Characteristics of Classical Swine Fever Virus Variants Derived from Live Attenuated GPE− Vaccine Seed

The GPE− strain is a live attenuated vaccine for classical swine fever (CSF) developed in Japan. In the context of increasing attention for the differentiating infected from vaccinated animals (DIVA) concept, the achievement of CSF eradication with the GPE− proposes it as a preferable backbone for a recombinant CSF marker vaccine. While its infectious cDNA clone, vGPE−, is well characterized, 10 amino acid substitutions were recognized in the genome, compared to the original GPE− vaccine seed. To clarify the GPE− seed availability, this study aimed to generate and characterize a clone possessing the identical amino acid sequence to the GPE− seed. The attempt resulted in the loss of the infectious GPE− seed clone production due to the impaired replication by an amino acid substitution in the viral polymerase NS5B. Accordingly, replication-competent GPE− seed variant clones were produced. Although they were mostly restricted to propagate in the tonsils of pigs, similarly to vGPE−, their type I interferon-inducing capacity was significantly lower than that of vGPE−. Taken together, vGPE− mainly retains ideal properties for the CSF vaccine, compared with the seed variants, and is probably useful in the development of a CSF marker vaccine.

Codon bias determines sorting of ion channel protein

The choice of codons can influence local translation kinetics during protein synthesis. The question of whether the modulation of polypeptide folding and binding to chaperons influences sorting of nascent membrane proteins remains unclear. Here, we use two similar K+ channels as model systems to examine the impact of codon choice on protein sorting. By monitoring transient expression of GFP tagged proteins in mammalian cells we find that targeting of one channel to the secretory pathway is insensitive to codon optimization. In contrast, sorting of the second channel to the mitochondria is very sensitive to codon choice. The protein with an identical amino acid sequence is sorted in a codon and cell cycle dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves together with a cell-state depending decoding mechanism as a secondary code for sorting intracellular proteins.

Malaria transmission assisted by interaction between Plasmodium α-tubulin-1 and Anopheles FREP1 protein

ABSTRACTPassage of Plasmodium through a mosquito midgut is essential for malaria transmission. FREP1, a peritrophic matrix protein in a mosquito midgut, binds to the parasite and mediates Plasmodium infection in Anopheles. The FREP1-mediated Plasmodium invasion pathway is highly conserved across multiple species of Plasmodium and Anopheles. Through pulldown, nine P. berghei proteins were co-precipitated with FREP1-conjugated beads. After cloning these nine genes from P. berghei and expressing them in insect cells, six of them were confirmed to interact with recombinant FREP1 protein. Among them, α-tubulin-1 and heat shock protein 70 (Hsp70) were highly conserved in Plasmodium species with >95% identity. Thus, P. falciparum α-tubulin-1 and Hsp70 were cloned and expressed in E. coli to stimulate antibody (Ab) in mice. Our results showed that anti-serum against P. falciparum α-tubulin-1 significantly inhibited P. falciparum transmission to An. gambiae, while Ab against P. falciparum Hsp70 serum did not. The polyclonal Ab against human α-tubulin did not interfere formation of ookinetes, however, significantly reduced the number of P. falciparum oocysts in An. gambiae midguts. Moreover, fluorescence microscope assays showed that anti-α-tubulin Ab bound to impermeable Plasmodium ookinete apical invasive apparatus. Therefore, we propose that the interaction between Anopheles FREP1 protein and Plasmodium α-tubulin-1 directs the ookinete invasive apparatus towards midgut peritrophic matrix for the efficient passage of the parasite. Anopheles FREP1 and Plasmodium α-tubulin-1 are potential targets for blocking malaria transmission to the mosquito host.AUTHOR SUMMARYThe molecular mechanisms of malaria transmission to mosquito are not well-understood. FREP1 proteins in mosquito midget PM has been proved to mediate malaria transmission by binding to parasite ookinetes. Here we reported that Plasmodium parasite α-tubulin-1 is an FREP1 binding partner. We initially identified the α-tubulin-1 through the FREP1-pulldown assay; Then we cloned P. falciparum α-tubulin-1, and demonstrated that the insect cell expressed recombinant Plasmodium α-tubulin-1 bound to FREP1 in vitro; Next, mouse anti-serum against P. falciparum α-tubulin-1 was found to inhibit P. falciparum transmission to An. gambiae. P. falciparum α-tubulin-1 shares >84% identical amino acid sequence with human α-tubulin, purified Ab against human α-tubulin significantly inhibited malaria transmission. Anti-human α-tubulin Ab did not interfere the gametocyte-to-ookinetes conversion. Final, we found that anti-α-tubulin Ab bound to the apical end of impermeable ookinetes. Structurally, ookinete invasive apparatus locates at the apical opening. Therefore, we propose that the interaction between Anopheles midgut FREP1 protein and Plasmodium apical α-tubulin-1 directs the ookinete invasive apparatus towards midgut PM for the efficient parasite invasion.

