Drug release properties of polyethylene-glycol-treated ciprofloxacin-Indion 234 complexes

The polyethylene glycol (PEG) treatment of ciprofloxacin-Indion 234 complex was aimed to retard rapid ion exchange drug release at gastric pH. Ciprofloxacin loading on Indion 234 was performed in a batch process, and the amount of K⁺ in Indion 234 displaced by drug with time was studied as equilibrium constant K_DM. Drug-resin complex (DRC) was treated with aqueous PEG solution (0.5%–2% wt/vol) of different molecular weights (MWs) for 2 to 30 minutes. The PEG-treated ciprofloxacin-Indion 234 complex was evaluated for particle size, water absorption time, and drug release at gastric pH. During drug loading on Indion 234, the equilibrium constant (K_DM) increased rapidly up to 20 minutes with efficient drug loading. Increased time of immersion of the drug resinate in PEG solutions significantly retained higher size particles upon dehydration. The larger DRC particles showed longer water absorption times owing to compromised hydrating power. The untreated DRC showed insignificant drug release in deionized water; while at gastric pH, ciprofloxacin release was complete in 90 minutes. A trend of increased residual particle size, proportionate increase in water absorption time, and hence the retardation of release with time of immersion was evident in PEG-treated DRC. The time of immersion of DRC in PEG-treated DRC. The time of immersion of DRC in PEG solution had predominant release retardant effect, while the effect of molecular weight of PEG was insignificant. Thus, PEG treatment of DRC successfully retards ciprofloxacin ion exchange release in acidic pH.     

Binding of bovine pancreatic trypsin inhibitor to trypsinogen: spectroscopic and volumetric studies

We have investigated the binding of bovine pancreatic trypsin inhibitor (BPTI) to bovine trypsinogen by combining ultrasonic velocimetry, high precision densimetry, and fluorescence spectroscopy. We report the changes in volume, adiabatic compressibility, van't Hoff enthalpy, entropy, and free energy that accompany the association of the two proteins at 25 degrees C and pH 8.0. We have used the measured changes in volume and compressibility in conjunction with available structural data to characterize the binding-induced changes in the hydration properties and intrinsic packing of the two proteins. Our estimate reveals that 110 +/- 40 water molecules become released to the bulk from the hydration shells of BPTI and trypsinogen. Furthermore, we find that the intrinsic coefficient of adiabatic compressibility of the two proteins decreases by 14 +/- 2%, which is suggestive of the binding-induced rigidification of the proteins' interior. BPTI-trypsinogen association is an entropy-driven event which proceeds with an unfavorable change in enthalpy. The favorable change in entropy results from partial compensation between two predominant terms. Namely, a large favorable change in hydrational entropy slightly prevails over a close in magnitude but opposite in sign change in configurational entropy. The reduction in configurational entropy and, consequently, protein dynamics is consistent with the observed decrease in intrinsic compressibility. In general, results of this work emphasize the vital role that water plays in modulating protein recognition events.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

Analysis of HIV-1 viral infectivity factor-mediated proteasome-dependent depletion of APOBEC3G: correlating function and subcellular localization

To study how HIV-1 viral infectivity factor (Vif) mediates proteasome-dependent depletion of host factor APOBEC3G, functional and nonfunctional Vif-APOBEC3G interactions were correlated with subcellular localization. APOBEC3G localized throughout the cytoplasm and co-localized with gamma-tubulin, 20 S proteasome subunit, and ubiquitin at punctate cytoplasmic bodies that can be used to monitor the Vif-APOBEC3G interaction in the cell. Through immunostaining and live imaging, we showed that a substantial fraction of Vif localized to the nucleus, and this localization was impaired by deletion of amino acids 12-23. When co-expressed, Vif exhibited more pronounced localization to the cytoplasm and reduced the total cellular levels of APOBEC3G but rarely co-localized with APOBEC3G at cytoplasmic bodies. On the contrary, Vif(C114S), which is inactive but continues to interact with APOBEC3G, stably associated with APOBEC3G in the cytoplasm, resulting in complete co-localization at cytoplasmic bodies and a dose-dependent exclusion of Vif(C114S) from the nucleus. Following proteasome inhibition, cytoplasmic APOBEC3G levels increased, and both proteins co-accumulated nonspecifically into a vimentin-encaged aggresome. Furthermore in the presence or absence of APOBEC3G, Vif localization was significantly altered by proteasome inhibition, suggesting that aberrant localization may also contribute to the loss of Vif function. Finally mutations at Vif Ile(9) disrupted the ability of Vif or Vif(C114S) to coimmunoprecipitate and to co-localize with APOBEC3G, suggesting that the N terminus of Vif mediates interactions with APOBEC3G. Taken together, these results demonstrate that cytoplasmic Vif-APOBEC3G interactions are required but are not sufficient for Vif to modulate APOBEC3G and can be monitored by co-localization in vivo.     

