Molecular structure of tetanus neurotoxin as revealed by Fourier transform infrared and circular dichroic spectroscopy

Secondary structure contents of tetanus neurotoxin have been estimated at neutral and acidic pH using circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. An analysis of the far-ultraviolet CD spectra of the neurotoxin dissolved in 50 mM citrate-phosphate buffer (pH 7.0) revealed 20.0 +/- 2.1% alpha-helix, 50.5 +/- 2.1% beta-pleated sheets, no beta-turns, and 29.5% random coils, which is at considerable variance with results from an earlier detailed study of tetanus neurotoxin's secondary structures (J.P. Robinson, L.A. Holladay, J.H. Hash and D. Puett, J. Biol. Chem. 257 (1982) 407). However, the alpha-helix content estimated in this study is consistent with the earlier studies of Robinson et al. (J.P. Robinson, L.A. Holladay, J.B. Picklesimer and D. Puett, Mol. Cell. Biochem. 5 (1974) 147; J.P. Robinson, J.B. Picklesimer and D. Puett, J. Biol. Chem. 250 (1975) 7435) and with the study by Lazarovici et al. (P. Lazarovici, P. Yanai and E. Yavin, J. Biol. Chem. 262 (1986) 2645), although other secondary structural features do not agree with those of the previous studies. Secondary structure estimation from Fourier transform infrared spectra in both amide I and amide III frequency regions revealed 22-23% alpha-helix, 49-51% beta-pleated sheets and 27-28% random coils, indicating a good correlation with the secondary structure content estimated from CD analysis. Lowering of the pH of the neurotoxin to 5.5 or 4.0 did not result in any noticeable change in the overall secondary structures. However, there were significant pH-induced variations observed in the individual curve-fitted FT-IR bands in the amide III frequency region. For example, the 1302 cm-1 band (relative area, 4.2%) observed at pH 7.0 was shifted to 1297 cm-1 (relative area, 2.2%) at pH 5.5, and the relative area of the band at 1316-1317 cm-1 (alpha-helix) increased by approx. 40%. This study suggests that contrary to earlier reports, tetanus neurotoxin is a beta-pleated sheet dominated structure, and although lower pH does not change the overall contents of the secondary structures, significant conformational alterations are observed.     

Clinical and experimental mycotic keratitis caused by Aspergillus terreus and the effect of subconjunctival oxiconazole treatment in the animal model

Aspergillus terreus was isolated from a case of Keratomycosis. The patient, a 50 year old, female presented with a large corneal ulcer with hypopyon. The direct microscopic examination of the scrapings revealed hyaline, thin, septate and branched hyphae. In vitro some antimycotics (amphotericin B,5-fluorocytosine, oxiconazole, amorolfine and ketoconazole) were tested against A. terreus by agar dilution method. Ketoconazole with MIC of 3 gmg/ml after 7 days of incubation was most effective followed by oxiconazole (10g/ml). Experimental corneal ulcer was produced by injecting intralamellary 0.1 ml of the spore suspension containing 10×10₆ cfu/ml into the eyes of previously immunocompressed albino rabbits. Histopathologic examination showed infiltration and large destruction of the corneal stroma. Subconjunctival oxiconazole therapy exhibited complete cure. Based on our findings, a clinical evaluation of oxiconazole in human keratomycosis seems to be justified.     

Facilitating care of patients with HIV infection by hospital and primary care teams.

To complement the role of primary care teams working with patients with HIV disease and AIDS within greater London and to ease the load on the special hospital units a home support team was developed. It comprises six specialist nurses, a general practitioner trained medical officer, and a receptionist and is funded from regional and district sources and charities. A nurse is available for out of hours and emergency weekend calls, with support from the patient''s general practitioner or the attached medical officer. During the first 18 months 249 patients were seen; the mean duration of care was five months. Nearly a third (18/50, 30%) of patients who were terminally ill died at home. The team''s activities included practical nursing care, emotional support for carers and patients, and advice and guidance to primary care teams. Problems in providing care in patients'' homes included issues relating to confidentiality and 24 hour cover. With the increasing incidence of HIV infection the home support team may be a useful model for care of large numbers of patients with symptomatic HIV disease, especially in large urban areas.     

