Synthesis and transport of the organic matrix of the spicules in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary The sequence of the synthesis and transport of the organic matrix of spicules has been elucidated in the gorgonian Leptogorgia virgulata by use of ³H-aspartic acid as the tracer in electron-microscopic autoradiography. The entire process of matrix synthesis and transport takes approximately 2 h. It seems that the protein moiety of the organic matrix is synthesized in the RER prior to 5 min following the initial 10 min incubation in the tracer. At the 5 min chase the label is moving from the RER to the Golgi complexes where the carbohydrate moiety of the matrix is presumed to be synthesized. At the 5 to 15 min chases the label is transported out of the Golgi complexes via Golgi vesicles. This phase continues for 30 min. From 60 to 120 min the ³H-aspartic acid moves to the spicules. After 120 min the majority of the label has moved into the spicules. Silver grain counts over both multivesicular and electron-dense bodies remain at relatively low and constant levels over 4 h indicating that neither organelle is involved in the synthesis and transport of the organic matrix.Contribution No 512; Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina 29208, USA     

Clinical effectiveness of biologics in clinical practice

TNF inhibitors are currently considered both effective and cost-effective in patients with active rheumatoid arthritis (RA), particularly in patients who have not responded fully to methotrexate. There is substantial doubt about the cost-effectiveness of TNF inhibitors as initial treatment for active RA. New data from the National Data Bank for Rheumatic Diseases now question the current consensus in methotrexate failures. The data suggest that in routine clinical practice TNF inhibitors provide only modest incremental benefits over best conventional therapy. If confirmed, these observational studies suggest that the economic argument underpinning the widespread use of TNF inhibitors in established RA is unsustainable.     

Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression.

Analysis of a T-cell antigen receptor (TCR) alpha promoter from a variable gene segment (V) revealed a critical GT box element which is also found in upstream regions of several V alpha genes, TCR enhancer, and regulatory elements of other genes. This element is necessary for TCR gene expression and binds several proteins. These GT box-binding proteins were identified as members of a novel Sp1 multigene family. Two of them, which we term Sp2 and Sp3, were cloned. Sp2 and Sp3 contain zinc fingers and transactivation domains similar to those of Sp1. Like Sp1, Sp2 and Sp3 are expressed ubiquitously, and their in vitro-translated products bind to the GT box in TCR V alpha promoters. Sp3, in particular, also binds to the Sp1 consensus sequence GC box and has binding activity similar to that of Sp1. As the GT box has also previously been shown to play a role in gene regulation of other genes, these newly isolated Sp2 and Sp3 proteins might regulate expression not only of the TCR gene but of other genes as well.     

Scleroblast cultures from the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Scleroblasts were separated from fragmented tissue of growing tips ofLeptogorgia virgulata and cultured using a modification of the technique of Rannou. Replacement of fetal bovine serum with horse serum seemed to increase scleroblast viability. Cell adhesion occurred from 14 to 43 d. Cultured scleroblasts demonstrated cell aggregation, spicule formation, and extrusion of spicules into the external medium. Cells showing spicules in the process of being extruded appeared on the average after 24 d of culture. Variability among cultures was marked with respect to both division and spicule formation. Healthy cultures were maintained for more than 4 mo. This work was supported by National Science Foundation grants PCM8201389 and DCB8502698. This is contribution No. 674 of Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina.     

The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

The promotive effect of smoke derived from burnt native vegetation on seed germination of Western Australian plants

Exposure of dormant seed to cold smoke derived from burnt native vegetation had a positive influence on germination in one or more seed provenances in 45 out of 94 species of native Western Australian plants that are normally hard to germinate. When tested under controlled conditions some species showed earlier germination in smoke treatments than controls; in others smoke-treated seeds continued to germinate for several weeks after controls had achieved full germination. In the remainder, treated and control seeds germinated to similar time schedules. A group of 23 species which responded positively had previously been recorded as extremely difficult or impossible to germinate using conventional techniques. These included members of the genera Geleznowia (Rutaceae), Hibbertia (Dilleniaceae), Stirlingia (Proteaceae), Verticordia (Myrtaceae), Actinostrobus (Cupressaceae) and Pimelea (Thymelaeaceae). Both large- and small-seeded species were encountered amongst the positively responding taxa, which encompassed representatives of 15 families and 26 genera of dicotyledons, 5 families and 8 genera of monocotyledons and the gymnosperm Actinostrobus acuminatus. Sowing seeds on smoke-fumigated filter papers or watering with aqueous eluates of smoke elicited similar degrees of stimulation of germination, as did exposure to gaseous smoke in a readily germinating species Anigozanthos manglesii (Haemodoraceae) and the normally intractable species Lysinema ciliatum (Epacridaceae). Exposing recently burnt and unburnt natural bushland sites to smoke, smoked water or smoked dry sand elicited a significant germination response in 15 species. Over one third of the species sampled in the burnt site exhibited germination additional to that caused by the fire. Data are discussed in relation to previous germination studies on Australian and other taxa.     

Manganese as a calcium probe: Electron paramagnetic resonance and nuclear magnetic resonance spectroscopy of intact cells

Summary When Lettree cells are exposed to Mn²⁺, the cation becomes associated with cells in two ways: in a relatively loose and mobile manner that gives a six-line EPR spectrum designated Mn _b ^*, and in an immobile, relatively tight manner that gives no detectable EPR spectrum, designated Mn _b . Mn _b ^* is probably on the surface of cells; most Mn _b is probably inside cells. NMR measurements of Lettree cell suspensions show two water proton relaxation rates and confirm the existence of cell-associated Mn. Human erythrocytes, on the other hand, bind no Mn²⁺ under these conditions, as judged by EPR and NMR measurements.Virally-treated Lettree cells show an increase in Mn _b (but not in Mn _b ^*). They also show a third water proton relaxation rate.