Molecular Simulation-based Combinatorial Modeling and Antioxidant Activities of Zingiberaceae Family Rhizomes.

OBJECTIVE: The main aim of this scientific report was to investigate a series of phytochemicals in silico and the pharmacology of four plants found at higher altitude in the ginger family, Zingiberaceae (incl. Costaceae) from North-East India, particularly Sikkim. First, the goal was to determine the biological activities of the four herbs (used under Zingiberaceae family) using antioxidant assays to identify the best species. Second, previously reported compounds in litero were subsequently screened for their anticancerous activities using in silico methods. MATERIALS AND METHODS: Using the methanolic extracts of herbs, quantitative detection of phytochemicals such as total phenols and total flavonoids was detected, and the free radical scavenging activity was also studied using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. Docking process was studied, using Discovery Studio version 3.5, to identify suitable molecules at the protein-binding sites through annealing and genetic simulation algorithms. Grids centered on active sites were obtained with spacing of 54 × 55 × 56, and 0.503 grid spacing was calculated. The methods adopted and used in this study were comparisons of Global and Local Search Methods to determine the parameters such as maximum number of 250,000 energy evaluations as well as generations of 27,000, followed by mutation and crossover rates of 0.02 and 0.80. The number of docking runs was set to 10. Molecular dynamics study was done to check the stability of the complex. RESULTS: Among all the genus of Zingiberaceae family investigated in this study, Curcuma angustifolia and Hedychium sp. exhibited the highest 537 ± 12.45; 292 ± 9.16 mg gallic acid equivalent/g total polyphenols and 38 ± 1.54; 75 ± 6.75 mg quercetin equivalent/g flavonoids, respectively. Depending on the concentration, the Hedychium sp. extract exerted the highest scavenging activity on DPPH radical (IC50 36.4 µg/mL). In silico result demonstrated that the synergetic effects of ß-phellandrene with other compounds might be responsible for its anticancerous activity. ß-phellandrene and farnesene epoxide showed bonding with Leu298, Ala302, Met336, Leu339, Leu343, Phe356, Ala302, Glu305, Met340, Leu343, Arg346, Phe356, Ile373, Ile376, Leu380, His475, Leu476, and Leu491. CONCLUSION: Based on the current available literature, this is the first study to understand the interaction of compounds found in the rhizomes of Zingiberaceae family. SUMMARY: The aqueous methanolic extract of Zingiberaceae family Curcuma angustifolia and Hedychium sp. has potent antioxidant activity as assessed by 2,2-diphenyl-1-picryl-hydrazyl assaysHedychium sp. is understood to possess more active compounds than other varietiesIn silico studies indicated synergetic effects of ß-phellandrene and other compounds for its anticancerous activity. Abbreviations used: CADD: Computer-aided drug designing; ROS: Reactive oxygen species; ADMET: Absorption, distribution, metabolism, and excretion-toxicity; FeCl3: Ferric chloride; DPPH: 2,2-diphenyl-1-picryl-hydrazyl; NaNO2: Sodium nitrite; TCA: Trichloroacetic acid; K2HPO4: Di-potassium hydrogen phosphate; H2O2: Hydrogen peroxide; KH2PO4: Potassium di-hydrogen phosphate, K2Fe (CN)6: Potassium ferricyanide; KOH: Potassium hydroxide; NaOH: Sodium hydroxide; Na2CO3: Sodium carbonate; CH3COONa: Sodium acetate; AlCl3: Aluminum chloride.

Impact of dental neglect score on oral health among patients receiving fixed orthodontic treatment: A cross-sectional study.

OBJECTIVE: Maintenance of meticulous oral health practices is critical for patients who are under orthodontic treatment as failure to do so can result in deterioration of periodontal health. Thus, the present study was commenced to assess dental negligence and oral health status among patients undergoing orthodontic treatment using dental neglect scale (DNS) questionnaire. MATERIALS AND METHODS: The present cross-sectional study was planned and carried out among the 40 patients undergoing fixed orthodontic treatment. The study comprised of two questionnaires, one was close-ended questionnaire which consisted of questions regarding patient practice in maintenance of oral health and other questionnaire comprised of DNS followed by examination of oral hygiene status using Oral Hygiene Index Simplified. Data so obtained were subjected to analysis using SPSS version 20 and Chi-square test was used to statistically analyze data with P < 0.05 regarded as a statistically significant value. RESULTS: The present study revealed that 63% among the studied orthodontic patients brushed once daily, 26% brushed twice daily, and 11% brushed thrice. About one-fourth was using brush with soft bristles and only 9% among the respondents used interdental aids. Data revealed positive correlation between DNS and oral hygiene index-simplified score with P < 0.05. CONCLUSION: The present study found that less frequency of brushing, rinsing mouth, and eating sticky and hard food can be attributed to self-neglect of the orthodontic patients.

