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Understanding the basis of biological diversity remains a central problem in evolutionary biology. Using microbial systems, adaptive diversification has been studied in (a) spatially heterogeneous environments, (b) temporally segregated resources, and (c) resource specialization in a homogeneous environment. However, it is not well understood how adaptive diversification can take place in a homogeneous environment containing a single resource. Starting from an isogenic population of yeast Saccharomyces cerevisiae, we report rapid adaptive diversification, when propagated in an environment containing melibiose as the carbon source. The diversification is driven due to a public good enzyme α-galactosidase, which hydrolyzes melibiose into glucose and galactose. The diversification is driven by mutations at a single locus, in the GAL3 gene in the S. cerevisiae GAL/MEL regulon. We show that metabolic co-operation involving public resources could be an important mode of generating biological diversity. Our study demonstrates sympatric diversification of yeast starting from an isogenic population and provides detailed mechanistic insights into the factors and conditions responsible for generating and maintaining the population diversity.

Melibiose is widely used as a functional carbohydrate. Whole-cell biocatalytic production of melibiose from raffinose could reduce its cost. However, characteristics of strains for whole-cell biocatalysis and mechanism of such process are unclear. We compared three different Saccharomyces cerevisiae strains (liquor, wine, and baker’s yeasts) in terms of concentration variations of substrate (raffinose), target product (melibiose), and by-products (fructose and galactose) in whole-cell biocatalysis process. Distinct difference was observed in whole-cell catalytic efficiency among three strains. Furthermore, activities of key enzymes (invertase, α-galactosidase, and fructose transporter) involved in process and expression levels of their coding genes ( suc2 , mel1 , and fsy1 ) were investigated. Conservation of key genes in S. cerevisiae strains was also evaluated. Results show that whole-cell catalytic efficiency of S. cerevisiae in the raffinose substrate was closely related to activity of key enzymes and expression of their coding genes. Finally, we summarized characteristics of producing strain that offered advantages, as well as contributions of key genes to excellent strains. Furthermore, we presented a dynamic mechanism model to achieve some mechanism insight for this whole-cell biocatalytic process. This pioneering study should contribute to improvement of whole-cell biocatalytic production of melibiose from raffinose.

Parkinson’s disease (PD) is a common neurodegenerative disease characterized by progressive loss of dopaminergic (DAergic) neurons in ventral brain. A disaccharide trehalose has demonstrated the potential to mitigate the DAergic loss in disease models for PD. However, trehalose is rapidly hydrolyzed into glucose by trehalase in the intestine, limiting its potential for clinical practice. Here we investigated the neuroprotective potentials of two trehalase-indigestible analogs, lactulose and melibiose, in sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Treatment with MPTP generated significant motor deficits, inhibited dopamine levels and down-regulated dopamine transporter (DAT) in striatum. Expression levels of genes involved in anti-oxidative stress pathways, including superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H dehydrogenase (NQO1) were also down-regulated, while expressions of oxidative stress marker 4-hydroxynonenal (4-HNE), microglial activation marker ionized calcium-binding adapter molecule 1 (IBA1) and astrocyte activation marker glial fibrillary acidic protein (GFAP) in ventral midbrain were up-regulated following MPTP treatment. MPTP also reduced the activity of autophagy, evaluated by autophagosomal marker microtubule-associated protein 1 light chain 3 (LC3)-II. Lactulose and melibiose significantly rescued motor deficits, increased dopamine in striatum, reduced levels of 4-HNE, IBA1 and GFAP, up-regulated SOD2, NRF2 and NQO1 levels, as well as LC3-II/LC3-I ratio in ventral midbrain with MPTP treatment. Our findings indicate the potential of lactulose and melibiose to protect DAergic neurons in PD.

Glycation is a non-enzymatic process involving the reaction of reducing sugars or reactive oxoaldehyde with proteins, lipids or nucleic acids, which results in the formation of advanced glycation end products (AGEs). The presented work discusses the glycation process in people with advanced stage of type 1 or type 2 diabetes. The concentration of different AGEs and their receptors for 58 serum samples was determined by ELISA and by spectrofluorimetric methods. In addition to fluorescent low molecular weight and protein-bound AGEs, we have also marked a new class of AGEs: melibiose-derived glycation product (MAGE). Our attention was also focused on the two groups of AGEs receptors: scavenger receptors (SR-A and SR-B) and RAGE. The correlation between the SR-AI scavenging receptors concentration and the fluorescence of AGEs as well as diabetes biological markers: GFR, creatinine contentration and HbA1c was demonstrated. A relationship between the concentration of AGEs and their receptors was also found in serum sample of patients treated with the metformin and aspirin. Furthermore, the concentration of SR-AI scavenger and the fluorescence of total AGEs was significantly lower in treated patients than in non treated patients. AGEs have also been found to contribute to the development of cardiovascular disease, atherosclerosis and diabetic complications, what could be deduced from the correlation of AGEs level and HDL cholesterol or uric acid level. Thus, it was confirmed that AGEs are involved in the pathomechanism of diabetes and other degenerative diseases. Nowadays, it is believed that AGEs due to the long time remaining in the body may be an important diagnostic marker. Their determination may allow monitoring the progression of the disease and the effectiveness of the therapy.

Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of d-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s−1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M−1 s−1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides d-(+)-galactose, the purified enzyme also acted against d-(+)-raffinose, α-d-(+)-melibiose, and methyl-α-d-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.

Objective Isofloridoside, a galactose-containing heteroside derived from marine red algae, has potential applications as a sweetener because it can activate the sweetness receptors T1R2/T1R3. The purpose of this study was to investigate the effects of isofloridoside on the growth and sucrose-dependent biofilm formation of the cariogenic bacterium Streptococcus mutans , to evaluate its potential as a caries-preventing agent. Results The results showed that S. mutans did not grow when isofloridoside was the sole carbon source. Isofloridoside inhibited sucrose-dependent biofilm formation by S. mutans in a concentration-dependent manner, similar to galactose and glucose, but unlike melibiose and galactose-containing disaccharides. Biofilm inhibition induced by isofloridoside was associated with inhibition of glucosyltransferase activity. Isofloridoside exhibited biofilm inhibition comparable to xylitol, an alternative sugar known to inhibit biofilm formation. The differential effects of isofloridoside and melibiose on biofilm formation may result from the structural differences that affect their interactions with S. mutans enzymes. These findings highlight the potential of galactose and its polysaccharides as regulators of S. mutans biofilm formation, and suggest that isofloridoside is a promising alternative sweetener for caries prevention.

Melibiose-derived AGE (MAGE) is an advanced glycation end-product formed in vitro in anhydrous conditions on proteins and protein-free amino acids during glycation with melibiose. Our previous studies revealed the presence of MAGE antigen in the human body and tissues of several other species, including muscles, fat, extracellular matrix, and blood. MAGE is also antigenic and induces generation of anti-MAGE antibody. The aim of this paper was to identify the proteins modified by MAGE present in human body fluids, such as serum, plasma, and peritoneal fluids. The protein-bound MAGE formed in vivo has been isolated from human blood using affinity chromatography on the resin with an immobilized anti-MAGE monoclonal antibody. Using mass spectrometry and immunochemistry it has been established that MAGE epitope is present on several human blood proteins including serum albumin, IgG, and IgA. In serum of diabetic patients, mainly the albumin and IgG were modified by MAGE, while in healthy subjects IgG and IgA carried this modification, suggesting the novel AGE can impact protein structure, contribute to auto-immunogenicity, and affect function of immunoglobulins. Some proteins in peritoneal fluid from cancer patients modified with MAGE were also observed and it indicates a potential role of MAGE in cancer.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by selective dopaminergic (DAergic) neuronal degeneration in the substantia nigra (SN) and proteinaceous α-synuclein-positive Lewy bodies and Lewy neuritis. As a chemical chaperone to promote protein stability and an autophagy inducer to clear aggregate-prone proteins, a disaccharide trehalose has been reported to alleviate neurodegeneration in PD cells and mouse models. Its trehalase-indigestible analogs, lactulose and melibiose, also demonstrated potentials to reduce abnormal protein aggregation in spinocerebellar ataxia cell models. In this study, we showed the potential of lactulose and melibiose to inhibit α-synuclein aggregation using biochemical thioflavin T fluorescence, cryogenic transmission electron microscopy (cryo-TEM) and prokaryotic split Venus complementation assays. Lactulose and melibiose further reduced α-synuclein aggregation and associated oxidative stress, as well as protected cells against α-synuclein-induced neurotoxicity by up-regulating autophagy and nuclear factor, erythroid 2 like 2 (NRF2) pathway in DAergic neurons derived from SH-SY5Y cells over-expressing α-synuclein. Our findings strongly indicate the potential of lactulose and melibiose for mitigating PD neurodegeneration, offering new drug candidates for PD treatment.

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.Guan and Hariharan report two crystal structures of melibiose transporter MelB in complex with substrate analogs, nitrophenyl-galactoside, and dodecyl-melibioside. Both structures revealed similar specific site for sugar recognition and resolved the cation-binding pocket, advancing the understanding of MelB and related transporters.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.