Additional Methionine Research Articles

Mutations in the promoter region of methionine transporter gene metM (Rv3253c) confer para-aminosalicylic acid (PAS) resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is a significant global public health threat. Despite the long-standing use of para-aminosalicylic acid (PAS) as a second-line anti-TB drug, its resistance mechanism remains unclear. In this study, we isolated 90 mutants of PAS-resistant Mycobacterium tuberculosis (MTB) H37Ra in 7H11 solid medium and performed whole-genome sequencing, gene overexpression, transcription level comparison and amino acid level determination in MTB, and promoter activity by β-galactosidase assays in Mycobacterium smegmatis to elucidate the mechanism of PAS resistance. Herein, we found that 47 of 90 (52.2%) PAS-resistant mutants had nine different mutations in the intergenic region of metM (Rv3253c) and Rv3254. Beta-galactosidase assays confirmed that mutations increased promoter activity only for metM but not Rv3254. Interestingly, overexpression of MetM or its M. smegmatis homolog (MSMEI_1796) either by its promoter in metM's direction or by exogenous expression in MTB induced PAS resistance in a methionine-dependent manner. Therefore, drug susceptibility results for the metM promoter mutants can be misleading when using standard 7H10 or 7H9 medium, which lacks methionine. At the metabolism level, PAS treatment led to higher intracellular methionine levels in the mutants than the wild type, antagonizing PAS and conferring resistance. Furthermore, 12 different mutations in the metM promoter were identified in clinical MTB strains. In summary, we found a novel mechanism of PAS resistance in MTB. Mutations in the metM (Rv3253c) promoter upregulate metM transcription and elevate intracellular methionine, which antagonize PAS. Our findings shed new light on the mechanism of PAS resistance in MTB and highlight issues with the current PAS susceptibility culture medium.IMPORTANCEAlthough para-aminosalicylic acid (PAS) has been used to treat TB for more than 70 years, the understanding of PAS resistance mechanisms is still vague, living gaps in our ability to predict resistance and apply PAS effectively in clinical practice. This study aimed to address this knowledge gap by inducing in vitro PAS resistance in Mycobacterium tuberculosis (MTB) using 7H11 medium and discovering a new PAS resistance mechanism. Our research revealed that spontaneous mutations occurring in the promoter region of the methionine transporting gene, metM, can upregulate the expression of metM, resulting in increased intracellular transport of methionine and consequently high-level resistance of Mycobacterium tuberculosis to PAS. Notably, this resistance phenotype cannot be observed when using the commonly recommended 7H10 medium, possibly due to the lack of additional methionine supply compared with that when using the 7H11 medium. Mutations on the regulatory region of metM were also found in some clinical MTB strains. These findings may have important implications for the unexplained PAS resistance observed in clinical settings and provide insight into the failures of PAS treatment. Additionally, they underscore the importance of considering the choice of culture media when conducting drug susceptibility testing for MTB.

Effects of copper, zinc, and manganese source and inclusion during late gestation on beef cow-calf performance, mineral transfer, and metabolism.

