Owing to its traditional applications, the current study focuses on Ajuga parviflora (A. parviflora) leaves extract for phytochemical and pharmacological analysis. The principle constituents were identified through gas chromatography (GC), and gas chromatography/mass spectroscopy (GC/MS), these includes phthalic acid, squalene, α-tocopherol, vitamin E, phytol, 2-methylenecholestan-3-ol, stigmasterol, cholest-22-ene-21-ol and 3,5-dehydro-6-methoxy. Hepatoprotective effect of A. parviflora was evaluated through isoniazid and rifampicin (INH and RFP) induced hepatotoxicity in rat. Animals in group A were treated with INH and RFP 50 mg/kg. Animals in group B, C, and D were pre-treated with A. parviflora extract at 100, 200 and 300 mg/kg dose prior drug administration. A. parviflora extract at 200 and 300 mg/kg in group C and D significantly reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and bilirubin (p<0.001) as compare to group B (100mg/kg). Total protein (TP) was also significantly (p<0.01) reduced in group C and D at dose of 200 and 300 mg/kg, respectively. The extract pre-treated animals with (A. parviflora, 200, and 300 mg/kg) showed that the epithelium of the central portal vein is intact with replete glucagon. The pre-treatment with A. parviflora protected the liver from INH and RFP induced hepatotoxicity. The results of pre-treated animals with A. parviflora 200, and 300 mg/kg dose prettily revert the severely disturb parameters like, cytolysis, lymphocytic infiltration, and lymphoid aggregate in portal vein and hydropic degeneration. The decrease peroxisome proliferator-receptor activator-δ (PPAR-δ) gene expression by INH, and RFP was significantly up regulated by A. parviflora extract in pre-treated animals at 200 and 300 mg/kg dose. These findings provide baseline pharmacological uses of A. parviflora in liver disorders. Further investigations are required for identification and isolation of biologically active components responsible for pharmacological activity.

(<em>b</em>)O<em>b</em>jective:</<em>b</em>) To study the effects of sevoflurane on reproductive function and its main mechanism of action in male rats.(<em>b</em>)Materials and methods:</<em>b</em>) Forty adult male Sprague-Dawley rats were divided into 4 groups and exposed to 0, 50, 300 and 1800 ppm of sevoflurane, respectively. After 15 days, the serum levels of sex hormones and inflammatory factors were detected using enzyme-linked immunosor<em>b</em>ent assay. Left testis was taken for conventional histopathological examination and TUNEL staining. Right testis was used for sperm production and daily sperm count were evaluated daily. Johnsen score was used to categorize the spermatogenesis. The expression of related genes in the hypothalamic-pituitary-gonadal axis were analyzed <em>b</em>y quantitative real time polymerase chain reaction (qRT-PCR).(<em>b</em>)Results:</<em>b</em>) Exposure to sevoflurane increased the levels of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1), decreased the content of serum testosterone (T), reduced the concentration of testicular sperm, the production of daily sperm and Johnsen score, and damaged vas deferens in a dose dependent manner. In addition, chronic exposure to sevoflurane down-regulated transcription of gonadotropin-releasing hormone (GnRH) and kisspeptin (Kiss)-1 as well as its receptor GPR54 in hypothalamus, attenuated GnRH receptor and LH-<em>β</em> mRNA levels, <em>b</em>ut increased FSH-<em>β</em> mRNA in pituitary gland, and enhanced mRNA of LH receptor and FSH receptor, <em>b</em>ut decreased <em>INH</em>-α and <em>INH</em>-<em>β</em>A mRNA levels in testes.(<em>b</em>)Discussion and conclusions:</<em>b</em>) Sevoflurane induces disorders of spermatogenesis and causes testicular injury. The underlying mechanism may <em>b</em>e related to the im<em>b</em>alance of sex hormones in the hypothalamic-pituitary-gonadal axis.

The developmental toxicities of five test compounds including carbon tetrachloride, urethane, phenacetin, parathion, and chloroform, were evaluated using Frog Embryo Teratogenesis Assay--Xenopus (FETAX), with minor modification. Post-isolation mixtures of differently-induced rat liver microsomes (phenobarbital- (PB), beta-naphthoflavone- (beta-NF), and isoniazid- (INH)-induced preparations) were co-cultured directly with X. laevis embryos. Results from these studies suggest that the Aroclor 1254-induced MAS could effectively be replaced by a mixed lot of PB-, beta-NF-, and INH-induced rat liver microsomes. Each of the test materials were found to be developmentally toxic when bioactivated by the mixed MAS.

