Quantification of intact 146S foot-and-mouth disease antigen for vaccine production by a double antibody sandwich ELISA using monoclonal antibodies

A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed to quantify 146S antigen of foot-and-mouth disease virus (FMDV) strain A10 Holland grown in suspension cultures of surviving bovine tongue epithelium. When virus harvests were incubated with trypsin--which affects VP1, the most immunogenic structural protein of FMDV--the concentration of 146S antigen as determined by ELISA was reduced by greater than 90%. Therefore, the test detected essentially only those virus particles with intact VP1. When the test was compared with the sucrose density gradient method, concentrations of 146S antigen correlated well (r = 0.87). The rate of variation in both tests was the same. In contrast to the sucrose density gradient method, the DAS-ELISA can simultaneously quantify 146S antigen in many samples, and also indicates when VP1 of 146S particles has disintegrated by the action of proteases.     

Stable transformation of tomato cell cultures after bombardment with plasmid and YAC DNA

Summary Stable transformants were obtained after microprojectile particle bombardment of tomato cell suspensions (Lycopersicon esculentum cv VFNT Cherry and L. pennellii). The suspensions were bombarded with tungsten particles coated with either plasmid (6.3 kb) or yeast artificial chromosome (YAC) (80 kb) DNA containing the ß-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. The YAC DNA contained an insert of approximately 50 kb of DNA from VFNT Cherry. L. pennellii suspensions were more amenable to transformation than VFNT Cherry; more kanamycin-resistant calli were recovered from L. pennelli after bombardment with plasmid DNA, and only L. pennellii cells produced transformants after bombardment with YAC DNA. DNA gel blot analysis confirmed the presence of the nptll and GUS genes. This analysis also confirmed the integration of YAC DNA into the genome of the kanamycin-resistant calli and suggested that the level of intactness of the integrated YAC DNA was fairly high in four of the five transformants examined. Microprojectile bombardment of regenerable cultures with YACs may ultimately aid in map-based cloning of agriculturally-important genes.Abbreviations YAC yeast artificial chromosome - MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-3-acetic acid - GUS ß-glucuronidase - nptII neomycin phosphotransferase II     

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis.

Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes O1K-, O4K2, O26K60, O75K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacarylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidogylcan-bound matrix protein I of E. coli O26K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II of E. coli O26K60 in both their unmodified and their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.     

Timing and size of blooms of the ctenophore Mnemiopsis leidyi in relation to temperature in Narragansett Bay, RI

The ctenophore Mnemiopsis leidyi is at the northern extreme of its geographic range in Narragansett Bay, an estuary on the northeast coast of the United States. Blooms have typically been observed in late summer and fall according to records from 1950 to 1979. We document an expansion of the seasonal range of this important planktonic predator to include springtime blooms during the 1980s and 1990s. This shift to an earlier seasonal maximum is associated with increasing water temperature in Narragansett Bay. Temperatures in spring have risen, on average, 2 °C from 1950 to 1999 with warm years being associated with the positive phase of the North Atlantic Oscillation. During 1999, M. leidyi appeared earlier in spring and was more abundant than during any previous year for which records are available. Changes in the seasonal pattern and abundance of this predator are likely to have important effects on planktonic ecosystem dynamics of Narragansett Bay. These include reduction of zooplankton abundance in spring followed by increases in size and frequency of summer phytoplankton blooms. Earlier blooms of M. leidyi may also reduce survival of eggs and larvae of fish because, as in 1999, they coincide with the period of peak spawning.     

Microbialite response to an anthropogenic salinity gradient in Great Salt Lake,Utah