Effects of Culture Media on Phytophthora palmivora Growth, α-elicitin Production and Toxicity to Dendrobium

Four culture media were evaluated for their ability to induce Phytophthora palmivora growth and produce culture filtrate (CF), and to determine a CF concentration and culture period effective for in vitro screening of black rot resistance in Dendrobium cv. ‘Earsakul’. Mycelial fresh weights of P. palmivora cultured in potato dextrose broth (PDB; the most commonly used medium for fungi), pea sucrose broth (PSB; a medium frequently used for Phytophthora spp.), and Murashige and Skoog broth (MSB; the most popular plant tissue culture medium) were found to be significantly higher than that in the newly developed modified oat meal broth (MOMB). When the total proteins of CFs were analysed with SDS-PAGE, a protein band of 10.5 kDa MW was found in CFs from all media with the highest level in PSB. LC-MS/MS analysis identified this protein as α-elicitin that had an identical amino acid sequence to the α-elicitin hibernalin of P. hibernalis and syringicin from P. syringae. The optimum conditions for in vitro selection of Dendrobium for black rot resistance using α-elicitin-containing CFs were also determined by evaluating the CF toxicity on Dendrobium protocorm-like bodies (PLBs) when cultured in all media supplemented with 0, 30, 50 and 100% CFs for seven, 14 and 21 d. The levels of PLB necrosis varied according to medium types, CF concentrations and culture periods. The maximum percentage of PLB necrosis (100%) was obtained in PSB supplemented with 50 and 100% CFs, and the severity of PLB necrosis was highest when treated with 100% CF for 14 and 21 d.

A phase I pharmacokinetics trial comparing PF-05280014 (a potential biosimilar) and trastuzumab in healthy volunteers (REFLECTIONS B327-01).

171 Background: PF-05280014, a proposed biosimilar to trastuzumab, has an identical amino acid sequence and similar physicochemical and in vitro functional properties to trastuzumab. This study was designed to demonstrate PK similarity of PF-05280014 to trastuzumab from the US (trastuzumab-US) and EU (trastuzumab-EU), and between the licensed drugs. Safety and immunogenicity were also evaluated. Methods: In this double-blind trial (NCT01603264), 105 healthy male volunteers, 18-55 years old were randomized 1:1:1 to receive a single 6 mg/kg IV dose of PF-05280014, trastuzumab-US or trastuzumab-EU. All subjects provided informed consent. PK, safety and immunogenicity assessments were conducted for 70 days. PK similarity for a given test-to-reference comparison was considered to be demonstrated if the 90% CI of the test-to-reference ratio of the AUC from time 0 to the last time point (AUCT) and maximum concentration (Cmax) were within 80% – 125%. Results: The baseline demographics for the 101 subjects evaluable for PK were similar among 3 treatment arms. The 3 study drugs exhibited similar characteristics of target-mediated disposition and similar PK parameters (Table). The 90% CI for the ratios of Cmax, AUCT, and AUC0-∞were within 80% – 125% for the comparisons of PF-05280014 to trastuzumab-EU or trastuzumab-US, and trastuzumab-EU to trastuzumab-US. Adverse events (AE) were similar for the 3 arms with treatment-related AEs reported by 71.4%, 68.6%, and 65.7% subjects in the PF-05280014, trastuzumab-EU and trastuzumab-US, respectively. No serious AEs were reported. Only 4 subjects had treatment interruptions; 2 discontinued. Only 1 subject (trastuzumab-EU) developed anti-drug antibodies after dosing. Conclusions: This study demonstrates PK similarity of PF-05280014 to both trastuzumab-US and trastuzumab-EU and of trastuzumab-EU to trastuzumab-US. The 3 study drugs also showed similar safety profiles. Clinical trial information: NCT01603264. [Table: see text]

A phase I pharmacokinetics trial comparing PF-05280014 (a potential biosimilar) and trastuzumab in healthy volunteers (REFLECTIONS B327-01).

612 Background: PF-05280014, a proposed biosimilar to trastuzumab, has an identical amino acid sequence and similar physicochemical and in vitro functional properties to trastuzumab. This study was designed to demonstrate PK similarity of PF-05280014 to trastuzumab from the US (trastuzumab-US) and EU (trastuzumab-EU), and between the licensed drugs. Safety and immunogenicity were also evaluated. Methods: In this double-blind trial (NCT01603264), 105 healthy male volunteers, 18-55 years old were randomized 1:1:1 to receive a single 6 mg/kg IV dose of PF-05280014, trastuzumab-US or trastuzumab-EU. All subjects provided informed consent. PK, safety, and immunogenicity assessments were conducted for 70 days. PK similarity for a given test-to-reference comparison was considered to be demonstrated if the 90% CI of the test-to-reference ratio of the AUC from time 0 to the last time point (AUCT) and maximum concentration (Cmax) were within 80% – 125%. Results: The baseline demographics for the 101 subjects evaluable for PK were similar among 3 treatment arms. The 3 study drugs exhibited similar characteristics of target-mediated disposition and similar PK parameters (Table). The 90% CI for the ratios of Cmax, AUCT, and AUC0-∞were within 80% – 125% for the comparisons of PF-05280014 to trastuzumab-EU or trastuzumab-US, and trastuzumab-EU to trastuzumab-US. Adverse events (AE) were similar for the 3 arms with treatment-related AEs reported by 71.4%, 68.6%, and 65.7% subjects in the PF-05280014, trastuzumab-EU and trastuzumab-US, respectively. No serious AEs were reported. Only 4 subjects had treatment interruptions; 2 discontinued. Only 1 subject (trastuzumab-EU) developed anti-drug antibodies after dosing. Conclusions: This study demonstrates PK similarity of PF-05280014 to both trastuzumab-US and trastuzumab-EU and of trastuzumab-EU to trastuzumab-US. The three study drugs also showed similar safety profiles. Clinical trial information: NCT01603264. [Table: see text]

D-Amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties

The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution.

