BackgroundNumerous strategies have been proposed to decrease the treatment time a patient requires in orthodontic treatment. Recently, a number of device-accelerated therapies have emerged in orthodontics. Photobiomodulation is an emerging area of science that has clinical applications in a number of human biological processes. The aim of this study was to determine if photobiomodulation reduces the treatment time in the alignment phase of orthodontic treatment.MethodsThis multicenter clinical trial was performed on 90 subjects (73 test subjects and 17 controls), and Little's Index of Irregularity (LII) was used as a measure of the rate of change of tooth movement. Subjects requiring orthodontic treatment were recruited into the study, and the LII was measured at regular time intervals. Test subjects used a device which produced near-infrared light with a continuous 850-nm wavelength. The surface of the cheek was irradiated with a power density of 60 mW/cm2 for 20 or 30 min/day or 60 min/week to achieve total energy densities of 72, 108, or 216 J/cm2, respectively. All subjects were fitted with traditional orthodontic brackets and wires. The wire sequences for each site were standardized to an initial round alignment wire (014 NiTi or 016 NiTi) and then advanced through a progression of stiffer arch wires unit alignment occurred (LII < 1 mm).ResultsThe mean LII scores at the start of the clinical trial for the test and control groups were 6.35 and 5.04 mm, respectively. Multi-level mixed effect regression analysis was performed on the data, and the mean rate of change in LII was 0.49 and 1.12 mm/week for the control and test groups, respectively.ConclusionsPhotobiomodulation produced clinically significant changes in the rates of tooth movement as compared to the control group during the alignment phase of orthodontic treatment.

A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry. S. clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a ␤-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin.Streptomyces clavuligerus produces several secondary metabolites with interesting pharmacological activities. It synthesizes the ␤-lactam antibiotic cephamycin C, the ␤-lactamase inhibitor clavulanic acid, and several antifungal compounds with a clavam structure (for reviews, see references 3 and 15). The clavulanic acid biosynthesis pathway has several steps in common with the pathway for clavam biosynthesis (18,19). In addition to the compounds indicated above, S. clavuligerus produces the antibiotics holomycin and tunicamycin (10). Holomycin is a compound with pyrrothine structure, while tunicamycin is a glucosamine-containing antibiotic. This wealth of genetic information for the biosynthesis of secondary metabolites is characteristic of some Streptomyces species (4, 22).S. clavuligerus is an excellent model for the study of the relationships between the regulatory mechanisms controlling the biosynthesis of the different secondary metabolites produced by these microorganisms. Formation of clavulanic acid is controlled by a LysR-type regulatory protein encoded by the claR gene. Formation of both clavulanic acid and cephamycin C in S. clavuligerus is controlled by the positive autoregulatory protein CcaR (25, 32). Mutant strains with disruption in ccaR do not express the claR gene (26), although this control is not exerted directly by the CcaR regulatory protein and appears to involve a cascade mechanism (32). The control of the formation of cephamycin C and/or clavulanic acid by CcaR or ClaR is exerted at the transcription level (1, 25).However, the ccaR::aph S....

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.

The ATP-binding cassette transporter ABCG2 restricts the exposure of certain drugs and natural compounds in different tissues and organs. Its expression in the mammary gland is induced during lactation and is responsible for the active secretion of many compounds into milk, including antimicrobial agents. This particular function of ABCG2 may affect drug efficacy against mastitis and the potential presence of drug residues in the milk. Previous in vitro and in vivo studies showed increased transport of several compounds, including fluoroquinolones, by the bovine ABCG2 Y581S polymorphism. Our main purpose was to study the potential effect of this bovine ABCG2 polymorphism on the secretion into milk of the antimicrobial danofloxacin administered at the therapeutic dose of 6mg/kg used for mastitis treatment. In addition, the effect of this polymorphism on the relative mRNA and protein levels of ABCG2 by quantitative real-time PCR and Western blot were studied. Danofloxacin 18% (6mg/kg) was administered to 6 Y/Y homozygous and 5 Y/S heterozygous cows. Danofloxacin levels in milk and milk-to-plasma concentration ratios were almost 1.5- and 2-fold higher, respectively, in Y/S cows compared with the Y/Y cows, showing a higher capacity of this variant to transport danofloxacin into milk. Furthermore, the higher activity of this polymorphism is not linked to higher ABCG2 mRNA or protein levels. These results demonstrate the relevant effect of the Y581S polymorphism of the bovine ABCG2 transporter in the secretion into milk of danofloxacin after administration of 6mg/kg, with potentially important consequences for mastitis treatment and for milk residue handling.

