S9 Protein Research Articles

In Vitro to In Vivo Scalars for Drug Clearance in Nonalcoholic Fatty Liver and Steatohepatitis.

In vitro-in vivo extrapolation (IVIVE) allows prediction of clinical outcomes across populations from in vitro data using specific scalars tailored to the biologic characteristics of each population. This study experimentally determined scalars for patients with varying degrees of nonalcoholic fatty liver disease (NAFLD), ranging from fatty liver to nonalcoholic steatohepatitis (NASH) and cirrhosis. Microsomal, S9, and cytosol fractions were extracted from 36 histologically normal and 66 NAFLD livers (27 nonalcoholic fatty liver [NAFL], 13 NASH, and 26 NASH with cirrhosis). Corrected microsomal protein per gram liver (MPPGL) progressively decreased with disease severity (26.8, 27.4, and 24.3 mg/g in NAFL, NASH, and NASH/cirrhosis, respectively, compared with 35.6 mg/g in normal livers; ANOVA, P < 0.001). Homogenate, S9, and cytosolic protein showed a consistent trend of decline in NASH/cirrhosis relative to normal control (post-hoc t test, P < 0.05). No differences across the groups were observed in homogenate, S9, cytosolic, and microsomal protein content in matched kidney samples. MPPGL-based scalars that combine protein content with liver size revealed that the reduction in MPPGL in NAFL and NASH was compensated by the reported increase in liver size (relative scalar ratios of 0.96 and 0.99, respectively), which was not the case with NASH/cirrhosis (ratio of 0.63), compared with healthy control. Physiologically based pharmacokinetics-informed global sensitivity analysis of the relative contribution of IVIVE scalars (hepatic CYP3A4 abundance, MPPGL, and liver size) to variability in exposure (area under the curve) to three CYP3A substrates (alprazolam, midazolam, and ibrutinib) revealed enzyme abundance as the most significant parameter, followed by MPPGL, whereas liver volume was the least impactful factor. SIGNIFICANCE STATEMENT: Nonalcoholic fatty liver disease-specific scalars necessary for extrapolation from in vitro systems to liver tissue are lacking. These are required in clearance prediction and dose selection in nonalcoholic fatty liver and steatohepatitis populations. Previously reported disease-driven changes have focused on cirrhosis, with no data on the initial stages of liver disease. The authors obtained experimental values for microsomal, cytosolic, and S9 fractions and assessed the relative impact of microsomal scalars on predicted exposure to substrate drugs using physiologically based pharmacokinetics.

Alleviation of banded leaf and sheath blight disease incidence in maize by bacterial volatile organic compounds and molecular docking of targeted inhibitors in Rhizoctonia solani.

Rhizoctonia solani (RS) is a pathogenic fungus that affects maize (Zea mays L.) plants and causes banded leaf and sheath blight (BLSB) with severe consequences leading to significant economic losses. Contrarily, rhizobacteria produce numerous volatile organic compounds (VOCs) that help in devising the environment-friendly mechanism for promoting plant growth and stress alleviation without having physical contact with plants. In the present study, 15 rhizobacterial strains were tested for their antagonism against RS. The antagonistic potential of VOCs of the tested plant growth-promoting rhizobacteria (PGPR) strains ranged from 50% to 80% as compared to the control (without PGPR). Among these 15 strains, the maximum (80%) antagonistic activity was exhibited by Pseudomonas pseudoalcaligenes SRM-16. Thus, the potential of VOCs produced by P. pseudoalcaligenes SRM-16 to alleviate the BLSB disease in maize was evaluated. A pot experiment was conducted under greenhouse conditions to observe the effect of VOCs on disease resistance of BLSB-infected seedlings. Overall, maize seedlings exposed to VOCs showed a significant increase in disease resistance as indicated by a reduced disease score than that of unexposed infected plants. The VOCs-exposed maize exhibited lower (11.6%) disease incidence compared to the non-inoculated maize (14.1%). Moreover, plants exposed to VOCs displayed visible improvements in biomass, photosynthetic pigments, osmoregulation, and plant antioxidant and defense enzyme activities compared to the healthy but unexposed seedlings. Simultaneous application of RS and VOCs enhanced superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), phenylalanine ammonia lyase (PAL), ascorbate peroxidase (APX), and polyphenol oxidase (PPO) activities by 96.7%, 266.6%, 313.7%, 246.6%, 307%, and 149.7%, respectively, in the roots and by 81.6%, 246.4%, 269.5%, 269.6%, 329%, and 137.6%, respectively, in the shoots, relative to those of the control plants. The binding affinity of the VOCs (2-pentylfuran, 2,3-butanediol, and dimethyl disulfide) with CRZ1 and S9 protein receptors of RS was assessed by deploying in silico methods. Overall, 2-pentylfuran exhibited a binding affinity with both the selected receptors of RS, while 2,3-butanediol and dimethyl disulfide were able to bind S9 protein only. Hence, it can be deduced that S9 protein receptors are more likely the target RS receptors of bacterial VOCs to inhibit the proliferation of RS.

