Conservation of the ST6Gal I gene and its expression in the mammary gland

Milk sialoglycoconjugates can protect the gastrointestinal tract of the suckling neonate by competitively binding to invading pathogens and promoting growth of beneficial flora, and their potential role in postnatal brain development is of particular interest in human infant nutrition. Although the concentration and the distribution of sialoglycoconjugates have been extensively studied in the milk of various species, the investigation of sialyltransferase gene expression in the mammary gland, in the context of lactation, has been limited. The sialyltransferase enzyme ST6Gal I transfers sialic acid from CMP-sialic acid to type 2 (Galβ1,4GlcNAc) free disaccharides or the termini of N- or O-linked oligosaccharides using an α2,6-linkage. Expression of the ST6Gal I gene is primarily regulated at the level of transcription through the use of several cell and development- specific promoters, producing transcripts with divergent 5′ untranslated regions (UTR). In the mouse mammary gland, the novel 5′UTR exon (L) appears to be associated with a drastic increase in ST6Gal I gene expression during lactation. We find that rats also possess an exon (L), suggesting conservation of this regulatory mechanism in rodents. In contrast, an exon (L)-containing transcript was not detected in the lactating bovine or human mammary gland. We also observed a trend of increasing ST6Gal I gene expression in the bovine mammary gland, culminating in involution. This is in contrast to species such as mice where the greatest change in ST6Gal I gene expression occurs between pregnancy and lactation, suggesting different roles in rodents vs. other mammals for α2,6-sialylated oligosaccharides present in milk.

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish, Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish,<i> Cherax destructori> (the yabby), we report the first detection of Cytochrome <i>bi> (Cyt <i>bi>) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt <i>bi> gene from 14 individuals of <i>C. destructori> using polymerase chain reaction (PCR) with primers designed from conserved regions of <i>Penaeus monodoni> and <i>Drosophila melanogaster i>mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt <i>bi> sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.

Elevated hypothalamic beacon gene expression in Psammomys obesus prone to develop obesity and type 2 diabetes

Objective: To investigate hypothalamic <i>beaconi> gene expression at various developmental stages in genetically selected diabetes-resistant and diabetes-prone <i>Psammomys obesusi>. In addition, effects of dietary energy composition on beacon gene expression were investigated in diabetes-prone <i>P. obesusi>. Methods: Hypothalamic <i>beaconi> gene expression was measured using Taqman&Ocirc; fluorogenic PCR in 4-, 8- and 16-week-old animals from each genetically selected line. Results: Expression of <i>beaconi> was elevated in the diabetes-prone compared with diabetes-resistant <i>P.i> <i>obesusi> at 4 weeks of age despite no difference in body weight between the groups. At 8 weeks of age, hypothalamic <i>beaconi> gene expression was elevated in diabetes-prone animals fed a high-energy diet, and was correlated with serum insulin concentration. Conclusion: <i>P. obesusi> with a genetic predisposition for the development of obesity and type 2 diabetes have elevated hypothalamic <i>beaconi> gene expression at an early age. Overexpression of beacon may contribute to the development of obesity and insulin resistance in these animals.

Calpain 3 gene expression in skeletal muscle is associated with body fat content and measures of insulin resistance

OBJECTIVE: To investigate whether skeletal muscle gene expression of <i>calpain 3i> is related to obesity and insulin resistance.

DESIGN: Cross-sectional studies in 27 non-diabetic human subjects and in <i>Psammomys obesusi>, a polygenic animal model of obesity and type 2 diabetes.

MEASUREMENTS: Expression of <i>CAPN3i> in skeletal muscle was measured using Taqman fluorogenic PCR. In the human subjects, body composition was assessed by DEXA and insulin sensitivity was measured by euglycemic-hyperinsulinemic clamp. In <i>Psammomys obesusi>, body composition was determined by carcass analysis, and substrate oxidation rates, physical activity and energy expenditure were measured by whole-body indirect calorimetry.

