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OBJECTIVE - To determine whether mutations in different Bardet-Biedl syndrome (BBS) genes result in different ocular phenotypes.
METHODS - Thirty-seven patients from 31 families were enrolled who met the clinical criteria for BBS and for whom a BBS mutation had been identified. Seventeen patients harbored mutations in BBS1, 10 in BBS10, and 10 in other genes (BBS2, BBS3, BBS5, BBS7, and BBS12). All the patients underwent ocular examination; 36 patients had computerized full-field electroretinograms (ERGs).
RESULTS - Visual acuity was significantly better in BBS1 patients than in patients with other BBS mutations (P=.01), and a larger proportion of BBS1 patients had good (≥20/50) visual acuity (P=.01). The ERG amplitudes were significantly higher in BBS1 patients than in patients with other BBS mutations in response to 0.5-Hz and 30-Hz flashes (P<.001 for both). All the BBS1 patients harbored at least 1 missense mutation compared with only 45% of patients with mutations in other BBS genes (P<.001); the rest harbored only null alleles. However, multivariate analysis demonstrated that visual acuity or ERG amplitude did not depend on the type of mutation present (missense or null) when controlling for BBS gene. Prevalences of bone spicule pigmentation and cataract were comparable in BBS subtypes.
CONCLUSIONS - Patients with BBS1 mutations had a milder phenotype than did patients with mutations in other BBS genes. Clinically, this manifested as significantly better visual acuity and larger ERG amplitudes.
CLINICAL RELEVANCE - These phenotypic differences can help guide genetic testing and genetic counseling for patients with this syndrome.
OBJECTIVE - Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here.
RESEARCH DESIGN AND METHODS - Muscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n = 8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry-based quantification using normalized spectral abundance factors.
RESULTS - Of 1,218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by ANOVA (P < 0.05). Analysis of protein sets revealed patterns of decreased abundance in mitochondrial proteins and altered abundance of proteins involved with cytoskeletal structure (desmin and alpha actinin-2 both decreased), chaperone function (TCP-1 subunits increased), and proteasome subunits (increased).
CONCLUSIONS - The results confirm the reduction in mitochondrial proteins in insulin-resistant muscle and suggest that changes in muscle structure, protein degradation, and folding also characterize insulin resistance.
A wide array of proteins in signal transduction pathways depend on Hsp90 and other chaperone components for functional maturation, regulation, and stability. Among these Hsp90 client proteins are steroid receptors, members from other classes of transcription factors, and representatives of both serine/threonine and tyrosine kinase families. Typically, dynamic complexes form on the client protein, and these consist of Hsp90- plus bound co-chaperones that often have enzymatic activities. In addition to its direct influence on client folding, Hsp90 locally concentrates co-chaperone activity within the client complex, and dynamic exchange of co-chaperones on Hsp90 facilitates sampling of co-chaperone activities that may, or may not, act on the client protein. We are just beginning to understand the nature of biochemical and molecular interactions between co-chaperone and Hsp90-bound client. This review focuses on the differential effects of Hsp90 co-chaperones toward client protein function and on the specificity that allows co-chaperones to discriminate between even closely related clients.
We examined the biogenesis of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) in vitro and in vivo. pVHL formed a complex with the cytosolic chaperonin containing TCP-1 (CCT or TRiC) en route to assembly with elongin B/C and the subsequent formation of the VCB-Cul2 ubiquitin ligase. Blocking the interaction of pVHL with elongin B/C resulted in accumulation of pVHL within the CCT complex. pVHL present in purified VHL-CCT complexes, when added to rabbit reticulocyte lysate, proceeded to form VCB and VCB-Cul2. Thus, CCT likely functions, at least in part, by retaining VHL chains pending the availability of elongin B/C for final folding and/or assembly. Tumor-associated mutations within exon II of the VHL syndrome had diverse effects upon the stability and/or function of pVHL-containing complexes. First, a pVHL mutant lacking the entire region encoded by exon II did not bind to CCT and yet could still assemble into complexes with elongin B/C and elongin B/C-Cul2. Second, a number of tumor-derived missense mutations in exon II did not decrease CCT binding, and most had no detectable effect upon VCB-Cul2 assembly. Many exon II mutants, however, were found to be defective in the binding to and subsequent ubiquitination of hypoxia-inducible factor 1alpha (HIF-1alpha), a substrate of the VCB-Cul2 ubiquitin ligase. We conclude that the selection pressure to mutate VHL exon II during tumorigenesis does not relate to loss of CCT binding but may reflect quantitative or qualitative defects in HIF binding and/or in pVHL-dependent ubiquitin ligase activity.
Previous studies with a mycobacterial heat shock protein (hsp-65) have demonstrated some efficacy using cationic liposome-mediated gene transfer in murine i.p. sarcoma models. To further analyze the efficacy of hsp-65 immunotherapy in clinically relevant models of localized cancer, immunocompetent mice bearing i.p. murine mesothelioma were treated with four i.p. doses of a cationic lipid complexed with plasmid DNA (pDNA) containing hsp65, LacZ, or a null plasmid. We observed >90% long-term survival (median survival, 150 days versus approximately 25 days, treated versus saline control, respectively) in a syngeneic, i.p. murine mesothelioma model (AC29). Long-term survivors were observed in all groups treated with lipid complexed with any pDNA. Lipid alone or DNA alone provided no demonstrable survival advantage. In a more aggressive i.p. model of mesothelioma (AB12), we observed >40% long-term survival in groups treated with lipid:pDNA complexes, again irrespective of the transgene. To ask whether these antitumor effects had led to an adaptive immune response against the tumor cell, we rechallenged long-term survivors in both murine models s.c. with the parental tumor cell line. Specific, long-lasting systemic immunity against the tumor was readily demonstrated in both models (AB12 and AC29). Consistent with these results, splenocytes from long-term survivors specifically lysed the parental tumor cell lines. Depleting the CD8+ T-cells from the splenocyte pool eliminated this lytic activity. Lipid:pDNA treatment of athymic, SCID, and SCID/Beige mice bearing a murine i.p. mesothelioma (AC29) resulted in only a slight survival advantage, but there were no long-term survivors. Treatment of immunocompetent mice depleted of specific immune effector cells demonstrated roles for CD8+ and natural killer cells. Although the exact mechanism(s) responsible for these antitumor effects is unclear, the results are consistent with roles for both innate and adaptive immune responses. An initial tumor cell killing stimulated by cationic lipid:pDNA complexes appears to be translated into long-term, systemic immunity against the tumor cell. These results are the first to demonstrate that adaptive immunity against a tumor cell can be induced by the administration of lipid:pDNA complexes. Multiple administrations of cationic lipid complexed with pDNA lacking an expressed transgene could provide a promising generalized immune-mediated modality for treating cancer.