The lectins, a class of proteins that occur widely in animals, plants, fungi, lichens and microorganisms, are known for their ability to specifically bind to carbohydrates. Plant lectins can be ...classified into 12 families including the Galanthus nivalis agglutinin (GNA)-related lectin superfamily, which is widespread among monocotyledonous plants and binds specifically to mannose, a behavior that confers remarkable anti-tumor, anti-viral and insecticidal properties on these proteins. The present study characterized a mitogenic lectin from this family, called tarin, which was purified from the crude extract from taro (Colocasia esculenta). The results showed that tarin is a glycoprotein with 2–3% carbohydrate content, composed of least 10 isoforms with pIs ranging from 5.5 to 9.5. The intact protein is a heterotetramer of 47kDa composed of two non-identical and non-covalently associated polypeptides, with small subunits of 11.9kDa and large subunits of 12.6kDa. The tarin structure is stable and recovers or maintains its functional structure following treatments at different temperatures and pH. Tarin showed a complex carbohydrate specificity, binding with high affinity to high-mannose and complex N-glycans. Many of these ligands can be found in viruses, tumor cells and insects, as well as in hematopoietic progenitor cells. Chemical modifications confirmed that both conserved and non-conserved amino acids participate in this interaction. This study determined the structural and ligand binding characteristics of a GNA-related lectin that can be exploited for several different purposes, particularly as a proliferative therapeutic molecule that is able to enhance the immunological response.
•Tarin is a glycoprotein with at least 10 isoforms with pIs ranging from 5.5 to 9.5.•It is a heterotetramer of 47kDa containing two non-covalently associated monomers.•Tarin functional structure is stable at different temperatures and pH.•This lectin shows a complex specificity toward high-mannose and complex N-glycans.•Conserved and non-conserved amino acids participate in the binding activity.
•Crude taro extract promoted total splenocytes proliferation in vitro and in vivo.•Inoculation of CTE in BALB/c and C57BL/6 resulted in B220+ splenocytes increase.•The extract caused in vivo ...reduction of B220+IgM+ and B220+IgM from bone-marrow.•Non-conventional B cells from C57BL/6 were also stimulated by TCE treatment.•Taro corms are a natural source of immunostimulatory proteins.
Besides its nutritional value, taro (Colocasia esculenta) is traditionally used as a medicinal plant and provides bioactive compounds with important biological properties. This study evaluated the in vitro and in vivo immunomodulatory potential of the protein extract from taro corms on the haematopoietic cells of C57BL/6 and BALB/c mice. The crude taro extract (CTE) stimulated the in vitro proliferation of mice splenocytes in a dose-dependent manner in both mice strains. The intraperitoneal inoculation of CTE induced splenomegaly and the proliferation of total spleen and bone-marrow cells. Additionally, CTE promoted in vivo B220+ splenocyte proliferation, which was accompanied by a reduction of mature (B220+ IgM+) and immature (B220+ IgM−) B cells in the bone marrow. The production of Antibody by B220+ splenocytes indicated the activation of B1 cells by the CTE only in C57BL/6. CTE represents a powerful source of immunostimulatory proteins, new candidates as additives for food and pharmaceutical industries.
Tarin, the Colocasia esculenta lectin from the superfamily of α-d-mannose-specific plant bulb lectins, is a tetramer of 47 kDa composed of two heterodimers. Each heterodimer possesses homologous ...monomers of ~11.9 (A chain) and ~12.7 (B chain) kDa. The structures of apo and carbohydrate-bound tarin were solved to 1.7 Å and 1.91 Å, respectively. Each tarin monomer forms a canonical β-prism II fold, common to all members of Galanthus nivalis agglutinin (GNA) family, which is partially stabilized by a disulfide bond and a conserved hydrophobic core. The heterodimer is formed through domain swapping involving the C-terminal β-strand and the β-sheet on face I of the prism. The tetramer is assembled through the dimerization of the B chains from heterodimers involving face II of each prism. The 1.91 Å crystal structure of tarin bound to Manα(1,3)Manα(1,6)Man reveals an expanded carbohydrate-binding sequence (QxDxNxVxYx
WX) on face III of the β-prism. Both monomers possess a similar fold, except for the length of the loop, which begins after the conserved tyrosine and creates the binding pocket for the α(1,6)-terminal mannose. This loop differs in size and amino-acid composition from 10 other β-prism II domain proteins, and may confer carbohydrate-binding specificity among members of the GNA-related lectin family.
Preparation of RNA free from DNA is a critical step before performing RT-PCR assay. Total RNA isolated from several sources, including those obtained from Saccharomyces cerevisiae, using routine ...methodologies are frequently contaminated with DNA, which can give rise to amplification products that mimic the amplicons expected from the RNA target.
We investigated the efficiency of two DNase I based protocols for eliminating DNA contaminations from RNA samples obtained from yeast cells. Both procedures are very efficient in eliminating DNA contamination from RNA samples and entail three main steps, which involve treating of RNA samples with DNase I, inhibition of the enzyme by EDTA and its subsequent inactivation at 65 degrees C. The DNase I treated samples were further purified with phenol: chloroform followed by precipitation with ice-cold ethanol (protocol I) or, alternatively, they were directly used in RT-PCR reactions (protocol II). Transcripts from ACT1, PDA1, CNA1, CNA2, TPS1 and TPS2 analyzed after each treatment showed that all mRNAs tested can be amplified if total RNA was extracted and purified after DNase I treatment, however, only TPS1, TPS2 and ACT1 mRNAs were amplified without extraction/purification step.