To determine effects of Cu, Zn, and Mn source and inclusion during late gestation, multiparous beef cows [n = 48; 649 ± 80 kg body weight (BW); 5.3 ± 0.5 body condition score (BCS)] were individually-fed hay and supplement to meet or exceed all nutrient recommendations except Cu, Zn, and Mn. From 91.2 ± 6.2 d pre-calving to 11.0 ± 3.2 d post-calving, cows received: no additional Cu, Zn, or Mn (control, CON), sulfate-based Cu, Zn, and Mn (inorganic, ITM) or metal methionine hydroxy analogue chelates (MMHAC) of Cu, Zn, and Mn at 133% recommendations, or a combination of inorganic and chelated Cu, Zn, and Mn (reduce and replace, RR) to meet 100% of recommendations. Data were analyzed with treatment and breeding group (and calf sex if P < 0.25 for offspring measures) as fixed effects, animal as experimental unit, and sampling time as a repeated effect for serum, plasma, and milk measures over time. Post-calving cow liver Cu was greater (P ≤ 0.07) in MMHAC compared with all other treatments. Calves born to RR had greater (P ≤ 0.05) liver Cu than ITM and CON, and MMHAC had greater (P = 0.06) liver Cu than CON. Liver Mn was less (P ≤ 0.08) for RR calves than all other treatments. Calf plasma Zn was maintained (P ≥ 0.15) from 0 to 48 h of age in ITM and MMHAC but decreased (P ≤ 0.03) in CON and RR. Gestational cow BW, BCS, and metabolites were not affected (P ≥ 0.13) by treatment, but gestational serum thiobarbituric acid reactive substances (TBARS) were greater (P = 0.01) for CON than MMHAC. Treatment did not affect (P ≥ 0.13) calf birth size, vigor, placental size and minerals, or transfer of passive immunity. Neonatal calf serum Ca was greater (P ≤ 0.05) for MMHAC than all other treatments; other calf serum chemistry and plasma cortisol were not affected (P ≥ 0.12). Pre-suckling colostrum yield, and lactose concentration and content, were greater (P ≤ 0.06) for MMHAC compared with ITM and RR. Colostral triglyceride and protein concentrations were greater (P ≤ 0.08) for RR than MMHAC and CON. Cow lactational BW and BCS, milk yield and composition, and pre-weaning calf BW and metabolism were not affected (P ≥ 0.13) by treatment. Lactational serum TBARS were greater (P = 0.04) for RR than CON at day 35 and greater (P ≤ 0.09) for MMHAC at day 60 than all other treatments. Source and inclusion of Cu, Zn, and Mn altered maternal and neonatal calf mineral status, but calf size and vigor at birth, passive transfer, and pre-weaning growth were not affected in this study.

Sequence and phylogenetic analysis of a novel alphaendornavirus, the first virus described from the oomycete plant pathogen Phytophthora heveae

Here, we report the discovery and complete genome sequence of a novel virus, designated as “Phytophthora heveae alphaendornavirus 1” (PhAEV1), from a single isolate of the plant pathogenic oomycete Phytophthora heveae (kingdom Stramenipila) isolated from a tropical evergreen lowland rainforest in northern Vietnam. PhAEV1 was detected by both cellulose affinity chromatography of dsRNA and high-throughput sequencing of total RNA, and its presence and sequence were confirmed by RT-PCR and Sanger sequencing. The PhAEV1 genome, 12,820 nucleotides (nt) in length, was predicted to encode a single large polyprotein with the catalytic core domain of viral (superfamily 1) RNA helicase (HEL, amino acid [aa] positions 1,287-1,531), glycosyltransferase (GT, aa positions ca. 2,800-3,125), and RNA-directed RNA polymerase (RdRp, aa positions 3,875-4,112). PhAEV1 is the most similar to Phytophthora cactorum alphaendornavirus 3, sharing 39.4% and 39.1% nt and aa sequence identity, respectively. In addition to the first 5′-terminal AUG codon, three additional in-frame methionine codons were found in close proximity (nt 14-16, 96-98, and 176-178), suggesting potential additional translation initiation sites. Conserved RdRp motifs (A-E) similar to those detected in related endornaviruses were identified in PhAEV1, as well as in several previously described alphaendornaviruses from other Phytophthora species in which these motifs had not been identified previously. Phylogenetic analysis showed that PhAEV1 clusters with members of the genus Alphaendornavirus in the family Endornaviridae and is basal to two other alphaendornaviruses described from another oomycete, Phytophthora cactorum. PhAEV1 is the first virus reported in P. heveae.

Protective effect of methionine on the intestinal oxidative stress and microbiota change induced by nickel.

Nickel is a common heavy metal pollutant in industrial areas and can cause oxidative damage to human and animal organs. As an essential amino acid with antioxidant function, methionine (Met) may protect the body from the oxidative stress induce by nickel, however, there is not enough research to study in this aspect. The study aims at investigating the effect of Met on the nickel-induced intestinal oxidative stress and further detected the gut microbiota changes. Mice were gavaged with quantitative NiCl2 (1.6mg/ml, 0.25ml) and fed with different doses of methionine in each group. The contents of intestinal oxidation product and antioxidant enzymes were determined by different biochemical quantitative methods, and the data showed that NiCl2 increased the content of intestinal oxidation product (MDA), and the antioxidant enzymes (GSH-Px, GR, SOD and CAT) were decreased. But this situation was alleviated in the group fed with additional methionine solution (0.5mg/ml). In addition, we detected changes in the gut microbiota using high-throughput sequencing, the results showed that the structure of intestinal flora was disturbed by NiCl2, but methionine restored the germs with antioxidant capacity. Based on the results, we speculate that methionine can alleviate the impact of NiCl2 on the intestinal by enhancing the activity of antioxidant enzymes and the number of gut bacteria with anti-oxidation, suggesting that methionine as a nutritional additive may have the potential to treat nickel poisoning.

