colonies from homogenized mycelium or from colonies stored at 4°, SC3 transcription was restored. However, after prolonged growth SC3 silencing was again observed. Introduction of a promoterless SC3 fragment into wild type gave less SC3 silencing.

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Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽

10.1182/blood-2012-07-441824 ◽

2012 ◽

Vol 120 (16) ◽

pp. 3336-3344 ◽

Cited By ~ 36

Author(s):

Anu Laitala ◽

Ellinoora Aro ◽

Gail Walkinshaw ◽

Joni M. Mäki ◽

Maarit Rossi ◽

...

Keyword(s):

Cultured Cells ◽

Mrna Level ◽

Hypoxia Inducible Factor ◽

Mrna Levels ◽

Wild Type ◽

Α Subunit ◽

Hepcidin Expression ◽

In The Wild ◽

Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

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BCL2 protein expression parallels its mRNA level in normal and malignant B cells

Blood ◽

10.1182/blood-2004-01-0243 ◽

2004 ◽

Vol 104 (9) ◽

pp. 2936-2939 ◽

Cited By ~ 17

Author(s):

Yulei Shen ◽

Javeed Iqbal ◽

James Z. Huang ◽

Guimei Zhou ◽

Wing C. Chan

Keyword(s):

B Cells ◽

Protein Expression ◽

B Cell ◽

Posttranscriptional Regulation ◽

Cell Lymphoma ◽

Dominant Role ◽

Mrna Level ◽

B Cell Lymphoma ◽

Transcriptional Level ◽

Bcl2 Protein

Abstract The regulation of B-cell lymphoma 2 (BCL2) protein expression in germinal center (GC) B cells has been controversial. Previous reports have indicated posttranscriptional regulation plays a dominant role. However, a number of recent studies contradicted these reports. Using real-time polymerase chain reaction (PCR) and Standardized Reverse Transcriptase-PCR (StaRT-PCR), we measured the level of mRNA expression in GC, mantle zone (MNZ), and marginal zone (MGZ) cells from laser capture microdissection. Both quantitative RT-PCR measurements of microdissected GC cells from tonsils showed that GC cells had low expression of BCL2 transcripts commensurate with the low protein expression level. These results are in agreement with microarray studies on fluorescence-activated cell sorter (FACS)-sorted cells and microdissected GC cells. We also examined BCL2 mRNA and protein expression on a series of 30 cases of diffuse large B-cell lymphoma (DLBCL) and found, in general, a good correlation. The results suggested that BCL2 protein expression is regulated at the transcriptional level in normal B cells and in the neoplastic cells in most B-cell lymphoproliferative disorders.

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Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6303 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6303-6310 ◽

Cited By ~ 37

Author(s):

L Yu ◽

M A Gorovsky

Keyword(s):

Protein Sequence ◽

Tetrahymena Thermophila ◽

Histone H3 ◽

Constitutive Expression ◽

Histone Variants ◽

Wild Type ◽

Ciliated Protozoan ◽

Knockout Mutant ◽

Double Knockout ◽

Constitutive Synthesis

Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown. Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells. Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes. This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants. Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena. Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable. Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth. In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not. These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.

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Abstract MP45: Deletion Of The Transient Receptor Vanilloid-1 (TRPV1) Channel In Rats Attenuates 2 Kidney 1 Clip Hypertension

Hypertension ◽

10.1161/hyp.78.suppl_1.mp45 ◽

2021 ◽

Vol 78 (Suppl_1) ◽

Author(s):

Sean D Stocker ◽

Leon J DeLalio

Keyword(s):