Although the effect of volatile organic compounds (VOCs) on the oxidation of dissolved sulfur dioxide by oxygen has been the subject of many investigations, this is the first study which examines the effect of a large number of precisely 16 hydroxy compounds. The kinetics both in the absence and the presence of VOCs was defined by rate laws (A and B): -d[S(IV)]dt = R₀ = k₀[S(IV)] (A) -d[S(IV)]dt = R(i) = k(i)[S(IV)] (B) where R₀ and k₀ are the initial rate and first-order rate constant, respectively, in the absence of VOCs, R(i), and k(i) are the initial rate and the first-order rate constant, respectively, in the presence of VOCs, and [S(IV)] is the concentration of dissolved sulfur dioxide, sulfur(IV). The nature of the dependence of k(i) on the concentration of inhibitor, [Inh], was defined by Eq. (C). [k(i) = k₀/(1 + B[Inh]) (C) where B is an empirical inhibition parameter. The values of B have been determined from the plots of 1/k(i) versus [Inh]. Among aliphatic and aromatic hydroxy compounds studied, t-butyl alcohol and pinacol were without any inhibition effect due to the absence of secondary or tertiary hydrogen. The values of inhibition parameter, B, were related to k(inh), the rate constant for the reaction of SO₄(-) radical with the inhibitor, by Eq. (D). B = (9 ± 2) x 10⁻⁴ x k(inh) (D) Equation (D) may be used to calculate the values of either of B or k(inh) provided that the other is known. The extent of inhibition depends on the value of the composite term, B[Inh]. However, in accordance with Eq. (C), the extent of inhibition would be sizeable and measurable when B[Inh]>> 0.1 and oxidation of S(IV) would be almost completely stopped when B[Inh] ≥ 10. B[Inh] value can be used as a guide whether the reaction step: SO4 (-) + organics → SO₄(2-) + non-chain products: should be included in the multiphase models or not.

BACKGROUND

The penicillin therapy of β hemolytic streptococcal pharyngitis has aided in the decrease of rheumatic heart disease (RHD) in developing countries. Tunisia is an endemic area, however, and incidence of RHD is weakly documented. We aimed at establishing the standardized incidence rate (SIR) of RHD in Monastir governorate and at determining RHD prevalence among hospitalized patients in two cardiology departments.

METHODS

From the regional register of Monastir Hospital morbidity, we have selected newly diagnosed patients with RHD, residents of Monastir, and hospitalized to the 2 cardiology departments between 2000 and 2013 (2001 not included).

RESULTS

We studied 676 newly admitted patients. We estimate 1060 to be the number of new annual RHD cases in Tunisia. The SIR per 105 person-years was 10.97, being 9.3 in men and 19.1 in women, respectively. We have notified a negative trend of crude incidence rate/105 Inhabitants (Inh) (CIR) (r=-0.23, p<10-3), and a strong positive correlation between age and CIR/105 Inh (r=0.989, p<10-4). RHD lethality was 1%. We have registered 728 hospitalizations for RHD, representing 2.5% of all cardiology hospitalizations [95% CI: 2.3-2.7%], with a prevalence for 13.3% for women aged 15-29years. The median hospital stay was 9days (IQR: 5-15).

CONCLUSIONS

Our results confirm the RHD incidence decrease, consistent with epidemiological transition in Tunisia. We have also emphasized on the close trend of RHD with age and the predominance of RHD among women especially at the procreation age.

Multidrug-resistant Mycobacterium tuberculosis infection is now world wide health problem. However, according to the recent advances of molecular biological technics, some of the genetic mechanisms of drug-resistance of M. tuberculosis has been uncovered. Generally, drug-resistance of M. tuberculosis was caused by point mutations in chromosomal gene. In isoniazid (INH) resistant M. tuberculosis, mutations and genetic deletions in catalase-peroxidase gene (katG), inhA gene, or alkyl hydroperoxide reductase gene were reported. We also found that about 15% of INH-resistant M. tuberculosis isolates lacked katG gene, and these isolates showed highly resistance to INH with MIC>> or = 64 micrograms/ml. On the other hand, mutations and other genetic alterations in RNA polymerase beta subunit gene (rpoB) were the major mechanisms of resistance to rifampicin (RFP) with high frequencies of 90% or more. Our evaluation of the relationship between RFP susceptibility and genetic alteration in rpoB gene also showed that 95% of RFP-resistant M. tuberculosis isolates involved genetic alterations in 69 bp core region of rpoB gene. Moreover, these genetic alterations in rpoB gene were suspected as the resistant mechanism to other rifamycin antituberculosis drugs, such as rifabutin and KRM-1648. In addition, it was reported that point mutations in 16S rRNA gene (rrs) and ribosomal protein S12 gene (rpsL) induced M. tuberculosis as streptomycin (SM) resistant phenotype. We analyzed genetic alternations in rpsL gene of clinically isolates of M. tuberculosis, about 60% of SM resistant isolates were shown point mutation in this gene ant they were all high SM-resistant with MIC>> or = 256 micrograms/ml. Furthermore, nicotinamidase (pncA) gene, DNA gyrase A subunit (gyrA) gene, and embB gene were reported as the responsible gene to pyrazinamide-, quinolone- and ethambutol-resistance, respectively. Although all mechanisms of drug-resistance were still unclear, these informations are very useful and helpful for development of rapid diagnosis system of drug-resistant M. tuberculosis.