The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.Streptomyces clavuligerus produces clavulanic acid (CA), an important ␤-lactamase inhibitor synthesized by the condensation of arginine (23,27), and glyceraldehyde-3-phosphate to form carboxyethylarginine (12, 18). In wild-type strains, arginine is a limiting precursor for clavulanic acid biosynthesis (23). Studies of the arginine gene cluster have been highly interesting as a way of understanding the channeling of this precursor into clavulanic acid (20,21).The presence of a cyclic arginine pathway in Streptomyces species has been reported previously (20). The S. clavuligerus arginine gene cluster contains an argJ gene encoding a protein of 39,733 Da, similar to other ArgJ proteins, as deduced from the genome sequences of Streptomyces coelicolor (74.9% identical amino acids), Mycobacterium tuberculosis (47.8% identity), and Corynebacterium glutamicum (40.7% identity). ArgJ recycles the acetyl group from N-acetylornithine to L-glutamic acid in the first step of the pathway. The argJ gene complements an Escherichia coli XSD2 mutant lacking N-acetylornithinase activity, indicating that the ornithine N-acetyltransferase (OAT) activity encoded by argJ is able to complement the N-acetylornithinase activity encoded by the argE gene of the lineal arginine pathway in E. coli.In the clavulanic acid gene cluster of S. clavuligerus, the oat2 gene (initially described as ORF6), located downstream of the pah gene, encodes a protein of 41,607 Da with a high similarity to ornithine acetyltransferases (9). ArgJ and Oat2 share 31.1% amino acid identity over the entire sequence. When oat2 was subcloned and transformed into E. coli XSD2, the argE mutation of this strain was complemented, indicating that the oat2 promoter was expressed in E. coli; moreover, the transformants showed a strong OAT activity but no ornithinase activity (20). A putative ARG box, which is characteristic of genes tha...

Flunixin meglumine is a nonsteroidal anti-inflammatory drug (NSAID) widely used in veterinary medicine. It is indicated to treat inflammatory processes, pain, and pyrexia in farm animals. In addition, it is one of the few NSAIDs approved for use in dairy cows, and consequently gives rise to concern regarding its milk residues. The ABCG2 efflux transporter is induced during lactation in the mammary gland and plays an important role in the secretion of different compounds into milk. Previous reports have demonstrated that bovine ABCG2 Y581S polymorphism increases fluoroquinolone levels in cow milk. However, the implication of this transporter in the secretion into milk of anti-inflammatory drugs has not yet been studied. The objective of this work was to study the role of ABCG2 in the secretion into milk of flunixin and its main metabolite, 5-hydroxyflunixin, using Abcg2 (2/2) mice, and to investigate the implication of the Y581S polymorphism in the secretion of these compounds into cow milk. Correlation with the in vitro situation was assessed by in vitro transport assays using Madin-Darby canine kidney II cells overexpressing murine and the two variants of the bovine transporter. Our results show that flunixin and 5-hydroxyflunixin are transported by ABCG2 and that this protein is responsible for their secretion into milk. Moreover, the Y581S polymorphism increases flunixin concentration into cow milk, but it does not affect milk secretion of 5-hydroxyflunixin. This result correlates with the differences in the in vitro transport of flunixin between the two bovine variants. These findings are relevant to the therapeutics of anti-inflammatory drugs.

