Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy.

Bilayer-to-nonbilayer phase transitions in phospholipids occur by means of poorly characterized intermediates. Many have proposed that membrane fusion can also occur by formation of these intermediates. Structures for such intermediates were proposed in a recent theory of these transition mechanisms. Using time-resolved cryo-transmission electron Microscopy (TRC-TEM), we have directly visualized the evolution of inverted phase micro-structure in liposomal aggregates. We have identified one of the proposed intermediates, termed an interlamellar attachment (ILA), which has the structure and dimensions predicted by the theory. We show that ILAs are likely to be the structure corresponding to "lipidic particles" observed by freeze-fracture electron microscopy. ILAs appear to assemble the inverted cubic (III) phase by formation of an ILA lattice, as previously proposed. ILAs are also observed to mediate membrane fusion in the same systems, on the same time scale, and under nearly the same conditions in which membrane fusion was observed by fluorescence methods in earlier studies. These earlier studies indicated a linkage between a membrane fusion mechanism and III phase formation. Our micrographs suggest that the same intermediate structure mediates both of those processes.     

Pertussis toxin inhibits enkephalin stimulation of GTPase of NG108-15 cells

In neuroblastoma-glioma (NG108-15) hybrid cells, opiates inhibit adenylate cyclase and stimulate a low Km GTPase. It has been postulated that the stimulation of GTPase plays a role in opiate inhibition of adenylate cyclase (Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4185-4189). Treatment of NG108-15 cells with pertussis toxin attenuates receptor-mediated inhibition of adenylate cyclase. The toxin acts by catalyzing the ADP-ribosylation of a 41,000-dalton substrate believed to be a part of the receptor-adenylate cyclase complex. We have found that toxin treatment of NG108-15 results in inhibition of the opiate-stimulated GTPase. The concentration of toxin required for inhibition of this GTPase was similar to that needed for both attenuation of opiate inhibition of adenylate cyclase and ADP ribosylation of the 41,000-dalton substrate. Inhibition of the opiate-induced GTPase by pertussis toxin in isolated membranes required NAD, consistent with the hypothesis that this effect of the toxin resulted from ADP ribosylation of a protein component of the system. Since the opiate-stimulated GTPase is believed to play a role in the receptor-mediated decrease in adenylate cyclase activity, inhibition of this GTPase may be an important part of the mechanism by which the toxin interferes with opiate action on adenylate cyclase.     

Differences among the polyadenylated RNA sequences of human leucocyte populations: an approach to the objective classification of human leukaemias

We have constructed a complementary DNA (cDNA) library representing expressed sequences of the white blood cells from a patient with chronic granulocytic leukaemia. The library was screened by colony hybridization of 32P-labelled cDNAs synthesized from the polyadenylated RNAs of the white blood cells from patients with chronic granulocytic or chronic lymphocytic leukaemia. The autoradiographic patterns were compared and 70 recombinants were selected to comprise a panel which distinguished between these two types of leukaemia. Hybridization of this panel with complementary DNAs transcribed from the polyadenylated RNAs of a variety of normal and neoplastic leucocyte populations showed that the RNA sequences in high abundance in leucocytes from chronic granulocytic leukaemias differ quite radically from those in other leucocytes. The patterns of hybridization seen when this panel was challenged with cDNAs representing the RNAs of normal and leukaemic leucocyte populations were sufficiently different to distinguish clearly the peripheral blood leucocytes of chronic granulocytic leukaemias from other populations of white blood cells, both normal and leukaemic. We suggest that this approach might provide additional markers useful in the classification of the acute leukaemias, especially the undifferentiated leukaemias whose identification by conventional methods is uncertain.     

Influence of photoperiod and medium formulation on peanut somatic embryogenesis

Somatic embryos were produced from peanut (Arachis hypogaea L.) immature zygotic cotyledons. Comparisons were made of the level of -naphthaleneacetic acid during induction, nitrogen formulation of the medium, and photoperiod. Over 70% embryogenesis was obtained regardless of NAA level used. Percent embryogenesis and number of embryos were markedly lower in explants induced on NAA compared to 2,4-D. Embryo production was not greatly affected by either the use of Murashige & Skoog versus Finer & Nagasawa salts or light versus dark culture conditions. However, embryo morphology was noticeably affected by photoperiod. Embryos produced under a 16 h photoperiod were tough, woody and difficult to separate for subsequent germination and conversion. Those produced under a 0-h photoperiod were succulent and pliable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - MS Murashige & Skoog (1962) - B5 Gamborg et al. (1968) - picloram 4-amino-3,5,6-trichloropicolinic acid - FN Finer & Nagasawa (1988) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

