The effect of different dietary levels of dl-methionine and dl-methionine hydroxy analogue on the antioxidant and immune status of young turkeys

The hypothesis postulating that the antioxidant and immunological effects of dietary methionine (Met) in young turkeys (1–8 weeks of age) can be differentiated by level and source of Met was investigated in this study. A total of 544 female Hybrid Converter turkeys were divided into four groups and fed diets in which Met content was tailored through supplementation with dl-methionine (dl-Met) or dl-methionine hydroxy analogue (MHA) to levels recommended by NRC (1994) (Groups dl-Met_L and MHA_L) and exceeding them by 50% (Groups dl-Met_H and MHA_H). Regardless of its source, the increased dietary Met content led to significantly higher body weight gains but had no effect on feed conversion rates. Moreover, an increased Met content lowered lipid peroxide concentrations in breast meat and increased selected indicators of the plasma antioxidant status like uric acid levels, activity of superoxide dismutase (SOD), glutathione (GSH) concentrations, the ferric-reducing ability of plasma (FRAP), increased immunoglobulin A (IgA) plasma levels and decreased interleukin 6 levels. In comparison with dl-Met, MHA decreased the activities of SOD and catalase, and GSH concentrations in plasma. A dosage by source interaction revealed that the lower MHA content was associated with the lowest plasma GSH concentrations, FRAP values and activities of SOD and catalase. The higher dietary MHA level resulted for most parameters similar values, except for a decrease in lipid peroxide concentrations and an increase in plasma IgA levels. It can be concluded that an increased dietary dl-Met and MHA content (about 150% of the recommendations given by NRC 1994) not only increased the growth rate of young turkeys but also improved their antioxidant status. MHA appears to be a less desirable source of dietary Met for young turkeys when the inclusion level of Met matches the current recommendations. Therefore, a further debate is needed to establish the dietary requirements for Met in poultry.     

The effect of dietary methionine levels on fattening performance and selected blood and tissue parameters of turkeys

A total of 490 eight-week-old female Hybrid Converter turkeys (body weight 4.11 ± 0.03 kg) were divided into 5 groups with 7 replicates of 14 birds each. For 8 weeks, basal diets were supplemented with methionine (Met) at following levels (weeks 9–12/weeks 13–16 of age): Group 1 – 0.34/0.29%, Group 2 – 0.39/0.34%, Groups 3 and 4 – 0.45/0.38% and 0.51/0.41%, respectively, Group 5 – 0.58/0.47%. Only in the first feeding phase the body weight gain (BWG) was affected by Met levels with the significantly highest BWG in Group 3. No treatment effects were found for feed conversion ratio, carcass yield, carcass composition and meat colour. The blood superoxide dismutase activity was significantly highest in Groups 2 and 3. The concentrations of reduced glutathione in the liver were linearly increased (p = 0.018), whereas the ratio of reduced glutathione to oxidised glutathione was highest in Group 3 (quadratic contrast, p = 0.004). It can be concluded that turkeys from Group 3 (Met levels age depending 15% and 10% above recommendations by NRC) were characterised by a well-balanced physiological response. Attention should be paid to the immune response of birds to higher dietary Met levels: plasma IgA concentrations decreased, whereas IL-6 and TNF-α levels increased in turkeys fed diets with the highest Met content.     

<Emphasis Type="Italic">M31R</Emphasis> and <Emphasis Type="Italic">R335C</Emphasis> polymorphic variants of the <Emphasis Type="Italic">IL8RA</Emphasis> gene in Russian and Buryat patients with atopic bronchial asthma