Dietary supplementation of methionine mitigates oxidative stress in broilers under high stocking density

We aimed to investigate whether dietary supplementation of methionine could mitigate intestinal oxidative injury in broilers under high stocking density (HSD). In the grower phase (d 22–42), 576 broilers with similar body weight were randomly chosen and divided into 8 groups in a 2 × 4 factorial experiment. Two different stocking densities (14 and 20 broilers per m2) were tested with 4 different methionine levels: 0.35%, 0.4%, 0.45%, or 0.5%. Intestinal morphological and oxidative stress markers were assessed at the end of the test period. The results showed that mortality of broilers was significantly higher in the HSD group fed 0.35% methionine diet than the other groups, which was reversed by supplementation with 0.40% to 0.50% methionine. HSD significantly decreased feed intake and daily weight gain. HSD treatment significantly decreased T-AOC, activity of GPX (P < 0.01) and increased the level of PCO (P < 0.01), MDA (P = 0.052) of plasma. The decreased glutathione peroxidase activity in the liver and jejunum caused by HSD was alleviated by additional methionine. Supplementation of methionine increased the ration of GSH/GSSG in the plasma. The jejunum villus height and ratio of villus height to crypt depth under low stocking density conditions with 0.40% methionine diet were the highest, whereas the 0.45% methionine group was the highest under HSD conditions. Thus, additional dietary supplementation of methionine mitigates oxidative stress in broilers under HSD conditions and 0.40% to 0.45% methionine can be applied in cage rearing broiler production for amelioration of oxidative stress caused by HSD.

The Effect of Amino Acids Lysine and Methionine on the Carcass of Native Chickens

This study aims to determine the effect of 1% lysine, 0.5% methionine and the combination of lysine and methionine (1% + 0.5%) in the ration on the weight gain of carcass parts of native chickens. The design used in this study was a completely randomized design (CRD) consisting of 4 treatments and 3 replications, while the treatments were R0 (control) native chickens that were not given additional lysine and methionine, R1 native chickens were given feed containing lysine 1 %, R2 of free-range chickens given feed containing 0.5% methionine, R3 of native chickens given feed containing lysine and methionine (1% + 0.5%). The parameters observed were the weight gain in the chest, thighs, wings, and back. The data obtained were analyzed by analysis of variance. The results showed that the provision of rations which were added with the combination of amino acids lysine and methionine had a very significant effect (P&gt; 0.01) on the weight gain of the carcass, thighs and backs of native chickens aged 10 weeks. Based on the results of this study it can be concluded that the combination of the amino acids lysine and methionine in the ration gave the best effect on the weight gain of carcass parts of native chicken aged 10 weeks. Based on the results of the study, it is suggested to add the combination of lysine (1%) and methionine (0.5%) in the native chicken ration.&#x0D; &#x0D; Keywords: native chickens, carcass weight, lysine, methionine

Assessment of calcium oxalate crystal inhibition potential, antioxidant activity and amino acid profiling in horse gram (Macrotyloma uniflorum): high altitude farmer’s varieties

