An Ancient Origin for the Enigmatic Flat-Headed Frogs (Bombinatoridae: Barbourula) from the Islands of Southeast Asia

Background

The complex history of Southeast Asian islands has long been of interest to biogeographers. Dispersal and vicariance events in the Pleistocene have received the most attention, though recent studies suggest a potentially more ancient history to components of the terrestrial fauna. Among this fauna is the enigmatic archaeobatrachian frog genus Barbourula, which only occurs on the islands of Borneo and Palawan. We utilize this lineage to gain unique insight into the temporal history of lineage diversification in Southeast Asian islands.

Methodology/Principal Findings

Using mitochondrial and nuclear genetic data, multiple fossil calibration points, and likelihood and Bayesian methods, we estimate phylogenetic relationships and divergence times for Barbourula. We determine the sensitivity of focal divergence times to specific calibration points by jackknife approach in which each calibration point is excluded from analysis. We find that relevant divergence time estimates are robust to the exclusion of specific calibration points. Barbourula is recovered as a monophyletic lineage nested within a monophyletic Costata. Barbourula diverged from its sister taxon Bombina in the Paleogene and the two species of Barbourula diverged in the Late Miocene.

Conclusions/Significance

The divergences within Barbourula and between it and Bombina are surprisingly old and represent the oldest estimates for a cladogenetic event resulting in living taxa endemic to Southeast Asian islands. Moreover, these divergence time estimates are consistent with a new biogeographic scenario: the Palawan Ark Hypothesis. We suggest that components of Palawan''s terrestrial fauna might have “rafted” on emergent portions of the North Palawan Block during its migration from the Asian mainland to its present-day position near Borneo. Further, dispersal from Palawan to Borneo (rather than Borneo to Palawan) may explain the current day disjunct distribution of this ancient lineage.     

Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.     

Response of antioxidant systems in oxygen deprived suspension cultures of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The effect of oxygen deprivation (anoxia) on the antioxidant system in suspension culture of anoxia-intolerant Malaysian rice mutants cells was examined. Abiotic stresses have been reported to adversely affect cell division, damage cellular and organelle membranes. The signaling defense mechanisms, such as molecular and biochemical aspects responding to stress have been proven to be very complex, and still largely untapped. The objective of this study was to determine the potential involvement of activated oxygen species, such as superoxide dismutase, catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase which occur in cells of rice plants exposed to anoxia stress in two Malaysian rice mutants, MR219-4 and MR219-9, and rice cultivar FR13A which is known to be tolerant to anoxia stress during 5–30 days of exposure. The antioxidative enzymes were decreased for MR219-4 and MR219-9 mutants for CAT and APX activities, and increased in FR13A cultivar starting at 20 days in suspension culture compared to that of control. CAT and APX activities were maintained higher in anoxia condition for all mutants and cultivar. These findings suggested that anoxia stress in suspension cultures induced the level of H₂O₂ to toxic levels.     

Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms

Background

Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.

Methodology/Principal Findings

Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10⁻⁶) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10⁴ CFU/mL) than M. tuberculosis (42×10¹ CFU/mL) and M. bovis (35×10¹ CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.

Conclusions/Significance

These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.     

Use of the H,K-ATPase beta subunit to identify multiple sorting pathways for plasma membrane delivery in polarized cells

A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase beta subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the beta subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase beta subunits associate with the endogenous Na,K-ATPase alpha(1) subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase beta subunit with the Na,K-ATPase alpha(1) subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the beta subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase beta subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the beta subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric beta subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase beta subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis.     

Electromagnetic Interference in a Private Swimming Pool: Case report

Although current lead design and filtering capabilities have greatly improved, Electromagnetic Interference (EMI) from environmental sources has been increasingly reported in patients with Cardiac Implantable Electronic Device (CIED) [1]. Few cases of inappropriate intracardiac Cardioverter Defibrillator (ICD) associated with swimming pool has been described [2]. Here we present a case of 64 year old male who presented with an interesting EMI signal that was subsequently identified to be related to AC current leak in his swimming pool.     

The genus Thermocyclops (Crustacea: Copepoda) in Uzbekistan (Central Asia)

Five Thermocyclops species were found in Uzbekistan: T. crassus, T. dybowskii, T. rylovi, T. taihokuensis, and T. vermifer. The last species has been for the first time recorded in the region. The data on morphology, variability and distribution of these species in Uzbekistan are given. A key to determine to the five species is proposed.