To test the M31R and R335C polymorphisms of the Il8RA gene for association with atopic bronchial asthma (BA), the allele and genotype frequency distributions of the polymorphisms were studied in Russian patients from Moscow and Buryat patients from Ulan-Ude. The study involved two Russian groups, one including 291 DNA samples of patients with atopic BA, and the other, 266 DNA samples of healthy people. The two Buryat groups included 124 and 152 DNA samples from patients with atopic BA and healthy people, respectively. The M31R polymorphism proved to be associated with atopic BA in Russians. Allele Arg and genotype Met/Arg suggested a higher risk of BA (OR = 4.45, P = 0.003 and OR = 4.58, P = 0.003, respectively), while allele Met and genotype Met/Met were associated with a lower risk (OR = 0.22, P = 0.003 and OR = 0.22, P = 0.003, respectively). The R335C polymorphism was not associated with atopic BA in Russians and was in Buryats. Allele Arg and homozygous genotype Arg/Arg suggested a higher risk of the disease (OR = 3.06, P = 0.030 and OR = 3.20, P = 0.027, respectively), while allele Cys and genotype Arg/Cys suggested a lower risk (OR = 0.33, P = 0.030 and OR = 0.31, P = 0.027, respectively). The results support the role of the IL8RA gene in atopic BA.     

Alterations at the Cross-Bridge Level Are Associated with a Paradoxical Gain of Muscle Function In Vivo in a Mouse Model of Nemaline Myopathy

Nemaline myopathy is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. The first disease causing mutation (Met9Arg) was identified in the gene encoding α-tropomyosin_slow gene (TPM3). Considering the conflicting findings of the previous studies on the transgenic (Tg) mice carrying the TPM3 ^Met9Arg mutation, we investigated carefully the effect of the Met9Arg mutation in 8–9 month-old Tg(TPM3)^Met9Arg mice on muscle function using a multiscale methodological approach including skinned muscle fibers analysis and in vivo investigations by magnetic resonance imaging and 31-phosphorus magnetic resonance spectroscopy. While in vitro maximal force production was reduced in Tg(TPM3)^Met9Arg mice as compared to controls, in vivo measurements revealed an improved mechanical performance in the transgenic mice as compared to the former. The reduced in vitro muscle force might be related to alterations occuring at the cross-bridges level with muscle-specific underlying mechanisms. In vivo muscle improvement was not associated with any changes in either muscle volume or energy metabolism. Our findings indicate that TPM3(Met9Arg) mutation leads to a mild muscle weakness in vitro related to an alteration at the cross-bridges level and a paradoxical gain of muscle function in vivo. These results clearly point out that in vitro alterations are muscle-dependent and do not necessarily translate into similar changes in vivo.     

Tc-99m-labeled RGD-conjugated alpha-melanocyte stimulating hormone hybrid peptides with reduced renal uptake

The purpose of this study was to examine whether the replacement of the positively-charged Lys or Arg linker with a neutral linker could reduce the renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys^3,4,10, D-Phe⁷, Arg¹¹]α-MSH_3-13 {(Arg¹¹)CCMSH} through the neutral βAla or Ahx {aminohexanoic acid} linker (replacing the Lys or Arg linker) to generate novel RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH hybrid peptides. The receptor-binding affinity and cytotoxicity of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH were determined in B16/F1 melanoma cells. The melanoma targeting and imaging properties of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH and ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The replacement of the Lys or Arg linker with the βAla or Ahx linker retained nanomolar receptor-binding affinities and remarkable cytotoxicity of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH. The receptor-binding affinities of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH were 0.8?±?0.05 and 1.3?±?0.1 nM. Three-hour incubation with 0.1 µM of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH decreased the survival percentages of B16/F1 cells by 71 and 67 % as compared to the untreated control cells 5 days post the treatment. The replacement of the Arg linker with the βAla or Ahx linker reduced the non-specific renal uptake of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH and ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH by 62 and 61 % at 2 h post-injection. ^99mTc-RGD-βAla-(Arg¹¹)CCMSH displayed higher melanoma uptake than ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH at 0.5, 2, 4, and 24 h post-injection. Enhanced tumor to kidney uptake ratio of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH warranted the further evaluation of ¹⁸⁸Re-labeled RGD-βAla-(Arg¹¹)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.     

