Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.     

Aerial visual acuity in harbor seals (<Emphasis Type="Italic">Phoca vitulina</Emphasis>) as a function of luminance

In this study, we measured aerial visual acuity in harbor seals. As a first approach to the hypothesis that harbor seals can obtain acute aerial visual acuity mediated by the interaction of the vertical slit-shaped pupil and the corneal flattening although refractive measurements had revealed aerial myopia, visual acuity was tested as a function of luminance and pupil dilation. We analyzed aerial visual acuity (minimal resolvable stripe width) in three harbor seals in a two-alternative-forced-choice discrimination experiment. Our results further support the hypothesis that harbor seals possess an aerial visual acuity comparable to the acuity in clear waters if the vertical slit pupil does not exceed the zone of corneal flattening in bright light. When the pupil dilates with decreasing luminance, visual acuity decreases which might be due to deflected light from the stronger curved peripheral cornea.     

Pyrano[2,3-e]isoindol-2-ones,new angelicin heteroanalogues

A convenient synthesis of the pyrano[2,3-e]isoindol-2-one ring system, an heteroanalogue of angelicin, is reported. Our synthetic approach consists of the annelation of the pyran ring on the isoindole moiety using 5-dialkylamino- or 5-hydroxymethylene intermediates as building blocks. The photoantiproliferative activity of the new derivatives was studied. Some of them bearing the benzyl group at the 8 position were active with IC₅₀ in the micromolar range. Cell cytotoxicity involves apoptosis, alteration of cell cycle profile and membrane photodamage.     

Imidazo[1,2-a]pyrazine diaryl ureas: Inhibitors of the receptor tyrosine kinase EphB4

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent^® and Nexavar^®, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.     

Prokaryotic expression,in vitro folding,and molecular pharmacological characterization of the neuropeptide Y receptor type 2

G protein‐coupled receptors (GPCRs) are a class of membrane proteins that represent a major target for pharmacological developments. However, there is still little knowledge about GPCR structure and dynamics since high‐level expression and characterization of active GPCRs in vitro is extremely complicated. Here, we describe the recombinant expression and functional folding of the human Y₂ receptor from inclusion bodies of E. coli cultures. Milligram protein quantities were produced using high density fermentation and isolated in a single step purification with a yield of over 20 mg/L culture. Extensive studies were carried out on in vitro refolding and stabilization of the isolated receptor in detergent solution. The specific binding of the ligand, the 36 residue neuropeptide Y (NPY), to the recombinant Y₂ receptors in micellar form was shown by several radioligand affinity assays. In competition experiments, an IC₅₀ value in low nanomolar range could be determined. Further, a K_D value of 1.9 nM was determined from a saturation assay, where NPY was titrated to the recombinant Y₂ receptors. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Glyphosate Resistance as a Novel Select-Agent-Compliant,Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei

Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.Members of the genus Burkholderia, comprising more than 40 different species, are extremely diverse gram-negative, non-spore-forming bacilli. Many Burkholderia species exist as innocuous soil saprophytes or plant pathogens (47), while others cause human and animal diseases. Among these human and animal pathogens are the etiological agents of melioidosis (Burkholderia pseudomallei) and glanders (Burkholderia mallei) (9, 50, 51). Melioidosis is an emerging infectious disease generally considered endemic to Southeast Asia and Northern Australia (12). Positive diagnoses in many tropical countries around the world have expanded the global awareness of melioidosis (3, 15, 24, 25, 28, 35, 39, 42, 52). In contrast to the ubiquitous nature of B. pseudomallei, B. mallei is also a highly infectious agent causing glanders, a predominantly equine disease (34, 50). B. mallei, a clone derived from genomic downsizing of B. pseudomallei, has been used in biowarfare (17). This historical significance, along with the low infectious dose and the route of infection, has contributed to the decision by the Centers for Disease Control and Prevention (CDC) to classify these two microbes as category B select agents (43).Classification of B. pseudomallei as a select agent has stimulated interest and research into the pathogenesis of melioidosis, necessitating the development of appropriate tools for genetic manipulation. In the struggle to elucidate the molecular mechanisms of pathogenesis, selectable markers are indispensable genetic tools (45). Current CDC regulations prohibit the cloning of clinically important antibiotic resistance genes into human, animal, or plant select-agent pathogens if the transfer could compromise the ability to treat or control the disease. The only antibiotic markers currently approved for use in B. pseudomallei are based on resistance to aminoglycosides (gentamicin, kanamycin, and zeocin) (45). However, the efficacy of these markers is limited, due to high levels of aminoglycoside resistance inherent within the Burkholderia genus and high levels of spontaneous aminoglycoside resistance in B. pseudomallei (10, 19, 41). In addition, the use of aminoglycosides (e.g., gentamicin) for selection may require aminoglycoside efflux pump mutants (10, 33). Another potential drawback is that efflux pumps play a major role in bacterial physiology, and mutating them may change the pathogenic traits under investigation (7, 40). A more logical approach employs alternative, non-antibiotic-selectable markers conferring resistance to compounds that are not potentially important in clinical treatment.Very few non-antibiotic resistance markers have been utilized successfully for Burkholderia species. A non-antibiotic-selectable-marker based on tellurite resistance (Tel^r) has been successfully developed and used with Pseudomonas putida, Pseudomonas fluorescens, and Burkholderia thailandensis (2, 27, 44). The engineering of Tel^r-FRT (flip recombination target) cassettes, coupled to FRT sequences, could be used to generate unmarked mutations and allow recycling of the Tel^r selectable-marker (2). In addition, utilization of Flp-FRT resistance cassettes to generate mutants allows downstream modification and manipulation such as fusion integration (29). However, the disadvantage of the Tel^r-cassette is the number of genes required (kilA-telA-telB) and the large size (>3 kb), making it less likely to obtain PCR products for allelic replacement by natural transformation (46). Another potentially useful non-antibiotic-selectable marker is based on the bar gene, encoding resistance to bialaphos or its degradation product, phosphinothricin (PPT) (49). PPT inhibits glutamine synthetase in plants (48), starving the cell for glutamine, and the bar gene has been used successfully as a selection marker in gram-negative bacteria (21). For select-agent Burkholderia species, however, the PPT MIC was found to be greater than 1,024 μg/ml (M. Frazier, K. Choi, A. Kumar, C. Lopez, R. R. Karkhoff-Schweizer, and H. P. Schweizer, presented at the American Society for Microbiology Biodefense and Emerging Diseases