Electrochemical oxidation of tetracycline antibiotics using a Ti/IrO2 anode for wastewater treatment of animal husbandry.

In animal husbandry, antibiotics are widely used to treat and prevent diseases or to promote growth. The use of antibiotics for domestic animals enables to promote safety of livestock products and enhance productivity. Tetracycline antibiotics (TCs) are one of the primarily used groups of antibiotics for cattle and swine. However, the unintentional spreading of antibiotics from animal waste to the environment may leave out drug residues, promoting resistant strains of bacteria, and will adversely affect the ecosystem and human health. To prevent the spread of veterinary antibiotics in the environment, it is required to treat residual antibiotics in livestock wastewater. In this study, we investigated the electrochemical oxidation of TCs to treat livestock wastewater. The concentrations of TCs in aqueous solutions were reduced from 100 mg/L to less than 0.6 mg/L by 6 h of electrochemical treatment using a Ti/IrO2 anode with Na2SO4 electrolyte. The concentration of oxytetracycline (OTC) in livestock wastewater was also reduced from 100 mg/L to less than 0.7 mg/L by the same treatment. Thus, the electrochemical oxidation using a Ti/IrO2 anode with Na2SO4 electrolyte was found to be effective for degradation of TCs. The results suggest that the electrochemical oxidation method is a promising treatment for TCs in livestock wastewater.

Distinct C-terminus of the B subunit of factor XIII in a population-associated major phenotype: the first case of complete allele-specific alternative splicing products in the coagulation and fibrinolytic systems.

OBJECTIVES: The purpose of this study was to elucidate the molecular bases of the heterogeneity of the B subunit of coagulation factor XIII (FXIII-B), classified by isoelectric focusing into its three population-associated major phenotypes. METHODS AND RESULTS: By genetic sequencing and polymerase chain reaction (PCR)-restriction fragment length polymorphism analyses, a C-to-G change was identified in intron K for the Asian-associated major phenotype FXIII-B*3. A transcript containing the novel exon XII' was detected by reverse transcription PCR using hepatocyte cell lines with this allele. The exclusive existence of a novel C-terminal peptide in a homozygote of FXIII-B*3 was also detected by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The FXIII-B*3 isoform had a C-terminus 15 residues longer than the other isoforms, containing two additional basic amino acids and one extra acidic amino acid. Accordingly, the C-to-G nucleotide substitution created an efficient splice acceptor AG dinucleotide, which resulted in allele-specific alternative splicing in intron K. When compared with FXIII-B*1, the third major phenotype, FXIII-B*2, had an A-to-G change in exon III, converting His95 to Arg, and a rare phenotype, FXIII-B*4, had an A-to-T change in exon VII, converting Glu368 to Val. CONCLUSIONS: We found an extremely rare event of complete allele-specific alternative splicing for FXIII-B. The FXIII-B*3 isoform had a distinct C-terminal peptide, while the FXIII-B*2 and FXIII-B*4 isoforms had His95 to Arg and Glu368 to Val substitutions, respectively, which led to differential isoelectric points of these isoforms. Such variations in the amino acid sequence of FXIII-B may have profound effects on its structure-function relationship, plasma FXIII levels, and disease susceptibility.

Distribution of two Asian-related coding SNPs in the MC1R and OCA2 genes.

Very little is known about the genes and mechanisms affecting skin lightening in Asian populations. In this study, two coding SNPs, c.G1129A (R163Q) at the MC1R (melanocortin 1 receptor) gene and c.A1962G (H615R) at the OCA2 (oculocutaneous albinism type II) gene, were investigated in a total of 1,809 individuals in 16 populations from various areas. The Q163 and R615 alleles prevailed almost exclusively in East and Southeast Asian populations. Wright's F (ST) was 0.445 for R163Q and 0.385 for H615R among the 16 populations. The frequency of the Q163 allele was higher in Northeast Asians than in Southeast Asians. The frequency of the R615 allele was highest in South China and unlikely to be associated with levels of ultraviolet radiation. This allele may be a good marker to study the genetic affinity among East Asians because of its restricted distribution and marked difference in allele frequency.

