TCA-100 tumour chemosensitivity assay: Differences in sensitivity between cultured tumour cell lines and clinical studies

The BATLE LE TCA-100 tumour chemosensitivity assay has been used to evaluate chemotherapeutic drug sensitivity of cultured tumour cell lines. Studies were performed using test drug concentrations calibrated to discriminate sensitivity and resistance of clinical specimens. Strong sensitivity which appeared to be inconsistent with clinical experience was detected for some drugs and cell lines. Findings of strong sensitivity were consistent with basic differences between sensitivity testing cultured cell lines and clinical specimens. Results with cell lines frequently may not apply directly to clinical applications. Characterization of differences between cell lines and clinical specimens may assist in application of cell line findings to clinical trials.     

Identification of a sporulation-specific promoter regulating divergent transcription of two novel sporulation genes in Saccharomyces cerevisiae

Transposition of a new Drosophila retrotransposon was investigated. Total genomic Southern analysis and polytene in situ hybridizations in D. buzzatii strains and other related species using a 6 kb D. buzzatii clone (cDb314) showed a dispersed, repetitive DNA pattern, suggesting that this clone contains a transposable element (TE). We have sequenced the cDb314 clone and demonstrated that it contains all the conserved protein sequences and motifs typical of retrovirus-related sequences. Although cDb314 does not include the complete TE, the protein sequence alignment demonstrates that it includes a defective copy of a new long terminal repeat (LTR) retrotransposon, related to the gypsy family, which we have named Osvaldo. Using a D. buzzatii inbred line in which all insertion sites are known, we have measured Osvaldo transposition rates in hybrids between this D. buzzatii line and its sibling species D. koepferae. The results show that Osvaldo transposes in bursts at high rate, both in the D. buzzatii inbred line and in species hybrids.     

Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling

The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc.     

Thermostable β-Glycosidase from Sulfolobus Solfataricus

The Sulfolobus solfataricus β-glycosidase (Sβgly) is a thermostable and thermophilic glycosyl-hydrolase with broad substrate specificity. The enzyme hydrolizes β-D-gluco-, fuco-, and galactosides, and a large number of /Winked glycoside dimers and oligomers, linked β1-3, β1-4, and β1-6, It is able to hydrolize oligosaccharides with up to 5 glucose residues. Furthermore, it is also able to promote transglycosylation reactions. The corresponding gene has been cloned and overexpressed both in yeast and Escherichia coli. Based on sequence and functional data, the Sβgly has been assigned to the so-called BGA family of glycosyl-hydrolases, including β-glycosidases, β-galactosidases and phosho-β-galactosidases from mesophilic and thermophilic organisms of the three domains. The Sβgly has been crystallized and the resolution of its structure is in progress. Because of its special properties, the enzymes has considerable biotechnological potential.     

Evolutionary study of multigenic families mapping close to the human MHC class I region

Summary During a search for novel coding sequences within the human MHC class I region (chromosome 6p21.3), we found an exon (named B30-2) coding for a 166-amino-acid peptide which is very similar to the C-terminal domain of several coding sequences: human 52-kD Sjögren's syndrome nuclear antigen A/Ro (SS-A/Ro) and ret finger protein (RFP), Xenopus nuclear factor 7 (XNF7), and bovine butyrophilin. The first three of these proteins share similarities over the whole length of the molecule whereas butyrophilin is similar in the C-terminal domain. The N-terminal domain of butyrophilin is similar to rat myelin/oligodendrocyte glycoprotein (MOG) and chicken B blood group system (B-G) protein. These domains are components of a new subfamily of the immunoglobulin superfamily (IgSF). Butyrophilin is thus a mosaic protein composed of the MOG/B-G Ig-like domain and the C-terminal domain of 52-kD SS-A/Ro, RFP, and XNF7 (1330-2-like domain). Moreover, in situ hybridization shows that RFP, butyrophilin, and MOG map to the human chromosome 6p2l.3-6p22 region and are thus close to the MHC class I genes. It is therefore possible that the butyrophilin gene is the product of an exon shuffling event which occurred between ancestors of the RFP and MOG genes. To our knowledge, this is the first example of the colocalization of a chimeric gene and its putative progenitors. Finally, regulatory protein T-lymphocyte 1 (Rpt-1) shares similarities with the N-terminal halves of RFP, 52-kD SS-A/Ro, and XNF7, but not with the B30-2-like domain. We show that the ancestral Rpt-l gene evolved by overprinting. Correspondence to: P. Pontarotti     

Regulation of taurine transport in Ehrlich ascites tumor cells

Summary Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized. Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na, 1Cl, 1taurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the leukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A₂ is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD₄. The swelling-induced taurine efflux is also inhibited by addition of the 5-lipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-lipoxygenase pathway. LTD₄ seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the Cl^– channels, i.e., via a depolarization of the cell membrane.     

