I. Photoaffinity probes provide a general method to prepare synthetic peptide-conjugates

A general synthetic strategy is described for the preparation of peptide-conjugates where the peptides contain the NH₂ terminal, COOH terminal, or internal regions of the protein sequence. Glycoprotein D of herpes simplex virus type 1 is used as a representative protein. Ten-residue peptide fragments of the native sequence were synthesized using standard solid-phase methodology. Photoprobes stable to conditions of synthesis and HF cleavage were coupled directly to the protected-peptide resin during synthesis. This one-step procedure eliminates the potential modification of functional groups in the sequence of interest that can occur when using chemically labile bifunctional reagents. Since the photoprobe is inert until photolysis, the synthetic peptide-probe can be readily purified by high-performance liquid chromatography before cross-linking to the carrier molecule. The following photoprobe derivatives were investigated: thep-azidobenzoyl,p-nitrophenylalanyl, andp-benzoylbenzoyl groups. The benzophenone photoprobes were shown to give the highest incorporation of peptide-probe with the protein carrier over a wide range ofpH and solvent conditions. For solid-phase synthesis three benzophenone photoprobes can be used: benzoylbenzoic acid, benzoylbenzoylglycine, andN ^e-(4-benzoylbenzoyl)-N -t-butyloxycarbonyl-lysine.     

Increased cell surface hydrophobicity associated with possession of an additional surface protein by Aeromonas salmonicida

Abstract The cell surface of strains of Aeromonas salmonicida possessing an additional surface protein (A-protein) was shown to be more hydrophobic than strains devoid of this protein, using the techniques of phase partitioning, agglutination in the presence of ammonium sulphate and hydrophobic interaction chromatography.     

Mass mortality of Diadema antillarum on the Caribbean coast of Panama

The ecologically important sea urchin Diadema antillarum suffered mass mortalities in 1983, first noted in Panama and then reported from the rest of the Caribbean. We documented the effects of this mortality at two localities on the Atlantic coast of Panama, Punta Galeta and the San Blas Archipelago. At Punta Galeta, affected by the mortality in January 1983, the numbers of D. antillarum changed from an estimated 14,000 per ha in June 1982 to 0.5 per ha in May 1983; by February 1984 they had increased to 38 per ha. In the San Blas, where mass mortality started in April 1983, the number of D. antillarum in permanent quadrats on 8 reefs was reduced by an average of 94.2%. The average reduction in population density measured in transects on nine reefs was 98.9%. Data taken in permanent quadrats on four reefs in 1978, 1979 and 1980 indicate that population fluctuations of D. antillarum are normally much smaller, justifying the labeling of the 1983 event as mass mortality. Size structure of the San Blas populations was also affected; mean test diameter of D. antillarum on four reefs was reduced from 48.6 mm to 25.0 mm. Other echinoids (Echinometra viridis, E. lucunter, Lytechinus variegatus, L. williamsi, Eucidaris tribuloides, Tripneustes ventricosus, Clypeaster rosaceus and Echinoneus cyclostomus) suffered no ill effects at either Galeta or the San Blas; their population densities remained stable or increased. Density determinations of Diadema mexicanum at the island of Taboguilla on the Pacific side of Panama indicate that Diadema mass mortality did not extend to the eastern Pacific. Sea surface temperatures, tidal levels, rainfall and salinity showed no abnormal fluctuations during the time of D. antillarum mass mortality at Galeta, suggesting that mortality was not due to physical stress. The wide geographical spread and species-specificity of the mortality suggest a water-borne pathogen as the most likely causative agent. Recovery of D. antillarum populations is likely to be slow because there are few, if any, unaffected populations in the Caribbean to contribute larvae for the recolonization of depleted areas. The absence of D. antillarum will probably be reflected by changes in the algal, coral and echinoid communities, and by altered patterns of bioerosion.     

Predisposing factors in systemic and central nervous system candidiasis: Histopathological and cultural observations in the rat

Four atypical isolates of Microsporum canis, three from humans and one from a cat, were obtained from North-West London. These and a further human isolate were compared with each other and with a typical isolate of the fungus. Immediately after isolation the atypical isolates were very labile, but were stabilised after a few subcultures from selected sectors. The stable forms differed from each other, but all had a tendency to brown rather than yellow pigmentation, to feathery submerged mycelium and to abnormal macroconidia. The macroscopic appearance and texture of the colonies depended on the density, orientation and branching pattern of the submerged mycelium.In recent years similar brown, feathery forms of M. canis have been reported from monkeys but not from cats. It is suggested that all such isolates may be culturally stable forms of a very unstable strain, probably feline in origin, which has yet to be described.     

