[The influence of the vitamin D3 level in the blood serum of lactase gene polymorphism on the development of chronic polypous rhinosinusitis]. / Vliianie urovnia vitamina D3 v syvorotke krovi i polimorfizma gena laktazy na razvitie khronicheskogo rinosinusita s polipami.

The objective of the present study was to elucidate the possible correlations between the vitamin D3 level in the blood serum and lactase gene polymorphism (LCT-13910 T>C) in the patients presenting with chronic polypous rhinosinusitis (CPRS). The study included 50 patients with this condition and 14 subjects comprising the control group. The variants of lactase gene polymorphism (LCT-13910 T>C) were identified with the use of polymerase chain reaction (PCR) in real time. The total level of serum vitamin D3 (VD3) was determined by means of the immunochemical analysis (the electrochemiluminescence technique). In the group of patients presenting with chronic polypous rhinosinusitis, the level of VD3 in the blood serum ranged from 48 nm/l to 85 nm/l (mean 60 nm/l) compared with that in the patients of the control group (from 78 nm/l to 112 nm/l; mean 97 nm/l) . The level of vitamin D3 'below the normal values' was documented in 71% of the patients with CPRS in comparison with 7% in the control subjects. Lactase gene polymorphism (LCT-13910 CC, CT) suggesting pronounced and latent hypolactasia was identified in 94% of the patients with CPRS compared with 78.6% in the control group. The occurrence of the CC genotype in the patients of both study groups was virtually identical: 52% in the patients presenting with chronic polypous rhinosinusitis and 57% in the control group. CT polymorphism was identified in 42% of the patients with CPRS and in 21% of the control subjects. The significant difference between the patients of the two groups was documented for the occurrence of TT polymorphism: 6% among the patients with CPRS and 21% in the controls (i.e. much higher in the healthy subjects). There was no significant difference between the serum levels of vitamin D3 either among the patients with CPRS having LCT-13910 gene polymorphisms (CC, CT, TT) or among the control subjects. It is concluded that the study revealed the higher levels of vitamin D3 in the blood sera from the control subjects in comparison with that in the patients with chronic polypous rhinosinusitis. Moreover, the patients of the latter group more frequently exhibited the variant of the LCT CT-13910 gene polymorphism suggesting latent hypolactasia whereas the subjects comprising the control group more frequently had the variant of the LCT CT-13910 gene polymorphism indicative of the normal tolerance of lactose.

In Vitro Functional Analyses of Infrequent Nucleotide Variants in the Lactase Enhancer Reveal Different Molecular Routes to Increased Lactase Promoter Activity and Lactase Persistence.

The genetic trait that allows intestinal lactase to persist into adulthood in some 35% of humans worldwide operates at the level of transcription, the effect being caused by cis-acting nucleotide changes upstream of the lactase gene (LCT). A single nucleotide substitution, -13910 C>T, the first causal variant to be identified, accounts for lactase persistence over most of Europe. Located in a region shown to have enhancer function in vitro, it causes increased activity of the LCT promoter in Caco-2 cells, and altered transcription factor binding. Three other variants in close proximity, -13907 C>G, -13915 T>C and -14010 G>C, were later shown to behave in a similar manner. Here, we study four further candidate functional variants. Two, -14009 T>G and -14011 C>T, adjacent to the well-studied -14010 G>C variant, also have a clear effect on promoter activity upregulation as assessed by transfection assays, but notably are involved in different molecular interactions. The results for the two other variants (-14028 T>C, -13779 G>C) were suggestive of function, -14028*C showing a clear change in transcription factor binding, but no obvious effect in transfections, while -13779*G showed greater effect in transfections but less on transcription factor binding. Each of the four variants arose on independent haplotypic backgrounds with different geographic distribution.

Toxicological evaluation of lactase derived from recombinant Pichia pastoris.

A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD50) based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure) did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system.