Use of a halo frame for optimum intra- and post-operative management after scalp replantation/revascularization.

We present a new technique for stabilizing an avulsed scalp during and after replantation/revascularization. We used an aluminium "halo" frame with 4 screws. This technique can rigidly stabilize an avulsed scalp and eliminate the possibility of shearing/pressure necrosis. This device can make perioperative management easier and more comfortable for the patient and caregivers.

Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.

Characterization of the carbohydrate moiety is a critical measure of manufacturing process consistency of recombinant human Factor VIII (rFVIII) in Chinese-hamster ovary (CHO) cells. FVIII, a large (300 kDa) glycoprotein, is employed therapeutically for the correction of haemophilia A. While N-linked and O-linked oligosaccharides are found in this protein, the current study focuses on the N-linked oligosaccharides. The N-linked oligosaccharides from rFVIII were released using either peptide N-glycosidase F or endoglycosidase H, derivatized with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonate, and analysed by fluorophore-assisted carbohydrate electrophoresis (FACE). The electrophoretically resolved oligosaccharide bands were isolated and individual bands subjected to digestion with defined pools of exoglycosidases and re-electrophoresed on FACE sequencing gels. The resulting gel patterns were interpreted, based on band mobility shifts, to obtain the sequence structure of the oligosaccharides. A total of eight acidic and 12 neutral structures were identified, and the majority of the oligosaccharides (approximately 92%) were found to be sialylated. All of the major oligosaccharide structures found in CHO-cell-derived rFVIII have also been reported to be present in plasma-derived FVIII. Among the most abundant are disialylated, biantennary, core-fucosylated (approximately 40%), followed by trisialylated, triantennary, core-fucosylated and monosialo, biantennary, core-fucosylated structures (each approximately 18%). The Gal alpha 1-3Gal structures reported to be present in baby-hamster-kidney-cell-derived rFVIII were not found in the CHO-cell-derived protein. The glycosylation patterns were consistent in six random lots of rFVIII [coefficient of variation (%) 3-14] based on percentage lane luminance data of bands that represent approximately 98% of all asparagine-linked oligosaccharides.

Use of aspirin as the sole antiplatelet agent following prosthetic valve replacement in rheumatic heart disease.

Aspirin was administered as the sole antiplatelet agent in 147 patients following valve replacement, who were at low risk for thromboembolism. Of these, 67 underwent mitral valve replacement (MVR), 61 aortic valve replacement (AVR) and 19 combined aortic and mitral valve replacement (DVR). The mean follow up period was 6.63 years (range 1-14 years). The incidence of thromboembolic episodes (TEE) in patients following MVR, AVR, and DVR was 0.41, 0.80 and nil respectively. The TEE free survival at the first year follow-up was 98.4%, 99.3% and 100% in patients following MVR, AVR and DVR respectively. Fatal intracranial haemorrhage was not encountered. Valve thrombosis in this patient population was not seen. In conclusion, aspirin as the sole antiplatelet agent appears to be safe and effective following prosthetic valve replacement in selected patients. Further studies involving larger number of patients are necessary to confirm these results.

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate.

An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nM folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37 degrees C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23 +/- 0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37 degrees C remained as unmetabolized folic acid. Binding was also rapid at 0 degrees C but uptake at the plateau was only one-half the value obtained at 37 degrees C. Half-maximal saturation of the binding component (KD) occurred at a folate concentration of 0.065 nM at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (KD = 2.0 nM). 5-Methyltetrahydrofolate was also bound by this component (Ki = 13 nM at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (Ki = 45 nM) and methotrexate (Ki = 325 nM). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nM caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Natural feline leukemia virus variant escapes neutralization by a monoclonal antibody via an amino acid change outside the antibody-binding epitope.

We have molecularly cloned a natural variant of feline leukemia virus subtype B. This isolate is unique in that it is not neutralized by a monoclonal antibody which neutralized all other feline leukemia virus isolates tested, including members of the A, B, and C subtypes. Western immunoblotting indicated that the monoclonal antibody was less able to bind to the gp70 of the resistant isolate (designated lambda B1) than to the gp70s of susceptible viruses. Nucleotide sequence analysis of the envelope gene of lambda B1 revealed a high degree of homology with the susceptible Snyder-Theilen, Gardner-Arnstein, and Rickard subtype B isolates, including the presence of a 5-amino-acid minimal binding epitope required for binding by the neutralizing monoclonal antibody. The only change within the vicinity of this epitope was in a single nucleotide, and this difference changed a proline residue to leucine three amino acids from the N terminus of the binding epitope. Competitive binding studies with synthetic peptides indicated that substitution of leucine for proline resulted in a 10-fold decrease in the ability of the peptide to compete for antibody binding to native antigen. The results are consistent with the interpretation that this amino acid change lowers the affinity of antibody binding, resulting in failure of the antibody to neutralize the variant virus.

