Properties of ATP-dependent Phosphofructokinase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

ATP-dependent phosphofructokinase (ATP:D-fructose S-phosphate, 1-phosphotransferase, EC 2.7.1.11, PFK) from endosperm of developing wheat grains was purified to apparent homogeneity with about 45% recovery using ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sepharose CL-SB and Sephadex G-200. The purified enzyme with a molecular weight of about 182 kD, was a heterotetramer with subunit molecular weights ranging between 20 and 80 kD. The enzyme exhibited maximum activity at pH 7.9 and was highly specific for its substrates. The enzyme had absolute requirement for Mg2+. At pH 7.9, the Km values as determined by Lineweaver-Burk plots were 1.43 and 0.70 mM, respectively for fru-S-P and ATP. Fru-2, S-P2 had no effect on the activity of the enzyme. The enzyme was inhibited strongly by citrate, ADP, 3-PGA and PEP with Ki values of 2.40, 1.75, 2.10 and 0.80 mM, respectively. Citrate and PEP inhibited the enzyme competitively with respect to both fru-S-P and ATP. ADP and 3-PGA inhibited the enzyme non-competitively and competitively, respectively with respect to fru-S-P and in a mixed manner with respect to ATP. Hill plot values indicated co-operative interaction of citrate, 3-PGA and PEP with the enzyme.     

Adaptogenic activity of Indian Panax pseudoginseng

The crude extract and saponins of Indian pseudoginseng and saponins of Korean ginseng have been studied using a battery of biological tests in rats and mice. Indian pseudoginseng saponins were found to exhibit better activity than the Korean ginseng saponins in several tests employed. The results indicate a need for in-depth study of Indian pseudoginseng as an adaptogenic agent, after cultivation of the plant under controlled conditions.     

Kinetics and thermodynamic transitions of N-acetyl-β-D-glucosaminidase A and B in free and bound forms: Role of cellulose ion-exchangers

Summary The kinetic and thermodynamic properties of N-acetyl--D-glucosaminidase A (Hex A) and N-acetyl--D-D-glucosaminidase (Hex B) from goat testes were investigated in free and bound (after binding them on ion-exchangers such as DEAE- or CM-cellulose respectively) forms. The optimum pH of free Hex A and Hex B was at 4.2 and 5.4, whereas the bound forms showed the optimum pH at 4.0 and 5.2 respectively. While apparent K_m of free and bound Hex A (0.8 and 1.0 mM respectively) did not differ, the K_m of Hex B increased when bound on CM-cellulose (K_m of free Hex B = 0.96 mM versus bound Hex B = 1.6 mM). Though the free Hex A was more thermo-labile than the free Hex B, both isozymes, on insoluble matrices decayed at faster rates on heating. Activation analysis revealed that the energy of activation (E _infa ^supo ) for transition state of free Hex B (81 Kcal deg^–1 mole^–1) did not differ from E _infa ^supo of bound Hex B. On the other hand, E _infa ^supo of free Hex A declined from 77.2 to 71.1 Kcal deg^–1 mole^–1 when heat transitions were carried out in free and bound state respectively. Thermodynamic analysis suggested a change in entropy of activation (S^*) of free Hex A and Hex B as 200 and 211 eu respectively. While S^* of Hex B did not change after heat transitions, S^* of Hex A was 182.5 eu.     

Cyclic-amp control of some oxido-reductases during pine pollen germination and tube growth

Cyclic-AMP markedly increased the activities of peroxidase, malate dehydrogenase and succinate dehydrogenase but not glucose-6-phosphate dehydrogenase. Using inhibitors of protein and RNA synthesis, it was found that a part of enzyme activity increase caused by cyclic-AMP required fresh protein synthesis. The question of specificity of enzyme induction by cyclic-AMP has been examined.     

Ultraviolet-B Induced Changes in Ultrastructure and D1/D2 Proteins in Cyanobacteria Synechococcus sp. PCC 7942

Effects of ultraviolet-B (UV-B) irradiation on ultrastructure, total cellular protein, and PS2 proteins D1 and D2 of Synechococcus sp. PCC 7942 cells was studied. The scanning electron micrographs showed UV-B radiation induced bending of the cells. The transmission electron micrographs revealed disorganization and shift in thylakoid lamellar structure to one side of the cell. The cellular phycocyanin/chlorophyll ratio decreased with increasing UV-B treatment and due to this the colour of cells turned light-green. No apparent change in total cellular proteins was evident, but the contents of two major proteins of PS2, D1 and D2, showed decline due to UV-B irradiation, although to different extent.     

