Detection of phosphodiester adducts formed by the reaction of benzo[a]pyrene diol epoxide with 2'-deoxynucleotides using collision-induced dissociation electrospray ionization tandem mass spectrometry

In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE) with 2′-deoxynucleoside 3′-monophosphates. The B[a]PDE plus 2′-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M−H]⁻ consistent with B[a]PDE adducted (either on the base or phosphate group) 2′-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)−H]⁻, resulting from cleavage of the glycosidic bond between the 2′-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 2′-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 2′-deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)−H]⁻ and [(2′-deoxyribose+phosphate+B[a]Ptriol)−H]⁻, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 2′-deoxynucleotides.     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

Serum glucose-regulated protein 78 (GRP78) levels in COVID-19-associated mucormycosis: results of a case–control study

Mycopathologia - In experimental models, the expression of glucose-regulated protein 78 (GRP78) in endothelial cells played a role in the pathogenesis of mucormycosis. However, the role of GRP78 in...     

Tetramethylpyrazine Attenuates Cognitive Impairment Via Suppressing Oxidative Stress,Neuroinflammation, and Apoptosis in Type 2 Diabetic Rats

Cognitive dysfunction is an important complication observed in type 2 diabetes mellitus (T2DM) patients. Tetramethylpyrazine (TMP) is known to exhibit anti-diabetic and neuroprotective properties. Therefore, the present study aimed to investigate the possible therapeutic effects of TMP against type 2 diabetes-associated cognitive impairment in rats. High-fat diet (HFD) followed by a low dose of streptozotocin (35 mg/kg) was used to induce diabetes in Sprague–Dawley rats. TMP (20, 40, and 80 mg/kg) and Pioglitazone (10 mg/kg) were administered for 4 weeks. The Morris water maze (MWM) and novel objective recognition task (NOR) tests were used to assess memory function. Fasting blood glucose (FBG), lipid profile, HOMA-IR, glycosylated hemoglobin (HbA1c), and glucose tolerance were measured. Acetylcholinesterase (AChE) and choline acetytransferase (ChAT) activity, acetylcholine (ACh) levels, oxidative stress, apoptotic (Bcl-2, Bax, caspase-3), and inflammatory markers (TNF-α, IL-1β, and NF-kβ) were assessed. BDNF, p-AKT, and p-CREB levels were also measured. In the present work, we observed that treatment of diabetic rats with TMP alleviated learning and memory deficits, improved insulin sensitivity, and attenuated hyperglycemia and dyslipidemia. Furthermore, treatment with TMP increased BDNF, p-Akt, and p-CREB levels, normalized cholinergic dysfunction, and suppressed oxidative, inflammatory, and apoptotic markers in the hippocampus. Collectively, our results suggest that the TMP may be an effective neuroprotective agent in alleviating type 2 diabetes-associated cognitive deficits.

     

Development and molecular characterization of wheat--Aegilops kotschyi addition and substitution lines with high grain protein, iron, and zinc

Over two billion people, depending largely on staple foods, suffer from deficiencies in protein and some micronutrients such as iron and zinc. Among various approaches to overcome protein and micronutrient deficiencies, biofortification through a combination of conventional and molecular breeding methods is the most feasible, cheapest, and sustainable approach. An interspecific cross was made between the wheat cultivar 'Chinese Spring' and Aegilops kotschyi Boiss. accession 396, which has a threefold higher grain iron and zinc concentrations and about 33% higher protein concentration than wheat cultivars. Recurrent backcrossing and selection for the micronutrient content was performed at each generation. Thirteen derivatives with high grain iron and zinc concentrations and contents, ash and ash micronutrients, and protein were analyzed for alien introgression. Morphological markers, high molecular weight glutenin subunit profiles, anchored wheat microsatellite markers, and GISH showed that addition and substitution of homoeologous groups 1, 2, and 7 chromosomes of Ae. kotschyi possess gene(s) for high grain micronutrients. The addition of 1U/1S had high molecular weight glutenin subunits with higher molecular weight than those of wheat, and the addition of 2S in most of the derivatives also enhanced grain protein content by over 20%. Low grain protein content in a derivative with a 2S-wheat translocation, waxy leaves, and absence of the gdm148 marker strongly suggests that the gene for higher grain protein content on chromosome 2S is orthologous to the grain protein QTL on the short arm of group 2 chromosomes.     