Transport of folate compounds by leukemic cells. Evidence for a single influx carrier for methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblasts.

Influx kinetics and inhibitor specificity have been compared for the transport of methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblastoid cells. Influx of each folate compound proceeded with approximately the same Vmax, fluctuated in the same fashion with the ionic composition of the medium, and was blocked by low concentrations of an N-hydroxysuccinimide ester of methotrexate in both an anion-deficient buffer and in a buffered saline medium containing physiological concentrations of glucose and bicarbonate. Moreover, methotrexate influx was inhibited by 5-methyltetrahydrofolate and folate, and the inhibition constants (Ki) of the latter compounds were equivalent to their Kt values for half-maximal influx. Folate influx was likewise inhibited by methotrexate. The Ki for methotrexate was equivalent to its Kt for influx, and o-phthalate and phosphate each inhibited folate and methotrexate with the same degree of effectiveness. Various reversible and irreversible inhibitors reduced the influx of each folate substrate by greater than 90%, and the progression of inhibition in each case was indicative of a single uptake component. Folate influx exhibited the same high sensitivity to inhibitors of methotrexate influx when measurements were performed at folate concentrations near the Kt for influx (10-50 microM) or at concentrations approximating physiological conditions (5-20 nM). These results indicate that CCRF-CEM cells possess a single shared transport system for the uptake of methotrexate, 5-methyltetrahydrofolate, and folate and that other high- or low-affinity uptake processes are not present in these cells.

Folate transport in Lactobacillus salivarius. Characterization of the transport mechanism and purification and properties of the binding component.

Lactobacillus salivarius cells contain an inducible transport system for folate. Influx via this system is time- and temperature-dependent, requires glucose and glutamine for optimum activity, and is half-maximal at folate concentrations in the nanomolar range. The folate internalized after 30 min at 30 degrees C is not released from the cells by excess extracellular folate and is recovered in cell extracts primarily in metabolized forms. A membrane-associated folate-binding protein is also present in cells that have been induced to transport folate. This binding protein constitutes 1% of total cellular protein, exhibits a high affinity for folate (KD = 0.40 nM), and requires divalent cations for optimum binding activity. Folate binds rapidly to this protein, while the exchange of bound substrate with folate added subsequently is relatively slow and dependent on the metabolic state of the cell. The transport rate per binding site is 0.05/min at 30 degrees C. A comparison of substrate specificity showed that folate binding and transport are both inhibited to the same extent by several different folate compounds, and a parallel irreversible inhibition of both processes is observed by prior treatment of the cells with a carbodiimide-activated derivative of folic acid. Binding protein labeled covalently with [3H]folate and solubilized with Triton X-100 was purified by a fractionation procedure involving absorption and elution from microgranular silica and molecular sieve chromatography. The isolated protein appeared homogeneous by gel electrophoresis and had an apparent molecular weight of 21,000. Monoclonal antibodies to the folate transport protein of Lactobacillus casei showed a high degree of cross-reactivity to the isolated binding protein from L. salivarius, indicating that these proteins share common epitopes. These results suggest that folate uptake by L. salivarius proceeds via an abundant membrane-associated binding protein which facilitates the movement of folate across the membrane as an electroneutral complex with cations. The substrate then slowly dissociates from internalized binding sites and is metabolized sequentially to coenzyme forms and then to membrane-impermeable folylpolyglutamates.

Characterization of the individual transport routes that mediate the influx and efflux of methotrexate in CCRF-CEM human lymphoblastic cells.

