Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria

The hyperactive antifreeze protein from the beetle, Tenebrio molitor, is an 8.5-kDa, threonine-rich protein containing 16 Cys residues, all of which are involved in disulfide bonds. When produced by Escherichia coli, the protein accumulated in the supernatant in an inactive, unfolded state. Its correct folding required days or weeks of oxidation at 22 or 4 degrees C, respectively, and its purification included the removal of imperfectly folded forms by reversed-phase HPLC. NMR spectroscopy was used to assess the degree of folding of each preparation. One-dimensional (1)H and two-dimensional (1)H total correlation spectroscopy spectra were particularly helpful in establishing the characteristics of the fully folded antifreeze in comparison to less well-folded forms. The recombinant antifreeze had no free -SH groups and was rapidly and completely inactivated by 10 mM DTT. It had a thermal hysteresis activity of 2.5 degrees C at a concentration of 1 mg/ml, whereas fish antifreeze proteins typically show a thermal hysteresis of approximately 1.0 degrees C at 10-20 mg/ml. The circular dichroism spectra of the beetle antifreeze had a superficial resemblance to those of alpha-helical proteins, but deconvolution of the spectra indicated the absence of alpha-helix and the presence of beta-structure and coil. NMR analysis and secondary structure predictions agree with the CD data and are consistent with a beta-helix model proposed for the antifreeze on the basis of its 12-amino-acid repeating structure and presumptive disulfide bond arrangement.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Measurement of Visceral Fat: Should We Include Retroperitoneal Fat?

Objective

Whether retroperitoneal fat should be included in the measurement of visceral fat remains controversial. We compared the relationships of fat areas in peritoneal, retroperitoneal, and subcutaneous compartments to metabolic syndrome, adipokines, and incident hypertension and diabetes.

Methods

We enrolled 432 adult participants (153 men and 279 women) in a community-based cohort study. Computed tomography at the umbilicus level was used to measure the fat areas.

Results

Retroperitoneal fat correlated significantly with metabolic syndrome (adjusted odds ratio (OR), 5.651, p<0.05) and the number of metabolic abnormalities (p<0.05). Retroperitoneal fat area was significantly associated with blood pressure, plasma glycemic indices, lipid profile, C-reactive protein, adiponectin (r = −0.244, P<0.05), and leptin (r = 0.323, p<0.05), but not plasma renin or aldosterone concentrations. During the 2.94±0.84 years of follow-up, 32 participants developed incident hypertension. Retroperitoneal fat area (hazard ration (HR) 1.62, p = 0.003) and peritoneal fat area (HR 1.62, p = 0.009), but not subcutaneous fat area (p = 0.14) were associated with incident hypertension. Neither retroperitoneal fat area, peritoneal fat area, nor subcutaneous fat areas was associated with incident diabetes after adjustment.

Conclusions

Retroperitoneal fat is similar to peritoneal fat, but differs from subcutaneous fat, in terms of its relationship with metabolic syndrome and incident hypertension. Retroperitoneal fat area should be included in the measurement of visceral fat for cardio-metabolic studies in human.     

A complex family of highly heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio molitor.

We have previously identified a Thr- and Cys-rich thermal hysteresis (antifreeze) protein (THP) in the beetle Tenebrio molitor that has 10-100 times the freezing point depression activity of fish antifreeze proteins. Because this 8.4 kDa protein is significantly different in its properties from THP preparations previously reported from this insect, a thorough search was undertaken for other antifreeze types. Many active proteins were observed, but all appeared to be isoforms of the THP that differed in their number of 12-amino acid repeats (consensus sequence CTxSxxCxxAxT), amino acid substitutions, and N-linked glycosylation. Mass spectral analysis has matched most of these isoforms with cDNA sequences of 17 different clones from a larval fat body library that encode eight different mature THPs containing 84, 96, or 120 amino acids. Genomic Southern blots suggest there may be 30-50 tightly linked copies of the gene, which is a signature consistently seen with unrelated fish antifreeze protein genes, and one that has been associated with the need to rapidly increase gene product in response to climate change. A three-dimensional model is proposed for the fully disulfide-bonded structure of T. molitor THP, which can accommodate addition or deletion of 12-amino acid repeats. The structure is a beta-helix that places most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.     

