Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

Anti-inflammatory effect of l-cysteine (a semi-essential amino acid) on 5-FU-induced oral mucositis in hamsters

Oral mucositis is an inflammation of the oral mucosa mainly resulting from the cytotoxic effect of 5-fluorouracil (5-FU). The literature shows anti-inflammatory action of l-cysteine (l-cys) involving hydrogen sulfide (H₂S). In view of these properties, we investigate the effect of l-cys in oral mucositis induced by 5-FU in hamsters. The animals were divided into the following groups: saline 0.9%, mechanical trauma, 5-FU 60–40 mg/kg, l-cys 10/40 mg and NaHS 27 µg/kg. 5-FU was administered on days 1st to 2nd; 4th day excoriations were made on the mucosa; 5th–6th received l-cys and NaHS. For data analysis, histological analyses, mast cell count, inflammatory and antioxidants markers, and immunohistochemistry (cyclooxygenase-2(COX-2)/inducible nitric oxide synthase (iNOs)/H₂S) were performed. Results showed that l-cys decreased levels of inflammatory markers, mast cells, levels of COX-2, iNOS and increased levels of antioxidants markers and H₂S when compared to the group 5-FU (p < 0.005). It is suggested that l-cys increases the H₂S production with anti-inflammatory action in the 5-FU lesion.

     

Serological biomarker for assessing human exposure to Aedes mosquito bites during a randomized vector control intervention trial in northeastern Thailand

BackgroundAedes mosquitoes are vectors for several major arboviruses of public health concern including dengue viruses. The relationships between Aedes infestation and disease transmission are complex wherein the epidemiological dynamics can be difficult to discern because of a lack of robust and sensitive indicators for predicting transmission risk. This study investigates the use of anti-Aedes saliva antibodies as a serological biomarker for Aedes mosquito bites to assess small scale variations in adult Aedes density and dengue virus (DENV) transmission risk in northeastern Thailand. Individual characteristics, behaviors/occupation and socio-demographics, climatic and epidemiological risk factors associated with human-mosquito exposure are also addressed.MethodsThe study was conducted within a randomized clustered control trial in Roi Et and Khon Kaen provinces over a consecutive 19 months period. Thirty-six (36) clusters were selected, each of ten houses. Serological and entomological surveys were conducted in all houses every four months and monthly in three sentinel households per cluster between September 2017 and April 2019 for blood spot collections and recording concurrent immature and adult Aedes indices. Additionally, the human exposure to Aedes mosquito bites (i.e., Mosquito Exposure Index or MEI) was estimated by ELISA measuring levels of human antibody response to the specific Nterm-34 kDa salivary antigen. The relationships between the MEI, vector infestation indices (adult and immature stages) and vector DENV infection were evaluated using a two-level (house and individual levels) mixed model analysis with one-month lag autoregressive correlation.ResultsThere was a strong positive relationship between the MEI and adult Aedes (indoor and outdoor) density. Individuals from households with a medium mosquito density (mean difference: 0.091, p<0.001) and households with a high mosquito density (mean difference: 0.131, p<0.001) had higher MEI’s compared to individuals from households without Aedes. On a similar trend, individuals from households with a low, medium or high indoor Aedes densities (mean difference: 0.021, p<0.007, 0.053, p<0.0001 and 0.037, p<0.0001 for low, medium and high levels of infestation, respectively) had higher MEI than individuals from houses without indoor Aedes. The MEI was driven by individual characteristics, such as gender, age and occupation/behaviors, and varied according to climatic, seasonal factors and vector control intervention (p<0.05). Nevertheless, the study did not demonstrate a clear correlation between MEI and the presence of DENV-infected Aedes.ConclusionThis study represents an important step toward the validation of the specific IgG response to the Aedes salivary peptide Nterm-34kDa as a proxy measure for Aedes infestation levels and human-mosquito exposure risk in a dengue endemic setting. The use of the IgG response to the Nterm-34 kDa peptide as a viable diagnostic tool for estimating dengue transmission requires further investigations and validation in other geographical and transmission settings.     

Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Two types of binding sites for hexokinase, designated as Type A or Type B sites, have been shown to coexist on brain mitochondria. The ratio of these sites varies between species.HK1 attaches by reversibly binding to the voltage dependent anion channel (VDAC). Regarding the nature of hexokinase binding sites, we investigated if it was linked to distinct VDAC interactomes. We approached this question by 2D BN/SDS-PAGE of mitochondria, followed by mass spectrometry.Our results are consistent with the possibility that the ratio of Type A/Type B sites is due to differential VDAC interactions in bovine and rat neuronal cells.     

Vitamins K interact with N-terminus α-synuclein and modulate the protein fibrillization in vitro. Exploring the interaction between quinones and α-synuclein

In the last decades, a series of compounds, including quinones and polyphenols, has been described as having anti-fibrillogenic action on α-synuclein (α-syn) whose aggregation is associated to the pathogenesis of Parkinson’s disease (PD). Most of these molecules act as promiscuous anti-amyloidogenic agents, interacting with the diverse amyloidogenic proteins (mostly unfolded) through non-specific hydrophobic interactions. Herein we investigated the effect of the vitamins K (phylloquinone, menaquinone and menadione), which are 1,4-naphthoquinone (1,4-NQ) derivatives, on α-syn aggregation, comparing them with other anti-fibrillogenic molecules such as quinones, polyphenols and lipophilic vitamins. Vitamins K delayed α-syn fibrillization in substoichiometric concentrations, leading to the formation of short, sheared fibrils and amorphous aggregates, which are less prone to produce leakage of synthetic vesicles. In seeding conditions, menadione and 1,4-NQ significantly inhibited fibrils elongation, which could be explained by their ability to destabilize preformed fibrils of α-syn. Bidimensional NMR experiments indicate that a specific site at the N-terminal α-syn (Gly31/Lys32) is involved in the interaction with vitamins K, which is corroborated by previous studies suggesting that Lys is a key residue in the interaction with quinones. Together, our data suggest that 1,4-NQ, recently showed up by our group as a potential scaffold for designing new monoamine oxidase inhibitors, is also capable to modulate α-syn fibrillization in vitro.     

Territory Availability at Different Spatial Scales does not Affect Fighting Investment in Males of the Lycaenid Butterfly Strymon mulucha

In territorial species, rivals investment in fights over territories may increase when the availability of suitable areas for defense is low. This should occur because low territory availability may increase the costs to maintain and acquire territories. Although such process occurs in small spatial scales (local scale), territory availability in larger scales (regional scale) may also affect fighting investment, as losers should incur additional dispersing costs to find new territories. In this study, we used males of the hilltopping butterfly Strymon mulucha to evaluate the hypothesis that males should invest more in territorial fights when the costs to find new territories are higher (both at local and at regional scale). We timed male–male contests for territories located in 12 hilltops and measured male density per territory in each hilltop (local scale). We also quantified the distance between hilltops containing suitable areas for territories (regional scale). Male–male contests lasted 21 s on average, and copulations did not occur during the observations. The duration of contests was unrelated to the male density per territory or to the distance among hilltops, indicating that the investment in fights was unaffected by the availability of territorial sites, independent of the spatial scale. As male–male contests in S. mulucha are longer than the mean contest duration in other butterfly species and mating is extremely rare, we suggest that the value of each territory may be high enough to favor males that always invest as much as possible in contests.