Seeds of Macrotyloma uniflorum collected from different Himalayan regions of Himachal Pradesh, India were screened for inhibition potential of calcium oxalate crystals along with their antioxidant and amino acid profiling. Aqueous, ethanol, 50% ethanol extracts were tested using agar-gel overlay, nucleation, and artificial urine methods in comparison to marketed products. The aqueous extract of Sundernagar and Rampur seeds showed highest inhibition potential (9.0 ± 0.81mm) than alcohol and hydroalcohol. Sundernagar seeds (aqueous extract) also showed maximum inhibition (45 ± 1.2 and 24 ± 0.88%) in nucleation and artificial urine assay, respectively. The Tris-buffer (pH. 8.0) extract of dry seeds showed more inhibition with low protein than high protein germinated seeds. The findings revealed that horse gram protein is not alone responsible for CaOx-crystals inhibition but its secondary metabolites may also contribute. Among all samples, buffer and aqueous extracts showed maximum activity, followed by hydroalcohol and alcohol. Further, TLC and UPLC method were used to monitor the presence of amino acids in aqueous and Cystone extracts. Sundernagar seeds contain 11 amino acids with additional arginine, aspargine, and methionine. Moreover, higher antioxidant potential was observed in seeds of high altitude, which relates the factors of altitudinal variation in secondary metabolism of plants. Therefore, this study will help in the development of economical and more reliable plant based nutraceuticals for kidney stone problems and related health issues.

Cysteine and Methionine Biosynthetic Enzymes Have Distinct Effects on Seed Nutritional Quality and on Molecular Phenotypes Associated With Accumulation of a Methionine-Rich Seed Storage Protein in Rice.

Staple crops in human and livestock diets suffer from deficiencies in certain “essential” amino acids including methionine. With the goal of increasing methionine in rice seed, we generated a pair of “Push × Pull” double transgenic lines, each containing a methionine-dense seed storage protein (2S albumin from sunflower, HaSSA) and an exogenous enzyme for either methionine (feedback desensitized cystathionine gamma synthase from Arabidopsis, AtD-CGS) or cysteine (serine acetyltransferase from E. coli, EcSAT) biosynthesis. In both double transgenic lines, the total seed methionine content was approximately 50% higher than in their untransformed parental line, Oryza sativa ssp. japonica cv. Taipei 309. HaSSA-containing rice seeds were reported to display an altered seed protein profile, speculatively due to insufficient sulfur amino acid content. However, here we present data suggesting that this may result from an overloaded protein folding machinery in the endoplasmic reticulum rather than primarily from redistribution of limited methionine from endogenous seed proteins to HaSSA. We hypothesize that HaSSA-associated endoplasmic reticulum stress results in redox perturbations that negatively impact sulfate reduction to cysteine, and we speculate that this is mitigated by EcSAT-associated increased sulfur import into the seed, which facilitates additional synthesis of cysteine and glutathione. The data presented here reveal challenges associated with increasing the methionine content in rice seed, including what may be relatively low protein folding capacity in the endoplasmic reticulum and an insufficient pool of sulfate available for additional cysteine and methionine synthesis. We propose that future approaches to further improve the methionine content in rice should focus on increasing seed sulfur loading and avoiding the accumulation of unfolded proteins in the endoplasmic reticulum.Oryza sativa ssp. japonica: urn:lsid:ipni.org:names:60471378-2.

Prevotella intermedia produces two proteins homologous to Porphyromonas gingivalis HmuY but with different heme coordination mode.

As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfill this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination modes. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.

Effects of additional organic micro-minerals and methionine on carcass composition, gait score, bone characteristics, and osteochondrosis in replacement gilts of different growth rate

