Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes form bundles of four parallel alpha-helices. The central '0' layer of interacting amino acid side chains is highly conserved and contains one arginine and three glutamines, leading to the classification of SNAREs into R, Qa, Qb, and Qc-SNAREs. Replacing one of the glutamines with arginine in the yeast exocytotic SNARE complex is either lethal or causes a conditional growth defect that is compensated by replacing the R-SNARE arginine with glutamine. Using the yeast SNARE complex mediating traffic from the endoplasmic reticulum to the Golgi apparatus, we now show that functionally interacting SNAREs can be mapped by systematically exchanging glutamines and arginines in the '0' layer. The Q-->R replacement in the Qb-SNARE Bos1p has the strongest effect and can be alleviated by an Q-->R replacement in the R-SNARE Sec22p. Four Q residues in the central layer caused growth defects above 30 degrees C that were rescued by Q-->R substitutions in the Qa and Qc SNAREs Sed5p and Bet1p, respectively. The sec22(Q)/sed5(R) mutant is temperature sensitive and is rescued by a compensating R-->Q replacement in the R-SNARE Ykt6p. This rescue is attributed to the involvement of Sed5p and Ykt6p in a different SNARE complex that functions in intra-Golgi trafficking.

No abstract

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (D19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (D24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to av integrinexpressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies. Gene Therapy (2006) 13, 893-905.

PurposeDespite recommendations for 99mTc-tetrofosmin dual tracer imaging for hyperparathyroidism in current guidelines, no report was published on dual-isotope 99mTc-tetrofosmin and 123I sodium iodide single-photon-emission-computed-tomography (SPECT). We evaluated diagnostic accuracy and the impact of preoperative SPECT on the surgical procedures and disease outcomes.MethodsAnalysis of 70 consecutive patients with primary hyperparathyroidism and 20 consecutive patients with tertiary hyperparathyroidism. Imaging findings were correlated with surgical results. Concomitant thyroid disease, pre- and postoperative laboratory measurements, histopathological results, type and duration of surgery were assessed.ResultsIn primary hyperparathyroidism, SPECT had a sensitivity of 80% and a positive predictive value of 93% in patient-based analysis. Specificity was 99% in lesion-based analysis. Patients with positive SPECT elicit higher levels of parathyroid hormone and higher weight of resected parathyroids than SPECT-negative patients. Duration of parathyroid surgery was on average, approximately 40 minutes shorter in SPECT-positive than in SPECT-negative patients (89±46 vs. 129±41 minutes, p=0.006); 86% of SPECT-positive and 50% of SPECT-negative patients had minimal invasive surgery (p = 0.021). SPECT had lower sensitivity (60%) in patients with tertiary hyperparathyroidism; however, 90% of these patients had multiple lesions and all of these patients had bilateral lesions.ConclusionDual-isotope SPECT with 99mTc-tetrofosmin and 123I sodium iodide has a high diagnostic value in patients with primary hyperparathyroidism and allows for saving of operation time. Higher levels of parathyroid hormone and higher glandular weight facilitated detection of parathyroid lesion. Diagnostic accuracy of preoperative imaging was lower in patients with tertiary hyperparathyroidism.

No abstract

No abstract