scholarly journals Detection of a pheomelanin-like pigment by EPR spectroscopy in the mycelium of Plenodomus biglobosus

2020 ◽  
Author(s):  
Jan Pukalski ◽  
Natalia Marcol ◽  
Norbert Wolan ◽  
Przemysław Mieszko Płonka ◽  
Przemysław Ryszka ◽  
...  
Keyword(s):  
Epr Spectroscopy ◽  
Culture Medium ◽  
Epr Spectrum ◽  
Spin Resonance ◽  
Dark Pigmentation ◽  
Dark Culture ◽  
Host Parasite ◽  
Electron Paramagnetic

Melanin occurrence in Plenodomus biglobosus was investigated using electron paramagnetic (spin) resonance (EPR, ESR) spectroscopy. The fungus was isolated from living and dead leaves of European ash (Fraxinus excelsior L.). Dark pigmentation of P. biglobosus mycelium in vitro, especially on the reverse, was observed. The black coloration intensified with the age of the culture and inspired us to check if the analyzed fungus species synthesizes melanin. Melanin contains unpaired electrons, thus, EPR spectroscopy was applied, as a specific technique, to verify its presence in P. biglobosus. The EPR spectrum of the mycelium showed a very strong melanin signal, revealing pheomelanin-like features. Thus, the black pigment of P. biglobosus was clearly identified as melanin. However, no melanin was detected in the apparently dark culture medium even when zinc (II) acetate was added to increase the sensitivity of detection. Pheomelanin has many unusual biological functions but it is not commonly found in fungi. Detection of this type of melanin in P. biglobosus, which can be both endophytic or pathogenic, suggests a closer examination of the potential role of this melanin in host-parasite interaction.

Follicle-stimulating hormone-dependent estrogen secretion by rat Sertoli cells in vitro: Modulation by calcium

1991 ◽  
Vol 125 (3) ◽  
pp. 280-285 ◽  
Author(s):  
J. Alan Talbot ◽  
Ann Lambert ◽  
Robert Mitchell ◽  
Marek Grabinski ◽  
David C. Anderson ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Culture Medium ◽  
Follicle Stimulating Hormone ◽  
Dibutyryl Camp ◽  
Immature Rat ◽  
Estradiol Secretion ◽  
Old Rats ◽  
Stimulating Hormone

Abstract We have investigated the role of Ca2+ in the control of FSH-induced estradiol secretion by Sertoli cells isolated from 8-10 days old rats. Exogenous Ca2+ (4-8 mmol/1) inhibited FSH-stimulated E2 secretion such that, with 8 mmol/l Ca2+ and FSH (8 IU/l) E2 secretion decreased from 2091±322 to 1480±84 pmol/l (p<0.002), whilst chelation of Ca2+ in the culture medium with EGTA (3 mmol/l) increased E2 secretion from 360±45 to 1242±133 pmol/l) in the absence of FSH. Further, EGTA (3 mmol/l) markedly potentiated FSH (8 IU/l), forskolin (1 μmol/l) and dibutyryl cAMP (1 mmol/l)-stimulated E2 secretion. Addition of the Ca2+ ionophores, ionomycin (2-5 μmol/l) and A23187 (2 μmol/l), inhibited FSH (8 IU/l)-stimulated E2 secretion by >80%. The effect of ionomycin was totally reversible, whereas that of A23187 was irreversible. Ionomycin (5 μmol/l) had no effect on EGTA-induced E2 secretion in the absence of FSH, but reduced EGTA-provoked E2 secretion by 59% in the presence of FSH (8 IU/l). Similarly, forskolin- and dibutyryl cAMP-provoked E2 production was inhibited 46-50% by ionomycin (5 μmol/l). We conclude that FSH-induced E2 secretion from immature rat Sertoli cells is modulated by intra- and extracellular Ca2+.

Sources and implications of basal nitric oxide in spontaneous contractions of guinea pig taenia caeci

2003 ◽  
Vol 285 (4) ◽  
pp. G747-G753 ◽  
Author(s):  
Catalina Caballero-Alomar ◽  
Carmen Santos ◽  
Diego Lopez ◽  
M. Teresa Mitjavila ◽  
Pere Puig-Parellada
Keyword(s):  
Nitric Oxide ◽  
Guinea Pig ◽  
Inhibitory Effect ◽  
No Production ◽  
Paramagnetic Resonance ◽  
Synthase Inhibitor ◽  
Spontaneous Contractions ◽  
Electron Paramagnetic

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 10–4 M), the neuronal blocker tetrodotoxin (TTX, 10–6 M), or both on spontaneous contractions and on the production of basal NO. Both l-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by l-NAME and was inhibited by 38% by TTX. When tested together, l-NAME in the presence of TTX or TTX in the presence of l-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.

