Replacement Of Methionine Research Articles

Atomic Tailoring of Ubiquitin Side Chains Influences E2‐Mediated Ubiquitin Chain Formation

AbstractUbiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i. e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi‐milligram scales. While some modifications including N‐terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2 K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13‐mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein.

Rescue of Methionine Dependence by Cobalamin in a Human Colorectal Cancer Cell Line.

Methionine dependence is a characteristic of most cancer cells where they are unable to proliferate when the essential amino acid methionine is replaced with its precursor homocysteine in the growing media. Normal cells, on the other hand, thrive under these conditions and are referred to as methionine-independent. The reaction that adds a methyl group from 5-methyltetrahydrofolate to homocysteine to regenerate methionine is catalyzed by the enzyme methionine synthase with the cofactor cobalamin (vitamin B12). However, decades of research have shown that methionine dependence in cancer is not due to a defect in the activity of methionine synthase. Cobalamin metabolism has been tied to the dependent phenotype in rare cell lines. We have identified a human colorectal cancer cell line in which the cells regain the ability to proliferation in methionine-free, L-homocystine-supplemented media when cyanocobalamin is supplemented at a level of 1 µg/mL. In human SW48 cells, methionine replacement with L-homocystine does not induce any measurable increase in apoptosis or reactive oxygen species production in this cell line. Rather, proliferation is halted, then restored in the presence of cyanocobalamin. Our data show that supplementation with cyanocobalamin prevents the activation of the integrated stress response (ISR) in methionine-deprived media in this cell line. The ISR-associated cell cycle arrest, characteristic of methionine-dependence in cancer, is also prevented, leading to the continuation of proliferation in methionine-deprived SW48 cells with cobalamin. Our results highlight differences between cancer cell lines in the response to cobalamin supplementation in the context of methionine dependence.

Probable Novel APP Met671Leu Mutation in a Chinese Han Family with Early-Onset Alzheimer's Disease.

Familial Alzheimer's disease (AD) is a rare disease caused by autosomal-dominant mutations. APP (encoding amyloid precursor protein), PSEN1 (encoding presenilin 1), and PSEN2 (encoding presenilin 2) are the most common genes cause dominant inherited AD. This study aimed to demonstrate a Chinese early-onset AD pedigree presenting as progressive memory impairment, apraxia, visual-spatial disorders, psychobehavioral disorders, and personality changes with a novel APP gene mutation. The family contains four patients, three carries and three normal family members. The proband underwent brain magnetic resonance imaging (MRI), 18F-fludeoxyglucose positron emission tomography (18F-FDG-PET), cerebrospinal fluid amyloid detection, 18F-florbetapir (AV-45) Positron Emission Computed Tomography (PET) imaging, whole-exome sequencing and Sanger sequencing. Brain MRI images showed brain atrophy, especially in the entorhinal cortex, temporal hippocampus, and lateral ventricle dilation. The FDG-PET showed hypometabolism in the frontotemporal, parietal, and hippocampal regions. 18F-florbetapir (AV-45) PET imaging showed cerebral cortex Aβ protein deposition. The cerebrospinal fluid amyloid protein test showed Aβ42/Aβ40 ratio decreases, pathological phosphor-tau level increases. Whole-exome sequencing detected a new missense mutation of codon 671 (M671L), which was a heterozygous A to T point mutation at position 2011 (c.2011A > T) in exon 16 of the amyloid precursor protein, resulting in the replacement of methionine to Leucine. The co-separation analysis was validated in this family. The mutation was found in 3 patients, 3 clinical normal members in the family, but not in the other 3 unaffected family members, 100 unrelated normal subjects, or 100 sporadic patients with AD. This mutation was probably pathogenic and novel in a Chinese Han family with early-onset AD.

Characterization of methionine dependence in melanoma cells.

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of S-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium.

Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic E. coli BL-21 and auxotrophic E. coli B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b5 (b5M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic E. coli, ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and b5M46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70-80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg.

