Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis

The conservation of fold and chemistry of the enzymes associated with histidine biosynthesis suggests that this pathway evolved prior to the diversification of Bacteria, Archaea, and Eukaryotes. The only exception is the histidinol phosphate phosphatase (HolPase). So far, non-homologous HolPases that possess distinct folds and belong to three different protein superfamilies have been identified in various phylogenetic clades. However, their evolution has remained unknown to date. Here, we analyzed the evolutionary history of the HolPase from γ-Proteobacteria (HisB-N). It has been argued that HisB-N and its closest homologue d -glycero-d -manno-heptose-1,7-bisphosphate 7-phosphatase (GmhB) have emerged from the same promiscuous ancestral phosphatase. GmhB variants catalyze the hydrolysis of the anomeric d -glycero-d -manno-heptose-1,7-bisphosphate (αHBP or βHBP) with a strong preference for one anomer (αGmhB or βGmhB). We found that HisB-N from Escherichia coli shows promiscuous activity for βHBP but not αHBP, while βGmhB from Crassaminicella sp. shows promiscuous activity for HolP. Accordingly, a combined phylogenetic tree of αGmhBs, βGmhBs, and HisB-N sequences revealed that HisB-Ns form a compact subcluster derived from βGmhBs. Ancestral sequence reconstruction and in vitro analysis revealed a promiscuous HolPase activity in the resurrected enzymes prior to functional divergence of the successors. The following increase in catalytic efficiency of the HolP turnover is reflected in the shape and electrostatics of the active site predicted by AlphaFold. An analysis of the phylogenetic tree led to a revised evolutionary model that proposes the horizontal gene transfer of a promiscuous βGmhB from δ- to γ-Proteobacteria where it evolved to the modern HisB-N.     

Hatching plasticity in a Southeast Asian tree frog mitigates submergence-induced mortality

Environmentally cued hatching has been well-documented in amphibians in response to a wide range of abiotic and biotic factors. The hatching of terrestrial amphibian eggs in response to flooding may be basal within the group, but amphibian lineages in tropical Asia and sub-Saharan Africa have not received as much attention as their Neotropical counterparts. We investigated submergence-induced hatching in Feihyla hansenae, a Rhacophorid tree frog with terrestrial eggs. We quantified natural rates of clutch submergence at our study site in Thailand. Using submergence experiments, we found that embryos are capable of hatching early to escape flooding, and that failure to hatch results in mortality. Among the embryos that were able to hatch early, only the earliest, youngest hatchlings experienced a trade-off in body size that persisted for 6 days, while later, older hatchlings were not significantly smaller than spontaneous hatchlings under control conditions. By incorporating our natural and experimental data into Monte Carlo methods to simulate and compare survival probabilities with and without hatching plasticity, we found an overall 3.1% increase in submergence survival due to hatching plasticity. Our findings support the idea that flooding-induced hatching is widespread across amphibians with terrestrial eggs and highlight the importance of researching understudied tropical regions. As climate change is projected to affect rainfall patterns, the ability of embryos to escape abiotic egg-stage threats may be an indicator of species' ability to flexibly navigate a changing environment.     

Aluminum Toxicity: A Case Study on Tobacco (<i>Nicotiana tabacum</i> L.)

Aluminum is an abundant metal in the earth’s crust that turns out to be toxic in acidic environments. Many plants are affected by the presence of aluminum at the whole plant level, at the organ level, and at the cellular level. Tobacco as a cash crop (Nicotiana tabacum L.) is a widely cultivated plant worldwide and is also a good model organism for research. Although there are many articles on Al-phytotoxicity in the literature, reviews on a single species that are economically and scientifically important are limited. In this article, we not only provide the biology associated with tobacco Al-toxicity, but also some essential information regarding the effects of this metal on other plant species (even animals). This review provides information on aluminum localization and uptake process by different staining techniques, as well as the effects of its toxicity at different compartment levels and the physiological consequences derived from them. In addition, molecular studies in recent years have reported specific responses to Al toxicity, such as overexpression of various protective proteins. Besides, this review discusses data on various organelle-based responses, cell death, and other mechanisms, data on tobacco plants and other kingdoms relevant to these studies.     

