Structural features of cytochrome P450 1A associated with the absence of EROD activity in liver of the loricariid catfish Pterygoplichthys sp

The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acid substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrate.     

Cold unfolding of β-hairpins: a molecular-level rationalization

Isolated β-hairpins in water have a temperature dependence of their conformational stability qualitatively resembling that of globular proteins, showing both cold and hot unfolding transitions. It is shown that a molecular-level rationalization of this cold unfolding can be provided extending the approach devised for globular proteins (Graziano G. Phys Chem Chem Phys 2010; 12:14245-14252). The decrease in the solvent-excluded volume upon folding, measured by the decrease in the solvent accessible surface area, produces a gain in configurational/translational entropy of water molecules that is the main stabilizing contribution of the folded conformation. This always stabilizing Gibbs energy contribution has a parabolic-like temperature dependence in water and is exactly counterbalanced at two temperatures (i.e., the cold and hot unfolding temperatures) by the always destabilizing Gibbs energy contribution due to the loss in conformational degrees of freedom of the peptide chain.     

Caméras hybrides TEMP-TDM et semi-quantification du I-FPCIT : évaluation de l’apport de la TDM

Objectives

Tomoscintigraphy of dopamine transporters with ¹²³I-FP-CIT is nowadays essential to visualise impairment of nigro-striatal system for the diagnosis of parkinsonism and for the differential diagnosis of dementia. With the development of hybrid cameras (SPECT-CT), the CT contribution in nuclear neurology needs to be assessed in diagnostic and semi-quantification performances. The main purpose of our study is to compare attenuation correction using CT to attenuation correction using the linear algorithm of Chang. SPECT-CT with parallel collimation results were also weighed against fan beam collimation and the contribution of partial volume effect correction was studied in secondary objective.

Materials and methods

We used a trilinear phantom to define spatial resolution and an anthropomorphic striatal phantom to quantify the activity in striatal cavities. We compared the impact of attenuation and scatter correction on spatial resolution and semi-quantification in striatum. We performed the partial volume effect correction on reconstructed images according to the method of Rousset.

Results

Attenuation correction by CT did not improve significantly spatial resolution compared to the algorithm of Chang. The semi-quantification of ¹²³I-FPCIT in striata was not significantly different according to the various CA, but was significantly improved with CT attenuation and scatter correction. Partial volume effect correction improved the quantification from 40 to 60% in the striatal structures, when the activity was superior in at least twice the background noise.

Conclusion

SPECT-CT hybrid cameras increase spatial resolution and improve semi-quantification of ¹²³I-FPCIT because of CT attenuation and scatter correction. Another use of CT is the possibility of calibrating anatomic segmentation of striata for partial volume effect correction. Partial volume effect correction improves quantification and is essential for early diagnosis of nigro-striatal disease.     

Red porgy (Pagrus pagrus) larval feeding performance and behavior at the onset of exogenous feeding

The feeding performance and behavior at the onset of exogenous feeding, 3 to 4 days after hatching (DAH), were studied in red porgy Pagrus pagrus larvae. Similar feeding efficiency and intensity were achieved for two feeding treatments (live or freeze-dried rotifers) suggesting that prey movement is not decisive for their detection and capture and demonstrating that at first feeding red porgy larvae can ingest inert food. Larvae feeding performance was not affected by a diet shift between treatments. Based on maximum rotifers consumption and gut evacuation time at 18 °C, the daily ration was estimated as 14.035 μg, considering 14 h of feeding and a 25% egg:female rotifer ratio. Larval swimming activity measured by video recording showed a close association with gut fullness and similar swimming patterns for 3 and 4 DAH larvae. However, 20.3% larger mouth gape and 54.6% higher swimming speed of the older larvae should provide a better feeding performance and more energy needed for growth.     

Paretic muscle atrophy and non-contractile tissue content in individual muscles of the post-stroke lower extremity

Muscle atrophy is one of many factors contributing to post-stroke hemiparetic weakness. Since muscle force is a function of muscle size, the amount of muscle atrophy an individual muscle undergoes has implications for its overall force-generating capability post-stroke. In this study, post-stroke atrophy was determined bilaterally in fifteen leg muscles with volumes quantified using magnetic resonance imaging (MRI). All muscle volumes were adjusted to exclude non-contractile tissue content, and muscle atrophy was quantified by comparing the volumes between paretic and non-paretic sides. Non-contractile tissue or intramuscular fat was calculated by determining the amount of tissue excluded from the muscle volume measurement. With the exception of the gracilis, all individual paretic muscles examined had smaller volumes in the non-paretic side. The average decrease in volume for these paretic muscles was 23%. The gracilis volume, on the other hand, was approximately 11% larger on the paretic side. The amount of non-contractile tissue was higher in all paretic muscles except the gracilis, where no difference was observed between sides. To compensate for paretic plantar flexor weakness, one idea might be that use of the paretic gracilis actually causes the muscle to increase in size and not develop intramuscular fat. By eliminating non-contractile tissue from our volume calculations, we have presented volume data that more appropriately represents force-generating muscle tissue. Non-uniform muscle atrophy was observed across muscles and may provide important clues when assessing the effect of muscle atrophy on post-stroke gait.     

