Gut Dysbiosis and Hemodynamic Changes as Links of the Pathogenesis of Complications of Cirrhosis.

The aim was to evaluate the relationship between gut dysbiosis and hemodynamic changes (hyperdynamic circulation) in cirrhosis, and between hemodynamic changes and complications of this disease. This study included 47 patients with cirrhosis. Stool microbiome was assessed using 16S rRNA gene sequencing. Echocardiography with a simultaneous assessment of blood pressure and heart rate was performed to assess systemic hemodynamics. Patients with hyperdynamic circulation had more severe cirrhosis, lower albumin, sodium and prothrombin levels, higher C-reactive protein, aspartate aminotransferase and total bilirubin levels, and higher incidences of portopulmonary hypertension, ascites, overt hepatic encephalopathy, hypoalbuminemia, hypoprothrombinemia, systemic inflammation, and severe hyperbilirubinemia than patients with normodynamic circulation. Patients with hyperdynamic circulation compared with those with normodynamic circulation had increased abundance of Proteobacteria, Enterobacteriaceae, Bacilli, Streptococcaceae, Lactobacillaceae, Fusobacteria, Micrococcaceae, Intestinobacter, Clostridium sensu stricto, Proteus and Rumicoccus, and decreased abundance of Bacteroidetes, Bacteroidaceae, Holdemanella, and Butyrivibrio. The systemic vascular resistance and cardiac output values correlated with the abundance of Proteobacteria, Enterobacteriaceae, Bacilli, Streptococcaceae, Lactobacillaceae, Micrococcaceae, and Fusobacteria. Heart rate and cardiac output value were negatively correlated with the abundance of Bacteroidetes. The mean pulmonary artery pressure value was positively correlated with the abundance of Proteobacteria and Micrococcaceae, and negatively with the abundance of Holdemanella.

Effect of probiotics on hemodynamic changes and complications associated with cirrhosis: A pilot randomized controlled trial.

BACKGROUND: Bacterial translocation exacerbates the hyperdynamic circulation observed in cirrhosis and contributes to a more severe disease course. Probiotics may reduce bacterial translocation and may therefore be useful to redress the circulatory imbalance. AIM: To investigate the effect of probiotics on hemodynamic parameters, systemic inflammation, and complications of cirrhosis in this randomized placebo-controlled trial. METHODS: This single-blind randomized placebo-controlled study included 40 patients with Child-Pugh class B and C cirrhosis; 24 patients received probiotics (Saccharomyces boulardii) for 3 mo, and 16 patients received a placebo over the same period. Liver function and the systemic hemodynamic status were evaluated pre- and post-intervention. Echocardiography and simultaneous blood pressure and heart rate monitoring were performed to evaluate systemic hemodynamic indicators. Cardiac output and systemic vascular resistance were calculated. RESULTS: Following a 3-mo course of probiotics in comparison to the control group, we observed amelioration of hyperdynamic circulation [a decrease in cardiac output (P = 0.026) and an increase in systemic vascular resistance (P = 0.026)] and systemic inflammation [a decrease in serum C-reactive protein levels (P = 0.044)], with improved liver function [an increase in serum albumin (P = 0.001) and a decrease in the value of Child-Pugh score (P = 0.001)] as well as a reduction in the severity of ascites (P = 0.022), hepatic encephalopathy (P = 0.048), and cholestasis [a decrease in serum alkaline phosphatase (P = 0.016) and serum gamma-glutamyl transpeptidase (P = 0.039) activity] and an increase in platelet counts (P < 0.001) and serum sodium level (P = 0.048). CONCLUSION: Probiotic administration was associated with amelioration of hyperdynamic circulation and the associated complications of cirrhosis.

A Recent Ten-Year Perspective: Bile Acid Metabolism and Signaling.

Bile acids are important physiological agents required for the absorption, distribution, metabolism, and excretion of nutrients. In addition, bile acids act as sensors of intestinal contents, which are determined by the change in the spectrum of bile acids during microbial transformation, as well as by gradual intestinal absorption. Entering the liver through the portal vein, bile acids regulate the activity of nuclear receptors, modify metabolic processes and the rate of formation of new bile acids from cholesterol, and also, in all likelihood, can significantly affect the detoxification of xenobiotics. Bile acids not absorbed by the liver can interact with a variety of cellular recipes in extrahepatic tissues. This provides review information on the synthesis of bile acids in various parts of the digestive tract, its regulation, and the physiological role of bile acids. Moreover, the present study describes the involvement of bile acids in micelle formation, the mechanism of intestinal absorption, and the influence of the intestinal microbiota on this process.