Intraspecific diversity within Ganoderma lucidum in the production of laccase and Mn-oxidizing peroxidases during plant residues fermentation.

Comparison of the potential for laccase and Mn-oxidizing peroxidases synthesis by ten strains of Ganoderma lucidum, originating from different worldwide areas, during solid-state fermentation of selected plant raw materials was the aim of this study. The great intraspecific variability in the production of analyzed enzymes as well as the dependence of the enzyme activity on plant raw materials were reported. The strain HAI 957 was the best laccase producer in the presence of corn stem, as a unique carbon source (129.46 U/L). The highest level of Mn-dependent peroxidase activity was noted after wheat straw fermentation by G. lucidum HAI 246 (78.64 U/L), while the maximal versatile peroxidase production (59.72 U/L) was observed in strain HAI 957 in the medium with oak sawdust.

Intolerancia a la lactosa: en quién y por qué / Lactose intolerance

No disponible

Lactase production by solid-state cultivation of Kluyveromyces marxianus CDBBL 278 on an inert support: effect of inoculum, buffer, and nitrogen source.

A study was carried out to select the conditions for cultivation of Kluyveromyces marxianus CDBBL 278 in solid-state culture (SSC) using polyurethane foam (PUF) as an inert support. PUF was impregnated with culture media containing lactose (50 g/L) as the carbon and energy source. Evaluation of culture parameters during different growth phases was carried out by respirometry. The effect of inoculum level, buffer capacity of the medium, and nitrogen source upon the yield of biomass on lactose (Yx/s) and production of lactase and inulinase was investigated. The highest lactase titre was achieved with an inoculum level of 1 x 10(7) cells per gram of wet matter (gwm) and 20% of the total nitrogen source provided as urea. The best biomass yield (0.37) was obtained when less than 40% of the total nitrogen was provided as urea. Using potassium phosphate allowed 90% substrate consumption in 30 h. In the best conditions, intracellular lactase and extracellular inulinase activities of 1147.7 IU/gX and 241.6 IU/gX were obtained, respectively, with a lag phase of 13.8 h and a rate of respiratory activity (microCO2) of 0.23 +/- 0.01 h(-1). To our knowledge, this is the first report on lactase production by K. marxianus CDBBL 278 in SSC. This study gives basic information about biomass yield and enzyme production using lactose as the sole carbon source in SSC on an inert support.

Hypolactasia as a molecular basis of lactose intolerance.

Lactase-phlorizin hydrolase (LPH), a membrane-bound glycoprotein present in the luminal surface of enterocytes in the intestine is responsible for lactose intolerance, a phenomenon prevalent in humans worldwide. In the rodent intestine, the post-natal development of the LPH follows a specific pattern, such that the enzyme levels are high in the peri-natal period, but declines considerably upon maturation. The observed maturational decline in the LPH activity is very similar to adult-type hypolactasia observed in humans. Majority of the studies have been carried out using animal models or cell lines and a number of hypotheses have been put forward to explain the maturational decline of lactase activity such as: (a) decreased amount of lactase protein, (b) defect in post-translational modification of precursor lactase to the mature enzyme, and (c) synthesis of an inactive, high molecular weight lactase with altered glycosylation, however, the precise underlying mechanism of adult-type hypolactasia remains undefined. The present review describes the recent developments in understanding the regulation of lactase expression and the possible mechanism of adult-type hypolactasia, as a cause of lactose intolerance.

Mutations in the translated region of the lactase gene (LCT) underlie congenital lactase deficiency.

