Controlled drug release: new water-soluble prodrugs of an HIV protease inhibitor

We designed and synthesized a series of highly water-soluble prodrugs of an HIV protease inhibitor, KNI-727 (1), containing tandem-linked two auxiliary units, a solubilizing moiety and a self-cleavable spacer. Prodrugs with an ionized amino group at the solubilizing moiety exhibited a remarkable increase of water-solubility (>10(4) fold) compared to the parent drug 1. These prodrugs released I not enzymatically, but chemically via an intramolecular cyclization-elimination reaction through an imide formation in physiological conditions. Diversified rates of parent drug release were observed when the chemical structure of both the solubilizing and the spacer moieties were modified. This new approach for water-soluble prodrugs will enable to control chemically the release of parent drug as well as to maintain high water-solubility.     

Chloroplast inheritance patterns in Actinidia hybrids determined by single stranded conformation polymorphism analysis

The inheritance patterns of the chloroplast genomes of the Actinidia hybrids A. eriantha (male parent) x A. chinensis (female parent) and A. chinensis (male parent) x A. melanandra (female parent) were analyzed using single-strand conformation polymorphism (SSCP) analysis of the trnL-trnF and psbA-trnH intergenic spacers. This showed that the artificial hybrids between A. eriantha and A. chinensis all had the haplotype of their male parent. Alignment of the sequences of A. eriantha and A. chinensis revealed four substitutions and one insertion (GATTC) in trnL-trnF and two substitutions in psbA-trnH. In contrast, the haplotypes of the artificial hybrids between A. chinensis and A. melanandra had the same patterns as their female parent. Alignment of the entire region of A. chinensis and A. melanandra revealed 12 substitutions: 1 in trnL-trnF and 11 in psbA-trnH. However, no sequence variation in the trnL-trnF and psbA-trnH intergenic spacers was found. We have developed a simple screening method for detecting the inheritance patterns of Actinidia chloroplast DNA haplotypes using SSCP analysis of the trnL-trnF and psbA-trnH intergenic spacers. Our findings indicate that the inheritance of the chloroplast genome in Actinidia hybrids differs according to the species selected.     

Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae)

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.     

Structure-based design of nonnatural ligands for the HLA-B27 protein

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove, and thus decrease more or less the affinity of the altered epitopes to HLA-B*2705. Bifunctional spacers ((R)-3-hydroxybutyrate and beta-homoalanine combinations) not only bridges the two distant anchoring amino acids but also strongly interact with the binding cleft and lead to an increase in binding to the MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.     

Modular synthesis of non-peptidic bivalent NPY Y1 receptor antagonists

According to a 'bivalent ligand approach' to increase the affinity of the potent argininamide-type NPY Y(1) receptor antagonist BIBP-3226, dimeric ligands were synthesized in which two molecules of the parent compound were linked by different spacers via N(G)-acylation at the guanidino groups. A synthetic route for the preparation of the title compounds was developed, which includes a copper(I)-catalyzed azide alkyne cycloaddition as the key step. Three bivalent analogues of BIBP-3226 were prepared showing nanomolar antagonistic activity and binding affinity to the NPY Y(1) receptor (calcium assay on HEL cells, radioligand binding assay on SK-N-MC cells), but these ligands were not superior to the parent compound and there was no correlation with the length or the chemical nature of the spacer. A trivalent BIBP-3226 derivate showed, surprisingly, no affinity to the NPY Y(1) receptor at all.     

Synthesis and biological evaluation of novel C (7) modified chrysin analogues as antibacterial agents

The natural product, chrysin (5,7-dihydroxy flavone), obtained from Oroxylum indicum, exhibits numerous biological activities including anticancer, anti-inflammatory, and antiallergic activities. Three series of chrysin analogues were prepared, in which chrysin and heterocyclic moieties are separated by 3-carbon, 4-carbon, and 6-carbon spacers. All the derivatives were screened for antibacterial activity against a panel of susceptible and resistant Gram-positive and Gram-negative organisms. It was observed that most of the derivatives displayed significant activity as compared to their parent compound (chrysin).     

Evidence for maternal inheritance of mitochondrial DNA in allotetraploid.

The complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and red crucian carp were determined in this paper. We compared the complete mtDNA sequences between the allotetraploid and its female parent red crucian carp, and between the allotetraploid and its male parent common carp. The results indicated that the complete mtDNA nucleotide identity (99.7%) between the allotetraploid and its female parent red crucian carp was higher than that (89.0%) between the allotetraploid and its male parent common carp. Moreover, the analysis on the start and stop codons, overlaps and spacers, and phylogeny of the mt genomes indicated the genetic relationship between the allotetraploid and its female parent red crucian carp was closer than that between the allotetraploid and its male parent common carp. Our results indicated that the allotetraploid mt genome was strictly maternally inherited. Through maternal inheritance, the mt genome in the F(11) allotetraploid displayed extremely high similarity to that in the female parent red crucian carp after 11 generations (from F(1) to F(11) hybrids). Such results indicated that the F(11) allotetraploid possessed the stable inheritance characteristic. Thus the tetraploid stocks possessed the good base to form a new tetraploid species in the future. Since the establishment of the new tetraploid stocks has the great significance in analyzing evolutionary theory of vertebrate and in improving aquaculture industry, analysis of the mt genome and the elucidation of the variation of the mt genome in the allotetraploid and its parents proved that it was a useful genetic marker to monitor the variations in the progeny of the crosses.     

Structure-Based Design of Nonnatural Ligands for the HLA-B27 Protein

Abstract

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove, and thus decrease more or less the affinity of the altered epitopes to HLA-B*2705. Bifunctional spacers ((R)-3-hydroxybutyrate and β-homoalanine combinations) not only bridges the two distant anchoring amino acids but also strongly interact with the binding cleft and lead to an increase in binding to the MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.     