Galectin-3 Mediates Tumor Progression in Astrocytoma by Regulating Glycogen Synthase Kinase-3β Activity

Numerous studies have considered galectin-3 or Glycogen synthase kinase 3 beta (GSK3B) as a potential prognosis marker for various cancers. However, the correlation between the protein expression of galectin-3/GSK3B and the clinical parameters of astrocytoma has not been reported. This study aims to validate the correlation between the clinical outcomes and protein expression of galectin-3/GSK3B in astrocytoma. Immunohistochemistry staining was performed to detect galectin-3/GSK3B protein expression in patients with astrocytoma. The Chi-square test, Kaplan-Meier evaluation, and Cox regression analysis were used to determine the correlation between clinical parameters and galectin-3/GSK3B expression. Cell proliferation, invasion, and migration were compared between a non-siRNA group and a galectin-3/GSK3B siRNA group. Protein expression in galectin-3 or GSK3B siRNA-treated cells was evaluated using western blotting. Galectin-3 and GSK3B protein expression were significantly positively correlated with the World Health Organization (WHO) astrocytoma grade and overall survival time. Multivariate analysis revealed that WHO grade, galectin-3 expression, and GSK3B expression were independent prognostic factors for astrocytoma. Galectin-3 or GSK3B downregulation induced apoptosis and decreased cell numbers, migration, and invasion. siRNA-mediated gene silencing of galectin-3 resulted in the downregulation of Ki-67, cyclin D1, VEGF, GSK3B, p-GSK3B Ser9 (p-GSK3B S9), and β-catenin. In contrast, GSK3B knockdown only decreased Ki-67, VEGF, p-GSK3B S9, and β-catenin protein expression but did not affect cyclin D1 and galectin-3 protein expression. The siRNA results indicated that GSK3B is downstream of the galectin-3 gene. These data support that galectin-3 mediated tumor progression by upregulating GSK3B and β-catenin protein expression in glioblastoma. Therefore, galectin-3 and GSK3B are potential prognostic markers, and their genes may be considered to be anticancer targets for astrocytoma therapy.

Genomic features of in vitro selected mutants of Escherichia coli with decreased susceptibility to tigecycline

The increase in multidrug-resistant bacteria has reached an alarming rate globally, making it necessary to understand the underlying mechanisms mediating resistance in order to discover new therapeutics. Tigecycline (TGC) is a last-resort antimicrobial agent for the treatment of serious infections caused by extensively drug-resistant Enterobacteriaceae. The TGC-resistant Escherichia coli mutants were obtained by exposing three different TGC-susceptible isolates belonging to ST131 (n=2) and ST405 (n=1) to increasing concentrations of TGC. The genetic alterations associated with reduced susceptibility to TGC were identified using whole genome sequencing. The fitness cost of TGC resistance acquisition, as well as incidence of cross-resistance, was also investigated. The TGC minimum inhibitory concentrations (MICs) of in vitro selected mutants were elevated 8 to 32 times compared with ancestral strains. Inactivating mutations (frameshift and nonsense) or amino acid substitutions were identified in genes encoding proteins with diverse functions, including AcrAB efflux pump or its regulators (lon and marR), Lipopolysaccharides (LPS) inner core biosynthesis enzymes (waaQ and eptB), ribosomal S9 protein (rpsI), and RNA polymerase β subunit. In most cases (but not all), acquisition of TGC resistance was associated with a fitness cost. While TGC resistance development was associated with cross-resistance to other members of the tetracycline family and chloramphenicol, hypersensitivity to nitrofurantoin was identified among heptose III-less LPS mutants. TGC resistance among the studied mutants was found to be multifactorial with extrusion by efflux transports being the most common mechanism. The LPS inner core biosynthesis pathway, as well as ribosomal S9 protein, could be additional targets for TGC resistance.

