Bacteriological activity in unfiltered calf sera collected for tissue culture use

Summary Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found wereBacillus species and streptococci and 63% of the sera coagulatedLimulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain ofEscherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus.

HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka approximately 1x10(9) M-1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection.     

Snow depth manipulation and its influence on soil frost and water dynamics in a northern hardwood forest

Climate change will likelyresult in warmer winter temperatures leading toless snowfall in temperate forests. Thesechanges may lead to increases in soil freezingbecause of lack of an insulating snow cover andchanges in soil water dynamics during theimportant snowmelt period. In this study, wemanipulated snow depth by removing snow for twowinters, simulating the late development of thesnowpack as may occur with global warming, toexplore the relationships between snow depth,soil freezing, soil moisture, and infiltration.We established four sites, each with two pairedplots, at the Hubbard Brook Experimental Forest(HBEF) in New Hampshire, U.S.A. and instrumentedall eight plots with soil and snow thermistors,frost tubes, soil moisture probes, and soillysimeters. For two winters, we removed snowfrom the designated treatment plots untilFebruary. Snow in the reference plots wasundisturbed. The treatment winters (1997/1998 and1998/1999) were relatively mild, withtemperatures above the seasonal norm and snowdepths below average. Results show the treatedplots accumulated significantly less snow andhad more extensive soil frost than referenceplots. Snow depth was a strong regulator ofsoil temperature and frost depth at all sites.Soil moisture measured by time domainreflectometry probes and leaching volumescollected in lysimeters were lower in thetreatment plots in March and April compared tothe rest of the year. The ratio of leachatevolumes collected in the treatment plots tothat in the reference plots decreased as thesnow ablation seasons progressed. Our data showthat even mild winters with low snowfall,simulated by snow removal, will result inincreased soil freezing in the forests at theHBEF. Our results suggest that a climate shifttoward less snowfall or a shorter duration ofsnow on the ground will produce increases insoil freezing in northern hardwood forests.Increases in soil freezing will haveimplications for changes in soil biogeochemicalprocesses.     

Logistic population growth under random dispersal

Various diffusion processes employed for modelling logistic growth are briefly summarized. A discrete-time, discrete-state space stochastic process for population growth is proposed and analyzed with either Bose-Einstein or Maxwell-Boltzmann statistics for the distribution of offspring in available sites in a restricted region. A diffusion approximation is constructed, which differs from those previously employed. The logistic law is a natural deterministic analog of the diffusion process.     

Environmental variables affecting small-scale distributions of five rotifer species in Lancaster Lake,Michigan

The small-scale distributions of the rotifers Polyarthra vulgaris, Synchaeta stylata, Conochilus unicornis, Hexarthra mira, and Asplanchna priodonta were investigated in Lancaster Lake, Cheboygan County, Michigan, July 21, 1974. Measurements were taken for 13 depths (at 1-m intervals) and at 4 times of the day (1:00 AM, 7:00 AM, 1:00 PM, and 7:00 PM). In addition, the abundances of 6 crustacean, 2 planktonic dipteran, and 10 algal species, as well as temperature, light, oxygen, chlorophyll a, alkalinity, pH and free carbon dioxide were measured. Whereas abiotic factors appeared to control large scale occupation of the lake, and excluded most species from the deeper portions of the hypolimnion, small-scale distributional variation of the rotifers depended upon biotic interactions, particularly with the crustacean zooplankton.     

Curium(III) complexation with pyoverdins secreted by a groundwater strain of Pseudomonas fluorescens

Pyoverdins, bacterial siderophores produced by ubiquitous fluorescent Pseudomonas species, have great potential to bind and thus transport actinides in the environment. Therefore, the influence of pyoverdins secreted by microbes on the migration processes of actinides must be taken into account in strategies for the risk assessment of potential nuclear waste disposal sites. The unknown interaction between curium(III) and the pyoverdins released by Pseudomonas fluorescens (CCUG 32456) isolated from the granitic rock aquifers at the Äspö Hard Rock Laboratory (Äspö HRL), Sweden, is the subject of this paper. The interaction between soluble species of curium(III) and pyoverdins was studied at trace curium(III) concentrations (3 × 10^?7 M) using time-resolved laser-induced fluorescence spectroscopy (TRLFS). Three Cm³⁺—P. fluorescens (CCUG 32456) pyoverdin species, M_pH_qL_r, could be identified from the fluorescence emission spectra, CmH₂L⁺, CmHL, and CmL^?, having peak maxima at 601, 607, and 611 nm, respectively. The large formation constants, log β₁₂₁ = 32.50 ± 0.06, log β₁₁₁ = 27.40 ± 0.11, and log β₁₀₁ = 19.30 ± 0.17, compared to those of other chelating agents illustrate the unique complexation properties of pyoverdin-type siderophores. An indirect excitation mechanism for the curium(III) fluorescence was observed in the presence of the pyoverdin molecules.     

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin

Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 ?) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.     

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease.