Enzyme Vitamin Research Articles

Vitamin D status, vitamin D receptor, CYP2R1, and CYP24A1 profiles in children.

Vitamin D plays a major role in the musculoskeletal and immune system. Understanding the comprehensive mechanism of vitamin D receptors and the enzyme of vitamin D induction (CYP2R1) and inhibition (CYP24A1) in its metabolism is interesting. This study aims to understand vitamin D metabolism in Indonesian pediatrics, specifically in Jakarta, which has abundant sun exposure. A cross-sectional study with comparative, correlative, and multivariate analysis on vitamin D, vitamin D receptor, CYP2R1, and CYP24A1 levels was conducted on 46 children with no known morbidity. Subjects were mostly male (52.2%), age group of 2-6 years (34.8%), and had sufficient vitamin D status (43.5%, median 27.55 ng/mL). Age was found to have a negative correlation with vitamin D levels (p < 0.001; r = -0.625) and CYP2R1 (p = 0.035; r = -0.311). Significant positive associations were found between CYP24A1 and CYP2R1 (p = 0.046; r = 0.296). Participants aged 0-2 are more likely to have a higher level of vitamin D status compared to those aged >2 years (OR 42.092, 95% CI [4.532-390.914], p = 0.001). VDR levels were significantly lower in insufficient vitamin D levels than in the sufficient group (p = 0.018). VDR and vitamin D status had a positive relation (OR 7.023, 95% CI [1.864-26.453], p = 0.004). Vitamin D levels decrease with the increase in age. Vitamin D receptor level has an inline-level progression with vitamin D level. CYP2R1 and CYP24A1 suggest a directly proportional relationship. Vitamin D screening and supplementation in children older than 2 years old are suggested.

Genetics aspect of vitamin C (Ascorbic Acid) biosynthesis and signaling pathways in fruits and vegetables crops.

Vitamin C, also known as ascorbic acid, is an essential nutrient that plays a critical role in many physiological processes in plants and animals. In humans, vitamin C is an antioxidant, reducing agent, and cofactor in diverse chemical processes. The established role of vitamin C as an antioxidant in plants is well recognized. It neutralizes reactive oxygen species (ROS) that can cause damage to cells. Also, it plays an important role in recycling other antioxidants, such as vitamin E, which helps maintain the overall balance of the plant's antioxidant system. However, unlike plants, humans cannot synthesize ascorbic acid or vitamin C in their bodies due to the absence of an enzyme called gulonolactone oxidase. This is why humans need to obtain vitamin C through their diet. Different fruits and vegetables contain varying levels of vitamin C. The biosynthesis of vitamin C in plants occurs primarily in the chloroplasts and the endoplasmic reticulum (ER). The biosynthesis of vitamin C is a complex process regulated by various factors such as light, temperature, and plant hormones. Recent research has identified several key genes that regulate vitamin C biosynthesis, including the GLDH and GLDH genes. The expression of these genes is known to be regulated by various factors such as light, temperature, and plant hormones. Recent studies highlight vitamin C's crucial role in regulating plant stress response pathways, encompassing drought, high salinity, and oxidative stress. The key enzymes in vitamin C biosynthesis are L-galactose dehydrogenase (GLDH) and L-galactono-1, 4-lactone dehydrogenase (GLDH). Genetic studies reveal key genes like GLDH and GLDH in Vitamin C biosynthesis, offering potential for crop improvement. Genetic variations influence nutritional content through their impact on vitamin C levels. Investigating the roles of genes in stress responses provides insights for developing resilient techniques in crop growth. Some fruits and vegetables, such as oranges, lemons, and grapefruits, along with strawberries and kiwi, are rich in vitamin C. Guava. Papaya provides a boost of vitamin C and dietary fiber. At the same time, red and yellow bell peppers, broccoli, pineapple, mangoes, and kale are additional sources of this essential nutrient, promoting overall health. In this review, we will discuss a brief history of Vitamin C and its signaling and biosynthesis pathway and summarize the regulation of its content in various fruits and vegetables.

Vitamin D in the Context of Evolution.

