Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies

Complement component C5a binds C5a receptor (C5aR) and facilitates leukocyte chemotaxis and release of inflammatory mediators. We used neutrophils from human C5aR knock-in mice, in which the mouse C5aR coding region was replaced with that of human C5aR, to immunize wild-type mice and to generate high-affinity antagonist monoclonal antibodies (mAbs) to human C5aR. These mAbs blocked neutrophil migration to C5a in vitro and, at low doses, both prevented and reversed inflammatory arthritis in the murine K/BxN model. Of approximately 40 mAbs generated to C5aR, all potent inhibitors recognized a small region of the second extracellular loop that seems to be critical for regulation of receptor activity. Human C5aR knock-in mice not only facilitated production of high-affinity mAbs against an important human therapeutic target but were also useful in preclinical validation of the potency of these antagonists. This strategy should be applicable to other important mAb therapeutics.     

Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis

Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity.     

Facilitated leaky scanning and atypical ribosome shunting direct downstream translation initiation on the tricistronic S1 mRNA of avian reovirus

The S1 mRNA of avian reovirus is functionally tricistronic, encoding three unrelated proteins, p10, p17 and σC, from three sequential, partially overlapping open reading frames (ORFs). The mechanism of translation initiation at the 3′-proximal σC ORF is currently unknown. Transient RNA transfections using Renilla luciferase reporter constructs revealed only a modest reduction in reporter expression upon optimization of either the p10 or p17 start sites. Insertion of multiple upstream AUG (uAUG) codons in a preferred start codon sequence context resulted in a substantial retention of downstream translation initiation on the S1 mRNA, but not on a heterologous mRNA. The S1 mRNA therefore facilitates leaky scanning to promote ribosome access to the σC start codon. Evidence also indicates that σC translation is mediated by a second scanning-independent mechanism capable of bypassing upstream ORFs. This alternate mechanism is cap-dependent and requires a sequence-dependent translation enhancer element that is complementary to 18S rRNA. Downstream translation initiation of the tricistronic S1 mRNA is therefore made possible by two alternate mechanisms, facilitated leaky scanning and an atypical form of ribosome shunting. This dual mechanism of downstream translation initiation ensures sufficient expression of the σC cell attachment protein that is essential for infectious progeny virus production.     

Reduced Juvenile Long-Term Depression in Tuberous Sclerosis Complex Is Mitigated in Adults by Compensatory Recruitment of mGluR5 and Erk Signaling

Tuberous sclerosis complex (TSC) is a multisystem genetic disease that manifests with mental retardation, tumor formation, autism, and epilepsy. Heightened signaling through the mammalian target of rapamycin (mTOR) pathway is involved in TSC pathology, however it remains unclear how other signaling pathways are perturbed and contribute to disease symptoms. Reduced long-term depression (LTD) was recently reported in TSC mutant mice. We find that although reduced LTD is a feature of the juvenile mutant hippocampus, heightened expression of metabotropic glutamate receptor 5 and constitutively activated Erk signaling in the adult hippocampus drives wild-type levels of LTD. Increased mGluR5 and Erk results in a novel mTOR-independent LTD in CA1 hippocampus of adult mice, and contributes to the development of epileptiform bursting activity in the TSC2^+/− CA3 region of the hippocampus. Inhibition of mGluR5 or Erk signaling restores appropriate mTOR-dependence to LTD, and significantly reduces epileptiform bursting in TSC2^+/− hippocampal slices. We also report that adult TSC2^+/− mice exhibit a subtle perseverative behavioral phenotype that is eliminated by mGluR5 antagonism. These findings highlight the potential of modulating the mGluR5-Erk pathway in a developmental stage-specific manner to treat TSC.     

Predictive Validity and Classification Accuracy of ActiGraph Energy Expenditure Equations and Cut-Points in Young Children

Objectives

Evaluate the predictive validity of ActiGraph energy expenditure equations and the classification accuracy of physical activity intensity cut-points in preschoolers.

Methods

Forty children aged 4–6 years (5.3±1.0 years) completed a ∼150-min room calorimeter protocol involving age-appropriate sedentary, light and moderate-to vigorous-intensity physical activities. Children wore an ActiGraph GT3X on the right mid-axillary line of the hip. Energy expenditure measured by room calorimetry and physical activity intensity classified using direct observation were the criterion methods. Energy expenditure was predicted using Pate and Puyau equations. Physical activity intensity was classified using Evenson, Sirard, Van Cauwenberghe, Pate, Puyau, and Reilly, ActiGraph cut-points.

