Compartmental modeling of whole-body vitamin A kinetics in unsupplemented and vitamin A-retinoic acid-supplemented neonatal rats

Little is known about the contribution of different tissues to whole-body vitamin A (VA) kinetics in neonates. Here, we have used model-based compartmental analysis of tissue tracer kinetic data from unsupplemented (control) and VA-retinoic acid (VARA)-supplemented neonatal rats to determine VA kinetics in specific tissues under control and supplemented conditions. First, compartmental models for retinol kinetics were developed for individual tissues, and then an integrated compartmental model incorporating all tissues was developed for both groups. The models predicted that 52% of chylomicron (CM) retinyl ester was cleared by liver in control pups versus 22% in VARA-treated pups, whereas about 51% of VA was predicted to be extrahepatic in 4- to 6-day-old unsupplemented neonatal rats. VARA increased CM retinyl ester uptake by lung, carcass, and intestine; decreased the release into plasma of retinol that had been cleared by liver and lung as CM retinyl esters; stimulated the uptake of retinol from plasma holo-retinol binding protein into carcass; and decreased the retinol turnover out of the liver. Overall, neonatal VA trafficking differed from that previously described for adult animals, with a larger contribution of extrahepatic tissues to CM clearance, especially after VA supplementation, and a significant amount of VA distributed in extrahepatic tissues.     

A quantitative analysis of germ cells and the histone variants in the testes of vitamin A-deficient rats and during subsequent repletion with vitamin A

A quantitative analysis of the different types of germ cells present in the seminiferous tubules of vitamin A-deficient-retinoate maintained rats revealed that the number of pachytene spermatocytes and spermatogonia was greatly reduced in the deficient rats. Spermatids were virtually absent in the deficient tubules which contained mostly spermatogonia and preleptotene spermatocytes along with the Sertoli cells. There was no change in the number of Sertoli cells present in the tubules of deficient rats as compared to that of normal rats. Following supplementation of retinyl acetate to vitamin A-deficient-retinoate maintained rats, there was an immediate thinning of the germinal epithelium resulting from the sloughing off of the damaged spermatocytes which were beyond repair. However, after 12 days of vitamin A supplementation fresh batch of pachytene spermatocytes started appearing while by day 16 round spermatids could be seen. Analysis of the acid soluble proteins from nuclei on different types of Polyacrylamide gel electrophoretic systems has revealed that the levels of the testis specific histone variants Hlt, TH2A and TH2B, synthesized predominantly in the pachytene spermatocytes were greatly reduced in the testes of retinoate maintained rats. Following supplementation of retinyl acetate for either 4 days or 8 days the levels of these histone variants further decreased which correlated with the decrease in the number of pachytene spermatocytes. However, by day 12 of supplementation onwards, their levels started increasing and reached near normal levels by day 24 of vitamin A-supplementation     

Characterization of hepatic sexual dimorphism in Alb-DsRed2 transgenic rats

We previously created the Alb-DsRed2 transgenic (Tg) rat that specifically expresses the red fluorescent protein, DsRed2, in the liver. Herein, we demonstrate that the DsRed2 expression is sexually dimorphic and exhibits a male-specific pattern. The profiling of sexual dimorphism in DsRed2 expression during pre-pubertal development was investigated using an in vivo fluorescent imaging analysis. The DsRed2 expression decreased gradually in both sexes until 28 days after birth. While DsRed2 expression was not persistent in the female liver, the male hepatic expression increased again at 35 days. Sexual dimorphic DsRed2 expression did not change in gonadectomized male and female Tg-rats. However, female hepatic DsRed2 was induced 72 h after the hypophysectomy. Hepatocytes isolated from the female Tg-rats also revealed DsRed2 induction by 96 h in culture. These results suggest that the pituitary hormone suppresses the female hepatic DsRed2 expression causing the sexual dimorphism of DsRed2 expression.     

A problem in multivariate analysis of codon usage data and a possible solution

Multivariate analyses are often used to identify major trends of variation in synonymous codon usage among genes. These analyses need to be performed on properly normalized codon usage data to avoid biases masking this synonymous variation, i.e., gene length, amino acid usage, and codon degeneracy; however, previous studies have failed to do so. In this paper, we demonstrate that the use of alternative normalized data (called 'relative adaptiveness' in the literature) can avoid all these biases and furthermore, can identify more trends of variation among genes, including GC-ending codon usage, GT-ending codon usage, and gene expression level.     