Osteochondrosis (OC) is a multifactorial defective endochondral ossification that causes lameness and early culling in gilts and sows. Previous research suggested that nutrition and growth rate could influence OC development and progression. As part of a broader study [n = 360 gilts; 28.8 ± 8.8 kg body weight (BW)] designed to evaluate the effect of 4 dietary treatments: 1) basal diet (CON); 2) CON plus organic micro-minerals (MIN, copper, manganese and zinc at 10, 20 and 50 mg/kg, respectively); 3) additional methionine (MET, at 102% methionine:lysine); and, 4) the organic micro-minerals plus the additional methionine (MM), on lameness and performance, a sub-sample of 40 heavy replacement gilts (10 gilts/treatment, 171.5 ± 8.1 kg of BW) was used. Within treatment, gilts were classified for final average daily gain (ADG) as low (LG, 838 ± 36.3 g/day; n = 20) or high (HG, 922 ± 31.1 g/day; n = 20). Dietary treatment and growth classification were the fixed effects to evaluate gait, OC, tibia bending measures, metacarpal mineralization; and using computerized tomography, the carcass composition, bone size, and whole bone density (WBD). The WBD was expressed as volume of Hounsfield values (HU), where higher values indicate increased density. A porcine reproductive and respiratory syndrome virus outbreak occurred during this trial. It differentially affected MM gilt performance and consequently may have influenced the results for this treatment. Gilts fed MIN diet had 0.75 cm larger tibia than CON (P < 0.05), and 10% increase of WBD > 140 HU compared to CON and MET (P < 0.05). The volume of high dense bones (> 1000 HU) was also increased in MIN and MET compared with CON (P < 0.05). Tibia bending moment and breakage strength were greater (P < 0.05) in MIN than in CON, with MET and MM intermediate. Metacarpal ash, Ca, and P content, but not proportions, were higher in gilts fed MIN than CON (P < 0.05). Total score of OC lesions was lower in MM gilts compared to CON (P < 0.05). The OC total score increased with ADG from 35.8 to 109.8 kg BW (R2 = 0.10; P < 0.10). However, between 109.8 and 171.5 kg BW OC score increased with decreased ADG (R2 = 0.14; P < 0.05). In conclusion, supplementing growing gilts with MIN enhanced bone strength and bone density, MET increased the proportion of highly dense bone (>1000 HU), and MM dietary treatment reduced OC lesion score compared with CON.

Aggregation and Fibril Structure of AβM01-42 and Aβ1-42.

A mechanistic understanding of Aβ aggregation and high-resolution structures of Aβ fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using Aβ peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant Aβ1-42, sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-Aβ1-42 fusion protein in reasonable yield, with which we compared aggregation kinetics with AβM01-42 containing the additional methionine residue. The data revealed that Aβ1-42 and AβM01-42 aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both AβM01-42 and Aβ1-42 and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of AβM01-42 in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail.

Assessing the bioavailability of the Novel Met-Met product (AQUAVI® Met-Met) compared to dl-methionine (dl-Met) in white shrimp (Litopenaeus vannamei)

This trial was conducted to compare Novel Met-Met product (AQUAVI® Met-Met) and dl-methionine (dl-Met) regarding their availability to juvenile white shrimp. Eleven experimental diets were formulated (Basal, dl-Met 0.06, dl-Met 0.12, dl-Met 0.18, dl-Met 0.24, dl-Met 0.3, AQUAVI 0.06, AQUAVI 0.12, AQUAVI 0.18, AQUAVI 0.24, AQUAVI 0.3), the basal diet was formulated without any additional methionine addition; the other ten diets (dl-Met 0.06, dl-Met 0.12, dl-Met 0.18, dl-Met 0.24, dl-Met 0.3; AQUAVI 0.06, AQUAVI 0.12, AQUAVI 0.18, AQUAVI 0.24, AQUAVI 0.3) were formulated with five graded levels of methionine with two different methionine sources of dl-Met and AQUAVI® Met-Met, respectively. Each diet was fed at a restricted rate to four groups of 30 shrimp for 63days. The highest value of survival was observed with shrimp fed AQUAVI 0.24 diet, which was significantly higher than that of shrimp fed the basal, dl-Met 0.06 and dl-Met 0.18 (P<0.05) diets, but without statistical difference with shrimp fed other diets (P>0.05). The highest values of growth performance (WG and SGR) were observed with shrimp fed AQUAVI 0.12 diet, which were significantly higher than those of shrimp fed the basal, dl-Met 0.06 and dl-Met 0.18 (P<0.05) diets, but without statistical difference with shrimp fed other diets (P>0.05). A non-linear exponential regression model indicated that AQUAVI® Met-Met was found to be more bioavailable than dl-Met with calculated relative bioavailability values of 285.8%, 276.4% and 300.4% based on weight gain, specific growth rate and feed efficiency, respectively. These findings lead to the conclusion that differences in bioavailability do exist between sources of synthetic methionine, and AQUAVI® Met-Met is more available than DL-Met for L. vannamei.

Copper(I/II), α/β-Synuclein and Amyloid-β: Menage à Trois?

