Novel Purine Derivative ITH15004 Facilitates Exocytosis through a Mitochondrial Calcium-Mediated Mechanism

Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca2+ microdomain (HCMD) generated by Ca2+ entry through nearby voltage-activated calcium channels. HCMD is efficiently cleared by local mitochondria that avidly take up Ca2+ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na+/Ca2+ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K+-depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca2+ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca2+ concentrations ([Ca2+]c) K+-challenged BCCs, and (v) non-inactivated [Ca2+]c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca2+ handling during K+ depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.

Development of the composition and technology of a combined gel for the treatment of osteoarthritis with a pharmacological rationale for the content of components

Introduction. Osteoarthritis (OA) is the most common joint disease that affects more than 10 % of the world's population. More than 600 000 people are diagnosed for the first time each year, but these data do not reflect the true prevalence of the disease, since not all patients seek help from hospitals [1, 2].Aim. Pharmaceutical development of the composition and technology of a gel based on meloxicam, a purine derivative and an immunomodulating component for the treatment of OA with pharmacological substantiation of the content of active substances.Materials and methods. A combination of three active pharmaceutical substances was studied: a non-steroidal anti-inflammatory drug – meloxicam, a purine derivative and an original immunomodulator M. Sodium alginate, natrozole and xanthan gum were considered as gelling agents. Were identified two technological modes of obtaining a gel base. The concentrations of active substances were selected based on the results of preclinical studies. OA was modeled by the combined administration of 0.1 ml of a mixture of Freund's complete adjuvant with a 10 % talc suspension in isotonic sodium chloride solution in a ratio of 1 : 10 into the hock (tarsus) joint cavity. The criteria for choosing the optimal composition of the gel were the size of the damaged joint, exercise tolerance and the histological picture in comparison with intact and control animals. For quantitative data, sample mean values (M) and standard deviations (SD) were calculated. The results corresponded to the laws of normal distribution, statistical processing was carried out using one-way analysis of variance (One-Way ANOVA) using the GraphPad Prism 8.0.2 software, USA at the level of statistical significance of differences p < 0,05 и p < 0,001.Results and discussion. The composition was developed and the technology of the topical dosage form based on sodium alginate was proposed. Preclinical data indicate that the highest efficacy is achieved when using a formulation containing 3 % purine derivative, 5 % immunomodulator M and 0.5 % meloxicam. The developed composition for the effectiveness of suppressing the symptoms of OA showed results that exceeded the reference drug.Conclusion. An original combined agent for the treatment of OA has been developed. Due to the selected component composition, with greater efficiency, it was possible to reduce the dosage of meloxicam to 0.5 %, and the use of sodium alginate as a gelling agent contributed to the prolongation of the action of the gel and the subsequent reduction in the number of applications.

Empirical Evaluation and Prediction of Protein Requirements for Maintenance and Growth of 18–24 Months Old Thai Swamp Buffaloes

In some geographical areas and in certain breeding situations, the interpretation of increased gain in the bovine is difficult to investigate. Due to their inherent genetic variations, their energy and protein needs vary as a function of inherent genetic differences, making these requirements difficult to accurately assess in bull species, e.g., Thai swamp buffalo. The study aimed at investigating and predicting protein requirement systems, by the provision of an abundant energy intake of 2.2 Mcal/kg DM for the maintenance and growth of Thai swamp buffaloes using a comparative prolonged feeding trial for 90 days. Sixteen bull Thai swamp buffaloes at the initial (Age: 18–24 months; BW: 233 ± 25.0 kg) were assigned into four treatment groups, four buffaloes each, fed 5.4, 6.6, 8.5, and 10.5% DM crude protein (CP). CP intake, BW, and physiological fluid were determined. The net CP requirements for maintenance and growth of Thai swamp buffaloes were 5.41 g CP/kg W0.75 and 0.46 g CP/g average daily gain (ADG), respectively. Our results indicated that CP requirement increases when the BW increases. An increased dietary CP resulted in increased amounts of blood urine nitrogen (N), N absorption, total volatile fatty acid, urinary purine derivative, and the microbial N. Notably, the net CP requirement for growth of Thai swamp buffalo was higher than it reported in NRC, but the maintenance was lower.