Effects of dietary supplementation with herbal extract as methionine replacer on growth performance, meat composition, oxidative stability and liver gene expression in broiler chickens

Abstract In the present study, an herbal feed additive was tested for partial-to-complete replacement of synthetic methionine in poultry diets, along with its effects on performance, breast and thigh meat chemical composition, oxidative stability during refrigerated storage and the expression of five target genes in liver. In a 35 days trial, 600 one-day-old male chicks were randomly allocated to 4 groups with 10 replicates. Birds in the control group were fed a regular maize–soybean-based diet that covered DL-methionine needs while the second group (Meth40) was similar to control but contained only DL – Methionine at 40% of control diet. Diet in third group contained DLMethionine at 40% of control and the herbal feed additive Methiorep TMMeth40+Mrep) with extracts of Boerhavia diffusa, Azadirachta indica, Vigna mungo and Trigonella foenum-graecum. Diet of fourth group was formulated to totally replace DL Methionine by Methiorep TM (Mrep). Body weight gain and feed consumption were weekly recorded. At the end of the trial, all birds were slaughtered and 2 chickens per pen were selected for meat and liver sampling. The liver was tested for the expression of five target genes, namely Methionine synthase (MTR), Tyrosine aminotransferase (TAT), Spermidine synthase (SMS), Methionine sulfoxide reductase (MSRB1) and Betaine homocysteine S-methyltransferase (BHMT). The results showed that the Meth40 group had reduced body weight compared to the Meth40+Mrep group while the Control and Mrep groups had comparable weights. Feed intake and feed conversion ratio did not differ among the experimental groups. Carcass, breast and thigh meat yield were higher in the Mrep and the Control compared to Meth40 and Meth40+Mrep groups. Also, meat oxidation was significantly lower in herbal groups compared to the control group. After normalization to β-actin expression, quantitative real-time PCR analysis revealed an induction in the expression of MTR and SMS genes in the liver of both herbal treated groups. No changes were observed for the TAT, MSRB1and BHMT genes in the herbal treated groups compared to the control or the Meth40 group. In conclusion, herbal feed additives with specific plant extracts may be able to improve both growth performance and antioxidant activity of broiler chickens, phenolic content; yet, they may also support in amino acid efficient use of broiler.

Dietary methionine starvation impairs acute myeloid leukemia progression

AbstractTargeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, nuclear magnetic resonance−based metabolite quantifications and 13C-tracing, polysomal profiling, and chromatin immunoprecipitation sequencing, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis, and induced a cell cycle block. Reactive oxygen species levels were not increased following methionine depletion, and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.

Structural Characterization of Cytochrome c′β‐Met from an Ammonia-Oxidizing Bacterium

The ammonia-oxidizing bacterium Nitrosomonas europaea expresses two cytochromes in the P460 superfamily that are predicted to be structurally similar. In one, cytochrome (cyt) P460, the substrate hydroxylamine (NH2OH) is converted to nitric oxide (NO) and nitrous oxide (N2O) requiring a unique heme-lysyl cross-link in the catalytic cofactor. In the second, cyt , the cross-link is absent, and the cytochrome instead binds H2O2 forming a ferryl species similar to compound II of peroxidases. Here, we report the 1.80 Å crystal structure of cyt ─a well-expressed protein in N. europaea with a lysine to a methionine replacement at the cross-linking position. The structure of cyt is characterized by a large β-sheet typical of P460 members; however, several localized structural differences render cyt distinct. This includes a large lasso-like loop at the "top" of the cytochrome that is not observed in other structurally characterized members. Active site variation is also observed, especially in comparison to its closest homologue cyt from the methane-oxidizing Methylococcus capsulatus Bath, which also lacks the cross-link. The phenylalanine "cap" which is presumed to control small ligand access to the distal heme iron is replaced with an arginine, reminiscent of the strictly conserved distal arginine in peroxidases and to the NH2OH-oxidizing cytochromes P460. A critical proton-transferring glutamate residue required for NH2OH oxidation is nevertheless missing in the active site. This in part explains the inability of cyt to oxidize NH2OH. Our structure also rationalizes the absence of a methionyl cross-link, although the side chain's spatial position in the structure does not eliminate the possibility that it could form under certain conditions.

Potent SARS-CoV-2 mRNA Cap Methyltransferase Inhibitors by Bioisosteric Replacement of Methionine in SAM Cosubstrate.