Copper binding to α-synuclein (aS) and to amyloid-β (Ab) has been connected to Parkinson's and Alzheimer's disease (AD), respectively, because Cu ions can modulate the peptide aggregation, and these Cu ⋅ peptide complexes can catalyse the production of reactive oxygen species (ROS). In a significant proportion of AD brains, aggregation of aS and Ab has been detected, and it was proposed that Ab and aS interact with each other. Thus, we investigated the potential interactions of Ab and aS through their binding of copper(I) and copper(II). Additionally, β-synuclein (bS) was investigated, due to its additional methionine residue, a potential Cu(I) ligand. We found that: 1) the peptides containing the Cu-binding domains Ab1-16, aS1-15 and bS1-15 have similar affinities towards Cu(II) and towards Cu(I), with Ab1-16 being slightly stronger, 2) in the case of Cu(I), the additional Met residue in bS1-15 increased the affinity slightly, 3) the exchange of Cu(I/II) between the two peptides is rapid (≤ ms), 4) a/bS1-15 and Ab1-16 form a heterodimeric complex with Cu(II), 5) Cu(I) probably promotes a transient ternary complex, 6) the different Cu(I/II) coordination of Ab1-16, aS1-15 and bS1-15 impacts the capacity to produce ROS and to oxidise catechol, and 7) when Ab1-16, aS1-15 and Cu are present, the ROS production more closely resembles that by Ab1-16. The work gives insights into the coordination chemistry of these related peptides, and the relevance of coordination differences, the ternary complex and ROS production are discussed.

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

Expression and refolding of bioactive α-bungarotoxin V31 in E. coli

In order to obtain bioactive α-bungarotoxin (αBtx) using recombinant protein technique, a codon-optimized synthetic gene was expressed in fusion with the N-terminal 10-His-tag and C-terminal Strep-tag in Escherichia coli. Further optimization through site-directed mutagenesis enabled moderate expression of the protein without the N-terminal His-tag or the C-terminal Strep-tag. Two such recombinant αBtx (rαBtx) were obtained, both with an additional methionine and a glycine at the N-terminal and one with (G4S1)2-Strep-tag at the C-terminal. The rαBtx proteins were refolded using a novel protocol, which efficiently produced final products with activity similar to its natural counterpart. The protocol could easily be scale up, which produced 0.3–1mg of pure and highly active rαBtx per liter of E. coli culture.

Additional dietary tryptophan and methionine improves feed conversion efficiency and markers of inflammation in weaner pigs infected with Escherichia coli

Additional dietary tryptophan and methionine improves feed conversion efficiency and markers of inflammation in weaner pigs infected with Escherichia coli

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

Heat shock protein 70 (Hsp70) molecular chaperones play critical roles in protein homeostasis. In the budding yeast Saccharomyces cerevisiae, cytosolic Hsp70 interacts with up to three types of nucleotide exchange factors (NEFs) homologous to human counterparts: Sse1/Sse2 (Heat shock protein 110 (Hsp110)), Fes1 (HspBP1), and Snl1 (Bag-1). All three NEFs stimulate ADP release; however, it is unclear why multiple distinct families have been maintained throughout eukaryotic evolution. In this study we investigate NEF roles in Hsp70 cell biology using an isogenic combinatorial collection of NEF deletion mutants. Utilizing well characterized model substrates, we find that Sse1 participates in most Hsp70-mediated processes and is of particular importance in protein biogenesis and degradation, whereas Fes1 contributes to a minimal extent. Surprisingly, disaggregation and resolubilization of thermally denatured firefly luciferase occurred independently of NEF activity. Simultaneous deletion of SSE1 and FES1 resulted in constitutive activation of heat shock protein expression mediated by the transcription factor Hsf1, suggesting that these two factors are important for modulating stress response. Fes1 was found to interact in vivo preferentially with the Ssa family of cytosolic Hsp70 and not the co-translational Ssb homolog, consistent with the lack of cold sensitivity and protein biogenesis phenotypes for fes1Δ cells. No significant consequence could be attributed to deletion of the minor Hsp110 SSE2 or the Bag homolog SNL1. Together, these lines of investigation provide a comparative analysis of NEF function in yeast that implies Hsp110 is the principal NEF for cytosolic Hsp70, making it an ideal candidate for therapeutic intervention in human protein folding disorders.

