Evolutionary assembly of cooperating cell types in an animal chemical defense system

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Involvement of Kv1.3 and p38 MAPK signaling in HIV-1 glycoprotein 120-induced microglia neurotoxicity

Inflammatory responses mediated by activated microglia play a pivotal role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders. Studies on identification of specific targets to control microglia activation and resultant neurotoxic activity are imperative. Increasing evidence indicate that voltage-gated K⁺ (K_v) channels are involved in the regulation of microglia functionality. In this study, we investigated K_v1.3 channels in the regulation of neurotoxic activity mediated by HIV-1 glycoprotein 120 (gp120)-stimulated rat microglia. Our results showed treatment of microglia with gp120 increased the expression levels of K_v1.3 mRNA and protein. In parallel, whole-cell patch-clamp studies revealed that gp120 enhanced microglia K_v1.3 current, which was blocked by margatoxin, a K_v1.3 blocker. The association of gp120 enhancement of K_v1.3 current with microglia neurotoxicity was demonstrated by experimental results that blocking microglia K_v1.3 attenuated gp120-associated microglia production of neurotoxins and neurotoxicity. Knockdown of K_v1.3 gene by transfection of microglia with K_v1.3-siRNA abrogated gp120-associated microglia neurotoxic activity. Further investigation unraveled an involvement of p38 MAPK in gp120 enhancement of microglia K_v1.3 expression and resultant neurotoxic activity. These results suggest not only a role K_v1.3 may have in gp120-associated microglia neurotoxic activity, but also a potential target for the development of therapeutic strategies.     

Identification of a GM1-Binding Protein on the Surface of Murine Neuroblastoma Cells

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.     

Les Pseudholaster (Echinoidea,Holasteroida) du Crétacé de France

In the order of Holasteroida, the fossil record highlights a contradiction between the genus Pseudholaster that appears in the Aptian, whose plastron is prostostern close to the Jurassic ancestors and the genus Holaster, which appears in the Valanginian, whose meridostern plastron appears more derived. This inconsistency can be explained by the ignorance of the plastronal architecture on the part of the early authors. A review of the species of Pseudholaster from the Cretaceous period of France was therefore carried out. The objective was to statistically determine the discriminating morphological characters, and to study the modifications of the architecture of the interambulacrum 5 of the French species belonging to this genus, as well as to the species included in the genus Holaster incorrectly by earlier authors. This review of the species of the genus Pseudholaster begins with a study of the ontogeny of the species Holaster intermedius Münster in Goldfuss, 1826–1833, first representative of the genus Pseudholaster, which appears in the Hauterivian in the Parisian and Rhodano-vocontian basins. The modifications during growth concern the overall shape, but also the plastron architecture: the number of plastron plates increases while the number of plates located between the peristome and the periproct remains fixed. The plastron of this species is protosternal and not meridosternal as Lambert pointed out. The labrum is cupuliform in contact with the second sternal 5a2 by a narrow digitation. However, this arrangement differs from that observed on a protosternal breastplate. This apomorphism of the plastron plate pattern, called “labrotaxienne”, is found in all the Pseudholaster studied, and the study of the architecture of the interambulacrum 5 also reveals a gradual decrease in the number of preanal plates between the oldest (Hauterivian) and the younger (Cenomanian-Lower Turonian) species studied. Most of the French species have been revised, with some synonyms. A new species, P. neraudeaui, is the last known Pseudholaster dated from the upper Cenomanian and lower Turonian of southwestern France. Our study illustrates the evolution of the genus Pseudholaster between the Hauterivian and the early Turonian in France. The interest of the study is to show that the appearance of the genus Pseudholaster is older than that of the genus Holaster. Pseuholaster intermedius, of Hauterivian age, possesses a derived protostern plastron called here “labrotaxien” and not meridostern as defined historically by Lambert, and to reveal that the number of preanals decreases over geological time. This data is essential for future phylogenetic studies. On a palaeobiogeographical level, the study reveals the expansion of the genus Pseudholaster during early Cretaceous in western Europe, with diversification during the Albian, its disappearance during late Cenomanian in the Paris basin while it still persists in the Aquitain basin, its predilection for circalitoral environments.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 (∼ 90 μM). Their IC₅₀ values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon’s plot; however, the inhibition mechanism of endometase was noncompetitive with a K_i value of 240 μM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.     

