The effect of IBMX and prolactin on functional status of cryopreserved bull spermatozoa

Purpose: investigate the effect of IBMX (activator of protein phosphorylation) and prolactin (PRL) on the functional state of cryopreserved bovine spermatozoa using inhibitory analysis.Materials and methods. Frozen-thawed semen samples from 60 black-and-white bulls was used in the experiments. For capacitation, cells were incubated in Sp-TALP medium supplemented with 6 mg/ml bovine serum albumin and various compounds: an inductor of capacitation (IBMX at concentrations of 1 μM, 10 μM, 50 μM, 100 μM), hormone (PRL at concentrations of 1 ng, 10 ng, 50 ng, 100 ng) and inhibitors of protein kinases C (Ro 31-8220 at a concentration of 10 ng/ml) and protein kinase A (H-89 at a concentration of 10 μM). The incubation was carried out at 38°C in an atmosphere of 5% CO2, 98% humidity for 4 hours. The functional status of the cells was determined by the chlortetracycline test.Results. It was shown that IBMX at all experimental concentrations did not affect the post-ejaculatory maturation (capacitation and acrosome reaction) of spermatozoa, while all concentrations of PRL (1-100 ng/ml) promoted the acrosome reaction in capacitated cells. In the presence of a protein kinase A inhibitor, there was a decrease in number of capacitated and an increase in number of acrosome-reactive spermatozoa under the action of IBMX at a concentration of 100 μM and no changes under the action of a protein kinase C inhibitor. Also, in case of protein kinase C inhibition the PRL-related stimulation of the acrosome reaction was canceled, while the usage of H-89 did not affect the functional status of spermatozoa, mediated by PRL. Thus, the influence of IBMX and PRL on the processes of post-ejaculatory maturation in thawed bovine spermatozoa was studied using the inhibitory analysis.Conclusion. At the capacital stage, all studied IBMX concentrations did not affect the ratio of deconved cells with various functional status. Prode also contributed to the passage of the acrosomous reaction in the rolled spermatozoa after defrosting. Inhibition of protein kinase A when incubating cells with IBMX has mediated the processes of acrosomal exocytosis in ripped cells and did not affect this process under the action of the PRR, while the protein kinase inhibitor C changed the ratio of cells with various functional status in the direction of increasing the percentage of cells at the rate of occasion I did not participate in intracellular action provided IBMX on deconved cells.

N-Acyl Dopamines Induce Apoptosis in Endometrial Stromal Cells from Patients with Endometriosis

Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N-acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N-arachidonoyldopamine, N-docosahexaenoyldopamine, and N-oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.

Self-Organization Provides Cell Fate Commitment in MSC Sheet Condensed Areas via ROCK-Dependent Mechanism

Multipotent mesenchymal stem/stromal cells (MSC) are one of the crucial regulators of regeneration and tissue repair and possess an intrinsic program from self-organization mediated by condensation, migration and self-patterning. The ability to self-organize has been successfully exploited in tissue engineering approaches using cell sheets (CS) and their modifications. In this study, we used CS as a model of human MSC spontaneous self-organization to demonstrate its structural, transcriptomic impact and multipotent stromal cell commitment. We used CS formation to visualize MSC self-organization and evaluated the role of the Rho-GTPase pathway in spontaneous condensation, resulting in a significant anisotropy of the cell density within the construct. Differentiation assays were carried out using conventional protocols, and microdissection and RNA-sequencing were applied to establish putative targets behind the observed phenomena. The differentiation of MSC to bone and cartilage, but not to adipocytes in CS, occurred more effectively than in the monolayer. RNA-sequencing indicated transcriptional shifts involving the activation of the Rho-GTPase pathway and repression of SREBP, which was concordant with the lack of adipogenesis in CS. Eventually, we used an inhibitory analysis to validate our findings and suggested a model where the self-organization of MSC defined their commitment and cell fate via ROCK1/2 and SREBP as major effectors under the putative switching control of AMP kinase.

Deregulation of Ca2+-Signaling Systems in White Adipocytes, Manifested as the Loss of Rhythmic Activity, Underlies the Development of Multiple Hormonal Resistance at Obesity and Type 2 Diabetes

Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)–dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors’ interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5–15% vs. 30–80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity.