Biosynthesis of spectinomycin: heterologous production of spectinomycin and spectinamine in an aminoglycoside-deficient host, Streptomyces venezuelae YJ003

Aims:  To obtain spectinomycin and spectinamine by heterologous expression into the biosynthetic deoxysugar (desosamine) gene-deleted host Streptomyces venezuelae YJ003.
Methods and Results:  The 17-kb spectinomycin biosynthetic gene cluster from Streptomyces spectabilis ATCC 27741 was heterologously expressed into Streptomyces venezuelae YJ003. Furthermore, the speA , speB and spcS2 encoded in the spectinomycin biosynthetic gene cluster of cosmid pSPC8 were also heterologously characterized to be responsible for the production of spectinamine.
Conclusions:  The results of this study indicated that pSPC8 contains all the genes necessary for the biosynthesis of spectinomycin. We also concluded that SpeA, SpeB and SpcS2 are sufficient for the biosynthesis of spectinamine. We also verified that SpeB and SpcS2 show dual character in the biosynthetic pathway of spectinomycin in Streptomyces spectabilis .
Significance and Impact of the Study:  This is the report regarding the expression of a biosynthetic gene cluster that gives rise to the production of aminoglycoside antibiotics in Streptomyces venezuelae YJ003. Therefore, this work may serve as a foundation for further research on spectinomycin biosynthesis and other aminoglycosides.     

Effects of stent porosity on hemodynamics in a sidewall aneurysm model

Computation and experiment have been complementarily performed to study the fluid flow inside a stented lateral aneurysm anchored on the straight parent vessel. The implicit solver was based on the time-dependent incompressible Navier-Stokes equations of laminar flow. Solutions were generated by a cell-center finite-volume method that used second-order upwind and second-order center flux difference splitting for the convection and diffusion term, respectively. The second-order Crank-Nicolson method was used in the time integration term. Experimental techniques used were flow visualization (FV) and particle tracking velocimetry (PTV). Experimentally, the straight afferent vessel had an inner diameter 10mm. The diameters of the aneurysmal orifice, neck, and fundus were 14, 10, and 15 mm, respectively, and the distance between the orifice and dome measured 20mm. A 30 mm long helix-shaped stent was tested. Four stent porosities of 100%, 70%, 50%, and 25% were examined. Volume-flow rate waveform of a cerebral artery was considered with a maximum Reynolds number of 250 and Womersley number of 3.9. Results are presented in terms of the pulsatile main and secondary flow velocity vector fields, the volume inflow rates into the aneurysm, and the wall shear stress (WSS) and wall pressure at the aneurysm dome. Some comparisons of computed results with the present FV and PTV results and with the data available from the literature are also made. The maximum flow velocity inside the aneurysm ostium and the WSS in the dome region at the peak flow can, respectively, be suppressed to less than 5% of the parent vessel bulk velocity (or 20% of the unstented case) and 8% of the unstented case if the stent porosity is smaller than 40% (about the porosity of the two-layer stents). In general, the three-layer stents seem not as effective as the two-layer stents in reducing the magnitude of aneurysm inflow rate and WSS.     

Role of ERK signaling in the neuroprotective efficacy of magnesium sulfate treatment during focal cerebral ischemia in the gerbil cortex

Magnesium sulfate (MgSO4) ameliorates focal ischemia-induced neuronal death in the rat and gerbil models. However, the molecular mechanisms for this neuroprotection are not known. Focal cerebral ischemia was produced by unilateral occlusion of the right common carotid artery and the right middle cerebral artery (CCAO + MCAO) for 30 min or 60 min. Treatment with MgSO4 significantly increased the level of mitogen-activated protein kinase/extra-cellular signal-regulated kinase kinase 1/2 (MEK1/2), extra-cellular signal-regulated kinase 1/2 (ERK1/2), cyclic-AMP response element binding protein (CREB) phosphorylation and the anti-apoptotic protein Bcl-2 both in the non-ischemic (contralateral) and ischemic (ipsilateral) cortex. However, these effects were reversed by administration of U0126, a MEK kinase inhibitor. In the ipsilateral cortex, a significant increase in the level of the proapoptotic proteins Bax, Bad, BNIP3 and activated caspase 3 were detected at the end of focal ischemia compared to the non-ischemic cortex. Treatment of MgSO4 prevented these ischemia-induced activations of the death cascade. Collectively, these data indicate that the ERK-CREB-Bcl-2 signaling pathway might be involved in MgSO4-induced neuroprotection following focal ischemia. Moreover, MgSO4 treatment also resulted in a reduction in pro-apoptotic proteins. These results enhance our understanding on the role of MgSO4 in treating cerebral ischemia.