A new genus, Perplexacara, and new generic placements of species of Australian marsh beetles (Coleoptera: Scirtidae) based on morphology and molecular genetic data.

The Australian Scirtidae species previously identified as misplaced in the widespread genus Prionocyphon Redtenbacher are revisited as well as their possible relationship with the Australian genus Macrodascillus (Lea) using sequence data from the mitochondrial gene, cytochrome oxidase subunit 1 and two nuclear genes, elongation factor 1-alpha and Topoisomerase. The study confirmed the conclusion of Cooper et al. (2014) that the species did not belong in Prionocyphon. The study also included a species from each of three possibly related genera, Chameloscyphon Watts, Daploeuros Watts and Dasyscyphon Watts. Chameloscyphon huonensis Watts, Dasyscyphon victoriaensis Watts and Daploeuros lamingtonensis Watts were recovered as separate lineages with C. huonensis linking with Das. victoriaensis and Dap. lamingtonensis isolated. The species previously included in Prionocyphon were shown to belong in two genera, Macrodascillus and a new genus Perplexacara: Perplexacara caementum (Watts) new combination, P. latusmandibulara (Watts) new combination, P. macroflavida (Watts) new combination, Macrodascillus scalaris (Lea), M. insolitus (Watts) new combination and M. lamingtonensis (Watts) new combination.

Intragenomic internal transcribed spacer 2 variation in a genus of parasitoid wasps (Hymenoptera: Braconidae): implications for accurate species delimitation and phylogenetic analysis.

A recent DNA barcoding study of Australian microgastrines (Hymenoptera: Braconidae) sought to use next-generation sequencing of the cytochrome c oxidase subunit 1 (COI) barcoding gene region, the wingless (WG) gene and the internal transcribed spacer 2 (ITS2) to delimit molecular species in a highly diverse group of parasitic wasps. Large intragenomic distances between ITS2 variants, often larger than the average interspecific variation, caused difficulties in using ITS2 for species delimitation in both threshold and tree-based approaches, and the gene was not included in the reported results of the previous DNA barcoding study. We here report on the intragenomic, and the intra- and interspecies, variation in ITS2in the microgastrine genus Diolcogasterto further investigate the value of ITS2as a marker for species delimitation and phylogenetics of the Microgastrinae. Distinctive intragenomic variant patterns were found in different species of Diolcogaster, with some species possessing a single major variant, and others possessing many divergent variants. Characterizing intragenomic variation of ITS2is critical as it is a widely used marker in hymenopteran phylogenetics and species delimitation, and large intragenomic distances such as those found in this study may obscure phylogenetic signal.

Patterns of population genetic variation in sympatric chiltoniid amphipods within a calcrete aquifer reveal a dynamic subterranean environment.

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km(2) region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.

Processing of normal lysosomal and mutant N-acetylgalactosamine 4-sulphatase: BiP (immunoglobulin heavy-chain binding protein) may interact with critical protein contact sites.

The lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase) is essential for the sequential degradation of the glycosaminoglycans, dermatan and chondroitin sulphate and, when deficient, causes the lysosomal storage disorder mucopolysaccharidosis type VI. The cysteine at codon 91 of human 4-sulphatase was identified previously as a key residue in the active site of the enzyme and was mutated by site-directed mutagenesis to produce a 4-sulphatase in which cysteine-91 was replaced by a threonine residue (C91T). The C91T mutation caused a loss of 4-sulphatase activity, a detectable protein conformational change and a lower level of intracellular 4-sulphatase protein [Brooks, Robertson, Bindloss, Litjens, Anson, Peters, Morris and Hopwood (1995) Biochem. J. 307, 457-463]. In the present study, we report that C91T is synthesized normally in the endoplasmic reticulum as a 66 kDa glycosylated protein, which is very similar in size to wild-type 4-sulphatase. However, C91T neither underwent normal Golgi processing, shown by lack of modification to form mannose 6-phosphate residues on its oligosaccharide side chains, nor did it traffic to the lysosome to undergo normal endosomal-lysosomal proteolytic processing. Instead, C91T remained in an early biosynthetic compartment and was degraded. The molecular chaperone, immunoglobulin binding protein (BiP), was associated with newly-synthesized wild-type and mutant 4-sulphatase proteins for extended periods, but no direct evidence was found for involvement of BiP in the retention or degradation of the C91T protein. This suggested that prolonged association of mutant protein with BiP does not necessarily infer involvement of BiP in the quality control process, as previously implied in the literature. The predicted BiP binding sites on 4-sulphatase map to beta-strands and alpha-helices, which are co-ordinated together in the folded molecule, indicating that BiP interacts with critical protein folding or contact sites on 4-sulphatase.