Gut-liver axis in cirrhosis: Are hemodynamic changes a missing link?

Recent evidence suggests that the condition of the gut and its microbiota greatly influence the course of liver disease, especially cirrhosis. This introduces the concept of the gut-liver axis, which can be imagined as a chain connected by several links. Gut dysbiosis, small intestinal bacterial overgrowth, and intestinal barrier alteration lead to bacterial translocation, resulting in systemic inflammation. Systemic inflammation further causes vasodilation, arterial hypotension, and hyperdynamic circulation, leading to the aggravation of portal hypertension, which contributes to the development of complications of cirrhosis, resulting in a poorer prognosis. The majority of the data underlying this model were obtained initially from animal experiments, and most of these correlations were further reproduced in studies including patients with cirrhosis. However, despite the published data on the relationship of the disorders of the gut microbiota with the complications of cirrhosis and the proposed pathogenetic role of hemodynamic disorders in their development, the direct relations between gut dysbiosis and hemodynamic changes in this disease are poorly studied. They remain a missing link in the gut-liver axis and a challenge for future research.

Probiotics in hepatology: An update.

The gut-liver axis plays an important role in the pathogenesis of various liver diseases. Probiotics are living bacteria that may be used to correct disorders of this axis. Notable progress has been made in the study of probiotic drugs for the treatment of various liver diseases in the last decade. It has been proven that probiotics are useful for hepatic encephalopathy, but their effects on other symptoms and syndromes of cirrhosis are poorly studied. Their effectiveness in the treatment of metabolic associated fatty liver disease has been shown both in experimental models and in clinical trials, but their effect on the prognosis of this disease has not been described. The beneficial effects of probiotics in alcoholic liver disease have been shown in many experimental studies, but there are very few clinical trials to support these findings. The effects of probiotics on the course of other liver diseases are either poorly studied (such as primary sclerosing cholangitis, chronic hepatitis B and C, and autoimmune hepatitis) or not studied at all (such as primary biliary cholangitis, hepatitis A and E, Wilson's disease, hemochromatosis, storage diseases, and vascular liver diseases). Thus, despite the progress in the study of probiotics in hepatology over the past decade, there are many unexplored and unclear questions surrounding this topic.

Gut dysbiosis is associated with poorer long-term prognosis in cirrhosis.

BACKGROUND: Gut dysbiosis is common in cirrhosis. AIM: To study the influence of gut dysbiosis on prognosis in cirrhosis. METHODS: The case-control study included 48 in-patients with cirrhosis and 21 healthy controls. Stool microbiome was assessed using 16S ribosomal ribonucleic acid gene sequencing. We used modified dysbiosis ratio (MDR): [Bacilli (%) + Proteobacteria (%)]/[Clostridia (%) + Bacteroidetes (%)]. Patients with MDR more the median made up the group with severe dysbiosis, others did the group with non-severe dysbiosis. The follow-up period was 4 years. RESULTS: The mortality rate of patients with severe dysbiosis was significantly higher than that of patients with non-severe dysbiosis (54.2% vs 12.5%; P = 0.001). The presence of severe dysbiosis was independent risk factors for death [hazard ratio = 8.6 × (1.9-38.0); P = 0.005]. The abundance of Enterobacteriaceae (P = 0.002), Proteobacteria (P = 0.002), and Lactobacillaceae (P = 0.025) was increased and the abundance of Firmicutes (P = 0.025) and Clostridia (P = 0.045) was decreased in the deceased patients compared with the survivors. The deceased patients had a higher MDR value than the survivors [0.131 × (0.069-0.234) vs 0.034 × (0.009-0.096); P = 0.004]. If we applied an MDR value of 0.14 as the cutoff point, then it predicted patient death within the next year with a sensitivity of 71.4% and a specificity of 82.9% [area under the curve = 0.767 × (0.559-0.974)]. MDR was higher in patients with cirrhosis than in health controls [0.064 × (0.017-0.131) vs 0.005 × (0.002-0.007); P < 0.001], and in patients with decompensated cirrhosis than in patients with compensated cirrhosis [0.106 × (0.023-0.211) vs 0.033 × (0.012-0.074); P = 0.031]. MDR correlated negatively with prothrombin (r = -0.295; P = 0.042), cholinesterase (r = -0.466; P = 0.014) and serum albumin (r = -0.449; P = 0.001) level and positively with Child-Turcotte-Pugh scale value (r = 0.360; P = 0.012). CONCLUSION: Gut dysbiosis is associated with a poorer long-term prognosis in cirrhosis.