Viral mRNA cap methyltransferases (MTases) are emerging targets for the development of broad-spectrum antiviral agents. In this work, we designed potential SARS-CoV-2 MTase Nsp14 and Nsp16 inhibitors by using bioisosteric substitution of the sulfonium and amino acid substructures of the cosubstrate S-adenosylmethionine (SAM), which serves as the methyl donor in the enzymatic reaction. The synthetically accessible target structures were prioritized using molecular docking. Testing of the inhibitory activity of the synthesized compounds showed nanomolar to submicromolar IC50 values for five compounds. To evaluate selectivity, enzymatic inhibition of the human glycine N-methyltransferase involved in cellular SAM/SAH ratio regulation was also determined, which indicated that the discovered compounds are nonselective inhibitors of the studied MTases with slight selectivity for Nsp16. No cytotoxic effects were observed; however, this is most likely a result of the poor cell permeability of all evaluated compounds.

Differences in cell death in methionine versus cysteine depletion.

Restriction of the sulfur amino acids methionine and cysteine has recently been proposed as potential adjuvant therapy in cancer. While cysteine depletion has been associated with ferroptotic cell death, methionine depletion has not. We hypothesized that comparing the response of melanoma cell lines to depletion of the amino acids methionine and cysteine would give us insight into the critical role in cancer of these two closely related amino acids. We analyzed the response to three conditions: methionine depletion, methionine replacement with homocysteine, and cysteine depletion. In cancer cells, the transcription factor ATF4 was induced by all three tested conditions. The replacement of methionine with homocysteine produced a strong ferroptotic gene signature. We also detected an activation of the NRF2 antioxidant pathway by both methionine and cysteine depletion. Total glutathione levels were decreased by 42% in melanoma cells grown without methionine, and by 95% in cells grown without cysteine. Lipid peroxidation was increased in cells grown without cysteine, but not in cells grown without methionine. Despite the large degree of overlap in gene expression between methionine and cysteine depletion, methionine depletion and replacement of methionine with homocysteine was associated with apoptosis while cysteine depletion was associated with ferroptosis. Glutamine depletion produced comparable gene expression patterns and was associated with a 28% decrease in glutathione. Apoptosis was detected in these cells. In this experiment, a strong ATF4-driven ferroptotic gene signature was insufficient to induce ferroptosis without a concomitant profound decrease in glutathione levels.

32219 Differences in cell death in methionine versus cysteine depletion

ABSTRACT IMPACT: Reducing methionine levels has repeatedly been shown to reduce cancer growth in vivo, while at the same time increasing lifespan in healthy animals. However, the mechanisms behind the beneficial effects of methionine restriction are currently unknown. OBJECTIVES/GOALS: We hypothesized that comparing the response of a cancer cell line to depletion of the amino acids methionine and cysteine would give us insight into the critical role of these two closely related amino acids in cancer, and help advance methionine restriction on the translational science spectrum. METHODS/STUDY POPULATION: We used the human melanoma cell line A101D to analyze the response to three conditions: methionine depletion, methionine replacement with homocysteine, and cysteine depletion in vitro. We measured proliferation/viability, gene expression patterns, and glutathione levels. We also assessed ferroptotic versus apoptotic cell death. We then used a normal human fibroblast cell line to compare responses. RESULTS/ANTICIPATED RESULTS: The transcription factors ATF4 and NRF2 were activated by all three tested conditions in melanoma cells. Glutathione levels were decreased by ˜40% in cells grown without methionine, and by 95% in cells grown without cysteine. Lipid peroxidation was increased in cells grown without cysteine, but not in cells grown without methionine. Inhibiting ferroptotic cell death partially rescued proliferation in cysteine-depleted but not in methionine-depleted cells. Almost 70% of cells grown in methionine-depleted media stained positive for Annexin V, an indicator of apoptosis, compared to only 20% of cells grown in cysteine-depleted media. In normal cells, ferrostatin recued proliferation/viability to 86% of control levels in cysteine-depleted cells. Ferrostatin did not affect methionine-depleted normal cells. DISCUSSION/SIGNIFICANCE OF FINDINGS: These results indicate that methionine depletion leads to apoptosis, while cysteine depletion leads to ferroptosis. We found overlapping pathways activated by methionine and cysteine depletion at the gene expression levels, but divergences in cell death pathways ultimately activated.