BACKGROUND

GenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosis resistance to isoniazid (INH) and rifampicin (RMP), the two major anti-tuberculosis (TB) drugs. Identification of INH resistance is largely based on the occurrence of mutations in the katG gene, coding for the catalase-peroxidase, or in the promoter region of the inhA gene, coding for the NADH-dependent enoyl-ACP reductase. For testing the RMP resistance, mutations in the rpoB gene, coding for the RNA polymerase β subunit, particularly in the RMP resistance determining region (RRDR) of the gene are investigated. The GenoType MTBDRplus assay has been validated in several countries. The aim of this study was to evaluate the ability of the assay to detect INH and RMP resistance among strains of M. tuberculosis, isolated from Pakistani TB patients, and phenotypically identified as multidrug-resistant (MDR), that is resistant to both INH and RMP.

METHODS

The study included a collection of 100 MDR M. tuberculosis strains isolated from as many Pakistani TB patients over a 9-month period (i.e. between January and September 2014). Drug susceptibility testing was performed using the standard 1% proportion method on Löwenstein-Jensen medium, with INH and RMP critical concentrations of 0.2mg/L and 40mg/L, respectively. Genomic DNA was extracted by the cetyl-trimethyl ammonium bromide (CTAB) method. The GenoType MTBDRplus assay (Hain Lifescience, Germany) was done following the manufacturer's instructions.

RESULTS

In the katG gene, with MTBDRplus assay, a specific mutation associated with INH resistance (i.e. G944C transition, conferring Ser315Thr amino acid change) was detected in 66 (66%) of the strains. Thirty-four (34%) strains did not carry the katG mutation detected by the assay. Mutations in the mabA-inhA promoter region were detected in 10 (10%) strains (C-15T - in 10 strains, and T-8C - in 2 strains). Seventy-seven (77%) strains tested harboured a mutation in rpoB gene. Mutations in the rpoB gene were of four types: C1349T, A1304T, C1333G, and TC1324CA found in 63 (63%), 11 (11%), 8 (8%), and one (1%) strain, respectively. Of the 100 strains designated as MDR by the proportion method, GenoType MTBDRplus confirmed this phenotype in only 62 strains. The results of GenoType MTBDRplus and the conventional drug susceptibility method were consistent in 70% (70/100) for INH, and 77% (77/100) for RMP.

CONCLUSIONS

As evidenced in this study, the major concern with the GenoType MTBDRplus assay were false negative results. In comparison to conventional drug susceptibility testing, the assay was unable to detect 30 (30/100; 30%) strains resistant to INH and 23 (23/100; 23%) strains resistant to RMP. The GenoType MTBDRplus failed to identify 38 MDR (38/100; 38%) strains. Resistance in those strains probably originate from mutations in other codons and/or genes than those covered by the test. For detecting INH and RMP resistance in TB cases, especially in high TB incidence countries, such as Pakistan, molecular approaches should still be a complement rather than areplacement to conventional drug susceptibility testing.

OBJECTIVE

To determine whether liver cirrhosis patients are at higher risk for drug-induced hepatotoxicity (DIH) than control subjects during treatment for tuberculosis (TB) with standard short-course regimens containing isoniazid (INH), rifampicin (RMP), ethambutol (EMB) and/or pyrazinamide (PZA).

METHODS

Fifty liver cirrhosis patients with newly diagnosed active TB treated with INH, RMP, EMB and/or PZA were included in the study, along with 147 patients without liver disease selected as control subjects. DIH was defined as alanine aminotransferase (ALT)>> 120 IU/l with hepatitis symptoms or ALT>> 200 IU/l.

RESULTS

The aetiology of the liver cirrhosis patients consisted of alcoholic liver cirrhosis (n = 37, 74%), hepatitis B (n = 10, 20%) and hepatitis C (n = 3, 6%). The mean Child-Pugh score of all liver cirrhosis patients was 7.0 ± 1.2. DIH was more frequently found in liver cirrhosis patients, but the difference was not statistically significant (8.0% vs. 2.7%, P = 0.115). INH and RMP were successfully rechallenged and maintained until the end of treatment in three of four liver cirrhosis patients with DIH.

CONCLUSIONS

Although DIH developed more frequently in TB patients with liver cirrhosis, the apparent difference in the incidence of DIH did not achieve statistical significance. Most of the patients with DIH were successfully treated with a standard short-course regimen including INH and RMP.

<A<em>b</em>stractText>The widespread use of molecular, genotypic drug suscepti<em>b</em>ility tests (DSTs) for antitu<em>b</em>erculosis (anti-TB) drugs has led to the dilemma of interpreting discordant results <em>b</em>etween genotypic and conventional, phenotypic DSTs. We investigated the clinical characteristics, including treatment patterns and outcomes, of TB patients with a genotype-phenotype discrepancy in suscepti<em>b</em>ility to isoniazid (<em>INH</em>) or rifampicin (RIF).</A<em>b</em>stractText><p><div>(<em>b</em>)Methods</<em>b</em>)</div>We retrospectively reviewed the medical records of TB patients who had results for 2 DSTs (genotypic method, MTBDR<i>plus</i> test for <em>INH</em> and RIF, and phenotypic method) treated <em>b</em>etween August 2010 and Octo<em>b</em>er 2016 in a tertiary university hospital.</p><A<em>b</em>stractText>Among 1,069 TB patients, 63 (5.9%) had discrepant results for the 2 DSTs. Of the 57 multidrug-resistant (MDR) TB cases diagnosed <em>b</em>y either DST, 18 (31.6%) showed discordant results for <em>INH</em> or RIF. The most frequent pattern of discordance was genotypic suscepti<em>b</em>ility with phenotypic resistance to <em>INH</em>. RIF-discordant su<em>b</em>jects with genotypic resistance were more likely to have <em>b</em>een exposed previously to anti-TB drugs and to have an MDR TB diagnosis and concurrent <em>INH</em> resistance. Forty-five of the 54 patients managed in our hospital (83.3%) had a favora<em>b</em>le outcome with a mean treatment duration of 14.0 months. Ten of the 16 <em>INH</em>-discrepant patients with a genotypic mutation continued taking <em>INH</em>, <em>b</em>ut more than half patients in the RIF-discrepant group (8/14) with a genotypic mutation discontinued taking RIF.</A<em>b</em>stractText><A<em>b</em>stractText>Despite the low frequency, discordant results were o<em>b</em>tained <em>b</em>etween the genotypic and phenotypic DSTs for <em>INH</em> or RIF, especially for patients with MDR TB or <em>INH</em> resistance. Furthermore, it seemed that RIF discrepancy with a genotypic mutation might have a greater impact on the clinical outcome than <em>INH</em> discrepancy.</A<em>b</em>stractText>

The cytochrome P450 (CYP) subfamily responsible for ethosuximide metabolism was investigated by HPLC assay of ethosuximide incubations with isolated rat liver microsomes from control rats and from rats treated with inducing agents to enrich hepatic microsomes in selected CYP isoforms. Inducing agents included beta-naphthoflavone (BNF, CYP1A inducer), phenobarbital (PB, CYP2B/2C/3A), isoniazid (INH, CYP2E1), clotrimazole (CTZ, CYP3A), clofibrate (CLO, CYP4A), and an imidazole CTZ-analog known as CDD3543 (CYP3A). Incubations with BNF, INH, CTZ, and control microsomes showed significantly (p<0.05) more metabolite produced by CTZ microsomes vs. BNF, INH, and control microsomes at 10, 30, 60, and 120 min incubation. Ethosuximide metabolite levels generated by CTZ microsomes at 120 min were 36.5 times those of control microsomes. Correspondingly, ethosuximide concentrations were significantly (p<0.05) lower for incubations with the CTZ microsomes compared with BNF, INH, and control microsomes at 60 and 120 min. Sixty-minute incubations with all microsome groups exhibited significantly (p<0.05) higher metabolite formation rates (nmol/nmol CYP/min) for CTZ (11.8x control) and PB (9.6x control) microsomes vs. all other groups. Antibody inhibition experiments demonstrated ethosuximide metabolite levels for PB microsomes were not affected by CYP2B1 antibodies, whereas CYP3A2 antibodies reduced metabolite levels for both PB and CTZ microsomes by over 80%. These results indicate CYP3A is primarily responsible for ethosuximide metabolism in rats.