Further studies on the structural requirements of agents for immunotherapy of the guinea pig line-10 tumor

Summary We made a comparative study of the in vivo binding of immunoglobulins (Ig) to a polyoma virus-induced ascitic tumor propagated in syngeneic or allogeneic mice. The Ig coat was found to appear more rapidly and to be denser in H 2-incompatible than in H 2-compatible mice. This suggests that antibodies were fixed specifically on strong normal transplantation antigens (H-2) recognized as non-self by allogeneic mice. Experiments with mice in which immunosuppression had been achieved by means of X-irradiation confirmed that the Ig fixed on SEWA cells are actively bound antibodies. The only mice that could fix Ig on tumor cells were those that had been specifically immunized against cell surface antigens shared by SEWA cells before irradiation, while mice hyperimmunized against nonrelated antigens could not.In partial fulfilment of doctorate thesis requirements     

Arachidonic acid metabolism in rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells metabolized arachidonic acid through more than one enzymatic pathway. The major cyclooxygenase product was prostaglandin (PG) D₂ as established by chromatographic and chemical behavior and the effect on platelet aggregation. PGD₂ formation from exogenous arachidonic acid was inhibited by indomethacin, 1 μg/ml. RBL-1 incubated with exogenous arachidonic acid also formed SRS-A the synthesis of which was not inhibited by indomethacin. However, the SRS-A activity was blocked by the specific receptor antagonist FPL 55712. [¹⁴C]arachidonic acid was effectively incorporated into the phospholipids of RBL-1 cells. Challenge of such prelabelled cells or unlabelled cells with A 23187 caused release of PGD₂, SRS-A and another presently unidentified product. However, with A 23187 as a stimulus, the RBL-1 cyclo-oxygenase could not be blocked by low concentrations of indomethacin. This work further substantiates our earlier findings that SRS-A formed from arachidontic acid is not a cyclooxegenase product.     

Recovery,structure, and function of dog granulocytes after freeze-preservation with dimethylsulfoxide

The recovery, structure and function of dog granulocytes were determined before and after freeze-preservation. Leucocytes were isolated from defibrinated or anti-coagulated whole blood and subsequent erythrocyte sedimentation on a column of 2:1 dextran (6%)-isopaque (33.9%). Granulocytes isolated by these procedures were examined for changes in O₂ consumption associated with phagocytosis, in vitro directed migration (chemotaxis), bactericidal activity, and ultrastructure before and after freezing. Granulocytes were frozen in DMSO (7.5%) and autologous serum or HBSS-minus and 20% autologous serum at the rate of ?1 °C/min to ?80 °C and stored in liquid N₂ vapor.After freeze-preservation, O₂ consumption associated with phagocytosis was decreased by 54 and 64% for granulocytes isolated from defibrinated or from ACD-anticoagulated blood, respectively. Bactericidal activity is only slightly depressed in samples from either isolation method after freeze-preservation when compared to the prefreeze controls, but granulocytes isolated from defibrinated blood are significantly less effective in killing bacteria than those from ACD-anticoagulated blood. Chemotactic response after freeze-preservation was completely inhibited in granulocytes isolated from defibrinated blood. Exposure of granulocytes to ACD inhibited chemotaxis prior to freezing, but the granulocytes responded chemotactically after freeze-thaw and additional washing. The ultrastructure of granulocytes observed before and after freeze-thaw was similar for cells isolated by both methods. However, nuclear, cytoplasmic, and granular changes observed were slightly greater in granulocytes isolated from defibrinated blood. Dog granulocytes isolated by either method withstood freeze-preservation in DMSO to a degree not previously reported.It is concluded that dog granulocytes freeze-preserved by these methods are functional in vitro, but that phagocytic, directed migration, and bactericidal functions and ultrastructure are impaired to different degrees, according to the method of isolation and preparation for storage. These results indicate the need for continued investigation on the effects of storage variables on the preservation of granulocytes.     

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 m thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.