Antibody directed targeting of methotrexate-containing small unilamellar vesicles

Summary The potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked ³H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of ³H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG). Abbreviations used: DPPC, DL- -dipalmitoyl phosphatidylcholine; DTT, dithiothreitol; MTX, methotrexate; (MTX)SUV or MLV, MTX-containing SUV or MLV; MLV, multilamellar vesicle; NRG, normal rabbit immunoglobulin G; RAMG, rabbit antimelanoma IgG; SA, stearylamine; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate; SUV, small unilamellar vesicle; CHOL, cholesterol; LUV, large unilamellar vesicle; Ig, immunoglobulin; PDP-SA, N-[3-(2-pyridyldithio)-propinyl]stearylamine     

Effect of methionine-sulfoximine on nitrate uptake and photosynthesis in the cyanobacteriumAnabaena doliolum Bharadwaja

l-Methionine-dl-sulfoximine (MSX) stimulated nitrate uptake but inhibited¹⁴CO₂ fixation and O₂ evolution inAnabaena doliolum. Nitrate uptake was inhibited by ammonium (NH ₄ ⁺ ) in the absence of MSX, but not in the presence of MSX. Glutamine or a derivative of it appears to be the actual negative effector of nitrate utilization. In presence of nitrate, MSX-treated cells ofA. doliolum evolve more O₂ than do untreated cells. Our results suggest a close relation between photoassimilation of carbon and utilization of nitrogen.     

Effect of seasonal changes on cyanobacterial production and nitrogen-yield

A mixed culture of cyanobacteria (BGA) containingAulosira sp.,Aphanothece sp., andGloeotrichia sp. were grown throughout the year to assess the influence of seasonal variables on their biomass production and nitrogen (N)-yield under field conditions. The seasonal variables considered in this study, i.e., water temperature (maximum, minimum), solar radiation, sunshine hours, and rainfall, fluctuated widely. Attempts were made to establish a relationship between seasonal changes as independent variables and BGA productivity and N-yield as dependent variables. The analysis indicated that solar radiation was the prime factor. Estimates of BGA biomass production varied from 3.3 to 366.5 kg (dry wt)/ha/month, and N-yield ranged from 0.1 to 11.8 kg N/ha/month. The nitrogen accumulated during the study period was 71.2 kg N/ha. The variations explained by seasonal changes were 52.3 and 50.3% for biomass production and N-yield of BGA, respectively.     

Lectin mediated agglutination of Candida kefyr cells and their spheroplasts grown in sterol enriched growth medium and its correlation with lipid composition

Supplementation of the growth medium with erosterol, cholesterol and lanosterol enriched the Candida kefyr cells, presumably cell membranes with sterols. Sterol enriched C. kefyr cells showed a decrease in percentage of PHA and Con-A mediated agglutination. Sterol supplementation also increased the sterol: phospholipid ratio and in such cells unsaturated fatty acids predominated over saturated ones. The overall effect of these changes resulted in rigidifying the cell membranes as indicated by shift of break in Arrhenius plots of Mg2+ ATPase. This showed that lectin mediated agglutination of yeast cells may be affected by its membrane fluidity.     

Interaction of rat testis protein, TP, with nucleosome core particle

Circular dichroism studies have revealed that addition of testis specific protein, TP in vitro, to rat testes nucleosome core particle resulted in a decrease in the compaction of the core particle DNA. This was also corroborated by thermal denaturation analysis. Addition of TP to nucleosome core particle resulted in the conversion of a biphasic transition towards a single phase. However, at the same time there was a 20% reduction in the overall hyperchromicity of core particle DNA at core particle to TP molar ratios of 1:2 and 1:3. These observations along with our earlier report, showing the DNA melting properties of TP, suggest that TP may play an important role in the disassembly process of nucleosome core particle during spermiogenesis.     

Use of Brassica callus culture to induce sporulation in Alternaria brassicae (Berk.) Sacc.

A non-sporulating isolate of Alternaria brassicae, inoculated on callus culture of Brassica juncea cv. Kranti, colonized the callus and produced spores. When captafol, a fungicide, was added (100 mg/l) to the callus culture medium, if effectively checked fungal contamination and saprophytic growth of A. brassicae on culture medium, without adversely affecting callus growth or establishment of dual culture.     

Luteolytic action of two antiprogestational agents (RU-38486 and ZK-98734) in the rat

RU-38486 or ZK-98734 treatment (3 mg/day, s.c.) to intact or hysterectomized adult female rats on Days 5-7 post coitum induced changes characteristic of luteolysis. Ultrastructurally, the luteal cells exhibited an extensive vacuolization of the cytoplasm and perinuclear areas, degeneration of mitochondrial cristae, massive accumulation of lipid droplets, increase in number of lysosome like granules and heterochromatinization of the nucleus. In general, RU-38486 induced more marked degeneration of the luteal cells than did ZK-98734. There was also a significant decrease in peripheral plasma progesterone concentrations in treated rats. We suggest that these antiprogestagens act via inhibition of luteal function in addition to their antagonism at the uterine progesterone receptor level.