Human N-acetyltransferase 2 (NAT2) catalyzes the N-acetylation of numerous aromatic amine drugs such as sulfamethazine (SMZ) and hydrazine drugs such as isoniazid (INH). NAT2 also catalyzes the N-acetylation of aromatic amine carcinogens such as 2-aminofluorene and the O- and N,O-acetylation of aromatic amine and heterocyclic amine metabolites. Genetic polymorphism in NAT2 modifies drug efficacy and toxicity as well as cancer risk. Acetyltransferase catalytic activities and heat stability associated with six novel NAT2 haplotypes (NAT2*6C, NAT2*14C, NAT2*14D, NAT2*14E, NAT2*17, and NAT2*18) were compared with that of the reference NAT2*4 haplotype following recombinant expression in Escherichia coli. N-acetyltransferase activities towards SMZ and INH were significantly (p < 0.0001) lower when catalyzed by the novel recombinant human NAT2 allozymes compared to NAT2 4. SMZ and INH N-acetyltransferase activities catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.001) than catalyzed by NAT2 6C and NAT2 14E. N-Acetylation catalyzed by recombinant human NAT2 17 was over several hundred-fold lower than by recombinant NAT2 4 precluding measurement of its kinetic or heat inactivation constants. Similar results were observed for the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and the intramolecular N,O-acetylation of N-hydroxy-N-acetyl-2-aminofluorene. The apparent V max of the novel recombinant NAT2 allozymes NAT2 6C, NAT2 14C, NAT2 14D, and NAT2 14E towards AF, 4-aminobiphenyl (ABP), and 3,2'-dimethyl-4-aminobiphenyl (DMABP) were each significantly (p < 0.001) lower while their apparent K m values did not differ significantly (p>> 0.05) from recombinant NAT2 4. The apparent V max catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.05) than the apparent V max catalyzed by NAT2 6C and NAT2 14E towards AF, ABP, and DMABP. Heat inactivation rate constants for recombinant human NAT2 14C, 14D, 14E, and 18 were significantly (p < 0.05) higher than NAT2 4. These results provide further evidence of genetic heterogeneity within the NAT2 slow acetylator phenotype.

We observed a 13 year old Turkish boy suffering from chronic arthritis of the left knee since 1983. Clinical symptoms as slow progression of the disease and laboratory data including a positive HLA B 27 test suggests the diagnosis of juvenile chronic arthritis. A tuberculous arthritis was initially excluded by X-ray examination of the chest. The positive tuberculin test was attributed to the BCG vaccination. Because of insufficient response to antiinflammatory drug therapy a synovectomy was performed for diagnostic as well as therapeutic reasons. Histopathological results suggested sarcoidosis. A second synovial biopsy of the affected joint revealed granulomas combined with multiple necrosis typical for tuberculous infection. The animal experiments showed positive results. Tuberculostatic therapy was successful with INH, myambutol and rifampicin. Joint function and MRI results markedly improved.

Spreading lesions clinically resembling lymphangitic sporotrichosis developed on the right arm and chest of a 60-year-old man with chronic lymphocytic leukemia. Acid-fast bacilli were seen in exudates from lesions and in biopsies, and were cultured from them. The isolant grew initially as a yellowish-orange scotochromogen on Lowenstein-Jensen medium at room temperature and at 35 C., but failed to grow at 37 C. It failed to grow on 7-H-10 medium. On repeated subculturing over a 2-year period it gradually converted to a photochromogen. Histologically, there was ulceration with extensive acute and chronic inflammation with fibrosis. Organisms occurred intracellularly as dense, compact, cigar-like packets resembling lepara bacilli. The appeared to have a predilection for the nucleus. The patient was anergic to PPD S, B, Y and G, and lacked antibodies to BCG phosphoglycolipids. The mycobacteriosis was alleviated by combined INH and ethambutol therapy. The isolant was identified as a rough variant of Mycobacterium marinum. It may have been transmitted by an insect vector.

The anti-tuberculostatic drug, isoniazid (INH) was evaluated for its mutagenic potential using Salmonella plate tests and fluctuation assays with various strains of bacteria, and different metabolic activation systems. In the Salmonella plate test INH proved to be a weak directly-acting base-substitution mutagen which was detoxified by S9-mix. S. typhimurium TA 1530 and TA 1535 were the sensitive strains, and this result confirmed some of the published data. In the present studies mutagenic activity was further diminished in the presence of larger concentrations of rat liver S9-mix. Furthermore, the reduction in mutagenic activity was observed with S9-mix derived from untreated, Aroclor 1254-treated or phenobarbitone/beta-naphthoflavone treated rats. In direct contrast, using the microtitre fluctuation assay, the mutagenic activity of INH was elevated in the presence of rat liver S9-mix, and continued to increase with increasing S9-concentration. This result was obtained irrespective of the S9-source. S. typhimurium strains TA 1530, TA 1535 and his G46, and E. coli strains TA 85, TA 86 and WP2 uvrA were all sensitive to the mutagenicity of INH after metabolic activation. The primary step in the metabolic activation of INH in the fluctuation test was mediated by a cytosolic enzyme, and the activity of dapsone as a competitive substrate implicated the involvement of an N-acetyl transferase. The rapid diffusion of the cytosolic enzyme into the basal agar layer, or the non-specific binding of the enzyme (or the active mutagenic INH metabolite) to components of the agar, may explain the contradictory data obtained in the Salmonella plate test. The modifying effects of agar on the distribution of drug metabolising enzymes within liver S9 fractions should be carefully considered when evaluating data from Salmonella plate tests.