A short history of nuclear activation analysis

The major developments in the field of nuclear activation analysis, from 1936 to 1989, are discussed. The developments are grouped into five consecutive time periods. The impact of various scientists on the development of the field in the first 35 years is also discussed.     

Considering of certain relevant information in the analysis of contingency tables. A three-step procedure

A heuristic three-step procedure for analysing multidimensional contingency tables is given to meet the requirements of a mixed analysis from both hypotheses-ruled and data-ruled type. The first-step provides the structure of relationships among the attributes by fitting an appropriate unsaturated log-linear model to the data of the given contingency table. Restriction to elementary hierarchical models allows to get them by combining pairs of conditional independence. The result of the first step may be regarded as a certain validisation of real model ideas. In the second step the significant pairs of conditional dependence are analysed in regard to the levels of the condition complex. Only such significant pairs are to be considered, in general, where the condition complex does not include the response variable. The third-step may test special subtests in that significant two-dimensional tables found in step two or may extend the general statements by partitioning, the corresponding test statistics in additive components. Application examples demonstrate the general line of action.     

Comparing the success of different prediction software in sequence analysis: a review

The abundance of computer software for different types of prediction in DNA and protein sequence analyses raises the problem of adequate ranking of prediction program quality. A single measure of success of predictor software, which adequately ranks the predictors, does not exist. A typical example of such an incomplete measure is the so-called correlation coefficient. This paper provides an overview and short analysis of several different measures of prediction quality. Frequently, some of these measures give results contradictory to each other even when they relate to the same prediction scores.This may lead to confusion. In order to overcome some of the problems, a few new measures are proposed including some variants of a 'generalised distance from the ideal predictor score'; these are based on topological properties, rather than on statistics. In order to provide a sort of a balanced ranking, the averaged score measure (ASM) is introduced.The ASM provides a possibility for the selection of the predictor that probably has the best overall performance.The method presented in the paper applies to the ranking problem of any prediction software whose results can be properly represented in a true positive-false positive framework, thus providing a natural set-up for linear biological sequence analysis.     

A possible mechanism of metabolic regulation in Gibberella fujikuroi using a mixed carbon source of glucose and corn oil inferred from analysis of the kinetics data obtained in a stirrer tank bioreactor

A nonstructured model was used to study the dynamics of gibberellic acid production in a stirred tank bioreactor. Experimental data were obtained from submerged batch cultures of Gibberella fujikuroi (CDBB H‐984) grown in varying ratios of glucose‐corn oil as the carbon source. The nitrogen depletion effect was included in mathematical model by considering the specific kinetic constants as a linear function of the normalized nitrogen consumption rate. The kinetics of biomass growth and consumption of phosphate and nitrogen were based on the logistic model. The traditional first‐order kinetic model was used to describe the specific consumption of glucose and corn oil. The nitrogen effect was solely included in the phosphate and corn oil consumption and biomass growth. The model fit was satisfactory, revealing the dependence of the kinetics with respect to the nitrogen assimilation rate. Through simulations, it was possible to make diagrams of specific growth rate and specific rate of substrate consumptions, which was a powerful tool for understanding the metabolic interactions that occurred during the various stages of fermentation process. This kinetic analysis provided the proposal of a possible mechanism of regulation on growth, substrate consumptions, and production of gibberellic acid (GA₃) in G. fujikuroi. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1169–1180, 2013     

Stat3 is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that has been implicated as a potential carcinogen and epigenotoxicant. We have previously reported dose-dependent incidence of hepatic tumors in 10-month-old isogenic mice perinatally exposed to BPA. Here, we evaluated DNA methylation at 3 candidate genes (Esr1, Il-6st, and Stat3) in liver tissue of BPA-exposed mice euthanized at 2 time points: post-natal day 22 (PND22; n = 147) or 10-months of age (n = 78, including n = 18 with hepatic tumors). Additionally, DNA methylation profiles were analyzed at human homologs of murine candidate genes in human fetal liver samples (n = 50) with known liver tissue BPA levels. Candidate genes were chosen based on reported expression changes in both rodent and human hepatocellular carcinoma (HCC). Regions for bisulfite sequencing were chosen by mining whole genome next generation sequencing methylation datasets of both mice and human liver samples with known perinatal BPA exposures. One of 3 candidate genes, Stat3, displayed dose-dependent DNA methylation changes in both 10-month mice with liver tumors as compared to those without liver tumors and 3-week sibling mice from the same exposure study, implicating Stat3 as a potential epigenetic biomarker of both early life BPA exposure and adult disease in mice. DNA methylation profiles within STAT3 varied with liver tissue BPA level in human fetal liver samples as well, suggesting STAT3 may be a translationally relevant candidate biomarker. These data implicate Stat3 as a potential early life biomarker of adult murine liver tumor risk following early BPA exposure with early evidence of relevance to human health.     

