Angular variances for internal bond rotations of side chains in GXG-based tripeptides derived from (13)C-NMR relaxation measurements: Implications to protein folding

The study of backbone and side-chain internal motions in proteins and peptides is crucial to having a better understanding of protein/peptide "structure" and to characterizing unfolded and partially folded states of proteins and peptides. To achieve this, however, requires establishing a baseline for internal motions and motional restrictions for all residues in the fully, solvent-exposed "unfolded state." GXG-based tripeptides are the simpliest peptides where residue X is fully solvent exposed in the context of an actual peptide. In this study, a series of GXG-based tripeptides has been synthesized with X being varied to include all twenty common amino acid residues. Proton-coupled and -decoupled (13)C-nmr relaxation measurements have been performed on these twenty tripeptides and various motional models (Lipari-Szabo model free approach, rotational anisotropic diffusion, rotational fluctuations within a potential well, rotational jump model) have been used to analyze relaxation data for derivation of angular variances and motional correlation times for backbone and side-chain chi(1) and chi(2) bonds and methyl group rotations. At 298 K, backbone motional correlation times range from about 50 to 85 ps, whereas side-chain motional correlation times show a much broader spread from about 18 to 80 ps. Angular variances for backbone phi,psi bond rotations range from 11 degrees to 23 degrees and those for side chains vary from 5 degrees to 24 degrees for chi(1) bond rotations and from 5 degrees to 27 degrees for chi(2) bond rotations. Even in these peptide models of the "unfolded state," side-chain angular variances can be as restricted as those for backbone and beta-branched (valine, threonine, and isoleucine) and aromatic side chains display the most restricted motions probably due to steric hinderence with backbone atoms. Comparison with motional data on residues in partially folded, beta-sheet-forming peptides indicates that side-chain motions of at least hydrophobic residues are less restricted in the partially folded state, suggesting that an increase in side-chain conformational entropy may help drive early-stage protein folding. Copyright 1999 John Wiley & Sons, Inc.     

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.     

Is an elongated stylohyoid process prevalent in the elderly? A radiographic study in a Brazilian population

Background: Elongation and calcification of the stylohyoid ligament complex may be correlated with Eagle's syndrome. The styloid complex pathogenesis is still being debated. Objective: The aim of this study was to investigate the prevalence of stylohyoid ligament complex elongation in panoramic radiographs of 2252 patients in a Brazilian adult, partially edentulous population, of both sexes. Methods: The radiographs, taken in the Semiology Department at the Dental School in Brazil, were randomly selected from January 1997 to December 2000. The stylohyoid ligament complexes were measured from the cranial base up to the osseous tip of each process. Mineralisation of the complex of more than 25 mm in length on the radiograph was considered to be abnormal. The same operator made all the measurements. The lengths of the areas of mineralisation were recorded, and whether the condition was bilateral was noted. Results: This abnormality was present in both sexes. A calcified complex was found in 451 of the 2252 patients. The majority of these calcified complexes (n = 248; 54.9%) were bilateral. Three hundred and fifty‐six (39.5%) of them were longer than 25 mm (and so were abnormal), and the length varied from 26.1 to 65 mm. (average length = 27.8 mm). Forty (4.4%) of the abnormalities were longer than 50 mm, and of these 36 (90%) were in the 40–59 year age group. The mean length of a mineralised stylohyoid ligament complex in patients in the 60–79 year age group was 32.75 mm. Conclusion: The results suggested that an anatomical variant of the stylohyoid ligament complex was more frequently found in the elderly female population, although this abnormality was present in both sexes. There was a greater tendency for the abnormality to be present in patients between 60 and 79 years of age.     

Biochemical changes during the development of witches' broom: the most important disease of cocoa in Brazil caused by Crinipellis perniciosa

Witches' broom disease (WBD) is caused by the hemibiotrophic basidiomycete fungus Crinipellis perniciosa, which is one of the most important diseases of cocoa in the western hemisphere. In this study, the contents of soluble sugars, amino acids, alkaloids, ethylene, phenolics, tannins, flavonoids, pigments, malondialdehyde (MDA), glycerol, and fatty acids were analysed in cocoa (Theobroma cacao) shoots during the infection and development of WBD. Alterations were observed in the content of soluble sugars (sucrose, glucose, and fructose), asparagine and alkaloids (caffeine and theobromine), ethylene, and tannins. Ethylene and tannins increased prior to symptom development and declined with the death of the infected tissues. Furthermore, MDA and glycerol concentrations were higher in infected tissue than in the controls, while fatty acid composition changed in the infected tissues. Chlorophylls a and b were lower throughout the development of the disease while carotenoids and xanthophylls dropped in the infected tissue by the time of symptom development. These results show co-ordinated biochemical alterations in the infected tissues, indicating major stress responses with the production of ethylene. Ethylene levels are hypothesized to play a key role in broom development. Some of the other biochemical alterations are directly associated with ethylene synthesis and may be important for the modification of its effect on the infected tissues.     

The P. falciparum CSP repeat region contains three distinct epitopes required for protection by antibodies in vivo

Rare and potent monoclonal antibodies (mAbs) against the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) on infective sporozoites (SPZ) preferentially bind the PfCSP junctional tetrapeptide NPDP or NVDP minor repeats while cross-reacting with NANP central repeats in vitro. The extent to which each of these epitopes is required for protection in vivo is unknown. Here, we assessed whether junction-, minor repeat- and central repeat-preferring human mAbs (CIS43, L9 and 317 respectively) bound and protected against in vivo challenge with transgenic P. berghei (Pb) SPZ expressing either PfCSP with the junction and minor repeats knocked out (KO), or PbCSP with the junction and minor repeats knocked in (KI). In vivo protection studies showed that the junction and minor repeats are necessary and sufficient for CIS43 and L9 to neutralize KO and KI SPZ, respectively. In contrast, 317 required major repeats for in vivo protection. These data establish that human mAbs can prevent malaria infection by targeting three different protective epitopes (NPDP, NVDP, NANP) in the PfCSP repeat region. This report will inform vaccine development and the use of mAbs to passively prevent malaria.