Biotic and abiotic determinants of the formation of ant mosaics in primary Neotropical rainforests

1. Ants are widespread in tropical rainforests, including in the canopy where territorially dominant arboreal species represent the main part of the arthropod biomass. 2. By mapping the territories of dominant arboreal ant species and using a null model analysis and a pairwise approach this study was able to show the presence of an ant mosaic on the upper canopy of a primary Neotropical rainforest (c. 1 ha sampled; 157 tall trees from 28 families). Although Neotropical rainforest canopies are frequently irregular, with tree crowns at different heights breaking the continuity of the territories of dominant ants, the latter are preserved via underground galleries or trails laid on the ground. 3. The distribution of the trees influences the structure of the ant mosaic, something related to the attractiveness of tree taxa for certain arboreal ant species rather than others. 4. Small‐scale natural disturbances, most likely strong winds in the area studied (presence of canopy gaps), play a role by favouring the presence of two ant species typical of secondary formations: Camponotus femoratus and Crematogaster levior, which live in parabiosis (i.e. share territories and nests but lodge in different cavities) and build conspicuous ant gardens. In addition, pioneer Cecropia myrmecophytic trees were recorded.     

M(en)TORship lessons on life and death by the integrated stress response

Cells employ pro-survival and pro-adaptive pathways to cope with different forms of environmental stress. When stress is excessive, and the damage caused by it is unsustainable, cells engage pro-death pathways, which are in place to protect the host from the deleterious effects of harmed cells. Two important pathways that determine the balance between survival and death of stressed cells are the integrated stress response (ISR) and the mammalian target of rapamycin (mTOR), both of which converge at the level of mRNA translation. The two pathways have established avenues of communication to control their activity and determine the fate of stressed cells in a context-dependent manner. The functional interplay between the ISR and mTOR may have significant ramifications in the development and treatment of human diseases such as diabetes, neurodegeneration and cancer.     

MiR-204 regulates type 1 IP3R to control vascular smooth muscle cell contractility and blood pressure

MiR-204 is expressed in vascular smooth muscle cells (VSMC). However, its role in VSMC contraction is not known. We determined if miR-204 controls VSMC contractility and blood pressure through regulation of sarcoplasmic reticulum (SR) calcium (Ca²⁺) release. Systolic blood pressure (SBP) and vasoreactivity to VSMC contractile agonists (phenylephrine (PE), thromboxane analogue (U46619), endothelin-1 (ET-1), angiotensin-II (Ang II) and norepinephrine (NE) were compared in aortas and mesenteric resistance arteries (MRA) from miR-204^−/− mice and wildtype mice (WT). There was no difference in basal systolic blood pressure (SBP) between the two genotypes; however, hypertensive response to Ang II was significantly greater in miR-204^−/− mice compared to WT mice. Aortas and MRA of miR-204^−/− mice had heightened contractility to all VSMC agonists. In silico algorithms predicted the type 1 Inositol 1, 4, 5-trisphosphate receptor (IP₃R1) as a target of miR-204. Aortas and MRA of miR-204^−/− mice had higher expression of IP₃R1 compared to WT mice. Difference in agonist-induced vasoconstriction between miR-204^−/− and WT mice was abolished with pharmacologic inhibition of IP₃R1. Furthermore, Ang II-induced aortic IP₃R1 was greater in miR-204^−/− mice compared to WT mice. In addition, difference in aortic vasoconstriction to VSMC agonists between miR-204^−/− and WT mice persisted after Ang II infusion. Inhibition of miR-204 in VSMC in vitro increased IP₃R1, and boosted SR Ca²⁺ release in response to PE, while overexpression of miR-204 downregulated IP₃R1. Finally, a sequence-specific nucleotide blocker that targets the miR-204-IP₃R1 interaction rescued miR-204-induced downregulation of IP₃R1. We conclude that miR-204 controls VSMC contractility and blood pressure through IP₃R1-dependent regulation of SR calcium release.     

An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.