Congenital lactase deficiency (CLD) is a severe gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. We initially assigned the CLD locus by linkage and linkage disequilibrium on 2q21 in 19 Finnish families. Here we report the molecular background of CLD via characterization of five distinct mutations in the coding region of the lactase (LCT) gene. Twenty-seven patients out of 32 (84%) were homozygous for a nonsense mutation, c.4170T-->A (Y1390X), designated "Fin(major)." Four rare mutations--two that result in a predicted frameshift and early truncation at S1666fsX1722 and S218fsX224 and two point mutations that result in substitutions Q268H and G1363S of the 1,927-aa polypeptide--confirmed the lactase mutations as causative for CLD. These findings facilitate genetic testing in clinical practice and enable genetic counseling for this severe disease. Further, our data demonstrate that, in contrast to common adult-type hypolactasia (lactose intolerance) caused by a variant of the regulatory element, the severe infancy form represents the outcome of mutations affecting the structure of the protein inactivating the enzyme.

[Overexpression of Aspergillus candidus lactase and analysis of enzymatic properties].

The lactase gene lacb' from Aspergillus candidus was fused behind alpha-factor signal sequence in the Pichia pastoris expression vector pPIC9, then integrated into the genome of P. pastoris by recombination events. The P. pastoris recombinants for lactase overexpression were screened by enzyme activity analysis and SDS-PAGE. The lactase expressed in P. pastoris was glycosylated protein with an apparent molecular weight of 130 kD, while the deglycosylated lactase treated with Endo H had an apparent molecular weight of about 110 kD. The expression level of secreted lactase protein in recombinant P. pastoris was 6 mg/mL with enzymatic activity of 3600 U/mL in the 5 L fermenter, which was the highest among that of all kinds of recombinant strains reported now. The optimal pH and optimal temperature of the lactase are 5.2 and 60 degrees C. The Vmax, Km, and specific activity of the lactase are 3.3 micromol/min, 1.7 mmol/L and 706.5 +/- 2.6 U/mg, respectively. Compare to the lactase from Aspergillus oryzae ATCC 20423, the expressed lactase from A. candidus have better enzymatic properties including the high thermostability, high specific activity and wide pH range for enzyme reaction.

Developmental changes in intestinal brush border enzymes of rats prenatally exposed to ethanol.

The activities of lactase, sucrase, alkaline phosphatase (AP) and y-glutamyl transpeptidase (gamma-GTP) were studied in the intestinal brush border membranes of pups born to rat mothers exposed to ethanol (1 ml of 30% ethanol daily during gestation) at different days of postnatal development. The activities of lactase (at day 4-20) and sucrase (at day 20-30) were considerably reduced in response to prenatal exposure to ethanol, while AP (at day 4-30) and gamma-GTP activities were significantly enhanced (p < 0.05) at day 4, 8, 14 and 20, but there was no significant difference by day 30 of postnatal development. The observed changes in enzyme activities were corroborated by Western blot analysis of lactase, sucrase and AP. Kinetic studies revealed a change in Vmax without affecting apparent Km of enzymes under these conditions. The present findings suggest that in utero ethanol exposure to rats is embryotoxic and affects the postnatal development of various brush border enzymes, which persist long after the ethanol was withdrawn prior to birth.

Expression of sodium-glucose co-transporter and brush border disaccharidases in Giardia lamblia infected rat intestine.

The absorption of D-glucose and brush border membrane disaccharidases in the intestine of rat during infection by Giardia lamblia has been studied. The level of mRNA encoding Na+/glucose co-transporter (SGLT1) and brush border sucrase and lactase activities were also analyzed. At the peak of infection, i.e, day 7, 11 and 15 post-infection, there was a marked decrease in the signal of 4.5 kb and 2.8 kb mRNAs encoding SGTL1 compared to the controls. A similar decrease in sucrase and lactase mRNA's (6.5 kb and 6.8 kb respectively) was also observed under these conditions. This corresponds to observed decrease in the rate of Na(+)-dependent D-glucose uptake and low activities of brush border sucrase and lactase under these conditions. There was no change in Na(+)-independent D-glucose uptake in giardia infected rat intestine. These findings suggest that the down regulation of the expression of SGLT1 and brush border sucrase and lactase activities may be responsible for the observed malabsorption in G. lamblia infection.