Immune disorders and rheumatologic manifestations of viral hepatitis.

Infection with hepatotropic viruses is not limited to the liver and can lead to the development of various immunological disorders (the formation of cryoglobulins, rheumatoid factor, antinuclear antibodies, autoantibodies specific for autoimmune hepatitis and primary biliary cholangitis, and others), which can manifest as glomerulonephritis, arthritis, uveitis, vasculitis (cryoglobulinemic vasculitis, polyarteritis nodosa, Henoch-Schonlein purpura, isolated cutaneous necrotizing vasculitis), and other rheumatologic disorders, and be a trigger for the subsequent development of autoimmune hepatitis and primary biliary cholangitis. A further study of the association between autoimmune liver diseases and hepatotropic virus infection would be useful to assess the results of treatment of these associated diseases with antiviral drugs. The relationship of these immune disorders and their manifestations with hepatotropic viruses is best studied for chronic hepatitis B and C. Only isolated cases of these associations are described for hepatitis A. These links are least studied, and are often controversial for hepatitis E, possibly due to their relatively rare diagnoses. Patients with uveitis, glomerulonephritis, arthritis, vasculitis, autoimmune liver diseases should be tested for biomarkers of viral hepatitis, and if present, these patients should be treated with antiviral drugs.

Correlation of Objective Endpoints and Subjective Patient-Reported Outcomes in NAFLD Treatment with Essential Phospholipids: Real-World Data Based on Pooled Analysis of Observational Studies.

BACKGROUND: While no "gold-standard" pharmacotherapy for nonalcoholic fatty liver disease (NAFLD) is yet established, essential phospholipids (EPLs) are reported to decrease steatosis and improve laboratory parameters. OBJECTIVE: This analysis evaluated adherence and satisfaction with EPL treatment as patient-reported outcomes and their relationship with changes in laboratory and ultrasound parameters among Russian patients with NAFLD. METHODS: Data were pooled from three observational Russian studies-MANPOWER (2015-2016), LIDER 1 (2012-2013), and LIDER 2 (2013)-in which EPLs were used for at least 12 weeks in the treatment of liver diseases and which measured both subjective and objective endpoints. Only patients who had NAFLD were included in this analysis. The main endpoints were to determine treatment adherence and satisfaction with 12 weeks of EPL therapy, relationship between adherence/satisfaction and changes in the laboratory and ultrasound parameters. A secondary subgroup analysis was performed to identify patients with NAFLD who responded better (or worse) to 24 weeks of adjunctive EPL treatment. RESULTS: Overall, 3384 patients were included. A total of 82.2% of patients were adherent to 12 weeks of EPL treatment; high/very high satisfaction was reported by 15.3%/65.9% of clinicians and 15.9%/64.4% of patients. There was positive correlation between patients' adherence and satisfaction and significant improvement in laboratory (transaminases, lipid profile; p < 0.001) and ultrasound (steatosis, p < 0.001) parameters, and improvement in symptoms (p < 0.001) after 24 weeks of EPL. Male patients, patients with unhealthy lifestyles, and those with more comorbidities showed a better response in laboratory and ultrasound parameters. CONCLUSIONS: Patients with NAFLD treated with adjunctive EPL therapy in real-world clinical practice in Russia showed good treatment adherence and treatment satisfaction. Improvements in laboratory and ultrasound parameters, as well as dynamics of patient symptoms, were positively correlated with adherence and satisfaction.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver disease, and patients have a risk of liver cancer and needing transplantation. Patients with advanced NAFLD are usually recommended to use medication. Clinical trials in patients with NAFLD showed that essential phospholipids (EPLs) comprising phosphatidylcholine improved liver fat accumulation (known as steatosis), so treatment guidelines in several countries recommend they be added to the usual therapy. EPLs have been associated with both objective and subjective improvements. This real-world study evaluated three observational studies to assess how patient adherence and satisfaction with EPL treatment were related to changes in clinical parameters in 3384 Russian patients with NAFLD. Overall, 82.2% of patients were adherent with 12 weeks of EPL therapy, and almost two-thirds of patients (64.4%) and clinicians (65.9%) reported very high satisfaction with treatment. Patients' adherence and satisfaction increased alongside significant improvements in liver enzymes, lipid levels, liver fat content (steatosis), and fewer symptoms after 24 weeks of EPL therapy. Male patients, those with an unhealthy lifestyle, and those with other comorbid conditions had the best response to EPL therapy. This study shows that Russian patients with NAFLD have good adherence and satisfaction with EPL therapy in routine clinical practice and highlights the importance of adherence to EPL therapy in these patients.