The effect of different dietary levels of DL-methionine and DL-hydroxy analogue on the antioxidant status of young turkeys infected with the haemorrhagic enteritis virus

Background

The results of experiments involving broiler chickens and turkeys indicate that increased dietary methionine (Met) levels may improve the antioxidant protection of tissues in fast-growing birds. This is an important consideration since viral infections induce oxidative stress. The aim of this study was to verify the hypothesis that turkey diets with increased Met content can suppress oxidation processes induced by infection caused by the haemorrhagic enteritis virus (HEV), and that the noted effect is determined by the chemical form of this amino acid: DL-methionine (DLM) or DL-hydroxy analogue of Met (MHA).

Results

Dietary Met content above 40% higher than the level recommended by the NRC (1994) intensified lipid peroxidation in the small intestine, leading to an increase in malondialdehyde (MDA) and lipid peroxide (LOOH) levels, but it also stimulated antioxidant mechanisms in the blood and liver of turkeys infected with HEV. In comparison with DLM, MHA contributed to more severe symptoms of oxidative stress, such as elevated MDA levels in the intestines, and a decrease in glutathione peroxidase (GPx) activity and ferric-reducing ability of plasma (FRAP).

Conclusions

In HEV-infected turkeys, diets with increased Met content did not exert a clear antioxidant effect, which was noted in uninfected birds. The prooxidant activity of Met observed in the small intestinal wall was suppressed in the blood and liver of turkeys, most likely due to intensified synthesis of uric acid and glutathione. In comparison with MHA, DLM had a more beneficial influence on the analysed parameters of the redox status in the small intestine, blood and liver of turkeys.

     

Replacement of the Lys linker with an Arg linker resulting in improved melanoma uptake and reduced renal uptake of Tc-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptide

The purpose of this study was to reduce the non-specific renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide through structural modification or l-lysine co-injection. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys^3,4,10, d-Phe⁷, Arg¹¹] α-MSH_3-13 {(Arg¹¹)CCMSH} through the Arg linker (substituting the Lys linker) to generate a novel RGD-Arg-(Arg¹¹)CCMSH hybrid peptide. The melanoma targeting and pharmacokinetic properties of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The effect of l-lysine co-injection on the renal uptake was determined through the co-injection of l-lysine with ^99mTc-RGD-Arg-(Arg¹¹)CCMSH or ^99mTc-RGD-Lys-(Arg¹¹)CCMSH. Replacement of the Lys linker with an Arg linker exhibited a profound effect in reducing the non-specific renal uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH, as well as increasing the tumor uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH compared to ^99mTc-RGD-Lys-(Arg¹¹)CCMSH. ^99mTc-RGD-Arg-(Arg¹¹)CCMSH exhibited high tumor uptake (21.41 ± 3.74% ID/g at 2 h post-injection) and prolonged tumor retention (6.81 ± 3.71% ID/g at 24 h post-injection) in B16/F1 melanoma-bearing mice. The renal uptake values of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH were 40.14–64.08% of those of ^99mTc-RGD-Lys-(Arg¹¹)CCMSH (p <0.05) at 0.5, 2, 4 and 24 h post-injection. Co-injection of l-lysine was effective in decreasing the renal uptakes of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH by 27.7% and ^99mTc-RGD-Lys-(Arg¹¹)CCMSH by 52.1% at 2 h post-injection. Substitution of the Lys linker with an Arg linker dramatically improved the melanoma uptake and reduced the renal uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH, warranting the further evaluation of ¹⁸⁸Re-labeled RGD-Arg-(Arg¹¹)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.     