High-Yielding Lovastatin Producer Aspergillus terreus Shows Increased Resistance to Inhibitors of Polyamine Biosynthesis

The biosynthesis of pharmaceutically significant secondary metabolites in filamentous fungi is a multistep process that depends on a wide range of various factors, one of which is the intracellular content of polyamines. We have previously shown that in Aspergillus terreus lovastatin high-yielding strain (HY) exogenous introduction of polyamines during fermentation can lead to an increase in the production of lovastatin by 20–45%. However, the molecular mechanisms of this phenomenon have not been elucidated. In this regard, we carried out an inhibitory analysis at the key stage of polyamine biosynthesis, the conversion of L-ornithine to putrescine by the enzyme ornithine decarboxylase (ODC). A. terreus HY strain showed upregulation of genes for biosynthesis of polyamines, 3–10-fold, and increased resistance compared to the original wild-type strain upon inhibition of ODC on synthetic medium with 5 mM α-difluoromethylornithine (DFMO), by 20–25%, and 5 mM 1-aminooxy-3-aminopropane (APA), by 40–45%. The data obtained indicate changes in the metabolism of polyamines in A. terreus HY strain. The observed phenomenon may have a universal character among fungal producers of secondary metabolites improved by classical methods, since previously the increased resistance to ODC inhibitors was also shown for Acremonium chrysogenum, a high-yielding producer of cephalosporin C.

ROS in tobacco stigma exudate affect pollen proteome and provoke membrane hyperpolarization

1.AbstractROS are known to be accumulated in stigmas of different species and can possibly perform different functions in plant reproduction. Here we confirm the assumption that they affect pollen by altering ion transport through the plasma membrane; as a more deferred effect, pollen proteome is modified. We detected ROS in stigma exudate, found hyperpolarization in exudate-treated growing pollen tubes and used flow cytometry of pollen protoplasts to compare the effects of fresh exudate and exogenous H2O2 on pollen tube plasmalemma. Exudate causes plasmalemma hyperpolarization similar to the one provoked by H2O2, which is abolished by catalase treatment and ROS quencher MnTMPP. Inhibitory analysis indicates the participation of Ca2+- and K+-conducting channels in the observed hyperpolarization, linking obtained data with previous patch-clamp studies in vitro. For a deeper understanding of pollen response to ROS we analyzed proteome alterations in H2O2-treated pollen grains. We found 50 unique proteins and 20 differently accumulated proteins that are mainly involved in cell metabolism, energetics, protein synthesis and folding. Thus, pollen is getting ready for effective resource usage, construction of cellular components and rapid growth.HighlightsThe active substance in stigma exudate is H2O2H2O2 causes hyperpolarization mediated by the activation of cation channels.H2O2 affects pollen proteome; we found 50 unique proteins.

Analisis Daya Hambat Ekstrak Daun Sirih Hijau (Piper betle L.) terhadap Mikroorganisme Indikator Mastitis

Abstrak. Penelitian tentang Analisis Daya Hambat Ekstrak Daun Sirih Hijau (Piper betle L.) terhadap Mikroorganisme Indikator Mastitis telah dilaksanakan di Laboratorium Ilmu dan Teknologi Pengolahan Susu dan Laboratorium Analisis Pangan dan Hasil Pertanian yang dilaksanakan pada 7 September sampai dengan 10 November 2018. Penelitian ini terdiri dari 4 perlakuan dan 5 ulangan. Data yang diperoleh selanjutnya akan diolah menggunakan Rancangan Acak Lengkap, jika didapatkan hasil yang berbeda maka akan dilanjutkan dengan uji Duncan. Perlakuan R1 yaitu penambahan ekstrak daun sirih hijau 15%, R2 yaitu penambahan ekstrak daun sirih hijau 30%, R3 yaitu penambahan ekstrak daun sirih hijau 45% dan R4 yaitu penambahan ekstrak daun sirih hijau 60%. Hasil penelitian menunjukkan bahwa ekstrak daun sirih hijau dengan konsentrasi 15%, 30%, 45% dan 60% mampu menghambat E. coli dan S. aureus. Berdasarkanhasil penelitian ini dapat disimpulkan bahwa ekstrak daun sirih hijau mampu menghambat pertumbuhan E. coli dan S. aureus.Inhibiting Power Analysis of Green Betel Leaf Extract (Piper betle L.) against Microorganism Indicator MastitisAbstract. Research on Inhibitory Analysis of Green Betel Leaf Extract (Piper betle L.) against Mastitis causing Microorganism has been conducted in Milk Processing Science and Technology Laboratory and Food and Agricultural Product Analysis Laboratory held on September 7 to November 10, 2018. This research consisted of 4 treatments and 5 replications. The data obtained will then be processed using a Completely Randomized Design, if different results are obtained it will be followed by the Duncan Multiple Range Test. The treatment of R1 is the addition of 15% green betel leaf extract, R2, which is the addition of 30% green betel leaf extract, R3, namely the addition of 45% green betel leaf extract and R4 which is the addition of 60% green betel leaf extract. The results showed that green betel leaf extract with a concentration of 15%, 30%, 45% and 60% was able to inhibit E. coli and S. aureus. Based on the results of this study it can be concluded that green betel leaf extract can inhibit the growth of E. coli and S. aureus.