For at least 1.2 billion years, eukaryotes have been able to synthesize sterols and, therefore, can produce vitamin D when exposed to UV-B. Vitamin D endocrinology was established some 550 million years ago in animals, when the high-affinity nuclear receptor VDR (vitamin D receptor), transport proteins and enzymes for vitamin D metabolism evolved. This enabled vitamin D to regulate, via its target genes, physiological process, the first of which were detoxification and energy metabolism. In this way, vitamin D was enabled to modulate the energy-consuming processes of the innate immune system in its fight against microbes. In the evolving adaptive immune system, vitamin D started to act as a negative regulator of growth, which prevents overboarding reactions of T cells in the context of autoimmune diseases. When, some 400 million years ago, species left the ocean and were exposed to gravitation, vitamin D endocrinology took over the additional role as a major regulator of calcium homeostasis, being important for a stable skeleton. Homo sapiens evolved approximately 300,000 years ago in East Africa and had adapted vitamin D endocrinology to the intensive exposure of the equatorial sun. However, when some 75,000 years ago, when anatomically modern humans started to populate all continents, they also reached regions with seasonally low or no UV-B, i.e., and under these conditions vitamin D became a vitamin.

735 Antifibrogenic activities of novel vitamin D3 derivatives are altered in human fibroblasts expressing low vitamin D receptor or CYP27B1 enzyme

The vitamin D receptor (VDR) serves as a receptor for active vitamin D hydroxyderivatives. The formation of vitamin D hydroxyderivatives requires the presence of specific cytochrome P450 (CYP) enzymes for vitamin D metabolism. These hydroxyderivatives can be further hydroxylated by CYP27B1 at the C1α-position which increases their affinity for the VDR. However, the role of CYP27B1 in antiproliferative and antifibrotic activities of CYP11A1-derived D3-metabolites is unknown. We tested the action of chemically synthesized vitamin D derivatives: 20,23(OH2D3, 1,20(OH)2D3, or 1,20,23(OH)3D3 and compared to the action of 1,25(OH)2D3 or 20(OH)D3 (non-calcemic compound).

FGF23 and Vitamin D Metabolism.

ABSTRACTApart from its phosphaturic action, the bone‐derived hormone fibroblast growth factor‐23 (FGF23) is also an essential regulator of vitamin D metabolism. The main target organ of FGF23 is the kidney, where FGF23 suppresses transcription of the key enzyme in vitamin D hormone (1,25(OH)2D) activation, 1α‐hydroxylase, and activates transcription of the key enzyme responsible for vitamin D degradation, 24‐hydroxylase, in proximal renal tubules. The circulating concentration of 1,25(OH)2D is a positive regulator of FGF23 secretion in bone, forming a feedback loop between kidney and bone. The importance of FGF23 as regulator of vitamin D metabolism is underscored by the fact that in the absence of FGF23 signaling, the tight control of renal 1α‐hydroxylase fails, resulting in overproduction of 1,25(OH)2D in mice and men. During recent years, big strides have been made toward a more complete understanding of the mechanisms underlying the FGF23‐mediated regulation of vitamin D metabolism, especially at the genomic level. However, there are still major gaps in our knowledge that need to be filled by future research. Importantly, the intracellular signaling cascades downstream of FGF receptors regulating transcription of 1α‐hydroxylase and 24‐hydroxylase in proximal renal tubules still remain unresolved. The purpose of this review is to highlight our current understanding of the molecular mechanisms underlying the regulation of vitamin D metabolism by FGF23, and to discuss the role of these mechanisms in physiology and pathophysiology. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Overexpression of 4-hydroxyphenylpyruvate dioxygenase (IbHPPD) increases abiotic stress tolerance in transgenic sweetpotato plants

