Tessa N. Campbell

Gaucher Disease and Cancer: Concept and Controversy

Gaucher disease is an inherited disorder caused by a deficiency in the lysosomal hydrolase glucocerebrosidase. There is a wide spectrum of clinical presentations, with the most common features being hepatosplenomegaly, skeletal disease, and cytopenia. Gaucher disease has been classified into three broad phenotypes based upon the presence or absence of neurological involvement: Type 1 (nonneuronopathic), Type 2 (acute neuronopathic), and Type 3 (subacute neuronopathic). The two main treatment options include enzyme replacement therapy and substrate reduction therapy. Recently, discussion has escalated around the association of Gaucher disease and cancer, with conflicting reports as to whether Gaucher patients have an increased risk of malignancy. In this review, we present both the concept and controversy surrounding the association of Gaucher disease with cancer.

Distinct membrane compartmentalization and signaling of ephrin-A5 and ephrin-B1.

Eph receptor tyrosine kinases and their membrane-bound ligand ephrins form an essential cell communication system. Both ephrin classes have been shown to localize within cell surface lipid rafts, yet regulate different biological processes. In order to provide insight into this distinct behavior, we examined ephrin-A5 and B1 localization and signaling in murine fibroblasts and tissues. Results indicated that ephrin-A5 was constitutively present in detergent-resistant membrane fractions, while ephrin-B1 displayed translocation to membrane fractions upon stimulation. Ephrin-A5 and B1 were present in detergent-resistant membrane fractions with different buoyancies in vitro and in different raft fractions in vivo. Moreover, ephrin-A5 and B1 differentially influenced actin reorganization. Finally, microarray analysis revealed unique patterns of gene expression between the two ephrin classes. We thus demonstrate that distinct localization and compartmentalization provide insight into the subcellular basis for differential signaling observed in ephrin-A and B classes.

Gaucher disease and the synucleinopathies: refining the relationship

Gaucher disease (OMIM 230800, 230900, 231000), the most common lysosomal storage disorder, is due to a deficiency in the enzyme glucocerebrosidase. Gaucher patients display a wide spectrum of clinical presentation, with hepatosplenomegaly, haematological changes, and orthopaedic complications being the predominant symptoms. Gaucher disease is classified into three broad phenotypes based upon the presence or absence of neurological involvement: Type 1 (non-neuronopathic), Type 2 (acute neuronopathic), and Type 3 (subacute neuronopathic). Nearly 300 mutations have been identified in Gaucher patients, with the majority being missense mutations. Though studies of genotype-to-phenotype correlations have revealed significant heterogeneity, some consistent patterns have emerged to inform prognostic and therapeutic decisions. Recent research has highlighted a potential role for Gaucher disease in other comorbidities such as cancer and Parkinsons Disease. In this review, we will examine the potential relationship between Gaucher disease and the synucleinopathies, a group of neurodegenerative disorders characterized by the development of intracellular aggregates of α-synuclein. Possible mechanisms of interaction will be discussed.

Expression of two green fluorescent protein variants in citrate-buffered media in Pichia pastoris.