scholarly journals Malaria and red cell genetic defects

Blood ◽  
1989 ◽  
Vol 74 (4) ◽  
pp. 1213-1221 ◽  
Author(s):  
RL Nagel ◽  
EF Jr Roth
Keyword(s):  
Culture System ◽  
Potential Role ◽  
Dna Analysis ◽  
Red Cell ◽  
Metabolic Products ◽  
Recombinant Technology ◽  
Host Parasite ◽  
Made In

Abstract The study of inherited RBC resistance to malaria has increased our knowledge of the biochemistry and physiology of the host-parasite interaction and suggested potential sites for therapeutic intervention. Discovery by Jensen and Trager of the in vitro culture system for P falciparum has facilitated research in this area. Known RBC defects may affect invasion, growth, or merozoite liberation (Fig 1). Significant advances made in understanding mechanisms underlying protection against malaria should not obscure the fact that the data are far from complete. More knowledge is needed about the influence of the erythrocyte cytoskeleton on invasion and growth of parasites as well as the potential role of phospholipids, erythrocyte enzymes other than G6PD, or other metabolic products. Application of DNA analysis and recombinant technology may have an increasing impact on study of the interaction of RBC defects with malarial parasites.

scholarly journals 108 THE ROLE OF NITRIC OXIDE SYNTHASE IN IN VITRO DEVELOPMENT OF BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 204
Author(s):  
A.K. Kadanga ◽  
D. Tesfaye ◽  
S. Ponsuksili ◽  
K. Wimmers ◽  
M. Gilles ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Culture Medium ◽  
Bovine Embryos ◽  
Nos Inhibitor ◽  
Blastocyst Rate ◽  
Oxide Synthase ◽  
Vitro Development ◽  
Bovine Oocytes

Nitric oxide (NO) is a free radical that serves as a key-signal molecule in various physiological processes including reproduction. Four isoforms of nitric oxide synthase (NOS) have been characterized: endothelial (eNOS), inducible (iNOS), neuronal (nNOS), and mitochondrial (mtNOS). The first two isoforms are reported to be expressed in mouse follicles, oocytes, and pre-implantation embryos (Nishikimi A et al. 2001 Reproduction 122, 957–963). However, the role of any of these isoforms have not yet been investigated in bovine embryos. Here we aimed to examine the role of NOS in in vitro development of bovine embryos by treating embryos with NOS inhibitor, N-omega-L-nitro-arginine methyl esther (L-NAME), and examining the localization of the protein in pre-implantation embryos. Oocytes and embryos were grown in the media with NOS inhibitor added at a level of 0 mM (control), 1 mM, and 10 mM to either maturation or culture medium. Each experiment was conducted in four replicates each containing 100 oocytes for IVP. Cleavage and blastocyst rate were recorded at Days 2 and 7, respectively. Data were analyzed using the General Linear Model in SAS version 8.02 (SAS Institute, Inc., Cary, NC, USA) with the main factors being the level of L-NAME and the point of application. Pairwise comparisons were done using the Tukey test. Protein localization in bovine oocytes and embryos was performed by immunocytochemistry using eNOS- and iNOS-specific antibodies. Embryos were fixed in 3.7% paraformaldehyde, permeabilized in 0.1% Triton-X100, and washed three times in PBS supplemented with BSA. They were incubated with eNOS and iNOS primary antibody (1:200 dilutions) and washed before incubation with secondary antibody conjugated to FITC. After washing they were mounted on glass slides and examined under a confocal laser scanning microscope (Carl Zeiss Jena, Carl Zeiss AG, Oberkochen, Germany). In the controls the primary antibodies were omitted. As shown in the table below, the presence of L-NAME in the maturation medium significantly reduced the cleavage and blastocyst rate independent of the dosage applied. However the presence of L-NAME in the culture medium had an influence only on the blastocyst rate. The immunocytochemical staining results showed that both eNOS and iNOS are expressed in the cytoplasm of the MII oocytes, and during the pre-implantation stage the fluorescence signal was observed in nuclei and cytoplasm. However, the nuclear signal was much weaker. In conclusion, the present study is the first to determine the role of NO and to detect NOS protein in bovine oocytes and pre-implantation embryos. These results indicate that nitric oxide may play an important role as diffusible regulator of bovine oocyte maturation and preimplantation embryo development. Table 1. Effect of l-name addition in maturation or culture medium on embryo development

scholarly journals Ultrastructural study of degradation of calcium phosphate ceramic by human monocytes and modulation of this activity by HILDA/LIF cytokine.

