Characterization of the hamster genomic fragment cloned by TAR cloning technology with interspecific sequence information

CHO (Chinese Hamster ovary) cells are widely used for biotechnology and biomedical purposes, and now the EST library database of CHO cells is built. Based on this, the construction of the hamster genome library is under exertion. Though the transformation-associated recombination (TAR) cloning method is accounted as an innovative cloning technology without the construction of the genome library in human and mouse, there has been no trial to isolate the genomic fragment from hamster genome by TAR cloning. In this study, approximately 31 kb of hamster genomic fragment was isolated from the normal human/hamster mono-chromosomal somatic cell line (UV5HL9-5B) using universal hooks of rodent repeats sequence of B1 and B2 by TAR cloning. This fragment was analyzed by bioinformatics tools related to the genome alignment for the similarity analysis among rodent and primate, and was classified into rodents by phylogenetic analysis. One putative gene was found in this region which has homology with the human c14orf4 gene. A zinc finger protein domain was found in the translated hamster ORF. Therefore, we suggest that TAR cloning technique can be applied in CHO cells using mouse genomic information, and it can lead to the establishment of the hamster genome database.     

Determination of the baculovirus transducing titer in mammalian cells

Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments.     

Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process

A shortcut biological nitrogen removal (SBNR) utilizes the concept of a direct conversion of ammonium to nitrite and then to nitrogen gas. A successful SBNR requires accumulation of nitrite in the system and inhibition of the activity of nitrite oxidizers. A high concentration of free ammonia (FA) inhibits nitrite oxidizers, but unfortunately decreases the ammonium removal rate as well. Therefore, the optimal range of FA concentration is necessary not only to stabilize nitrite accumulation but also to achieve maximum ammonium removal. In order to derive such optimal FA concentrations, the specific substrate utilization rates of ammonium and nitrite oxidizers were measured. The optimal FA concentration range appeared to be 5–10 mg/L for the adapted sludge. The simulated results from the modified inhibition model expressed by FA and ammonium/nitrite concentrations were shown very similar to the experimental results.     

Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

Effect of germination temperature on characteristics of phytase production from barley

The effects of germination temperature on the growth of barley seedlings for phytase production were studied at 15, 20 and 25 degrees C for 6-10 days. The growth rate of the barley seedlings was increased as the germination temperature was increased. The initial rate of total protein production was closely coupled to that of the barley growth, and the rate of total protein production tended to increase as the germination temperature was increased. SDS-PAGE analysis of total protein from the barley seedlings showed time-dependent appearance and disappearance of protein bands. Although no significant phytase activity was detected at zero time of germination, a significant increase in phytase activity up to 7.9-fold occurred during the first several days of germination then decreased. Phosphate production (viz. phytate degradation) in the barley seedlings occurred rapidly at the beginning of germination. However, the rate of production continued to decrease with further germination. A time lag of about 1-2 days between the rate of total protein production and that of phytase production was observed. Unlike the extent of total protein production, that of phytase production was similar irrespective of germination temperature. Partial purification of a crude enzyme extract by hydrophobic interaction chromatography resulted in two phytase fractions (PI and PII). Zymogram analysis demonstrated that PI had two bands with molecular masses of about 66 and 123 kDa while PII had one band corresponding to a molecular mass of about 96 kDa. The optimal temperature for PI was found to be 55 degrees C, while it was 50 degrees C for PII. The enzyme fraction PI had a pH optimum at 6.0, whereas the optimum pH for PII was found to be 5.0. Addition of 0.1% (v/v) Tween 80 was found to increase enzyme activity significantly (i.e., 167% for PI and 137% for PII). Phytate in cereals including barley, rice, corn and soybean degraded effectively by the treatment of the barley phytases.     

Bone-induced streak artifact suppression in sparse-view CT image reconstruction

ABSTRACT: BACKGROUND: In sparse-view CT imaging, strong streak artifacts may appear around bony structures and they often compromise the image readability. Compressed sensing (CS) or total variation (TV) minimization-based image reconstruction method has reduced the streak artifacts to a great extent, but, sparse-view CT imaging still suffers from residual streak artifacts. We introduce a new bone-induced streak artifact reduction method in the CS-based image reconstruction. METHODS: We firstly identify the high-intensity bony regions from the image reconstructed by the filtered backprojection (FBP) method, and we calculate the sinogram stemming from the bony regions only. Then, we subtract the calculated sinogram, which stands for the bony regions, from the measured sinogram before performing the CS-based image reconstruction. The image reconstructed from the subtracted sinogram will stand for the soft tissues with little streak artifacts on it. To restore the original image intensity in the bony regions, we add the bony region image, which has been identified from the FBP image, to the soft tissue image to form a combined image. Then, we perform the CS-based image reconstruction again on the measured sinogram using the combined image as the initial condition of the iteration. For experimental validation of the proposed method, we take images of a contrast phantom and a rat using a micro-CT and we evaluate the reconstructed images based on two figures of merit, relative mean square error and total variation caused by the streak artifacts. RESULTS: The images reconstructed by the proposed method have been found to have smaller streak artifacts than the ones reconstructed by the original CS-based method when visually inspected. The quantitative image evaluation studies have also shown that the proposed method outperforms the conventional CS-based method. CONCLUSIONS: The proposed method can effectively suppress streak artifacts stemming from bony structures in sparse-view CT imaging.     

Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.     

Biomechanics of Larval Morphology Affect Swimming: Insights from the Sand Dollars Dendraster excentricus

Most planktonic larvae of marine invertebrates are denser than sea water, and rely on swimming to locate food, navigate advective currents, and avoid predators. Therefore, swimming behaviors play important roles in larval survival and dispersal. Larval bodies are often complex and highly variable across developmental stages and environmental conditions. These complex morphologies reflect compromises among multiple evolutionary pressures, including maintaining the ability to swim. Here, I highlight metrics of swimming performance, their relationships with morphology, and the roles of behavior in modulating larval swimming within biomechanical limits. Sand dollars have a representative larval morphology using long ciliated projections for swimming and feeding. Observed larval sand dollars fell within a narrow range of key morphological parameters that maximized their abilities to maintain directed upward movement over the most diverse flow fields, outperforming hypothetical alternatives in a numerical model. Ontogenetic changes in larval morphology also led to different vertical movements in simulated flow fields, implying stage-dependent vertical distributions and lateral transport. These model outcomes suggest a tight coupling between larval morphology and swimming. Environmental stressors, such as changes in temperature and pH, can therefore affect larval swimming through short-term behavioral adjustments and long-term changes in morphology. Larval sand dollars reared under elevated pCO(2) conditions had significantly different morphology, but not swimming speeds or trajectories. Geometric morphometric analysis showed a pH-dependent, size-mediated change in shape, suggesting a coordinated morphological adjustment to maintain swimming performance under acidified conditions. Quantification of the biomechanics and behavioral aspects of swimming improves predictions of larval survival and dispersal under present-day and future environmental conditions.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.