The Aequorea victoria green fluorescent protein (GFP) has become one of the most frequently employed molecular reporters. A variety of mutational methods have created a large number of GFP variants with differing pH sensitivity [1], differing spectra, and optimized expression [2]. Many of the recent mutational approaches have focused on species-specific codon optimization. First established in 1980 [3], the idea that organisms do not display a random pattern of synonymous codon usage has been validated by the large amount of recent genome sequencing projects [4]. A substantial number of studies have examined the effects of varying codon bias in heterologous protein expression systems under the assumption that disparate patterns of codon bias in the transgene and the expression host will have a significant impact on the levels of recombinant protein produced. It has been suggested that the human codon-optimized enhanced green fluorescent protein (EGFP; Clontech, Palo Alto, CA) is unsuitable for expression in yeast host systems due to such preferred codon discrepancy. However, several studies have demonstrated successful expression in yeast, mainly in Saccharomyces cerevisiae [5–7]. Therefore, we compared the expression of two GFP variants, red-shifted GFP (RSGFP from pRSGFP-C1; Clontech), and EGFP, in the methylotrophic yeast Pichia pastoris. Unlike EGFP, RSGFP is not human codon-optimized; however, like EGFP, RSGFP does have an excitation wavelength shifted toward the red end of the spectrum. Both variants also exhibit greater fluorescence intensity than wildtype GFP: EGFP fluoresces 35 times greater and RSGFP fluoresces 4–6 times greater. We chose to examine the expression of both variants in citrate-buffered rather than phosphate-buffered media to determine whether either variant could serve as a fusion partner with proteins reported to exhibit inhibition in the presence of phosphate. Glucocerebrosidase (acid b-glucosidase, EC 3.2.1.45) activity, for example, has been suggested to be inhibited by phosphate ions [8]. Based on the codon bias theory, we expected to see moderate amounts of RSGFP and minimal amounts of EGFP produced. To create the RSGFP and EGFP constructs, the variant cDNAs were PCR-amplified with the following primers: 50-TAGAATTCCCGGTCGCCACCATG-30 and50-TGTTACAGGGCCCGCGGTTCAGTCGAC-30 for RSGFP; 50-AACGGTCGAATTCATGGTGAGCA AGGG-30 and 50-TATGATCTGAATTGCCGGCCGC TTTACTT-30 for EGFP. PCR amplification was performed with an initial denaturation at 94 C for 2.5min, followed by 30 cycles of 94 C for 1min, 60 C (for RSGFP) or 56 C (for EGFP) for 1.5min, and 72 C for 1.5min. Reactions were carried out with 1ll of template (pRSGFP-C1 or pEGFP-N1 vector), 5ll of 10 PCR buffer, 5ll of 25mM MgCl2, 5ll of 2.5mM dNTPs, and 4 ll forward and reverse primers in a total volume of 50 ll. These amplified cDNAs were digested with EcoR1 and ligated into the vector pPICZaA (Invitrogen, San Diego, CA) prior to electroporation into bacterial cells. Transformantswere selected by resistance toZeocin (0.1mg/ml; Invitrogen) on low-salt Luria–Bertani plates (pH 7.5). Clones were confirmed by PCR amplification and sequenced to ensure the fidelity of the PCR and cloning steps. True positive pPICZaA–RSGFP and pPICZaA– EGFP clones were isolated, purified, and linearized at the BstX1 restriction site prior to electroporation into GS115 P. pastoris cells according to the Pichia Expression Manual protocol (Invitrogen). Transformants were Analytical Biochemistry 311 (2002) 193–195

Expression of Active α-N-Acetylglucosaminidase/TAT Chimerae in Cultured Spodoptera frugiperda Cells

We examined the production and secretion of fusion constructs containing alpha-N-acetylglucosaminidase, the enzyme deficient in Sanfilippo B, and either wildtype TAT or modified TAT in cultured Spodoptera frugiperda cells. All constructs exhibited successful expression of active enzyme, suggesting the future possibility of utilizing TAT/alpha-N-acetylglucosaminidase chimerae in enzyme replacement therapy.

Gaucher Disease and Dementia

Gaucher disease is due to a deficiency in the lysosomal hydrolase glucocerebrosidase. The disease has been further subdivided into three phenotypes based upon clinical symptoms of neurological involvement: Type 1 (non-neuronopathic), Type 2 (acute neuronopathic), and Type 3 (subacute neuronopathic). Types 2 and 3 Gaucher disease differ in both their average age of onset and rate of progression of clinical characteristics. Several disease models are currently available to study the underlying neurodegenerative mechanisms to identify key therapeutic targets. Recently, mutations in the glucocerebrosidase gene have also been identified as risk factors for two synucleinopathies, Parkinson’s disease, and dementia with Lewy bodies. Examination of the relationships among these diseases may provide new avenues for investigation and lead to the development of novel treatments for cognitive decline.