A railroad causeway across Great Salt Lake, Utah (GSL), has restricted water flow since its construction in 1959, resulting in a more saline North Arm (NA; 24%–31% salinity) and a less saline South Arm (SA; 11%–14% salinity). Here, we characterized microbial carbonates collected from the SA and the NA to evaluate the effect of increased salinity on community composition and abundance and to determine whether the communities present in the NA are still actively precipitating carbonate or if they are remnant features from prior to causeway construction. SSU rRNA gene abundances associated with the NA microbialite were three orders of magnitude lower than those associated with the SA microbialite, indicating that the latter community is more productive. SSU rRNA gene sequencing and functional gene microarray analyses indicated that SA and NA microbialite communities are distinct. In particular, abundant sequences affiliated with photoautotrophic taxa including cyanobacteria and diatoms that may drive carbonate precipitation and thus still actively form microbialites were identified in the SA microbialite; sequences affiliated with photoautotrophic taxa were in low abundance in the NA microbialite. SA and NA microbialites comprise smooth prismatic aragonite crystals. However, the SA microbialite also contained micritic aragonite, which can be formed as a result of biological activity. Collectively, these observations suggest that NA microbialites are likely to be remnant features from prior to causeway construction and indicate a strong decrease in the ability of NA microbialite communities to actively precipitate carbonate minerals. Moreover, the results suggest a role for cyanobacteria and diatoms in carbonate precipitation and microbialite formation in the SA of GSL.     

Cofactor‐induced reversible folding of Flavodoxin‐4 from Lactobacillus acidophilus

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN‐binding mechanism to flavodoxins‐4 were obtained from the NMR structures of the apo‐protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO ) and exhibits the characteristic flavodoxin fold, with a central five‐stranded parallel β–sheet and five α‐helices forming an α/β‐sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the β‐sheet and covers the FMN‐binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.     

Fractionation of the asymmetry current in the squid giant axon into inactivating and non-inactivating components

The operation of the voltage-sensitive sodium gating system in the nerve membrane involves conformational changes that are accompanied by small asymmetrical displacement currents. The asymmetry current may be divided into a component that is inactivated by positive voltage-clamp pulses, and recovers from inactivation with exactly the same time course as the sodium conductance, and one that is not inactivated. A method is described for recording the two components separately with the aid of an inactivating prepulse. They appear to have a marked difference in their rising phases, that of the non-inactivating component being just about as fast as the imposed step in membrane potential, while the inactivating component requires some tens of microseconds to reach its peak.     

Differential responses of freshwater wetland soils to sulphate pollution

Sulphate (SO₄ ^2-)reduction rates are generally low in freshwaterwetlands and are regulated by the scarceavailability of the ion. Increasedconcentrations of this electron acceptor due tosulphur (S) pollution of groundwater andsurface water may, however, lead to highSO₄ ^2- reduction rates now regulatedby the availability of appropriate electrondonors. Due to variations in this availability,the response to S pollution (e.g. from surfacewater or groundwater) is expected to differbetween soils. This hypothesis was tested inlaboratory mesocosm experiments by comparingtwo wetland soil types with distinctlydifferent humus profiles: a Hydromoder and aRhizomull type. In the first type, expected tohave a higher availability of degradable soilorganic matter (SOM), SO₄ ^2-availability appeared to be rate limiting forSO₄ ^2- reduction. In the Rhizomullsoils, in contrast, the electron acceptor didnot limit SO₄ ^2- reduction rates athigher concentrations. These differences inresponse could not, however, be attributed todifferences in the various SOM fractions or inSOM densities. Eutrophication and free sulphideaccumulation, two major biogeochemical problemscaused by SO₄ ^2- pollution, occurredin both types. The absolute extent ofphosphorus mobilisation was determined by theconcentration of this element in the soil (C/Pratio), while the level of sulphideaccumulation was governed by the concentrationof dissolved iron in the pore water. It wastherefore concluded that neither the humusprofile nor the concentrations of different SOMfractions in the soils are reliable indicatorsfor the sensitivity of wetland types to Spollution.     

Structure of the growing telomeres of Trypanosomes

We have developed a method for the molecular cloning of DNA adjacent to chromosome ends (telomeres). A recombinant DNA clone obtained from the telomeres of the protozoan Trypanosoma brucei contains large stretches of the repeat (CCCTAA)n. This repeat is flanked by a larger subtelomeric repeat (29 bp in one case). These repeats account for the presence of large DNA stretches not cut by restriction enzymes downstream of telomeric VSG genes. All telomeres analyzed thus far (more than 30) grow by approximately 6 bp per trypanosomal division and contract by occasional large deletions. Our results suggest that growth is due mainly to addition of CCCTAA units.