Prevalence of CYP2C19 gene mutations in patients with coronary heart disease and its biological activation effect in clopidogrel antiplatelet response

Objective: The purpose of this study was to investigate the effects of CYP2C19 gene mutations on clopidogrel antiplatelet activity in the patients with coronary heart disease treated by percutaneous coronary intervention. Methods: Patients with coronary heart disease, who hospitalized in the Second Affiliated Hospital of Nanchang University from March 2011 to June 2019, and healthy individuals with matching genetic background, gender, and age as controls were included in this study. Basic clinical data were analyzed and blood samples of all research subjects were obtained for extraction of DNA, and Sanger first-generation sequencing method was used to detect CYP2C19 gene mutation from full exon and exon and intron junction. CYP2C19 gene variations in patients with coronary heart disease were compared with the 1000 Genomes Browse database and the sequencing results of healthy controls to determine whether the gene variation was a genetic mutation or a genetic polymorphism. After that, PolyPhen-2 prediction software was used to analyze the harmfulness of gene mutations to predict the effect of mutations on protein function. The same dose of CYP2C19 wild-type plasmid and the CYP2C19 gene mutant plasmids were transfected into human normal liver cells HL-7702. After transfection of 24 h, the expression of CYP2C19 protease in each group was detected. The liver S9 protein was incubated with clopidogrel, acted on platelets to detect the platelet aggregation rate and the activity of human vasodilator-activated phosphoprotein (VASP). Results: A total of 1 493 patients with coronary heart disease (59.36%) were enrolled, the average age was (64.5±10.4) years old, of which 1 129 were male (75.62%). Meanwhile, 1 022 healthy physical examination volunteers (40.64%) were enrolled, and the average age was (64.1±11.0) years old, of which 778 were male (76.13%). A total of 5 gene mutations of CYP2C19 gene were identified in 12 patients (0.80%), namely, 4 known mutations T130K (1 case), M136K (6 cases), N277K (3 cases), V472I (1 case) and one new mutation G27V (1 case), no corresponding gene mutation was found in healthy controls. It was found that T130K and M136K were probably damaging, G27V was possibly damaging, and N277K and V472I were benign mutations. In vitro, we demonstrated that the platelet aggregation rate of the M136K gene mutation group was 24.83% lower than that of the wild type (59.58% vs. 34.75%; P<0.05), and the phosphorylated VASP level was 23.0% higher than that of the wild type (1.0 vs. 1.23; P<0.05). However, the platelet aggregation rate and phosphorylated VASP level were similar between of G27V, T130K, N277K, V472I gene mutation groups and wild type group (P>0.05). Conclusions: In this study, 5 gene mutations are defined in patients with coronary heart disease, namely G27V, T130K, M136K, N277K, V472I. In vitro functional studies show that CYP2C19 gene mutation M136K, as a gain-of-function gene mutation, can enhance the activation of CYP2C19 enzyme on clopidogrel, thereby inhibiting the platelet aggregation rate.

Aldehyde Oxidase Contributes to All-Trans-Retinoic Acid Biosynthesis in Human Liver.

All-trans-retinoic acid (atRA) is a critical endogenous signaling molecule. atRA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to atRA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to atRA formation in human liver. Human recombinant AOX formed atRA from retinaldehyde (Km ∼1.5 ± 0.4 µM; kcat ∼3.6 ± 2.0 minute-1). In human liver S9 fractions (HLS9), atRA formation was observed in the absence of NAD+, suggesting AOX contribution to atRA formation. In the presence of NAD+, Eadie-Hofstee plots of atRA formation in HLS9 indicated that two enzymes contributed to atRA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of atRA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg-1 S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg-1 S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of atRA in the presence of NAD+ correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to atRA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter atRA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to atRA and contributes to hepatic atRA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic atRA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic atRA concentrations and signaling and alter vitamin A homeostasis.

