Effects of photophase and altitude on oviposition rhythm of the himalayan strains of Drosophila ananassae

The effects of varying photophase and altitude of origin on the phase angle difference (Ψ) of the circadian rhythm of oviposition during entrainment to light-dark (LD) cycles and the aftereffects of such photophases on the period of the free-running rhythm (τ) in constant darkness (DD) were evaluated in two Himalayan strains of Drosophila ananassae, the high-altitude (HA) strain from Badrinath (5,123 m above sea level=ASL) and the low-altitude (LA) strain from Firozpur (179 m ASL). The Ψ (i.e., the hours from lights-on of the LD cycle to oviposition median) of both strains was determined in LD cycles in which the photophase at 100 lux varied from 6 to 18 h/24 h. The HA strain was entrained by all LD cycles except the one with 6 h photophase in which it was weakly rhythmic, but the LA strain was entrained by only three LD cycles with photophases of 10, 12, and 14 h, but photophases of 6, 8, 16, and 18 h rendered it arrhythmic. Lights-off transition of LD cycles was the phase-determining signal for both strains as oviposition medians of the HA strain occurred∼6 h prior to lights-off, while those of the LA strain occurred∼1 h after lights-off. The Ψ of the HA strain increased from∼2 h in 8 h photophase to∼11 h in 18 h photophase, while that of the LA strain increased from∼11 h in 10 h photophase to∼15 h in 14 h photophase. The aftereffects of photophase of the prior entraining LD cycles on τ in DD were determined by transferring flies from LD cycles to DD. The τ of the HA strain increased from∼19 to∼25 h when transferred to DD from LD 8:16 and LD 18:6 cycles, respectively, whereas the τ of the LA strain increased from∼26 to∼28 h when transferred to DD from LD 10:14 and LD 14:10 cycles, respectively. Thus, these results demonstrate that the photophases of entraining LD cycles and the altitude of origin affected several parameters of entrainment and the period of the free-running rhythm of these strains.     

Altitudinal variation in phase response curves for the Himalayan strains of Drosophila helvetica

In previous research, it was determined that the altitude of origin altered the parameters of photic entrainment and free-running rhythmicity of adult locomotor activity of the high-altitude Himalayan (haH) strain (Hemkund-Sahib, 4121 m above sea level) of Drosophila helvetica compared to the low-altitude Himalayan (laH) strain (Birahi, 1132 m above sea level) of the same species. The present study investigated whether the altitude of origin also affects the parameters of the light pulse phase response curve (PRC) of the adult locomotor activity rhythm of the haH strain. Light pulse PRCs were determined for both strains against the background of constant darkness. Although both were "weak" or type 1 PRCs, the PRC for the haH strain differed from that of the laH strain in three basic parameters. The PRC for the haH strain was of low amplitude, had a protracted dead zone, and showed a ratio of the advance to delay region (A/D>1), while the PRC of the laH strain was characterized by high amplitude, absence of dead zone, and a A/D ratio<1. The asymmetric PRCs of these strains might explain the process of photic entrainment to 24 h light-dark cycles, as the long period of the free-running rhythm (tau) of the haH strain is complemented with a larger advance portion of its PRC (A/D>1), whereas the short tau of the laH strain is matched with a larger delay portion of its PRC (A/D<1). Prolonged dead zone and low amplitude in the PRC of the haH strain imply that the photic sensitivity of this strain has been drastically diminished as an adaptation to environmental conditions at the altitude of its origin. While adults of this strain begin activity in very bright light in the forenoon due to non-permissible low temperature in the morning, the converse is true for the laH strain.     

Statistics of catalytic domain sequences in protein kinases vs non-kinases

The catalytic regions of Protein Kinases are known to have similarity in primary chains. However, it is not known whether there is a signature profile specific to a particular catalytic region? Whether the signature profile, if any, is unique to a protein kinases family in a particular species or in a group of species? We have attempted analyzing some of these aspects by statistical data mining using an authentic and exhaustive database of Protein Kinases. The results reveal interesting features and provide some new directions to look at their applications.     

Protease-catalyzed synthesis of the tripeptide CCK<Subscript>26–28</Subscript>, a fragment of CCK-8

Summary. Two enzymatically synthetic strategies of the tripeptide derivative PhAc-Asp(OMe)-Tyr-Met-OAl are reported. The second strategy gains the advantage of more economical starting materials, less reaction steps and a higher overall isolated yield of this tripeptide fragment over the first strategy. The effect of the acyl-donor ester concentration and structure, the C-α protecting group of the nucleophile, reaction media, enzyme and the carrier on the tripeptide derivative synthesis were studied. This tripeptide selected is a fragment of the cholecystokinin C-terminal octapeptide (CCK-8), a potential therapeutic agent in the control of gastrointestinal function and also a drug candidate for the treatment of epilepsy.     

Encapsulation of endoglucanase using a biopolymer Gum Arabic for its controlled release

Gum Arabic, a biodegradable natural polymer was used as a matrix to encapsulate endoglucanase from Thermomonospora sp. The modified enzyme retained complete biocatalytic activity and exhibited a shift in the optimum temperature [50-55 degrees C] and considerable increase in the pH and temperature stabilities as compared to the free enzyme. Encapsulation of the enzyme also protected the activity in presence of detergents and enhanced the shelf life. A 3-fold decrease in the initial rate of reaction indicated a controlled release of the enzyme conferring properties preferred for its potential application in the manufacture of detergents.     

