High concentration cultivation of Bifidobacterium bifidum in a submerged membrane bioreactor

In batch cultures, after 25 h, the maximum cell mass of Bifidobacterium bifidum BGN4 was 4.5 g/L, and the maximum cell count was 3.0 x 10(9) cfu/mL at pH 6.0 and 50 g/L sucrose. To increase the viable counts of bifidobacteria, cell retentive culture was applied using a submerged membrane bioreactor with suction and gas sparging. The maximum mass, count, and productivity of the cells after 36 h were 12.0 g/L, 2.2 x 10(10) cfu/mL, and 6.1 x 10(8) cfu/mL x h, respectively, at the feeding (dilution) rate of 120 mL/h (0.06 h-1) in the feeding medium. The accumulated levels of organic acids and ammonium ions at the end of the cultivation were 1.5 and 1.0 g/L, respectively. The viable counts and volumetric productivity of the cells after the cell retentive culture were 7.3- and 5.1-fold higher, respectively, than the values obtained during batch culture. These high viable counts and volumetric productivities were obtained by maintaining lower concentrations of organic acids and ammonium ions so that the growth of B. bifidum BGN4 was not inhibited. The submerged membrane bioreactor produced the highest viable counts of B. bifidum without membrane fouling and cell damage.     

Transmembrane domain-induced oligomerization is crucial for the functions of syndecan-2 and syndecan-4

The syndecans are known to form homologous oligomers that may be important for their functions. We have therefore determined the role of oligomerization of syndecan-2 and syndecan-4. A series of glutathione S-transferase-syndecan-2 and syndecan-4 chimeric proteins showed that all syndecan constructs containing the transmembrane domain formed SDS-resistant dimers, but not those lacking it. SDS-resistant dimer formation was hardly seen in the syndecan chimeras where each transmembrane domain was substituted with that of platelet-derived growth factor receptor (PDGFR). Increased MAPK activity was detected in HEK293T cells transfected with syndecan/PDGFR chimeras in a syndecan transmembrane domain-dependent fashion. The chimera-induced MAPK activation was independent of both ligand and extracellular domain, implying that the transmembrane domain is sufficient to induce dimerization/oligomerization in vivo. Furthermore, the syndecan chimeras were defective in syndecan-4-mediated focal adhesion formation and protein kinase Calpha activation or in syndecan-2-mediated cell migration. Taken together, these data suggest that the transmembrane domains are sufficient for inducing dimerization and that transmembrane domain-induced oligomerization is crucial for syndecan-2 and syndecan-4 functions.     

Isolation of a cDNA encoding the human GM2 activator protein

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.     

MOSFET-BJT hybrid mode of the gated lateral bipolar junction transistor for C-reactive protein detection

In this study, we propose a novel biosensor based on a gated lateral bipolar junction transistor (BJT) for biomaterial detection. The gated lateral BJT can function as both a BJT and a metal-oxide-semiconductor field-effect transistor (MOSFET) with both the emitter and source, and the collector and drain, coupled. C-reactive protein (CRP), which is an important disease marker in clinical examinations, can be detected using the proposed device. In the MOSFET-BJT hybrid mode, the sensitivity, selectivity, and reproducibility of the gated lateral BJT for biosensors were evaluated in this study. According to the results, in the MOSFET-BJT hybrid mode, the gated lateral BJT shows good selectivity and reproducibility. Changes in the emitter (source) current of the device for CRP antigen detection were approximately 0.65, 0.72, and 0.80 μA/decade at base currents of -50, -30, and -10 μA, respectively. The proposed device has significant application in the detection of certain biomaterials that require a dilution process using a common biosensor, such as a MOSFET-based biosensor.     

Production of Recombinant Hinnavin II/MSH Hybrid in Insect Cells

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. For the purpose of this study, a cDNA encoding hinnavin II‐α‐melanocyte stimulating hormone (hin/MSH) hybrid was chemically synthesized, annealed, and then cloned into transfer vector pBacPAK 9 for expression in Sf21 insect cells. Recombinant hin/MSH (rhin/MSH) hybrid was efficiently produced in baculovirus expression vector system (BEVS) as a hybrid peptide. The antibacterial activity of the rhin/MSH hybrid was compared to that of the recombinant hinnavin II (rhin), using inhibition zone and overlay assay. This new recombinant hybrid peptide may serve as an attractive candidate for powerful novel class of antimicrobial pharmaceuticals.     