Assessment of capybara (Hydrochoerus hydrochaeris) populations in the wetlands of Corrientes,Argentina

Ten sampling sites were selected to represent six distinct habitat types used by capybaras (clean lagoons, dirty lagoons, cutwaters, fens and marshes, gallery forests, and erosion ditches). The sites were sampled during winter (July and August); densities were expressed as number of capybaras per linear km of shoreline (C/LKS). The sites were classified as protected from poachers (P), under light hunting pressure (LHP), and under heavy hunting pressure (HHP). Clean protected (P) lagoons had three times as many capybaras as LHP ones (30.7 and 10.9 C/LKS, respectively), and thirty times those under HHP (1.0 C/LKS). Protected marshes and dirty lagoons had even higher capybara densities (52.5 and 50.0 C/LKS, respectively). Gallery forests under LHP had low densities (6.3 C/LKS), and protected cutwaters intermediate densities (27.5 C/LKS). Erosion ditches had exceptionally high densities (900 C/LKS), probably because cattle were fenced out, reducing forage competition. These densities, when converted to the standard unit area measurement (individuals/ha), were similar to those obtained by other researchers in the Brazilian Pantanal, and somewhat smaller than those in the Venezuelan Llanos. Mean number of capybaras per group remained relatively constant in all habitats (averages ranged between 9.2 and 11.8 individuals/group) but its coefficient of variation was much higher in LHP sites, probably because social structure was altered severely by hunting. The overall ratio of young to adults and juveniles was 1:7.4. In one of the sites, 13 of 34 groups (38.2%) were with young (average of 17 capybaras per group, 4.7 of which were young), confirming that this species can reproduce all year long.Requests for reprints should be sent to: Dr. J. Rabinovich.     

Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation.

Tumor necrosis factor-alpha converting enzyme (TACE or ADAM17) is a member of the ADAM (a disintegrin and metalloproteinase) family of type I membrane proteins and mediates the ectodomain shedding of various membrane-anchored signaling and adhesion proteins. TACE is synthesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. We have identified the proprotein-convertases PC7 and furin to be involved in maturation of TACE. This maturation is negatively influenced by the phorbol ester phorbol-12-myristate-13-acetate (PMA), which decreases the cellular amount of the mature form of TACE in PMA-treated HEK293 and SH-SY5Y cells. Furthermore, we found that stimulation of protein kinase C or protein kinase A signaling pathways did not influence long-term degradation of mature TACE. Interestingly, PMA treatment of furin-deficient LoVo cells did not affect the degradation of mature TACE. By examination of furin reconstituted LoVo cells we were able to exclude the possibility that PMA modulates furin activity. Moreover, the PMA dependent decrease of the mature enzyme form is specific for TACE, as the amount of mature ADAM10 was unaffected in PMA-treated HEK293 and SH-SY5Y cells. Our results indicate that the activation of TACE by the proprotein-convertases PC7 and furin is very similar to the maturation of ADAM10 although there is a significant difference in the cellular stability of the mature enzyme forms after phorbol ester treatment.     

Complete subunit sequences, structure and evolution of the 6 x 6-mer hemocyanin from the common house centipede, Scutigera coleoptrata.

Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6 x 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5-55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 x 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata.     

Selenoprotein P associates with endothelial cells in rat tissues

 Selenoprotein P is an extracellular heparin-binding protein that has been implicated in protecting the liver against oxidant injury. Its location in liver, kidney, and brain was determined by conventional immunohistochemistry and confocal microscopy using a polyclonal antiserum. Selenoprotein P is associated with endothelial cells in the liver and is more abundant in central regions than in portal regions. It is also present in kidney glomeruli associated with capillary endothelial cells. Staining of selenoprotein P in the brain is also confined to vascular endothelial cells. The heparin-binding properties of selenoprotein P could be the basis for its binding to tissue. Its localization to the vicinity of endothelial cells is potentially relevant to its oxidant defense function. Accepted: 6 March 1997     