Results

The Pate equation significantly overestimated VO₂ during sedentary behaviors, light physical activities and total VO₂ (P<0.001). No difference was found between measured and predicted VO₂ during moderate-to vigorous-intensity physical activities (P = 0.072). The Puyau equation significantly underestimated activity energy expenditure during moderate-to vigorous-intensity physical activities, light-intensity physical activities and total activity energy expenditure (P<0.0125). However, no overestimation of activity energy expenditure during sedentary behavior was found. The Evenson cut-point demonstrated significantly higher accuracy for classifying sedentary behaviors and light-intensity physical activities than others. Classification accuracy for moderate-to vigorous-intensity physical activities was significantly higher for Pate than others.

Conclusion

Available ActiGraph equations do not provide accurate estimates of energy expenditure across physical activity intensities in preschoolers. Cut-points of ≤25counts⋅15 s⁻¹ and ≥420 counts⋅15 s⁻¹ for classifying sedentary behaviors and moderate-to vigorous-intensity physical activities, respectively, are recommended.     

Why are some animal populations unaffected or positively affected by roads?

In reviews on effects of roads on animal population abundance we found that most effects are negative; however, there are also many neutral and positive responses [Fahrig and Rytwinski (Ecol Soc 14:21, 2009; Rytwinski and Fahrig (Biol Conserv 147:87–98, 2012)]. Here we use an individual-based simulation model to: (1) confirm predictions from the existing literature of the combinations of species traits and behavioural responses to roads that lead to negative effects of roads on animal population abundance, and (2) improve prediction of the combinations of species traits and behavioural responses to roads that lead to neutral and positive effects of roads on animal population abundance. Simulations represented a typical situation in which road mitigation is contemplated, i.e. rural landscapes containing a relatively low density (up to 1.86 km/km²) of high-traffic roads, with continuous habitat between the roads. In these landscapes, the simulations predict that populations of species with small territories and movement ranges, and high reproductive rates, i.e. many small mammals and birds, should not be reduced by roads. Contrary to previous suggestions, the results also predict that populations of species that obtain a resource from roads (e.g. vultures) do not increase with increasing road density. In addition, our simulations support the predation release hypothesis for positive road effects on prey (both small- and large-bodied prey), whereby abundance of a prey species increased with increasing road density due to reduced predation by generalist road-affected predators. The simulations also predict an optimal road density for the large-bodied prey species if it avoids roads or traffic emissions. Overall, the simulation results suggest that in rural landscapes containing high-traffic roads, there are many species for which road mitigation may not be necessary; mitigation efforts should be tailored to the species that show negative population responses to roads.     

Dual processing of FAT1 cadherin protein by human melanoma cells generates distinct protein products

The giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less commonly FAT2, FAT3, and FAT4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that, like Drosophila FAT, human FAT1 is cleaved into a non-covalent heterodimer before achieving cell surface expression. The use of inhibitors also established that such cleavage requires the proprotein convertase furin. However, in melanoma cells, the non-cleaved proform of FAT1 is also expressed at the cell surface together with the furin-cleaved heterodimer. Moreover, furin-independent processing generates a potentially functional proteolytic product in melanoma cells, a persistent 65-kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. In vitro localization studies of FAT1 showed that melanoma cells display high levels of cytosolic FAT1 protein, whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. Such differences in protein distribution appear to reconcile with the different protein products generated by dual FAT1 processing. We suggest that the uncleaved FAT1 could promote altered signaling, and the novel products of alternate processing provide a dominant negative function in melanoma.     

Possible regulation of trehalose metabolism by methylation in Saccharomyces cerevisiae

