Biochemical Properties of Gastrokine-1 Purified from Chicken Gizzard Smooth Muscle

The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractile properties.

Background

GNK1 is thought to provide mucosal protection in the superficial gastric epithelium. However, the actual role of gastrokine-1 with regards to its known decreased expression in gastric cancer is still unknown. Recently, trefoil factors (TFF) were reported to have important roles in gastric epithelial regeneration and cell turnover, and could be involved in GNK1 interactions. The aim of this study was to evaluate the role and function of GNK1 in smooth muscle cells.

Methodology/Principal Findings

From fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions. The purified protein was used to produce polyclonal antibody and highlighted the smooth muscle cell distribution and F-actin association of GNK1 through a few different methods.

Conclusion/Significance

Altogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells. A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.     

Essential Oil Variation among Natural Populations of Lavandula multifida L. (Lamiaceae)

Volatiles from twelve wild Tunisian populations of Lavandula multifida L. growing in different bioclimatic zones were assessed by GC (RI) and GC/MS. Thirty‐six constituents, representing 83.48% of the total oil were identified. The major components at the species level were carvacrol (31.81%), β‐bisabolene (14.89%), and acrylic acid dodecyl ester (11.43%). These volatiles, together with α‐pinene, were also the main compounds discriminating the populations. According to these dominant compounds, one chemotype was revealed, a carvacrol/β‐bisabolene/acrylic acid dodecyl ester chemotype. However, a significant variation among the populations was observed for the majority of the constituents. A high chemical‐population structure, estimated both by principal component analysis (PCA) and unweighted pair group method with averaging (UPGMA) cluster analysis based on Euclidean distances, was observed. Both methods allowed separation of the populations in three groups defined rather by minor than by major compounds. The population groups were not strictly concordant with their bioclimatic or geographic location. Conservation strategies should concern all populations, because of their low size and their high level of destruction. Populations exhibiting particular compounds other than the major ones should be protected first.     

Chemical Composition of the Leaf and Flower Essential Oils of Tunisian Lavandula dentata L. (Lamiaceae)

Essential oils of Lavandula dentata, a Tunisian native plant, were isolated from leaves and flowers by hydrodistillation in a Clevenger‐type apparatus and characterized by GC‐FID and GC/MS analyses. The average essential oil yields, means of five replicates, were higher for the flowers (8.60 mg/g) than for the leaves (6.56 mg/g). A total of 72 compounds were identified, accounting for 98.1 and 97.7% of the total oil composition of the leaves and flowers, respectively. The main essential oil constituents were 1,8‐cineole, camphor, and L ‐fenchone, accounting for 33.54, 18.89, and 8.36% in the leaf oils and for 19.85, 23.33, and 7.13% in the flower oils, respectively. Besides this quantitative variation, the results also showed considerable qualitative variation between the essential oils of the two plant parts analyzed. These differences might be adaptative responses to ecological exigencies.     

Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., “MTMRs”). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.     

Genetic diversity of Lavandula multifida L. (Lamiaceae) in Tunisia: implication for conservation

The genetic variation within and among eight Tunisian natural populations of Lavandula multifida L., from different bioclimatic zones was assessed using random amplified polymorphic DNA (RAPDs). Of a total of 97 generated bands from seven selected primers, 84 bands were polymorphic. The genetic diversity within a population was high and varied according to the populations (0.308 < H’ < 0.459) without relationships to altitudes or pluviothermic indices of sites. The genetic differentiation among populations was high (G_ST = 0.395 and Φ_ST = 0.318). All population pairs were significantly differentiated. Among populations, within ecological groups genetic structure was high (0.219); whilst among them it was low (Φ_CT = 0.049; P < 0.05). The correlation between Φ_ST and geographic distance matrices among pairs of populations was not significant, suggesting that genetic connectivity between populations has a stochastic component at all spatial scales. The neighbour‐joining cluster analysis showed that individuals from each population clustered together. UPGMA cluster analysis showed that population groupings are not strictly in accordance with bioclimates or geographic location. The genetic differentiation in L. multifida could have occurred at local scales because of genetic drift. Efforts should be made to protect all populations. The maintenance of substantial population size should be initiated via fencing and controlling collection to restore the regeneration of populations.     

