Nmr relaxation studies on the hydrate layer of intrinsically unstructured proteins

Intrinsically unstructured/ disordered proteins (IUPs) exist in a disordered and largely solvent- exposed, still functional, structural state under physiological conditions. As their function is often directly linked with structural disorder, understanding their structure-function relationship in detail is a great challenge to structural biology. In particular, their hydration and residual structure, both closely linked with their mechanism of action, require close attention. Here we demonstrate that the hydration of IUPs can be adequately approached by a technique so far unexplored with respect to IUPs, solid-state NMR relaxation measurements. This technique provides quantitative information on various features of hydrate water bound to these proteins. By freezing nonhydrate ( bulk) water out, we have been able to measure free induction decays pertaining to protons of bound water from which the amount of hydrate water, its activation energy, and correlation times could be calculated. Thus, for three IUPs, the first inhibitory domain of calpastatin, microtubule-associated protein 2c, and plant dehydrin early responsive to dehydration 10, we demonstrate that they bind a significantly larger amount of water than globular proteins, whereas their suboptimal hydration and relaxation parameters are correlated with their differing modes of function. The theoretical treatment and experimental approach presented in this article may have general utility in characterizing proteins that belong to this novel structural class

Three-dimensional dynamic morphology of the mitral valve in different forms of mitral valve prolapse - potential implications for annuloplasty ring selection.

BACKGROUND: Real-time three-dimensional transesophageal echocardiography has increased our understanding of the distinct pathomechanisms underlying functional, ischaemic or degenerative mitral regurgitation. However, potential differences in dynamic morphology between the subtypes of degenerative mitral prolapse have scarcely been investigated. METHODS: In order to compare the dynamic behavior of the different phenotypes of degenerative mitral valve prolapse, real-time three-dimensional transesophageal echocardiography recordings of 77 subjects, 27 with Barlow disease (BD), 32 with Fibroelastic deficiency (FED) and 18 normal controls (NC) were analysed. RESULTS: Geometric annular and valvular parameters of the myxomatous patients were significantly larger compared to controls (BD vs. FED vs. NC 3D annular area: 15 +/- 2.8 vs. 13.3 +/- 2.4 vs. 10.6 +/- 2.3cm(2), all p < 0.01). Beside similar ellipticity, BD annuli were significantly flatter compared to FED. Myxomatous annuli appeared less dynamic than normals, with decreased overall 3D area change, however only the BD group differed from NC significantly (BD vs. FED vs. NC normalized 3D area change 4.40 vs. 6.81 vs. 9.69 %; BD vs. NC p = 0.000; FED vs. NC p = not significant, BD vs. FED p = 0.025). CONCLUSION: BD and FED differ not only in terms of valve morphology, but also annular dynamics. Both pathologies are characterized by annular dilatation. However, in BD the annulus is remarkably flattened and hypodynamic, whereas in FED its saddle-shape and contractile function is relatively preserved. These features might influence the choice of repair technique and the selection of annuloplasty ring

CYP24A1 inhibition facilitates the anti-tumor effect of vitamin D3 on colorectal cancer cells

AIM: The effects of vitamin D3 have been investigated on various tumors, including colorectal cancer (CRC). 25-hydroxyvitamin-D3-24-hydroxylase (CYP24A1), the enzyme that inactivates the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25-D3), is considered to be the main enzyme determining the biological halflife of 1,25-D3. During colorectal carcinogenesis, the expression and concentration of CYP24A1 increases significantly, suggesting that this phenomenon could be responsible for the proposed efficacy of 1,25-D3 in the treatment of CRC. The aim of this study was to investigate the anti-tumor effects of vitamin D3 on the human CRC cell line Caco-2 after inhibition of the cytochrome P450 component of CYP24A1 activity. METHODS: We examined the expression of CYP24A1 mRNA and the effects of 1,25-D3 on the cell line Caco-2 after inhibition of CYP24A1. Cell viability and proliferation were determined by means of sulforhodamine-B staining and bromodeoxyuridine incorporation, respectively, while cytotoxicity was estimated via the lactate dehydrogenase content of the cell culture supernatant. CYP24A1 expression was measured by realtime reverse transcription polymerase chain reaction. A number of tetralone compounds were synthesized to investigate their CP24A1 inhibitory activity. RESULTS: In response to 1,25-D3, CYP24A1 mRNA expression was enhanced significantly, in a time- and dose-dependent manner. Caco-2 cell viability and proliferation were not influenced by the administration of 1,25-D3 alone, but were markedly reduced by coadministration of 1,25-D3 and KD-35, a CYP24A1-inhibiting tetralone. Our data suggest that the mechanism of action of co-administered KD-35 and 1,25-D3 does not involve a direct cytotoxic effect, but rather the inhibition of cell proliferation. CONCLUSION: These findings demonstrate that the selective inhibition of CYP24A1 by compounds such as KD-35 may be a new approach for enhancement of the anti-tumor effect of 1,25-D3 on CRC

Crossover of texture and morphology in (Ti1 − xAlx)1 − yYyN alloy films and the pathway of structure evolution

In our earlier published work, we have shown that there is a composition range of the (Ti 1 − x Al x ) 1 − y Y y N alloy films (0.72 b Ti/Al b 0.88) deposited at oblique vapour beam incidence and 500 °C (corresponding to zone T) in which mixed cubic TiN (c-TiN) and wurtzite AlN (w-AlN) structures were formed together with an unusual complex texture. The texture of c-TiN phase changed from 〈001〉 to b111N at a certain thickness forming a definite crossover. Moreover the c-TiNb111N and the w-AlN〈0001〉 crystals were epitaxially related with axes tilted to the direction of the vapour beam. Based on a comprehensive transmission electron microscopy (TEM) and diffraction (XRD and selected area electron diffraction (SAED)) structure and morphology analysis, we discovered the details of this exotic structure making it possible to construct the complex pathway of structure evolution including the formation of the w-AlN phase and the change of the dominating texture of c-TiN phase with thickness in dependence of the Ti/Al ratio and the deposition parameters. This pathway could be deduced from the fundamental phenomena of structure formation and may be generalised for multi-component thin film systems. A composition structure zone model has been also proposed for the (Ti 1 − x Al x ) 1 − y Y y N thin film system in the 0 b x b 1 composition range

Crossover of texture and morphology in (Ti 1 − x Al x ) 1 − y Y y N alloy films and the pathway of structure evolution

In our earlier published work, we have shown that there is a composition range of the (Ti 1 − x Al x ) 1 − y Y y N alloy films (0.72 b Ti/Al b 0.88) deposited at oblique vapour beam incidence and 500 °C (corresponding to zone T) in which mixed cubic TiN (c-TiN) and wurtzite AlN (w-AlN) structures were formed together with an unusual complex texture. The texture of c-TiN phase changed from 〈001〉 to b111N at a certain thickness forming a definite crossover. Moreover the c-TiNb111N and the w-AlN〈0001〉 crystals were epitaxially related with axes tilted to the direction of the vapour beam. Based on a comprehensive transmission electron microscopy (TEM) and diffraction (XRD and selected area electron diffraction (SAED)) structure and morphology analysis, we discovered the details of this exotic structure making it possible to construct the complex pathway of structure evolution including the formation of the w-AlN phase and the change of the dominating texture of c-TiN phase with thickness in dependence of the Ti/Al ratio and the deposition parameters. This pathway could be deduced from the fundamental phenomena of structure formation and may be generalised for multi-component thin film systems. A composition structure zone model has been also proposed for the (Ti 1 − x Al x ) 1 − y Y y N thin film system in the 0 b x b 1 composition range