The structure-activity relationship of the series of non-peptide small antagonists for p56lck SH2 domain

The antagonists for the SH2 domain are regarded as novel therapeutic candidates for cancer, autoimmune disease, and chronic inflammatory disease. Previously, we identified rosmarinic acid (alpha-o-caffeoyl-3,4-dihydroxyphenyl-lactic acid; RosA) from Prunella vulgaris as an antagonist for the p56lck SH2 domain by screening natural products. RosA not containing phosphotyrosine surrogate had a considerable inhibitory activity for T-cell antigen receptor (TCR)-induced interleukin (IL)-2 expression, and subsequent T-cell proliferation in vitro cell assay. To investigate the structure-activity relationship of RosA and to identify a novel p56lck SH2 antagonist with more potent in vitro T-cell inhibitory activity, we synthesized several analogs of RosA by using rational design. All synthesized compounds were tested in vitro binding activity for the SH2 domain and in vitro T-cell inhibitory activity. All four hydroxyl groups of RosA were essential for binding with the p56lck SH2 domain and T-cell inhibitory activity. Unexpectedly, conformationally less constrained analogs 4 and 9 showed a more potent binding affinity for the SH2 domain than that of RosA, and chirality of the analog did not play an important role in protein binding. We successfully identified several RosA analogs with a more potent T-cell inhibitory activity than that of RosA. Overall results revealed important structural requirements of the p56lck SH2 antagonists for in vitro T-cell inhibitory activity and in vitro protein binding activity.     

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10 ns, 150 kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by ∼2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10 ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA-protein complexes.     

Role of a conserved salt bridge between the PAS core and the N-terminal domain in the activation of the photoreceptor photoactive yellow protein

The effect of ionic strength on the conformational equilibrium between the I(2) intermediate and the signaling state I(2)' of the photoreceptor PYP and on the rate of recovery to the dark state were investigated by time-resolved absorption and fluorescence spectroscopy. With increasing salt concentration up to approximately 600 mM, the recovery rate k(3) decreases and the I(2)/I(2)' equilibrium (K) shifts in the direction of I(2)'. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl(2), MgSO(4)) salts show that the low salt effect depends on the ionic strength and not on the cation or anion species. These observations can be described over the entire ionic strength range by considering the activity coefficients of an interdomain salt bridge. At low ionic strength the activity coefficient decreases due to counterion screening whereas at high ionic strength binding of water by the salt leads to an increase in the activity coefficient. From the initial slopes of the plots of log k(3) and log K versus the square root of the ionic strength, the product of the charges of the interacting groups was found to be -1.3 +/- 0.2, suggesting a monovalent ion pair. The conserved salt bridge K110/E12 connecting the beta-sheet of the PAS core and the N-terminal domain is a prime candidate for this ion pair. To test this hypothesis, the mutants K110A and E12A were prepared. In K110A the salt dependence of the I(2)/I(2)' equilibrium was eliminated and of the recovery rate was greatly reduced below approximately 600 mM. Moreover, at low salt the recovery rate was six times slower than in wild-type. In E12A significant salt dependence remained, which is attributed to the formation of a novel salt bridge between K110 and E9. At high salt reversal occurs in both mutants suggesting that salting out stabilizes the more compact I(2) structure. However, chaotropic anions like SCN shift the I(2)/I(2)' equilibrium toward the partially unfolded I(2)' form. The salt linkage K110/E12 stabilizes the photoreceptor in the inactive state in the dark and is broken in the light-induced formation of the signaling state, allowing the N-terminal domain to detach from the beta-scaffold PAS core.     

DNA bending induced by carbocyclic sugar analogs constrained to the north conformation

DNA bending caused by introduction of carbocyclic sugars constrained to the north conformation was studied, using explicit solvent molecular dynamic (MD) simulations. The native Drew-Dickerson (DD) dodecamer and its three modifications containing north carbocyclic sugars in the 7th (T7*), 8th (T8*) or both 7th and 8th (T7T8*) nucleotide positions were examined. Introduction of the carbocyclic sugar results in A-form conformations for the alpha, beta, chi, zeta, and sugar pucker backbone parameters in the modified nucleotides. Increased steric repulsion between the sugar and its parent base in the modified oligonucleotides impacts the roll and cup dinucleotide step parameters, increasing the bending of the oligomer axis. Increased buckling of the substituted nucleotides disrupts the usual stabilizing base stacking interactions. The level of overall bending depends on the number and position of carbocyclic sugars introduced in the DNA sequence. Single sugar substitutions are unable to induce substantial bending due to the neighboring unmodified nucleotides counterbalancing the distortion. Significant bending can, however, be induced by two consecutive north sugars (T7T8*), which is in agreement with experimental results. The modified oligomers populate a wide range of bend angles, indicating that they maintain flexibility in the bent state. The present results suggest that insertion of carbocyclic sugars into DNA or RNA duplexes can be used to engineer bending of the duplexes without impacting the electrostatic or chemical properties of the phosphodiester backbone, thereby serving as excellent tools for experimental elucidation of nucleic acid structure-function relationships.