SphK1 mediates hepatic inflammation in a mouse model of NASH induced by high saturated fat feeding and initiates proinflammatory signaling in hepatocytes

Steatohepatitis occurs in up to 20% of patients with fatty liver disease and leads to its primary disease outcomes, including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Mechanisms that mediate this inflammation are of major interest. We previously showed that overload of saturated fatty acids, such as that which occurs with metabolic syndrome, induced sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine-1-phosphate (S1P). While data suggest beneficial roles for S1P in some contexts, we hypothesized that it may promote hepatic inflammation in the context of obesity. Consistent with this, we observed 2-fold elevation of this enzyme in livers from humans with nonalcoholic fatty liver disease and also in mice with high saturated fat feeding, which recapitulated the human disease. Mice exhibited activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, SphK1-null mice were protected from these outcomes. Studies in cultured cells demonstrated saturated fatty acid induction of SphK1 message, protein, and activity, and also a requirement of the enzyme for NFκB signaling and increased mRNA encoding TNFα and MCP1. Moreover, saturated fat-induced NFκB signaling and elevation of TNFα and MCP1 mRNA in HepG2 cells was blocked by targeted knockdown of S1P receptor 1, supporting a role for this lipid signaling pathway in inflammation in nonalcoholic fatty liver disease.     

Diversity analysis of methanogens in rumen of Bubalus bubalis by 16S riboprinting and sequence analysis

The molecular diversity of rumen methanogens was investigated by 16S rDNA gene library prepared from the rumen contents obtained from Murrah buffaloes in India. Genomic DNA was isolated from adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the positive clones were selected assuming based on blue-white screening and sequenced. Positive clones were reamplified and the resulting PCR products were further subjected to Amplified Ribosomal DNA Restriction Analysis (ARDRA) by using HaeIII enzyme. A total of 108 clones were examined, and the analysis revealed 16 phylotypes. Out of sixteen phylotypes, nine phylotypes belong to the uncultured group of methanogens, and the rest of seven phylotypes belong to the order Methanomicrobiales, Methanococcales and Methanobacteriales. Out of the 108 rDNA clones, 66 clones which constitute 61.1% of the total clone representing 9 phylotypes, show less than 97% sequence similarity with any of the cultured strain of methanogens. The second largest group of clones (24 clones) represented by four phylotypes show a sequence similarity ranging from 91% to 99% with Methanomicrobium mobile strain of methanogens. The third group of 16S rDNA clones clustered along with M. burtonii strain of methanogens. This group consists of 6 clones and constitutes about 5.5% of the total clones and represented by only single phylotype. Fourth and fifth clusters of 16S rDNA clones consist of 5 and 7 clones respectively, and these were matched with Methanobrevibacter gottschalkii and Methanobrevibacter rumanatium strain of methanogens and constitute about 4.6% and 6.4% of the total clones.     

Stabilization of the catalytic thiolate in a mammalian glutaredoxin: structure, dynamics and electrostatics of reduced pig glutaredoxin and its mutants