Degradation of Dithiocarbamate Fungicide Polycarbamate in Upland Soils

Oligo-L-methionine ethylester (OMOEt) prepared by the papain-catalyzed oligomerization of L-methionine ethylester (MetOEt) is a mixture of pentamer to dodecamer and has nearly the same supplementary effect as free methionine (Met) for the growth of rats when added to a low casein diet, but its supplementary effect to a low-soy protein isolate (SPI) diet is not consistent and depends on the degree of polymerization. Rats were fed for 2wk with an 8% casein or 10% SPI diet supplemented with 0.3% L-Met, each chemically synthesized Met_nOEt with a polymerization degree (n) of 6, 7, 8, or 9, or with OMOEt prepared by papain-catalyzed polymerization of MetOEt. Met₆OEt, Met₇OEt, and Met₈OEt had nearly the same supplementary effect on the growth of rats, as did free Met, both with the 8% casein and 10% SPI diets. The supplementary effect of Met₉OEt was not significantly lower than that of Met when added to the 8% casein diet, but was significantly lower when added to the 10% SPI diet. The digestibility of Met₉OEt supplemented to the 8% casein and 10% SPI diets was 50.5% and 35.6%, respectively. It appears likely that there is a gap in the bioavailability of oligomethionine between the octamer and nonamer when added to a low-protein diet, probably due to the rigidity of the structure increasing with the polymerization degree by α-helix formation. Although the differences in absorption rate of Met from OMOEt for a short time after feeding has been related to the different effects of supplemented OMOEt, the absorption rate of OMOEt for 30 min after feeding was not considered to be the main cause of the differential effects of OMOEt in this experiment.     

Linker modification reduced the renal uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to examine the biodistribution of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH in B16/F1 melanoma-bearing C57 mice to determine whether the replacement of the Lys linker with an Arg linker could decrease the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH. ^99mTc-RAD-Arg-(Arg¹¹)CCMSH exhibited rapid and high tumor uptake (17.98 ± 4.96% ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice. As compared to ^99mTc-RAD-Lys-(Arg¹¹)CCMSH, the replacement of the Lys linker with an Arg linker dramatically decreased the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH by 68%, 62%, 73% and 64% at 0.5, 2, 4 and 24 h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2 h post-injection using ^99mTc-RAD-Arg-(Arg¹¹)CCMSH as an imaging probe.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

低蛋白质日粮对断奶仔猪生长相关激素和肠道微生物区系的影响

【目的】在饲喂低蛋白质日粮条件下,探究断奶仔猪生长相关激素、回肠和盲肠微生物组成及其代谢产物的变化。【方法】选取体重相近杜长大断奶仔猪54头,随机平均分为3组,每组18头,分别饲喂含20%(NP组)、17%(MP组)和14%(LP组)粗蛋白日粮,平衡日粮中的赖氨酸、蛋氨酸、苏氨酸和色氨酸,于试验第10、25和45天每组屠宰6头,采血测定血常规和生长相关激素;于第45天采集回肠和盲肠食糜,分析微生物及其代谢产物。【结果】与NP组相比,第25和45天时MP和LP组尿素氮水平显著降低(P0.05),第25天时LP组甘油三脂含量、第45天时LP组胆固醇含量显著增加(P0.05)。各时间点血液胰高血糖素、胰岛素、生长激素、T3和T4在3组之间差异均不显著。门水平上,回肠和盲肠中的微生物均以厚壁菌门占主导地位,但各组间差异不显著;随日粮蛋白质含量降低,乳酸杆菌属呈上升趋势,严格梭菌属呈下降趋势,但差异不显著。降低日粮蛋白质含量显著减少了回肠和盲肠中氨氮的产量(P0.05)。【结论】断奶仔猪日粮蛋白质降低3或6个百分点不影响机体生长相关激素的分泌,但能降低血液尿素氮和肠道内氨氮的浓度,对肠道有益菌乳酸杆菌属的相对丰度有一定的提高作用。这说明低蛋白质日粮能提高断奶仔猪对饲料氮源的利用率,且有利于肠道健康。     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Role of dietary lysine, methionine, and arginine in the regulation of hypercholesterolemia in rabbits

