Ng free b2-m rises 30 to 40 fold [17]. Furthermore, it is worth noting that both collagen, which is structurally similar to the human counterpart, and glycosaminoglycans are highly represented in the basement membrane of the C. elegans muscle system [18] and are potent promoters of b2-m amyloidogenesis under physiological like conditions [19]. To recapitulate the aggregation process occurring in mammals, we expressed the b2-m isoforms in C. elegans under the control of a body-wall muscle promoter. Here we show that both the P32G replacement and DN6 truncation remarkably exacerbate the behavioural defects that the expression of wild type human b2-m causes in transgenic worms. Mutated and truncated species of b2-m had a greater propensity to form in vivo soluble AZ-876 price oligomeric species than the wild type protein, thus, indicating that the toxicity of these proteins was strictly related to their sequence and aggregation propensity. To determine whether these new transgenic nematodes might be applied to the screening of compounds that counteract b2-m amyloidogenesis and amyloid toxicity, we MedChemExpress Sudan I tested their response to tetracyclines, which have been already reported to inhibit, in vitro, the b2-m aggregation [20]. These drugs are emerging antiamyloidogenic compounds and, their ability to counteract the aggregation of various amyloidogenic proteins, including TTR [21], and interact in vitro and in vivo with Ab oligomers has been already described [22].Materials and Methods Construction of C. elegans transgenic strainsTransgenic C. elegans strains were engineered to express human wild type b2-m and two isoforms, P32G and DN6, under the control of the body-wall muscle-specific unc-54 promoter/enhancer. Minigenes encoding wild type b2-m and DN6 were assembled in two steps. Sequence coding for signal peptide containing compatible cohesive ends (forward sequence: 59-CTAGCAAAAATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTGGTAC-39; reverse sequence: 59-CAGCCTCCAGGCCAGAAAGAGAGAGTAGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATTTTTG-39) was inserted between the unique NheI and KpnI sites of pPD30.38 vector (Addgene) [5]. Subsequently, wild type b2-m and DN6 sequences (obtained from the plasmids pHN1 and pET11a, respectively) were amplified by using b2-m cDNA as template and the oligonucleotide primers 59-GGGGGTACCATCCAGCGTACTCCAAAG-39 for the full length, 59-GGGGGTACCATTCAGGTTTACTCACGTC-39 for the truncated species and, 39CCCGAGCTCTTACATGTCTCGATCCCAC-59 for both species. The amplified DNA was inserted between the unique KpnI and SacI sites of pPD30.38 previously engineered with the signal peptide. To obtain P32G b2-m plasmid, a site-directed mutagenesis of wild type b2-m engineered plasmid pPD30.38 was performed, using the following primers: 59-CTATGTGTCTGGGTTTCATGGATCCGACATTGAAGTTGAC-39 and 59-GTCAACTTCAATGTCGGATCCATGAAACCCAGACACATAG-39. A pPD30.38 plasmid containing only the signal peptide was created as control. DNA sequencing was carried out to confirm that all subcloned plasmids were correct. Transgenes were introduced into MT309 multivulva C. elegans strain (Caenorhabditis Genetics Center, CGC, University of Minnesota, USA) by gonad microinjection of a DNA solution containing 25 ng/ml of the b2-m construct together with 20 ng/ml of ttx-3::rfp and 30 ng/ ml of plin-15(+) as marker plasmids. Multiple extra-chromosomal lines were established based on both the fluorescent marker and the disappearance of the multivulva phenotype. The transgenic worms mainta.Ng free b2-m rises 30 to 40 fold [17]. Furthermore, it is worth noting that both collagen, which is structurally similar to the human counterpart, and glycosaminoglycans are highly represented in the basement membrane of the C. elegans muscle system [18] and are potent promoters of b2-m amyloidogenesis under physiological like conditions [19]. To recapitulate the aggregation process occurring in mammals, we expressed the b2-m isoforms in C. elegans under the control of a body-wall muscle promoter. Here we show that both the P32G replacement and DN6 truncation remarkably exacerbate the behavioural defects that the expression of wild type human b2-m causes in transgenic worms. Mutated and truncated species of b2-m had a greater propensity to form in vivo soluble oligomeric species than the wild type protein, thus, indicating that the toxicity of these proteins was strictly related to their sequence and aggregation propensity. To determine whether these new transgenic nematodes might be applied to the screening of compounds that counteract b2-m amyloidogenesis and amyloid toxicity, we tested their response to tetracyclines, which have been already reported to inhibit, in vitro, the b2-m aggregation [20]. These drugs are emerging antiamyloidogenic compounds and, their ability to counteract the aggregation of various amyloidogenic proteins, including TTR [21], and interact in vitro and in vivo with Ab oligomers has been already described [22].Materials and Methods Construction of C. elegans transgenic strainsTransgenic C. elegans strains were engineered to express human wild type b2-m and two isoforms, P32G and DN6, under the control of the body-wall muscle-specific unc-54 promoter/enhancer. Minigenes encoding wild type b2-m and DN6 were assembled in two steps. Sequence coding for signal peptide containing compatible cohesive ends (forward sequence: 59-CTAGCAAAAATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTGGTAC-39; reverse sequence: 59-CAGCCTCCAGGCCAGAAAGAGAGAGTAGCGCGAGCACAGCTAAGGCCACGGAGCGAGACATTTTTG-39) was inserted between the unique NheI and KpnI sites of pPD30.38 vector (Addgene) [5]. Subsequently, wild type b2-m and DN6 sequences (obtained from the plasmids pHN1 and pET11a, respectively) were amplified by using b2-m cDNA as template and the oligonucleotide primers 59-GGGGGTACCATCCAGCGTACTCCAAAG-39 for the full length, 59-GGGGGTACCATTCAGGTTTACTCACGTC-39 for the truncated species and, 39CCCGAGCTCTTACATGTCTCGATCCCAC-59 for both species. The amplified DNA was inserted between the unique KpnI and SacI sites of pPD30.38 previously engineered with the signal peptide. To obtain P32G b2-m plasmid, a site-directed mutagenesis of wild type b2-m engineered plasmid pPD30.38 was performed, using the following primers: 59-CTATGTGTCTGGGTTTCATGGATCCGACATTGAAGTTGAC-39 and 59-GTCAACTTCAATGTCGGATCCATGAAACCCAGACACATAG-39. A pPD30.38 plasmid containing only the signal peptide was created as control. DNA sequencing was carried out to confirm that all subcloned plasmids were correct. Transgenes were introduced into MT309 multivulva C. elegans strain (Caenorhabditis Genetics Center, CGC, University of Minnesota, USA) by gonad microinjection of a DNA solution containing 25 ng/ml of the b2-m construct together with 20 ng/ml of ttx-3::rfp and 30 ng/ ml of plin-15(+) as marker plasmids. Multiple extra-chromosomal lines were established based on both the fluorescent marker and the disappearance of the multivulva phenotype. The transgenic worms mainta.
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