Dietary Methionine and Total Sulfur Amino Acid Restriction in Healthy Adults.

OBJECTIVES: Dietary restriction of methionine (Met) and cysteine (Cys) delays the aging process and aging-related diseases, improves glucose and fat metabolism and reduces oxidative stress in numerous laboratory animal models. Little is known regarding the effects of sulfur amino acid restriction in humans. Thus, our objectives were to determine the impact of feeding diets restricted in Met alone (MetR) or in both Met and Cys (total sulfur amino acids, SAAR) to healthy adults on relevant biomarkers of cardiometabolic disease risk. DESIGN: A controlled feeding study. SETTING AND PARTICIPANTS: We included 20 healthy adults (11 females/9 males) assigned to MetR or SAAR diet groups consisting of three 4-wk feeding periods: Control period; low level restriction period (70% MetR or 50% SAAR); and high level restriction period (90% MetR or 65% SAAR) separated by 3-4-wk washout periods. RESULTS: No adverse effects were associated with either diet and level of restriction and compliance was high in all subjects. SAAR was associated with significant reductions in body weight and plasma levels of total cholesterol, LDL, uric acid, leptin, and insulin, BUN, and IGF-1, and increases in body temperature and plasma FGF-21 after 4 weeks (P<0.05). Fewer changes occurred with MetR including significant reductions in BUN, uric acid and 8-isoprostane and an increase in FGF-21 after 4 weeks (P<0.05). In the 65% SAAR group, plasma Met and Cys levels were significantly reduced by 15% and 13% respectively (P<0.05). CONCLUSION: These results suggest that many of the short-term beneficial effects of SAAR observed in animal models are translatable to humans and support further clinical development of this intervention.

Genetic polymorphisms and antiviral activity in the bovine MX1 gene.

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.

Male hybrid sterility of mice with the genomic region of the KitW mutation and the KitS allele from Mus spretus.

Two congenic strains, C57BL-KitW and C57BL-KitS, were generated. The KitW allele originated from strain WB-KitW and the KitS allele from Mus spretus. The KitW/KitS males showed hybrid sterility with small testes, but the females were fertile. The development of the seminiferous tubules of KitW/KitS males stopped before the spermatocyte stage and they were almost free of sperm. The Kit gene is located at position 42 on chromosome 5. We investigated in the C57BL-KitS congenic strain which part of the chromosomal region adjacent to the KitS allele is introduced from SPR into a C57BL background. The region between positions 42 and 44 was derived from SPR. Eleven amino acid substitutions of the KitS cDNA were detected by comparison with the sequence data of the +Kit cDNA from C57BL; seven were in the extracellular domain, one in the transmembrane domain, two in the kinase I domain, and one in the carboxy-terminal tail. The Kit mRNA derived from both KitW and KitS alleles was expressed in the sterile testes of KitW/KitS males.

The roles of Nramp1 and Tnfa genes in nitric oxide production and their effect on the growth of Salmonella typhimurium in macrophages from Nramp1 congenic and tumor necrosis factor-alpha-/- mice.

The macrophages from Nramp1 congenic mice and tumor necrosis factor (TNF)-alpha(-/-) mice were used to examine the functions of Nramp1 and Tnfa genes in nitric oxide (NO) production and Salmonella typhimurium infection. It was confirmed that the level of inducible NO synthase (iNOS)-mediated NO production in Nramp1(r) peritoneal macrophages was generally higher than that of Nramp1(s) macrophages after stimulation by interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) alone or in combination. Nramp1 mRNA expression in both Nramp1 congenic macrophages was constitutive notwithstanding cytokine stimulation. During infection with S. typhimurium strain 6203, Nramp1(r) macrophages produced a lower amount of NO because of an initial strong reaction and unsustained iNOS gene expression as compared with Nramp1(s) macrophages. An inhibitory effect of the Nramp1(r) gene on bacterial replication was also observed during the early stage of S. typhimurium infection, whereas the effect of TNF-alpha occurred later. NO production and iNOS expression in TNF-alpha(-/-) macrophages were not detected from the start of the bacterial infection or at 24 h after infection. We also observed that S. typhimurium strain 6203 grew more profoundly without TNF-alpha, especially in Nramp1(s) macrophages. These data, therefore, demonstrate that there is cooperation of the Nramp1 and Tnfa genes in NO production and a growth inhibitory effect in response to S. typhimurium infection.

Analysis of genetic factors associated with nitric oxide production in mice.

Different mouse strains possessing the Nramp1r allele, which were theoretically expected to have relatively high nitric oxide (NO) production after cytokine stimulation, were used to analyze the genetic factors associated with NO production. After gamma interferon and lipopolysaccharide stimulation, the strains NZB/N, DBA/2N, AKR/N, and A/J showed significantly low NO production; NJL, 129/J, MOG, SJL/J, CBA/N, and NOD/Shi had moderate amounts; and C3H/He and SPR had the highest levels as compared to the other mice. The F1 progeny of A/J x C3H/He and AKR/N x C3H/He showed significantly higher NO production, whereas the F1 progeny of DBA/2N x C3H/He produced a relatively low amount. Furthermore, the backcross progeny from their F1 showed variations in NO production, and therefore it was speculated that the regulation of NO production is polygenic. Genetic typing experiments related to the NO production in the backcross progeny demonstrated significant deviations to some genetic microsatellite markers. Sequencing of the iNOS promoter regions of the Nramp1r strains to examine the relationship with NO production revealed that MOG and SPR strains had substitutions within the NF-kappaB and the gamma-IRE transcription binding factor, respectively.