Intradermal or oral delivery of GAD-encoding genetic vaccines suppresses type 1 diabetes

Genetic vaccines are promising candidates for prevention of type 1 diabetes, an autoimmune disease resulting from cell-mediated destruction of pancreatic beta cells. It is known that the prophylactic effect and immune responses induced by administration of a genetic vaccine can depend on site of delivery. In the work presented here, we used the NOD mouse model for type 1 diabetes to evaluate different routes of delivery for DNA vaccines coding for the beta-cell antigen glutamic acid decarboxylase (GAD). Plasmid DNA coding for intracellular or secreted GAD was given via either the intramuscular (i.m.), intradermal (i.d.), or oral route, using, respectively, 300, 100, or 300 micro g DNA per mouse. Results indicated that both i.d. and oral delivery of GAD-encoding DNA were more effective than i.m. delivery for disease suppression. In addition, cytokine-specific ELISpot analysis indicated that immune responses induced by the different immunization protocols were more dependent on the cellular localization of GAD antigen than on the delivery route, while ELISA of anti-GAD serum antibody isotypes indicated that i.d. delivery of DNA was most likely to induce a Th2-like response. Our results suggest that i.d. or oral delivery of a genetic vaccine for type 1 diabetes might be preferable over the i.m. route in a future clinical setting.     

The significance of key regulators of apoptosis in the development and prognosis of prostate carcinoma. II. Products of suppressor genes Rb and PTEN, CDKI, Fas

The molecular basis for the transition of carcinoma of the prostate from androgen-dependent to androgen-independent growth is largely unknown. Currently for example, it is not clear whether the androgen-independent phenotype is a result of selection of a subgroup of genetically distinct prostate tumour cells which are already hormone-resistant or a genetic adaptation of prostate tumour cells to the hormone therapy itself. It has also been established that prostate tumour transformation is a result of homeostatic control defects, a line of thinking directed toward elucidating the apoptotic profile of prostate tumour cells that may be important in determining prognosis, response to therapy and illness progression. Main consideration in this part of rewiev is given to the role of tumour suppressor genes pRb and PTEN and also the natural inhibitors of cyclin dependent kinases - proteins p21(Waf1/Cip1) and p27(Kip1). Attention is also given to the role of FAS-mediated pathways in apoptosis induction.     

Multiple response elements and differential p53 binding control Perp expression during apoptosis

The p53 tumor suppressor gene responds to cellular stress by activating either cell cycle arrest or apoptosis. A growing number of target genes involved in each of these pathways have been identified. However, the mechanism by which the apoptosis versus arrest decision is made remains to be elucidated. Perp is a proapoptotic target gene of p53 expressed to high levels in apoptotic cells compared with those undergoing cell cycle arrest. This pattern of expression is unusual among p53 target genes, many of which are induced to similar levels during arrest and apoptosis. Here, we describe the regulation of the Perp gene by p53 through at least three response elements in the Perp promoter and first intron. These sites are occupied in vivo in E1A-expressing mouse embryo fibroblasts undergoing apoptosis but not cell cycle arrest, in contrast to the p21 5' response element, which is occupied during both. The apoptosis-deficient p53 point mutant, p53V143A, displays a selective deficit in binding to the Perp elements, demonstrating that p53 can distinguish between Perp and p21 at the level of DNA binding. These results provide mechanistic insight into the selective expression of Perp during apoptosis and may provide a useful model for studying the p53-dependent cell cycle arrest versus apoptosis decision.     

Unusual binding properties of the SH3 domain of the yeast actin-binding protein Abp1: structural and functional analysis

Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain solved at 1.3-A resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands.     

Insulin fails to alter plasma LCFA metabolism in muscle perfused at similar glucose uptake