Heart Rate ◽

Renovascular Hypertension ◽

Sensory Nerve ◽

Experimental Models ◽

Sensory Nerves ◽

Male Rats ◽

Wild Type ◽

Renal Nerve ◽

Trpv1 Channels ◽

Arterial Blood

Renal denervation lowers arterial blood pressure (ABP) in both clinical populations and multiple experimental models of hypertension. This therapeutic effect is partly attributed to the removal of overactive renal sensory nerves that increase sympathetic efferent activity and ABP. Renal sensory nerves highly express TRPV1 channels, and administration of the TRPV1 agonist capsaicin increases renal sensory nerve activity. However, the extent by which TRPV1 channels directly contribute to renal nerve dependent models of hypertension has not been tested. To test this hypothesis, we generated a novel TRPV1 -/- rat using CRISPR/Cas9 and deletion of exon 3. Male and female TRPV1 -/- and wild-type littermates (8-12 weeks) were instrumented with telemetry. At 2 weeks later, renovascular hypertension via renal stenosis was produced by placement of a PTFE cuff (0.16 x 0.22 inches, 1mm long) around the right renal artery. Male TRPV1 -/- and wild-type rats had no differences in baseline mean ABP (99±2 vs 98±3 mmHg, respectively; n=7-9) or heart rate (390±7 vs 400±8 bpm, respectively). Renal stenosis significantly increased mean ABP in both groups; however, mean ABP was significantly lower at Day 28 in male TRPV1 -/- versus wild-type rats (125±8 vs 155±2 mmHg, respectively: P<0.01). Ganglionic blockade with chlorisondamine (2.5mg/kg, sc) at Day 28 produced a smaller fall in mean ABP of male TRPV1 -/- versus wild-type rats (-53±4 vs -86±3 mmHg, respectively; P<0.001). On the other hand, female TRPV1 -/- and wild-type rats had no differences in baseline mean ABP (102±2 vs 104±1 mmHg, respectively; n=6-9) or heart rate (419±8 vs 410±7 bpm, respectively). Renal stenosis significantly increased mean ABP in both groups; however, there were no differences at Day 28 between female TRPV1 -/- versus wild-type rats (117±8 vs 122±6 mmHg, respectively). Moreover, the increase in mean ABP was smaller in females versus males. The ganglionic blocker chlorisondamine produced similar depressor responses in female TRPV1 -/- versus wild-type rats (-64±7 vs -65±7 mmHg, respectively). These findings illustrate a sex difference in renovascular hypertension in rats, but importantly indicate that TRPV1 channels contribute to the established phase of renovascular hypertension in male rats.

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REACTIVE CHANGES OF LIVER AND KIDNEY HISTOLOGICAL ELEMENTS IN MURINE MODELS OF SEPSIS CAUSED BY ADMINISTRATION OF DIFFERENT STRAINS TO MICE

Морфология ◽

10.34922/ae.2020.158.4.010 ◽

2020 ◽

pp. 66-71

Author(s):

Татьяна Геннадьевна Боровая ◽

Владимир Григорьевич Жуховицкий ◽

Мария Николаевна Черкасова

Keyword(s):

