The effect of supercooling on the developmental capacity of mouse embryos.

The effect of supercooled storage (at subzero temperatures without ice formation) on compacted mouse morulae and early blastocysts was studied. The embryos were equilibrated with one of three storage solutions containing 1, 3, or 6% each of methanol and glycerol and cooled to -2, -5, -10, or -15 degrees C and stored for up to 24 h to assess the effect of subzero storage at different temperatures and concentrations of the permeating cryoprotectants on embryo survival. Early blastocysts showed substantially greater survival than morulae and, in general, survival of embryos of either stage increased with the concentration of cryoprotectant, while the proportion of embryos surviving decreased with decreasing storage temperature and with increased duration of storage.     

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers

Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the -helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to -polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D) and perpendicular (D) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P to L/P 15 results in a decrease of the rotational diffusion coefficient D from 3.4 × 10⁷ to 1.9 × 10⁷ s^–1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters P₂ and P₄, and the rotational diffusion coefficient D of the fluorescent probe. The order parameters P₂ and P₄ increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35. The rotational diffusion coefficient D of the DPH-propionic acid probe decreases on incorporating M13 coat protein, in accordance with the ESR results. It is concluded that M13 coat protein in the -monomeric state is not able to produce a long living lipid boundary shell and consequently an immobilization of the lipids. An overall effect on the lipids is induced, resulting in a reduction in the dynamics and an increase in average lipid order. The hydrophobic region of M13 coat protein is proposed to perfectly match the lipid bilayer, resulting in a relatively small distortion of the bilayer structure of the lipid system.     

Multilaboratory evaluation of methods for detecting enteric viruses in soils.

Two candidate methods for the recovery and detection of viruses in soil were subjected to round robin comparative testing by members of the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group. Selection of the methods, designated "Berg" and "Goyal," was based on results of an initial screening which indicated that both met basic criteria considered essential by the task group. Both methods utilized beef extract solutions to achieve desorption and recovery of viruses from representative soils: a fine sand soil, an organic muck soil, a sandy loam soil, and a clay loam soil. One of the two methods, Goyal, also used a secondary concentration of resulting soil eluants via low-pH organic flocculation to achieve a smaller final assay volume. Evaluation of the two methods was simultaneously performed in replicate by nine different laboratories. Each of the produced samples was divided into portions, and these were respectively subjected to quantitative viral plaque assay by both the individual, termed independent, laboratory which had done the soil processing and a single common reference laboratory, using a single cell line and passage level. The Berg method seemed to produce slightly higher virus recovery values; however, the differences in virus assay titers for samples produced by the two methods were not statistically significant (P less than or equal to 0.05) for any one of the four soils. Despite this lack of a method effect, there was a statistically significant laboratory effect exhibited by assay titers from the independent versus reference laboratories for two of the soils, sandy loam and clay loam.     

Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments

Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions.     

Total in vitro maturation of the Saccharomyces cerevisiae a-factor lipopeptide mating pheromone

The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is post-translationally modified in a manner analogous to that of the ras proto-oncogene product. A consensus C-terminal amino acid sequence, -CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification. Recently, the RAM/DPR1 gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor. In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events. This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras -CAAX sequences. Such agents would be potential anti-Ras-related cancer therapeutic drugs.     

ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates

The liver is the major organ which eliminates leukotriene C4 (LTC4) and other cysteinyl leukotrienes from the blood circulation into bile. Transport of LTC4 was studied using inside-out vesicles enriched in canalicular and sinusoidal membranes from rat liver. The incubation of canalicular membrane vesicles with [3H]LTC4 in the presence of ATP resulted in an uptake of LTC4 into vesicles. The initial rate of ATP-stimulated LTC4 uptake was about 40-fold higher in canalicular than in sinusoidal membrane vesicles. When liver plasma membrane vesicles were incubated in the absence of ATP, an apparent transient uptake of LTC4 was observed which was temperature-dependent and not affected by the osmolarity. This indicates that LTC4 was bound to proteins on the surface of plasma membrane vesicles. Two proteins with relative molecular weights of 17,000 and 25,000 were detected by direct photoaffinity labeling as major LTC4-binding proteins. One protein (Mr 25,000) was ascribed to subunit 1 (Ya) of glutathione S-transferase which was associated with the membrane. LTD4, LTE4, N-acetyl-LTE4, and omega-carboxy-N-acetyl-LTE4 were also transported into liver plasma membrane vesicles in an ATP-dependent manner with initial rates relative to LTC4 (1.0) of 0.46, 0.11, 0.35, and 0.22, respectively. Mutual competition between the cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione for uptake indicated that they are transported by a common carrier. Apparent Km values of the transport system for LTC4, LTD4, and N-acetyl-LTE4 were 0.25, 1.5, and 5.2 microM, respectively. The ATP-dependent transport of LTC4 into vesicles was not inhibited by doxorubicin, daunorubicin, or verapamil, or by the monoclonal antibody C219, suggesting that the transport system differs from P-glycoprotein. Liver plasma membrane vesicles prepared from mutant rats deficient in the hepatobiliary excretion of cysteinyl leukotrienes lacked the ATP-dependent transport of cysteinyl leukotrienes and S-(2,4-dinitrophenyl)-glutathione. These results demonstrate that the ATP-dependent carrier system is responsible for the transport of cysteinyl leukotrienes and glutathione S-conjugates from the hepatocytes into bile.     

In vivo synthesis of 6-azauridine 5'-triphosphate and incorporation of 6-azauridine into RNA of germinating wheat embryonic axes

The cytostatic effect of 6-azauridine on cell growth is generally regarded to be a consequence of the inhibition of de novo pyrimidine biosynthesis by the metabolite, 6-azauridine 5'-monophosphate. We show here that wheat embryonic axes further metabolize 6-azauridine to the 5'-triphosphate and incorporate the analogue into RNA, thus offering an alternative mechanism for growth inhibition. At a level of 6-azauridine required to maximally inhibit UTP biosynthesis, the ratio of 6-azaUTP to UTP is about 2:1 and substitution of 6-azauridine for uridine in new RNA is on the order of 1 in 18. The new metabolites of 6-azauridine are identified by high pressure and thin layer chromatography coupled with enzyme treatments.     

Genetics of morphological variation in geographically distant populations of the sea urchin,Arbacia punctulata (Lamarck)

Individuals of Arbacia punctulata (Lamarck) from Woods Hole, Massachusetts, and the northeastern Gulf of Mexico were reared from fertilized eggs through metamorphosis under comparable laboratory conditions. Interpopulation differences in spine length development were significant between pure-bred offsprings of these two widely separated geographic areas. Spine lengths of hybrid urchins were intermediate to pure-bred animals. Interpopulation differentiation of specific portions of the genome is proposed to account for the observed phenotypic variation in spine length.     

Purification of murine oncornaviral phosphoproteins using alkyl agarose derivatives..

Methods for the purification of both murine mammary tumor (type B) and murine leukemia (type C) oncornaviral phosphoproteins are described, in which chromatography on alkyl-agarose derivatives is used as the primary fractionation step. Gel filtration or ion exchange chromatography on phosphocellulose was the only subsequent step required for the purification of the type B and type C viral proteins, respectively. The two-step protocols also resulted in the co-purification of a low molecular weight core protein from each virus. Recoveries of the viral proteins purified by this method, based on per cent contribution of individual polypeptides to total virion proteins, were 70% or greater. Radioimmunocompetition analysis of the purified murine mammary tumor virus major core protein as well as analysis of the RNA binding properties of purified low molecular weight type C virus proteins suggests that neither antigenic reactivity nor specific RNA binding characteristics are altered by the purification protocols. The availability of these procedures should aid studies on the possible function and immunochemical properties of the native murine oncornaviral phosphoproteins and may also be extended to the purification of other oncornaviral proteins.