Research Meeting, Washington, DC, 2007). We have found the effective concentration of PPT for B. pseudomallei and B. mallei to be ∼2.5% (25,000 μg/ml [data not shown]). The high concentration of PPT required for selection in these species may be costly, considering that purified PPT costs ∼$380 per g. Therefore, further development of non-antibiotic resistance markers, as well as a more economical source of herbicide for use with restricted select-agent species, is needed.Work by Castle et al. (5) generated a highly active glyphosate N-acetyltransferase (GAT) enzyme for plant engineering, making it possible to utilize the gat gene as an effective non-antibiotic resistance marker for bacterial selection with glyphosate (GS). The commonly used herbicide GS inhibits the 5-enolpyruvylshikimate-3-phospate synthase (EPSPS) of plants through competition with phosphoenolpyruvate for overlapping binding sites on EPSPS (14), depriving plants of three aromatic amino acids (Fig. ​(Fig.1).1). Since humans and animals obtain tryptophan and phenylalanine (giving rise to tyrosine) through dietary intake, GS is relatively nontoxic. Like plants, bacteria must make these amino acids, when they are lacking, from basic precursors. GS has been found to be inhibitory to a variety of bacteria, including Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, and Bradyrhizobium japonicum (16, 55), while other bacterial strains are able to metabolize low concentrations of GS (26, 31). Although B. pseudomallei has been reported to have two genes (glpA and glpB) for GS degradation and metabolism (38), our searches of all available genomes of Burkholderia species in GenBank yielded no glpA or glpB genes within this genus. GS resistance by bacteria has been documented through EPSPS target mutations or GS detoxification mechanisms (36). However, these mechanisms did not confer resistance to relatively high GS concentrations. More recently, directed evolution of the gat gene, based on various bacterial gat sequences and selection in E. coli, yielded a very active GAT protein sequence with an efficiency increase of nearly 4 orders of magnitude (5), holding promise as an appropriate non-antibiotic resistance marker for select-agent species.Open in a separate windowFIG. 1.(A) A 946-ml bottle of the “superconcentrated” herbicide Roundup used in this study, available for ∼$50 from most local hardware stores and garden or farm supply centers. The active ingredient, 50% GS, is indicated on the label, and the chemical structure of GS is shown. GAT, encoded by the gat gene, catalyzes the inactivation of GS via N acetylation. (B) Pathways of aromatic amino acid biosynthesis. GS inhibits the enzyme EPSPS, which is required for the biosynthesis of aromatic amino acids, thus starving bacteria for tyrosine, phenylalanine, and tryptophan. PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle.Here we engineered and tested a novel non-antibiotic-selectable-marker (gat) for use in the select agent B. pseudomallei. GS is the active ingredient in Roundup, which was used for selection (Fig. ​(Fig.1).1). The effective compound GS is readily available, inexpensive, relatively nontoxic, very soluble, and not clinically important, and it yields tight selection. The engineered gat marker (563 bp) was optimized for Burkholderia codon usage and adapted (with a Burkholderia rpsL promoter) for use in the select agent B. pseudomallei. Effective concentrations of GS for several species of Burkholderia, including the select agents B. pseudomallei and B. mallei, were determined. Using the gat gene, we created deletion mutants of the essential B. pseudomallei asd and B. pseudomallei dapB (asd_Bp and dapB_Bp) genes (encoding aspartate-semialdehyde dehydrogenase and dihydrodipicolinate reductase, respectively) in two wild-type B. pseudomallei strains. The Δasd_Bp mutant of B. pseudomallei showed a phenotypic defect consistent with the lack of diaminopimelate (DAP) for cell wall cross-linking. Complementation of the B. pseudomallei Δasd_Bp mutant with the asd_Bp gene located on a site-specific transposon, mini-Tn7-bar, was successful by using an inexpensive source of PPT for selection.     