These experiments were conducted to see whether the hypercholesterolemia produced by a diet enriched in lysine (Lys) and methionine (Met) can be reproduced by feeding these amino acids separately, and whether dietary arginine (Arg) counteracts their hypercholesterolemic effects. Another aim was to investigate the mechanisms involved in modulations of serum cholesterol levels by these amino acids. The results of this study, which were in agreement with the results of earlier experiments in our laboratory, showed that feeding a low-fat, cholesterol-free, semipurified amino acid diet enriched with Lys + Met to rabbits caused a marked increase in serum total and low density lipoprotein cholesterol and apolipoprotein B levels, whereas a similar diet enriched in essential ketogenic amino acids (EketoAA) resulted in a more moderate increase in these parameters. Supplementing the diet with either Lys or Met alone was also less effective in inducing hypercholesterolemia than increasing levels of both amino acids. Dietary Arg partially counteracted the hypercholesterolemic effect of Lys + Met but not that of the EketoAA or of Lys and Met fed separately. The growth performance of rabbits fed the Lys + Met diet was inferior to that of those fed the other diets. Liver total phospholipid levels and the ratio of phosphatidylcholine to phosphatidylethanolamine were higher in rabbits fed the Lys + Met-enriched diet than in those animals fed a diet in which Arg was supplemented. In conclusion, our results indicate that high levels of both Lys and Met are needed to cause a maximum elevation of serum cholesterol and that the moderately antihypercholesterolemic effect of Arg is seen only when both amino acids are supplemented. They also suggest that these essential amino acids may affect cholesterol metabolism partly through alteration of liver phospholipids.     

Structural and functional characterization of a triple mutant form of S100A7 defective for Jab1 binding

S100A7 (psoriasin) is a calcium‐ and zinc‐binding protein implicated in breast cancer. We have shown previously that S100A7 enhances survival mechanisms in breast cells through an interaction with c‐jun activation domain binding protein 1 (Jab1), and an engineered S100A7 triple mutant (Asp⁵⁶Gly, Leu⁷⁸Met, and Gln⁸⁸Lys—S100A7₃) ablates Jab1 binding. We extend these results to include defined breast cancer cell lines and demonstrate a disrupted S100A7₃/Jab1 phenotype is maintained. To establish the basis for the abrogated Jab1 binding, we have recombinantly produced S100A7₃, demonstrated that it retains the ability to form an exceptionally thermostable dimer, and solved the three dimensional crystal structure to 1.6 Å. Despite being positioned at the dimer interface, the Leu⁷⁸Met mutation is easily accommodated and contributes to a methionine‐rich pocket formed by Met¹², Met¹⁵, and Met³⁴. In addition to altering the surface charge, the Gln⁸⁸Lys mutation results in a nearby rotameric shift in Tyr⁸⁵, leading to a substantially reorganized surface cavity and may influence zinc binding. The final mutation of Asp⁵⁶ to Gly results in the largest structural perturbation shortening helix IV by one full turn. It is noteworthy that position 56 lies in one of two divergent clusters between S100A7 and the functionally distinct yet highly homologous S100A15. The structure of S100A7₃ provides a unique perspective from which to characterize the S100A7‐Jab1 interaction and better understand the distinct functions between S100A7, and it is closely related paralog S100A15.     

Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors

Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2'' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr₄-Ile₅ bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes.     