Identification of Ser-Pro and Thr-Pro Phosphorylation Sites in Chicken Neurofilament-M Tail Domain

Abstract: The tail domain of the midsize chicken neurofilament polypeptide (NF-M) contains several different types of Ser-Pro and Thr-Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [³²P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF-M was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys-C, or endoprotease Glu-C, and peptides separated by two-dimensional high-voltage electrophoresis and thin-layer chromatography. ³²P-labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser-Pro and Thr-Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys-Ser-Pro sites and at least 16 of the 17 Lys-Xaa-Xaa-Ser/Thr-Pro repeats. In addition, a novel Ser-Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF-M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino-terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF-M tail.     

Endogenous prostaglandin secretion during cloprostenol-induced abortion in mares

Repeated administration of prostaglandin is the treatment of choice for the termination of pregnancy in mares more than 40 days pregnant. Even though it is well documented that PGF-2 or analogue needs to be administered every 12–24 h for successful induction of abortion, little is known about the underlying endocrine changes and the mechanism by which abortion occurs. The aim of this study was to characterize the changes in PGF-2, progesterone and estrogen secretion during prostaglandin-induced abortion. Six mares, 82–102 days pregnant, were treated daily with 250 μg cloprostenol, blood was collected at 1-h intervals until fetal expulsion and pregnancy examination was performed daily. Four mares, 92–97 days pregnant, received no treatment but were subjected to the same hourly blood collections and daily genital examinations described for cloprostenol-treated mares for 3 days. Mean time from first cloprostenol administration until fetal expulsion was 48.6 ± 5.6 h and required 2.8 ± 0.2 cloprostenol administrations. In all mares, progesterone concentrations decreased in a near linear manner after the first cloprostenol administration and were invariably low (1.3 ± 0.2 ng ml⁻¹, mean ± SEM) at the time of fetal expulsion. Mean estrogen secretion remained unchanged until 5 h before fetal expulsion and then decreased rapidly to non-pregnant levels. Endogenous PGF-2 secretion rate increased with each cloprostenol administration and culminated in sustained PGF-2 secretion which persisted until fetal expulsion was completed. From these results we conclude that cloprostenol-induced abortion is associated with endogenous PGF-2 secretion, fetal expulsion coincides with sustained PGF-2 secretion and low progesterone concentrations and plasma estrogen concentrations remain unchanged until hours before fetal expulsion.     

Chemical characterization of two lipopolysaccharide species isolated from Rhizobium loti NZP2213

Phenol-water extraction of Rhizobium loti NZP2213 cells allowed a simultaneous isolation of two structurally different lipopolysaccharides, from the aqueous (LPS-W) and phenol (LPS-P) phase that differed in their sodium doexycholate-PAGE pattern and composition. LPS-W showed a profile indicating an R-type LPS; LPS-P had a cluster of poorly resolved bands in the high-molecular-weight region. LPS-P contained large amounts of 6-deoxy-l-talose (6dTal), and a small amount of 2-O-methyl-6-deoxy-talose (molar ratio 30:1), both of which were completely absent in LPS-W. Methylation analysis gave only one major product, 2,4-di-O-methyl-6dTal, indicating that the O-chain is composed of a homopolymer of 1,3-linked 6dTal, having the methylated 6dTal (2-O-me-6dTal) probably localized at the non-reducing end of the O-chain. This homopolymeric O-chain was additionally O-acetylated, as evidenced by GC-MS and by ¹³C NMR analysis. The lipid A moieties of both LPS-W and LPS-P showed almost identical composition, with six, different 3-OH fatty acids and with two, so far not described, long-chain 4-oxo-fatty acids, all being amide-linked, and with 27-OH-28:0 as the main ester-linked fatty acid. Lipid A was of the lipid A_DAG-type, i.e., having a (phosphorylated) 2,3-diamino-2,3-dideoxy-d-glucose-containing lipid A backbone. Lipid A_DAG is widespread among species of the -2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.