Selenium Biofortification: Roles, Mechanisms, Responses and Prospects.

The trace element selenium (Se) is a crucial element for many living organisms, including soil microorganisms, plants and animals, including humans. Generally, in Nature Se is taken up in the living cells of microorganisms, plants, animals and humans in several inorganic forms such as selenate, selenite, elemental Se and selenide. These forms are converted to organic forms by biological process, mostly as the two selenoamino acids selenocysteine (SeCys) and selenomethionine (SeMet). The biological systems of plants, animals and humans can fix these amino acids into Se-containing proteins by a modest replacement of methionine with SeMet. While the form SeCys is usually present in the active site of enzymes, which is essential for catalytic activity. Within human cells, organic forms of Se are significant for the accurate functioning of the immune and reproductive systems, the thyroid and the brain, and to enzyme activity within cells. Humans ingest Se through plant and animal foods rich in the element. The concentration of Se in foodstuffs depends on the presence of available forms of Se in soils and its uptake and accumulation by plants and herbivorous animals. Therefore, improving the availability of Se to plants is, therefore, a potential pathway to overcoming human Se deficiencies. Among these prospective pathways, the Se-biofortification of plants has already been established as a pioneering approach for producing Se-enriched agricultural products. To achieve this desirable aim of Se-biofortification, molecular breeding and genetic engineering in combination with novel agronomic and edaphic management approaches should be combined. This current review summarizes the roles, responses, prospects and mechanisms of Se in human nutrition. It also elaborates how biofortification is a plausible approach to resolving Se-deficiency in humans and other animals.

Identification of a novel c.1A>G variant of GDAP1 gene in a pedigree affected with autosomal recessive fibula atrophy

To explore the genetic basis for a pedigree affected with Charcot-Marie-Tooth (CMT) disease through high-throughput sequencing. Potential variants of the genes associated with CMT were screened by next-generation sequencing (NGS) of the members of the pedigree. NGS has revealed that the two affected sisters both harbored homozygous c.1A>G variant of the GDAP1 gene, which caused replacement of the first amino acid Methionine by Valine (p.Met1Val). Their parents were both carriers of the heterozygous c.1A>G variant. The variant was unreported previously and has an extremely low frequency in the population. Meanwhile, one of the sisters and the mother also carried heterozygous c.710A>T variant of the BAG3 gene. The homozygous c.1A>G variant of the GDAP1 gene probably underlay the CMT in both children. Above result has enabled clinical diagnosis and genetic counseling for this pedigree.

Phenotypic and genotypic analysis of a pedigree affected with hereditary protein C deficiency due to heterozygous deletional mutation of PROC gene

To analyze the phenotype and genetic variants of a pedigree affected with hereditary protein C (PC) deficiency. The protein C activity (PC:A) of the proband and her family members (a four-generation pedigree including 11 individuals) were tested by chromogenic substrate method, and the protein C antigen (PC:Ag) was detected with an enzyme-linked immunosorbent assay(ELISA). The 9 exons and flanking sequences of the protein C (PROC) gene were amplified by PCR and directly sequenced. Suspected mutation was validated by clone sequencing and in other members of the family. MutationTaster and ClustalX-2.1-win was used to analyze the pathogenicity and conservation of the mutation site,respectively. Three-dimentional protein model and amino acids interaction were analyzed with Swiss-PdbViewer software. The PC: A and PC: Ag of the proband were decreased to 46% (reference range: 70%-130%) and 50% (referencerange:70%-140%), respectively. Her grandmother,aunt, cousin and younger brother also showed declined PC:A and PC:Ag by approximately 50%. Genetic analysis revealed that the above individuals have all carried a deletional mutation c.1212-1212delG (p.Met364TrpfsX15) in exon 9 of the PROC gene which can cause replacement of Methionine at position 364 by Tryptophan and alteration of 15 downstream amino acids, and produce a premature stop codon at position 378. The score of MutationTaster was 1.000, indicating that the variant is pathogenic. Conservative analysis showed that the 15 altered amino acids are located in a conserved region across nine homologous species. Protein model analysis showed that the mutation has disrupted a catalytic domain of protein C thereby affected its function. The heterozygous c.1212-1212delG deletional mutation in exon 9 of the PROC gene, which was unreported previously,probably accounts for the decrease of PC:A and PC:Ag in this pedigree.

