Influence of CYP3A4, CYP3A5 and MDR-1 polymorphisms on tacrolimus pharmacokinetics and early renal dysfunction in liver transplant recipients

Objectives

Tacrolimus is a widely used immunosuppressive drug in organ transplantation. The oral bioavailability of tacrolimus varies greatly between individuals and depends largely on the activity of both the cytochrome P450 3A (CYP3A) subfamily and P-glycoprotein (P-gp). The possible influence of single nucleotide polymorphisms (SNPs) of CYP3A subfamily and P-gp (MDR-1) in liver transplant recipients has recently been indicated as one of the most important variables affecting the pharmacokinetics of tacrolimus and the renal injury induced by tacrolimus.

Methods

A total of 216 liver transplant recipients were enrolled in this study. The recipients' mean follow-up time was 52 mo (range from 16 to 96 mo). All liver transplant recipients were all in a stable stage with normal serum creatinine (SCr). All liver transplant recipients treated with tacrolimus were genotyped for CYP3A5 (6986A>G), CYP3A4 intron 6 (CYP3A4*22), MDR-1 exon 26 (3435C>T) and exon 12 (1236 C>T) SNPs by HRM analysis (high-resolution melting curve analysis). Recipients were defined as the early renal injury by the elevation of different microproteins in the urine including microalbumin (MA), urine immunoglobulin G (IGU), urine transferrin (TRU) and α1-microglobulin (A1M).

Results

The daily dose of tacrolimus was higher for recipients with CYP3A5*1/*1 (AA) genotype than those with CYP3A5*3/*3 (GG) genotype [3.0 (2.0–4.0) versus 2.0 (1.5–2.5) mg/d, P < 0.05]. The concentration/dose ratio of recipients with CYP3A5*1 homozygotes was lowest compared to recipients with CYP3A5*3/*3 and CYP3A5*1/*3 genotypes. Furthermore, the recipients carrying CYP3A5*3 allele were associated with increased risk of early renal glomerular injury compared to the recipients carrying CYP3A5*1 allele (P = 0.01). MDR-1 polymorphisms were not related with tacrolimus pharmacokinetics and early renal injury.

Conclusion

CYP3A5 6986A>G genetic polymorphism affected daily dose requirements, concentration and nephrotoxicity of tacrolimus. Screening for this single nucleotide polymorphism before the transplantation might be helpful for the selection of adequate initial daily dose and to achieve the desired immunosuppression outcome.     

Description of Tropicibacter mediterraneus sp. nov. and Tropicibacter litoreus sp. nov.

Four strains (M15∅_3, M17^T, M49 and R37^T) were isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. Together with an older preserved isolate (strain 2OM6) from cultured oysters at Vinaroz, Castellón, Spain, the strains were thoroughly characterized in a polyphasic study and were placed phylogenetically within the Roseobacter clade in the family Rhodobacteraceae. Highest 16S rRNA sequence similarities of the five strains to the types of any established species corresponded to Tropicibacter multivorans (95.8–96.4%), Phaeobacter inhibens (95.9–96.3%) and Phaeobacter gallaeciensis (95.9–96.2%). On the other hand, whole genome (ANI) and protein fingerprinting (MALDI-TOF) data proved: (i) non clonality among the strains, and (ii) the existence of two genospecies, one consisting of strains M15∅_3, M17^T, M49 and 2OM6 and another one consisting of strain R37^T. Phenotypic traits determined allow differentiating both genospecies from each other and from closely related taxa. In view of all data collected we propose to accommodate these isolates in two species as members of the genus Tropicibacter, Tropicibacter mediterraneus sp. nov. (type strain M17^T = CECT 7615^T = KCTC 23058^T) and Tropicibacter litoreus sp. nov. (type strain R37^T = CECT 7639^T = KCTC 23353^T).     

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.     