Hydroxylamine Analogue of Agmatine: Magic Bullet for Arginine Decarboxylase

The biogenic polyamines, spermine, spermidine (Spd) and putrescine (Put) are present at micro-millimolar concentrations in eukaryotic and prokaryotic cells (many prokaryotes have no spermine), participating in the regulation of cellular proliferation and differentiation. In mammalian cells Put is formed exclusively from L-ornithine by ornithine decarboxylase (ODC) and many potent ODC inhibitors are known. In bacteria, plants, and fungi Put is synthesized also from agmatine, which is formed from L-arginine by arginine decarboxylase (ADC). Here we demonstrate that the isosteric hydroxylamine analogue of agmatine (AO-Agm) is a new and very potent (IC50 3•10−8 M) inhibitor of E. coli ADC. It was almost two orders of magnitude less potent towards E. coli ODC. AO-Agm decreased polyamine pools and inhibited the growth of DU145 prostate cancer cells only at high concentration (1 mM). Growth inhibitory analysis of the Acremonium chrysogenum demonstrated that the wild type (WT) strain synthesized Put only from L-ornithine, while the cephalosporin C high-yielding strain, in which the polyamine pool is increased, could use both ODC and ADC to produce Put. Thus, AO-Agm is an important addition to the set of existing inhibitors of the enzymes of polyamine biosynthesis, and an important instrument for investigating polyamine biochemistry.

IgG-Dependent Dismutation of Superoxide in Patients with Different Types of Multiple Sclerosis and Healthy Subjects

This work is the first to demonstrate that class G immunoglobulins (IgGs) in patients with multiple sclerosis and healthy individuals have the ability to catalyze the dismutation reaction of the superoxide anion radical. Thus, superoxide dismutase (SOD) activity is an intrinsic property of antibodies, which is confirmed by a number of stringent criteria. SOD activity of IgGs in patients with multiple sclerosis statistically significantly exceeds such activity in healthy individuals by 2-4 times. Moreover, the maximum activity has been registered in patients with relapsing remitting multiple sclerosis. The kinetic characteristics of the SOD reaction of IgGs are several orders of magnitude lower than those for the SOD enzyme but do not differ between patients with multiple sclerosis and healthy individuals. Consequently, abzymes with SOD activity have a lower catalysis rate than that of the enzymes and form a stronger complex with the substrates. Inhibitory analysis showed that this activity is inhibited by classical metal-dependent SOD inhibitors. The activity of IgGs was inhibited by classical metal-dependent inhibitors EDTA and TETA (triethylenetetramine). Also, high catalase activity of IgGs was detected in these patients. We suggest that these abzymes help protect the body from oxidative stress.

Selection of Phylloplane and Endophyte Microbes as Biocontrol for Rubber Leaf Fall Disease (Corynespora cassiicola)

<p><em>Leaf fall disease in rubber caused by </em>Corynespora cassiicola<em> </em><em>fungi significantly decreases rubber productivity. </em>C. cassiicola<em> causes leaves to fall all year round</em><em>, a delay in the tapping of immature rubber plants, yield decrease of producing plants, and even death of susceptible clones. The study aimed to obtain phylloplane and endophytic microbes potentially to inhibit the disease, was conducted from January to December 2016. The study used randomized complete design to assess antagonistic fungi and phylloplane and endophytic bacterias toward </em>C. cassiicola<em> in isolates obtained through exploration in </em><em>West Java and West Kalimantan. Pathogen isolation showed </em>Corynespora sp<em> with pale brown color, single conidia which slightly bended, shaped like a stick that is swollen at the base, with 2–14 septa. Inhibitory analysis found 42 fungi isolates and 19 bacteria isolates potentially inhibiting </em>C. cassiicola<em>. </em><em>Six fungi isolates have an inhibitory ability of ≥90%, consisting of two phylloplane fungi isolates (DTJF11 and CPSR7) and four endophytic fungi (CEBPM15, CEBPM23, CEBPM27, and CEPR9) with lysis, mycoparasitism, competition, and antibiosis inhibitory mechanism. The identification showed fungi isolate of DTJF11 is classified as </em>Trichoderma asperellum<em>, CPSR7 as </em>Talaromyces pinophilus<em>, and CEBPM15</em><em> as </em>Amanita tenuifolia<em>. </em><em>Potential bacterial isolates as biological agents are BP7, L3, BP3, BP4, BP5 and BP6 isolates, which have inhibitory power of 28.54%–40.94%, with antibiosis inhibition mechanism.</em></p>