Assessment of the diversity of comfrey (Symphytum officinale L. and S. × uplandicum Nyman)

Comfrey Symphytum officinale L. (true comfrey) and S. × uplandicum Nyman (a hybrid between S. asperum Lepech × S. officinale L., Russian comfrey) are used externally for the treatment of pain, inflammation and swelling of muscles and joints in degenerative arthritis, in acute myalgia, sprains, contusions and strains after accidents. Besides plant secondary compounds associated with beneficial activities (e.g. rosmarinic acid and allantoin) comfrey forms also toxic pyrrolizidine alkaloids (PA). To improve further breeding and study the genetic relationships of a comfrey collection, a sample set of 219 S. officinale and 5 hybrid plants of S. × uplandicum were analysed with 34 SNP markers by KASP (Kompetitive Allele Specific PCR), developed from a next generation sequencing approach of three different individuals of S. officinale. In parallel, the plants were analysed for the polyphenol rosmarinic acid, the purine derivative allantoin, and the toxic pyrrolizidine alkaloids. Besides the two beneficial compounds, further 13 polyphenols and 10 purine derivatives were determined. The plants were grouped into six distinct genetic clusters. Rosmarinic acid was not linked to any of the clusters, while one cluster was distinctively different for some compounds, amongst them allantoin and globoidnan A. Also linked to a certain genetic cluster was a low content of PA, which could become a valuable gene pool for minimizing PA content by breeding. A subset of the samples was analysed in a second year again where rosmarinic acid and allantoin showed a medium stability, while globoidnan A was completely unstable.

Empirical Evaluation and Prediction of Protein Requirements for Maintenance and Growth of 18-24 Months old Thai Swamp Buffaloes

Interpretation of increased gain in the bovine animals is difficult to be validated due to inherent genetic variation to meet their requirement for energy and protein, and those may relate to the bull species, e.g., Thai swamp buffalo. Therein, the study aimed at investigating and predicting protein requirement systems, with providing abundant energy intake 2.20 Mcal/kg DM for maintenance and growth of Thai swamp buffaloes using the comparative prolonged feeding trial for 90 days. Sixteen bull Thai swamp buffaloes at the initial (Age: 18-24 months; BW: 233 &plusmn; 25.0 kg) were as-signed into four treatment groups, four buffaloes each, fed 5.42, 6.96, 8.94, and 10.71% DM crude protein (CP). CP intake, BW, and physiological fluid were determined. The net CP requirements for maintenance and growth of Thai swamp buffaloes were 5.41 g CP/kg W0.75 and 0.46 g CP/g average daily gain (ADG), respectively. Our results indicated that CP requirement increases, when BW increases. An increased dietary CP resulted in an increased number at blood urine nitrogen (N), N absorption, total volatile fatty acid, urinary purine derivative, and the microbial N. Notably, the net CP requirement for growth of Thai swamp buffalo was higher than it reported in NRC, but the maintenance was lower.

Effect of storage of wet brewer’s grains with incremental levels of salt on apparent total-tract nutrient digestibility and purine derivative excretion in dairy heifers

Objectives of this study were to evaluate apparent total-tract nutrient digestibility and purine derivative (PD) excretion in dairy heifers limit-fed diets containing wet brewer’s grains (WBG) treated with salt. A 12-wk replicated 4×4 Latin square was conducted using 8 Holstein heifers of 224.5 ± 19.4 d of age, and body weight (BW) of 219.2 ± 28.1 kg (mean ± SD). Fresh WBG were treated with 0%, 0.8%, 1.6%, and 2.4% salt and stored for 4 d before being fed. Salt was added either to the WBG or separately to equalize the amount of salt in the diet. The diet contained 9% grass silage, 47% corn silage, 19% corn meal, 17.6% WBG and salt, 2% soybean meal (SBM), and 3% mineral mix. Diets were formulated to be limit-fed at 2.15% of BW, provide 14% crude protein (CP) and 2.27 Mcal metabolizable energy (ME)/kg of dry matter (DM). Heifers were adapted to diets for 14 d followed by a 7-d collection period. Dry matter intake (DMI) was recorded daily during the collection week while BW was recorded once a week. Urine and fecal samples were collected during the last 4 d of the collection period. Acid insoluble ash was used as an internal marker to determine apparent nutrient digestibility. Weight loss of WBG during storage was determined from d 1 to d 11 and initial and final yeast and mold counts were determined. Final yeast counts were similar among treatments while final mold counts tended to be lesser (P = 0.07) for the 0.8 and 1.6% salt treatments. Urinary volume was similar among treatments while allantoin (P = 0.14), and uric acid (P &lt; 0.01) and total PD excretion tended to increase (P = 0.13) quadratically. Dry matter intake was varied by treatment (linear, quadratic, and cubic effects P &lt; 0.01). Heifers fed the 0.8% treatment had the least DMI. Non-fiber carbohydrate (NFC) digestibility linearly decreased (P &lt; 0.04) as salt increased. Digestibilities of DM, and organic matter (OM), tended to decrease (P &lt; 0.10) with increasing levels of salt added to WBG. Fat digestibility was quadratic with the greatest value for the 1.6% treatment. Treating WBG with salt reduced its deterioration based on lesser mold counts for the 0.8 and 1.6% treatments. These treatments had resulted in greater fat digestibility and tended to have increased PD excretion suggesting improved microbial protein synthesis.

