Identification of actin as an estradiol 17-beta-stimulated protein in the human placenta

Addition of estradiol 17-beta to first trimester human placental minces resulted in an increased synthesis of a protein of apparent molecular weight 45 kDa. The specific involvement of estrogen in the stimulation of this protein was established by demonstrating a reduction in the level of this protein by the addition of CCS 16949 A, an inhibitor of aromatase, a key enzyme in the biosynthesis of estradiol 17-beta and ICI 182,780, an estrogen receptor antagonist. The protein was purified to homogeneity and N-terminal sequencing of two of the internal peptides obtained by enzymatic digestion of the protein, as well as the absence of a free N-terminal indicated that it could be actin. This was confirmed by Western blotting using commercially available actin antiserum. The role of estradiol 17-beta in the stimulation of actin synthesis in human placenta was also established by monitoring the quantitative inhibition of DNase I by actin.

Dynamics of ribonuclease A and ribonuclease S: Computational and experimental studies

RNase S is a complex consisting of two proteolytic fragments of RNase A: the S peptide (residues 1-20) and S protein (residues 21-124). RNase S and RNase A have very similar X-ray structures and enzymatic activities. previous experiments have shown increased rates of hydrogen exchange and greater sensitivity to tryptic cleavage for RNase S relative to RNase A. It has therefore been asserted that the RNase S complex is considerably more dynamically flexible than RNase A. In the present study we examine the differences in the dynamics of RNaseS and RNase A computationally, by MD simulations, and experimentally, using trypsin cleavage as a probe of dynamics. The fluctuations around the average solution structure during the simulation were analyzed by measuring the RMS deviation in coordinates. No significant differences between RNase S and RNase A dynamics were observed in the simulations. We were able to account for the apparent discrepancy between simulation and experiment by a simple model, According to this model, the experimentally observed differences in dynamics can be quantitatively explained by the small amounts of free S peptide and S protein that are present in equilibrium with the RNase S complex. Thus, folded RNase A and the RNase S complex have identical dynamic behavior, despite the presence of a break in polypeptide chain between residues 20 and 21 in the latter molecule. This is in contrast to what has been widely believed for over 30 years about this important fragment complementation system.

Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a process 5'-3' single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg2+ or Mn2+ and inhibited by Cd2+ suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays, Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interactio between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways.

Xylan-Degrading Enzymes from the Thermophilic Fungus Humicola Zanuginosa (Griffon and Maublanc) Bunce: Action Pattern of Xylanase and beta-Glucosidase on Xylans, Xylooligomers and Arabinoxylooligomers

The mode of action of xylanase and beta-glucosidase purified from the culture filtrate of Humicola lanuginosa (Griffon and Maublanc) Bunce on the xylan extracted from sugarcane bagasse and on two commercially available larchwood and oat spelt xylans, on xylooligomers and on arabinoxylooligomers was studied. While larchwood and oat spelt xylans were hydrolyzed to the same extent in 24 h, sugarcane bagasse xylan was hydrolyzed to a lesser extent in the same period. It was found that the rate of hydrolysis of xylooligomers by xylanase increased with increase in chain length, while beta-glucosidase acted rather slowly on all the oligomers tested. Xylanase exhibited predominant ''endo'' action on xylooligomers attacking the xylan chain at random while beta-glucosidase had ''exo'' action, releasing one xylose residue at a time. On arabinoxylooligomers, however, xylanase exhibited ''exo'' action. Thus, it appears that the presence of the arabinose substituent has, in some way, rendered the terminal xylose-xylose linkage more susceptible to xylanase action. It was also observed that even after extensive hydrolysis with both the enzymes, substantial amounts of the parent arabinoxylooligomer remained unhydrolyzed together with the accumulation of arabinoxylobiose. It can therefore be concluded that the presence of the arabinose substituent in the xylan chain results in linkages that offer resistance to both xylanase and beta-glucosidase action.

Ultrasound-assisted surface engineering of pharmaceutical powders

Effective processing of powdered particles can facilitate powder handling and result in better drug product performance, which is of great importance in the pharmaceutical industry where the majority of active pharmaceutical ingredients (APIs) are delivered as solid dosage forms. The purpose of this work was to develop a new ultrasound-assisted method for particle surface modification and thin-coating of pharmaceutical powders. The ultrasound was used to produce an aqueous mist with or without a coating agent. By using the proposed technique, it was possible to decrease the interparticular interactions and improve rheological properties of poorly-flowing water-soluble powders by aqueous smoothing of the rough surfaces of irregular particles. In turn, hydrophilic polymer thin-coating of a hydrophobic substance diminished the triboelectrostatic charge transfer and improved the flowability of highly cohesive powder. To determine the coating efficiency of the technique, the bioactive molecule β-galactosidase was layered onto the surface of powdered lactose particles. Enzyme-treated materials were analysed by assaying the quantity of the reaction product generated during enzymatic cleavage of the milk sugar. A near-linear increase in the thickness of the drug layer was obtained during progressive treatment. Using the enzyme coating procedure, it was confirmed that the ultrasound-assisted technique is suitable for processing labile protein materials. In addition, this pre-treatment of milk sugar could be used to improve utilization of lactose-containing formulations for populations suffering from severe lactose intolerance. Furthermore, the applicability of the thin-coating technique for improving homogeneity of low-dose solid dosage forms was shown. The carrier particles coated with API gave rise to uniform distribution of the drug within the powder. The mixture remained homogeneous during further tabletting, whereas the reference physical powder mixture was subject to segregation. In conclusion, ultrasound-assisted surface engineering of pharmaceutical powders can be effective technology for improving formulation and performance of solid dosage forms such as dry powder inhalers (DPI) and direct compression products.

Inhibition of Peroxidase-Catalyzed Iodination by Cephalosporins: Metallo-beta-Lactamase-Induced Antithyroid Activity of Antibiotics

Broad-spectrum antibiotics with heterocyclic side chains strongly inhibit peroxidase-catalyzed iodination in the presence of metallo--lactamase. This suggests that antibiotic resistance due to hydrolysis of the -lactam ring in antibiotics would have negative effects on thyroid activity.

Reducing Browning of Fresh-cut ‘Maha’ Carambola with Chemical Additives and Low-oxygen Atmospheres

The greatest attraction to using carambola (Averrhoa carambola L.) in the fresh-cut market is the star shape that the fruit presents after a transverse cut. Carambola is well-suited for minimal processing, but cut surface browning is a main cause of deterioration. This problem is exacerbated as a result of mechanical injuries occurring during processing and is mainly induced by the leakage of phenolic compounds from the vacuole and subsequent oxidation by polyphenol oxidase (PPO) (Augustin et al., 1985). The use of browning inhibitors in processed fruits is restricted to compounds that are non-toxic, ‘wholesome’, and that do not adversely affect taste and flavour (Gil et al., 1998). In the past, browning was mainly controlled by the action of sulphites, but the use of this compound has declined due to allergic reactions in asthmatics (Weller et al., 1995). The shelf life of fresh-cut products may be extended by a combination of oxygen exclusion and the use of enzymatic browning inhibitors. The objectives of this work were to determine the effects of: (1) post-cutting chemical treatments of ascorbic, citric, oxalic acids, and EDTA-Ca; (2) atmospheric modification; and (3) combinations of the above, on the shelf life of carambola slices based on appearance, colour and polyphenol oxidase activity