Shigella dysenteriae type 1 toxin induced lipid peroxidation in enterocytes isolated from rabbit ileum

To evaluate the role of reactive oxygen species (ROS) in Shigella dysenteriae 1 toxin (STx) mediated intestinal infection, the ligated rabbit small intestinal loops were injected with STx. The enterocytes isolated from STx treated rabbit ileal loops had a significantly higher level of lipid peroxidation as compared to enterocytes isolated from control rabbit ileum. To study the role of second messengers in STx mediated intestinal damage, the in vivo and in vitro effects of modulators of lipid peroxidation of enterocytes were used. The presence of Ca²⁺-ionophore A23187 enhanced the extent of lipid peroxidation in enterocytes isolated from the control and STx treated rabbit ileum. However, l-verapamil only marginally decreased the lipid peroxidation level of enterocytes isolated from STx treated rabbit ileum. The in vitro effect of modulators was in agreement with in vivo studies. Dantrolene significantly decreased the extent of lipid peroxidation of enterocytes isolated from STx treated rabbit ileum. PMA significantly increased the lipid peroxidation level of enterocytes isolated from control ileum. However, PMA could not further enhance the lipid peroxidation level of enterocytes isolated from STx treated rabbit ileum. The presence of H-7 significantly decreased the extent of lipid peroxidation of enterocytes isolated from STx treated rabbit ileum. In vitro effect of PMA and H-7 was in agreement with that of in vivo findings. The role of arachidonic acid metabolites, prostaglandins (PGs), in mediating STx induced lipid peroxidation was also studied. The presence of indomethacin (a PG synthesis inhibitor) significantly decreased the lipid peroxidation induced by STx. These findings suggest that lipid peroxidation induced by STx is mediated through cytosolic calcium. The increase in (Ca²⁺)_i leads to activation of PKC.A significant decrease in the enterocyte levels of antioxidant enzymes superoxide dismutase, catalase and reduced glutathione in STx treated rabbit ileum as compared to control was seen. A significant decrease in vitamin E levels was also observed. This suggests that there is decreased endogenous intestinal protection against ROS in STx mediated intestinal infection which could contribute to enterocyte membrane damage that ultimately leads to changes in membrane permeability and thus to fluid secretion.     

Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. × 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. × 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (V_max) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of a-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes.     

Factors influencing older people's self reported use of dental services in the UK

Objectives: This study was designed to determine the use of dental services and factors associated with their use among the United Kingdoms' older population. Design: A national study involving 1,116 older people (aged 60 or older). Setting: Home Interview s were undertaken exploring the time and reason for last dental visit. In addition, socio-demographic characteristics and proxy oral health measures (self-reported number of teeth and edentulous status) of the respondents were collected. Results: Forty seven percent (528) claimed they visited the dentist within the past year, 10% (116) claimed that the reason for their last visit was because of a dental emergency, 43% (484) were classified as “regular attenders” - having attended the dentist within the past year for a non dental emergency. Bivariate analysis identified that regular dental attendance was associated with age (P<0.01), social class (P<0.01), income level (P<0.01), educational attainment (P<0.01), self-reported number of teeth possessed (P<0.01) and edentulous status (P<0.01). In regression analysis, self reported edentulous status and number of teeth possessed emerged as the most important factors in determining service utilisation. Possessing a full denture was associated with a 6-fold decrease, having accounted for other factors, in the likelihood of attending the dentist within the past year for a non dental-emergency (OR=0.15, CI 0.10,0.21). Conclusion: Less than half of the sample population were “regular dental attenders”, their attendance was associated with a number of socio-demographic and oral health factors. In particular, edentulous state was a major factor associated with their use of services.     

Nifedipine loaded chitosan microspheres: characterization of internal structure

In the present investigation, a simple technique was employed to obtain cross-sections of unloaded and nifedipine loaded chitosan microspheres. Microspheres, adhering to a polymerized resin block, were cut with an ultramicrotome and viewed with a scanning electron microscope. Unloaded microspheres exhibited a uniform dense matrix structure while crystals of nifedipine were clearly visible in the drug-loaded microspheres. At 2% drug loading, however, no crystals could be seen in the microspheres indicating that either the drug was molecularly dispersed or dissolved in the matrix at this concentration. This was confirmed by powder X-ray diffractometry studies where no peak due to crystalline nifedipine was observed. At high Span 85 concentration (1.5% w/v), the external surface of the microspheres collapsed, but the internal structure remained dense. When the drug was dispersed in the chitosan solution with stirring during preparation, the entrapment was good and the shape of the crystals was changed. The internal structure of the microspheres following dissolution exhibited the presence of pores.     

Establishment of genetic fidelity of In vitro-raised Lilium bulblets through rapd markers

Summary Random amplified polymorphic DNA (RAPD) markers were utilized for screening the clonal fidelity of in vitro-raised bulblets of Lilium sp. (Asiatic hybrids) produced through adventitious mode of propagation. The first set of 14 bulblets was randomly chosen from about 175 micropropagated bulblets regenerated from 1×1 cm² bulbscale segments (explants) of cultivar ‘Gran Paradiso’ after four subcultures (after 6 mo.). The second set of 15 bulblets was selected again randomly after 12 subcultures (after one and a half years) when the bulblets were to be taken out for transplantation. Of the 20 primers used to screen the samples, only 14 primers gave clear reproducible bands. The 14 primers produced a total of 163 (an average of 11.6 bands per primer) scorable bands. Analysis of individual primers revealed the RAPD patterns produced were all shared by both the in vitro-raised bulblets (randomly selected after four and 12 subcultures) and the mother bulb. There was no variation observed within the tissue culture-raised progenies. Thus, our results show that adventitiously propagated in vitro bulblets of Asiatic hybrids of lilies are clonally uniform and stable.