Pseudomonas Aeruginosa ◽

Morphological Changes ◽

Blood Stasis ◽

Intraperitoneal Administration ◽

Liver Parenchyma ◽

Experimental Models ◽

Control Group ◽

Reactive Changes ◽

Liver And Kidney ◽

Different Strains

Цель - выявление реактивных изменений гистологических элементов печени и почек у мышей в экспериментальных моделях сепсиса, вызванного штаммами 1840 и 1623 Pseudomonas aeruginosa (PsA1840, 1623). Материал и методы. Сепсис моделировали на двух группах половозрелых самцов мышей линии C57Bl/6 интраперитонеальным введением Pseudomonas aeruginosa. Животным 1-й группы (8 особей) вводили штамм 1840, животным 2-й группы (12 особей) - штамм 1623. Контрольная группа состояла из 3 животных. Перед началом опыта штаммы тестировали на присутствие генов экзотоксинов U, S, T, Y (ExoU, ExoS, ExoT, ExoY) с помощью полимеразной цепной реакции (ПЦР). Для визуализации продуктов ПЦР применяли электрофорез в горизонтальном 1,5 % агарозном геле. Животных вскрывали на терминальной стадии сепсиса. Серийные парафиновые срезы печени и почек толщиной 4 мкм окрашивали гематоксилином - эозином, анализировали особенности гистоструктуры органов и фотографировали в световом микроскопе «AxioPlus» (фирма Zeiss, Германия). Результаты. Штамм PsA 1840, имеющий ген exoU, вызывал выраженные деструктивные изменения пластинок гепатоцитов и замещение участков паренхимы печени гомогенным эозинофильным веществом. Присутствовали признаки стаза крови в синусоидных капиллярах, расширение и тромбоз центральных вен, немногочисленные скопления лейкоцитов. Морфологические изменения нефронов состояли в локальных деструктивных изменениях проксимальных канальцев на периферии коркового вещества почек. При введении PsA1623, имеющего ген exoS, возникали массовая гибель почечных телец и дегенерация канальцев нефронов. В печени дольковая гистоархитектура в основном сохранялась. Выводы. Предполагается связь выявленных различий в реактивных изменениях гистологических элементов печени и почек в подопытных группах с особенностями геномов штаммов PsA, использованных для моделирования сепсиса. Objective - to identify reactive changes of liver and kidney histological elements in experimental models of sepsis in mice caused by 1840 and 1623 Pseudomonas aeruginosa strains (PsA1840, 1623). Material and methods. Sepsis was modeled in two groups of mature male C57Bl/6 mice by intraperitoneal administration of Pseudomonas aeruginosa. Strain 1840 was administered to animals of the first group (n=8), animals of the second group (n=12) were administered strain 1623; the control group consisted of 3 animals. Before the experiment, the strains were tested for the presence of genes of exotoxins U, S, T, Y (ExoU, ExoS, ExoT, ExoY) using polymerase chain reaction (PCR). Electrophoresis in horizontal 1,5 % agarose gel was used to visualize PCR products. The animals were euthanized at the terminal stage of sepsis. The extracted liver and kidneys were fixed according to the generally accepted histological method, and embedded into paraffin blocks. Serial 4 μm thick sections of organs were stained with hematoxylin and eosin, analyzed and photographed using «AxioPlus» light microscope (Zeiss, Germany). Results. Strain PsA 1840, carrying the gene of exotoxin U (ExoU), caused severe destructive changes of hepatocytes plates and the replacement of the liver parenchyma with homogeneous eosinophilic substance. There were signs of blood stasis in sinusoidal capillaries, expansion and thrombosis of central veins, a few accumulations of leukocytes. Morphological changes of nephrons consisted of local destructive changes in the proximal tubules at the periphery of kidney cortical substance. After the introduction of PsA1623, carrying the gene of exotoxin S (ExoS), the massive death of renal corpuscles and degeneration of nephron tubules were registered. However, the lobular histoarchitecture in the liver remained mostly unaltered. Conclusions. It is supposed that there is a possible connection of the observed differences in reactive changes of liver and kidney histological elements in two experimental groups with genome features of PsA strains used for the sepsis modeling.

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Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2521-2532.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2521-2532 ◽

Cited By ~ 69

Author(s):

C. Lange ◽

D. Rittmann ◽

V. F. Wendisch ◽

M. Bott ◽

H. Sahm

Keyword(s):

Corynebacterium Glutamicum ◽

Expression Profiling ◽

Large Subunit ◽

Mrna Level ◽

Acetohydroxyacid Synthase ◽

Limiting Factor ◽

Unexpected Result ◽

Dependent Manner ◽

Wild Type ◽

Concentration Dependent Manner

ABSTRACT Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvA ΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l-isoleucine could relieve the valine effect on VAL1 whereas l-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

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Identification of Gαs messenger ribonucleic acid splice variants in human granulosa cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0180027 ◽

1997 ◽

Vol 18 (1) ◽

pp. 27-35 ◽

Cited By ~ 2

Author(s):

G N Europe-Finner ◽

E Cartwright ◽

J Bellinger ◽

H J Mardon ◽

D H Barlow ◽

...

Keyword(s):

Granulosa Cells ◽

Ovarian Function ◽

Splice Variants ◽

Consensus Sequence ◽

Phosphorylation Site ◽

Follicular Development ◽

Protein Isoforms ◽

Mrna Isoforms ◽

Messenger Ribonucleic Acid

ABSTRACT Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

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