Conjugates of the fungal cytotoxin illudin M with improved tumour specificity

A simplified procedure for the isolation of gram quantities of illudin M from culture broths of basidiomycete Omphalotus olearius is described. Esters of illudin M with docosahexaenoic acid, chlorambucil, demethylcantharidinic acid (endothall) and 2,2'-bipyridyl-5,5'-dicarboxylic acid were synthesised and tested for cytotoxicity and induction of apoptosis in two clinically relevant tumour cell lines (Panc-1 pancreas carcinoma and HT-29 colon carcinoma) and in non-malignant human foreskin fibroblasts. The demethylcantharidin and the bipyridine conjugates retained the cytotoxicity of the parent illudin M while displaying an improved specificity for the tumour cells over the fibroblasts.     

Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity.

Asparagine-linked N-glycosylation is an essential protein modification occurring in all eukaryotic cells. The central step is the co-translational transfer of the core oligosaccharide assembled on the lipid carrier dolichol phosphate to selected Asn-X-Ser/Thr residues of nascent polypeptide chains in the endoplasmic reticulum. This reaction is catalyzed by the enzyme N-oligosaccharyl transferase. In yeast, Wbp1p is an essential component of this enzyme. Using a high copy number suppression approach, the SWP1 gene was isolated as an allele specific suppressor of a wbp1 mutation. Swp1p is a 30 kDa type I transmembrane protein and essential for cell viability. Similar to Wbp1p, depletion of Swp1p results in reduced N-oligosaccharyl transferase activity in vivo and in vitro. Wbp1p and Swp1p can be chemically cross-linked, suggesting that both proteins are essential constituents of the N-oligosaccharyl transferase complex.     

Improvement of the pre-deployment net closure procedure used with opening/closing plankton nets

The construction of a closing device to be used during the deploymentof phyto-zooplankton nets utilizing General Oceanics-type opening/closingmechanisms is described. This device (the cowl) decreases systemset-up time, decreases contamination from ambient particleswhile waiting for deployment, protects the net from mechanicaltears during descent, and increases system reliability. ¹This research is supported by the National Science FoundationGrant No. OCE-8003200     

Pleuropulmonary manifestations of hepatic amebiasis.

Pleuropulmonary manifestations of hepatic amebiasis occurred in 30 patients; 18 (60%) presented with at least 1 pulmonary complaint and 10 (33%) had multiple pulmonary symptoms. In 14 patients (47%), abnormalities were found on examination of the chest. In 16 chest roentgenograms (53%), there was at least 1 abnormality: right-sided pleural effusion (9 patients) and elevated right hemidiaphragm (8 patients) were the most common. All patients were treated with metronidazole (Flagyl) and had resolution of the amebic liver abscess and pulmonary disease. Pleuropulmonary disease is a common complication of amebic liver abscess. The clinical presentation and chest roentgenograms are virtually diagnostic and obviate the need for invasive procedures to confirm the diagnosis. Pleuropulmonary disease resolves with amebicidal treatment of the hepatic abscess.     

A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation.

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.     

Detection of an Ss-like protein in the sera of inbred and wild rats

A normal serum protein that crossreacts with rabbit anti-mouse Ss serum was isolated by alternating gel nitration and ion exchange chromatography from the inbred Long-Evans (LGE) rat strain. Rabbit antisera prepared against this protein detected it in the sera of all inbred and individual wild rats tested. The close physical and immunochemical similarity between this protein and the mouse C4 component of complement (Ss protein) indicates that this protein may represent the rat homolog of the mouse C4. Quantitative differences in the level of the Ss-like rat protein, comparable to those seen in Ss low mice, were not observed in 25 inbred strains or 22 individual wild rats. These quantitative results were supported by functional assays for total hemolytic complement and individual C2, C3, and C4 complement components. Sixteen inbred strains were examined and all had normal levels of activity for each of the assays.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Ligand blotting with 125I-fluoresceinamine-heparin

A highly sensitive method for ligand blotting with heparin has been developed. This ligand-blotting method is successful largely due to the ability to prepare heparin derivatives of high radiospecific activity. Heparin was modified with fluoresceinamine according to the method of C.G. Glabe, P.K. Harty, and S.D. Rosen [1983) Anal. Biochem. 130, 287-294), and this fluoresceinamine-derivatized heparin can be radioiodinated to a specific activity of 100,000 cmp/ng of uronic acid. This is a 500-fold increase in specific activity over Bolton-Hunter-modified heparin, as prepared by A.D. Cardin, K.R. Witt, and R.L. Jackson [1984) Anal. Biochem. 137, 368-373). 125I-Fluoresceinamine-derivatized heparin retains its ability to interact specifically with heparin-binding proteins such as human protease nexin-I and antithrombin III. 125I-Fluoresceinamine-derivatized heparin can be used to visualize and quantify heparin binding proteins on nitrocellulose. Protease nexin-I can be visualized at the nanogram level. In addition, ligand blotting with 125I-fluoresceinamine heparin can be combined with Cleveland digestion (D.W. Cleveland, S. Fisher, M.W. Kirschner, and U.K. Laemmli (1977) J. Biol. Chem. 252, 1102-1106) in order to identify heparin binding fragments of proteins with heparin binding domains.     

First record of bat-pollination in the species-rich genus Tillandsia (Bromeliaceae)

Background and Aims

Bromeliaceae is a species-rich neotropical plant family that uses a variety of pollinators, principally vertebrates. Tillandsia is the most diverse genus, and includes more than one-third of all bromeliad species. Within this genus, the majority of species rely on diurnal pollination by hummingbirds; however, the flowers of some Tillandsia species show some characteristics typical for pollination by nocturnal animals, particularly bats and moths. In this study an examination is made of the floral and reproductive biology of the epiphytic bromeliad Tillandsia macropetala in a fragment of humid montane forest in central Veracruz, Mexico.

Methods

The reproductive system of the species, duration of anthesis, production of nectar and floral scent, as well as diurnal and nocturnal floral visitors and their effectiveness in pollination were determined.

Key Results

Tillandsia macropetala is a self-compatible species that achieves a higher fruit production through outcrossing. Nectar production is restricted to the night, and only nocturnal visits result in the development of fruits. The most frequent visitor (75 % of visits) and the only pollinator of this bromeliad (in 96 % of visits) was the nectarivorous bat Anoura geoffroyi (Phyllostomidae: Glossophaginae).

Conclusions

This is the first report of chiropterophily within the genus Tillandsia. The results on the pollination biology of this bromeliad suggest an ongoing evolutionary switch from pollination by birds or moths to bats.     

Discovery of veterinary antiparasitic agents in the 21st Century: A view from industry

Discovery of antiparasitic agents is a challenging process, requiring discovery of molecules with the ability to kill parasites but not their hosts. Customer preference is for fewer doses and ease of application, but this is not always compatible with reduced withdrawal times, human food safety and/or user safety. This article describes some of the difficulties faced by researchers in the search for new antiparasitic agents, while highlighting advances that may improve the discovery process and the chance of success in discovering novel drugs.