Abstract A06: Differential response to autophagy in primary versus melanoma cells

Abstract Autophagy is a cellular recycling mechanism triggered by nutrient scarcity. Upregulation of autophagy is associated with lifespan extension, DNA repair, and improved cancer immune recognition. Cancer cells can also utilize this mechanism to cope with nutrient-limited conditions. Chloroquine, an inhibitor of autophagy, has shown promising results alone and in combination with other anticancer agents in various types of cancer. However, several promoters of autophagy have also been shown to display potent anticancer properties. To better understand the relationship between autophagy and cancer, we tested promoters and inhibitors of autophagy in a metastatic melanoma cell line (B16F10) and compared the results with primary human dermal fibroblasts (HDF). After 48h, all tested promoters and inhibitors of autophagy decreased the number of live cells in the B16F10 melanoma cell line compared to untreated controls. In primary cells, promotion of autophagy with methionine restriction (25 μM versus 100 μM in control), complete methionine replacement with homocysteine, and 50 μM of the polyamine spermidine did not affect the number of live cells. Sulforaphane (10 μM) and serum starvation significantly decreased the number of live primary cells. Inhibition of autophagy with 6.25 μM chloroquine did not affect primary cell numbers, while there was a significant decrease in cell numbers associated with a dose of 10 μM of chloroquine and 0.1 μM bafilomycin A1. Cell numbers remained unaffected by longer incubations (96h with replenishment after 48h) of HDF cells with methionine restriction, replacement of methionine with homocysteine, and 6.25 μM chloroquine. These data suggest that cancer cells are more sensitive to both promotion and inhibition of autophagy than primary cells. Interesting compound-specific differences were noted in primary cells. Ongoing preclinical studies evaluate the potential of modulating autophagy as an adjuvant cancer therapy for immune checkpoint inhibitors in metastatic melanoma. Citation Format: Lauren C. Morehead, Alan J. Tackett, Isabelle R. Miousse. Differential response to autophagy in primary versus melanoma cells [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A06.

Effects of Dietary Rumen-Protected Betaine on Lactation Performance and Serum Metabolites of Mid-lactation Holstein Dairy Cows

This experiment was conducted to investigate the effects of dietary rumen-protected betaine (RPB) supplementation, as partial replacement for methionine, on the lactation performance of mid-lactation dairy cows. A total of 36 Holstein dairy cows were randomly assigned to three groups [control, 20 g/day RPB, or 15 g/day rumen-protected methionine (RPM)]. The experiment was conducted over 9 weeks, with the first week for adaptation. Blood metabolites were analyzed with metabolomics in the control and RPB groups. The results revealed that the milk yield and milk protein content were higher in cows fed RPB and RPM compared to those in the control group. Concentrations of nine metabolites differed between cows in the RPB and control groups. These metabolites were mainly concentrated in six pathways, such as arginine synthesis and proline degradation and cyanoamino acid synthesis. This study revealed that RPB can spare methionine and improve lactation performance of dairy cows fed with diets moderately deficient in methionine.

Further epidemiological survey for atovaquone resistant related gene of Babesia gibsoni in Japan during 2015-2018.

A single-nucleotide polymorphism causing the replacement of methionine with isoleucine (M121I) in cytochrome b of Babesia gibsoni has been reported to reduce the susceptibility to atovaquone (ATV) in B. gibsoni infection. In our previous study, B. gibsoni with M121I was suggested to exist in nature. Thus, further examinations were performed. In total, 105 genomic DNA samples from B. gibsoni-infected dogs were collected from western (98 samples from 15 prefectures) and eastern areas (7 samples from 4 prefectures) in Japan. The M121I variant population was identified using allele-specific real-time PCR: it was then detected in nine samples (8.57%), which was higher than that in the previous study (4.11%). Although there are unclear points, such as the history of ATV usage, careful attention should be given to emerging ATV resistance.