Tocopherols are lipid-soluble compounds regarded as vitamin E compounds and they function as antioxidants in scavenging lipid peroxyl radicals and quenching reactive oxygen species (ROS). In our previous studies, we isolated five tocopherol biosynthesis genes from sweetpotato (Ipomoea batatas [L.] Lam) plants including 4-hydroxyphenylpyruvate dioxygenase (IbHPPD). HPPD is the first regulatory enzyme in vitamin E biosynthesis and serves to catalyze in the first steps α-tocopherol and plastoquinone biosynthesis by converting 4-hydroxyphenylpyruvate (HPP) to homogentisic acid (HGA). In this study, we generated transgenic sweetpotato plants overexpressing IbHPPD under the control of cauliflower mosaic virus (CaMV) 35S promoter (referred to as HP plants) via Agrobacterium-mediated transformation to understand the function of IbHPPD in sweetpotato. Three transgenic lines (HP3, HP14 and HP15) with high transcript levels of IbHPPD were selected for further characterization. Compared with non-transgenic (NT) plants, HP plants exhibited enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses. In addition, HP plants showed increased tolerance to the herbicide sulcotrione, which is involved in the inhibition of the HPPD. Interestingly, after stress treatments, HP plants also showed higher abscisic acid (ABA) contents than NT plants. Under dehydrated condition, HP plants displayed an elevated α-tocopherol content to 19–27% in leaves compared with NT plants. These results indicate that increased abiotic stress tolerance in HP plants is related to inducing enhancement of α-tocopherol and ABA contents.

Targeting multiple enzymes in vitamin K metabolism for anticoagulation

Vitamin K antagonists (VKAs), such as warfarin, are oral anticoagulants used to treat and prevent pulmonary embolism, deep vein thrombosis, and complications associated with atrial fibrillation and heart valve replacement. VKAs are effective against blood clotting where blood flow is relatively slow, such as in veins, in dysfunctional atria, and behind heart valves. These drugs, however, have a narrow therapeutic window and require frequent anticoagulation monitoring and dose adjustments. To overcome these problems, direct oral anticoagulants (DOACs) targeting factor Xa and thrombin have been developed, with the advantages of less monitoring, fixed dosing, predictable responses, and fewer food interactions.

Impact of vitamin D3 Nanoemulsion on spermatogenesis and antioxidant enzymes in Vitamin D deficient induced albino male rats.

Vitamin D deficiency is common with several effects that include nonskeletal impacts. Many studies showed associations between Vitamin D deficiency and oxidative statues with disturbed testicles in male rats (Rattus norvegicus) which might be improved by vitamin D. The aim of this work is to find the effects of nanoemulsion pea protein isolates and novel vitamin D carrier, developed by sonication and pH shifting of pea protein isolate nanoemulsion on the spermatogenesis in induced vitamin D deficient male rats.&nbsp; Thirty male albino rats distributed into 5 groups, sufficient control was fed with normal diet,&nbsp; deficient control&nbsp; was treated with 100 mg/kg Rimfapicin and 50 mg/kg Isoniazed for three weeks, pea protein isolate&nbsp; group :vitamin D deficient rats treated daily with 60 mg/ml/kg nanoemulsion pea protean isolate, Vitamin D deficient rats were treated with vitamin D (54 mcg/ml/ kg), and pea protean isolate + vitamin D group: Vitamin D deficient rats were treated by nanoemulsion pea protein isolate (60 mg/dl/kg) plus Vitamin D (54 mcg/ml/ kg) for four weeks. The serum used for Vitamin D and total testosterone assay, after estimation of sperm count and morphology. Homogenized testes used for determination of Catalase and Glutathione peroxidase. The results revealed that vitamin D and supplementations each one alone have a positive significant effects on spermatogenesis. The nanoemulsion pea protein isolate +vitamin D resulted in a significant increase in the levels of vitamin D of total testosterone more than the supplementation of vitamin D and nanoemulsion pea protein isolate alone. Also sperm count, normal sperm morphology, as well as catalase and Glutathione peroxidase improved significantly in this group. In conclusion we demonstrated vitamin D nanoemulsion as more efficient formulation with more prominent effects on spermatogenesis in induced vitamin D deficient rats.

Effect on malondialdehyde, superoxide dismutase and reduced glutathione and its metabolizing enzymes in vitamin D deficient newly diagnosed Type- 2 diabetic patients.

