Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.     

A fiber-bed bioreactor for anchorage-dependent animal cell cultures: part II. Scaleup potential

A simple hydrodynamic model is introduced to describe the airlift fiber-bed bioreactor, which can enhance the volumetric productivity of anchorage-dependent animal cell cultures. By applying the model, liquid flow rates and volumetric mass transfer coefficients are predicted and are in agreement with experimental measurements. Consequently, the optimal reactor configuration giving the maximal oxygen supply is derived. Also, theoretical scaleup potential of this concentric internal loop reactor is considered for volumes ranging from 10 to 67,000 L with which cell densities of 5.1 x 10(7) and 1.2 x 10(7) cells/cm(3), respectively, can be maintained.     

Using marker gene analysis instead of mixed lymphocyte reaction assay for identification of functional CD4+FOXP3+ regulatory T cells

Objective

To establish a quick analytical method using quantitative PCR for marker gene analysis to identify the functions of iTreg cells and subsequently curtail the harvest time for iTreg cells.

Results

The data from the marker gene analysis indicated that varying proportions of iTreg cells could reveal the various expression levels of these genes. FoxP3 expression increased to a considerable degree. By using the same iTreg population, the mixed lymphocyte reaction assay was conducted for 5 days. The suppression percentage of T-cells was dependent on the proportion of iTreg cells, indicating that gene expression levels can represent the biological functions of iTreg cells. By using human peripheral blood mononuclear cells for Treg cell induction, the marker gene expression analysis showed a difference between iTreg cells and uninduced T cells.

Conclusion

Marker gene analysis requires only 1 day to identify the functions of human iTreg cells can save time in clinical application and might prevent graft-versus-host disease occurrence effectively.

     

The structural proteins of infectious pancreatic virus are not glycosylated.

The major capsid protein, VP2, of infectious pancreatic necrosis virus, a nonenveloped icosahedral virus, contains six N-glycosylation consensus sequences (Asn-X-[Thr/Ser]). Since VP2 contains the major virus-neutralizing epitopes, the possible role for glycosylation in capsid formation and antigenicity was examined. The carbohydrate content of the virion proteins was determined by chemical detection, pulse-chase experiments,[3H]mannose labeling, and alteration of protein migration on sodium dodecyl sulfate-polyacrylamide gels after tunicamycin treatment. No glycosylation of any virion protein was observed when the carbohydrate nature of the glycoprotein of infectious hematopoietic necrosis virus was detected.     

Effect of triton X-100 on alkaline lipase production by Pseudomonas pseudoalcaligenes F-111

Summary An extracellular alkaline lipase was evaluated from Pseudomonas pseudoalcaligenes F-111, which was isolated from soil using a rhodamine B agar plate with Na₂CO₃. For enzyme production, olive oil, soymeal, triton X-100 and sodium ion were found to be essential. The addition of triton X-100 increased the alkaline lipase by 50- fold. The effect of additives as well as the development of suitable culture conditions for lipase production is discussed.     

Development of an In Planta system to monitor phosphorus status by agroinfiltration and agroinjection

Plant and Soil - An optimal supply of phosphate (Pi) fertilizer is important to ensure crop yield and quality and to maintain agricultural sustainability. In this study, an in planta monitoring...     

Impact of Mated Female Nonproductive Days in Breeding Herd after Porcine Epidemic Diarrhea Virus Outbreak

Porcine epidemic diarrhea virus (PEDV) is an important pathogen that has a significant economic impact on the swine industry by imposing a high rate of mortality in suckling piglets. However, limited information on the productivity values of gilts and sows infected with PEDV is available. Here, we evaluate the productivity index in gilts and sows during the 1-year period before (19 January 2013 to 18 January 2014) and after (19 January 2014 to 18 January 2015) a PEDV outbreak from a 2000-sow breeding herd in Taiwan. The farrowing rate (FR), return rate (RR), total pigs born per litter (TB), pigs born alive per litter (BA), weaning pigs per litter (WPL), pre-weaning mortality, percentage of sows mated by 7 days after weaning, weaning to first service interval (WFSI), mated female nonproductive days (NPDs), replacement rate of sows and sow culling rate were compared using productive records. The FR (-9.6%), RR (+9.8%), TB (-1.6), BA (-1.1), WPL (-1.1), sows mated by 7 days after weaning (-6.9%), WFSI (+0.8 days), NPDs (+6.9 days) and sow culling rate (+7.2%) were significantly different between the 1-year pre-PEDV outbreak period and the post-PEDV outbreak period. Impacts of the PEDV infection on the reproductive performance were more severe in pregnant gilts than in sows. In conclusion, these findings indicate that the outbreak of PEDV caused an increase in the rate of NPDs in breeding herds.     

Physicochemical characterization of α-crystallins from bovine lenses: Hydrodynamic and conformational properties

A detailed investigation of hydrodynamic and conformational behavior has been made of the HMα-crystallin and α-crystallins of bovine lens. Results from this study indicated that HMα (high-molecular-weight α-crystallin) and α (low-molecular-weight α-crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HMα with an average sedimentation coefficient of about 50 S and a more homogeneous system of α-crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant β-sheet conformation for α-crystallin, yet a highly asymmetrical and aggregated form for HMα. The conformational stability of α-crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native α-crystallin. Conformational flexibility of α-crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on α-crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HMα from α-crystallin. The comparison of conformational properties between HMα and α-crystallin strongly indicated that HMα is a denatured form of α-crystallin.