A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli

Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC₅₀ was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli.

     

Amino acid spacing in isotachophoresi; on polyacrylamide gel: a critical evaluation.

Isotachophoresis of colored model proteins was carried out on polyacrylamide gel, using the stack of a multiphasic buffer system computed on the basis of the Jovin theory and operative at pH 10.4. The stack was elongated by milligram loads of various amino acids per analytical gel. The extent of the stack was determined by chemical localization of the leading and trailing constituents. The isotachophoretic nature of the stack was ascertained by determining the positions of all protein specles under study as intermediate between the leading and trailing constituents. The shallow pH gradient across the extended stack was measured. Spacing of proteins in isotachophoresis is restricted, with regard to constituent multiplicity and constituent load, by the practical limitations of electrophoresis time and gel length. Therefore, spacing by a small number of constituents at relatively low loads was attempted. Amino acids were chosen as spacers because they can be selected conveniently, in radioactively labeled form, for the specific separation between pairs of proteins with particular mobilities. In the particular buffer system used, lysine, histidine, serine, and threonine were found to be effective spacers between bovine serum albumin (BSA) and hemoglobins, while no amino acids effectively spaced between hemoglobins A and S. It is concluded that specific spacing in isotachophoresis on polyacrylamide gel (ITPPA) can be useful in improving resolution. However, in practice, few spacers exist in the mobility range of proteins, the positions of which relative to specific proteins could be convenlently located on the basis of isotope analysis or other assays. Mixtures of multiple spacers with undefined mobilities appear applicable as “blind spacers” only at concentrations so low that their effectiveness is annulled.     

Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

Background  

Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.     

Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification

Background  

PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.     

CRISPR-mediated host genomic DNA damage is efficiently repaired through microhomology-mediated end joining in Zymomonas mobilis

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against mobile genetic elements(MGEs) through uptake of invader-derived spacers. De novo adaptation samples spacers from both invaders and hosts, whereas primed adaptation shows higher specificity to sample spacers from invaders in many model systems as well as in the subtype I-F system of Zymomonas mobilis. Self-derived spacers will lead to CRISPR self-interference. However, our in vivo study demonstrated that this species used the microhomology-mediated end joining(MMEJ) pathway to efficiently repair subtype I-F CRISPR-Cas system-mediated DNA breaks guided by the self-targeting spacers. MMEJ repair of DNA breaks requires direct microhomologous sequences flanking the protospacers and leads to DNA deletions covering the protospacers. Importantly, CRISPR-mediated genomic DNA breaks failed to be repaired via MMEJ pathway in presence of higher copies of short homologous DNA. Moreover, CRISPR-cleaved exogenous plasmid DNA was failed to be repaired through MMEJ pathway, probably due to the inhibition of MMEJ by the presence of higher copies of the plasmid DNA in Z. mobilis. Our results infer that MMEJ pathway discriminates DNA damages between in the host chromosome versus mobile genetic element(MGE) DNA, and maintains genome stability post CRISPR immunity in Z. mobilis.     

Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system

The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme.     

Recent hybrid speciation in Cardamine (Brassicaceae) – conversion of nuclear ribosomal ITS sequences in statu nascendi

 The very recent allopolyploid speciation of Cardamine insueta and Cardamine schulzii is well documented. We used this system for a further understanding of the evolution of the internal transcribed spacers (ITS) of nuclear ribosomal DNA in recently formed hybrids. The ITS sequencing of the two parent species and the alloploid offspring suggests a synopsis of the types of ITS evolution, reported so far in the literature. We detected homogenization to one parental ITS type with a very strong bias to the maternal sequence. Nevertheless, maintainance of both parental ITS sequences in the alloploids was also recorded. Our data suggest: (1) rapid genomic change in newly formed alloploids, and (2) a multiple origin of C. insueta and C. schulzii. Received: 24 August 1998 / Accepted: 8 September 1998     

Sequence Variation and Comparison of the 5S rRNA Sequences in <Emphasis Type="Italic">Allium</Emphasis> Species and their Chromosomal Distribution in Four <Emphasis Type="Italic">Allium</Emphasis> Species

The gene structure and sequence diversity of 5S rRNA genes were analyzed in 13 Allium species. While the lengths and sequences of the coding gene segments were conserved, the spacers were highly variable and could be characterized as either short (213–404 bp) or long (384–486 bp) spacers. The short spacers were further classified into five subtypes (SS-I to SS-V) and the long spacers into four subtypes (LS-I to LS-IV). The short spacers were more conserved than were the long spacers. There was a sequence duplication of 85 bp in SS-III that distinguished it from SS-II. The coding sequences of the 5S rRNA genes started with CGG and ended with either CCC or TCC. Both long and short spacers started with TTTT at their 5′-ends. However, the long spacers ended with a 3′-TGA sequence, whereas the short spacers terminated with various sequences, such as TTA, ATA, or TGA. GC content ranged from 27 to 41% in whole repeats, and the GC content in the long spacers was lower than in the short spacers. The nucleotide diversity in the coding regions was lower than in the spacers, and the nucleotide diversity in the coding regions did not correlate with that of the spacers. FISH analysis confirmed that each Allium species has either short spacers or long spacers. Although chromosomal locations of the 5S rRNA genes in Allium wakegi confirmed the allodiploid nature of A. cepa and A. fistulosum, spacer sequences revealed the absence of SS-II in A. cepa and in A. wakegi. The current study demonstrated that the 5S rRNA genes diverged in early stages in Allium species differentiation except of the allodiploid A. wakegi.