The bovine adenosine triphosphate-binding cassette transporter G2 (ABCG2/breast cancer resistance protein) polymorphism Tyr581Ser (Y581S) has recently been shown to increase in vitro transepithelial transport of antibiotics. Since this transporter has been extensively related to the active secretion of drugs into milk, the potential in vivo effect of this polymorphism on secretion of xenobiotics in livestock could have striking consequences for milk production, the dairy industry, and public health. Our purpose was to study the in vivo effect of this polymorphism on the secretion of danofloxacin, a widely used veterinary antibiotic, into milk. Danofloxacin (1.25 mg/kg) was administered to six Y/Y 581 homozygous and six Y/S 581 heterozygous lactating cows, and plasma and milk samples were collected and analyzed by high-performance liquid chromatography. No differences were found in the pharmacokinetic parameters of danofloxacin in plasma between the two groups of animals. In contrast, Y/S heterozygous cows showed a 2-fold increase in danofloxacin levels in milk. In addition, the pharmacokinetic elimination parameters, mean residence time and elimination half-life, were significantly lower in the milk of the animals carrying the Y/S polymorphism. These in vivo results are in agreement with our previously published in vitro data, which showed a greater capacity of the S581 variant in accumulation assays, and demonstrate, for the first time, an important effect of the Y581S single-nucleotide polymorphism on antibiotic secretion into cow milk. These findings could be extended to other ABCG2 substrates, and may be relevant for the treatment of mastitis and for the design of accurate and novel strategies to handle milk residues. IntroductionThe ATP-binding cassette (ABC) transporter ABCG2 (breast cancer resistance protein) is expressed in a wide range of tissues and organs, including the intestine, liver, blood-brain barrier, and mammary gland (van Herwaarden and Schinkel, 2006). It affects the bioavailability of its substrates and mediates the active secretion of xenobiotics and several vitamins in milk (van Herwaarden et al., 2007). The occurrence of drug residues in milk could lead to the development of bacterial resistance, allergies, or hypersensitivity reactions in consumers (McManaman and Neville, 2003). On the other hand, effective treatments for mastitis may require a considerable transfer of drugs into milk (Escudero et al., 2007). All of these issues are of great concern for general public health, the dairy industry, and veterinary therapeutics. Hence, identification of relevant factors for the transfer of drugs into milk in livestock constitutes a priority.Changes in the expression and/or the function of ABCG2 can lead to dramatic variations in the pharmacokinetics and secretion of its substrates into milk Ni et al., 2010). Several single-nucleotide polymorphisms (SNPs) have been studied in human ABCG2, and of these, Gln141Lys (Q141K) is one of the most important, causing impaired urate transport wh...

In the present study, the effects of orally administered beta-casomorphins (beta-CM) and met-enkephalin on postprandial plasma somatostatin-like immunoreactivity (SLI) were assessed in conscious dogs. The intragastric instillation of a liver extract-sucrose test meal containing 12 mg beta-CM or 10 mg met-enkephalin, respectively, augmented the postprandial rise of peripheral vein plasma SLI levels significantly. This effect was inhibited by the additional administration of the specific opiate-receptor antagonist, naloxone. When liver extract-sucrose was dissolved in fresh bovine milk the increase of plasma SLI levels was significantly greater than liver extract-sucrose dissolved in water. This milk-induced augmentation of SLI levels was also reduced by naloxone. Since these opiate-active compounds have an influence upon insulin release when given iv, the effect of beta-CM-7, beta-CM-5, beta-CM-4 beta-CM-4-amide, and met-enkephalin on SLI levels was assessed during their iv infusion at a rate of 1 nmol/kg . h during an iv background infusion of a glucose-amino acid mixture. The infusion of beta-CM-5 elicited a stimulation of peripheral vein SLI levels, whereas the infusion of met-enkephalin resulted in a significant decrease of SLI levels. beta-CM-7, beta-CM-4, and beta-CM-4-amide had no effect on plasma SLI levels at the dose employed. The present data demonstrate that in dogs the ingestion of opiate-active peptide stimulates postprandial SLI release, indicating that nutrient-contained opiate-active material (exorphins) might participate in the regulation of postprandial gastrointestinal endocrine function.