The transport routes utilized by CCRF-CEM human lymphoblastic cells for the influx and efflux of methotrexate have been analyzed. Evidence was obtained for a single influx route for methotrexate: (a) Influx at 2 microM [3H]methotrexate was inhibited completely by high concentrations of unlabeled methotrexate, o-phthalate, and bromosulfophthalein, and the inhibition profile with each anion was monophasic; and (b) Pretreatment of the cells with an N-hydroxysuccinimide ester of methotrexate also blocked influx, and this inhibition was complete over a range of substrate concentrations from 2 to 50 microM. Influx was also saturable and proceeded with a maximum rate (Vmax) of 4.3 pmol/min/mg protein (at 37 degrees C) and with a Kt of 0.8 microM in an anion-deficient buffer and 4.6 microM in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonate-buffered saline. The ratio of Vmax to the amount of carrier protein (0.3 pmol/mg protein) gave a turnover number for the transport system of 14.3/min. In contrast to influx, methotrexate efflux proceeded via three routes which could be separated by their sensitivity to specific inhibitors. The major portion of efflux occurred via the methotrexate influx carrier, the identity of which was established from its sensitivity to the N-hydroxysuccinimide ester of methotrexate and by its requirement for anions in the external medium. Methotrexate, adenosine monophosphate, and phosphate each stimulated efflux via this route and this stimulation was half-maximal at anion concentrations that approximated their Ki values for inhibition of methotrexate influx. A second efflux route was identified by its sensitivity to bromosulfophthalein. This route was relatively inactive and did not fluctuate significantly upon addition of various anions, glucose, or metabolic inhibitors. The third route was quantitated by its sensitivity to probenecid and its activity was increased in saline buffers and upon addition of glucose and was inhibited by oligomycin. Similar transport routes for methotrexate are present in L1210 mouse cells, although these two cell lines can be distinguished by the amount of transport protein and by the activity of the bromosulfophthalein-sensitive efflux route for methotrexate.

Kinetic evidence for two interconvertible forms of the folate transport protein from Lactobacillus casei.

Lactobacillus casei cells contain a folate transport protein which exhibits a high affinity for folate. The dissociation constant (KD) for folate derived from binding parameters at the steady state (at 0 degree C) is 0.4 nM at pH 7.5 and 0.1 nM at pH 6.0. In the present study, folate binding to this protein at pH 7.5 (and 0 degree C) was shown to follow second-order kinetics and to proceed with an association constant (k+1) of 4.9 X 10(7) liter/mol per min. K+1 was not affected by preincubation conditions which alter the energetic state of the cell. Measurements on the extent of binding showed further that (at 0 degree C) essentially all unoccupied folate-binding sites reside at or are readily accessible to the outer surface of the membrane. In contrast, after saturating the binding site with [3H]folate, the first-order rate constant (k-1) for dissociation of the bound substrate (at 0 degree C) was found to vary substantially with the conditions employed. k-1 was 0.028/min in freshly harvested cells, but it increased by 2.8-fold in cells preincubated at 23 degrees C for 60 min and by 5.4-fold in isolated membranes. In addition, the faster rate observed in preincubated cells (k-1 = 0.077/min) returned to a slower rate after brief exposure of the cells to pH 6.0 (k-1 = 0.041/min), glucose (k-1 = 0.050/min), or both (k-1 = 0.012/min). k-1 was twofold lower at pH 6.0 than at pH 7.5 and was less dependent on the preincubation conditions, although it also increased substantially (5.5-fold) when the cells were converted to plasma membranes. The proposed explanation for these results is that folate transport protein of L. casei exists in two forms which can be distinguished by the accessibility of the binding site to the external medium and whose amounts are dependent upon the presence of bound folate, the pH, and the energetic state of the cell. It is suggested that these forms are transport proteins with binding sites oriented towards the inner and outer surfaces of the membrane.

Differential interaction of cations with the thiamine and biotin transport proteins of Lactobacillus casei.

Lactobacillus casei cells contain separate and specific binding proteins which mediate the cellular uptake of thiamine and biotin. In buffered salt solutions, these proteins exhibit a very high affinity for their vitamin substrate. Dissociation constants (Kd values) at pH 7.5 are 0.03 and 0.15 nM for thiamine and biotin, respectively. Optimal binding of biotin requires the presence of cations. This cation dependence is substantial since the Kd for biotin is 60-fold higher in a buffer containing 0.1 mM K-Hepes, compared with a buffer composed of 50 mM K-Hepes and 5 mM MgCl2. Measurements of Kd versus cation concentration showed that Mg2+ is 300-fold more effective than K+ in promoting biotin binding. The extent of cation dependence decreases as the pH is reduced from 7.5 to 5.0, suggesting that protons can partially fulfill the cation requirement. In contrast, binding of thiamine to the thiamine transport protein shows no dependence on the ionic composition of the medium. These results suggest that the transport protein for the anionic vitamin, biotin, contains a binding site for cations. Cotransport of both the vitamin and cation into the cell might then occur during the normal transport cycle, allowing the cellular uptake of the vitamin to occur against the membrane potential. Conversely, the cationic vitamin, thiamine, does not appear to be transported into the cell as a complex with other ions.