Residue-Specific Incorporation of Noncanonical Amino Acids for Protein Engineering.

The incorporation of noncanonical amino acids has given protein chemists access to an expanded repertoire of amino acids. This methodology has significantly broadened the scope of protein engineering allowing introduction of amino acids with non-native functionalities, such as bioorthogonal reactive handles (azides and alkynes) and hydrophobic fluorinated side chains. Here, we describe the efficient residue-specific replacement of methionine by azidonorleucine in an engineered green fluorescent protein using a bacterial expression system to introduce a single reactive site for the strain-promoted azide-alkyne cycloaddition.

Partial Replacement of Dietary Methionine with Betaine and Choline in Heat-Stressed Broiler Chickens.

We conducted two trials to evaluate the methionine-sparing effects of choline (Chol) and betaine (Bet), and their effects on growth performance and blood antioxidative potential in heat-stressed broiler chickens fed methionine (Met)-deficient diets. We used 360 1-day-old broiler chicks (Ross 308) in a completely randomized study with 5 replicate pens of 12 birds each. After Day 21, we raised the temperature to 35±3°C using an automated air-forced heater for 12 hours/day from 8 am to 8 pm to expose the birds to heat stress. In Trial 1, the treatments comprised a negative control (control-; 1200 mg/kg Met-deficient), a positive control (control+; recommended level of Met), 280Chol (control- plus 280 mg/kg Chol), 560Chol (control- plus 560 mg/kg Chol), 320Bet (control- plus 320 mg/kg Bet), and 640Bet (control- plus 640 mg/kg Bet); and in Trial 2, the treatments comprised a negative control (control-), a positive control (control+), 140Chol+160Bet (control- plus 140 mg/kg Chol and 160 mg/kg Bet), 280Chol+160Bet (controlplus 280 mg/kg Chol and 160 mg/kg Bet), 140Chol+320Bet (control- plus 140 mg/kg Chol and 320 mg/kg Bet), and 280Chol+320Bet (control- plus 280 mg/kg Chol and 320 mg/kg Bet). Compared with the other treatments, the feed conversion ratio (FCR) was improved in the 280Chol and control+ groups in Trials 1 and 2 (P<0.05). In Trial 2, the cost of meat production for the entire experimental period (1–42 days) was higher in the 140Cho+320Bet-fed birds than in the other birds (P<0.05), except the control- birds. Supplementing diets with 280 mg/kg of Chol significantly reduced the serum concentration of uric acid compared with the control+ group (P<0.05). Our results indicate that the Met requirements of heat-stressed broiler chickens can be reduced by 20% (1200 mg/kg) if the diet is supplemented with 280 mg/kg of Chol.

Effects of Replacement of Methionine in Diets with Taurine on Growth Performance and Blood Index in Broilers.

We studied effects of replacement of methionine with taurine on growth performance and blood index of AA+ broilers. Six hundred 1 day broilers were divided into 5 groups, with 3 replicates of 40 broilers in each. The experiment lasted for 42 days.The control group were fed on formulated diets containing 2% methionine; the other groups were offered feed with equal nitrogen and calories to the control group, but contained 25, 50, 75 and 100% taurine in place of methionine.Compared with the control group, no significant differences were observed in growth performance of 1-21 days broilers, or the serum LDL-C, TC, IgG and SOD of the experimental groups (P> 0.05). ADG and F/G from days 1-42, ADG, ADFI and F/G from days 22-42 were significantly different between the experimental groups and the control group (P < 0.05). ADFI and Mortality in 50, 75 and 100% taurine groups were significantly different compared with the control group (P < 0.05). IgM and GSH-PX of 50 and 75% taurine groups were significantly different compared with the control group (P < 0.05). Serum HDL-C, T-AOC levels in 50, 75 and 100% taurine groups were significantly different compared with the control group (P < 0.05). Based on the quadratic regression analysis, the best replacement ratios were 58%, 61% and 61% on days 1-21, 22-42, and 1-42, respectively. In conclusion, appropriate levels of taurine supplement can improve growth performance, immune system, T-AOC, and lipid metabolism.