Identification and in silico molecular modelling study of newly isolated Bacillus subtilis SI-18 strain against S9 protein of Rhizoctonia solani

The numerous bioactive components from Bacillus subtilis are commonly used as antimicrobial agents for reducing plant diseases caused by fungal pathovars. In this study, we isolated and identified B. subtilis SI-18 strain from twenty isolates of rhizosphere soil through morphological and molecular approaches, and explored its inhibitory activities against Rhizoctonia solani. According to morphological features and 16S rRNA and gyrB gene sequence analysis, B. subtilis SI-18 strain was identified. Additionally, the culture filtrate of B. subtilis SI-18 resulted in the suppression of R. solani mycelium growth and material leakage from the cells. Then, we have performed homology modelling and molecular docking study of S9 protein from R. solani where three potential compounds (D1, D2, and D3) were identified among 134 antimicrobial compounds derived from B. subtilis group based on higher binding energy and interaction at the active grove of the target protein. The D1 compound creates alkyl bond at Val48 whereas D2 also binds with Val48 by creating hydrogen bond. On the other hand, two hydrogen bonds were observed at Val48 and Ile52 by D3, which might be responsible for possible blocking of the target S9 protein of R. solani. To validate the docking study and understand the change in drug-ligand conformation, molecular dynamics simulation was assessed where rigid conformation was found for D1, D2 and D3 complexes. Moreover, ADMET study confirms that no toxicity and carcinogenicity were found for screened compounds. Based on our studies, we demonstrated that compounds D1, D2, and D3 derived from B. subtilis can be a potential inhibitor of S9 protein of R. solani that might be a possible strategy for fungal disease prevention.

Optimization of a pre-metabolization procedure using rat liver S9 and cell-extracted S9 in the Ames fluctuation test

Many environmental pollutants pose a toxicological hazard only after metabolic activation. In vitro bioassays using cell lines or bacteria have often no or reduced metabolic activity, which impedes their use in the risk assessment. To improve the predictive capability of in vitro assays, external metabolization systems like the liver S9 fraction are frequently combined with in vitro toxicity assays. While it is typical for S9 fractions that samples and testing systems are combined in the same exposure system, we propose to separate the metabolism step and toxicity measurement. This allows for a modular combination of metabolic activation by enzymes isolated from rat liver (S9) or a biotechnological alternative (ewoS9R) with in vitro bioassays that lack metabolic capacity. Benzo(a)pyrene and 2-aminoanthracene were used as model compounds to optimize the conditions for the S9 metabolic degradation/activation step. The Ames assay with Salmonella typhimurium strains TA98 and TA100 was applied to validate the set-up of decoupling the S9 activation/metabolism from the bioassay system. S9 protein concentration of 0.25 mgprotein/mL, a supplement of 0.13 mM NADPH and a pre-incubation time of 100 min are recommended for activation of samples prior to dosing them to in vitro bioassays using the regular dosing protocols of the respective bioassay. EwoS9R performed equally well as Moltox S9, which is a step forward in developing true animal-free in vitro bioassays. After pre-incubation with S9 fraction, chemicals induced bacteria revertants in both the TA98 and the TA100 assay as efficiently as the standard Ames assay. The pre-incubation of chemicals with S9 fraction could serve for a wide range of cellular in vitro assays to efficiently combine activation and toxicity measurement, which may greatly facilitate the application of these assays for chemical hazard assessment and monitoring of environmental samples.

