Isomaltose production by modification of the fructose-binding site on the basis of the predicted structure of sucrose isomerase from "Protaminobacter rubrum"

"Protaminobacter rubrum" sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a (325)RLDRD(329) motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other alpha-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg(325) and Arg(328) in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R(325)Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter(-1) and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35 degrees C. Maximum isomaltose production (75.7 g liter(-1)) was recorded at 35 degrees C, and its yield for the consumed sucrose was 0.61 g g(-1) with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.     

PKR regulates B56(alpha)-mediated BCL2 phosphatase activity in acute lymphoblastic leukemia-derived REH cells

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic, scaffold, and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56alpha is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56alpha at serine 28 in vitro, but it has been unclear how PKR might regulate the BCL2 phosphatase. In the present study, PKR regulation of B56alpha in REH cells was examined, because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56alpha-mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56alpha phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells, as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56alpha proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56alpha protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2alpha activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56alpha, because wild-type but not mutant S28A B56alpha promoted mitochondrial PP2A activity. Cells expressing wild-type B56alpha but not S28A B56alpha were sensitized to etoposide. These results suggest that PKR regulates B56alpha-mediated PP2A signaling in REH cells.     

Paraoxonase and Arylesterase Levels in Behcet’s Disease and Their Relations with the Disease Activity

The aim of this study was to determine the paraoxonase (PON) and arylesterase (ARE) enzyme activity levels in Behcet’s disease (BD) and to investigate whether they are associated with the disease activity. Twenty-six patients (study group) with active BD and 28 healthy controls (control group) were included in this study. While the patients who had at least one of the symptoms related to genital ulcer, skin lesions, active uveitis, arthritis, thrombophlebitis, or central nervous system involvement in addition to oral ulcers were considered as the active group, the patients who did not show clinical symptoms in the last one month due to the medical treatment were considered as the inactive group in the clinical evaluation of patients with BD. The PON and ARE levels were found to be significantly lower in the study group than the control group (p < 0.05). The PON levels of the active and inactive groups were 96.23 ± 57.84 and 112.2 ± 65.14, respectively. The ARE levels of the active and inactive groups were 30.49 ± 5.81 and 30.85 ± 6.40, respectively. No significant correlations were found between clinical findings and the activity levels of PON and ARE in the active patient group (p > 0.05). The activities of the antioxidant PON and ARE enzymes are reduced in BD. Therefore, it may be useful to add antioxidant therapy to the conventional treatment of the disease.     

Prevalence of known mutations in the MEFV gene in a population screening with high rate of carriers

The Familial Mediterranean Fever (FMF) shows an autosomal recessive pattern of inheritance and affects certain ethnic groups. Disease is caused by mutations in MEFV gene and more than 180 mutations have been defined in affected individuals. Current study aimed to determine the frequency-type of the mutations for MEFV gene in Sivas??middle Anatolian city. The cohort was composed of 3340 patients. MEFV gene mutations were studied by multiplex PCR based reverse hybridization stripAssay method. Patients?? clinical features were; family history: 68%, erysipelas-like erythema: 17.6%, fever: 89.9%, abdominal pain: 84.2%, peritonitis: 90.2%, arthritis: 33%, pleuritis: 14.2%, parental consanguinity: 21.2%. Current results revealed that M694V is the most frequent mutation (43.12%), followed by E148Q (20.18), M680I(G/C) (15.00%) and V726A (11.32%). The study population has a high rate of carriers and the E148Q mutation frequency was found to be highest when compared to the other regions of Turkey and other Mediterranean groups.     

Growth response of Pinus densiflora seedlings to different fertilizer compound ratios in a recently burned area in the eastern coast of Korea

This study was conducted to determine fertilizer compound ratios suitable for soil conditions and tree seedling growth in recently burned areas in Korean forests. Currently, the conventional fertilizer ratio applied to undisturbed forests in Korea is N:P:K 3:4:1. In this study, Japanese red pine (Pinus densiflora S. et Z.) seedlings planted in the burned area were fertilized over four growing seasons with the following NPK compound ratios: unfertilized (CON), 3:4:1, 6:4:1, 3:8:1, and 3:4:2. Fertilization generally increased current-year needle nutrient concentrations of the seedlings, and the chlorophyll a:b ratio in CON was significantly lower than in all fertilized plots. Fertilization significantly affected the growth of the pine seedlings, which had 71–87 % more height growth and 29–67 % increased root collar diameter (RCD) compared to CON. The increases in height and RCD were significantly higher with the 6:4:1 and 3:8:1 ratios than with the 3:4:1 ratio. The 3:4:2 and 3:4:1 fertilizer ratios had no effect on the RCD growth of seedlings. This suggests that the early growth of pine seedlings could be improved by providing high N and P supplies to areas affected by forest fires rather than the conventional fertilizer ratio of 3:4:1 in Korean forest soils. Therefore, application of the suitable fertilization ratio may be a very effective way to reduce reforestation cost as well as to shorten restoration period.     

Genome structure and origin of nontoxigenic strains of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> of El Tor biovar with different epidemiological significance

Intraspecies genetic differentiation of nontoxigenic strains of Vibrio cholerae of El Tor biovar containing one of the key pathogenicity genes, tcpA, is studied along with the phylogenetic relationships between these strains and toxigenic isolates. Comparative analysis of the whole genome nucleotide sequences demonstrates for the first time that ctxA^–tcpA⁺ strains vary considerably and can be clustered into two separate groups, the CTX_φ–RS1_φ⁺VPI⁺VSP⁺/CTX_φ–RS1_φ–VPI⁺VSP⁺ isolates and the CTX_φ–RS1_φ–VPI⁺VSP^– isolates, differing in their epidemiological significance. In the course of model experiments, it is established that nontoxigenic potentially epidemic CTX_φ–RS1_φ⁺VPI⁺VSP⁺/CTX_φ–RS1_φ–VPI⁺VSP⁺ isolates are derivatives of toxigenic strains. The results of whole genome SNP analysis of 35 Vibrio cholerae strains confirm these data and indicate genetic remoteness of nontoxigenic CTX_φ–RS1_φ–VPI⁺VSP^– strains both from the potentially epidemic strains and from the toxigenic isolates. It is found that the genomes of the CTX_φ–RS1_φ–VPI⁺VSP^– strains contain unique SNPs which are characteristic of them alone. The new data on the structure of the genome of nontoxigenic strains with different epidemiological significance may be further used for their genetic differentiation.     

Novel regenerable potassium-based dry sorbents for CO2 capture at low temperatures

A novel potassium-based dry sorbent (KZrI) was developed for CO₂ capture at a low temperature range between 50 °C and 200 °C. The CO₂ absorption and regeneration properties of this novel regenerable potassium-based dry sorbent were measured in a fixed-bed reactor during multiple absorption/regeneration cycles at low temperature conditions (CO₂ absorption at 50–100 °C and regeneration at 130–200 °C). The total CO₂ capture capacity of the KZrI sorbent was maintained during the multiple CO₂ absorption/regeneration cycles. The XRD patterns and FTIR analyses of the sorbents after CO₂ absorption showed the KHCO₃ phase only except for the ZrO₂ phase used as support. Even after 10 cycles, any other new structures resulting from the by-product during CO₂ absorption were not observed. This phase could be easily converted into the original phase during regeneration, even at a low temperature (130 °C). The KZrI sorbent developed in this study showed excellent characteristics in CO₂ absorption and regeneration in that it satisfies the requirements of a large amount of CO₂ absorption (91.6 mg CO₂/g sorbent) and the complete regeneration at a low temperature condition (1 atm, 150 °C) without deactivation.