Effectiveness of phosphatidylcholine as adjunctive therapy in improving liver function tests in patients with non-alcoholic fatty liver disease and metabolic comorbidities: real-life observational study from Russia.

Objective: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of abnormal results of liver function tests. Earlier research showed that polyenylphosphatidylcholine (PPC) has hepatoprotective effects and thus can be used for the treatment of NAFLD and the prevention of its progression. Accordingly, the aim of this observational study was to evaluate if PPC administered as adjunctive therapy in routine clinical practice can effectively improve liver function tests of NAFLD in Russian patients with associated metabolic comorbidities. Design: A total of 2843 adult patients with newly diagnosed NAFLD, who had a least one of four comorbidities, namely, overweight/obesity, hypertension, type 2 diabetes mellitus, and hypercholesterolaemia, and who were prescribed 1.8 g/day of PPC as an adjunctive treatment to standard care, were enrolled during 2015-2016. Laboratory data were collected at baseline and 12 and 24 weeks of the study, and included liver function tests (aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT)), fasting plasma glucose, and lipid profile. Results: Overall, 2263 patients (79.6%) had at least two metabolic comorbidities associated with NAFLD, and overweight/obesity was the most common comorbidity reported in 2298 (80.8%) patients. At 24 weeks, there was a significant decrease in liver enzyme levels (all p<0.001 compared with baseline). Across the four comorbidity subgroups, there was a mean drop of ALT levels ranging from 19.7 to 22.0 U/L, AST from 16.9 to 18.4 U/L, and GGT from 17.2 to 18.7 U/L. Similar findings were reported in subgroups with either one, two, three, or four comorbidities, with a significant decrease in liver enzyme levels ranging from 18.4 to 22.4 U/L for ALT, 14.8 to 18.7 U/L for AST, and 15.5 to 19.5 U/L for GGT. Conclusions: Adjuvant treatment with PPC resulted in consistent improvements in liver enzymes in patients with newly diagnosed NAFLD and associated metabolic comorbidities. Trial registration number: NCT00063622.

Effectiveness of phosphatidylcholine in alleviating steatosis in patients with non-alcoholic fatty liver disease and cardiometabolic comorbidities (MANPOWER study).

