In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

Background

The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro.

Methodology

Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture.

Principal Findings

The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line.

Conclusions

The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs).     

Caenorhabditis elegans ZC376.5 encodes a tRNA (m2/2G(26))dimethyltransferance in which (246)arginine is important for the enzyme activity

It has been estimated that eukaryotes carry more than 50 genes for tRNA modifying enzymes. Of the few so far identified most come from yeast, a lower eukaryote. In Saccharomyces cerevisiae, the TRM1 gene is a nuclear gene encoding the tRNA(m2/ 2G(26))dimethyltransferase, which catalyses the formation of the N2, N2-dimethylguanosine at position 26 in tRNA. We have isolated and characterized the corresponding gene ZC376.5 in Caenorhabditis elegans. Via RTPCR the cDNA sequence of the full length ZC376.5 has now been cloned, expressed in Escherichia coli and demonstrated to encode a tRNA(m2/2G(26))dimethyltransferase that produces dimethyl-G26 in vivo and in vitro with tRNA from yeast and bacteria as substrates. This is the first example of a complete gene sequence coding for a tRNA modifying enzyme from a multicellular organism. A point mutation in exon IV in the C. elegans genome sequence coding for the tRNA(m2/2G(26))methyltransferase that substituted arginine246 for glycine eliminated the modification activity. Exchanging the corresponding lysine residue in the yeast Trm1p for alanine caused a severe loss of activity, indicating that the identity of the amino acid at this position is important for enzyme activity.     

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.     

Transgenic rice as a novel production system for <Emphasis Type="Italic">Melanocarpus</Emphasis> and <Emphasis Type="Italic">Pycnoporus</Emphasis> laccases

Laccases have numerous biotechnological applications, among them food processing. The widespread use of laccases has increased the demand for an inexpensive and safe source of recombinant enzyme. We explored the use of a rice-based system for the production of two fungal laccases derived from the ascomycete Melanocarpus albomyces and the basidiomycete Pycnoporus cinnabarinus. High-expression levels of active recombinant laccases were achieved by targeting expression to the endosperm of rice seeds. The laccase cDNAs were fused to a plant-derived signal sequence for targeting to the secretory pathway, and placed under the control of a constitutive seed-specific promoter fused to an intron for enhanced expression. This construct enabled the recovery of on average 0.1-1% of soluble laccase in total soluble proteins (TSP). The highest yields of recombinant laccases obtained in rice seeds were 13 and 39 ppm for riceMaL and ricePycL, respectively. The rice-produced laccases were purified and characterized. The wild-type and the recombinant proteins showed similar biochemical features in terms of molecular mass, pI, temperature and optimal pH and the N-terminus was correctly processed. Although presenting lower kinetic parameters, the rice-produced laccases were also suitable for the oxidative cross-linking of a food model substrate [maize-bran feruloylated arabinoxylans (AX)].     

A new role of Deinococcus radiodurans RecD in antioxidant pathway

In Deinococcus radiodurans, RecBCD holoenzyme is not intact because of the absence of RecB and RecC, but a RecD-like protein does indeed exist. In this work, D. radiodurans recD disruptant was constructed and its possible biological functions were investigated. The results showed that disruption of the recD gene of D. radiodurans resulted in a remarkably increased sensitivity to hydrogen peroxide but had no apparent effect on the resistance to gamma and UV radiation. Furthermore, complementation experiments showed that Escherichia coli RecD, helicase domain or N-terminal domain of D. radiodurans RecD could not individually restore the resistant phenotype to hydrogen peroxide of the recD disruptant, whereas the complete D. radiodurans RecD protein could. Further studies showed that D. radiodurans RecD took part in antioxidant process by stimulating catalase activity and reactive oxygen species scavenging activity in D. radiodurans. These results suggest that D. radiodurans RecD has a new role in the antioxidant pathway.     

Origin and differentiation of endemism in the flora of China

The present paper analyzed 239 endemic genera in 67 families in the flora of seed plants in China.The results showed that there are five families containing more than ten endemic genera,namely,Gesneriaceae (27),which hereafter refers to the number of endemic genera in China,Composite (20),Labiatae (12),Cruciferae (11),and Umbelliferae (10),15 families with two endemic genera,and another 30 families with only one endemic genus.Four monotypic families (Ginkgoaceae,Davidiaceae,Eucommiaceae and Acanthochlamydaceae)are the most ancient,relict and characteristic in the flora of seed plants in China.Based on integrative data of systematics,fossil history,and morphological and molecular evidence of these genera,their origin,evolution and relationships were discussed.In gymnosperms,all endemic genera are relicts of the Arctic-Tertiary flora,having earlier evolutionary history,and can be traced back to the Cretaceous or to the Jurassic and even earlier.In angiosperms,the endemic genera are mostly relicts,and are represented in all lineages in the"Eight-Class System ofClassification of Angiosperms",and endemism can be found in almost every evolutionary stage of extant angiosperms.The relict genera once occupied huge areas in the northern hemisphere in the Tertiary or the late Cretaceous,while neo-endemism mostly originated in the late Tertiary.They came from Arctic-Tertiary,Paleo-tropical-Tertiary and Tethys-Tertiary florisitic elements,and the blend of the three elements with many genera of autochthonous origin.The endemism was formed when some dispersal routes such as the North Atlantic Land Bridge,and the Bering Bridge became discontinuous during the Tertiary,as well as the climate change and glaciations in the late Tertiary and the Quaternary.Therefore,the late Tertiary is the starting point of extant endemism of the flora in China.     