Quantitative in vitro studies of antioxidant activities have been performed under aerobic conditions. However, since the biological system has lower oxygen tension, the effectiveness of antioxidants may be considerably different in vivo. alpha-Tocopherol, in vivo the most active tocopherol, is a very poor antioxidant in vitro. To clarify these points, the radical-scavenging activities of vitamin E (Toc) (alpha-, beta-, gamma- and delta-tocopherols) and ubiquinone were evaluated by the induction period method from the kinetics of polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2'azobisisobutyronitrile (AIBN) (alkyl radical, R*), or benzoyl peroxide (BPO) (peroxyl radical, PhCOO*) under nearly anaerobic conditions. The ratio of the rate constant of inhibition to that of propagation (k(inh)/k(p)) for Toc was about 10 in a system with a molar ratio of AIBN to Toc of 100:1, whereas in the corresponding BPO system k(inh)/k(p) declined in the order alpha (47)>> beta (15)>> gamma (10)>> delta (7). In contrast, with AIBN the number of free radicals trapped by the phenolic moiety (n) declined in the order delta (3.0)>> gamma (2.5)>> alpha (2.2)>> beta (1.6), whereas with BPO n declined in the order delta (1.9)>> gamma (1.4)>> beta (1.0)>> alpha (0.3). A similar tendency was found in systems with a molar ratio of 10:1. Also, ubiquinone-10 showed radical-scavenging activity, although the n (0.02) was much less than that for Toc. The low n value for alpha-Toc (n = 0.3) may be attributed to the formation of stable alpha-Toc during the induction period. With a n = about 1 for beta- and gamma-Toc, a dimerization coupling of Tocs is suggested. Thus, the radical-scavenging activity is affected by the number and position of the methyl groups in the benzene nucleus of the various tocopherol compounds.

OBJECTIVE

To study the molecular basis of preventive effect of human apolipoprotein-1 (h-apoA-1) on vascular smooth muscle cell (vSMC) proliferation and lipid deposition induced by oxidized low density lipoprotein (ox-LDL).

METHODS

Smooth muscle cells originated from the aortae of h-apo-A-1 transgenic mice were cultured and divided into 2 groups, one group was stimulated by ox-LDL (tester) and the other group was used as control (driver). Subtractive hybridization was used to enrich the genes differentially expressed in the vSMCs induced by ox-LDL. A subtractive library was thus established and confirmed by colony hybridization in situ and dot blot analysis. 15 clones out of the 57 differentially expressed clones were randomly chosen foe sequencing and homology analysis. The whole-length cDNA library of vSMC induced by ox-LDL was established using SMART technique.

RESULTS

Three expression sequence tags (EST), all correlated with immune system, were confirmed: C1-inhibitor (C1-INH), lectin, and T cell receptor beta. The whole-length cDNA library contained 1.5 x 10(6) pfu/ml primary recombinants with insertions 0.5 - 3 kb in length.

CONCLUSIONS

The 3 EST may be involved in the mechanism of atherogenesis by ox-LDL and the mechanism of the function of h-ApoA-1 in retarding the progression of atherogenesis induced by ox-LDL.

BACKGROUND

The influence of estradiol (E2) on granulosa cell (GC) function has not been tested clinically in women with polycystic ovary syndrome (PCOS). The objective of this study is to determine if E2 influences GC responses to FSH in women with PCOS.

METHODS

This is a two phase, single cohort study conducted over a 2-year period at a single academic center. Nine women with PCOS according to NIH criteria. In Phase 1, FSH stimulation of GC responses as measured by E2 and Inhibin B (Inh B) were assessed before and at 5 and 6 weeks after GnRH agonist administration. In Phase 2, the same protocol was employed with the addition of an aromatase inhibitor (letrozole, LET) administered daily beginning at week 4 for 2 weeks.

RESULTS

In Phase 1, recovery of FSH, E2 and Inh B from ovarian suppression occurred at 5 and 6 weeks after GnRH agonist injection and preceded resumption of LH and androgen secretion. In Phase 2, hormone recovery after GnRH agonist was characterized by elevated FSH and suppressed E2 levels whereas recovery of LH and androgen levels were unchanged. In Phase 1, FSH stimulated E2 and Inh B responses were unaltered during recovery from ovarian suppression. In Phase 2, E2 and Inh B fold changes after FSH were significantly reduced at weeks 5 (p < 0.04) and 6 (p < 0.01), respectively.

CONCLUSIONS

In anovulatory women with PCOS, chronic, unopposed E2 secretion may contribute, at least in part, to enhanced ovarian responsiveness to FSH.