Hyper-IgD and periodic fever syndrome: a new MVK mutation (p.R277G) associated with a severe phenotype

Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS; MIM# 260920) is a rare recessively-inherited autoinflammatory condition caused by mutations in the MVK gene, which encodes for mevalonate kinase, an essential enzyme in the isoprenoid pathway. HIDS is clinically characterized by recurrent episodes of fever and inflammation. Here we report on the case of a 2 year-old Portuguese boy with recurrent episodes of fever, malaise, massive cervical lymphadenopathy and hepatosplenomegaly since the age of 12 months. Rash, arthralgia, abdominal pain and diarrhea were also seen occasionally. During attacks a vigorous acute-phase response was detected, including elevated erythrocyte sedimentation rate, C-reactive protein, serum amyloid A and leukocytosis. Clinical and laboratory improvement was seen between attacks. Despite normal serum IgD level, HIDS was clinically suspected. Mutational MVK analysis revealed the homozygous genotype with the novel p.Arg277Gly (p.R277G) mutation, while the healthy non-consanguineous parents were heterozygous. Short nonsteroidal anti-inflammatory drugs and corticosteroid courses were given during attacks with poor benefits, whereas anakinra showed positive responses only at high doses. The p.R277G mutation here described is a novel missense MVK mutation, and it has been detected in this case with a severe HIDS phenotype. Further studies are needed to evaluate a co-relation genotype, enzyme activity and phenotype, and to define the best therapeutic strategies.     

Influence of rootstocks on growth,yield, fruit quality and leaf mineral element contents of pear cv. ‘Santa Maria’ in semi-arid conditions

Background

Rootstocks play an essential role to determining orchard performance of fruit trees. Pyrus communis and Cydonia oblonga are widely used rootstocks for European pear cultivars. The lack of rootstocks adapted to different soil conditions and different grafted cultivars is widely acknowledged in pear culture. Cydonia rootstocks (clonal) and Pyrus rootstocks (seedling or clonal) have their advantages and disadvantages. In each case, site-specific environmental characteristics, specific cultivar response and production objectives must be considered before choosing the best rootstock. In this study, the influence of three Quince (BA 29, Quince A = MA, Quince C = MC) and a local European pear seedling rootstocks on the scion yield, some fruit quality characteristics and leaf macro (N, P, K, Ca and Mg) and micro element (Fe, Zn, Cu, Mn and B) content of ‘Santa Maria’ pear (Pyrus communis L.) were investigated.

Results

Trees on seedling rootstock had the highest annual yield, highest cumulative yield (kg tree⁻¹), largest trunk cross-sectional area (TCSA), lowest yield efficiency and lowest cumulative yield (ton ha⁻¹) in the 10^th year after planting. The rootstocks had no significant effect on average fruit weight and fruit volume. Significantly higher fruit firmness was obtained on BA 29 and Quince A. The effect of rootstocks on the mineral element accumulation (N, K, Ca, Mg, Fe, Zn, Cu, Mn and B) was significant. Leaf analysis showed that rootstocks used had different mineral uptake efficiencies throughout the early season.

Conclusion

The results showed that the rootstocks strongly affected fruit yield, fruit quality and leaf mineral element uptake of ‘Santa Maria’ pear cultivar. Pear seedling and BA 29 rootstock found to be more prominent in terms of several characteristics for ‘Santa Maria’ pear cultivar that is grown in highly calcareous soil in semi-arid climate conditions. We determined the highest N, P (although insignificant), K, Ca, Mg, Fe and Cu mineral element concentrations on the pear seedling and BA 29 rootstocks. According to the results, we recommend the seedling rootstock for normal density plantings (400 trees ha⁻¹) and BA 29 rootstock for high-density plantings (800 trees ha⁻¹) for ‘Santa Maria’ pear cultivar in semi-arid conditions.     

Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke

Rapamycin has been demonstrated to exhibit neuroprotective functions via the activation of autophagy in a cerebral ischemia model. However, the involvement of mitophagy in this process and its contribution to the protection of mitochondrial function remains unknown. The present study explored the characteristics of mitophagy after cerebral ischemia and the effect of rapamycin on mitochondrial function. Male Sprague–Dawley rats underwent transient middle cerebral artery occlusion (tMCAO). Neurological deficits scores; infarct volumes; mitophagy morphology; and the levels of malondialdehyde (MDA), adenosine triphosphate (ATP) and mitochondrial membrane potentials (Δψm) were examined. The expression of LC3, Beclin-1 and p62 in the mitochondrial fraction combined with transmission electronic microscopy were used to explore mitophagic activity after ischemia. We also blocked autophagosome formation using 3-methyladenine (3-MA) to check the linkage between the mitochondrial protective effect of rapamycin and enhanced mitophagy. We observed that rapamycin significantly enhanced mitophagy, as evidenced by the increase in LC3-II and Beclin-1 expression in the mitochondria and p62 translocation to the mitochondria. Rapamycin reduced infarct volume, improved neurological outcomes and inhibited mitochondrial dysfunction compared with the control animals (p < 0.05). However, these protective effects were reversed by 3-methyladenine treatment after rapamycin. The present study indicates that rapamycin treatment attenuates mitochondrial dysfunction following cerebral ischemia, which is linked to enhanced mitophagy.     

