Rapid separation of low molecular weight solutes from liposomes without dilution

Liposomes can be separated from low molecular weight solutes on minicolumns of Sephadex G-50 made from the barrels of 1- or 5-ml plastic syringes. Excess fluid is first removed from the Sephadex beads by centrifugation and a mixture of liposomal entrapped and free solute is applied to the column bed. The centrifugation is repeated forcing the liposomal material through the column into a test tube while the free solute is quantitatively retained in the Sephadex. The procedure is applicable to a variety of solutes and 92 to 100% recovery is achieved for both charged and neutral liposomes. This technique has advantages over other methods for separating extraliposomal solutes from liposomes. Numerous samples can be processed simultaneously within minutes with no dilution of the liposomal preparation. Nonentrapped solute within the Sephadex can be easily recovered in a small volume of water or buffer.     

Distribution of Bdellovibrio bacteriovorus in sewage works, river water, and sediments.

Bdellovibrio was found in all liquid phases of the sewage works examined. The predator was also found in all the river sediments and sewage-polluted river waters examined but could not be found in some unpolluted river waters. Bdellovibrio was able to multiply on the high numbers of bacteria present in the aerobic percolating filter film but could not survive in anaerobic sludge. Similarly, the predator was present in the aerobic surface layers of river sediments but not in the anaerobic bottom layers. The major source of Bdellovibrio in the polluted rivers examined were sewage works effluents, and numbers in both river water and sediment were correlated with river water quality. It was unlikely that Bdellovibrio was important in reducing numbers of other bacteria in either sewage or river sediment.     

Trends in tuna carbon isotopes suggest global changes in pelagic phytoplankton communities

Considerable uncertainty remains over how increasing atmospheric CO₂ and anthropogenic climate changes are affecting open‐ocean marine ecosystems from phytoplankton to top predators. Biological time series data are thus urgently needed for the world's oceans. Here, we use the carbon stable isotope composition of tuna to provide a first insight into the existence of global trends in complex ecosystem dynamics and changes in the oceanic carbon cycle. From 2000 to 2015, considerable declines in δ¹³C values of 0.8‰–2.5‰ were observed across three tuna species sampled globally, with more substantial changes in the Pacific Ocean compared to the Atlantic and Indian Oceans. Tuna recorded not only the Suess effect, that is, fossil fuel‐derived and isotopically light carbon being incorporated into marine ecosystems, but also recorded profound changes at the base of marine food webs. We suggest a global shift in phytoplankton community structure, for example, a reduction in ¹³C‐rich phytoplankton such as diatoms, and/or a change in phytoplankton physiology during this period, although this does not rule out other concomitant changes at higher levels in the food webs. Our study establishes tuna δ¹³C values as a candidate essential ocean variable to assess complex ecosystem responses to climate change at regional to global scales and over decadal timescales. Finally, this time series will be invaluable in calibrating and validating global earth system models to project changes in marine biota.     

Defining natural factors that stimulate and inhibit cellulose:xyloglucan hetero-transglucosylation

Certain transglucanases can covalently graft cellulose and mixed-linkage β-glucan (MLG) as donor substrates onto xyloglucan as acceptor substrate and thus exhibit cellulose:xyloglucan endotransglucosylase (CXE) and MLG:xyloglucan endotransglucosylase (MXE) activities in vivo and in vitro. However, missing information on factors that stimulate or inhibit these hetero-transglucosylation reactions limits our insight into their biological functions. To explore factors that influence hetero-transglucosylation, we studied Equisetum fluviatile hetero-trans-β-glucanase (EfHTG), which exhibits both CXE and MXE activity, exceeding its xyloglucan:xyloglucan homo-transglucosylation (XET) activity. Enzyme assays employed radiolabelled and fluorescently labelled oligomeric acceptor substrates, and were conducted in vitro and in cell walls (in situ). With whole denatured Equisetum cell walls as donor substrate, exogenous EfHTG (extracted from Equisetum or produced in Pichia) exhibited all three activities (CXE, MXE, XET) in competition with each other. Acting on pure cellulose as donor substrate, the CXE action of Pichia-produced EfHTG was up to approximately 300% increased by addition of methanol-boiled Equisetum extracts; there was no similar effect when the same enzyme acted on soluble donors (MLG or xyloglucan). The methanol-stable factor is proposed to be expansin-like, a suggestion supported by observations of pH dependence. Screening numerous low-molecular-weight compounds for hetero-transglucanase inhibition showed that cellobiose was highly effective, inhibiting the abundant endogenous CXE and MXE (but not XET) action in Equisetum internodes. Furthermore, cellobiose retarded Equisetum stem elongation, potentially owing to its effect on hetero-transglucosylation reactions. This work provides insight and tools to further study the role of cellulose hetero-transglucosylation in planta by identifying factors that govern this reaction.     

Molecular evidence that the deadliest sea snake Enhydrina schistosa (Elapidae: Hydrophiinae) consists of two convergent species

We present a striking case of phenotypic convergence within the speciose and taxonomically unstable Hydrophis group of viviparous sea snakes. Enhydrina schistosa, the ‘beaked sea snake’, is abundant in coastal and inshore habitats throughout the Asian and Australian regions, where it is responsible for the large majority of recorded deaths and injuries from sea snake bites. Analyses of five independent mitochondrial and nuclear loci for populations spanning Australia, Indonesia and Sri Lanka indicate that this ‘species’ actually consists of two distinct lineages in Asia and Australia that are not closest relatives. As a result, Australian “E. schistosa” are elevated to species status and provisionally referred to Enhydrina zweifeli. Convergence in the characteristic ‘beaked’ morphology of these species is probably associated with the wide gape required to accommodate their spiny prey. Our findings have important implications for snake bite management in light of the medical importance of beaked sea snakes and the fact that the only sea snake anti-venom available is raised against Malaysian E. schistosa.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

The Synthesis,Fluorescence and Antiviral Studies of 3′-Amino-2′,3′-Dideoxythymidine/substituted 10-Cyano-9-isothiocyanatoanthracene Adducts

Abstract

Adducts of 3′-amino-2′,3′-dideoxythymidine and various methoxy-substituted 10-cyano-9-isothiocyanatoanthracenes were prepared for use as fluorescent-tagged molecular probes. The thymidine/anthracene adducts were subjected to antiviral assays to determine if the adducts possessed antiviral activity.