Background: India, with 32 million diabetic individuals, currently has the highest incidence of diabetes worldwide. Recently, World Health Organization (WHO) reported that these numbers are predicted to increase to 80 million by the year 2030. Vitamin D regulates glucose metabolism and oxidative stress is a well-established factor of this multi-factorial disease. Aim: Present, study was designed to evaluate certain oxidative stress markers like malondialdehyde, superoxide dismutase, reduced glutathione, glutathione reductase, Glutathione Peroxidase (GPx) along with total antioxidant activity in vitamin D deficient newly diagnosed Type 2 diabetics. Methods: 150 vitamin D deficient newly diagnosed type 2 diabetics’ and equal number of healthy subjects of both genders were recruited. Fasting blood was collected for evaluation of glucose, 25(OH) D and oxidative stress markers. Results: Significant (P<0.01) increase in malondialdehyde by 51.03% while a significant decrease in oxidative stress markers like SOD, GSH, GR, GPx and total antioxidant activity by 27.91 (P<0.05), 26.14% (P<0.05), 32.04% (P<0.01), 25.41% (P<0.05) and 35.85% (P<0.01) respectively was recorded in vitamin D deficient newly diagnosed type 2 diabetics with respect to healthy controls. Conclusions: A fore mentioned observations suggested that vitamin-D deficient newly diagnosed Type-2 diabetics of North-West Indian’s are associated with oxidative stress, a hallmark of various diseases like Diabetes Mellitus, cardiovascular diseases, osteoporosis etc. Therapeutic interventions in combinations of lifestyle and dietary modification might be beneficial to prevent further risk of development of Diabetes Mellitus and cardiovascular diseases like atherosclerosis in North-West Indians.

Genetic variants of VDR and CYP2R1 affect BMI independently of serum vitamin D concentrations

BackgroundVitamin D metabolism and obesity have been linked by several studies, however the reason for this association is unclear. Our objective was to investigate potential correlations between genetic variants in key enzymes of vitamin D metabolism and the body mass index on a representative and random sample of Hungarian adults.MethodsAltogether 462 severely vitamin D deficient individuals were studied at the end of winter in order to decrease environmental and maximize any relevant genetic effect. Furthermore, participants with lifestyle factors known to affect vitamin D homeostasis were also excluded. We selected 23 target SNPs in five genes that encode key proteins of vitamin D metabolism (NADSYN1, GC, CYP24A1, CYP2R1, VDR).ResultsVariants in 2 genetic polymorphisms; rs2853564 (VDR) and rs11023374 (CYP2R1) showed a significant association with participants‘ BMI. These associations survived further adjustment for total-, free-, or bioactive-25(OH) vitamin D levels, although the variance explained by these 2 SNPS in BMI heterogeneity was only 3.2%.ConclusionOur results show two novel examples of the relationship between genetics of vitamin D and BMI, highlighting the potential role of vitamin D hormone in the physiology of obesity.

Genome editing of upstream open reading frames enables translational control in plants.

Translational regulation by upstream open reading frames (uORFs) is becoming established as a general mechanism for controlling the amount of protein that is synthesized from downstream primary ORFs (pORFs). We found that genome editing of endogenous uORFs in plants enabled the modulation of translation of mRNAs from four pORFs that are involved in either development or antioxidant biosynthesis. A single-guide RNA that targeted the region harboring a uORF initiation codon can produce multiple mutations. Following uORF editing, we observed varying amounts of mRNA translation in four pORFs. Notably, editing the uORF of LsGGP2, which encodes a key enzyme in vitamin C biosynthesis in lettuce, not only increased oxidation stress tolerance, but also increased ascorbate content by ∼150%. These data indicate that editing plant uORFs provides a generalizable, efficient method for manipulating translation of mRNA that could be applied to dissect biological mechanisms and improve crops.