Generation of bivalent and bispecific kringle single domains by loop grafting as potent agonists against death receptors 4 and 5

Bivalent or bispecific binding activity of proteins has been mainly achieved by assembling two or more domains in a single molecule. Here we report bivalent/bispecific single-domain proteins based on the kringle domain (KD), which has a cystine knot structural motif and is highly tolerant of sequence modifications. KD has seven loops protruding from the core fold into two largely opposite directions, dubbed loop cluster regions (LCRs) 1 and 2. Mutational analysis of previously isolated agonistic KD variants against human death receptors (DRs) 4 and 5 revealed that they can simultaneously recognize two target molecules of DR4 and/or DR5 via the two independent binding sites of LCR1 and LCR2. Binding loop mapping of yeast-surface-displayed KD mutants identified high-affinity target binding loops in LCR2, which were then grafted into conformationally compatible loops located on the opposite side of LCR1 within the same or different KD variants to generate bivalent/bispecific KD variants against DR4 and/or DR5 with improved affinity. The loop-grafted bivalent/bispecific KD variants showed enhanced cell-death-inducing activity of tumor cells compared with their monovalent/monospecific and bivalent/monospecific counterparts, demonstrating an advantage of bispecific targeting to both DR4 and DR5 over the targeting of only one of the two pro-apoptotic receptors. Our results suggest that the KD with the two independent binding surfaces for target recognition is an appropriate scaffold for the development of bivalency and/or bispecificity by loop grafting on the single domain, which offers a distinct advantage over other protein scaffolds with a single binding surface.     

Role of interaction of XPF with RPA in nucleotide excision repair

Nucleotide excision repair (NER) is a very important defense system against various types of DNA damage, and it is necessary for maintaining genomic stability. The molecular mechanism of NER has been studied in considerable detail, and it has been shown that proper protein-protein interactions among NER factors are critical for efficient repair. A structure-specific endonuclease, XPF-ERCC1, which makes the 5′ incision in NER, was shown to interact with a single-stranded DNA binding protein, RPA. However, the biological significance of this interaction was not studied in detail. We used the yeast two-hybrid assay to determine that XPF interacts with the p70 subunit of RPA. To further examine the role of this XPF-p70 interaction, we isolated a p70-interaction-deficient mutant form of XPF that contains a single amino acid substitution in the N-terminus of XPF by the reverse yeast two-hybrid assay using randomly mutagenized XPF. The biochemical properties of this RPA-interaction-deficient mutant XPF-ERCC1 are very similar to those of wild-type XPF-ERCC1 in vitro. Interestingly, expression of this mutated form of XPF in the XPF-deficient Chinese hamster ovary cell line, UV41, only partially restores NER activity and UV resistance in vivo compared to wild-type XPF. We discovered that the RPA-interaction-deficient XPF is not localized in nuclei and the mislocalization of XPF-ERCC1 prevents the complex from functioning in NER.     

Multiple factors insulate Msh2-Msh6 mismatch repair activity from defects in Msh2 domain I

DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2-Msh6 binds mispairs and small insertion/deletion loops, and Msh2-Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2-Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2-Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2-Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1-Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.     

阔叶红松林森林资源可持续利用方案

天保工程实施后,为了促进次生林向原始阔叶红松林恢复,不采红松只采伐阔叶树种的经营方式在长白山地区被广泛应用,部分林区陷入可采伐资源匮乏的困境。为了探究阔叶红松林森林资源可持续利用方案,针对红松(蓄积)比例不同的阔叶红松林次生林,利用林木材积生长方程与保留系数模型,模拟了预设经营方案下林分总蓄积量与可采蓄积量动态变化。研究结果表明,在禁止采伐红松的经营方式下,红松(蓄积)比例较高的次生林将无法达到森林资源可持续利用的目标,次生林的经营方案需要根据林分中红松(蓄积)比例不同而区别制定:对于红松蓄积低于40%的次生林,推荐不采伐红松、20%采伐强度、40a周期的经营方案;对于红松蓄积高于40%的次生林,推荐可以采伐红松、20%采伐强度、30a周期的经营方案。另外,可采蓄积量的恢复期比总蓄积量的恢复期更长,以可采蓄积恢复期作为评价指标,确定采伐周期,更有利于森林资源的可持续利用。