Crystal structure of ß1→6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α-galactose.

In a search for better comprehension of ß-galactosidase function and specificity, we solved the crystal structures of the GH42 ß-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme kcat without significant modification in its Km , whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with ß1→6 and ß1→3 bonds, while binding of the substrates with ß1→4 linkages is less favorable. DATABASE: Structural data are available in the PDB under the accession numbers 4UZS and 4UCF.

In vitro and in vivo biological effects of novel arylimidamide derivatives against Trypanosoma cruzi.

Chagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi, remains a serious public health problem in several Latin American countries. The available chemotherapies for CD have limited efficacy and exhibit undesirable side effects. Aromatic diamidines and arylimidamides (AIAs) have shown broad-spectrum activity against intracellular parasites, including T. cruzi. Therefore, our aim was to evaluate the biological activity of eight novel AIAs (16DAP002, 16SAB079, 18SAB075, 23SMB022, 23SMB026, 23SMB054, 26SMB070, and 27SMB009) against experimental models of T. cruzi infection in vitro and in vivo. Our data show that none of the compounds induced a loss of cellular viability up to 32 µM. Two AIAs, 18SAB075 and 16DAP002, exhibited good in vitro activity against different parasite strains (Y and Tulahuen) and against the two relevant forms of the parasite for mammalian hosts. Due to the excellent selective indexes of 18SAB075, this AIA was moved to in vivo tests for acute toxicity and parasite efficacy; nontoxic doses (no-observed-adverse-effect level [NOAEL], 50 mg/kg) were employed in the tests for parasite efficacy. In experimental models of acute T. cruzi infection, 18SAB075 reduced parasitemia levels only up to 50% and led to 40% protection against mortality (at 5 mg/kg of body weight), being less effective than the reference drug, benznidazole.

Activity of Debaryomyces hansenii UFV-1 α-galactosidases against α-D-galactopyranoside derivatives.

α-D-Galactopyranosides were synthesized and their inhibitory activities toward the Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases were evaluated. Methyl α-D-galactopyranoside was the most potent inhibitor compared to the others tested, with K(i)(') values of 0.82 and 1.12 mmolL(-1), for extracellular and intracellular enzymes, respectively. These results indicate that the presence of a hydroxyl group in the C-6 position of α-D-galactopyranoside derivatives is important for the recognition by D. hansenii UFV-1 α-galactosidases.

Improvement of SCP production and BOD removal of whey with mixed yeast culture

This research emphasizes on single cell protein (SCP) production and Biochemical Oxygen Demand (BOD) removal from whey with mixed yeast culture. For this purpose, 11 yeast strains were isolated from dairy products (M1-M11) and the strains were identified by morphological and physiological properties. These yeast strains were tested for their ability to reduce the BOD and to produce SCP from whey. Among these strains, K. lactis (M2) had the most SCP production from whey with the yield of 11.79 g/l. Ammonium sulphate as nitrogen source had an increasing effect on biomass yield. The mixed culture of the isolated yeast strains with Saccharomyces cerevisiae was used in order to increase the biomass yield and BOD removal. The highest biomass yield (22.38 g/l) and reduction of initial BOD from 30000 to 3450 mg/l were obtained with the mixed culture of K. lactis (M2) and S. cerevisiae.

[Adaptation of rats, after weaning, to a diet with a high concentration of lactose. I. Evaluation of water and diet ingestion]. / Adaptação de ratos, após desmame, à ração com concentração elevada de lactose. I. Avaliação da ingestão de água e ração.

Twelve female Wistar rats, weaned at 21 days, were allocated in two groups: Lactose Group composed by six rats receiving a normal laboratory diet added with 25 g of lactose for each 100 g of final mixture, and the Saccharose Group that was fed with the same diet but with the lactose being replaced by saccharose in the same proportion; both groups were studied for 28 days. The rats were weighed at the beginning of the experiment and on the 7th, 14th, 21st and 28th days; the relative ingestion of water and diet was evaluated for each animal on the same days except for the first. During the observation period weight gain was significantly lower on the Lactose Group compared to the Saccharose Group, although this difference became less evident towards the end of the experiment. In the Lactose Group the diet ingestion was higher on the 21st and 28th days opposed to the water ingestion which was higher on the 7th and 14th days. The results here presented suggest that, in spite of the ontogenic fall of intestinal lactase in rats, these animals can, even after weaning, accommodate to high doses of dietary lactose, by using adaptative pathways which deserve further investigation.

Relationship between changes in lactase activity and monosaccharide uptake in the small intestine of the rat during development.

The relationship between changes in intestinal lactase activity and monosaccharide uptake was studied in rats of different ages using the technique of intestinal everted sacs. In the postweaning period there is a sharp decrease in the rate of glucose and galactose uptake by the small intestinal mucosa. This change occurs simultaneously with a decrease in lactase activity in the tissue. The kinetic analysis showed a lower Vmax for monosaccharide uptake in the 50-days-old rats as compared with the fifteen-days-old animals, with no change in the apparent Kt. The addition of Tris+ (40 mM) to the incubation media was found to produce a decrease in Vmax for monosaccharide uptake only in the suckling rats but not in the 50-days-old animals; the K1 remained unchanged. Tris+, at the concentration used had previously been shown to completely inhibit lactase activity in homogenates of small intestinal mucosa. These observations lead us to postulate the lactase in the suckling rat might possess a translocating role for sugar, in addition to its hydrolytic function. The decrease in sugar uptake after weaning could be explained by the disappearance of this role.

The acro-osteolysis syndrome: Morphologic and biochemical studies.

The acro-osteolysis syndrome consists of dissolution of terminal phalanges of the hands and feet, dolichocephaly with multiple wormian bones, delayed closure of cranial sutures, absence of frontal sinuses, a prominent occipital ridge, skeletal demineralization, vertebral and extremity fractures, joint laxity, and coarse hair. Studies of bone morphology reveal diminished bone density and bone formation. Osteoblasts have widely dilated smooth endoplasmic reticulum. It is postulated that an abnormality of a structural protein is the pathogenic basis of this disease.