Structural and Functional Insights into the Catalytic Inactivity of the Major Fraction of Buffalo Milk Xanthine Oxidoreductase

Background

Xanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species.

Methods and Findings

Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k _cat/K _m) of 0.11 sec⁻¹ µM⁻¹ of buffalo XO was 8–10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε_{430 nm}) of 46,000 M⁻¹ cm⁻¹ suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD⁺ entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum.

Conclusion

A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way.     

Phylogenetic Patterns in the Microbial Response to Resource Availability: Amino Acid Incorporation in San Francisco Bay

Aquatic microorganisms are typically identified as either oligotrophic or copiotrophic, representing trophic strategies adapted to low or high nutrient concentrations, respectively. Here, we sought to take steps towards identifying these and additional adaptations to nutrient availability with a quantitative analysis of microbial resource use in mixed communities. We incubated an estuarine microbial community with stable isotope labeled amino acids (AAs) at concentrations spanning three orders of magnitude, followed by taxon-specific quantitation of isotopic incorporation using NanoSIMS analysis of high-density microarrays. The resulting data revealed that trophic response to AA availability falls along a continuum between copiotrophy and oligotrophy, and high and low activity. To illustrate strategies along this continuum more simply, we statistically categorized microbial taxa among three trophic types, based on their incorporation responses to increasing resource concentration. The data indicated that taxa with copiotrophic-like resource use were not necessarily the most active, and taxa with oligotrophic-like resource use were not always the least active. Two of the trophic strategies were not randomly distributed throughout a 16S rDNA phylogeny, suggesting they are under selective pressure in this ecosystem and that a link exists between evolutionary relatedness and substrate affinity. The diversity of strategies to adapt to differences in resource availability highlights the need to expand our understanding of microbial interactions with organic matter in order to better predict microbial responses to a changing environment.     

An unexpected role for caspase-2 in neuroblastoma

Caspase-2 has been implicated in various cellular functions, including cell death by apoptosis, oxidative stress response, maintenance of genomic stability and tumor suppression. The loss of the caspase-2 gene (Casp2) enhances oncogene-mediated tumorigenesis induced by E1A/Ras in athymic nude mice, and also in the Eμ-Myc lymphoma and MMTV/c-neu mammary tumor mouse models. To further investigate the function of caspase-2 in oncogene-mediated tumorigenesis, we extended our studies in the TH-MYCN transgenic mouse model of neuroblastoma. Surprisingly, we found that loss of caspase-2 delayed tumorigenesis in the TH-MYCN neuroblastoma model. In addition, tumors from TH-MYCN/Casp2^−/− mice were predominantly thoracic paraspinal tumors and were less vascularized compared with tumors from their TH-MYCN/Casp2^+/+ counterparts. We did not detect any differences in the expression of neuroblastoma-associated genes in TH-MYCN/Casp2^−/− tumors, or in the activation of Ras/MAPK signaling pathway that is involved in neuroblastoma progression. Analysis of expression array data from human neuroblastoma samples showed a correlation between low caspase-2 levels and increased survival. However, caspase-2 levels correlated with clinical outcome only in the subset of MYCN-non-amplified human neuroblastoma. These observations indicate that caspase-2 is not a suppressor in MYCN-induced neuroblastoma and suggest a tissue and context-specific role for caspase-2 in tumorigenesis.The caspase family of cysteine proteases are highly conserved regulators of cell death by apoptosis.¹ In addition to their pro-apoptotic function, many caspases also have non-apoptotic roles in other physiological processes, such as inflammation, necrosis and tumor suppression.^{2, 3, 4} The most highly conserved caspase, caspase-2, has recently been demonstrated to function in the cellular stress response, protection against ageing, maintenance of genome stability and in tumor suppression.^{2, 5, 6, 7, 8}The tumor suppressor function of caspase-2 was first demonstrated using E1A/Ras-transformed caspase-2-deficient mouse embryonic fibroblasts (MEFs), which showed an increased tumorigenic potential in athymic nude mice.⁷ Further supporting evidence came from experiments demonstrating that caspase-2 deficiency enhances B-cell lymphoma development in Eμ-Myc transgenic mice⁷ and mammary carcinomas in MMTV/c-neu mice,⁹ suggesting that caspase-2 prevents oncogene-induced lymphomas and epithelial tumors. Importantly, tumor suppression by caspase-2 is also evident in the non-oncogene-driven Atm^−/− thymoma mouse model.¹⁰Given its role in apoptosis, the tumor suppression function of caspase-2 was thought to be associated with this role, via the elimination of mutagenic or potentially tumorigenic cells. Recent studies have now indicated that the role of caspase-2 may extend beyond apoptosis and that its tumor suppression function may, in part, be mediated by maintaining genomic stability and/or the oxidative stress response. Caspase-2-deficient MEFs and tumor cells from Eμ-Myc/Casp2^−/−, MMTV/c-neu/Casp2^−/− and Atm^−/−;Casp2^−/− mice all display aberrant proliferation, and increased genomic instability^{6, 9, 10} and indicate that caspase-2 is important for the maintenance of genome stability. Importantly, the role of caspase-2 in maintaining genomic stability in primary cells appears to be required for its tumor suppressor function.¹⁰Genomic instability is a hallmark of cancer¹¹ and the overexpression of Myc family oncoproteins is commonly associated with genomic instability and a wide spectrum of human cancers.^{12, 13, 14} Interestingly, a common feature of the oncogene-induced tumor models used in the study of caspase-2 tumor suppressor function is the overexpression of c-Myc¹⁵ or aberrant c-Myc signaling.^{16, 17, 18} Given the role of Myc proteins as key mediators of genomic instability as well as cell proliferation, cell growth and DNA damage, we were interested in further assessing whether caspase-2 can promote tumor suppression in other MYC-dependent mouse tumor models. We used the MYCN mouse model of neuroblastoma (TH-MYCN mouse), in which MYCN is constitutively expressed under the control of the rat tyrosine hydroxylase (TH) promoter leading to neural crest cell-specific expression and early-onset neuroblastoma.¹⁹ Amplification of MYCN occurs in ∼20% of human neuroblastomas and high MYCN protein levels are strongly associated with tumor progression and poor clinical outcome.^{20, 21} Thus, the TH-MYCN transgenic mouse model recapitulates many clinical features of aggressive neuroblastomas in humans and provides a powerful model of preclinical neuroblastoma.^{19, 22}MYCN-mediated neuroblastoma onset and progression is commonly associated with additional genetic events, including the expression of the key genes including Odc1, Mrp1, SirT1 and Ras.^{23, 24, 25} A recent study has found that caspase-8 is in fact a potent suppressor of neuroblastoma, with the loss of caspase-8 expression occurring in ∼70% of neuroblastoma patients.^{26, 27} Interestingly, the loss of caspase-8 also promotes bone marrow metastasis in the TH-MYCN neuroblastoma mouse model.^{26, 27} The role of other caspases in neuroblastoma has not previously been examined, and given the function of caspase-2 in tumor suppression, provided additional relevance in assessing its role in this model.This study shows that caspase-2 is not able to suppress neuroblastoma development in TH-MYCN mice. In contrast to a role for caspase-2 as a tumor suppressor, our findings demonstrate that loss of caspase-2 somewhat delays neuroblastoma onset in mice. Interestingly, expression array data from human neuroblastoma show a strong correlation between low caspase-2 levels and improved outcome. Our data demonstrate that the tumor suppressor function of caspase-2 is not specific to Myc-mediated oncogenesis and that its role is likely to be tissue- and/or context-specific.     

