Protein folding determinants: structural features determining alternative disulfide pairing in alpha- and chi/lambda-conotoxins

Alpha-conotoxins isolated from Conus venoms contain 11-19 residues and preferentially fold into the globular conformation that possesses a specific disulfide pairing pattern (C1-3, C2-4). We and others isolated a new family of chi-conotoxins (also called lambda conotoxins) with the conserved cysteine framework of alpha-conotoxins but with alternative disulfide pairing (C1-4, C2-3) resulting in the ribbon conformation. In both families, disulfide pairing and hence folding are important for their biological potency. By comparing the structural differences, we identified potential structural determinants responsible for the folding tendencies of these conotoxins. We examined the role of conserved proline in the first intercysteine loop and the conserved C-terminal amide on folding patterns of synthetic analogues of ImI conotoxin by comparing the isoforms with the regiospecifically synthesized conformers. Deamidation at the C-terminus and substitution of proline in the first intercysteine loop switch the folding pattern from the globular form of alpha-conotoxins to the ribbon form of chi/lambda-conotoxins. The findings are corroborated by reciprocal folding of CMrVIA chi/lambda-conotoxins. Substitution of Lys-6 from the first intercysteine loop of CMrVIA conotoxin with proline, as well as the inclusion of an amidated C-terminal shifted the folding preference of CMrVIA conotoxin from its native ribbon conformation toward the globular conformation. Binding assays of ImI conotoxin analogues with Aplysia and Bulinus acetylcholine binding protein indicate that both these substitutions and their consequent conformational change substantially impact the binding affinity of ImI conotoxin. These results strongly indicate that the first intercysteine loop proline and C-terminal amidation act as conformational switches in alpha- and chi/lambda-conotoxins.     

Accelerated exchange of exon segments in Viperid three-finger toxin genes (<Emphasis Type="Italic">Sistrurus catenatus edwardsii</Emphasis>; Desert Massasauga)

Background  

Snake venoms consist primarily of proteins and peptides showing a myriad of potent biological activities which have been shaped by both adaptive and neutral selective forces. Venom proteins are encoded by multigene families that have evolved through a process of gene duplication followed by accelerated evolution in the protein coding region.     

Synthesis of all stereoisomers of KRN7000, the CD1d-binding NKT cell ligand

KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release Th1 and Th2 cytokines. In an effort to understand the structure–activity relationships, we have carried out the synthesis of a complete set of the eight KRN7000 stereoisomers, and their biological activities have been examined.     

In vitro embryo development and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes recovered from slaughterhouse ovaries or live animals by ovum pick-up

The present study was undertaken to determine whether the source of oocytes (ovum pick up versus slaughterhouse ovaries) affected in vitro embryo production and embryo survival (as measured by blastocyst hatching rates) following vitrification in buffaloes (Bubalus bubalis). Oocytes recovered from live buffaloes (n=6) by ovum pick up (OPU) and by manual aspiration from slaughterhouse ovaries were in vitro matured, fertilized and cultured to blastocyst stage under same culture conditions. Vitrification of blastocysts was carried out in two steps at 24 degrees C. Embryos were equilibrated in 10% EG+10% DMSO+0.3 M sucrose in base medium for 4 min. Subsequently, the embryos were transferred into 25% EG+25% DMSO+0.3 M sucrose in base medium for 45 s and then the embryos were loaded into straws and immersed in liquid nitrogen. Following warming, blastocysts were cultured in vitro for 48 h to assess hatching. Oocytes derived from live animals by OPU resulted in a significantly higher blastocyst yield then those derived from slaughterhouse ovaries (30.6+/-4.3 versus 18.5+/-1.8). Blastocyst hatching rates following vitrification of buffalo embryos produced from the oocytes collected from live animals by OPU was significantly higher than the oocytes collected from slaughterhouse ovaries (52.8+/-4.2 versus 40.2+/-4.4). In conclusion, the present study showed that source of oocytes (OPU versus slaughterhouse ovaries) affects the in vitro embryo development and blastocyst hatching rates following vitrification of embryos in buffaloes.     

Role of accelerated segment switch in exons to alter targeting (ASSET) in the molecular evolution of snake venom proteins

Background  

Snake venom toxins evolve more rapidly than other proteins through accelerated changes in the protein coding regions. Previously we have shown that accelerated segment switch in exons to alter targeting (ASSET) might play an important role in its functional evolution of viperid three-finger toxins. In this phenomenon, short sequences in exons are radically changed to unrelated sequences and hence affect the folding and functional properties of the toxins.     

The emerging roles of nitric oxide (NO) in plant mitochondria

In recent years nitric oxide (NO) has been recognized as an important signal molecule in plants. Both, reductive and oxidative pathways and different subcellular compartments appear involved in NO production. The reductive pathway uses nitrite as substrate, which is exclusively generated by cytosolic nitrate reductase (NR) and can be converted to NO by the same enzyme. The mitochondrial electron transport chain is another site for nitrite to NO reduction, operating specifically when the normal electron acceptor, O₂, is low or absent. Under these conditions, the mitochondrial NO production contributes to hypoxic survival by maintaining a minimal ATP formation. In contrast, excessive NO production and concomitant nitrosative stress may be prevented by the operation of NO-scavenging mechanisms in mitochondria and cytosol. During pathogen attacks, mitochondrial NO serves as a nitrosylating agent promoting cell death; whereas in symbiotic interactions as in root nodules, the turnover of mitochondrial NO helps in improving the energy status similarly as under hypoxia/anoxia. The contribution of NO turnover during pathogen defense, symbiosis and hypoxic stress is discussed in detail.     

Isolation,expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic? Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.     

Full Genome Characterization of the Culicoides-Borne Marsupial Orbiviruses: Wallal Virus,Mudjinbarry Virus and Warrego Viruses

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell ‘T2’ and core-surface ‘T13’ proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.