Native and recombinant Pg-AMP1 show different antibacterial activity spectrum but similar folding behavior

Glycine-rich proteins (GRPs) derived from plants compose a family of proteins and peptides that share a glycine repeat domain and they can perform diverse functions. Two structural conformations have been proposed for GRPs: glycine loops arranged as a Velcro and an anti-parallel β-sheet with several β-strands. The antimicrobial peptide Pg-AMP1 is the only plant GRP with antibacterial activity reported so far and its structure remains unclear. Recently, its recombinant expression was reported, where the recombinant peptide had an additional methionine residue at the N-terminal and a histidine tag at the C-terminal (His6-tag). These changes seem to change the peptide's activity, generating a broader spectrum of antibacterial activity. In this report, through ab initio molecular modelling and molecular dynamics, it was observed that both native and recombinant peptide structures were composed of an N-terminal α-helix and a dynamic loop that represents two-thirds of the protein. In contrast to previous reports, it was observed that there is a tendency to adopt a globular fold instead of an extended one, which could be in both, glycine loops or anti-parallel β-sheet conformation. The recombinant peptide showed a slightly higher solvated potential energy compared to the native form, which could be related to the His6-tag exposition. In fact, the His6-tag could be mainly responsible for the broader spectrum of activity, but it does not seem to cause great structural changes. However, novel studies are needed for a better characterization of its pharmacological properties so that in the future novel drugs may be produced based on this peptide.

Possible Effects of Delivering Methionine to Broilers in Drinking Water at Constant Low and High Environmental Temperatures

An experiment was conducted to study the effects of water-soluble DL-methionine supplied through water and feed on the performance and carcass yield of broilers housed at two constant temperatures from 21 to 42 days of age. Birds were housed in two rooms (240 birds per room) with temperatures set at 21±2 and 30±2°C, respectively. Birds were divided into five groups of equal number within each room and fed five different diets (G1-G5). A low-methionine basal diet without supplemental methionine was fed to group 1 (G1). The basal diet was fortified with 0.025% or 0.050% methionine, either in feed (G2 and G3, respectively), or in a water solution (G4 and G5, respectively). Mortality was not significantly altered by any dietary treatment. Neither feed nor water intake was affected adversely by DL-methionine inclusion in drinking water. Housing at high temperature showed deleterious effect on birds’ weight gain. Additional methionine intake both in feed and water was associated with significantly heavier body weight, weight gain and feed conversion ratio, than the basal diet at low and high environmental temperature. Carcass yields, as a percentage of live body weight, were not affected by dietary treatment. The results indicate that, under the experimental conditions, DL-methionine provided in drinking water can be effectively assimilated by broilers, at least from 21 to 42 days of age.

Studies on the expression and processing of human proinsulin derivatives encoded by different DNA constructs

A synthetic gene encoding human proinsulin, containing Escherichia coli preferred codons, with an additional N-terminal methionine, was used for the expression, of M-proinsulin and construction of nine derivatives. No improvement in expression was noted, relative to that of M-proinsulin, when the 5′- of the gene was appended to codons for seven amino acids of a well expressed E. coli protein (threonine dehydrogenase), or the constructs contained multiple copies of the proinsulin gene. That in the latter constructs only the gene adjacent to the prometer sequence is expressed, was shown by a construct containing a proinsulin gene followed by that for interferon α-2b. With the latter construct, the proinsulin was, predominantly, expressed. The availability of data on the constructs prompted, subjecting these to analysis by two models designed to predict the expression of proteins from the sequences, of putative mRNA, around the start of translation but no significant relationship was noted. In all cases the proteins were expressed as inclusion bodies, which were refolded to give products of desired masses and successfully converted into insulin derivatives. Of all the constructs containing a trypsin sensitive site before phenylalanine (F), the N-terminal sequence, MKR↓F, was most efficiently processed, by a cocktail of trypsin and buffalo carboxypeptidase B, to give insulin with the removal of the N-terminus linker as well as the C-peptide in a single step, without cleaving the trypsin sensitive K29T30 peptide bond.