Rabip4' is an effector of rab5 and rab4 and regulates transport through early endosomes

We describe the characterization of an 80-kDa protein cross-reacting with a monoclonal antibody against the human La autoantigen. The 80-kDa protein is a variant of rabip4 with an N-terminal extension of 108 amino acids and is expressed in the same cells. For this reason, we named it rabip4'. rabip4' is a peripheral membrane protein, which colocalized with internalized transferrin and EEA1 on early endosomes. Membrane association required the presence of the FYVE domain and was perturbed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Expression of a dominant negative rabip4' mutant reduced internalization and recycling of transferrin from early endosomes, suggesting that it may be functionally linked to rab4 and rab5. In agreement with this, we found that rabip4' colocalized with the two GTPases on early endosomes and bound specifically and simultaneously to the GTP form of both rab4 and rab5. We conclude that rabip4' may coordinate the activities of rab4 and rab5, regulating membrane dynamics in the early endosomal system.     

DNA Barcodes of Rosy Tetras and Allied Species (Characiformes: Characidae: Hyphessobrycon) from the Brazilian Amazon Basin

DNA barcoding can be an effective tool for fast and accurate species-level identification based on sequencing of the mitochondrial cytochrome c oxidase subunit (COI) gene. The diversity of this fragment can be used to estimate the richness of the respective species. In this study, we explored the use of DNA barcoding in a group of ornamental freshwater fish of the genus Hyphessobrycon. We sequenced the COI from 10 species of Hyphessobrycon belonging to the “Rosy Tetra Clade” collected from the Amazon and Negro River basins and combined our results with published data. The average conspecific and congeneric Kimura 2-parameter distances were 2.3% and 19.3%, respectively. Six of the 10 species were easily distinguishable by DNA barcoding (H. bentosi, H. copelandi, H. eques, H. epicharis, H. pulchrippinis, and H. sweglesi), whereas the remaining species (H. erythrostigma, H. pyrrhonotus, H. rosaceus and H. socolofi) lacked reciprocal monophyly. Although the COI gene was not fully diagnostic, the discovery of distinct evolutionary units in certain Hyphessobrycon species under the same specific epithet as well as haplotype sharing between different species suggest that DNA barcoding is useful for species identification in this speciose genus.     

Microbial Ecology and Association of Bacillus thuringiensis in Chicken Feces Originating from Feed

To explain the association of Bacillus thuringiensis (Bt) with animal feces, an ecological analysis in chickens was conducted by introducing a cry ^? strain marked by production of green fluorescent protein (GFP). After feeding with the tagged Bt strains, the feces of the tested chickens were collected at different times, isolated, and the morphology of Bt was observed. It was shown that Bt strain HD-73GFP in spore form could be isolated from feces of chickens for a period of 13 d, and then it disappeared thereafter. Bt could be detected only up to day 4 (but not thereafter), when chickens were fed with vegetative cells of HD-73GFP. To confirm the source of newly isolated strains, the gfp gene was examined by polymerase chain reaction (PCR), which showed that all the isolated strains harbored the marker gene. Recent data from isolation and PCR had suggested that fecal Bt strains had originated from food. Chicken tissues were thus dissected to isolate Bt strains and to investigate whether Bt could be located in vivo. Bt was located within the duodenum in spore form. Compared to the morphology of the isolated strains at different growth times, the growth rates of all the tested Bt had little changes when passing through the digestive system to the feces. Dissection of the chickens confirmed that Bt was safe for the tested animal.     

Synthesis and Some Properties of Modified Oligonucleotides. II. Oligonucleotides Containing 2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl Pyrimidine Nucleosides

Abstract

In order to find the effects of unnatural nucleosides on the stability of duplex, several oligonucleotides containing 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-uracil(FAU),-cytosine (FAC) and -thymine (FMAU) were synthesized by two alternative approaches: phosphoramidite method on an ABI 392 synthesizer and H-phosphonate procedure on our GeneSyn I universal module synthesizer. It was shown from the melting profiles that the presence of FMAU has a large stabilizing effect on the duplex. Replacement of thymidine with FAU, or deoxycytidine with FAC resulted in the formation of less stable duplexes. Temperature-dependent CD spectroscopy demonstrated that the structures of the fluorine containing oligomers are very similar to those of unmodified oligomers.     