Distribution of the F374 allele of the SLC45A2 (MATP) gene and founder-haplotype analysis.

The membrane-associated transporter protein (MATP) plays an important role in melanin synthesis. The L374F mutation in the SLC45A2 gene encoding MATP has been suggested to be associated with skin colour in major human populations. In this study more detailed distribution of the F374 allele was investigated in 1649 unrelated subjects from 13 Eurasian populations and one African population. The highest allele frequency was observed in Germans (0.965); French and Italians showed somewhat lower frequencies; and Turks had an intermediate value (0.615). Indians and Bangladeshis from South Asia were characterized by low frequencies (0.147 and 0.059, respectively). We also found the F374 allele in some East and Southeast Asian populations, and explained this by admixture. Haplotype analysis revealed that the haplotype diversity was much lower in Germans than in Japanese, and suggest that the L374F mutation occurred only once in the ancestry of Caucasians. The large differences in distribution of the F374 allele and its haplotypes suggest that this allele may be an important factor in hypopigmentation in Caucasian populations.

Polymerase chain reaction-based analysis using deaminated DNA of dodecamer expansions in CSTB, associated with Unverricht-Lundborg myoclonus epilepsy.

Progressive myoclonus epilepsy of the Unverricht-Lundborg type is an autosomal recessive disorder that is characterized clinically by myoclonic seizures and ataxia. The majority of affected individuals carry repeat expansions of a dodecamer in the promoter region of the cystatin B gene. The unusually high GC content of this tract is refractory to conventional polymerase chain reaction (PCR), and, as a result, a circumventive procedure involving the deamination of DNA with sodium bisulfite has been proposed. This study evaluates the effectiveness of this deamination modification for the detection of dodecamer repeat variants. An analysis of 258 healthy Japanese individuals revealed an allele with four copies of the dodecamer repeat with a frequency of 0.01, in addition to the more commonly observed two and three copy repeat alleles. Homozygous repeat expansions 600 and 680 base pairs in length were detected in the analyses of two affected individuals. For these cases, sequencing, along with an alternative PCR-stutter formation, revealed 41 and 48 copies, respectively, of the dodecamer repeat. The complete conversion of C to T was observed in the expanded tracts, indicating that no methylation occurred at the CpG sites. Based on these results, it was concluded that the use of deaminated DNA allows for a precise analysis of consecutive GC tracts.

Effects of yeast culture and galacto-oligosaccharides on ruminal fermentation in holstein cows.

Four nonlactating, ruminally cannulated Holstein cows were used in a 4 x 4 Latin square design, balanced for residual effects, to evaluate the effects of supplementing dairy cow diets with yeast culture (Trichosporon sericeum; YC), galacto-oligosaccharides (GOS), or the mixture of YC and GOS on ruminal fermentation, microbial N supply, in situ degradation, and energy and nitrogen metabolism. Treatments were arranged in a 2 x 2 factorial as follows: 1) basal diet, 2) basal diet plus 10 g/d YC, 3) basal diet plus 2% GOS, 4) basal diet plus a mixture of 10 g/d YC and 2% GOS. Nitrogen losses in urine were lower, and retained N was higher, for cows supplemented with a mixture of YC and GOS. Ruminal pH was lower in cows supplemented with GOS alone compared with other treatments. Total VFA concentration was higher in cows fed control and GOS-supplemented diets than in those fed YC containing diets. The molar proportion of propionate was higher, and the molar proportion of acetate was lower, in cows fed control diets. Microbial N supply was higher in cows fed control diets. There were no major positive effects of supplements observed in this study. However, supplementation of a mixture of YC and GOS had a tendency for synergistic effects on N metabolism and in situ degradation of a soluble fraction of oat straw DM and CP of concentrates compared with supplementation of YC or GOS alone.

The human complement component C1R gene: the exon-intron structure and the molecular basis of allelic diversity.