RESULTS: In human subjects, <i>calpain 3i> gene expression was negatively correlated with total (<i>Pi>=0.022) and central abdominal fat mass (<i>Pi>=0.034), and with blood glucose concentration in non-obese subjects (<i>Pi>=0.017). In <i>Psammomys obesusi>, <i>calpain 3i> gene expression was negatively correlated with circulating glucose (<i>Pi>=0.013) and insulin (<i>Pi>=0.034), and with body fat mass (<i>Pi>=0.049). Indirect calorimetry revealed associations between calpain 3 gene expression and carbohydrate oxidation (<i>Pi>=0.009) and energy expenditure (<i>Pi>=0.013).

CONCLUSION/INTERPRETATION: Lower levels of expression of <i>calpain 3i> in skeletal muscle were associated with reduced carbohydrate oxidation and elevated circulating glucose and insulin concentrations, and also with increased body fat and in particular abdominal fat. Therefore, reduced expression of <i>calpain 3i> in both humans and <i>Psammomys obesui><i>si> was associated with phenotypes related to obesity and insulin resistance.

The brachyspira hyodysenteriae ftnA gene : DNA vaccination and real-time PCR quantification of bacteria in a mouse model of disease

The nucleotide sequence of the <i>Brachyspira hyodysenteriae ftnAi> gene, encoding a putative ferritin protein (FtnA), was determined. Analysis of the sequence predicted that this gene encoded a protein of 180 amino acids. RT-PCR and Western blot showed that the <i>ftnAi> gene was expressed in <i>B.i> <i>hyodysenteriaei>, and evidence suggests that FtnA stores iron rather than haem. ftnA was delivered as DNA and recombinant protein vaccines in a mouse model of <i>B. hyodysenteriaei> infection. Vaccine efficacy was monitored by caecal pathology and quantification of <i>B. hyodysenteriaei> numbers in the caeca of infected mice by real-time PCR.

Reduced plasma free fatty acid availability during exercise: effect on gene expression

Endurance exercise transiently increases the mRNA of key regulatory proteins involved in skeletal muscle metabolism. During prolonged exercise and subsequent recovery, circulating plasma fatty acid (FA) concentrations are elevated. The present study therefore aimed to determine the sensitivity of key metabolic genes to FA exposure, assessed in vitro using L6 myocytes and secondly, to measure the expression of these same set of genes in vivo, following a single exercise bout when the post-exercise rise in plasma FA is abolished by acipimox. Initial studies using L6 myotubes demonstrated dose responsive sensitivity for both PDK4 and PGC-1α mRNA to acute FA exposure in vitro. Nine active males performed two trials consisting of 2 h exercise, followed by 2 h of recovery. In one trial, plasma FA availability was reduced by the administration of acipimox (LFA), a pharmacological inhibitor of adipose tissue lipolysis, and in the second trial a placebo was provided (CON). During the exercise bout and during recovery, the rise in plasma FA and glycerol was abolished by acipimox treatment. Following exercise the mRNA abundance of PDK4 and PGC-1α were elevated and unaffected by either acipimox or placebo. Further analysis of skeletal muscle gene expression demonstrated that the CPT I gene was suppressed in both trials, whilst UCP-3 gene was only modestly regulated by exercise alone. Acipimox ingestion did not alter the response for both CPT I and UCP-3. Thus, this study demonstrates that the normal increase in circulating concentrations of FA during the later stages of exercise and subsequent recovery is not required to induce skeletal muscle mRNA expression of several proteins involved in regulating substrate metabolism.

Genetic polymorphisms of cytochrome P450 2B6 gene in Han Chinese

Cytochrome P450 (CYP2B6) is an important enzyme that metabolizes more than eight compounds and about 3.0% of therapeutic drugs. The genetic polymorphisms of <i>CYP2B6i> have earlier been studied in Caucasian, Japanese and Korean, but the data are lacking for Han Chinese. The aim of this study was to investigate the frequencies of allelic variants of <i>CYP2B6i> in healthy Han Chinese and compare with those in other ethnic groups reported in the literature. Polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) method was used to test the five common non-synonymous single nucleotide polymorphisms (SNPs) of <i>CYP2B6i> gene, namely, 64C > T, 516G > T, 777C > A, 785A > G and 1459C > T in unrelated healthy Han Chinese (<i>ni> = 193). The study demonstrated that the frequencies of 64C > T, 516G > T, 777C > A, 785A > G and 1459C > T SNPs in Han Chinese were 0.03, 0.21, 0, 0.28 and 0.003, respectively. The frequencies of all five SNPs tested in female were higher than those in male, but the statistical difference was insignificant (<i>Pi> > 0.05). Compared to the data reported in the literature, the frequencies of common <i>CYP2B6i> allelic variants in Chinese are similar to those of other Asian populations including Japanese and Korean, but markedly different from those in Caucasians. These results indicate the presence of marked ethnic difference in <i>CYP2B6i> SNP frequencies between Chinese and Caucasian. Further studies are required to explore the impact of these SNPs of <i>CYP2B6 i>gene on the clinical response (efficacy and toxicity) to drugs that are substrates for CYP2B6 in patients.

