Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols

Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.     

Significant increase in plasma 4β-hydroxycholesterol concentration in patients after kidney transplantation

Several previous studies have shown that renal failure decreases not only renal elimination but also metabolic clearance of drugs, particularly those metabolized by CYP3A. However, whether recovery of renal function results in recovery of hepatic CYP3A activity remains unknown. In this study, we evaluated the effect of renal function on CYP3A activity after kidney transplantation in patients with end-stage renal disease (ESRD) by measuring the change in CYP3A activity using plasma concentration of 4β-hydroxycholesterol as a biomarker. The study enrolled 13 patients with ESRD who underwent the first kidney allograft transplantation. Morning blood samples were collected before and 3, 7, 10, 14, 21, 30, 60, 90, 120, 150 and 180 days after kidney transplantation. Plasma concentration of 4β-hydroxycholesterol was measured using GC-MS. Compared with before kidney transplantation, creatinine clearance increased significantly from day 3 after kidney transplantation and stabilized thereafter. Plasma concentration of 4β-hydroxycholesterol was elevated significantly on days 90 and 180 after kidney transplantation. In conclusion, this study suggests the recovery of CYP3A activity with improvement in renal function after kidney transplantation in patients with ESRD.     

Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites

Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.     

The Arabidopsis dwarf1 mutant is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis

Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22alpha-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism.     

Increased level of apolipoprotein B mRNA in the liver of ventromedial hypothalamus lesioned obese rats

The mRNA level of apolipoprotein B (apoB), which is a principal protein component of nascent very low density lipoprotein (VLDL), was determined in parallel with the measurement of acetyl-coenzyme A (Ac-CoA) carboxylase activity in the liver of ventromedial hypothalamus (VMH) lesioned obese rats. Eight weeks after the electrolysis of the bilateral VMH, the level of apoB mRNA in the VMH-lesioned rats was about 1.5-fold higher than that in the sham-operated rats, indicating increased apoB synthesis in the liver of the VMH-lesioned obese rats. The activity of Ac-CoA carboxylase, which is a rate-limiting enzyme for the fatty acid biosynthesis, was about 1.8-fold higher in the VMH-lesioned rats. These observations indicated that VLDL synthesis is increased in the liver of VMH-lesioned obese rats.     

Breast Milk Is Not a Significant Source for Early Epstein-Barr Virus or Human Herpesvirus 6 Infection in Infants: A Seroepidemiologic Study in 2 Endemic Areas of Human T-Cell Lymphotropic Virus Type I in Japan

In order to evaluate the possibility of Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) transmission via breast milk, a total of 331 serum specimens collected from bottle-fed and breast-fed children and their mothers, in 2 endemic areas of human T-cell lymphotropic virus type I (HTLV-I) in Japan, were assayed for antibodies to EBV and HHV-6. The seroprevalences of EBV and HHV-6 were over 95% both in the mothers of bottle-fed children and in those of breast-fed children. The seroprevalence of EBV at 12–23 months of age was 54.5% (36/66) and 55.8% (24/43) in breast-fed children and bottle-fed children, respectively. The seroprevalence of HHV-6 at 12–23 months of age was 90.9% (60/66) and 93.0% (40/43) in breast-fed children and bottle-fed children, respectively. No difference was observed between the seroprevalences of EBV and HHV-6 in breast-fed and bottle-fed children at 12–23 months of age. Our seroepidemiologic data indicate that breast milk is not a significant source of early EBV or HHV-6 infection in infancy.     

The conversion of L-lysine to saccharopine and alpha-aminoadipate in mouse

Saccharopine [?-N-(l-glutaryl-2)-l-lysine] has been found to occur in normal, untreated mouse liver. The pool of saccharopine as well as that of α-aminoadipate become labeled shortly after the administration of l-lysine-U-¹⁴C into intact mouse. In vitro experiments using the mouse liver homogenate have shown that l-lysine is converted to saccharopine in the presence of α-ketoglutarate and NADPH, and saccharopine to α-aminoadipate in the presence of NAD⁺. The oxidation of α-aminoadipic-δ-semialdehyde (Δ¹-piperideine-6-carboxylate), the proposed reaction product of saccharopine cleavage, to α-aminoadipate is effected by either NAD⁺ or NADP⁺.     

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes ^1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement ^3,4. The resulting hematomas and lacerations cause a vascular response ^3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury ^6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure ⁹. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals ^10-12, which ultimately result in long-term neurological disabilities ^13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration ¹. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue ^1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure ^16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull ¹⁸. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury ¹⁹. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation ²⁰.We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.