Physiological and morphological leaf trait variations in two Apennine plant species in response to different altitudes

Morphological and physiological traits of Crepis pygmaea L. subsp. pygmaea and Isatis apennina Ten. ex Grande growing at different altitudes in the Gran Sasso Massif (Abruzzo, Italy) were analyzed. The two populations of C. pygmaea and I. apennina growing at the highest altitude (C _p2 and I _p2 at 2,310 m a.s.l. and 2,350 m a.s.l., respectively) had a lower leaf mass area (LMA) than the two populations growing at the lowest altitude (C _p1 and I _p1 at 2,250 m a.s.l. and 2,310 m a.s.l., respectively). Leaf tissue density (LTD) had the same LMA trend, decreasing 23 and 10% in C. pygmaea and I. apennina, respectively, from the highest to the lowest altitude. C. pygmaea and I. apennina had the highest photosynthetic rates (P _N) in July decreasing on an average 17 and 30%, respectively, in August and 50 and 38%, respectively, in September. Leaf respiration (R) in I _p1 and I _p2 had the same trend as C _p1 and C _p2, showing the highest rates in September. Global warming could drive C. pygmaea and I. apennina toward higher altitudes in the Gran Sasso Massif. Nevertheless, C. pygmaea with the higher plasticity index (PI) both at physiological and at morphological levels (0.50 and 0.35, respectively) might have a competitive advantage over I. apennina over the long term.     

A new family of Cdc42 effector proteins, CEPs, function in fibroblast and epithelial cell shape changes

Cdc42, a Rho GTPase, regulates the organization of the actin cytoskeleton by its interaction with several distinct families of downstream effector proteins. Here, we report the identification of four new Cdc42-binding proteins that, along with MSE55, constitute a new family of effector proteins. These molecules, designated CEPs, contain three regions of homology, including a Cdc42 binding domain and two unique domains called CI and CII. Experimentally, we have verified that CEP2 and CEP5 bind Cdc42. Expression of CEP2, CEP3, CEP4, and CEP5 in NIH-3T3 fibroblasts induced pseudopodia formation. Fibroblasts coexpressing dominant negative Cdc42 with CEP2 or expressing a Cdc42/Rac interactive binding domain mutant of CEP2 did not induce pseudopodia formation. In primary keratinocytes, CEP2- and CEP5-expressing cells showed reduced F-actin localization at the adherens junctions with an increase in thin stress fibers that extended the length of the cell body. Keratinocytes expressing CEPs also showed an altered vinculin distribution and a loss of E-cadherin from adherens junctions. Similar effects were observed in keratinocytes expressing constitutively active Cdc42, but were not seen with a Cdc42/Rac interactive binding domain mutant of CEP2. These results suggest that CEPs act downstream of Cdc42 to induce actin filament assembly leading to cell shape changes.     

Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.     

La flora del litorale pescarese come indicatore biologico dello stato ambientale e delle sue trasformazioni

Abstract

The littoral flora of Pescara as a biological indicator of the environmental status and its modifications.—The flora of the Pescara littoral, and the vegetation, limited now to restricted areas in the outskirts, mainly near the mouth of the Saline river, and to the dunes, have been studied.

Three hundred and six entities have been recorded and the abundance, the distribution, the biological forms and the phytogeographical elements have been described.

The biological spectrum (P = 6, 14%; Ch = 5,41%; H = 32,5%; G = 10,46%; T = 45,49%) confirms a marked Mediterranean character.

The considerable amount of trees and shrubs is due to the introduction of breakwinds and ornamental species.

The analysis of the chorological elements confirms the dominance of the Mediterranean species; the ruderal polycore entities with the occasional naturalized alien species, reach about 25%. At Pescara their remarkable presence, which gives to the landscape the feature of a slum area, is the result of a great environmental degradation due to a deep anthropisation. The entities previously recorded for Pescara are also reported; probable causes of the disappearance of some species or of immediate danger for others, have been indicated according to U.I.C.N.

Si riferisce su uno studio sulla flora del litorale Pescarese e sulle suo medificazioni, con cenni sulla vegetazione naturale residua, limitata a ristrette fasce dunose alla periferia di Pescara ed allo ambiente deltiziale del fiume Saline. Delle entità censite (306) si riportano: la distribuzione nel territorio, l'abbondanza, la forma biologica e l'elemento fitogeografico. Preliminarmente, inoltre, sono accennate le caratteristiche climatiche della zona.