Hepatic Clearance Binding Terms of Hydrophobic Organic Chemicals in Rainbow Trout: Application of a Streamlined Sorbent-Phase Dosing Method

Knowledge of the freely dissolved chemical fraction in liver S9 incubation medium (ϕS9) and blood plasma (ϕP), often expressed as the ϕP/ϕS9 ratio or fU, plays a central role in in vitro–in vivo extrapolation of biotransformation rates for hydrophobic organic chemicals (HOCs) in fish. However, there are very few empirical fU data for adequately assessing the bioaccumulation potential of HOCs (i.e., log KOW ≥ 4) from in vitro biotransformation rates. In this study, we present a novel streamlined approach for obtaining the fU values of HOCs and produce some of the first empirical fU data for rainbow trout. We show that fU is relatively constant for substances with a log KOW of ≥4 and appears to be largely controlled by the relative differences in protein and lipid contents in blood plasma and liver S9 incubation media. Predictive fU models are in reasonable agreement with empirical observations. Empirical fU values for HOCs with a log KOW of ≥4 ranged between 0.015 and 0.096 with an average fU value of 0.044 at an S9 protein concentration of 1 mg/mL. This range of fU values may provide reasonable preliminary estimates for the extrapolation of biotransformation rates measured in trout liver S9 bioassays.

Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber)

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RT-qPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species.

Detoxification of 1,2-dihydroxy- 4-allylbenzene, a major phenolic compound in Piper betle, through glucuronidation using S9 protein of rat liver (Sprague Dawley)

1,2-dihydroxy- 4-allylbenzene (DHAB) is a major compound in Piper betle leaf and also a metabolite of safrole metabolism. Epidemilogic studies showed people who have betel quid chewing habit are related to the incidence of oral submucous fibrosis and/or tumor formation. The aims of this research was to study the detoxification through the glucuronidation of DHAB using S9 protein of male rat liver ( Sprague Dawley) and a co-factor of uridin 5′-diphosphoglucuronide acid (UDPGA). The results showed that glucuronidation of DHAB using S9 protein of rat liver resulted two isomers of glucuronide metabolites due to the availibility of two active hydroxyl groups that one of them can bind glucuronide but not two of hydroxyl at the same time.

A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe

In the present study, we established a quantitative western blotting method to measure the expression level of recombinant serine hydrolases based on their catalytic mechanism. Fluorophosphonate (FP)-biotin was selected as a universal probe to quantify their expression levels, since FP moiety irreversibly inhibits serine hydrolases through strong stoichiometric binding to active serine residue. The linearity of detection using FP-biotin was assessed on three serine hydrolases; human carboxylesterase (CES) 1, butyrylcholinesterase and porcine liver esterases (PLE). Similar response signals were obtained from the equimolar concentrations of these enzymes and excellent linearity was observed at the range of 0.4–3.4pmol/lane (r2>0.99). Accuracy and precision of the proposed method were proved using PLE with recovery of 97.1–107.2% and relative standard deviation of 5.56%. PLE was selected as a calibration standard because of its high stability and commercial availability. As an application of the developed method, we measured the expression levels of four recombinant CES isozymes from human and cynomolgus macaque in S9 fraction of HEK293 cell homogenates. The expression levels of human CES1 and CES2, and cynomolgus macaque CES1 and CES2 were 2.51±0.1, 1.63±0.17, 0.79±0.09 and 1.37±0.13pmol/5μg S9 protein, respectively. Based on these determinations, their hydrolytic activities were accurately assessed. Cynomolgus CESs showed lower hydrolysis activities for p-nitrophenyl esters than human CESs. The hydrolase activities of CES2 isozymes were higher than CES1 in both species. Three to five folds faster hydrolysis for p-nitrophenyl butyrate than p-nitrophenyl acetate was observed in all CES isozymes except of cynomolgus CES1 that showed nearly same hydrolysis for both substrates. The provided method could be widely used for universal quantitative analysis of recombinant serine hydrolases.

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin, a pore-forming peptide

1. An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency.2. Addition of Mg2+ (to 1 mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow.3. When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout.4. These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.

The interaction pattern between a homology model of 40S ribosomal S9 protein of Rhizoctonia solani and 1-hydroxyphenaize by docking study.

1-Hydroxyphenazine (1-OH-PHZ), a natural product from Pseudomonas aeruginosa strain SD12, was earlier reported to have potent antifungal activity against Rhizoctonia solani. In the present work, the antifungal activity of 1-OH-PHZ on 40S ribosomal S9 protein was validated by molecular docking approach. 1-OH-PHZ showed interaction with two polar contacts with residues, Arg69 and Phe19, which inhibits the synthesis of fungal protein. Our study reveals that 1-OH-PHZ can be a potent inhibitor of 40S ribosomal S9 protein of R. solani that may be a promising approach for the management of fungal diseases.