1996 ◽  
Vol 44 (10) ◽  
pp. 1131-1140 ◽  
Author(s):  
M D Benahmed ◽  
D Heymann ◽  
M Berreur ◽  
M Cottrel ◽  
A Godard ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Culture Medium ◽  
Marked Decrease ◽  
Calcium Phosphate Ceramic ◽  
Multinuclear Cells ◽  
Residual Bodies ◽  
First Time

Biodegradation of ceramics in vivo is achieved essentially by monocytes and multinuclear cells (osteoclasts). Monocytes are the key element in this process because they intervene first at the biomaterial implantation site during inflammatory reaction. In this work, in vitro studies were conducted on an ultrastructural scale to determine the specific behavior of these cells with regard to a calcium phosphate (CaP) ceramic. Two types of phagocytosis were observed when cells came into contact with the biomaterial: either CaP crystals were taken up alone and then dissolved in the cytoplasm after disappearance of the phagosome membrane or they were incorporated together with large quantities of culture medium, in which case dissolution occurred after the formation of heterophagosomes. Phagocytosis of CaP coincided with autophagy and the accumulation of residual bodies in the cells. Addition of HILDA/LIF factor to these cultures induced a very marked decrease in phagocytotic activity directed at the capture of CaP crystals and culture medium. Autophagy was reduced, and residual bodies were rare or absent. This study specifies the role of monocytes in CaP biodegradation and demonstrates for the first time that HILDA/LIF has a biological effect on this cell line.

Macrophage Induced Effect of Particulate Silica on Rat Mesenchymal Stem Cells In Vitro

2008 ◽  
Vol 396-398 ◽  
pp. 123-126
Author(s):  
Timothy Wilson ◽  
Reeta Viitala ◽  
Mervi Puska ◽  
Mika Jokinen ◽  
Risto Penttinen
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Cultures ◽  
Culture Medium ◽  
Matrix Protein ◽  
Bioactive Glasses ◽  
Significant Difference ◽  
Silica Microparticles

The role of silica and macrophages in fibrosis is well documented, but in bone formation it is relatively unknown despite decades of research with bioactive glasses. In this study macrophages were isolated from rat peritoneal and then cultured for five days in the presence of two types of silica microparticles with different solubilities. After the fifth day the culture medium was collected, purified and used as an additive in bone marrow derived rat stem cell cultures. The stem cells were cultured for five days in α-mem containing only 0,5% of FCS, enabling cell survival but disrupting their proliferation. As controls, stem cells were also cultured in α-mem containing silica microparticles. At days one and five the amount of soluble collagen was assayed from the culture medium and the cells were counted. All stem cell cultures with macrophage medium additives were found to be proliferative, with statistically significant difference to controls. However, collagen was only produced in cultures containing medium from macrophages cultured with fast-dissolving silica microparticles. This suggests that silica can induce cell proliferation and extra cellular matrix protein secretion which is mediated by macrophages, and that the solubility of silica is also a major factor in this reaction.

scholarly journals Control of hsp70 RNA levels in human lymphocytes.

1987 ◽  
Vol 104 (2) ◽  
pp. 183-187 ◽  
Author(s):  
L Kaczmarek ◽  
B Calabretta ◽  
H T Kao ◽  
N Heintz ◽  
J Nevins ◽  
...  
Keyword(s):  
Culture Medium ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Peripheral Blood Mononuclear ◽  
Growth State ◽  
Steady State Levels ◽  
Rna Blots

The expression of a hsp70 gene in human cells has previously been shown to be related to the growth state of the cells. As an alternative to in vitro synchronization procedures, we have measured steady-state levels of the RNA for a heat-shock protein 70 (hsp70) in human peripheral blood mononuclear cells (PBMC) that are naturally quiescent in a G0 state. The probe used recognized, on RNA blots, one single band. The levels of this hsp70 RNA are elevated in circulating PBMC and decrease when the cells are incubated with serum, or phytohemagglutinin, or simply when they are incubated in culture medium. The levels of hsp70 RNA decrease within 30 min after in vitro culture, and are accompanied by an increase in the levels of c-fos RNA. These findings, together with other recent reports in the literature, suggest a possible role of the hsp70 proteins in the regulation of cell growth.

scholarly journals Monitoring the end of the in vitro phase of Anthurium andreanum Lindl. plantlets

2010 ◽  
Vol 22 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Giulio Cesare Stancato ◽  
Maria Luiza Sant’Anna Tucci
Keyword(s):  
Culture Medium ◽  
Soluble Sugars ◽  
Dry Mass ◽  
Total Soluble Sugars ◽  
Gas Exchanges ◽  
Single Action ◽  
Fluorescent Lamps ◽  
Anthurium Andreanum