The current study was undertaken to correlate post‐translational protein modification by methylation with the functionality of enzymes involved in trehalose metabolism in Saccharomyces cerevisiae. Trehalose is an economically important disaccharide providing protection against various kinds of stresses. It also acts as a source of cellular energy by storing glucose. Methyl group donor S‐adenosyl L ‐methionine (AdoMet) and methylation inhibitor‐oxidized adenosine (AdOx) were used for the methylation study. AdoMet delayed initial growth of the cells but the overall growth rate remained same suggesting its interference in G1 phase of the cell cycle. Metabolic‐altered enzyme activities of acid trehalase (AT), neutral trehalase (NT), and trehalose‐6‐phosphate synthase (TPS) were observed when treated with AdOx and AdoMet separately. A positive effect of methylation was observed in TPS, hence, it was purified in three different conditions, using AdoMet, AdOx, and control. Differences in mobility of methylated, methylation‐inhibited, and control TPS during acidic native gel electrophoresis confirmed the occurrence of induced methylation. Hydrolysis under alkaline pH conditions revealed that methylation of TPS was different than O‐methylation. MALDI‐TOF analysis of trypsin‐digested samples of purified methylated, methylation‐inhibited, and control TPS revealed that an increase of 18 Da mass in methylated peptides suggesting the introduction of methyl ester in TPS. Results of amino acid analysis corroborated the presence of methyl cysteine. The data presented here strongly suggests that trehalose production was enhanced due to methylation of TPS arising from carboxymethylation of cysteine residues. J. Cell. Physiol. 226: 158–164, 2010. © 2010 Wiley‐Liss, Inc.     

Oversulfated Chondroitin Sulfate Inhibits the Complement Classical Pathway by Potentiating C1 Inhibitor

Oversulfated chondroitin sulfate (OSCS) has become the subject of multidisciplinary investigation as a non-traditional contaminant in the heparin therapeutic preparations that were linked to severe adverse events. In this study, it was found that OSCS inhibited complement fixation on bacteria and bacterial lysis mediated by the complement classical pathway. The inhibition of complement by OSCS is not due to interference with antibody/antigen interaction or due to consumption of C3 associated with FXII-dependent contact system activation. However, OSCS complement inhibition is dependent on C1 inhibitor (C1inh) since the depletion of C1inh from either normal or FXII-deficient complement plasma prevents OSCS inhibition of complement activity. Surface plasmon resonance measurements revealed that immobilized C1inhibitor bound greater than 5-fold more C1s in the presence of OSCS than in presence of heparin. Although heparin can also inhibit complement, OSCS and OSCS contaminated heparin are more potent inhibitors of complement. Furthermore, polysulfated glycosaminoglycan (PSGAG), an anti-inflammatory veterinary medicine with a similar structure to OSCS, also inhibited complement in the plasma of dogs and farm animals. This study provides a new insight that in addition to the FXII-dependent activation of contact system, oversulfated and polysulfated chondroitin-sulfate can inhibit complement activity by potentiating the classical complement pathway regulator C1inh. This effect on C1inh may play a role in inhibiting inflammation as well as impacting bacterial clearance.     

Behavioral changes across the menstrual cycle in isosexual groups of bonnet macaques (Macaca radiata)

Behavioral changes were systematically recorded across menstrual cycles over a six-month period in two laboratory-housed isosexual triads ofMacaca radiata (bonnet macaque). The purpose of this study was to determine whether females of this species demonstrate premenstrual behavioral changes as reported for humans, or heightened levels of aggression during the first half of their cycle as reported in some species of Old World monkeys. A total of 34 menstrual cycles were divided into five segments including two follicular, one periovulatory, and two luteal phases. The mean frequencies of behaviors were analyzed according to phase, rank, and time since triad formation, for a total of 34 menstrual cycles. There was no evidence of an increase in spatial separation during the premenstrual phase or during any other phase of the menstrual cycle. Although contact aggression did not show an increase during the early follicular half of the cycle (phases 1 and 2) or during the phase of the cycle immediately preceding menses (phase 5), contact aggression did show two peaks: one in the early-mid luteal half of the cycle (phase 4) and a peak in the mid-late follicular (phase 2). The non-aggressive hierarchical behaviors did not follow the same pattern as contact aggression. Instead the distribution of these behaviors showed a pattern similar to that of estrogen levels across the cycle. Subjects’ location in pen also varied significantly according to cycle phase: subjects spent more time on perches during the premenstrual phase and more time on the pen floor during the periovulatory phase. The increase in contact aggressive behaviors during the early-mid follicular phase and the mid-late luteal phase does not suggest a simple hormonal correlate as these two phases are characterized by high levels of estrogen and progesterone, respectively. However, the distribution of non-aggressive hierarchical behaviors suggest that this category of agonistic interaction may be related to mating competition among females of this species. Results are discussed with reference to the social behavior and promiscuous mating strategy ofM. radiata. The findings in this present study are compared with previous studies utilizing other species of Old World monkeys. Differences in study design and group composition are considered as factors effecting discrepant results both within and between species.