Distinctive contributions of the ribosomal P-site elements m2G966, m5C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli

The accuracy of pairing of the anticodon of the initiator tRNA (tRNAfMet) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m2G966 (methylated by RsmD), m5C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNAfMet. We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA (tRNAfMet with CAU anticodon); CAC and CAU with tRNA; UAG with tRNA; UAC with tRNA; and AUC with tRNA using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

In vitro models of xenobiotic metabolism in trout for use in environmental bioaccumulation studies

1. In vitro screens are sought as informative, alternatives to the use of animals in vivo and to improve upon the current use of fish liver 9000 g supernatants (S9) in environmental risk assessment.2. The rates of ethoxyresorufin-O-deethylation (relative to S9 protein) measured under different conditions of culture of rainbow trout hepatocytes were significantly higher than those detected in S9, in the order of freshly isolated hepatocytes > 10-day spheroid cultures > primary hepatocytes in culture > S9. The percentage of conjugated metabolites was also similar between freshly isolated and spheroid cultured hepatocytes (9.9 and 13.5%).3. The rate of oxidation was enhanced (1.7 fold) when S9 was supplemented with cofactors for phase II conjugation but this was only approximately one tenth of the rate in freshly isolated hepatocytes (7.1 pmol/min/mg S9 protein equivalent).4. Hepatocytes also hydroxylated ibuprofen, producing two metabolites, in contrast to only one (identified as the 1-hydroxy derivative) using hepatic S9 fractions.5. Since the bioaccumulation potential of chemicals is often based on un-supplemented S9 in incubations ≥1 h when activity declines, it is recommended that predictability would be greatly improved through the use of hepatocyte spheroids, due to their maintenance of activity and longevity.

Immune response and protective efficacy of S9 ribosomal protein of Streptococcus pneumoniae in a model of sepsis

Vaccination is the most promising strategy to reduce the incidence of pneumococcal infection. Although there are vaccines available, all of them are based on polysaccharide antigens (conjugated or not). In addition to their high cost, those vaccines do not cover all serotypes. To overcome these hindrances, we evaluated the immunogenicity and the protective efficacy of the S9 ribosomal protein of Streptococcus pneumoniae with the aim of developing a protein-based vaccine in the future. The gene encoding the S9 ribosomal protein was cloned in pET21-a expression vector, and the recombinant S9 protein was used to immunize mice. Significantly higher levels of anti-S9 immunoglobulin G were achieved (with predominance of immunoglobulin G1) in comparison with the control. Antibodies elicited against S.pneumoniae protein extract in rabbit recognized the recombinant S9 protein by Western blot, thus demonstrating its immunogenicity. Moreover, mice immunized with recombinant S9 protein and challenged with a virulent strain of S.pneumoniae presented a significant reduction of bacteremia after 24h of infection as compared with the control. However, in the S9-immunized mice the onset of death was insignificantly delayed, but all of them died by the fourth day postinfection.

An Alternative Splice Variant of KCNMA1 Drives Breast Cancer Metastasis and Invasion.

