KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent regulatory subunit when expressed in plant cells

The Shaker potassium channels are tetrameric proteins formed by the assembly of four α-subunits. The oligomerization can occur among both homo- and hetero-α-subunits. KDC1 is a carrot Shaker-like potassium channel expressed in the epidermis of plantlet roots and the protoderm of somatic embryos. KDC1 was previously characterised electrophysiologically in CHO and Xenopus oocytes cells, but the experiments performed in these systems did not provide conclusive evidence that KDC1 forms a functional homomeric channel in plant cells. In this report, we show that KDC1 localizes to the plasma membrane of root cells in transgenic tobacco plants transformed with a KDC1∷GFP fusion construct. In tobacco mesophyll protoplasts, transiently transformed with KDC1∷GFP, KDC1 was present on the endomembrane and the protoplasts did not show any inward potassium current, as demonstrated by patch-clamp experiments. The co-expression of KDC1∷GFP with the Arabidopsis thaliana potassium channel AKT1 in tobacco mesophyll protoplasts has the effect of shifting KDC1 localization from endomembranes to the plasma membrane. Patch-clamp experiments performed on tobacco mesophyll protoplasts expressing AKT1 alone or in combination with KDC1∷GFP showed voltage-activated inward potassium currents with different properties. In particular, the addition of Zn²⁺ to the bath solution induced a clear decrease of the potassium currents in protoplasts transformed with AKT1 alone, whereas a current potentiation (indicative of KDC1 presence) was observed in protoplasts co-transformed with AKT1 + KDC1∷GFP. Split-Ubiquitin assay experiments performed in yeast cells confirmed the interaction between AKT1 and KDC1.     

The RhoA-associated protein Citron-N controls dendritic spine maintenance by interacting with spine-associated Golgi compartments

Dendritic spines are highly dynamic protuberances that are thought to be crucial for learning and memory. Although it is well known that actin filaments and membrane dynamics regulate spine plasticity, how these two events are linked locally is less clear. Here, we provide evidence that Citron-N (CIT-N), a binding partner of the small GTPase RhoA, is associated with the actin filaments and Golgi compartments of dendritic spines. We also show that CIT-N is required for recruiting F-actin and Golgi membranes at spines of in vitro-grown neurons. Studies in knockout mice show that this protein is essential for the maturation of dendritic spines. We suggest that CIT-N might function as a scaffold protein in spine organization through its ability to bind to Golgi membranes and by affecting actin remodelling.     

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.     

A Novel K+ Channel Expressed in Carrot Roots with a Low Susceptibility Toward Metal Ions

Kdc1 is a novel K⁺-channel gene cloned from carrot roots, and which is also present in cultured carrot cells. We investigated the characteristics of the ionic current elicited in Xenopus oocytes coinjected with KDC1 (K⁺-Daucus carota 1) and KAT1 (from Arabidopsis thaliana) RNA. Expressed heteromeric channels displayed inward-rectifying potassium currents whose kinetics, voltage characteristics, and inhibition by metal ions depended on KDC1:KAT1 ratios. At low KDC1:KAT1 ratios, Zn²⁺ inhibition of heteromeric K⁺ current was less pronounced compared to homomeric KAT1 channels, while at higher KDC1:KAT1 ratios, the addition of Zn²⁺ even produced an increase in current. Under the same conditions, the Ni²⁺ inhibition of the current was also reduced, but no current increase was observed. These effects might be explained by the unusual amino acid composition of the KDC1 protein in terms of histidine residues that are absent in the pore region, but abundant (four per subunit) in the proximity of the pore entrance. Channels like KDC1 could be at least partially responsible for the higher resistance of carrot cells in the presence of metals.     

Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae)

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.     

Multivariate analysis of metastasis risk in laryngeal carcinoma. I. Tumor factors

A multivariate analysis with a logistic regression model was performed in 181 cases of patients affected by laryngeal cancer, in order to evaluate the histologic tumor factors in predicting metastatic risk. The patterns used were: grading, localization, extension to the extralaryngeal structures, mode of growth. Nodal metastases are less influenced by the localization and the differentiation grade of the tumor. Cancer extended to the extralaryngeal tissues and with "infiltrating" type of growth are significantly correlated with nodal involvement. Multivariate analysis seems to be the best method to identify the exact value of each histological parameter used in prognosis of laryngeal carcinoma.     

Human erythrocytes glucose-6-phosphate dehydrogenase: Labelling of a reactive lysyl residue by pyridoxal-5′-phosphate

Reaction of glucose-6-phosphate dehydrogenase from human erythrocytes with pyridoxal-5′-phosphate causes 80% loss of activity. The substrate glucose-6-phosphate fully protects the enzyme against this inhibition, which is reversible upon dilution, but becomes irreversible after treatment with NaBH₄. We presume that pyridoxal-5′-phosphate forms with the enzyme a Schiff base which is reduced by NaBH₄. One mole of N-?-pyridoxyl-lysine is formed per mole of enzyme subunit when the remaining activity reaches its minimal level of 20%.