Abstract 402: Vitamin C Deficiency Impairs Cardiac Function and Post-infarction Survival in the Mouse

Introduction: L-gulonolactone oxidase (Gulo) is the rate limiting enzyme for Vitamin C (VitC) biosynthesis. Humans rely on dietary VitC for collagen synthesis, extracellular matrix formation, and tissue regeneration. VitC deficiency is an unrecognized condition and its role in cardiac homeostasis and post-acute myocardial infarction (AMI) remodeling is unknown. Hypothesis: Low levels of VitC impair cardiac function and tissue repair following AMI. Methods: Adult male Gulo -/- knockout mice (C57BL6 background, N=8) and control C57BL (N=8), which are able to synthesize VitC were used. VitC deficiency was maintained supplying low levels of VitC (30mg/l) to Gulo -/- mice in drinking water. Mice underwent M-mode and Doppler echocardiography to measure left ventricular (LV) diameters and wall thicknesses, fractional shortening (FS), E and A waves, E/A ratio, isovolumetric relaxation time (IRT) and myocardial performance index (MPI). Experimental AMI was induced by coronary artery ligation for 7 days. An additional group of Gulo -/- were mice supplemented with physiological levels of VitC (330 mg/l) and underwent AMI. Results: VitC deficient Gulo -/- mice exhibited significantly reduced LV wall thicknesses, reduced FS, and impaired diastolic function, measured as significantly reduced E/A ratio and longer IRT (Panel A, B &amp; C). Following AMI, 100% (8/8) of deficient Gulo -/- mice died within 5 days. Supplementation with physiological levels of VitC significantly improved survival after AMI (Panel D). Conclusion: VitC deficiency impairs systolic and diastolic function. Moreover, VitC is critical for the post-AMI survival.

Mutations in SDR9C7 gene encoding an enzyme for vitamin A metabolism underlie autosomal recessive congenital ichthyosis

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of hereditary skin disorder characterized by an aberrant cornification of the epidermis. ARCI is classified into a total of 11 subtypes (ARCI1-ARCI11) based on their causative genes or loci. Of these, the causative gene for only ARCI7 has not been identified, while it was previously mapped on chromosome 12p11.2-q13.1. In this study, we performed genetic analyses for three Lebanese families with ARCI, and successfully determined the linkage interval to 9.47 Mb region on chromosome 12q13.13-q14.1, which was unexpectedly outside of the ARCI7 locus. Whole-exome sequencing and the subsequent Sanger sequencing led to the identification of missense mutations in short chain dehydrogenase/reductase family 9C, member 7 (SDR9C7) gene on chromosome 12q13.3, i.e. two families shared an identical homozygous mutation c.599T > C (p.Ile200Thr) and one family had another homozygous mutation c.214C > T (p.Arg72Trp). In cultured cells, expression of both the mutant SDR9C7 proteins was markedly reduced as compared to wild-type protein, suggesting that the mutations severely affected a stability of the protein. In normal human skin, the SDR9C7 was abundantly expressed in granular and cornified layers of the epidermis. By contrast, in a patient’s skin, its expression in the cornified layer was significantly decreased. It has previously been reported that SDR9C7 is an enzyme to convert retinal into retinol. Therefore, our study not only adds a new gene responsible for ARCI, but also further suggests a potential role of vitamin A metabolism in terminal differentiation of the epidermis in humans.

Altered lipid metabolism in the aging kidney identified by three layered omic analysis.

Aging-associated diseases and their comorbidities affect the life of a constantly growing proportion of the population in developed countries. At the center of these comorbidities are changes of kidney structure and function as age-related chronic kidney disease predisposes to the development of cardiovascular diseases such as stroke, myocardial infarction or heart failure. To detect molecular mechanisms involved in kidney aging, we analyzed gene expression profiles of kidneys from adult and aged wild-type mice by transcriptomic, proteomic and targeted lipidomic methodologies. Interestingly, transcriptome and proteome analyses revealed differential expression of genes primarily involved in lipid metabolism and immune response. Additional lipidomic analyses uncovered significant age-related differences in the total amount of phosphatidylethanolamines, phosphatidylcholines and sphingomyelins as well as in subspecies of phosphatidylserines and ceramides with age. By integration of these datasets we identified Aldh1a1, a key enzyme in vitamin A metabolism specifically expressed in the medullary ascending limb, as one of the most prominent upregulated proteins in old kidneys. Moreover, ceramidase Asah1 was highly expressed in aged kidneys, consistent with a decrease in ceramide C16. In summary, our data suggest that changes in lipid metabolism are involved in the process of kidney aging and in the development of chronic kidney disease.