Interaction of hematopoietic progenitor kinase 1 and c-Abl tyrosine kinase in response to genotoxic stress

The c-Abl protein tyrosine kinase is activated by certain DNA-damaging agents and regulates induction of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). The hematopoietic progenitor kinase 1 (HPK1) has also been shown to act upstream to the SAPK/JNK signaling pathway. We report here that exposure of hematopoietic Jurkat T cells to genotoxic agents is associated with activation of HPK1. The results demonstrate that exposure of Jurkat cells to DNA-damaging agents is associated with translocation of active c-Abl from nuclei to cytoplasm and binding of c-Abl to HPK1. Our findings also demonstrate that c-Abl phosphorylates HPK1 in cytoplasm and stimulates HPK1 activity. The functional significance of the c-Abl-HPK1 interaction is supported by the demonstration that this complex regulates SAPK/JNK activation. Overexpression of c-Abl(K-R) inhibits HPK1-induced activation of SAPK/JNK. Conversely, the dominant negative mutant of HPK1 blocks c-Abl-mediated induction of SAPK/JNK. These findings indicate that activation of HPK1 and formation of HPK1/c-Abl complexes are functionally important in the stress response of hematopoietic cells to genotoxic agents.     

Sclerotinia sclerotiorum Thioredoxin1 (SsTrx1) is required for pathogenicity and oxidative stress tolerance

Sclerotinia sclerotiorum infects host plant tissues by inducing necrosis to source nutrients needed for its establishment. Tissue necrosis results from an enhanced generation of reactive oxygen species (ROS) at the site of infection and apoptosis. Pathogens have evolved ROS scavenging mechanisms to withstand host‐induced oxidative damage. However, the genes associated with ROS scavenging pathways are yet to be fully investigated in S. sclerotiorum. We selected the S. sclerotiorum Thioredoxin1 gene (SsTrx1) for our investigations as its expression is significantly induced during S. sclerotiorum infection. RNA interference‐induced silencing of SsTrx1 in S. sclerotiorum affected the hyphal growth rate, mycelial morphology, and sclerotial development under in vitro conditions. These outcomes confirmed the involvement of SsTrx1 in promoting pathogenicity and oxidative stress tolerance of S. sclerotiorum. We next constructed an SsTrx1‐based host‐induced gene silencing (HIGS) vector and mobilized it into Arabidopsis thaliana (HIGS‐A) and Nicotiana benthamiana (HIGS‐N). The disease resistance analysis revealed significantly reduced pathogenicity and disease progression in the transformed genotypes as compared to the nontransformed and empty vector controls. The relative gene expression of SsTrx1 increased under oxidative stress. Taken together, our results show that normal expression of SsTrx1 is crucial for pathogenicity and oxidative stress tolerance of S. sclerotiorum.     

Correlates of delayed disease progression in HIV-1-infected Kenyan children

Without treatment most HIV-1-infected children in Africa die before their third birthday (>89%) and long-term nonprogressors are rare. The mechanisms underlying nonprogression in HIV-1-infected children are not well understood. In the present study, we examined potential correlates of delayed HIV disease progression in 51 HIV-1-infected African children. Children were assigned to progression subgroups based on clinical characterization. HIV-1-specific immune responses were studied using a combination of ELISPOT assays, tetramer staining, and FACS analysis to characterize the magnitude, specificity, and functional phenotype of HIV-1-specific CD8(+) and CD4(+) T cells. Host genetic factors were examined by genotyping with sequence-specific primers. HIV-1 nef gene sequences from infecting isolates from the children were examined for potential attenuating deletions. Thymic output was measured by T cell rearrangement excision circle assays. HIV-1-specific CD8(+) T cell responses were detected in all progression groups. The most striking attribute of long-term survivor nonprogressors was the detection of HIV-1-specific CD4(+) Th responses in this group at a magnitude substantially greater than previously observed in adult long-term nonprogressors. Although long-term survivor nonprogressors had a significantly higher percentage of CD45RA(+)CD4(+) T cells, nonprogression was not associated with higher thymic output. No protective genotypes for known coreceptor polymorphisms or large sequence deletions in the nef gene associated with delayed disease progression were identified. In the absence of host genotypes and attenuating mutations in HIV-1 nef, long-term surviving children generated strong CD4(+) T cell responses to HIV-1. As HIV-1-specific helper cells support anti-HIV-1 effector responses in active disease, their presence may be important in delaying disease progression.