Human C1r is a component of the complement system, which is a major mediator of innate immunity. In this study we investigated the exon-intron organization of the human C1R gene, which spans 11 kb from the initiation codon to the stop codon, and is very similar in exon-intron structure to the C1S gene. Six common and rare alleles, C1R*1, C1R*2, C1R*5, C1R*8, C1R*9, and C1R*13, were characterized by five mutations at amino acid positions 114, 135, 146, 167 and 244, in exons 4, 5 and 7 where the CUB1, EGF and CUB2 domains are encoded, respectively. A comparison with the cDNA of the mouse C1r gene showed that C1R*2is likely to be an ancestral allele. In addition, nine nucleotide substitutions and one length polymorphism were found in introns 2, 3, 4, 8 and 10.

Multiplex amplified product-length polymorphism analysis for rapid detection of human mitochondrial DNA variations.

A number of mutations in coding and noncoding regions of mitochondrial DNA (mtDNA) have previously been studied. In the present study, we simultaneously typed six mutation sites in the coding region by use of amplified product-length polymorphism (APLP) analysis. The mtDNA variations of 2471 individuals from 20 populations of Japanese, Korean, Chinese, and German were examined and classified into 18 haplotypes. Two of these haplotypes, B1 (estimated ancestral haplotype) and C1, were distributed among all populations tested. However, the haplotypes A1, A2, B2, B3, and C2 were mostly restricted to the Mongoloid populations, whereas haplotypes B5 and C5 appeared almost exclusively in the German population. Phylogenetic analysis by the neighbor-joining method revealed that the Japanese populations were more closely related to each other than to the other East Asian populations surveyed. The multiplex APLP method is suitable for large-scale screening studies of mtDNA variability because it is both rapid and economical.

Characterization of genomic rearrangements of the alpha1-acid glycoprotein/orosomucoid gene in Ghanaians.

In this study, the structure of the alpha1-acid glycoprotein (AGP), or orosomucoid (ORM), gene was investigated in a Ghanaian mother and her child, who shared an unusual variant, ORM1 S2(C), found by isoelectric focusing. Three remarkable changes of nucleotide sequence were observed: (1) The two ORM1 alleles, ORMI*S and ORMI*S2(C), had the AGP2 gene-specific sequence at one and three regions, respectively, in exon 5 to intron 5. The variant allele originating from ORMi*S was characterized by a G-to-A transition, resulting in an amino acid change from valine to methionine, which is also detected in ORM1 F2, a form that is common in Europeans. (2) The AGP2 gene of the child, inherited from the father, was duplicated, as revealed by long-range polymerase chain reaction. (3) Three new mutations were observed in two exons of the AGP2 genes of the mother and child. All of these novel genomic rearrangements, which were not observed in Japanese subjects, may have arisen through point mutation, gene conversion, and unequal crossover events. It is likely that the rearrangement of the AGP gene has often occurred in Africans.

Haplotype analysis of the human alpha2-HS glycoprotein (fetuin) gene.

Alpha2-HS glycoprotein (AHSG), which is equivalent to fetuin in other species, is a protein found in human plasma. AHSG is polymorphic with two common alleles and many variants. To examine the intragenic haplotypes and their diversity at this locus, a contiguous genomic DNA sequence (10.3 kb) was analyzed in 20 samples (40 chromosomes), and haplotypes were determined for 309 subjects. Judging from the aligned nucleotide sequences and the conserved amino acid residues comparing human and chimpanzee AHSG, it was concluded that the type 1 allele is probably older and has evolved into four major suballeles. The type 2 allele was generated from one branch of the type 1 allele. AHSG*3 and *5 variants were each found to have a single nucleotide change in exon 7, resulting in the change of an amino acid residue from Arg299 to Cys and from Asp258 to Asn, respectively. It was noted that the AHSG*3 mutation gives rise to an additional cysteine residue, which possibly affects the conformation of the protein. The AHSG gene was found to have a low mutation rate and no apparent recombination events. Furthermore, the detected substitutions were nonhomogeneously distributed at this locus. In particular, four nonsynonymous substitutions were concentrated in the carboxyl-terminal domain.

A novel dimorphism in the human SRY gene: usefulness in human migration studies.