Growth and the cell cycle of the yeast Saccharomyces cerevisiae. I. Slowing S phase or nuclear division decreases the G1 cell cycle period

To determine changes in distribution or mobility of cell-surface glycoconjugates during myogenesis the binding of fluorescein-conjugated plant lectins to myoblasts and myotubes of the L6 rat skeletal muscle cell line has been studied. Binding has been carried out at 4 degrees C on either live or glutaraldehyde-fixed cells. Fluorescein conjugates of soybean agglutinin (Fl-SBA), wheat germ agglutinin (Fl-WGA), concanavalin A (Fl-conA) and Lens culinaris agglutinin (Fl-LCA) produced predominantly uniform fluorescence on both live and fixed myoblasts. On fixed myotubes, Fl-LCA, Fl-conA and Fl-SBA again produced predominantly uniform fluorescence, whereas Fl-WGA showed a pattern of diffuse, irregular spots in addition to uniform fluorescence. Fl-conA, Fl-LCA and Fl-WGA binding to live myotubes resulted in patterns quite similar to those on fixed myotubes; the only differences being the presence of weak patterns of diffuse spots with Fl-LCA and Fl-conA and an enhanced pattern of diffuse spots with Fl-WGA. Fl-SBA, however, showed a unique pattern on live myotubes which consisted of discrete, round spots and minimal uniform fluorescence. With shorter labeling times, Fl-SBA produced relatively more prominent uniform fluorescence on live myotubes. It appears, therefore, that the native distribution of SBA, conA and LCA-binding sites is similar and predominantly random on L6 myoblasts and myotubes, whereas some WGA-binding sites may be aggregated on myotubes. The results also suggest that SBA-binding sites readily cluster at 4 degrees C on myotubes but not myoblasts, whereas the other lectin sites undergo little or no redistribution on either cell type. Thus the mobility of SBA-binding sites may increase with differentiation.     

The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism

Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.     

Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag–PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag–PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]; however, previous studies suggested that HTLV-1 Gag–PM interactions and therefore virus assembly are less dependent on PI(4,5)P₂. We have recently shown that PI(4,5)P₂ binds directly to HTLV-1 unmyristoylated MA [myr(–)MA] and that myr(–)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P₂. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA–membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P₂–binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P₂. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.     

Adenosine triphosphate (ATP) hydrolysis promoted by cobalt(III).Participation of polynuclear metal complexes

Complex formation between ATP (adenosine 5′-triphosphate) and tn₂CO^III(aq) (tn = trimethylenediamine) and resulting hydrolysis of the ATP to ADP (adenosine 5′-diphosphate), AMP (adenosine 5′-monophosphate), PP_i (pyrophosphate), and P_i (orthophosphate) have been examined by means of ³¹P nmr. With ATP ～0.1 M and tn₂Co^III(aq) up to 0.3 M, complex formation was promoted by equilibrating solutions for a period at pH 4, after which hydrolysis was allowed to proceed at each of several pHs in the range 5 to 9 prior to quenching by addition of strong base. With ATP 0.01 M and tn₂Co^III(aq) up to 0.08 M, the above procedure was followed in some cases; in other experiments the pH of each ATP/tn₂Co^III(aq) solution was adjusted immediately to a value in the range 5 to 9 with the remainder of the procedure as before. In most cases the hydrolysis was at 25°C, but temperature dependence was also examined. The integrals for the β-phosphorus resonance have been used to analyze for ATP in the quenched solutions; independent measurements of ATP by an enzyme/spectrophotometric method (Bergmeyer) gave similar results. Cobalt to ATP molar ratios up to 1 produce tn₂Co^IIIATP as the predominant ATP complex; this 1:1 complex shows no detectable acceleration in hydrolysis compared to free ATP. Cobalt to ATP molar ratios of ?1 lead to complexes of type (tn₂Co^III)₂ATP and (tn₂Co^III)₃ATP, which exhibit greatly enhanced reactivity towards ATP hydrolysis. At a 2:1 molar ratio (0.1 or 0.01 M ATP), the enhancement is rate is ～10⁵ at pH 7 where the rate is a maximum (comparison for 25°C); at higher molar ratios the rate enhancements are even greater. The results support the view that effective metal ion catalysis of ATP hydrolysis requires formation of reactive species involving more than one metal ion per ATP.