A membrane protein primarily associated with the lysosomal compartment.

A monoclonal antibody designated MBR 39 has been generated against a membrane associated protein found selectively on lysosomes. MBR 39 reacts with the cytosolic face of the lysosome and was used to develop an organelle binding assay which reacted with high density organelles characteristic of lysosomes. These organelles contained lysosomal enzyme markers which included the integral membrane protein acetyl-CoA:alpha-glucosaminide N-acetyltransferase and the soluble lysosomal enzyme markers acid phosphatase (mature form), beta-hexosaminidase, arylsulfatase, and alpha-L-iduronidase. Under conditions which disrupt lysosomes the release of the latter soluble lysosomal enzymes was demonstrated from MBR 39 bound organelles. Immunoblots of MBR 39 with purified fibroblast lysosomal membrane, demonstrated reactivity with polypeptides of molecular mass 63 kDa (major species) and 73 kDa (minor species).

Juvenile form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). A C-terminal extension causes instability but increases catalytic efficiency of arylsulfatase B.

A deficiency of the enzyme arylsulfatase B results in the lysosomal storage disorder Maroteaux-Lamy syndrome or mucopolysaccharidosis type VI. Severe, intermediate and mild forms of this autosomal recessively inherited disease can be clinically differentiated. To determine the molecular defect in a patient with the intermediate form of the disorder, DNA fragments generated from the patient's mRNA by reverse transcription and subsequent amplification by the polymerase chain reaction were subcloned and sequenced. The mRNA transcribed from one allele contains a 244-base pair deletion causing a frameshift and a truncation of the open reading frame. The C-terminal third of the encoded mutant polypeptide has a nonsense sequence. This mutation is due to a deletion of exon 5 in this allele. A silent A to G transition at nucleotide 1191 was present in the same allele, and the second allele was characterized by a T to C transition at nucleotide 1600 causing a mutation of the translational stop codon to a glutamine codon (*534Q) and extending the encoded polypeptide by 50 amino acids. Stable expression of the *534Q allele in LTK- cells resulted in a mutant precursor 4 kDa larger than the wild-type precursor. The majority of the mutant precursor appears to be degraded before reaching the trans Golgi. This is consistent with an altered polypeptide structure, where a number of missing or masked epitopes were observed in an enzyme immunobinding assay using a panel of monoclonal antibodies. Immunoquantification analysis showed that epitopes were most likely masked, as missing epitopes could be reformed by binding the mutant protein to a polyclonal antibody of arylsulfatase B. It is suggested that the additional amino acids at the C terminus of the arylsulfatase B polypeptide induce a protein conformational change. *534Q mutant polypeptide escaping degradation is sorted to dense lysosomes. The mutant polypeptide has an approximately 9-fold higher catalytic efficiency than wild-type arylsulfatase B.

An improved method for the purification of IgG monoclonal antibodies from culture supernatants.

A method for the purification of mouse monoclonal antibodies from hybridoma culture supernatants is described. The protocol involves the use of a combination of three previously described methods for the concentration and purification of monoclonal antibodies. Firstly, hybridomas were grown in a Diacult dialysis system (Inter Med Laboratory, Denmark) to yield milligram quantities of monoclonal antibody in a culture supernatant. Monoclonal antibodies were then purified from the culture supernatant by precipitation with polyethylene glycol 6000 (PEG 6000) and finally reprecipitated using an ammonium sulphate procedure. The PEG 6000 treatment caused a density change in the ammonium sulphate immunoglobulin precipitate, and resulted in the formation of a pellicle which contained pure mouse monoclonal antibody. The protocol removed contaminating bovine serum immunoglobulin as well as other serum and cellular protein from the monoclonal antibody preparations.