A nucleotide polymorphism of C or T was detected at position 465 in the sex-determining region Y (SRY) gene. To evaluate the utility of this dimorphism in human population studies, the frequency and the frequency of the haplotype combined with the two polymorphic loci YAP and M9 were examined in a total of 130 unrelated Japanese and 130 unrelated German males. The T nucleotide was found in 24.6% (32/130) of the Japanese but not in any of the 130 German males. Accordingly, four of the eight possible combination haplotypes of SRY/YAP/M9 were identified in the Japanese population, but one of the four haplotypes comprising SRY(T) was absent in the German samples. This suggests that the C to T transition may be more recent than the YAP insertion or the M9 transversion and the change might have occurred in an ancestral Asian population. These results imply that the dimorphism at the SRY gene is one of the Y-linked markers useful for human population studies and also for ethnic identification of forensic samples.

A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers.

We present a simple and rapid polymerase chain reaction (PCR)-based technique, termed consumed allele-specific primer analysis (CASPA), as a new strategy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with several noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into the PCR products are determined. Thus, nucleotide substitutions are identified by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present method, the allele-specific primer only anneals with the target polymorphic site on the DNA, so it is not necessary to analyze the PCR products. Therefore, this method is only little affected by modification of the PCR products. The CASPA method is expected to be a useful tool for typing of SNPs.

Molecular characterization of four alpha-1-antitrypsin variant alleles found in a Japanese population: a mutation hot spot at the codon for amino acid 362.

In this study alpha-1-antitrypsin (AAT) phenotypes at the protease inhibitor (PI) locus were determined by isoelectric focusing of native and desialylated serum samples from 236 Japanese subjects living in the western part of Japan. The shifts in relative mobility between some PI types were observed before and after desialylation. This technique was useful in distinguishing between some PI M subtypes and variants. The molecular basis of four variant alleles, including two new alleles found in this study, was characterized: PI E(tokyo) [Lys(335)(AAG)--> Glu(GAG)] and PI N(nagato) [Leu(276)(CTG)-->Pro(CCG)] arose from PI M1(Val(213)) and PI M2, respectively. A new PI P(yonago) [Asp(19)(GAT)-->Ala(GCT)] originated from PI M1(Val(213)). A new PI M5(gunma) [Pro(362)(CCC)-->Ser(TCC)], arising from PI M3, was the sixth allele involving a mutation at codon 362, which is suggested to be a mutation hot spot. PI M5(gunma) was likely to show normal AAT levels and function although the mutations occurred near codon 358 for Met(358). The molecular basis of PI variant alleles found in Japanese was different from that reported in previous studies.

Laser soldering with light-intensity patterns reconstructed from computer-generated holograms.

We describe a laser soldering technology with diffractive optics. This technology provides efficient one-step laser illumination for components to be soldered. A phase-only computer-generated hologram set in a variable-focal-length optical configuration generates a diffraction pattern with the dimensions required for processing solder. We verified the effectiveness of the proposed scheme by sealing a ceramic package such that it could house a quartz device. The factors for successful soldering include alignment of the diffraction pattern to the work piece, the thermal properties of the materials involved, and the wavelength of the laser used to process the solder. The beam intensity across the diffraction pattern influences the process, and the 0th-order intensity should be minimized to prevent damage to the work piece.

[Application of the PCR-APLP method to determine ABO genotypes in forensic samples].

We carried out ABO genotyping of forensic samples by the amplified product length polymorphism (APLP) technique. We present two novel systems. One is termed as eight primers system, in which eight allele-specific primers are added into a single PCR reaction. Another is termed as six primers system. In both APLP systems, all alleles were clearly detected using DNA purified from forensic samples. In PCR amplification with direct addition of specimen, ABO genotyping was also possible to blood stain, seminal stain, blood, saliva and urine. Furthermore, ABO genotyping worked only to chimpanzee. This PCR-APLP method should be convepffnt and valuable for forensic practice.

The rearrangement of the human alpha(1)-acid glycoprotein/orosomucoid gene: evidence for tandemly triplicated genes consisting of two AGP1 and one AGP2.