The variety of functions performed by proteins of the thioredoxin superfamily, including glutaredoxins, involves the wide range of redox potential associated with the -Cys-X-X-Cys- motif found in their active sites. The determinants of these differences in redox potential are still obscure. A better understanding requires a detailed characterization of the reduced state of these enzymes, especially because the lowered pK(a) of the reduced N-terminal active-site cysteine is a key feature of these enzymes' chemistry, including their redox potential. Analysis of the factors controlling this pK(a) is complicated by the apparent structural heterogeneity of the reduced active sites across glutaredoxins. In this family, pig glutaredoxin (pGrx) was one of the first to be functionally characterized, including some intriguing mutagenesis data, but a structure of its reduced state has been lacking. We used long molecular dynamics simulations and electrostatic calculations to analyze the structure, dynamics and electrostatics of reduced pGrx and some of its mutants. Comparison with experimental data is drawn whenever possible. It is shown that a dynamic model is essential to capture the structural properties of the cationic side-chains around the -Cys22-Pro23-Phe24-Cys25- sequence in the pGrx active site. Examples include Arg26, which can swing to stack on this sequence, and Lys19 which can contact the thiolate. However, contrary to a commonly held hypothesis, these cationic side-chains provide little stabilization for the thiolate, implying that they affect the enzymatic activity via other mechanisms. The pK(a) value of nucleophilic cysteine 22 (pK(a)(22)) is dominated by local hydrogen-bonds, formed only in a well-defined active-site conformation, supported by a comparison between the calculated and experimental values of pK(a)(22). The edge of the aromatic ring of Phe24 is polar enough to contribute to stabilize the thiolate, consistent with the conserved aromatic side-chain at this position in the glutaredoxin motif. The locality and directionality of the hydrogen bonds in the active site suffice to explain the vast difference between the pK(a) values of its two cysteine residues. A control of the cysteine pK(a) values by local hydrogen bonds implies that the peripheral ionized side-chains can evolve independently of the maintenance of these pK(a) values, maybe guided instead by substrate recognition. Comparison with other glutaredoxins indicates that the calculated pK(a) values of the N-terminal cysteine are better conserved than those of the C-terminal cysteine. Overall, a methodological strategy to systematically compare all reduced enzymes of this family emerges.     

Role of maternal tissue in the synthesis of polyunsaturated fatty acids in response to a lipid-deficient diet during pregnancy and lactation in rats

During pregnancy and lactation, metabolic adaptations involve changes in expression of desaturases and elongases (Elovl2 and Elovl5) in the mammary gland and liver for the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic acid (AA) required for fetal and postnatal growth. Adipose tissue is a pool of LC-PUFAs. The response of adipose tissue for the synthesis of these fatty acids in a lipid-deficient diet of dams is unknown. The aim of this study was to explore the role of maternal tissue in the synthesis of LC-PUFAs in rats fed a low-lipid diet during pregnancy and lactation. Fatty acid composition (indicative of enzymatic activity) and gene expression of encoding enzymes for fatty acid synthesis were measured in liver, mammary gland and adipose tissue in rats fed a low-lipid diet. Gene expression of desaturases, elongases, fatty acid synthase (Fasn) and their regulator Srebf-1c was increased in the mammary gland, liver and adipose tissue of rats fed a low-lipid diet compared with rats from the adequate-lipid diet group throughout pregnancy and lactation. Genes with the highest (P < 0.05) expression in the mammary gland, liver and adipose tissue were Elovl5 (1333%), Fads2 (490%) and Fasn (6608%), respectively, in a low-lipid diet than in adequate-lipid diet. The percentage of AA in the mammary gland was similar between the low-lipid diet and adequate-lipid diet groups during the second stage of pregnancy and during lactation. The percentage of monounsaturated and saturated fatty acids was significantly (P < 0.05) increased throughout pregnancy and lactation in all tissues in rats fed a low-lipid diet than in rats fed an adequate-lipid diet. Results suggest that maternal metabolic adaptations used to compensate for lipid-deficient diet during pregnancy and lactation include increased expression of genes involved in LC-PUFAs synthesis in a stage- and tissue-specific manner and elevated lipogenic activity (saturated and monounsaturated fatty acid synthesis) of maternal tissues including adipose tissue.     

Comparing methods for metabolic network analysis and an application to metabolic engineering

Bioinformatics tools have facilitated the reconstruction and analysis of cellular metabolism of various organisms based on information encoded in their genomes. Characterization of cellular metabolism is useful to understand the phenotypic capabilities of these organisms. It has been done quantitatively through the analysis of pathway operations. There are several in silico approaches for analyzing metabolic networks, including structural and stoichiometric analysis, metabolic flux analysis, metabolic control analysis, and several kinetic modeling based analyses. They can serve as a virtual laboratory to give insights into basic principles of cellular functions. This article summarizes the progress and advances in software and algorithm development for metabolic network analysis, along with their applications relevant to cellular physiology, and metabolic engineering with an emphasis on microbial strain optimization. Moreover, it provides a detailed comparative analysis of existing approaches under different categories.