Development of multiple embryos in polyembryonic insertional mutant OsPE of rice

A T-DNA insertional mutant OsPE of rice gives twin and triplet seedlings in up to 20?% of the seeds. Detailed cytological and histological analysis of OsPE indicated normal male and female gametogenesis in the OsPE mutant. Confocal laser scanning microscopic (CLSM) analysis of the developing seeds of OsPE showed multiple embryo development in up to 60?% of the ovules. The multiple embryos, mostly twins and triplets, and rarely quadruplets, developed through sequential cleavage from a single zygotic embryo in each ovule. The reduced number of multiple seedlings compared with multiple embryos observed in CLSM study may be attributed to their inability to develop further due to competition in a single embryo sac. Key message Multiple seedlings in the OsPE mutant are due to sequential proliferation and cleavage of the zygotic embryos. The nucellar tissue was not involved in multiple embryo development.     

Conformational behavior of polypeptides derived through simultaneous global conservative site-directed mutagenesis of chymotrypsin inhibitor 2

The natural occurrence of conservative residue substitutions in proteins suggests that side-chain packing schemes in protein interiors can accommodate mutational replacements of residues by others of similar nature. To explore the extent to which such substitutions are tolerated, especially when introduced simultaneously and globally over the entire length of a polypeptide chain, we examined the conformational behavior of a model 65 residues-long protein, wild-type chymotrypsin inhibitor 2 (WTCI2), and two globally-mutated (GM) variants named GMCI2-1 and GMCI2-2, each incorporating 55 conservative residue substitutions. GMCI2-1, was soluble over a wide range of pH, and folded into a compact, spherical, monomer marked by (i) complete absence of surface hydrophobicity, (ii) a WTCI2-like betaII-type CD spectrum, (iii) high WTCI2-like thermal stability, and (d) 1D and 2D NMR spectra characteristic of folded protein structure. GMCI2-2 was insoluble over a wide range of pH, and could be solubilized only at pH 4.0, showing non-WTCI2-like far-UV CD spectra characterized by high helical content. These results tentatively indicate that polypeptides incorporating residues of identical nature at equivalent chain locations can show the potential to fold with similar characteristics. However, further detailed investigations would be required to determine whether indeed the structural fold of GMCI2-1 resembles that of WTCI2, and to evaluate the extent to which it does so.     

Microsatellite Marker Linked to a Leaf Rust Resistance Gene from Triticum monococcum L Transferred to Bread Wheat

Triticum monococcum L, a diploid wheat species closely related to the A genome of cultivated wheats, is highly resistant to leaf rust. A synthetic amphiploid, T. monococcum — T. durum was crossed with T. aestivum cv WL711, highly susceptible to leaf rust. Leaf rust resistant derivatives were selected among backcross generations with the recurrent parent WL711 and cytologically analysed. Chromosome number of the leaf rust resistant BC₁F₃ progenies varied from 39 to 44. Six leaf rust resistant and susceptible bulks from different BC₁F₃ progenies were analysed using 29 wheat microsatellite(WMS) markers already mapped on A genome of bread wheat and found polymorphic among parents. One T. monococcum specific allele of WMS gwm136 locus was found to be closely linked to the leaf rust resistance gene in all the resistant bulks. Differential chromosome number, frequency of univalents and multivalents, however, indicated that the critical T. monococcum chromosome might be present in addition to the A genome chromosomes of wheat, substituted either for the B or D genome chromosome of wheat or translocated to chromosome 1A of wheat in one or the other bulks. The association of the T. monococcum specific allele of WMS gwm136 locus to leaf rust resistance was further confirmed from bulked segregant analysis in BC₂F₁ generation.     

Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.