The breakdown of malathion in soil and soil components

The disappearance of the organophosphorus insecticide, malathion, from a silt loam soil and from its organic and inorganic components was examined. Half-lives and the time taken for 90% decomposition in nonsterile, sodium azide-treated, and 2.5 Mrad-irradiated soils were similar (3/4–1 1/2 days and 4–6 days, respectively) but breakdown in autoclaved soils was negligible. Decay in nonsterile sand, silt, and clay minus organic matter fractions was 3–6 times slower than that recorded in the original soil. Breakdown of malathion in the clay plus organic matter fraction (organo-mineral complex) was rapid (half-life, 1 day), as was the case in the separated organic matter (half-life, 1 3/4 days). Filter-sterilized organic matter was not as effective in catalyzing the breakdown of malathion (half-life, 4 days), and no loss occurred from any of the autoclaved components. Irradiation doses of 2.5 and 5.0 Mrad had little influence on the ability of soil to degrade malathion. Thereafter, increases up to 20 Mrad had a more drastic, though far from totally inhibitory, effect. Our results suggest that either the colloidal organic matter itself, or a fraction associated with it, is the most important single factor concerned with the rapid breakdown of malathion in the soil studied. Direct microbial metabolism is a slower process and may have a significant role in malathion disappearance in coarsetextured soils low in colloidal organic matter. The catalytic component of the organic matter is suggested to be a stable exoenzyme and is supportive of reports by other workers. The quantitative effect of organo-mineral complex (containing the active degradative ingredient) additions to sand and silt fractions on the rate of subsequent malathion decay is also described.     

Primary structure of kunitz-type trypsin inhibitor-2a (pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed

The primary structure of acidic trypsin inhibitor-2a (WBTI-2a,pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed was determined. This inhibitor consists of a single polypeptide chain of 180 amino acids including four half-cystine residues and has an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2a,pI 5.9, showed 84% identity to acidic trypsin inhibitor-2 (WBTI-2,pI 5.1) but only 57% identity to the basic trypsin inhibitor (WBTI-1,pI 8.9) and 50% identity to the chymotrypsin inhibitor of winged bean. The data indicate that winged bean seed contains a family of three Kunitz-type inhibitors which have about 50% identity.     

Platelet Recruitment Promotes Keratocyte Repopulation following Corneal Epithelial Abrasion in the Mouse

Corneal abrasion not only damages the epithelium but also induces stromal keratocyte death at the site of injury. While a coordinated cascade of inflammatory cell recruitment facilitates epithelial restoration, it is unclear if this cascade is necessary for keratocyte recovery. Since platelet and neutrophil (PMN) recruitment after corneal abrasion is beneficial to epithelial wound healing, we wanted to determine if these cells play a role in regulating keratocyte repopulation after epithelial abrasion. A 2 mm diameter central epithelial region was removed from the corneas of C57BL/6 wildtype (WT), P-selectin deficient (P-sel^-/-), and CD18 hypomorphic (CD18^hypo) mice using the Algerbrush II. Corneas were studied at 6h intervals out to 48h post-injury to evaluate platelet and PMN cell numbers; additional corneas were studied at 1, 4, 14, and 28 days post injury to evaluate keratocyte numbers. In WT mice, epithelial abrasion induced a loss of anterior central keratocytes and keratocyte recovery was rapid and incomplete, reaching ~70% of uninjured baseline values by 4 days post-injury but no further improvement at 28 days post-injury. Consistent with a beneficial role for platelets and PMNs in wound healing, keratocyte recovery was significantly depressed at 4 days post-injury (~30% of uninjured baseline) in P-sel^-/- mice, which are known to have impaired platelet and PMN recruitment after corneal abrasion. Passive transfer of platelets from WT, but not P-sel^-/-, into P-sel^-/- mice prior to injury restored anterior central keratocyte numbers at 4 days post-injury to P-sel^-/- uninjured baseline levels. While PMN infiltration in injured CD18^hypo mice was similar to injured WT mice, platelet recruitment was markedly decreased and anterior central keratocyte recovery was significantly reduced (~50% of baseline) at 4–28 days post-injury. Collectively, the data suggest platelets and platelet P-selectin are critical for efficient keratocyte recovery after corneal epithelial abrasion.