Small interfering RNA-mediated silencing of cytochromeP450 3A4 gene

RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the <i>CYP3A4i> expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with <i>CYP3A4i> gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced <i>CYP3A4i> mRNA levels by 65% and protein expression levels by 75%. All these siRNAs did not affect the expression of <i>CYP3A5i> at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, cyclophosphamide and ifosfamide, in CHL-3A4 cells, with the IC₅₀ increased from 55 to 210 &micro;M to >1000 &micro;M. Nifedipine at 5.78, 14.44, and 28.88 &micro;M was significantly (<i>Pi> < 0.01) depleted by approximately 100, 40, and 22%, respectively, in S9 fractions from CHL-3A4 cells compared with parental CHL-pIC19h cells. In addition, transfection of the CHL-3A4 cells with vectors expressing the 3A4III siRNAs almost completely inhibited CYP3A4-mediated nifedipine metabolism. This study demonstrated, for the first time, the specific suppression of <i>CYP3A4i> expression and function using vector-based RNAi technique. The use of RNAi is a promising tool for the study of cytochrome P450 family function.

Identification and disruption of the gene encoding the third member of the low-molecular-mass rhoptry complex in Plasmodium falciparum

The low-molecular-mass rhoptry complex of <i>Plasmodium falciparumi> consists of three proteins, rhoptry-associated protein 1 (RAP1), RAP2, and RAP3. The genes encoding RAP1 and RAP2 are known; however, the <i>RAP3i> gene has not been identified. In this study we identify the <i>RAP3i> gene from the <i>P. falciparumi> genome database and show that this protein is part of the low-molecular-mass rhoptry complex. Disruption of<i> RAP3i> demonstrated that it is not essential for merozoite invasion, probably because RAP2 can complement the loss of RAP3. RAP3 has homology with RAP2, and the genes are encoded on chromosome 5 in a head-to-tail fashion. Analysis of the genome databases has identified homologous genes in all <i>Plasmodium sppi>., suggesting that this protein plays a role in merozoite invasion. The region surrounding the<i> RAP3i> homologue in the <i>Plasmodium yoeliii> genome is syntenic with the same region in <i>P. falciparumi>; however, there is a single gene. Phylogenetic comparison of the RAP2/3 protein family from <i>Plasmodium sppi>. suggests that the RAP2/3 duplication occurred after divergence of these parasite species.

Expression of the male reproduction-related gene (Mar-Mrr) in the spermatic duct of the giant freshwater prawn, Macrobrachium rosenbergii

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, <i>Macrobrachium rosenbergiii>, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the <i>Mar-Mrri> gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The <i>Mar-Mrri> gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.

Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant

The <i>Saccharomyces cerevisiae RAD1i> and human <i>XPFi> genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination. An <i>Arabidopsis thalianai> gene (<i>AtRAD1i>) encoding the orthologous plant protein has been identified recently. Here we report the isolation of three structurally distinct <i>AtRAD1i> cDNAs from <i>A. thalianai> leaf tissue RNA. One of the isolates (<i>AtRAD1-1i>) corresponds to the cDNA previously shown to encode the full-length AtRad1 protein, whereas the other two (<i>AtRAD1-2, AtRAD1-3i>) differ slightly in size due to variations at the 5&prime; end of exon 6 or the 3&prime; end of exon 7, respectively. The sequence differences argue that these cDNAs were probably templated by mRNAs generated via alternative splicing. Diagnostic polymerase chain reaction pointed to the presence of the <i>AtRAD1-1i> and <i>AtRAD1-2i> but not <i>AtRAD1-3i> transcripts in bud and root tissue, and to a fourth transcript (<i>AtRAD1-4i>), having both alterations identified in <i>AtRAD1-2i> and <i>AtRAD1-3i>, in root tissue. However, the low frequency of detection of <i>AtRAD1-3i> and <i>AtRAD1-4i> makes the significance of these tissue-specific patterns unclear. The predicted <i>AtRad1-2i>, <i>AtRad1-3i> and <i>AtRad1-4i> proteins lack part of the region likely required for endonuclease complex formation. Expression of <i>AtRAD1-2i> and <i>AtRAD1-3i> in a yeast rad1 mutant did not complement the sensitivity to ultraviolet radiation or the recombination defect associated with the rad1 mutation. These results suggest that alternative splicing may modulate the levels of functional AtRad1 protein.