Lo spettro biologico (P = 6,14; Ch = 5,41; H = 32,5; G = 10,46; Th = 45,49%) conferma una spiccata tendenza mediterranea. La rimarchevole presenza di emicrittofite e geofite è in dipendenza di canali e zone acquitrinose; la non trascurabile quantità di alberi ed arbusti va attribuita non tanto alla consistenza arborea naturale, bensì alla introduzione di specie frangivento ed ornamentali.

Viene riportato poi il quadro degli elementi corologici, nel quale predominano le entità mediterranee s.1. (50,54%).

Una considerazione a parte meritano le entità policore legate agli ambienti ruderali e le avventizie, componenti che, nell'insieme, assommano a ben il 25% circa. A Pescara la loro massiccia presenza conferisce al paesaggio un'impronta di ambiente ruderale ed è conseguenza dell'alto grado di degradazione ambientale determinato dalla massiccia antropizzazione.

Si riportano, infine, le entità gia segnalate per Pescara e non rinvenute, indicando per ciascuna la probabile causa che ne ha determinato la scomparsa, e quelle in pi[ugrave] immediato pericolo di estinzione, con a fianco di ognuna la causa del pericolo, secondo i criteri dell'U.I.C.N.     

Molecular organization of the cullin E3 ligase adaptor KCTD11

The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (lKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that lKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and lKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than lKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and lKCTD11 and their individual domains in Escherichia coli yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both lKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for lKCTD11 BTB and for its complex with Cul3. These analyses indicate that a stable lKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in lKCTD11-Cul3 recognition.     

Sulfolobus solfataricus thiol redox puzzle: characterization of an atypical protein disulfide oxidoreductase

Protein disulfide oxidoreductases (PDOs) are proteins involved in disulfide bond formation playing a crucial role in adaptation to extreme environment. This paper reports the functional and structural characterization of Sso1120, a PDO from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was expressed in Escherichia coli and purified to homogeneity. The functional characterization showed that the enzyme has reductase activity, as tested by insulin assay, but differently from the other PDOs, it does not present isomerase activity. In addition it is able to form a redox couple with the thioredoxin reductase that could be used in undiscovered pathways. The protein revealed a melting point of around 90 °C in CD spectroscopy-monitored thermal denaturation and high denaturant resistance. The X-ray crystallographic structure was solved at 1.80 Å resolution, showing differences with respect to other PDOs and an unexpected similarity with the N-terminal domain of the alkyl hydroperoxide reductase F component from Salmonella typhimurium. On the basis of the reported data and of bioinformatics and phylogenetic analyses, a possible involvement of this atypical PDO in a new antioxidant system of S. solfataricus has been proposed.     

Molecular recognition of Cullin3 by KCTDs: Insights from experimental and computational investigations

Recent investigations have highlighted a key role of the proteins of the KCTD (K-potassium channel tetramerization domain containing proteins) family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptor in E3 ligases during protein ubiquitination. Here we present a characterization of the KCTD5-Cullin3 interactions which are mediated by the KCTD5 BTB domain. Isothermal titration calorimetry experiments reveal that KCTD5 avidly binds the Cullin3 (Cul3). The complex presents a 5:5 stoichiometry and a dissociation constant of 59 nM. Molecular modeling and molecular dynamics simulations clearly indicate that the two proteins form a stable (KCTD5–Cul3)₅ pinwheel-shaped heterodecamer in which two distinct KCTD5 subunits cooperate in the binding of each cullin chain. Molecular dynamics simulations indicate that different types of interactions contribute to the stability of the assembly. Interestingly, residues involved in Cul3 recognitions are conserved in the KCTD5 orthologs and paralogs implicated in important biological processes. These residues are also rather well preserved in most of the other KCTD proteins. By using molecular modeling techniques, the entire ubiquitination system including the E3 ligase, the E2 conjugating enzyme and ubiquitin was generated. The analysis of the molecular architecture of this complex machinery provides insights into the ubiquitination processes which involve E3 ligases with a high structural complexity.     

Cullin 3 Recognition Is Not a Universal Property among KCTD Proteins

Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6^BTB and KCTD11^BTB bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12^BTB and KCTD15^BTB do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of α2β3 loop between KCTD11^BTB and KCTD12^BTB is sufficient to abolish the ability of KCTD11^BTB to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.     

Serotonin receptor of type 6 (5-HT6) in human prefrontal cortex and hippocampus post-mortem: An immunohistochemical and immunofluorescence study

Given the paucity of data on the distribution of serotonin (5-HT) receptors of type 6 (5-HT₆) in the human brain, the aim of this study was to investigate their distribution in postmortem human prefrontal cortex, striatum and hippocampus by either immunohistochemical or immunofluorescence techniques.