Objective: The concept of using naturally occurring compounds such as polyenylphosphatidylcholine (PPC) as an adjunctive therapy to treat non-alcoholic fatty liver disease (NAFLD) and alleviate or reverse hepatic steatosis appears a very attractive option for liver protection. We aim to evaluate if PPC adjunctive therapy can effectively improve the ultrasonographic features of NAFLD in routine clinical practice in Russian patients with cardiometabolic comorbidities. Design: This 24-week, observational, prospective study was carried out in 174 medical sites across 6 federal districts of Russia. A total of 2843 adult patients with newly diagnosed NAFLD, who had a least one of four comorbidities, namely overweight/obesity, hypertension, type 2 diabetes mellitus and hypercholesterolaemia, and who received PPC as an adjunctive treatment to standard care, were enrolled. The assessment of liver ultrasonography was qualitative. Results: Overall, 2263 (79.6%) patients had at least two metabolic comorbidities associated with NAFLD, and overweight/obesity was the most common comorbidity reported in 2298 (80.8%) patients. Almost all study participants (2837/2843; 99.8%) were prescribed 1.8 g of PPC administered three times daily. At baseline, the most frequently identified abnormalities on ultrasound were liver hyperechogenicity (84.0% of patients) and heterogeneous liver structure (62.9%). At 24 weeks, a significant (p<0.05) improvement in liver echogenicity and in liver structure was observed in 1932/2827 (68.3%) patients (95% CI 66.6% to 70.1%) and in 1207/2827 (42.7%) patients (95% CI 40.9% to 44.5%), respectively. The analysis of ultrasonographic signs by number of comorbidities revealed similar findings-liver echogenicity improved in 67.2%-69.3% and liver structure in 35.6%-45.3% of patients depending on the number of comorbidities. Conclusion: This study showed that PPC adjunctive therapy may be useful in improving the ultrasonographic features of NAFLD in patients with associated cardiometabolic comorbidities. It also supports evidence regarding the role of PPC in the complex management of NAFLD.

Real-world comorbidities and treatment patterns among patients with non-alcoholic fatty liver disease receiving phosphatidylcholine as adjunctive therapy in Russia.

OBJECTIVE: Previous research conducted in Russia showed that the number of patients with non-alcoholic fatty liver disease (NAFLD) and associated metabolic comorbidities is large. We conducted an observational study to describe the management of NAFLD in patients with metabolic syndrome in Russia. DESIGN: A total of 2843 adult patients from 174 medical sites across 6 federal districts of Russia with newly diagnosed NAFLD, who had at least one of four comorbidities, namely overweight/obesity, hypertension, type 2 diabetes mellitus, and hypercholesterolaemia, and who received phosphatidylcholine (PPC) as an adjunctive treatment to standard care, were enrolled during 2015-2016. RESULTS: Overall, 2263 patients (79.6%) had at least two metabolic comorbidities associated with NAFLD; overweight/obesity was the most common comorbidity reported in 2298 patients (80.8%). Simple steatosis was the most frequently identified clinical form of NAFLD, diagnosed in 2128 patients (74.9%). Among hypertensive patients, ACE inhibitors, statins, and sartans were most commonly prescribed. Biguanides were administered in more than half of diabetic patients. In patients with overweight/obesity and hypercholesterolaemia, statins were the most frequently prescribed medications. Almost all patients (2837/2843; 99.8%) were treated with 1.8 g of PPC three times per day. PPC therapy was associated with a 90.5% 6-month compliance rate, high treatment satisfaction, and a favourable safety profile. However, almost 15% of diabetic patients and 40% of overweight/obese patients received no further treatment. CONCLUSIONS: In Russia, patients with newly diagnosed NAFLD represent a population heavily burdened by comorbidities, mainly overweight/obesity and hypercholesterolaemia. A significant part of these patients did not receive a comprehensive pharmacotherapy, highlighting the existing unmet need in the current management of NAFLD patients with metabolic syndrome in Russia.

Platelet count to spleen diameter ratio non-invasively identifies severe fibrosis and cirrhosis in patients with autoimmune hepatitis.

BACKGROUND AND AIM: Non-invasive markers are essential to assess the progression of chronic liver diseases to fibrosis/ cirrhosis and the effectiveness of therapeutic strategies. The aim of this study was to evaluate the ability of non-invasive markers to identify significant fibrosis, severe fibrosis, and cirrhosis in patients with autoimmune hepatitis (AIH). METHODS: Seventy-six patients with AIH were enrolled in the study and analyzed for the following parameters of liver fibrosis: Fibrosis 4 score (FIB-4), aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (AAR), AST to platelet count ratio (APRI), and platelet count to spleen diameter (PC/SD) ratio. All patients underwent liver biopsy. The diagnostic accuracy of tests was evaluated by the area under the receiver operating characteristic curve (AUROC). RESULTS: Among the 76 AIH patients, 55 (72.3%) had significant fibrosis (≥ F2), 37 (48.7%) had severe fibrosis (≥ F3), and 29 (38.2%) had cirrhosis (F4). PC/SD ratio (AUROC = 0.840) was superior to AAR (AUROC = 0.756), FIB-4 (AUROC = 0.702), and APRI (AUROC = 0.626) in discriminating between mild and significant fibrosis (≥ F2). The AUROCs of PC/SD ratio, FIB-4, AAR, and APRI were 0.884, 0.742, 0.731, and 0.707, respectively, for severe fibrosis (≥ F3); 0.968, 0.795, 0.744, and 0.723, respectively, for cirrhosis (F4). PC/SD ratio correctly identified 85.1% of patients with severe fibrosis, and 89.6% of patients with cirrhosis. CONCLUSIONS: PC/SD ratio proved to be a simple non-invasive tool to correctly identify AIH patients with severe fibrosis and cirrhosis, thereby reducing the need for a liver biopsy in these patients.