BACKGROUND

NCT02389088.

Abnormal porphyrin metabolism can be induced by several chemicals. To investigate the synergistic effect on porphyrinopathy of isonicotinic acid hydrazide (INH) with a low concentration of griseofulvin (GF), the two chemicals were given to mice simultaneously. INH was added to drinking water at a concentration of 0.05%. GF was mixed with feed at a concentration of 0.1%. The mice (dd-y strain) were divided into four groups. Those in group A were fed normally. Group B received only 0.1% GF, group C received only 0.05% INH, and group D received both 0.1% GF and 0.05% INH. The treatment was continued for 13 to 30 weeks. After treatment, the mice were anesthetized and sacrificed. The liver and whole blood were taken for analysis of porphyrins. The results revealed a slight elevation of erythrocytic porphyrins in the groups treated with 0.1% GF or 0.05% INH alone and remarkable abnormalities in the hepatic and erythrocytic porphyrin levels of the group simultaneously treated with both chemicals. These results show that INH itself may have a small potential for the induction of porphyric abnormalities, and that the administration of INH with a low concentration of GF induces marked porphyrinopathy in dd-y strain mice.

BACKGROUND

The use of a bioartificial liver with pig cells for the treatment of fulminant hepatic failure will require research on the plasma complement regulatory proteins of the pig, because the liver produces most of the complement components and plasma complement regulatory proteins. In our previous study, the pig C1 esterase inhibitor (C1-INH), which functions as an inhibitor of the complement reaction in the first step of the classical pathway in the fluid phase, was cloned and some relevant features of the molecule were characterized, especially its cross-species regulation, in comparison with human C1-INH. In a further analysis, the species specificity of C1-INH was examined, using pig endothelial cells (PEC) and several types of sera.

METHODS

The cDNA of pig C1-INH was used to produce the membrane type pC1-INH, pC1-INH-PI, and inserted into the cloning site of pCXN2 (chicken beta actin promoter). The pCX/pCl-INH-PI plasmid was then transfected into PEC to establish stable PEC with pCl-INH-PI. The expression of the pCl-INH-PI was evaluated by a FACS analysis, and complement-dependent cell lysis with human, dog, rabbit, and mouse sera was then assessed.

RESULTS

The transfectant with pig Cl-INH-PI showed a high level of expression on PEC. The PEC transfectants showed an inhibitory effect on complement-dependent PEC lysis. Pig Cl-INH did not show the same suppressive effect for each serum. However, considering the alternative pathway activation of each serum on the pig cell membrane, it can be concluded that pCl-INH has a relatively small species restriction.

CONCLUSIONS

Pig Cl-INH, having a similar structure to human Cl-INH, shows a strong complement regulatory function on other species sera.

As an attractive drug-target, retinoic acid receptor-related orphan receptor-gamma-t (RORγt) has <em>b</em>een employed widely to develop clinically relevant small molecular modulators as potent therapy for autoimmune disease and cancer, <em>b</em>ut its molecular mechanism of action (MOA) remains unclear. In the present study, we designed and discovered two novel RORγt ligands that are similar in structure, <em>b</em>ut different in efficacy. Using fluorescence resonance energy transfer (FRET) assay, compound (<em>b</em>)1</<em>b</em>) was identified as an agonist with an EC<su<em>b</em>)50</su<em>b</em>) of 3.7 μM (max. act.: 78%), while compound (<em>b</em>)2</<em>b</em>) as an inverse agonist with an IC<su<em>b</em>)50</su<em>b</em>) value of 2.0 μM (max. <em>inh</em>.: 61%). We performed molecular dynamics (MD) simulations, and elucidated the MOA of RORγt agonist and inverse agonist. Through the analyses of our MD results, we found that, after RORγt is <em>b</em>ound with the agonist (<em>b</em>)1</<em>b</em>), the side chain of Trp317 stays in the <i>gauche</i>- conformation, and thus helps to form the hydrogen <em>b</em>ond, His479-Trp502, and a large hydropho<em>b</em>ic network among H11, H11', and H12. All these interactions sta<em>b</em>ilize the H12, and helps the receptor recruit the coactivator. When the RORγt is <em>b</em>ound with the inverse agonist (<em>b</em>)2</<em>b</em>), the side chain of Trp317 is forced to adopt the <i>trans</i> conformation, and these presumed interactions are partially destroyed. Taken together, the critical role of residue Trp317 could <em>b</em>e viewed as the driving force for the activation of RORγt.

