Sequential and compartmentalized action of Rabs,SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV–membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.     

Cellular delivery and enhanced anticancer activity of berberine complexed with a cationic derivative of γ–cyclodextrin

A cationic derivative of γ-cyclodextrin (GCD) modified with propylenediamine (PDA) was synthesized. It was shown that the derivative (GCD–PDA) is mucoadhesive and resistant to the digestion with?∝-amylase indicating that it may constitute an efficient oral delivery vehicle. GCD-PDA formed an inclusion complex with berberine (BBR), an alkaloid displaying a multitude of beneficial physiological effects. The complexed BBR penetrates a lipid membrane easier than the free one. Both uncomplexed BBR and that complexed with GCD-PDA was delivered to normal (NMuMG) and cancerous (4T1) murine mammary gland cells. In the normal cells both free and complexed BBR was homogeneously dispersed in the cytoplasm and was nontoxic up to 131?μM. In the cancerous cells uncomplexed BBR was also homogeneously dispersed but it was toxic to about 25% of cells at 131?μM, while the GCD-PDA/BBR complex was preferably localized in lysosomes and its toxicity doubled at this concentration compared to that of free BBR. Moreover, free BBR and GCD-PDA/BBR showed even more efficient inhibitory effect against murine melanoma (B16-F10) cells than against 4T1 cells.     

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.     

Sampling bias created by ampicillin in isolation media for Aeromonas

Members of the bacterial genus Aeromonas are widely isolated from aquatic environments and studied in part for their ability to act as opportunistic pathogens in a variety of animals. All aeromonads, with the exception of Aeromonas trota, are generally thought to be resistant to ampicillin, so the antibiotic is frequently added to isolation medium as a selective agent. In this study, 282 aeromonads from environmental sources were isolated on a medium without ampicillin and their resistance to ampicillin determined. Of the 104 of these isolates that were judged to be independent (nonredundant), 18 (17.3%) were susceptible to ampicillin. A chi-square analysis was performed to determine the impact of ampicillin use on enumerating Aeromonas species from environmental samples. Our results indicate that, when ampicillin is used as a selective agent, a significant portion of the aeromonad population in at least some environments can be omitted from isolation.     

Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis

In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.     

Effects of climate change and horticultural use on the spread of naturalized alien garden plants in Europe

Climate warming is supposed to enlarge the area climatically suitable to the naturalization of alien garden plants in temperate regions. However, the effects of a changing climate on the spread of naturalized ornamentals have not been evaluated by spatially and temporarily explicit range modelling at larger scales so far. Here, we assess how climate change and the frequency of cultivation interactively determine the spread of 15 ornamental plants over the 21st century in Europe. We coupled species distribution modelling with simulations of demography and dispersal to predict range dynamics of these species in annual steps across a 250 × 250 m raster of the study area. Models were run under four scenarios of climate warming and six levels of cultivation intensity. Cultivation frequency was implemented as size of the area used for planting a species. Although the area climatically suitable to the 15 species increases, on average, the area predicted to be occupied by them in 2090 shrinks under two of the three climate change scenarios. This contradiction obviously arises from dispersal limitations that were pronounced although we assumed that cultivation is spatially adapting to the changing climate. Cultivation frequency had a much stronger effect on species spread than climate change, and this effect was non‐linear. The area occupied increased sharply from low to moderate levels of cultivation intensity, but levelled off afterwards. Our simulations suggest that climate warming will not necessarily foster the spread of alien garden plants in Europe over the next decades. However, climatically suitable areas do increase and hence an invasion debt is likely accumulating. Restricting cultivation of species can be effective in preventing species spread, irrespective of how the climate develops. However, for being successful, they depend on high levels of compliance to keep propagule pressure at a low level.     

Ultrastructure of hematopoietic and stromal cells of the subendosteal region of the bone marrow

Using electron microscopic cytochemistry and immunoelectron microscopy, the ultrastructure of bone marrow (BM) cells of the subendosteal region with a high colony-forming (CFUs) ability was studied. In comparison with the central part of BM, the subendosteal region of CBA and BALB/c mice contains a higher number of lymphocyte-like mononuclears, bearing an antigen, common with the brain surface one but negative for peroxidase and acid and alkaline phosphatase. The ultrastructure of these cells is similar to that of presumptive hematopoietic stem cells. In the subendosteal region mononuclears are concentrated with the lower nucleo-cytoplasmic ratio, a feston-like line of the nucleus and more numerous organoids. These cells are characteristic of BM myeloid islands composed of granulocytes being on various stages of differentiation, and of reticular cells positive for alkaline phosphatase.     

Haptoglobin glycoforms in a case of carbohydrate-deficient glycoprotein syndrome

Alterations in haptoglobin (Hp) glycosylation were examined in the plasma of the first patient with carbohydrate-deficient glycoprotein syndrome (CDGS) who was described in Poland. Hp concentration in the CDGS patient plasma was low (240mg/l) and the Hp phenotype was shown to be 2-2. Three glycoforms of the Hp subunit were observed in SDS-PAGE in CDGS. The densitometric analysis and molecular weight determinations suggested that 50% of glycoforms were fully glycosylated; 30% contained three out of four and 20% only two out of four glycan units compared to those that are present in Hp derived from healthy people. Results with lectins (concanavalin A and Sambucus nigra, Maackia amurensis and Alleuria aurantia agglutinins) indicate that all three glycoforms of subunit of CDGS-Hp contained biantennary complex glycans terminated with 2,6 bound sialic acid, but without fucose or 2,3 linked sialic acid. Hp glycosylation abnormalities described in this work suggest that this case was a type I carbohydrate-deficient glycoprotein syndrome.     

AMP-deaminase from normal and cirrhotic human liver: A comparative study

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.