Estimulation of autotrophy in in vitro plantlets could be achieved through changes in the culture medium, or by changing the traditional hermetic caps by one that could allow gas exchanges between the culture and the environment. Besides that, the use of lamps with distinct emission spectrum irradiaction has propitiated successful results. This work was carried out aiming to evaluate the either the combined or the single action of some factors that can induce autotrophy on in vitro A. andraeanum cv. Eidibel plantlets. 3 sucrose concentrations were used: 0, 15 and 60 mM and for each one, to kinds of flasks according to the cap ventilation: under (0.038 L.h-1) and without ventilation. Flasks were kept under cold light fluorescent lamps or under gro-lux lamps. At the end of the experiment showing the highest shoot dry mass treatment was 60 mM, under ventilation and gro-lux, and the treatment which accumulate root dry mass to a lesser extent were 0 mM with ventilation and cold light and 15 mM without ventilation and cold light. In average, treatments with higher sucrose content in the culture medium, that is, 60 mM, under gro-lux lamps, presented the highest chlorophyll a, b and total contents, than those under cold lamp. Steps of carbohydrates metabolism could be associated with the total soluble sugars (sucrose and reducing sugars) levels, highlighting the steps where nutrient requirements were higher, showing the role of the plantlets sink.

scholarly journals 316EFFECTS OF ESTRADIOL-17² AND PROGESTERONE SUPPLEMENT ON THE RESUMPTION OF MEIOSIS OF CANINE OOCYTES MATURED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 277
Author(s):  
M.K. Kim ◽  
Y.H. Fibrianto ◽  
H.J. Oh ◽  
G. Jang ◽  
K.S. Lee ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Linear Models ◽  
Uv Light ◽  
Germinal Vesicle ◽  
Domestic Animal ◽  
Estradiol 17Β

In the bitch, oocytes are ovulated at the germinal vesicle (GV) stage and mature in the isthmus of the oviduct around 3 days after ovulation, it is not known what elements trigger the release of this meiotic arrest. Canine IVM has shown limited success with maturation rates, usually around 20% (MII) (Farstad W, 2000 Anim. Reprod. Sci. 60–61, 375–387). Estrogen and progesterone are suggested to play a significant role in causing oocyte resumption of meiosis and progression to MII stage. The purpose of this study was to investigate the role of estradiol-17β (E2) and progesterone (P4) during in vitro maturation of canine oocytes in serum-free tissue culture medium (TCM)-199. Canine oocytes collected from bitches were categorized into three groups based on estrous stages, follicular, luteal, or anestrus, at routine ovariohystrectomy. Oocytes were cultured in vitro in TCM-199 supplemented with E2, P4 or E2+P4 according to experimental design at 39°C in 5% CO2 and O2. After 72h of maturation culture, oocytes were denuded, fixed in a 3.7% paraformaldehyde solution for 10min, stained with Hoechst 33342 in glycerol, and observed under the UV light. Three groups of oocytes were cultured in TCM-199 supplemented with different concentrations (0, 0.1, 1.0 or 2.0μgmL−1) of E2 (Experiment 1, n=898, replications: 5) or P4 (0, 0.5, 1.0 or 2.0μgmL−1, Experiment 2, n=734, replications: 5). Multiple comparisons were implemented using Generalized Linear Models in the SAS 8.12 program. The rates of oocyte maturation to MII stage were higher (P&lt;0.05) in follicular stage oocytes cultured with 2μgmL−1 E2 (17.9%) compared to other supplement groups (0 to 7.6%). No differences (P&lt;0.05) in rate of MII stage oocytes among P4 supplement groups were observed. In Experiment 3, to investigate the combined effects of E2 and P4 on in vitro maturation, three groups of oocytes were cultured in TCM-199 supplemented with 2μgmL−1 E2 and various concentration of P4 (0, 0.5, 1.0 or 2.0μgmL−1, Experiment 3, n=1613, replications: 5). The rate of oocyte maturation to MII stage (11.5%) was higher (P&lt;0.05) in follicular stage oocytes cultured with 2μgmL−1 E2+2.0μgmL−1 P4 supplement compared to other supplement groups (0 to 6.4%). In conclusion, the present study demonstrated that E2 supplement in the culture medium increased maturation of canine oocyte to MII stage and that supplement of P4 alone did not promote oocyte maturation. However, P4 supplemented with E2 further promoted oocyte maturation in the follicular stage compared to E2 supplement alone, indicating that P4 acts synergistically with E2 on canine oocyte maturation in the presence of E2. From our results, we conclude that canine oocytes are exposed to high levels of P4 during maturation due to the preovulatory luteinization of canine follicles which gives rise to high intrafollicular as well as intratubal P4 concentrations-this is very different from the situation in oocytes from other domestic animal species. This study was supported by Biogreen 21-1000520030100000.

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