Cloning, expression and preliminary application of a alpha-hydroxynitrile lyase from cassave]

alpha-Hydroxynitrile lyase (ME-HNLs, E.C. 4.1.2.3.37) from the cyanogenic crop cassava(Manihot esculentz, Crantz) catalyze the condensation of hydrocyanic acid and aldehydes or ketone into (s)-cyanohydrins, which are valuable starting material for various optically active compounds, such as pharmaceuticals and agrochemicals. The cDNA of a ME-HNL were obtained by RT-PCR and cloned. The sequencing result for the cDNA showed that the sequence encoded for the ME-HNL was inconsistent with all those which are published, such as hnl10, hnl24, hnl4. The full sequence analysis demonstrated that the cloned cDNA was about 75.2%, 79.8%, 99.2% homologous to other three reported HNL genes from cassava, respectively, among which the last was the same to the cloned gene except the five base substitution at the site 142, 337, 476, 634 and 636, respectively. The two base substitutions lead to change the amino acid sequence, i.e., Ser113-->Gly113, Phe158-->Tyr158. To construct the recombinant plasmid pET30a-hnl, the cDNA was inserted into an expression vector pET30a. After transformation of pET30a-hnl and induction with IPTG, the ME-HNL was efficiently expressed in E. coli. BL21 (DE3) and reached over 2100 units/L of culture with the specific activity 8.5 u/mg protein. By one simple treatment, incubating 10 minutes at 70 degrees C, the recombinant ME-HNL may be used as an catalyst for production of (S)-mandelonitrile with enantiomeric excess of 95.2% and 98.2% yield.     

Variation of phenotype,ploidy level,and organogenic potential of in vitro regenerated polyploids of <Emphasis Type="Italic">Pyrus communis</Emphasis>

A wide range of phenotypic variation was observed among neopolyploids obtained from the diploid pear cultivar ‘Fertility’ by in vitro colchicine treatment. The variant plantlets had alterations in leaf characteristics. Neopolyploids had significantly different ratios of leaf length to leaf width compared to the diploid control. Shoot regeneration from leaf explants and rooting ability from in vitro shoots of neopolyploids was examined. Regeneration frequencies of shoots from leaf explants of seven of the nine neopolyploids were significantly decreased compared to the diploid control. The organogenic potential of neopolyploids was highly genotype-dependent for both shoots and roots. Tetraploid clone 4x − 4 failed to regenerate shoots from leaf explants and the pentaploid clone 5x − 2 failed to root from in vitro shoots. The results suggest that polyploidization caused the decrease in or loss of in vitro organogenic potential. Regenerated shoots derived from neopolyploids showed different phenotypes, depending on the ploidy of the donor plant.     

Purification of lysozyme by affinity-based reversed micellar two-phase extraction

A dye-affinity reversed micellar system was used for lysozyme purification from a crude solution of chicken egg white. The dye-affinity reversed micelles consisted of Cibacron Blue F-3GA (CB; 0.1 mM) modified lecithin (50 g/l) in n-hexane. Starting with a crude egg white solution containing lysozyme of 0.0381 mg/mg protein, lysozyme purity was increased by 16 to 20 times, reached 0.62 to 0.76 mg/mg protein. The affinity micellar system was recycled and used three times. Addition of polyoxyethylene (20) sorbitan trioleate (Tween 85) as a cosurfactant could increase the capacity of the affinity-based reversed micelles. A lysozyme recovery yield of over 70% was obtained at a forward aqueous phase pH of 9.16 using the reversed micelles additionally containing 20 g/l of Tween 85.     

Observing the Temperature Dependent Transition of the GP2 Peptide Using Terahertz Spectroscopy

The GP2 peptide is derived from the Human Epidermal growth factor Receptor 2 (HER2/nue), a marker protein for breast cancer present in saliva. In this paper we study the temperature dependent behavior of hydrated GP2 at terahertz frequencies and find that the peptide undergoes a dynamic transition between 200 and 220 K. By fitting suitable molecular models to the frequency response we determine the molecular processes involved above and below the transition temperature (T _D). In particular, we show that below T _D the dynamic transition is dominated by a simple harmonic vibration with a slow and temperature dependent relaxation time constant and that above T _D, the dynamic behavior is governed by two oscillators, one of which has a fast and temperature independent relaxation time constant and the other of which is a heavily damped oscillator with a slow and temperature dependent time constant. Furthermore a red shifting of the characteristic frequency of the damped oscillator was observed, confirming the presence of a non-harmonic vibration potential. Our measurements and modeling of GP2 highlight the unique capabilities of THz spectroscopy for protein characterization.     

Second to fourth digit ratio and the sporting success of sumo wrestlers

The second (index finger) to fourth (ring finger) digit length ratio (2D:4D) is known to be a putative marker of prenatal exposure to testosterone. It has been reported that fetal and adult testosterone may be critical for development of physical and mental traits such as cardiovascular system, reaction time, aggressiveness and masculinity. Testosterone-driven attributes are associated with success in male-to-male physical competition, which may be proxied by ability in sports. Many researchers have found that 2D:4D is sexually dimorphic and is a negative correlate of athletic performance. This study aims to investigate the associations of 2D:4D with measures of power as another possible testosterone-associated trait using ability in sumo wrestling as a proxy for male physical competitiveness. The measures of sumo performance comprised the sumo ranks and winning percentages of 142 Japanese professional sumo wrestlers. We found that sumo wrestlers with low 2D:4D had higher sumo ranks and better winning records. The significant negative associations between 2D:4D and the athletic prowess of sumo wrestlers provide further evidence of the possible link between high testosterone levels and muscle strength. The relatively small effect sizes found in this study, however, imply that 2D:4D may be a weaker predictor for sports requiring explosive power than for those requiring endurance.     

Novel Inhibitors of Staphylococcus aureus Virulence Gene Expression and Biofilm Formation

Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.     

Lactoferrin activates plasma membrane oxidase and Na+/H+ antiport activity.

Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.     

Immunohistochemical Identification of Met-Enkephalin in Digestive System and Its Effect on Digestive Enzyme Activities of the Scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

The study was designed to determine whether methionine-enkephalin (M-ENK) was present in the digestive system of the scallop Chlamys farreri and investigate the effects of M-ENK on the activity of amylase, protease and lipase in the digestive system of C. farreri. The results indicated that M-ENK-like material was present in the epithelium and connective tissue of labial palps, mouth labia, stomach, intestine, rectum, and hepatopancreas of the scallop C. farreri. Moreover, it was also found that many isolated small cells showing M-ENK-like immunoreactivity were scattered in the epithelial layer of intestine, and many isolated big epithelial cells showing M-ENK-like immunoreactivity were scattered in tubules of hepatopancreas of the scallop. The activity levels of amylase and lipase in crystalline style, hepatopancreas and intestine were enhanced at 1 h after injection of exogenous M-ENK into adductor muscle of the scallops, whereas protease activity levels were significantly suppressed. Our report constitutes the first characterization of M-ENK in the digestive system of scallop C. farreri and investigates the effects of M-ENK on the activities of digestive enzymes of mollusk for the first time. The results suggest an involvement of M-ENK in the functional regulation of the digestive system of C. farreri.