Molecular analyses of <Emphasis Type="Italic">Ficus erecta</Emphasis> and its allies within the subsection <Emphasis Type="Italic">Frutescentiae</Emphasis> (Moraceae)

Ficus (Moraceae) is a keystone group in tropical and subtropical forests with remarkable diversity of species and taxonomical challenges as a consequence of fig–pollinator coevolution. Ficus subsect. Frutescentiae includes about 30 species that are predominantly shrubs or small trees with Terminalia branching. Many of these species are difficult to delimit morphologically, and the group includes a tangle of uncertain taxa and incorrectly applied names. We conducted a phylogenetic analysis with internal and external transcribed spacer data (ITS and ETS) and data from 18 polymorphic microsatellite loci to evaluate the species status of the most perplexing members of this subsection. The results confirm the monophyly of subsect. Frutescentiae, with F. pedunculosa as sister to the rest. The F. erecta complex comprises approximately 17 taxa: F. erecta, F. abelii, F. boninsimae, F. nishimurae, F. iidaiana, F. gasparriniana var. laceratifolia, F. gasparriniana var. viridescens, F. pyriformis, F. stenophylla, F. fusuiensis, F. fengkaiensis, F. sinociliata, F. tannoensis, F. vaccinioides, F. formosana, F. pandurata, and F. periptera. The last five of these were supported as good species, while the others were not well supported by the present evidence. Evidence also supported the status of the non-F. erecta complex species including. F. pedunculosa, F. ischnopoda, F. heteromorpha, and F. variolosa. Ficus filicauda and F. neriifolia are possibly conspecific. The species status of F. potingensis should be restored and it should be treated as a member of section Eriosycea. Identification of the remaining taxa (F. gasparriniana var. esquirolii, F. ruyuanensis, F. daimingshanensis, F. chapaensis, F. changii, F. trivia, and F. tuphapensis) and their relationships to the F. erecta complex were not clarified. As a whole, only ten species in this subsection are confirmed, one is excluded, one is synonymous, and the others are either unresolved or short of samples. There appears to be a consistent genetic background among these unresolved groups, which suggests that repeated hybridization (as a result of pollinator host shifts) has filled up the interspecific gaps during the fig–pollinator coevolution process.     

Phylogenetic relationships and divergence dates of softshell turtles (Testudines: Trionychidae) inferred from complete mitochondrial genomes

The softshell turtles (Trionychidae) are one of the most widely distributed reptile groups in the world, and fossils have been found on all continents except Antarctica. The phylogenetic relationships among members of this group have been previously studied; however, disagreements regarding its taxonomy, its phylogeography and divergence times are still poorly understood as well. Here, we present a comprehensive mitogenomic study of softshell turtles. We sequenced the complete mitochondrial genomes of 10 softshell turtles, in addition to the GenBank sequence of Dogania subplana, Lissemys punctata, Trionyx triunguis, which cover all extant genera within Trionychidae except for Cyclanorbis and Cycloderma. These data were combined with other mitogenomes of turtles for phylogenetic analyses. Divergence time calibration and ancestral reconstruction were calculated using BEAST and RASP software, respectively. Our phylogenetic analyses indicate that Trionychidae is the sister taxon of Carettochelyidae, and support the monophyly of Trionychinae and Cyclanorbinae, which is consistent with morphological data and molecular analysis. Our phylogenetic analyses have established a sister taxon relationship between the Asian Rafetus and the Asian Palea + Pelodiscus + Dogania + Nilssonia + Amyda, whereas a previous study grouped the Asian Rafetus with the American Apalone. The results of divergence time estimates and area ancestral reconstruction show that extant Trionychidae originated in Asia at around 108 million years ago (MA), and radiations mainly occurred during two warm periods, namely Late Cretaceous–Early Eocene and Oligocene. By combining the estimated divergence time and the reconstructed ancestral area of softshell turtles, we determined that the dispersal of softshell turtles out of Asia may have taken three routes. Furthermore, the times of dispersal seem to be in agreement with the time of the India–Asia collision and opening of the Bering Strait, which provide evidence for the accuracy of our estimation of divergence time. Overall, the mitogenomes of this group were used to explore the origin and dispersal route of Trionychidae and have provided new insights on the evolution of this group.