Abstract Background: We have recently shown that KCNMA1, which encodes the pore forming α-subunit of large-conductance calcium-activated voltage-sensitive potassium (BKCa) channel, plays a highly critical role in breast tumor cells metastatic to brain and somewhat less critical role in primary breast cancer cell metastasis. The resulting functional heterogeneity of BKCa channels is attributed generally to alternative splicing of KCNMA1 that generate BKCa channel isoform with altered channel properties. We have identified a KCNMA1 splice variant (KCNMA1vE22) expressed in breast cancer cell line (MDA-MB-361) metastatic to brain with a deletion of 108 bp in exon 22 between the S9 and S10 protein subunit (C-terminus). In this study we investigated the biological function of KCNMA1vE22 in vitro and in vivo.Methods: We validated the results from the breast tumor cell lines data with human normal breast tissue, primary, non-metastatic and metastatic breast tumors by RT-PCR. The KCNMA1vE22 was identified from A-172 tumor cells and cloned into mammalian expression vector (pcDNA6). MCF-7 cells (non-metastatic, null type for KCNMA1vE22) and HEK cells (null type for KCNMA1 and KCNMA1vE22) were transfected with pcDNA6/KCNMA1vE22 and stable clones selected. The expression of KCNMA1 and KCNMA1vE22 was confirmed using RT-PCR, western blot and qPCR. The biological role of the splice variant was studied using parental and transfected MCF-7 cells by cell proliferation, matrigel invasion, transendothelial migration and membrane potential assays in vitro and tumorogenecity using s.c xenograft mouse model.Results: Based on the preliminary screening of breast tumor samples, KCNMA1vE22 was undetectable in primary as well as systemic metastatic breast tumor samples. Stably transfected HEK and MCF-7 cells showed 2.5 to 3-fold increase in KCNMA1 mRNA expression, which corroborated with the increased protein levels and BKCa channel activity. In addition MCF-7 cells expressing KCNMA1vE22 showed a eight-fold increase in invasion while a three-fold increase in TEM was observed compared to parental MCF-7 cells. A marked increase in the proliferation rate of KCNAM1vE22-transfected cells compared to MCF-7 alone was observed, suggesting that KCNAM1vE22 has a profound effect on breast cancer cell proliferation. The in vitro results were validated by subcutaneous mouse model experiment. We found statistically significant increase in mean tumor volume in mice with KCNMA1vE22 transfected compared with parental MCF-7 cells.Conclusions: Our results clearly show that KCNMA1vE22 promotes breast cancer cell invasion, and possibly metastasis to brain. Perhaps the discovery and validation of brain specific metastasis-associated KCNMA1 alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. The variant KCNMA1vE22 that we have discovered potentially may fill the gap to serve as a new generation of biomarker of breast cancer metastasis to brain. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6166.

Liver microsomes and S9 from rainbow trout (Oncorhynchus mykiss): Comparison of basal‐level enzyme activities with rat and determination of xenobiotic intrinsic clearance in support of bioaccumulation assessment

Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6beta-hydroxylase, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro-to- in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 +/- 5.1 (mean +/- standard deviation throughout) and 95.9 +/- 11.9 mg/g, respectively. Intrinsic clearance (CL(int)) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic clearance (CL(H)) using the well-stirred liver model, but their CL(H) values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CL(int) values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CL(H) and bioconcentration factor prediction.

KsgA mutations confer resistance to kasugamycin in Neisseria gonorrhoeae

The aminoglycoside antibiotic kasugamycin (KSG) inhibits translation initiation and thus the growth of many bacteria. In this study, we tested the susceptibilities to KSG of 22 low-passage clinical isolates and 2 laboratory strains of Neisseria gonorrhoeae. Although the range of KSG minimum inhibitory concentrations (MICs) was narrow (seven-fold), clinical isolates and laboratory strains fell into three distinct classes of KSG sensitivity, susceptible, somewhat sensitive and resistant, with MICs of 30, 60–100 and 200 μg/mL, respectively. Two genes have previously been shown to be involved in bacterial KSG resistance: rpsI, which encodes the 30S ribosomal subunit S9 protein; and ksgA, which encodes a predicted dimethyltransferase. Although sequencing of rpsI and ksgA from clinical isolates revealed polymorphisms, none correlated with the MICs of KSG. Ten spontaneous KSG-resistant (KSG R) mutants were isolated from laboratory strain FA1090 at a frequency of <4.4 × 10 −6 resistant colony-forming units (CFU)/total CFU. All isolated KSG R variants had mutations in ksgA, whilst no mutations were observed in rpsI. ksgA mutations conferring KSG resistance included four point mutations, two in-frame and one out-of-frame deletions, one in-frame duplication and two frame-shift insertions. These data show a narrow range of susceptibilities for the clinical isolates and laboratory strains examined; moreover, the differences in MICs do not correlate with nucleotide polymorphisms in rpsI or ksgA. Additionally, spontaneous KSG R mutants arise by a variety of ksgA mutations.