In this report, we describe the first case ever reported in the literature, of an inhibin-A (INHA) and inhibin-B (INHB) producing fibrothecoma. A post-menopausal woman was referred to our unit because of follicle stimulating hormone (FSH) level below the reference interval for postmenopausal women. By contrast luteinizing hormone, hCG, and estradiol levels were within normal range. This discrepancy suggested the secretion of FSH inhibitory factors. INHB and INHA levels were markedly elevated for age, 475 pg/mL and 100 pg/mL, respectively. Ultrasonography and MRI showed a pelvic mass of indeterminate nature. Abnormal inhibin secretion is generally observed in granulosa cell tumors. In this case this etiology was unlikely because of low estradiol and AMH levels. Surgical exploration revealed a 10 cm mass of the left ovary proven histologically to be an ovarian fibrothecoma (OFT). After tumor removal, INHB and INHA levels decreased rapidly. Only three cases of OFT with an important secretion of INHB have been reported to date. INHA secretion has never been associated with OFT. There is a need to develop coupled hormone and imaging strategies to diagnose the source of INH secretion in case of FSH/LH discrepancy.

The objective of this study was to determine whether neutralizing endogenous inhibin would affect ovulation rate and serum concentrations of FSH, LH, estradiol-17 beta, and progesterone in gilts. At wk 0, during their second postpubertal estrous cycle, gilts (195 +/- 2.4 d of age) were given a primary immunization against the 1-26 gly-tyr NH-terminal amino acid sequence of bovine inhibin-alpha conjugated to human alpha globulin (INH; n = 10) or against human alpha globulin alone (control; n = 10). The primary immunization mixed with Freund's complete adjuvant contained .915 mg of the inhibin peptide. Booster immunizations in Freund's incomplete adjuvant contained .3 and .183 mg of the inhibin peptide and were given at wk 8 and 12, respectively. Free, unconjugated inhibin was given to INH gilts at 16 wk. Blood samples for determination of hormones were collected every 4 h beginning on d 15 of the first estrous cycle beyond wk 16 (first cycle) and continuing until d 5 of the second estrous cycle following wk 16 (second cycle). Ovulation rate was estimated by laparoscopy during the second cycle. Antibody titers were estimated by determining the percentage of [125I]-INH bound by serum diluted 1:4,000. The antibody titers were 17 +/- 2, 22 +/- 3, and 9 +/- 1% at wk 9, 17, and 23 for INH gilts, respectively, and 0% at all times for control gilts. Duration of three consecutive estrous cycles terminating with the first experimental cycle did not differ between treatments (INH, 20.7 +/- .3 vs control, 20.4 +/- .3 d).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytogenetic effects of three combinations of anti-tubercular drugs were evaluated on human lymphocytes in vivo and were compared with controls of two types: (1) newly diagnosed tuberculosis patients before starting therapy and (2) individuals from the general population. The drugs used were: isoniazid (INH), thiacetazone (TAZ), para-aminosalicylic acid (PAS), and streptomycin (SM). These drugs were tested in the following combinations: (a) INH + TAZ + SM, (b) INH + PAS + SM, (c) INH + SM. The frequency of chromosome aberrations was significantly increased in patients treated with both the triple drug combinations, i.e., with INH + TAZ + SM and INH + PAS + SM, whereas patients treated with INH + SM did not exhibit an increase in the frequency of chromosome aberrations as compared to the controls. Although both the triple drug combinations were clastogenic, none of the three drug combinations tested induced an increase in the frequency of sister chromatid exchanges (SCEs). In other words, the mechanisms leading to SCEs and chromosome aberrations may be different. SM appeared to depress the mitotic index in patients treated with INH + SM and INH + PAS + SM, though it was found to possess a mild anti-clastogenic effect. INH + TAZ + SM, on the other hand, enhanced the mitotic index. This enhanced mitotic index was probably due to the presence of TAZ.

Me-β-cyclodextrin (Me-βCD) and HP-β-cyclodextrin (HP-βCD) inclusion complexes with isoniazid (INH) were prepared with the aim of modulating the physicochemical and biopharmaceutical properties of the guest molecule, a well-known antibuberculosis drug. The architectures of the complexes were initially proposed according to NMR data Job plot and ROESY followed by density functional theory (DFT) calculations of (1)H NMR spectra using the PBE1PBE functional and 6-31G(d,p) basis set, including the water solvent effect with the polarizable continuum model (PCM), for various inclusion modes, providing support for the experimental proposal. An analysis of the (1)H NMR chemical shift values for the isoniazid (H6',8' and H5',9') and cyclodextrins (H3,5) C(1)H hydrogens, which are known to be very adequately described by the DFT methodology, revealed them to be extremely useful, promptly confirming the inclusion complex formation. An included mode which describes Me-βCD partially enclosing the hydrazide group of the INH is predicted as the most favorable supramolecular structure that can be used to explain the physicochemical properties of the encapsulated drug. Antibacterial activity was also evaluated, and the results indicated the inclusion complexes are a potential strategy for tuberculosis treatment.