Circumventing the Crabtree effect: forcing oxidative phosphorylation (OXPHOS) via galactose medium increases sensitivity of HepG2 cells to the purine derivative kinetin riboside

Small-molecule compound-based therapies have provided new insights into cancer treatment against mitochondrial impairment. N6-furfuryladenosine (kinetin riboside, KR) is a purine derivative and an anticancer agent that selectively affects the molecular pathways crucial for cell growth and apoptosis by interfering with mitochondrial functions and thus might be a potential mitotoxicant. Metabolism of cancer cells is predominantly based on the Crabtree effect that relies on glucose-induced inhibition of cell respiration and thus on oxidative phosphorylation (OXPHOS), which supports the survival of cancer cells in metabolic stress conditions. The simplest way to circumvent this phenomenon is to replace glucose with galactose in the culture environment. Consequently, cells become more sensitive to mitochondrial perturbations caused by mitotoxicants. In the present study, we evaluated several cellular parameters and investigated the effect of KR on mitochondrial functions in HepG2 cells forced to rely mainly on OXPHOS. We showed that KR in the galactose environment is a more potent apoptosis-inducing agent. KR decreases the mitochondrial membrane potential, reduces glutathione level, depletes cellular ATP, and induces reactive oxygen species (ROS) production in the OXPHOS state, leading to the loss of cell viability. Taken together, these results demonstrate that KR directly acts on the mitochondria to limit their function and that the sensitivity of cells is dependent on their ability to cope with energetic stress.

Diurnal and dietary impacts on estimating microbial protein flow from urinary purine derivative excretion in beef cattle1

Two experiments were conducted to determine the effects of diet composition and time of urine spot sampling on estimates of urinary purine derivative (PD) excretion. In Exp. 1, 116 individually fed crossbred heifers (407 ± 32 kg) were arranged in a randomized block design (82 d). Treatments were arranged in a 2 × 3 factorial design, with two urine spot sample collection times (0700 and 1700 hours; AM and PM) and three diets: 85% steam-flaked corn (SFC); 85% SFC + 1.5% urea (UREA); or 25% SFC, 30% wet corn gluten feed, and 30% corn bran (BYPROD). In Exp. 2, six ruminally and duodenally fistulated steers (474 ± 37 kg) were arranged in a replicated 3 × 3 Latin square design, with dietary treatments identical to Exp. 1 (63 d). Treatment diets were selected to result in varied amounts of microbial crude protein (MCP) in order to evaluate the accuracy of using estimates of urinary PD excretion to predict MCP. Urine spot samples were collected at 0700, 1200, 1700, and 2200 hours. No urine collection time × diet interactions occurred (P &gt; 0.20) for any variable in either experiment. In Exp. 1, dry matter intake (DMI) was greatest with BYRPOD (10.40 kg/d) and lowest with SFC (7.90 kg/d; P &lt; 0.05). Feed efficiencies were greatest for UREA (0.182) and least for SFC (0.141; P &lt; 0.05). Urinary PD:creatinine (PD:C) ratio was greatest for BYPROD (1.25) and least for SFC (0.94; P &lt; 0.05). Urine spot sampling time had a significant (P &lt; 0.05) impact on PD:C, 1.03 for AM and 1.22 for PM samples. In Exp. 2, DMI was greater (P &lt; 0.05) with BYPROD than with SFC and tended (P = 0.07) to be greater with BYPROD than with UREA. Ruminal pH was greatest for BYPROD (5.94; P &lt; 0.05). Flow of MCP was 636, 829, and 1,056 g/d for SFC, UREA, and BYPROD, with BYPROD being greater (P &lt; 0.05) than SFC and tending (P = 0.06) to be greater than UREA. Urinary PD:C was greater (P &lt; 0.05) for BYPROD than SFC and tended (P = 0.09) to be greater for UREA than SFC. Urinary PD:C increased linearly (P &lt; 0.05) with sampling time. Diets formulated to affect DMI and MCP flow resulted in differences in urinary PD excretion, and these results related well with MCP flow estimated from duodenal purines. Collecting spot samples of urine later in the day resulted in greater estimates of urinary PD excretion; purine and PD flows appear to increase with time after one morning feeding per day. This method is well suited to evaluating relative differences between treatments but should not be extrapolated to assume absolute values.