DNA Sequence Analysis of rdxA andfrxA from 12 Pairs of Metronidazole-Sensitive and -Resistant Clinical Helicobacter pyloriIsolates

ABSTRACT We previously reported that inactivation of rdxAand/or frxA converted Helicobacter pylorifrom metronidazole sensitive to metronidazole resistant. To examine the individual roles of rdxA and frxA in the development of metronidazole resistance in H. pylori, we examined the status of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant H. pylori isolates obtained following unsuccessful therapy containing metronidazole. Arbitrary primed fingerprinting analyses revealed that the genotypes of 11 sensitive and resistant pairs of strains were essentially identical. Amino acid sequence identities of RdxA and FrxA from the 14 metronidazole-sensitive isolates ranged from 92 to 98% and 95 to 98%, respectively, compared to that of H. pylori J99 (MIC, 1 μg/ml). All strains with high-level metronidazole resistance (MICs, 128 μg/ml) contained premature truncation of both RdxA and FrxA caused by nonsense and/or frameshift mutations. Strains with intermediate resistance to metronidazole (MICs, 32 to 64 μg/ml) contained a single premature truncation and/or altered RdxA and FrxA caused by nonsense, frameshift, and unique missense mutations. The low-level metronidazole-resistant strains (MICs, 8 μg/ml) contained unique missense mutations in FrxA but no specific changes in RdxA. The results demonstrate that alterations in both the rdxA and frxA genes are required for moderate and high-level metronidazole resistance and that metronidazole resistance that develops during anti-H. pylori therapy containing metronidazole is most likely to involve a single sensitive strain infection rather than a coinfection with a metronidazole-resistant strain.

Ultraviolet and violet receptors express identical mRNA encoding an ultraviolet-absorbing opsin: identification and histological localization of two mRNAs encoding short-wavelength-absorbing opsins in the retina of the butterfly Papilio xuthus

This paper describes the primary structures of two opsins of short-wavelength-absorbing visual pigments deduced from the mRNA sequences in the retina of the Japanese yellow swallowtail butterfly Papilio xuthus. A phylogenetic analysis of the amino acid sequences indicates that one of these visual pigments is of the ultraviolet-absorbing type and that the other is of the blue-absorbing type. We identified the photoreceptor cells that express these mRNAs by histological in situ hybridization. The mRNA of the ultraviolet type is expressed in two distinct photoreceptor types previously identified as ultraviolet and violet receptors, providing the first molecular biological evidence that different types of spectral receptor probably express a visual pigment with an identical amino acid sequence. The mRNA of the blue type is expressed exclusively in cells classified as blue receptors.

Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide

To purify novel ligands for the corticotrophin-releasing factor binding protein (CRF-BP) from ovine brain, whole brain was homogenised in methanol and the supernatant extracted on Sep-pak C18 cartridges followed by a preliminary HPLC step. Three peaks of ovine CRF-BP ligand activity were detected in the HPLC fractions, the first two of which were also detected by a specific corticotrophin-releasing factor two-site immunoradiometric assay, the third peak being detected by a human CRF-BP ligand assay, which will not detect ovine CRF. Human CRF-BP ligand-containing fractions were further purified by affinity chromatography on a human recombinant CRF-BP column with two additional HPLC steps. The human CRF-BP ligand was found to: (a) possess a molecular mass of 4707 Daltons, (b) have an N-terminal amino acid sequence (5 residues) identical to rat urocortin, (c) be detected by a specific urocortin radioimmunoassay, (d) have high affinity for both the human and ovine CRF-BPs and (e) be present in many regions of the ovine brain. Additionally, a 300 bp cDNA fragment sharing 83% homology with the rat urocortin gene was cloned from ovine brain, the product of which was predicted to have an identical amino acid sequence to that of rat urocortin. These pieces of information confirmed the identity of the human CRF-BP ligand as an ovine urocortin. The specially developed CRF-BP ligand assays showed that the rank orders of affinity of the CRF family members for human CRF-BP were: carp urotensin-1>>human CRF=rat/ovine urocortin>human urocortin>>frog sauvagine>>ovine CRF, and those for the ovine CRF-BP were: carp urotensin-1> human CRF=rat/ovine urocortin>human urocortin> frog sauvagine>>ovine CRF. This study describes a successful technique for the purification and detection of peptide ligands for the CRF-BP. We conclude that urocortin is the principal ligand for the CRF-BP in ovine brain and we could find no evidence for a centrally located mammalian sauvagine-like peptide.