The human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is controlled by the two tandemly arranged genes, AGP1 and AGP2. The further duplication of the AGP1 gene has been suggested by a few duplicated ORM1 locus haplotypes including ORM1*F1. S and ORM1*B9. S, detected by isoelectric focusing. To clarify the triplication of the AGP gene, 39 DNA samples from Japanese subjects were studied by the long-range PCR of intergenic regions. The analysis of PCR products showed that the tandemly triplicated genes, AGP1A-AGP1B-AGP2, occurred on about 20% of chromosomes. These composites were divided into ORM1A*F1-ORM1B*S-ORM2*M and ORM1A*B9-ORM1B*S-ORM2*M by allelic variations. Furthermore, the former was classified into a few haplotypes by three synonymous sequence variations, which might have arisen through gene conversion-like events. The recombination breakpoints existed between the 5' flanking region and intron 2 of the AGP1B gene. Thus, it is likely that the rearrangement of the AGP gene has often occurred.

DXS10011: a hypervariable tetranucleotide STR polymorphism on the X chromosome.

The locus DXS10011 is a polymorphic system with a tetranucleotide repeat sequence located on the human X chromosome. The distribution of allele frequencies was examined in 334 Japanese and 171 German individuals and a total of 36 alleles was detected in the two population groups. This STR polymorphism will be a useful marker for linkage analysis.

Improved haplotype analysis of human myelin basic protein short tandem repeat loci.

We report an improved haplotype analysis of the human myelin basic protein gene (MBP) short tandem repeat (STR) polymorphism. The polymorphic G-->A transition and 2 conventional STR polymorphisms, MBPA and MBPB, were simultaneously determined by an amplified product length polymorphism technique. After the MBPC fragments containing MBPA and MBPB were amplified, the linkage of these 2 STR loci was determined by a second amplification, using polymerase chain reaction (PCR) technique, of the isolated MBPC fragments. The present haplotype analysis dispensed with family studies for the haplotyping of MBPA and MBPB. Polymorphisms of the MBP loci studied in German and Japanese populations showed a high genomic variation. Haplotype analysis of the MBP loci showed distinct differences between the German and the Japanese populations. Consequently, haplotype analysis of the MBP loci promises to be useful in forensic identification and paternity testing.

Optical resolution of (+/-)-1-aryl-1-alkanols using enantioselective transesterification by lipases.

Enantioselective transesterification of 1-phenyl-1-alkanols (PhCH(OH)(CH2)n-2CH3; n = 2, 3, 4, 5, 6, 9, 12, 18) with vinyl acetate catalyzed by lipases in benzene has been studied to find the catalyst which is generally used for preparing optically active 1-phenyl-1-alkanols having various alkyl chains. Amongst lipases examined (lipases LIP, PS, AK, CAL and RML), the lipase from Pseudomonas aeruginosa (LIP) is the best catalyst which shows high reactivity and enantioselectivity and low substrate specificity. The rate of the LIP-catalyzed transesterification decreases with increasing the alkylchain length till n = 4. The catalysis of LIP recovers again toward the alkanols with n = 5-18. Other lipases do not exhibit such an effect of alkyl-chain length and show very poor or no catalysis for the alkanols with n > or = 4. LIP is also the best catalyst for the enantioselective transesterification of 1-(1-naphthyl)-, 1-(2-naphthyl)- and 1-(1-pyrenyl)-1-propanols. Each optically pure 1-aryl-1-alkanol was isolated by the present method.

Molecular analysis of the human orosomucoid gene ORM1*Q0köln responsible for incompatibility in a German paternity case.

In a German paternity test, an alleged father was excluded only by reverse homozygosity of ORM1 phenotypes (mother ORM1 S, child ORM1 S and alleged father ORM1 F1) out of the 28 classical and DNA markers investigated. Without the ORM1 system the biostatistical probability of paternity was calculated to exceed 99.999%. The intensity of the immunoprinted bands of the ORM1 protein for the child and alleged father after isoelectric focusing appeared to be reduced to about half. To identify a possible null allele, gene-specific amplification followed by single-strand conformation polymorphism and sequencing analyses were carried out. Deletion of one of the two copies of a 4 bp direct repeat sequence (GTCT) in exon 4 of the consensus sequence of ORM1*F1 was observed in the child and alleged father. Thus, the sharing of a rare mutant gene, ORM1*Q0köln, increased the probability of paternity.