High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.

The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.     

A subunit-sized butyrylcholinesterase present in high concentrations in pooled rabbit serum

A butyrylcholinesterase of mol.wt. approx. 83000 was observed in pooled rabbit serum. The enzyme was named monomeric butyrylcholinesterase to distinguish it from the larger oligomeric butyrylcholinesterase of horse and human serum whose subunits are the same size as the monomeric enzyme. The active-site concentration of monomeric butyrylcholinesterase in the pooled serum was 0.18mum, which is five times the concentration of butyrylcholinesterase in pooled horse serum. This was surprising, since the horse serum is regarded as a rich source of butyrylcholinesterase, whereas rabbit serum is not generally thought to contain significant amounts of any butyrylcholinesterase. The explanation, in large part, was the relatively low k(cat.) of the monomeric enzyme, which was approx. 57s(-1) with butyrylthiocholine as substrate and is one-thirtieth of the comparable k(cat.) of horse butyrylcholinesterase. The substrate specificity of monomeric butyrylcholinesterase also differed significantly from that of horse and human butyrylcholinesterase. For example, with the monomeric enzyme, the hydrolysis of 1mm-acetylthiocholine was only 4% the rate for 1mm-butyrylthiocholine, whereas human and horse butyrylcholinesterases hydrolysed 1mm-acetylthiocholine at 50% of the rate for 1mm-butyrylthiocholine. Moreover, monomeric butyrylcholinesterase generally hydrolysed aromatic esters more rapidly than choline esters, whereas the reverse is true of the butyrylcholinesterases. To facilitate the study of monomeric butyrylcholinesterase, it was separated from the larger butyrylcholinesterase and acetylcholinesterase, also present in rabbit serum, and purified 89-fold by fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography.     

Oögenesis in a marine teleost,Blennius pholis L.

Summary Oögenesis in the oviparous marine teleost, Blennius pholis L., is examined. Eleven developmental stages are identified by ultrastructural observations when changes in the distributions of the organelles and inclusions are described. An exogenous source for the protein yolk precursors is indicated, but less clear is the endogenous contribution. Changes in the follicle epithelium are described together with the formation of the zona which is considered to be follicular in origin. Two types of follicle cell are distinguished and these probably function differently in the process of zona formation. The zona becomes divided into the externa and interna, the latter probably resulting from the chemical ordering by disulphide bonding of the proteinaceous material of the former.We are indebted to Professor E.W. Knight-Jones in whose department the work was carried out, and to the Natural Environment Research Council for support for one of us (S.E.S.).     

Quercetin inhibits hexose transport in a human diploid fibroblast

Summary The flavonol quercetin, a phloretin analog, inhibits transport of 2-deoxyglucose and 3-O-methylglucose in a cultured human diploid fibroblast. This inhibition is related to transport itself and not to the reported effects of flavonoids on membrane-bound ATPases. From concentration-inhibition curves at several pH's we conclude that uncharged (acid) quercetin (pK=7.65) is the inhibitory form of the molecule (K _I =10m). Quercetin, unlike phloretin, is rapidly degraded in 0.1n NaOH; the degradation products are weakly inhibitory to hexose transport.     

SMITHSONIELLA GEN. NOV., A POSSIBLE EVOLUTIONARY LINK BETWEEN THE MULTICELLULAR AND SIPHONOUS HABITS IN THE ULVOPHYCEAE,CHLOROPHYTA

A low relief, green turf-forming alga of a heterotrichous habit was discovered in the coral reef microcosm, Museum of Natural History, Smithsonian Institution. Erect filaments bore lateral, specialized sporangia and together with basal filaments possessed septal plugs between adjacent cells, grossly similar to the “pit connections” of red algae. Data are presented which: 1) establish the identity of our plant with a plant recently described as Pilinia earleae Gallagher et Humm from the Florida Gulf coast; 2) support our establishment of the new genus Smithsoniella and our transfer of P. earleae to this new taxon. Additional data on pigmentation and cytology are related to the fine structure of other selected green algae to develop and test three hypotheses, viz. Smithsoniella earleae represents either: 1) a symbiotic association between a green and a red alga; 2) an alga which belongs to either the Ulotrichales, Chaetophorales or the Chroolepidales; or 3) an alga representing an evolutionary link between filamentous forms of the Ulvophyceae and members of the coenocytic siphonalean complex (e.g., Codiales or Caulerpales) of the Chlorophyta. Data refute hypotheses 1 and 2 but do lend support to the third hypothesis.     

Parallel usage of medicinal plants by africans and their caribbean descendants

Cultivated plants are cited by anthropologists as important indicators of man’s past. Medicinal species, to a large extent, have been overlooked even though in some cases these plants represent some of the social and cultural traditions of the people who use them. A number of cultivated plants have been traced from the Old World to the New World and are generally believed to have been carried there by European explorers and early settlers. However, some evidence has been accumulating to indicate that there may have been contacts other than by European colonists. One trade route that has been neglected is that of the slave trade from west Africa to the Caribbean. Three plant species,Citrus aurantifolia, Ricinus communis andAbrus precatorius, may exemplify the role and use of this route. They also indicate the migration and assimilation of west African Fulani, Hausa, and Mandingo cultures and Obeah religion into Caribbean society.     

Infection of human natural killer (NK) cells with replication-defective human T cell leukemia virus type I provirus. Increased proliferative capacity and prolonged survival of functionally competent NK cells.

Human T-cell leukemia virus type I (HTLV-I) can infect a variety of human cell types, but only T lymphocytes are efficiently immortalized after HTLV-I infection. This study reports an attempt to infect and to immortalize NK cells with HTLV-I. Co-cultivation of freshly isolated NK cells with a HTLV-I-producing T cell line did not result in NK cell infection. However, NK cells activated with an anti-CD16 mAb and co-cultivated with a HTLV-I-producing T cell line were reproducibly infected by HTLV-I. HTLV-I infection was documented in NK cell lines and clones by the detection of defective integrated provirus by both Southern blot and polymerase chain reaction analysis. Although HTLV-I-infected NK cells produced viral proteins, they did not produce infectious viral particles. HTLV-I-infected NK cells were phenotypically indistinguishable from their uninfected counterparts (CD16+, CD2+, CD56+, CD3-). They also retained the ability to mediate both natural and antibody-dependent cell cytotoxicity. The IL-2-dependent proliferation of HTLV-I-infected NK cells was significantly greater than that of uninfected NK cells. The doubling time of this infected population was reduced from 9 days to 3 days, and the overall survival of the culture in the absence of restimulation was extended from 5 wk to 18 wk. Unlike T lymphocytes, HTLV-I-infected NK cells were not immortal, implying a fundamental difference between these two lymphocyte populations.