Myotubularin phosphoinositide phosphatases: cellular functions and disease pathophysiology

The myotubularin family of phosphoinositide phosphatases includes several members mutated in neuromuscular diseases or associated with metabolic syndrome, obesity, and cancer. Catalytically dead phosphatases regulate their active homologs by heterodimerization and potentially represent key players in the phosphatase-kinase balance. Although the enzymatic specificity for phosphoinositides indicates a role for myotubularins in endocytosis and membrane trafficking, recent findings in cellular and animal models suggest that myotubularins regulate additional processes including cell proliferation and differentiation, autophagy, cytokinesis, and cytoskeletal and cell junction dynamics. In this review, we discuss how myotubularins regulate such diverse processes, emphasizing newly identified functions in a physiological and pathological context. A better understanding of myotubularin pathophysiology will pave the way towards therapeutic strategies.     

Genetic diversity and population structure in Tunisian Lavandula stoechas L. and Lavandula multifida L. (Lamiaceae)

The genetic diversity and population structure of 20 Tunisian Lavandula stoechas L. and Lavandula multifida L. populations, from different bioclimates, were analysed by starch gel electrophoresis using seven isozymes. The genetic diversity within populations varied according to species. Variation in L. multifida was higher than that observed for L. stoechas, and exclusive alleles were detected for taxa.

A high differentiation among populations, for each species, estimated by Wright's F-statistics was revealed. The genetic structure of populations from the same bioclimate was substantial. Nei's, R. [1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583–590] genetic distance among pairs of populations was low. The UPGMA cluster analysis of genetic distance values revealed that populations for each species were not strictly clustered together according to bioclimate or geographic proximity.

For each species, the low genetic divergence among populations and their substantial structure indicate their recent fragmentation due to anthropic pressures. The dendrogram generated from pairwise genetic distance among all populations showed two distinct clusters each corresponding to one species. The high genetic divergence between the two species, based on isozymes, corroborates their taxonomic status, as previously reported using morphological traits. The strategy for the management and conservation of populations should be made for each taxa according to its level of diversity and bioclimate.     

The myotubularin–amphiphysin 2 complex in membrane tubulation and centronuclear myopathies

Myotubularin (MTM1) and amphiphysin 2 (BIN1) are two proteins mutated in different forms of centronuclear myopathy, but the functional and pathological relationship between these two proteins was unknown. Here, we identified MTM1 as a novel binding partner of BIN1, both in vitro and endogenously in skeletal muscle. Moreover, MTM1 enhances BIN1‐mediated membrane tubulation, depending on binding and phosphoinositide phosphatase activity. BIN1 patient mutations induce a conformational change in BIN1 and alter its binding and regulation by MTM1. In conclusion, we identified the first molecular and functional link between MTM1 and BIN1, supporting a common pathological mechanism in different forms of centronuclear myopathy.     

Protective Action of Spermine and Spermidine against Photoinhibition of Photosystem I in Isolated Thylakoid Membranes

The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O₂ uptake rates, P700 photooxidation and the accumulation of superoxide anions (O₂ ⁻) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O₂ ⁻ generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O₂ ⁻ generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer.     

Mechanism of Interaction of Al3+ with the Proteins Composition of Photosystem II

The inhibitory effect of Al3+on photosystem II (PSII) electron transport was investigated using several biophysical and biochemical techniques such as oxygen evolution, chlorophyll fluorescence induction and emission, SDS-polyacrylamide and native green gel electrophoresis, and FTIR spectroscopy. In order to understand the mechanism of its inhibitory action, we have analyzed the interaction of this toxic cation with proteins subunits of PSII submembrane fractions isolated from spinach. Our results show that Al ³⁺, especially above 3 mM, strongly inhibits oxygen evolution and affects the advancement of the S states of the Mn₄O₅Ca cluster. This inhibition was due to the release of the extrinsic polypeptides and the disorganization of the Mn4O5Ca cluster associated with the oxygen evolving complex (OEC) of PSII. This fact was accompanied by a significant decline of maximum quantum yield of PSII (F_v/F_m) together with a strong damping of the chlorophyll a fluorescence induction. The energy transfer from light harvesting antenna to reaction centers of PSII was impaired following the alteration of the light harvesting complex of photosystem II (LHCII). The latter result was revealed by the drop of chlorophyll fluorescence emission spectra at low temperature (77 K), increase of F₀ and confirmed by the native green gel electrophoresis. FTIR measurements indicated that the interaction of Al ³⁺ with the intrinsic and extrinsic polypeptides of PSII induces major alterations of the protein secondary structure leading to conformational changes. This was reflected by a major reduction of α-helix with an increase of β-sheet and random coil structures in Al ³⁺-PSII complexes. These structural changes are closely related with the functional alteration of PSII activity revealed by the inhibition of the electron transport chain of PSII.