Role of regulatory agencies

In this paper the authors discuss the role of regulation in assuring blood safety. After an overview of the subject by a leading expert, examples are provided of regulatory systems for blood transfusion services in several countries and regions. Additionally, the perspective of WHO is given on the essential role of national regulatory authorities in assuring the quality of national blood programmes.Collectively, the sections of this paper afford an opportunity for readers to make comparisons among different regulatory frameworks and to "benchmark" among the existing systems. Despite many differences in approach, a clear pattern emerges of worldwide efforts to strengthen blood regulatory systems.     

Archaeal ApbC/Nbp35 Homologs Function as Iron-Sulfur Cluster Carrier Proteins

Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.Members of the Archaea produce many proteins that require iron-sulfur cluster cofactors, including redox proteins, aconitase-like dehydratases, radical S-adenosylmethionine enzymes, and RNA polymerase (9, 13, 18, 32). Methanogenic archaea are obligate anaerobes, and many heterotrophic archaea grow anaerobically, indicating that oxidative stress has not limited the proliferation of iron-sulfur proteins in these lineages. Archaea must have a mechanism to assemble Fe-S clusters, but many members lack homologs of the known bacterial and eukaryotic Nif or Isc systems, suggesting that an alternative system is present (see Table S1 in the supplemental material) (14, 15).Some euryarchaea have homologs of the bacterial genes iscS (encoding cysteine desulfurase) and iscU (encoding a scaffold protein). Yet many other archaea, including the euryarchaea Pyrococcus furiosus, Methanocaldococcus jannaschii, and Methanococcus maripaludis, plus most crenarchaea, either lack a homologous cysteine desulfurase gene or have no homologs of A-type or U-type scaffold genes. Due to their sulfide-rich environments, it is reasonable that the anaerobic archaea may use an inorganic sulfur source to assemble Fe-S clusters. Most archaeal genome sequences do carry homologs of the sufBC genes that are part of the alternative Suf system for Fe-S cluster biosynthesis (36). Biochemical studies have shown that SufC is an ABC-type ATPase and that SufB is a persulfide acceptor that may act as a site for Fe-S cluster assembly (20). The SufB and SufC proteins interact, and SufB stimulates the ATPase activity of SufC. We hypothesize that the Archaea share a common mechanism for Fe-S cluster biosynthesis, supplemented with genes acquired by horizontal gene transfer in some lineages.A screen for Salmonella enterica bacteria defective in thiamine biosynthesis identified lesions in the apbC locus (28) that compromised Fe-S metabolism (33). An abpC mutant cannot grow with tricarballylate as a carbon and energy source, which may be due to a defect in assembling or repairing [4Fe-4S] clusters in the membrane-bound TcuB protein (21, 22). ApbC is a 40-kDa cytoplasmic protein with Walker A and B nucleotide-binding domains and two conserved carboxy-terminal cysteine residues separated by two amino acids (Cys-X-X-Cys). Mutational analyses have shown that ApbC proteins with directed changes in the Cys-X-X-Cys or Walker A motifs are not active in vivo (6). Suppressor analysis allowed the conclusion that a degree of functional redundancy between ApbC and the Fe-S scaffold protein IscU exists (4, 38). Although purified ApbC does not contain iron or sulfur, biochemical studies showed that ApbC can bind an Fe-S cluster and rapidly transfer it to an apoprotein (5).It is thought that in eukaryotes, Fe-S clusters are assembled by the mitochondrial iron-sulfur cluster (ISC) system (23). The clusters are transported into the cytosol and delivered by the cytosolic iron-sulfur protein assembly system. Two components of this system, Nbp35 and Cfd1, are homologs of bacterial ApbC (Fig. ​(Fig.1)1) and act as intermediate Fe-S cluster-trafficking proteins in the cytosol (16, 27, 30). Electron paramagnetic resonance, Mössbauer, and absorbance spectra of the Saccharomyces cerevisiae, human, and Arabidopsis Nbp35 holoproteins suggest that these holoproteins form dimers with stable amino-terminal [4Fe-4S] clusters and a shared carboxy-terminal [4Fe-4S] cluster (10, 34).Open in a separate windowFIG. 1.A protein sequence alignment of bacterial, archaeal, and eukaryotic ApbC/Nbp35 homologs was constructed using the ClustalW program (version 1.83) (37). The sequence of the S. enterica serovar Typhimurium protein (ApbC; RefSeq accession no. NP_461098.1) is shown without the amino-terminal domain that is not homologous to the amino-terminal domains of the archaeal and eukaryotic proteins. The archaeal homologs are from S. solfataricus (SSO0460; accession no. NP_341994.1), P. furiosus (PF1145; accession no. NP_578874.1), Methanosarcina acetivorans (MA4246; accession no. NP_619111.1), M. jannaschii (MJ0283; accession no. NP_247256.1), and M. maripaludis (MMP0704; accession no. NP_987824.1). The two paralogs from S. cerevisiae are Nbp35 (accession no. NP_011424.1) and Cfd1 (accession no. NP_012263.1). Conserved amino acid residues are shown in white on a black background. Similar residues are shown in black on a gray background. The four conserved amino-terminal cysteine residues shared by the MMP0704 and Nbp35p proteins are boxed. Asterisks above the sequences indicate MMP0704 residues replaced by mutagenesis in this study. A vertical bar indicates the N termini of the truncated proteins MJ0283(19-290) and MMP0704(20-289).Archaeal homologs of bacterial ApbC and eukaryotic Nbp35 are underannotated as nucleotide-binding proteins or misannotated as cobyrinic acid a,c-diamide synthases in sequence databases. The hallmarks of the Nbp35 sequences are an amino-terminal ferredoxin-like domain, an ATP-binding motif, and two conserved carboxy-terminal cysteine residues that are believed to bind an Fe-S cluster. The amino-terminal ferredoxin-like domain is absent in the ApbC family of proteins. The ApbC and Nbp35 proteins belong to a large superfamily of P-loop-containing nucleoside triphosphate hydrolases that also includes the bacterial MinD and CooC proteins. The M. maripaludis MMP0704 protein shows approximately 40% amino acid identity to both the S. enterica ApbC and S. cerevisiae Nbp35 proteins (Fig. ​(Fig.1).1). However, the MMP0704 protein also shows 30% sequence identity to two paralogous proteins from M. maripaludis. The genome sequence of M. maripaludis encodes at least nine paralogs, although only the MMP0704 protein contains the conserved cysteine residues found in most ApbC/Nbp35 proteins.The experiments described herein identified the first archaeal proteins that form functional Fe-S carrier proteins. The apbC/NBP35 homologs from M. maripaludis (MMP0704), M. jannaschii (MJ0283), and Sulfolobus solfataricus (SSO0460) allowed an S. enterica strain with an apbC null mutation to grow on tricarballylate. Genetic studies showed that the Walker A box and at least one cysteine residue from the Cys-X-X-Cys motif were required for in vivo functionality. The unique amino-terminal ferredoxin-like domains of the MMP0704 and MJ0283 proteins were not required. Purified MMP0704 proteins bound Fe-S clusters. Orthologs of ApbC/Npb35 proteins were found in all of the available genomes of free-living archaea, identifying this protein family as an ancient part of the Fe-S assembly system that evolved before the divergence of Archaea and Eukarya.