Anti-HIV therapy with AZT prodrugs: AZT phosphonate derivatives, current state and prospects.

IMPORTANCE OF THE FIELD: AIDS, a disease caused by human immunodeficiency virus, was called 'plague of the twentieth century'. 3'-Azido-3'-deoxythymidine (AZT), the first compound approved for the treatment of HIV, is still a mandatory component of treatment schemes. However, its toxicity stimulated a search for new agents. AREAS COVERED IN THIS REVIEW: This review presents the history and current state of the design of AZT prodrugs based on its phosphonate derivatives. WHAT THE READER WILL GAIN: Although every effort was made to include as many AZT structures bearing phosphonate residues and demonstrate the variety they offer, we also concentrated on the studies performed in our laboratory. Special attention was also paid to AZT 5'-H-phosphonate (phosphazide, Nikavir) approved in the Russian Federation as a drug for the prevention and treatment of HIV infection. TAKE HOME MESSAGE: The prodrug strategy applied to AZT phosphonate derivatives enriched chemistry, biology and medicine not only with new knowledge, methods and structures, but also with a new anti-HIV drug Nikavir. Currently, study of another phosphonate, AZT 5'-aminocarbonylphosphonate, is underway. Slow release of AZT following oral administration and penetration into cells, decreased toxicity and the lack of cumulative properties make the compounds of this group promising as extended-release forms of AZT.

5'-aminocarbonyl phosphonates as new zidovudine depot forms: antiviral properties, intracellular transformations, and pharmacokinetic parameters.

The main disadvantages of 3'-azido-3'-deoxythymidine (zidovudine, AZT), the most common anti-HIV drug, are toxicity and a short half-life in the organism. The introduction of an H-phosphonate group into the AZT 5' position resulted in significant improvement of its therapeutic properties and allowed a new anti-HIV drug, Nikavir (AZT H-phosphonate). In this work, we described a new group of AZT derivatives, namely, AZT 5'-aminocarbonylphosphonates. The synthesized compounds displayed antiviral properties in cell cultures infected with HIV-1 and the capacity to release the active nucleoside in animals (rabbits and dogs) in a dose-dependent manner. The compounds were less toxic in MT-4 and HL-60 cell cultures and experimental animals compared with AZT. Major metabolites found in MT-4 cells after their incubation with AZT 5'-aminocarbonylphosphonate 1 were AZT and AZT 5'-phosphate (25 and 55%, respectively). Among the tested compounds, phosphonate 1 was the most effective AZT donor, and its longest t(1/2) and T(max) values in the line phosphonate 1--AZT H-phosphonate--AZT imply that compound 1 is an extended depot form of AZT. Although bioavailability of AZT after oral administration of phosphonate 1 was lower than those of AZT H-phosphonate and AZT (8 against 14 and 49%), we expect that this reduction would not cause essential decrease of antiviral activity but noticeably decrease toxicity as a result of gradual accumulation of AZT in blood and the absence of sharp difference between C(max) and C(min). Such a combination of properties makes the compounds of this group promising for further studies as extended-release forms of AZT.

The human vomeronasal type-1 receptor family--detection of volatiles and cAMP signaling in HeLa/Olf cells.

