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A localized PCR inhibitor in a porcelain crab suggests a protective role.

El-Maklizi MA, Ouf A, Ferreira A, Hedar S, Cruz-Rivera E - PeerJ (2014)

Bottom Line: By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure.Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound.The identity of the inhibitory molecule remains unknown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, The American University in Cairo , New Cairo , Egypt.

ABSTRACT
A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

No MeSH data available.


Related in: MedlinePlus

Relative melanin content of samples.Absorbance of samples at 320 nm to assess melanin content (N = 4). Bars represent means + 1SE. Statistical analysis was performed with one-way ANOVA.
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fig-8: Relative melanin content of samples.Absorbance of samples at 320 nm to assess melanin content (N = 4). Bars represent means + 1SE. Statistical analysis was performed with one-way ANOVA.

Mentions: The inhibitor was heat stable as shown by an experiment in which foregut extracts from three individuals crabs were heated for an hour at 99 °C before adding them to the PCR reactions. The addition of these boiled extracts to fish DNA blocked amplification with 18s, while fish DNA without any foregut extract added, amplified at the expected band size (Fig. 7). This suggests that the inhibitor is not a protein with a complex structure that would undergo denaturation. The inhibitor also did not react as expected from a melanin-like compound (Dörrie et al., 2006)). When extracts from all three main sections of the crab gut, gills, crab muscle and fish muscle were spectrophotometrically evaluated, no significant differences in absorbance were observed (Fig. 8). In fact, despite sometimes large variance, absorbance for most samples ranged from 0–0.05 and was not statistically different than zero (P ≥ 0.219, Mann–Whitney U tests). One-way ANOVA showed no significant difference between any of the six treatment groups in terms of potential melanin content (P = 0.284; Fig. 7).


A localized PCR inhibitor in a porcelain crab suggests a protective role.

El-Maklizi MA, Ouf A, Ferreira A, Hedar S, Cruz-Rivera E - PeerJ (2014)

Relative melanin content of samples.Absorbance of samples at 320 nm to assess melanin content (N = 4). Bars represent means + 1SE. Statistical analysis was performed with one-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260131&req=5

fig-8: Relative melanin content of samples.Absorbance of samples at 320 nm to assess melanin content (N = 4). Bars represent means + 1SE. Statistical analysis was performed with one-way ANOVA.
Mentions: The inhibitor was heat stable as shown by an experiment in which foregut extracts from three individuals crabs were heated for an hour at 99 °C before adding them to the PCR reactions. The addition of these boiled extracts to fish DNA blocked amplification with 18s, while fish DNA without any foregut extract added, amplified at the expected band size (Fig. 7). This suggests that the inhibitor is not a protein with a complex structure that would undergo denaturation. The inhibitor also did not react as expected from a melanin-like compound (Dörrie et al., 2006)). When extracts from all three main sections of the crab gut, gills, crab muscle and fish muscle were spectrophotometrically evaluated, no significant differences in absorbance were observed (Fig. 8). In fact, despite sometimes large variance, absorbance for most samples ranged from 0–0.05 and was not statistically different than zero (P ≥ 0.219, Mann–Whitney U tests). One-way ANOVA showed no significant difference between any of the six treatment groups in terms of potential melanin content (P = 0.284; Fig. 7).

Bottom Line: By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure.Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound.The identity of the inhibitory molecule remains unknown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, The American University in Cairo , New Cairo , Egypt.

ABSTRACT
A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

No MeSH data available.


Related in: MedlinePlus