Arg64β3‐Adrenoceptor Variant and the Components of Energy Expenditure

Objective: To determine Trp64Arg β₃‐adrenoceptor genotype‐specific differences in the components of energy expenditure. Hypothesis: We hypothesized that resting metabolic rate (RMR) and physical activity levels would be lower and that thermic effect of feeding (TEF) would be higher in those with the Arg64 allele. Research Methods and Procedures: RMR and TEF were measured by indirect calorimetry, physical activity by questionnaire, and total energy expenditure by the doubly labeled water method. Genotype‐specific measures were compared using ANOVA and analysis of covariance (ANCOVA). Results: RMR in Arg64 homozygotes was significantly lower than in Trp64 homozygotes [Arg64, 1373 ± 259 kcal/d (n = 15) vs. Trp64Arg, 1538 ± 238 kcal/d (n = 25) vs. Trp64, 1607 ± 290 kcal/d (n = 22); p < 0.01]. TEF was significantly higher in Arg64 homozygotes compared with Trp64 homozygotes (Arg64, 359 ± 28 kcal/d; Trp64Arg, 322 ± 22 kcal/d; and Trp64, 279 ± 23 kcal/d; p < 0.05). No differences were identified between genotypes in physical activity or in total energy expenditure. Discussion: Our results suggest that the Arg64 β₃‐adrenoceptor allele contributes significantly to the genetic variability in both RMR and TEF.     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Differential effects of ruminally protected amino acids on fattening of fallow deer in two culling periods

A well-balanced amino acid profile in the feedstuffs for livestock is essential to support adequate growth. This is well studied for monogastric species but still not well understood for ruminants and especially for the most unconventional species, like cervids. This study investigated the influence of ruminally protected lysine (Lys) and methionine (Met) supplementation during the fattening period, as well as two slaughter seasons (late autumn (LA) v. late winter (LW)), on the growth, carcass traits, body condition and blood plasma metabolites of fallow deer (Dama dama). Forty-five yearlings of fallow deer bucks were allocated into three groups, balanced by weight (40.2 ± 2.7 kg). The deer were pasture-fed and supplemented with barley, free-choice mineral premix, silage during the winter period, and varying levels of ruminally protected Lys and Met: no amino acids (Control), 9 g/day of Lys, and 9 g/day of Lys plus 3 g/day of Met (Lys+Met). Animals were slaughtered in two separate seasons, LA (six animals per group), and LW (nine animals per group). Animals culled in LA had higher average daily weight gain than LW (P = 0.002), due to the reduced growth during winter typical for seasonal cervids in temperate zone, mediated by the photoperiod. Dressing percentage was significantly higher in LW and in the Lys+Met group (P = 0.002). Body condition score (P = 0.024), kidney fat index (KFI) (P = 0.005), and internal fat (P < 0.001) increased significantly with Lys+Met supplementation. During LW, KFI (P = 0.004) and kidney fat (P = 0.001) were also significantly higher than in LA. Blood creatinine concentration increased significantly for deer receiving Lys (P = 0.002) and Lys+Met (P < 0.001). Also, triglycerides level increased in Lys group (P < 0.001). These findings highlight the effects of Lys and Met supplementation on the growth and internal fat storage for winter survival, suggesting a different use of the supplemented resource according to the season. Also, the observed effects on protein and fat metabolism of fallow deer may influence the production of farmed animals, and offer interesting insights about the physiology of the species.     

Rumen escape of methionine and lysine administered intraruminally to growing double-muscled Belgian Blue bulls

In many dietary conditions, methionine (Met) and lysine (Lys) are the most limiting amino acids (AA) for ruminants. The AA protected from ruminal fermentation are not commercially available, with the exception of Met which is not always economical, especially for meat production. This study measured ruminal escape of free Met and Lys supplemented intraruminally to fast growing bulls. Six double-muscled Belgian Blue bulls, fed a high concentrate diet and fitted with a rumen cannula, received free Met (40 g x d(-1)) and free Lys (60 g x d(-1)), individually or simultaneously, in a duplicated Latin square design. The mean ruminal escape of Met and Lys reached 37 and 45% respectively, and did not differ if administered separately or together. Plasma Lys and Met concentrations were increased by 504 and 126%, respectively. Substantial proportions of free AA escaped ruminal fermentation and were available for absorption from the small intestine when they were administered at physiologically high levels.