Peptide synthesis using proteases dissolved in organic solvents

Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of k_cat/K_m for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH₂ to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH₂ that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.     

Regulation of Mitochondrial Iron Import through Differential Turnover of Mitoferrin 1 and Mitoferrin 2

Mitoferrin 1 and mitoferrin 2 are homologous members of the mitochondrial solute carrier family. Mitoferrin 1 is required for mitochondrial iron delivery in developing erythrocytes. Here we show that mitoferrin 1 and mitoferrin 2 contribute to mitochondrial iron delivery in a variety of cells. Reductions in mitoferrin 1 and/or mitoferrin 2 levels by RNA interference result in decreased mitochondrial iron accumulation, heme synthesis, and iron-sulfur cluster synthesis. The ectopic expression of mitoferrin 1 in nonerythroid cells silenced for mitoferrin 2 or the expression of mitoferrin 2 in cells silenced for mitoferrin 1 restored heme synthesis to “baseline” levels. The ectopic expression of mitoferrin 2, however, did not support hemoglobinization in erythroid cells deficient in mitoferrin 1. Mitoferrin 2 could not restore heme synthesis in developing erythroid cells because of an inability of the protein to accumulate in mitochondria. The half-life of mitoferrin 1 was increased in developing erythroid cells, while the half-life of mitoferrin 2 did not change. These results suggest that mitochondrial iron accumulation is tightly regulated and that controlling mitoferrin levels within the mitochondrial membrane provides a mechanism to regulate mitochondrial iron levels.Iron is a required element for all eukaryotes, but iron can be toxic at high concentrations. Consequently, the cellular acquisition of iron is highly regulated, as is the concentration of free iron in biological fluids. The regulation of iron concentration is extended to cellular organelles that either store or utilize iron. Mitochondria utilize iron for the synthesis of heme and iron-sulfur (Fe-S) clusters. These prosthetic groups are used within the mitochondria and are exported for use by cytosolic and nuclear proteins. The mechanisms that regulate mitochondrial iron levels are not known, although it is clear that mitochondrial iron levels must be regulated. For example, the loss of function mutations in genes that encode enzymes required for Fe-S cluster synthesis or the Atm1 transporter that exports Fe-S clusters, results in excessive mitochondrial iron accumulation in yeast and humans (for a review, see reference 11).The mechanisms that regulate mitochondrial iron pools are not well defined. Mitochondrial iron pools might be regulated at the level of import. Mitoferrin 1 (Mfrn1) has been shown to be required for mitochondrial iron import in developing erythroid cells. A mutation in zebrafish Mfrn1 (frascati) or the deletion of mouse Mfrn1 leads to defects in hemoglobinization due to a deficit in mitochondrial iron uptake (17). The phenotype of frascati zebrafish is restricted to developing red blood cells; other cell types showed no evidence of a mitochondrial iron phenotype. Mfrn1 has a paralogue, Mfrn2, and both genes have homologues MRS3 and MRS4 in Saccharomyces cerevisiae. Yeast with deletions of MRS3 and MRS4 grows poorly under low iron conditions due to impaired mitochondrial iron acquisition (5, 10, 13, 23). In yeast, the expression of Mfrn1 or Mfrn2 in Δmrs3 Δmrs4 cells can correct the poor growth under low iron conditions. The expression of either mouse or zebrafish Mfrn1 as a transgene in frascati zebrafish corrected the hemoglobin deficiency in cells, but the expression of Mfrn2 did not (17). These observations raise three questions. (i) What is the role of Mfrn2 in mitochondrial iron metabolism? (ii) Is iron transport into mitochondria regulated? (iii) If Mfrn2 transports iron into the mitochondria of vertebrate cells, why doesn''t Mfrn2 rescue the mitochondrial defect in Mfrn1-deficient zebrafish?Here, we show that Mfrn1 and Mfrn2 can transport iron into the mammalian mitochondria of nonerythroid cells. The ectopic expression of either Mfrn1 or Mfrn2 can restore mitochondrial iron transport in cells silenced for Mfrn2 and -1, respectively, but ectopic expression has little effect on increasing mitochondrial iron levels above the baseline values. Mitochondrial iron levels do not increase over the baseline because the levels of Mfrns are regulated posttranslationally. Mfrn1 accumulates in the mitochondria of developing red blood cells as a result of an increased protein half-life. In contrast, Mfrn2 does not accumulate in developing red blood cells or other cells, as the half-life of Mfrn2 protein remains constant.     

