Evidence of a Recessively Inherited CCN3 Mutation as a Rare Cause of Early-Onset Parkinsonism.

The study of consanguineous families has provided novel insights into genetic causes of monogenic parkinsonism. Here, we present a family from the rural Khyber Pakhtunkhwa province, Pakistan, where three siblings were diagnosed with early-onset parkinsonism. Homozygosity mapping of two affected siblings and three unaffected family members identified two candidate autozygous loci segregating with disease, 8q24.12-8q24.13 and 9q31.2-q33.1. Whole-exome sequence analysis identified a single rare homozygous missense sequence variant within this region, CCN3 p.D82G. Although unaffected family members were heterozygous for this putative causal mutation, it was absent in 3,222 non-Parkinson's disease (PD) subjects of Pakistani heritage. Screening of 353 Australian PD cases, including 104 early-onset cases and 57 probands from multi-incident families, also did not identify additional carriers. Overexpression of wild-type and the variant CCN3 constructs in HEK293T cells identified an impaired section of the variant protein, alluding to potential mechanisms for disease. Further, qPCR analysis complemented previous microarray data suggesting mRNA expression of CCN3 was downregulated in unrelated sporadic PD cases when compared to unaffected subjects. These data indicate a role for CCN3 in parkinsonism, both in this family as well as sporadic PD cases; however, the specific mechanisms require further investigation. Additionally, further screening of the rural community where the family resided is warranted to assess the local frequency of the variant. Overall, this study highlights the value of investigating underrepresented and isolated affected families for novel putative parkinsonism genes.

Biotechnological production of hyaluronic acid: a mini review.

Hyaluronic acid (HA) is a polysaccharide found in the extracellular matrix of vertebrate epithelial, neural and connective tissues. Due to the high moisture retention, biocompatibility and viscoelasticity properties of this polymer, HA has become an important component of major pharmaceutical, biomedical and cosmetic products with high commercial value worldwide. Currently, large scale production of HA involves extraction from animal tissues as well as the use of bacterial expression systems in Streptococci. However, due to concerns over safety, alternative sources of HA have been pursued which include the use of endotoxin-free microorganisms such as Bacilli and Escherichia coli. In this review, we explore current knowledge of biosynthetic enzymes that produce HA, how these systems have been used commercially to produce HA and how the challenges of producing HA cheaply and safely are being addressed.

Structure determination of human semaphorin 4D as an example of the use of MAD in non-optimal cases.

Semaphorins are an important class of signalling molecules involved in axon guidance, immune function and angiogenesis. They are characterized by having an extracellular sema domain of about 500 residues. The steps involved in the determination of the structure of human semaphorin 4D are described here as a case study of selenium MAD phasing in a difficult case with low symmetry, moderate diffraction and low selenium content. A particular feature of this study was the large number of diffraction images required to give data of sufficient quality for structure determination and these data are re-analyzed here to investigate the effects of radiation damage on eventual data quality and to suggest strategies for successful MAD phasing in similar difficult cases.

The sema domain.

The sema domain was first defined from sequence by Kolodkin and colleagues in the early 1990s, and constitutes the distinctive structural and functional element of semaphorins, their plexin receptors and the receptor tyrosine kinases MET and RON, three protein families with major roles in development, tissue regeneration and cancer. Recently determined crystal structures of two semaphorins (SEMA3A and SEMA4D) and the MET receptor have shown that the sema domain consists of a highly conserved variant form of the seven-blade beta-propeller fold. The structures, however, also suggest differences between these families with respect to the mode of dimerisation and the regions of the domain involved in ligand-receptor interactions. This reflects the considerable plasticity and adaptation of the sema domain in order to meet different binding requirements, properties that may underlie the vast array of ligand-receptor specificities and functions of the semaphorin superfamily.

The ligand-binding face of the semaphorins revealed by the high-resolution crystal structure of SEMA4D.

Semaphorins, proteins characterized by an extracellular sema domain, regulate axon guidance, immune function and angiogenesis. The crystal structure of SEMA4D (residues 1-657) shows the sema topology to be a seven-bladed beta-propeller, revealing an unexpected homology with integrins. The sema beta-propeller contains a distinctive 77-residue insertion between beta-strands C and D of blade 5. Blade 7 is followed by a domain common to plexins, semaphorins and integrins (PSI domain), which forms a compact cysteine knot abutting the side of the propeller, and an Ig-like domain. The top face of the beta-propeller presents prominent loops characteristic of semaphorins. In addition to limited contact between the Ig-like domains, the homodimer is stabilized through extensive interactions between the top faces in a sector of the beta-propeller used for heterodimerization in integrins. This face of the propeller also mediates ligand binding in integrins, and functional data for semaphorin-receptor interactions map to the equivalent surface.