Abstract 5056: PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells

Abstract Dysregulation of vitamin D signaling and metabolism enzymes is a frequent change in many cancers and may confound approaches to vitamin -D based therapies. The mechanisms by which aberrant vitamin D signaling and metabolism lead to resistance to vitamin D action are poorly understood. The kidney plays a critical role in vitamin D synthesis and metabolism; in renal cell carcinoma (RCC) cells vitamin D metabolism is dysregulated. Recently, we discovered that an oncogenic protein kinase PIM-1 is involved in vitamin D-induced expression of 24-hydroxylase, a key catalytic enzyme of vitamin D encoded by CYP24A1 gene, in bladder and prostate cancer. To study the impact of PIM1 on vitamin D signaling and vitamin D-mediated anti-tumor activity in RCC, we analyzed 4 human renal cell carcinoma cell lines (Caki-1, ACHN, A498 and 786-O) and 25 samples of human RCC tumor tissue and 13 benign kidney tissues (including 10 matched tumor and benign tissue samples) by qRT-PCR, immunohistochemistry (IHC), Western blot analysis, MTT and clonogenic assays. VDR mRNA and protein were detected in all RCC cell lines. IHC showed that VDR was strongly expressed in the normal kidney tissues, but decreased in RCC tissues. As expected, VDR expression was significantly increased by treatment with 1,25-dihydroxyvitamin D3 (1,25D3) in RCC cell lines. MTT and clonogenic assays showed that 1,25D3 inhibited RCC cell growth (p&amp;lt;0.05). We also noted a significant increase of CYP24A1 expression induced by vitamin D3 (p&amp;lt;0.0001); and effect which would be expected to promote the catabolism of 1,25D3. qRT-PCR and Western blot analysis demonstrated that PIM-1 is expressed in RCC cells. PIM-1 inhibition by siRNA or PIM-1 inhibitor JP_11646 significantly reduced 1,25D3-induced CYP24A1 expression (p&amp;lt;0.05). Therefore, we hypothesized that targeting PIM-1 kinase activity with a PIM-1 inhibitor may be an effective strategy to reduce 1,25D3 metabolism, thereby enhancing vitamin D-mediated anti-tumor activity. Inhibition of PIM1 kinase activity by JP_11646 reduced CYP24A1 expression at the transcriptional level in RCC cells. We further evaluated the efficacy of the PIM1 inhibitor alone or in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP_11646 reduced cell growth in RCC cell lines in a dose-dependent manner as measured by MTT and clonogenic assays. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in RCC cells (p&amp;lt;0.05). This study indicates that PIM-1 is involved in the regulation of vitamin D signaling in RCC. Inhibition of PIM kinase activity combined with vitamin D may be an attractive new therapeutic strategy for RCC. Citation Format: Wei Luo, Yingyu Ma, Carmen Baldino, Justin Caserta, Swathi Ramakrishnan, Pili Roberto, Candace Johnson, Donald Trump. PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2015-5056

Cloning of a functional 25‐hydroxyvitamin D‐1α‐hydroxylase in zebrafish (Danio rerio)

Activation of precursor 25-hydroxyvitamin D3 (25D) to hormonal 1,25-dihydroxyvitamin D3 (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). To establish new models for assessing the physiological importance of the 1α-hydroxylase-25D-axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α-hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5-150 nM) or active 1,25D (0.1-10 nM) induced dose responsive expression (15-95-fold) of the vitamin D-target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full-length zebrafish cyp27b1 cDNA was then generated using RACE and RT-PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC-8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25-fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism.

Involvement of VKORC1 in the inhibition of calcium oxalate crystal formation in HK-2 cells

The vitamin K epoxide reductase complex subunit 1 (VKORC1), the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate (CaOx) crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORC1shRNA-2 as a PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC1 transfection group than in the pFLAG-CMV-7.1 control group (P<0.01). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate (COM) crystal medium for 48 h was 14±4 per field (100×) in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field (100×) in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group (P<0.05). The expression levels of VKORC1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field (100×) in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group and 24±6 per field (100×) in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). These findings suggested that the VKORC1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx.