Although more laborious and requiring a higher initial amount of material, the inclusion of an extraction and purification step allows to prepare RNA samples that are free from DNA and from low molecular contaminants and can be applied to amplify any Saccharomyces cerevisiae mRNA by RT-PCR.
O objetivo foi utilizar métodos complementares de diagnóstico (histopatológicos, bacteriológicos e moleculares), no julgamento de lesões suspeitas de tuberculose observadas durante a inspeção post ...mortem de rotina em abatedouros. Foi acompanhado o abate e a inspeção de 41.193 bovinos, sadios ao exame ante mortem, em sete abatedouros no estado de Mato Grosso. Carcaças de 198 (0,48%) animais apresentaram lesões, sendo 182 (92,0%) classificadas como granulomatosas ou piogranulomatosas na avaliação histopatológica. Entretanto, na baciloscopia, não foi evidenciada a presença de bacilo álcool-ácido resistente (BAAR). Mycobacterium bovis foi isolado em três (1,5%) lesões, provenientes de linfonodos retrofaringeanos de bovinos com até três anos de idade. Quando usado a PCR múltipla (m-PCR) diretamente nos fragmentos de tecido, detectou-se a presença de DNA de M. bovis em 14 (7,0%) lesões, incluindo as três amostras identificadas na análise bacteriológica. O julgamento das lesões pelo exame macroscópico concordou em 93,0% (184/198) com os resultados obtidos por meio da PCR. A fim de evitar equívocos durante a avaliação, principalmente das lesões paucibacilares, como as encontradas neste estudo, recomenda-se a utilização de testes complementares rápidos e confirmatórios. A m-PCR, associada à inspeção post mortem de rotina, demonstrou ser uma técnica promissora para a vigilância da tuberculose bovina em abatedouros, contribuindo para o sucesso do programa de erradicação da tuberculose bovina.
When different strains of Saccharomyces cerevisiae grown at 23 degrees C were transferred to 36 degrees C, trehalose and glycogen were accumulated. Glycogen accumulation was less extensive and its ...synthesis started at least 15 min after initiation of trehalose synthesis. The steady-state intracellular concentration of trehalose increased simultaneously with the activities of the enzymes trehalose-6P synthase. UDPG-pyrophosphorylase phosphoglucomutase and trehalase. A small but significant change was observed in hexokinase activity. Our results directly implicate isoform PII of hexokinase and the minor isoform of phosphoglucomutase in the pathway of trehalose formation during heat-shock. We also showed that the major isoform of phosphoglucomutase increased in activity but was not essential for trehalose accumulation. Studies with the glucose uptake system indicated that trehalose accumulation could be primarily determined by intracellular availability of substrates due to the increase in the rate of glucose uptake. The increased uptake appears to have two components: a kinetic effect of temperature upon glucose transporters and an increase in the numbers of molecules of the transporters. probably mediated by synthesis de novo.
Three bacterial strains were isolated from the activated sludge system of petroleum refinery wastewater, identified by partial sequencing of 16S rDNA, and classified as Acinetobacter genomospecies 3, ...Bacillus pumilus, and Bacillus flexus. The degradation efficiency of aromatic hydrocarbons was evaluated by gas chromatography with a flame ionization detector. In a mineral medium containing anthracene and phenanthrene and the consortium of microorganisms, the removal efficiency was 96% and 99%, respectively, after 30 days. The good rate of hydrocarbon degradation proves the operational efficiency of the microbial consortium in treating effluents containing these compounds.
UDPG-pyrophosphorylase (EC 2.7.7.9) from
Saccharomyces cerevisiae was studied and the presence of isoforms investigated. Its activity was monitored during growth of cultures in rich media containing ...glucose, galactose, sucrose, maltose or glycerol as carbon sources. The results suggest that UDPG-pyrophoshorylase is subject to both catabolite repression and catabolite inactivation. The inactivation process seems to be complex, in order to produce maximum inactivation, glucose and ammonium sulfate must be added together. Addition of glucose or ammonium sulfate separately produced little effect upon enzyme activity. Adsorption to and elution from a DEAE-Sephacel column of a crude protein extract prepared from yeast cell, collected in stationary phase from a glucose medium showed three activity peaks, which we denominated isoform I, II and III. Isoform I is constitutive, it was the only form present during exponential growth on glucose medium, and did not suffer any alteration after glucose exhaustion, heat shock or by growing cells on maltose. On the other hand, isoforms II and III were shown to be repressed by glucose, and induced by heat shock. Furthermore, isoform II of UDPG-pyrophosphorylase was present together with isoform I when yeast cells were grown on maltose. The presence of a
MAL4
C allele rendered isoform II constitutive. Interestingly a
gal3 mutant strain had low UDPG-pyrophosphorylase activity and isoforms I and II were not expressed. These results are discussed in relation to trehalose metabolism.
CNA1 and
CNA2 encode isoforms of the catalytic subunit of calcineurin, a Ser/Thr-specific phosphoprotein phosphatase regulated by Ca
2+/calmodulin. The relative abundance of both transcripts was ...evaluated during growth of
Saccharomyces cerevisiae in glucose by reverse-transcription polymerase chain reaction using
PDA1 mRNA as a novel internal standard.
CNA1 and
CNA2 were concomitantly transcribed with different average expression ratios at the exponential and stationary growth phases and both showed a remarkable drop in the expression at diauxie. Prolonged hyper-osmotic shock resulted in a moderate induction of
CNA1, whereas
CNA2 expression was not affected.
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