Isolation and Molecular Characterization of Five Marine Cyanophages Propagated on Synechococcus sp. Strain WH7803

Five marine cyanophages propagated on Synechococcus sp. strain WH7803 were isolated from three different oceanographic provinces during the months of August and September 1992: coastal water from the Sargasso Sea, Bermuda; Woods Hole harbor, Woods Hole, Mass.; and coastal water from the English Channel, off Plymouth Sound, United Kingdom. The five cyanophage isolates were found to belong to two families, Myoviridae and Styloviridae, on the basis of their morphology observed in the transmission electron microscope. DNA purified from each of the cyanophage isolates was restricted with a selection of restriction endonucleases, and three distinguishably different patterns were observed. DNA isolated from Myoviridae isolates from Bermuda and the English Channel had highly related restriction patterns, as did DNA isolated from Styloviridae isolates from Bermuda and the English Channel. DNA isolated from the Myoviridae isolate from Woods Hole had a unique restriction pattern. The genome size for each of the Myoviridae isolates was ca. 80 to 85 kb, and it was ca. 90 to 100 kb for each of the Styloviridae isolates. Southern blotting analysis revealed that there was a limited degree of homology among all cyanophage DNAs probed, but clear differences were observed between cyanophage DNA from the Myoviridae and that from the Styloviridae isolates. Polypeptide analysis revealed a clear difference between Myoviridae and Styloviridae polypeptide profiles, although the major, presumably structural, protein in each case was ca. 53 to 54 kDa.     

Modelling C3 photosynthesis: A sensitivity analysis of the photosynthetic carbon-reduction cycle

A model of the C ₃ photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations c _i intercellular CO₂ concentration - C _infP ^supJ control coefficient for enzyme P with respect to flux J - DHAP dihydroxyacetonephosphate - E4P erythrose 4-phosphate - F6P fructose 6-phosphate - FBP fructose 1,6-bisphosphate - FBPase fructose 1,6-bisphosphatase - G3P glyceraldehyde 3-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - Pi inorganic phosphate - PCR photosynthetic carbon reduction - PGA 3-phosphoglyceric acid - PPFD photosynthetically active photon flux density - R _n ^J response coefficient for effector n with respect to flux J - R5P ribose 5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - Ru5P ribulose 5-phosphate - RuBP ribulose 1,5-bisphosphate - S7P sedoheptulose 7-phosphate - SBP sedoheptulose 1,7-bisphosphate - SBPase sedoheptulose 1,7-bisphosphatase - SPS sucrose-phosphate synthase - Xu5P xylulose 5-phosphate - _n ^P elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated.     

Localization of spectrin isoforms in the adult mouse heart

The distribution of two isoforms of spectrin in the adult mouse heart was investigated by Western blotting and immunocytochemistry by use of monospecific antibodies to erythrocyte spectrin and nonerythroid brain spectrin (240/235). Western blotting revealed proteins analogous to both isoforms of -spectrin in adult heart. Light-microscopic immunocytochemistry indicated that erythroid spectrin was distributed throughout the myocardium, with immunofluorescence localized to plasma membranes, Z-lines, and intercalated discs. Antibodies to brain spectrin (240/235) exhibited staining throughout the heart, with a generally diffuse distribution except for the prominent immunoreactivity associated with the intercalated discs. Nonerythroid spectrin immunofluorescence was detected in the endothelial cells of the endocardium and the mesothelial cell lining of the epicardium. Erythrocyte spectrin was not detected in the endocardium or the epicardium. The identification and localization of spectrin isoforms in the mammalian heart suggest the importance of spectrin proteins in the structural integrity and proper function of cardiac cells and tissues. This is the first demonstration of two different -spectrin subunits in the mammalian heart.