Effect of vitamin E and zinc supplementation on liver enzymatic profile of pre- and post-partum Sahiwal cows

The study was conducted to elucidate the changes in circulatory plasma levels of hepatic enzymes in vitamin E and zinc supplemented periparturient Sahiwal cows. Sahiwal cows (18) were selected from the institute herd, apparently healthy and in advanced state of pregnancy. The cows were randomly divided into 3 groups 6 cows in each, viz. control, treatment 1 (T1) and treatment 2 (T2). Control group was fed a control diet as practiced for pregnant cows in NDRI dairy farm. The treatment 1 (T1) group was supplemented with zinc @ 60 ppm/day/cow and treatment 2 (T2) group was supplemented a mixture of vitamin E @ 1000 IU/day/animal and zinc @ 60 ppm/ day/cow with control diet during day 60 prepartum to day 90 postpartum. Plasma was analyzed for alkalline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). ALP levels were differing significantly in all 3 groups. ALT level was significantly lower in treatment 2 as comparison to its level of both control and treatment 1 animals. AST level was also significantly lower in treatment 2 as comparison to its level of both control and treatment 1 animals. The study indicated that vitamin E and zinc supplementation lowers these hepatic enzymes in blood plasma, prevents oxidative damage of liver and improves the health condition of liver.

Hesperidin increases intestinal β,β-carotene 15-15′ mono-oxygenase 1 (BCMO1) activity in Mongolian gerbils (Meriones unguiculatus) fed with β-carotene-free diet

β,β-Carotene 15-15’ mono-oxygenase 1 (BCMO1) is a key enzyme in vitamin A (VitA) metabolism in mammals. Dietary compounds, such as carotenoids and polyphenols, were reported to influence BCMO1 activity. The aim of this study was to evaluate the effect of hesperidin (Hes), on the VitA bioefficacy of β-carotene (Bc) from orange-fleshed sweet potato, using Mongolian gerbils, focussing on BCMO1 activity. Gerbils (n=50) depleted in VitA were divided into five groups fed with basal diet containing 3% white- or orange-fleshed sweet potatoes supplemented or not with Hes. Liver BCMO1 activity was low, with no significant differences between groups. Interestingly, intestinal mucosal BCMO1 activity was significantly higher in the gerbils fed without Bc or VitA than those fed with a VitA/Bc-supplemented diet. Finally, our results show that, under a low VitA status, Hes dramatically stimulated intestinal BCMO1 activity, an effect that could possibly be related to its action as an agonist of PPARγ.

The evolution and functional divergence of the beta-carotene oxygenase gene family in teleost fish—Exemplified by Atlantic salmon

In mammals, two carotenoid cleaving oxygenases are known; beta-carotene 15,15'-monooxygenase (BCMO1) and beta-carotene 9',10'-oxygenase (BCO2). BCMO1 is a key enzyme in vitamin A synthesis by symmetrically cleaving beta-carotene into 2 molecules of all-trans-retinal, while BCO2 is responsible for asymmetric cleavage of a broader range of carotenoids. Here, we show that the Atlantic salmon beta-carotene oxygenase (bco) gene family contains 5 members, three bco2 and two bcmo1 paralogs. Using public sequence databases, multiple bco genes were also found in several additional teleost species. Phylogenetic analysis indicates that bco2a and bco2b originate from the teleost fish specific genome duplication (FSGD or 3R), while the third and more distant paralog, bco2 like, might stem from a prior duplication event in the teleost lineage. The two bcmo1 paralogs (bcmo1 and bcmo1 like) appear to be the result of an ancient duplication event that took place before the divergence of ray-finned (Actinopterygii) and lobe-finned fish (Sarcopterygii), with subsequent nonfunctionalization and loss of one Sarcopterygii paralog. Gene expression analysis of the bcmo1 and bco2 paralogs in Atlantic salmon reveals regulatory divergence with tissue specific expression profiles, suggesting that the beta-carotene oxygenase subtypes have evolved functional divergences. We suggest that teleost fish have evolved and maintained an extended repertoire of beta-carotene oxygenases compared to the investigated Sarcopterygii species, and hypothesize that the main driver behind this functional divergence is the exposure to a diverse set of carotenoids in the aquatic environment.