Delayed egg‐laying and shortened incubation duration of Arctic‐breeding shorebirds coincide with climate cooling

Biological impacts of climate change are exemplified by shifts in phenology. As the timing of breeding advances, the within‐season relationships between timing of breeding and reproductive traits may change and cause long‐term changes in the population mean value of reproductive traits. We investigated long‐term changes in the timing of breeding and within‐season patterns of clutch size, egg volume, incubation duration, and daily nest survival of three shorebird species between two decades. Based on previously known within‐season patterns and assuming a warming trend, we hypothesized that the timing of clutch initiation would advance between decades and would be coupled with increases in mean clutch size, egg volume, and daily nest survival rate. We monitored 1,378 nests of western sandpipers, semipalmated sandpipers, and red‐necked phalaropes at a subarctic site during 1993–1996 and 2010–2014. Sandpipers have biparental incubation, whereas phalaropes have uniparental incubation. We found an unexpected long‐term cooling trend during the early part of the breeding season. Three species delayed clutch initiation by 5 days in the 2010s relative to the 1990s. Clutch size and daily nest survival showed strong within‐season declines in sandpipers, but not in phalaropes. Egg volume showed strong within‐season declines in one species of sandpiper, but increased in phalaropes. Despite the within‐season patterns in traits and shifts in phenology, clutch size, egg volume, and daily nest survival were similar between decades. In contrast, incubation duration did not show within‐season variation, but decreased by 2 days in sandpipers and increased by 2 days in phalaropes. Shorebirds demonstrated variable breeding phenology and incubation duration in relation to climate cooling, but little change in nonphenological components of traits. Our results indicate that the breeding phenology of shorebirds is closely associated with the temperature conditions on breeding ground, the effects of which can vary among reproductive traits and among sympatric species.     

Characterization of cis-acting elements in light regulation of the nuclear gene encoding the A subunit of chloroplast isozymes of glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana.

We have characterized cis-acting elements involved in light regulation of the nuclear gene (GapA) encoding the A subunit of chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in Arabidopsis thaliana. Our results show that a 1.1-kb promoter fragment of the GapA gene is sufficient to confer light inducibility and organ specificity in transgenic Nicotiana tabacum (tobacco) plants, using the beta-glucuronidase gene of Escherichia coli as the reporter gene. Deletion analysis indicates that the -359 to -110 bp region of the GapA gene is necessary for light responsiveness. Within this region there are three copies of a decamer repeat (termed the Gap box) having the consensus sequence 5'-CAAATGAA(A/G)A-3', which has not been characterized in the promoter regions of other light-regulated genes. A deletion (to -247) producing loss of one copy of these elements from the GapA promoter reduces light induction by two- to threefold compared with a promoter deletion (to -359) with all three Gap boxes present, while deletion of all three Gap boxes (to -110) abolishes light induction completely. Gel mobility shift experiments using tobacco nuclei as the source of nuclear proteins show that GapA promoter fragments that contain these repeats bind strongly to a factor in the nuclear extract and that binding can be abolished by synthetic competitors consisting only of a monomer or dimer of the Gap box. Furthermore, a trimer, dimer, and monomer of the Gap box show binding activity and, like the authentic GapA promoter-derived probes, show binding activities that are correlated with Gap box copy number. These results strongly suggest that these repeats play important roles in light regulation of the GapA gene of A. thaliana.     

Nucleotide sequence of the gene for aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1 and characteristics of the deduced primary structure of the enzyme

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile [Matsuzawa, H., Hamaoki, M. & Ohta, T. (1983) Agric. Biol. Chem. 47, 25-28]. The gene encoding aqualysin I was cloned into Escherichia coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the NH2-terminal sequence previously reported and the determined amino acid sequences, including the COOH-terminal sequence, of the tryptic peptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28,350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin-type serine proteases, and 43% identity with proteinase K, 37-39% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. The nucleotide sequence of the cloned DNA (1105 nucleotides) revealed that it contains the entire gene encoding aqualysin I and one open reading frame without a translational stop codon. Therefore, aqualysin I was considered to be produced as a large precursor, which contains a NH2-terminal portion, the protease and a COOH-terminal portion. The G + C content of the coding region for aqualysin I was 64.6%, which is lower than those of other Thermus genes (68-74%). The codon usage in the aqualysin I gene was rather random in comparison with that in other Thermus genes.     

Systematic Modeling Approach to Selective Plugging Using In Situ Bacterial Biopolymer Production and Its Potential for Microbial-enhanced Oil Recovery

This study presents a systematic modeling approach for examining the efficiency of the MEOR process based on in situ selective plugging by bacterial biopolymer production and optimization of the nutrient injection strategy to yield the maximum oil recovery. This study focuses on modeling in situ selective plugging by the bacterial biopolymer dextran that is generated by Leuconostoc mesenteroides. Bacterial growth and dextran generation were described by a stoichiometric equation and kinetic reactions using batch model simulation. Based on the parameters for permeability reduction obtained from the sandpack model, the MEOR process was implemented in a pilot-scale system that included a highly permeable thief zone in a low-permeability reservoir. The base MEOR design yielded a 61.5% improvement of the recovery factor compared to that obtained with waterflooding. The parametric simulations revealed that the recovery efficiency was influenced by the amount of dextran, as well as the distribution of dextran, and thus, the injection strategy is critical for controlling the dextran distribution. By incorporating the results from the sensitivity analysis and optimization to determine the optimal design parameters, a 36.7% improvement of the oil recovery was achieved with the optimized MEOR process in comparison with the base case.