Insulin has been shown to alter long-chain fatty acid (LCFA) metabolism and malonyl-CoA production in muscle. However, these alterations may have been induced, in part, by the accompanying insulin-induced changes in glucose uptake. Thus, to determine the effects of insulin on LCFA metabolism independently of changes in glucose uptake, rat hindquarters were perfused with 600 microM palmitate and [1-(14)C]palmitate and with either 20 mM glucose and no insulin (G) or 6 mM glucose and 250 microU/ml of insulin (I). As dictated by our protocol, glucose uptake was not significantly different between the G and I groups (10.3 +/- 0.6 vs. 11.0 +/- 0.5 micromol x g(-1) x h(-1); P > 0.05). Total palmitate uptake and oxidation were not significantly different (P > 0.05) between the G (10.1 +/- 1.0 and 0.8 +/- 0.1 nmol x min(-1) x g(-1)) and I (10.2 +/- 0.6 and 1.1 +/- 0.2 nmol. min(-1) x g(-1)) groups. Preperfusion muscle triglyceride and malonyl-CoA levels were not significantly different between the G and I groups and did not change significantly during the perfusion (P > 0.05). Similarly, muscle triglyceride synthesis was not significantly different between groups (P > 0.05). These results demonstrate that the presence of insulin under conditions of similar glucose uptake does not alter LCFA metabolism and suggest that cellular mechanisms induced by carbohydrate availability, but independent of insulin, may be important in the regulation of muscle LCFA metabolism.     

Feasibility of assessing the carcinogenicity of neutrons among neutron therapy patients

Nuclear workers, oil well loggers, astronauts, air flight crews, and frequent fliers can be exposed to low doses of neutrons, but the long-term human health consequences of neutron exposure are unknown. While few of these exposed populations are suitable for studying the effects of neutron exposure, patients treated with neutron-beam therapy might be a source of information. To assess the feasibility of conducting a multi-center international study of the late effects of neutron therapy, we surveyed 23 cancer centers that had used neutron beam therapy. For the 17 responding institutions, only 25% of the patients treated with neutrons (2,855 of 11,191) were alive more than 2 years after treatment. In a two-center U.S. pilot study of 484 neutron-treated cancer patients, we assessed the feasibility of obtaining radiotherapy records, cancer incidence and other follow-up data, and of estimating patient organ doses. Patients were treated with 42 MeV neutrons between 1972 and 1989. Applying a clinical equivalence factor of 3.2 for neutrons, total average organ doses outside the treatment beam ranged from 0.14 to 0.29 Gy for thyroid, 0.40 to 2.50 Gy for breast, 0.63 to 2.35 Gy for kidney, and 1.12 to 1.76 Gy for active bone marrow depending upon the primary cancer treatment site. We successfully traced 97% of the patients, but we found that patient survival was poor and that chemotherapy was not confirmable in a quarter of the patients. Based on our findings from the international survey and the feasibility study, we conclude that a large investigation could detect a fivefold or higher leukemia risk, but would be inadequate to evaluate the risk of solid cancers with long latent periods and therefore would likely not be informative with respect to neutron-related cancer risk in humans.     

PARK3 influences age at onset in Parkinson disease: a genome scan in the GenePD study

Parkinson disease (PD) is a late-onset neurodegenerative disorder. The mean age at onset is 61 years, but the disease can range from juvenile cases to cases in the 8th or 9th decade of life. The parkin gene on chromosome 6q and loci on chromosome 1p35-36 and 1p36 are responsible for some cases of autosomal recessive early-onset parkinsonism, but they do not appear to influence susceptibility or variability of age at onset for idiopathic PD. We have performed a genomewide linkage analysis using variance-component methodology to identify genes influencing age at onset of PD in a population of affected relatives (mainly affected sibling pairs) participating in the GenePD study. Four chromosomal loci showed suggestive evidence of linkage: chromosome 2p (maximum multipoint LOD [MaxLOD] = 2.08), chromosome 9q (MaxLOD = 2.00), chromosome 20 (MaxLOD = 1.82), and chromosome 21 (MaxLOD = 2.21). The 2p and 9q locations that we report here have previously been reported as loci influencing PD affection status. Association between PD age at onset and allele 174 of marker D2S1394, located on 2p13, was observed in the GenePD sample (P=.02). This 174 allele is common to the PD haplotype observed in two families that show linkage to PARK3 and have autosomal dominant PD, which suggests that this allele may be in linkage disequilibrium with a mutation influencing PD susceptibility or age at onset of PD.     

Molecular features of the copper binding sites in the octarepeat domain of the prion protein

Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH approximately 6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.     

Preparation and investigation of antibacterial carbohydrate-based surfaces

Surfaces bearing carbohydrate units have been modified in a two-step process to incorporate functionalities (lipophilic with polycationic units) that bear antibacterial activity. The effectiveness of these modified surfaces for antibacterial action against a series of seven Gram-positive and Gram-negative bacteria are reported.