On the causes of trends in the seasonal amplitude of atmospheric CO2

No consensus has yet been reached on the major factors driving the observed increase in the seasonal amplitude of atmospheric CO₂ in the northern latitudes. In this study, we used atmospheric CO₂ records from 26 northern hemisphere stations with a temporal coverage longer than 15 years, and an atmospheric transport model prescribed with net biome productivity (NBP) from an ensemble of nine terrestrial ecosystem models, to attribute change in the seasonal amplitude of atmospheric CO₂. We found significant (p < .05) increases in seasonal peak‐to‐trough CO₂ amplitude (AMP_P‐T) at nine stations, and in trough‐to‐peak amplitude (AMP_T‐P) at eight stations over the last three decades. Most of the stations that recorded increasing amplitudes are in Arctic and boreal regions (>50°N), consistent with previous observations that the amplitude increased faster at Barrow (Arctic) than at Mauna Loa (subtropics). The multi‐model ensemble mean (MMEM) shows that the response of ecosystem carbon cycling to rising CO₂ concentration (eCO₂) and climate change are dominant drivers of the increase in AMP_P‐T and AMP_T‐P in the high latitudes. At the Barrow station, the observed increase of AMP_P‐T and AMP_T‐P over the last 33 years is explained by eCO₂ (39% and 42%) almost equally than by climate change (32% and 35%). The increased carbon losses during the months with a net carbon release in response to eCO₂ are associated with higher ecosystem respiration due to the increase in carbon storage caused by eCO₂ during carbon uptake period. Air‐sea CO₂ fluxes (10% for AMP_P‐T and 11% for AMP_T‐P) and the impacts of land‐use change (marginally significant 3% for AMP_P‐T and 4% for AMP_T‐P) also contributed to the CO₂ measured at Barrow, highlighting the role of these factors in regulating seasonal changes in the global carbon cycle.     

Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC₅₀ = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.     

Production and utilization of acetate in mammals

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.     

Retrospective study of the prevalence of high-risk human papillomaviruses among Croatian women

The infection with Human papillomavirus (HPV) is the necessary cause for cervical cancer. There are at least 15 High-Risk (HR) HPV types that are significantly associated with progression of cervical intraepithelial neoplasia to cervical cancer. Since previous studies showed that the prevalence of HPV in cervical cancers varies among different geographic regions, we wanted to investigate the prevalence of HPV types in Croatia, especially low abundant HR HPV types. By means of consensus primers directed polymerase chain reaction (PCR), we analysed cervical DNA samples of 2,136 Croatian women, mostly with abnormal cervical smears, in order to detect the presence of HPV Type-specific primers were then used to determine Low-Risk (LR) HPV types 6/11 and HR HPV types 16, 18, 31, 33, 45, 52 and 58. Out of 2,136 specimens, 1,255 (58.8%) were positive for HPV More than half of positive samples were typed (64.5%) and 35.5% still remained untyped. Multiple HPV infections were found in 10.3% of the cases. The most prevalent type, including both single and multiple infections, was HPV16 with the prevalence of 15.9%, followed by HPV types 31, 6/11, 33, 18, 52, 45 and 58 with 8.7%, 7.1%, 4.5%, 3.8%, 2.3%, 1.2% and 1.1%, respectively. The significant increase of frequency from Low-grade Squamous Intraepithelial Lesions (LSIL) to High-grade Squamous Intraepithelial Lesions (HSIL) was observed for HR HPV types 16, 18, 31 and 33 but not 45, 52 and 58. The frequency of unknown HPV types was almost the same in cervical specimens of women with LSIL and those with HSIL, 19.8% and 21.1%, respectively. The prevalence of HPV infection rate decreased significantly with patient age from 68.5% (age group 12 to 24 years) to 38.8% (age group 45 to 54 years). But, in women aged 55 or older the overall prevalence increased to 56.6%. Our results indicate that prevalence of HR HPV types in Croatia is similar to other countries. We suggest that HPV positive women in Croatia should be closely monitored by typing for HR HPV types: 16, 18, 31, 33, 45, 52 and 58.     

Segmentation and Tracking of Adherens Junctions in 3D for the Analysis of Epithelial Tissue Morphogenesis

Epithelial morphogenesis generates the shape of tissues, organs and embryos and is fundamental for their proper function. It is a dynamic process that occurs at multiple spatial scales from macromolecular dynamics, to cell deformations, mitosis and apoptosis, to coordinated cell rearrangements that lead to global changes of tissue shape. Using time lapse imaging, it is possible to observe these events at a system level. However, to investigate morphogenetic events it is necessary to develop computational tools to extract quantitative information from the time lapse data. Toward this goal, we developed an image-based computational pipeline to preprocess, segment and track epithelial cells in 4D confocal microscopy data. The computational pipeline we developed, for the first time, detects the adherens junctions of epithelial cells in 3D, without the need to first detect cell nuclei. We accentuate and detect cell outlines in a series of steps, symbolically describe the cells and their connectivity, and employ this information to track the cells. We validated the performance of the pipeline for its ability to detect vertices and cell-cell contacts, track cells, and identify mitosis and apoptosis in surface epithelia of Drosophila imaginal discs. We demonstrate the utility of the pipeline to extract key quantitative features of cell behavior with which to elucidate the dynamics and biomechanical control of epithelial tissue morphogenesis. We have made our methods and data available as an open-source multiplatform software tool called TTT (http://github.com/morganrcu/TTT)