Sucrose Octaacetate Chemical Kinetics and Shelf Lives at Various Formulation pHs

Developing pediatric friendly dosage forms is a high priority worldwide. Sucrose octaacetate (SOA) has been recommended for use as a surrogate for bitter tasting active pharmaceutical ingredients. Even though SOA has found a number of human use applications and has been employed for decades, there are no rigorous chemical kinetic studies reported. A recently reported SOA stability-indicating method was used to perform SOA chemical kinetic and stability studies. As part of the chemical kinetic study, reaction order, activation energies, extrapolated rate constants, pH–rate profiles at 4 and 25°C, and estimated shelf lives at 4 and 25°C at different buffer pHs are provided. The estimated SOA shelf lives at 25°C and pHs 4.00, 5.20, and 6.00 were 25.3, 114, and 27.4 days, respectively. At 4°C, SOA’s estimated shelf lives were 0.478, 5.26, and 1.47 years at pHs 4.00, 5.20, and 6.00, respectively. SOA can be formulated at pHs 4 to 6 and stored at 25°C for short-duration (less than 25 days) uses such as a bitter tasting surrogate for fundamental taste mechanism studies or brief taste masking assessment clinical studies. For longer term solution studies, like being used as a bitter tasting control for blinded clinical trials, SOA should be formulated at the optimum pH of 5.40 and refrigerated at 4°C for maximum stability. The reported data can be used as a starting point for developing stable SOA formulations and estimating shelf life.     

Structural and Functional Properties of Exopolysaccharide Excreted by a Novel <Emphasis Type="Italic">Bacillus anthracis</Emphasis> (Strain PFAB2) of Hot Spring Origin

Exopolysaccharide produced by a unique avirulent Bacillus anthracis strain PFAB2 of hot spring origin has been characterized and its functional properties are investigated which is a first report. Maximum yield of EPS is 7.66 g/l with 2% glucose and 1% peptone as optimum carbon and nitrogen source respectively. The EPS is found to be a homopolymer consisting of only glucose as principle monosaccharide component. Through ¹H NMR study, different dextran-like proton peaks are observed. Molecular weight of the EPS resembles low molecular weight bacterial origin polysaccharides. Melting transition of the EPS has started after 276 °C which indicates good thermal stability. The EPS also shows potent antioxidant activity in terms of DPPH and ABTS mediated free radical scavenging property compared to standard ascorbic acid. Emulsifying property of the EPS is also observed and has shown good emulsification of vegetable oils. The polysaccharide forms a thermo resistant gel during the heating phase, with G′ higher than G″ indicating excellent shear-thinning behaviour and viscoelastic nature of the EPS.