High affinity human antibody fragments to dengue virus non-structural protein 3

Background

The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5′ triphosphatase domain which forms the remainder of the 618-aa long protein.

Methodology/Principal Findings

In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531) within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells.

Conclusions/Significance

Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.     

Melt solidification technique: Incorporation of higher wax content in ibuprofen beads

The purpose of this study was to achieve incorporation of a higher amount of wax during the preparation of ibuprofen beads by a melt solidification technique for better integrity and prolonged drug release by using a combination of waxes. A mixture of cetyl alcohol (CA) and palmitic acid (PA) was used to improve the matrix integrity and drug release. The effect of variables such as CA, PA, and speed of agitation were studied using 3³ factorial design. Yield, crushing strength, and drug release were analyzed using response surface methodology. The in vitro dissolution test did not show any significant improvement in the drug release. Scanning electron microscopy (SEM) showed that beads were spherical with a smooth surface, but after dissolution became rough and porous. Differential scanning calorimetry (DSC) studies showed that different solidification and erosion properties of waxes are responsible for the inability of waxes to retard drug release even at higher concentration.     

Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine epsilon-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.     

Design and evaluation of deformable talc agglomerates prepared by crystallo-Co-agglomeration technique for generating heterogenous matrix

The crystallo-co-agglomeration technique was used to design directly compressible and deformable agglomerates of talc containing the low-dose drug bromhexine hydrochloride (BXH). The process of agglomeration involved the use of dichloromethane as a good solvent and bridging liquid for BXH, water as a poor solvent, talc as diluent, and Tween 80 to aid dispersion of BXH and diluent into the poor solvent. Hydroxypropyl methylcellulose (50 cps) 4% wt/wt was used to impart the desired mechanical strength and polyethylene glycol 6000 5% wt/wt was used to impart the desired sphericity to the agglomerates. Clarity of the supernatant was considered an endpoint for completion of the agglomeration process. The drug-to-talc ratio in optimized batch 1 (BT1) and batch 2 (BT2) was kept at 1:15.66 and 1:24, respectively. The spherical agglomerates obtained were evaluated for topographic, micromeritic, mechanical, deformation, compressional, and drug release properties. The agglomeration yield and drug entrapment for both batches were above 94% wt/wt. Crushing strength and friability studies showed good handling qualities of agglomerates. Heckel plot studies showed low mean yield pressure and high tensile strength, indicating excellent compressibility and compactibility of agglomerates. Diametral and axial fracture of compacts showed deformation of agglomerates revealing formation of a heterogeneous compact. Drug release was sustained for 9 hours and 5 hours from BT1 and BT2, respectively, in 0.1N HCl. Hence, the crystallo-co-agglomeration technique can be successfully used for obtaining spherical, deformable, and directly compressible agglomerates, generating a heterogeneous matrix system and providing sustained drug release. Published: July 27, 2007     

Simultaneous determination of cinnamaldehyde, eugenol and piperine by HPTLC densitometric method

An HPTLC densitometric method for the simultaneous determination of cinnamaldehyde and eugenol as well as trace amounts of piperine in pepper-contaminated cinnamon was developed. The applicability of the method was tested with cinnamon bark powder adulterated with pepper powder, cinnamon oil, clove powder, clove oil and a commercial preparation containing cinnamaldehyde and eugenol. The method was validated for specificity, precision, accuracy and robustness. The method was found to be precise for different concentrations of cinnamaldehyde, eugenol and piperine. The accuracy of the method was checked by conducting a recovery study at three different levels. The linearity was found to be in the ranges 52.54-735.56, 533.2-8531.2 and 50-300 ng/spot, respectively, with correlation coefficients of 0.9985 +/- 0.04, 0.9982 +/- 0.06 and 0.9937 +/- 0.11 for cinnamaldehyde, eugenol and piperine.