A zeolite as a tool for successful refolding of PEGylated proteins and their reassembly with tertiary structures

In the present study, we demonstrated zeolites' potential contribution to establish a method for preparing successfully refolded and reassembled PEGylated protein nanoparticles without the use of protein denaturants through the proteins' reassembly process. At first, the PEGylated nanoparticles are disassembled into identical PEGylated protein subunits by means of protein denaturants, and then the denatured subunits are adsorbed to zeolites. After the complete removal of denaturants, high-molecular-weight poly(ethylene glycol) (PEG) molecules are added to a solution where the zeolites suspend. Consequently, the PEGylated proteins are gradually reassembled into nanoparticles because the subunits are desorbed from the zeolites by the steric hindrance of the added PEG molecules. The present study reveals that PEGylated encapsulin was reassembled and hollow encapsulin nanoparticles were obtained. The results clearly demonstrate the usefulness of zeolites as a tool for the successful refolding of PEGylated proteins and their reassembly with tertiary structures.     

An Archaeal Glutamate Decarboxylase Homolog Functions as an Aspartate Decarboxylase and Is Involved in β-Alanine and Coenzyme A Biosynthesis

β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5′-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4′-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.     

Reevaluation of erythropoietin production by the nephron

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.     

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.     

Genetic diversity of Microcystis cyanophages in two different freshwater environments

Bacteriophages rapidly diversify their genes through co-evolution with their hosts. We hypothesize that gene diversification of phages leads to locality in phages genome. To test this hypothesis, we investigated the genetic diversity and composition of Microcystis cyanophages using 104 sequences of Ma-LMM01-type cyanophages from two geographically distant sampling sites. The intergenetic region between the ribonucleotide reductase genes nrdA and nrdB was used as the genetic marker. This region contains the host-derived auxiliary metabolic genes nblA, an unknown function gene g04, and RNA ligase gene g03. The sequences obtained were conserved in the Ma-LMM01 gene order and contents. Although the genetic diversity of the sequences was high, it varied by gene. The genetic diversity of nblA was the lowest, suggesting that nblA is a highly significant gene that does not allow mutation. In contrast, g03 sequences had many point mutations. RNA ligase is involved in the counter-host’s phage defense mechanism, suggesting that phage defense also plays an important role for rapid gene diversification. The maximum parsimony network and phylogenic analysis showed the sequences from the two sampling sites were distinct. These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes.     

Hybridization between Japanese and North American Chlamydomonas reinhardtii (Volvocales,Chlorophyceae)

The microalga Chlamydomonas reinhardtii is a model organism whose whole genome has been sequenced. Although considered a cosmopolitan species, only eastern North American isolates of C. reinhardtii were available before 2010, when new Japanese isolates were reported. In the study describing the new Japanese isolates, zygote formation between Japanese and North American strains was shown, but germination was not demonstrated. In this study, the germination of intercontinental hybrid zygotes was examined using wild‐type Japanese strains and mutant American strains that cannot utilize nitrate. Several clonal progeny strains were established, and the progeny strains were screened based on mating type and nitrate utilization to confirm their hybrid nature. The establishment of four intercontinental hybrid strains with different phenotypic combinations was confirmed by sequencing mating type‐specific and nitrate reductase‐related genes. The potential for hybrid formation between Japanese and North American strains suggests the existence of a worldwide mating population of C. reinhardtii.     

Importance of Fatty Acid Compositions in Patients with Peripheral Arterial Disease

Objective

Importance of fatty acid components and imbalances has emerged in coronary heart disease. In this study, we analyzed fatty acids and ankle-brachial index (ABI) in a Japanese cohort.

Methods

Peripheral arterial disease (PAD) was diagnosed in 101 patients by ABI ≤0.90 and/or by angiography. Traditional cardiovascular risk factors and components of serum fatty acids were examined in all patients (mean age 73.2±0.9 years; 81 males), and compared with those in 373 age- and sex-matched control subjects with no evidence of PAD.

Results

The presence of PAD (mean ABI: 0.71±0.02) was independently associated with low levels of gamma-linolenic acid (GLA) (OR: 0.90; 95% CI: 0.85–0.96; P = 0.002), eicosapentaenoic acid∶arachidonic acid (EPA∶AA) ratio (OR: 0.38; 95% CI: 0.17–0.86; P = 0.021), and estimated glomerular filtration rate (OR: 0.97; 95% CI: 0.96–0.98; P<0.0001), and with a high hemoglobin A1c level (OR: 1.34; 95% CI: 1.06–1.69; P = 0.013). Individuals with lower levels of GLA (≤7.95 µg/mL) and a lower EPA∶AA ratio (≤0.55) had the lowest ABI (0.96±0.02, N = 90), while the highest ABI (1.12±0.01, N = 78) was observed in individuals with higher values of both GLA and EPA∶AA ratio (P<0.0001).

Conclusion

A low level of GLA and a low EPA∶AA ratio are independently associated with the presence of PAD. Specific fatty acid abnormalities and imbalances could lead to new strategies for risk stratification and prevention in PAD patients.     

Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples.     

Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals

Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.