Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.

A resposta oxidativa em corações de camundongos é modulada por background genético / The oxidative response of mouse hearts is modulated by genetic background

FUNDAMENTO: o tabagismo apresenta importante papel sobre as doenças cardiovasculares, entretanto permanecem pouco compreendidos os motivos pelos quais alguns seres humanos as desenvolvem e outros não. OBJETIVO: nosso objetivo foi analisar o perfil redox do coração de diferentes linhagens de camundongos após exposição à fumaça de cigarro. MÉTODOS: camundongos machos suíços (n = 10), C3H (n = 10), BALB/c (n = 10) e C57BL/6 (n = 10) foram expostos à fumaça de cigarro (12 cigarros/dia), enquanto os respectivos controles (n = 10) ao ar ambiente por 60 dias. Após sacrifício, o coração foi retirado para análises bioquímicas. RESULTADOS: embora o conteúdo de malondialdeído não tenha aumentado em nenhum grupo, a atividade da catalase diminuiu no grupo suíço (p < 0,05), BALB/c (p < 0,05) quando comparados aos respectivos grupos-controle, enquanto a mieloperoxidase diminuiu no grupo C3H (p < 0,05) e C57BL/6 (p < 0,001) quando comparados aos respectivos grupos controle. O conteúdo de glutationa reduzida diminuiu nos grupos suíço, C3H, C57BL/6 (p < 0,05) e no grupo BALB/c (p < 0,001) quando comparados com os respectivos controles. Observamos aumento do conteúdo da glutationa oxidada no grupo Suíço (p < 0,05) e diminuição nos grupos C3H (p < 0,05) e BALB/c (p < 0,001) quando comparados aos respectivos grupos-controle. A razão glutationa reduzida/ glutationa oxidada apresentou redução nos grupos suíço e C57BL/6 (p < 0.05) quando comparados aos grupos controle. CONCLUSÃO: o background genético nos camundongos pode influenciar na resposta antioxidante após a exposição à fumaça de cigarro e parece ser um fator determinante para o desequilíbrio redox no suíço e C57BL/6. Compreender as respostas antioxidantes e do background genético C3H e BALB/c podem fornecer importantes informações quanto à resistência cardíaca a fumaça de cigarro.

BACKGROUND: Smoking plays an important role in cardiovascular diseases. However, the reasons why some individuals develop those diseases and others do not remain to be explained. OBJECTIVE: This study aimed at assessing the redox profile of the heart of different mouse strains after exposure to cigarette smoke. METHODS: Male mice of the Swiss (n = 10), C3H (n = 10), BALB/c (n = 10) and C57BL/6 (n = 10) strains were exposed to cigarette smoke (12 cigarettes/day), while their respective controls (n = 10) were exposed to ambient air for 60 days. After being euthanized, their heart was removed for biochemical analyses. RESULTS: Although the malondialdehyde content did not increase in any of the groups, catalase activity decreased in the Swiss (p < 0.05) and BALB/c (p < 0.05) strain mice as compared with their respective control groups, while myeloperoxidase decreased in the C3H (p < 0.05) and C57BL/6 (p < 0.001) strain mice as compared with their respective control groups. The reduced glutathione content decreased in the Swiss, C3H, C57BL/6 (p < 0.05) and BALB/c (p < 0,001) strain mice as compared with their respective control groups. Regarding reduced glutathione content, an increase was observed in the Swiss strain mice (p < 0.05), while a decrease was observed in the C3H (p < 0.05) and BALB/c (p < 0.001) strain mice as compared with their respective control groups. The reduced glutathione/reduced glutathione ratio showed a reduction in the Swiss and C57BL/6 (p < 0.05) strain mice as compared with their respective control groups. CONCLUSIONS: The genetic background of mice can influence the antioxidant response after exposure to cigarette smoke and seems to be a determinant factor for redox imbalance in Swiss and C57BL/6 strain mice. Understanding antioxidant responses and genetic background of C3H and BALB/c strain mice might provide important information regarding cardiac resistance to cigarette smoke.

Molecular genetics of hereditary thyroid disease due to a defect in the thyroglobulin or thyroperoxidase synthesis

1. Hereditary goiter and the various degrees of thyroid hypofunction are the result of structural changes in the thyroglobulin (Tg) or thyroperoxidase (TPO) proteins, the inability to couple iodotyrosines or defective iodination, impairing or substantially altering the synthesis of T4 and T3. 2. The first nmutations in the Tg and TPO genes responsable for human cases of dys-hormonogenesis have been described. The mutation in two siblings with hereditary goiter and marked impairment of Tg synthesis was a cytosine to thymine transition creating a stop codon at postion 1510. The point mutation is removed by the preferential accumulation of a 171-nt deleted Tg mRNA. In another subject, molecular studies revealed that exon 4 was missing from the major Tg transcript due to a cytosine to guanine transversion at postion minus 3 in the acceptor splice site of intron 3. 3. Genomic DNA studies identified a duplication of a 4-base sequence in the eight exon of the TPO gene. Interestingly, besides abolishing the enzymatic activity by disrupting the reading frame of the messenger RNA and introducing stop codons, the GGCC duplication also unmasks a cryptic acceptor splice site in exon 9. 4. In conclusion, the identification of different molecular defects provied evidence that hereditary goiter associated with abnormal Tg or TPO synthesis is caused by heterogeneous genetic alterations