Complete mitochondrial DNA sequences of the decapod crustaceans pseudocarcinus gigas (Menippidae) and macrobrachium rosenbergii (Palaemonidae)

The complete mitochondrial DNA sequence was determined for the Australian giant crab <i>Pseudocarcinns i>gigas (Crustacea: Decapoda: Menippidae) and the giant freshwater shrimp <i>Macrobrachium rosenbergiii> (Crustacea: Decapoda: Palaemonidae). The Pse gigas and Mrosenbergii mitochondrial genomes are circular molecules, 15,515 and 15,772 bp in length, respectively, and have the same gene composition as found in other metazoans. The gene arrangement of <i>M. rosenbergiii> corresponds with that of the presumed ancestral arthropod gene order, represented by <i>Limulus polyphemusi>, except for the position of the tRNA^Leu(UUR) gene. The<i> Pse. gigasi> gene arrangement corresponds exactly with that reported for another brachyuran, Portunus trituberculatus, and differs from the M. rosenbergii gene order by only the position of the tRNA^His gene. Given the relative positions of intergenic nonoding nucleotides, the “duplication/random loss” model appears to be the most plausible mechanism for the translocation of this gene. These data represent the first caridean and only the second brachyuran complete mtDNA sequences, and a source of information that will facilitate surveys of intraspecific variation within these commercially important decapod species.

Characterization of haemolytic phospholipase A2 activity in clinical isolates of Campylobacter concisus

A membrane-bound, haemolytic phospholipase A2 (PLA2) activity was detected in clinical strains of Campylobacter concisus isolated from children with gastroenteritis. The clinical strains were assigned into two molecular groups (genomospecies) based on PCR amplification of their 23S rDNA. This calcium-dependent, heat-stable, haemolytic PLA2 activity was detected in strains from both genomospecies. A crude haemolysin extract (CHE) was initially prepared from cellular outer-membrane proteins of these isolates and was further fractionated by ultrafiltration. The haemolytic activity of the extracted fraction (R30) was retained by ultrafiltration using a 30 kDa molecular mass cut-off filter, and was designated haemolysin extract (HE). Both CHE and HE had PLA2 activity and caused stable vacuolating and cytolytic effects on Chinese hamster ovary cells in tissue culture. Primers for the conserved region of <i>pldAi> gene (phospholipase A gene) from <i>Campylobacter colii> amplified a gene region of 460 bp in all tested isolates, confirming the presence of a homologous PLA gene sequence in <i>C. i>concisus. The detection of haemolytic PLA₂ activity in <i>C.i> concisus indicates the presence of a potential virulence factor in this species and supports the hypothesis that <i>C.i> concisus is a possible opportunistic pathogen.

Functional studies on the Wilson copper P-Type ATPase and toxic milk mouse mutant

The Wilson protein (WND; ATP7B) is an essential component of copper homeostasis. Mutations in the <i>ATP7Bi> gene result in Wilson disease, which is characterised by hepatotoxicity and neurological disturbances. In this paper, we provide the first direct biochemical evidence that the WND protein functions as a copper-translocating P-type ATPase in mammalian cells. Importantly, we have shown that the mutation of the conserved Met1386 to Val, in the <i>Atp7Bi> for the mouse model of Wilson disease, toxic milk (tx), caused a loss of Cu-translocating activity. These investigations provide strong evidence that the toxic milk mouse is a valid model for Wilson disease and demonstrate a link between the loss of catalytic function of WND and the Wilson disease phenotype.