The human genome harbors 5 remnant genes coding for vomeronasal type-1 receptors, compared with 187 of such receptors in mice. In rodents, vomeronasal type-1 receptors are typically expressed in the vomeronasal organ. They are believed to be highly selective and sensitive pheromone detectors, as may be inferred from one receptor, V1rb2, responding to picomolar concentrations of the mouse pheromone 2-heptanone. Expression patterns, ligands, and signal transduction of human vomeronasal type-1 receptors have, however, remained largely obscure. Our aim was to deorphan and functionally characterize these 5 putative human pheromone receptors. Here, we report functional expression for all 5 receptors in HeLa/Olf cells. The recombinant, N-terminally tagged receptors expressed at the plasma membrane of HeLa/Olf cells and responded differentially to 19 of 140 odorants in a combinatorial way. C9-C10 aliphatic alcohols or aldehydes emerged as the best agonists at submicromolar concentrations above human odorant thresholds. Surprisingly, and in contrast to mouse V1rb2, all human vomeronasal type-1 receptors activated cAMP signaling via G protein alphaolf, when expressed in HeLa/Olf cells. While a biological function of human vomeronasal type-1 receptors is still elusive, our data show that their major functional characteristics are similar to those of odorant receptors.

5'-carbamoylphosphonyl-[6-3H]-AZT as a tool for studying metabolic transformations of the nonradioactive counterpart, an inhibitor of HIV replication.

An effective synthesis of 5'-carbamoylphosphonyl-[6-3H]-AZT was developed from [6-3H]-AZT. For the synthesized compound, chemical and enzymatic stability were determined and its penetration across HL-60 cell membranes was studied.

Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2.

An interaction of odorants with olfactory receptors is thought to be the initial step in odorant detection. However, ligands have been reported for only 6 out of 380 human olfactory receptors, with their structural determinants of odorant recognition just beginning to emerge. Guided by the notion that amino acid positions that interact with specific odorants would be conserved in orthologs, but variable in paralogs, and based on the prediction of a set of 22 of such amino acid positions, we have combined site-directed mutagenesis, rhodopsin-based homology modelling, and functional expression in HeLa/Olf cells of receptors OR1A1 and OR1A2. We found that (i) their odorant profiles are centred around citronellic terpenoid structures, (ii) two evolutionary conserved amino acid residues in transmembrane domain 3 are necessary for the responsiveness of OR1A1 and the mouse ortholog Olfr43 to (S)-(-)-citronellol, (iii) changes at these two positions are sufficient to account for the differential (S)-(-)-citronellol responsiveness of the paralogs OR1A1 and OR1A2, and (iv) the interaction sites for (S)-(-)-citronellal and (S)-(-)-citronellol differ in both human receptors. Our results show that the orientation of odorants within a homology modelling-derived binding pocket of olfactory receptor orthologs is defined by evolutionary conserved amino acid positions.

Synthesis of novel alkyl triphosphates and their substrate properties toward terminal deoxynucleotidyltransferase.

Novel triphosphate derivatives bearing bulky or small groups at alpha-position attached to the triphosphate residue through linkers of different structures and lengths were synthesized and studied as substrates toward terminal deoxynucleotidyltransferase. The substrate efficacy depends on the structure of substituents, linker length, and nature of metal activator. The replacement of hydrophobic groups by small substituents decreased the substrate efficacy by about 20 times in respect to hydrophobic residues. The dependence on metal activator is the following: Co(2+) > Mn(2+) >> Mg(2+). The model of interaction of alkyl triphosphates with linkers of different lengths bearing TdT active site is presented.

AZT 5'-Cholinephosphate as an anti-HIV agent: the study of biochemical properties and metabolic transformations using its 32P-labelled counterpart.

Biochemical and metabolic transformations of 3'-azido-3'-deoxythymidine 5'-choline phosphate (1) were studied using its 32P-labelled counterpart for the evaluation of possible reasons for its enhanced anti-HIV activity. An effective synthesis of 32P-labelled 1 with a specific activity >1,000 Ci/mmol was developed by esterification of 32P-phosphoric acid with choline in the presence of BrCN followed by the coupling of the resulting choline phosphate with 3'-azido-3'-deoxythymidine (AZT). Chemical and enzymatic stabilities of 1 as well as the dynamics of penetration through HL-60 cell membranes were studied at the concentrations comparable to its antiviral concentrations. The products of intracellular transformations of the studied nucleotide were identified.

Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels.

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG/ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.