Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.     

Maximization of Candida utilis cytochromes by pulse additions of ethanol to continuous cultures

Candida utilis was grown with glucose as growth-limiting carbon source in batch culture, steady-state continuous culture, and non-steady-state continuous culture. High cytochrome concentrations were routinely recorded in cells harvested in the latter stages of batch culture. They were occasionally recorded in cells from imprecisely controlled steady-state cultures, but precise control of the steady-state dissolved oxygen tension stabilized the cytochromes at relatively low levels. Controlled non-steady-state continuous cultures, imposed by pulse additions of ethanol, routinely produced cells with high cytochrome concentrations. A mechanism is proposed whereby maintenance of cytochrome derepression in continuous culture is dependent upon indefinitely prolonging an “overshoot” response in gene expression.     

Effects of dinocap on otolith development: evaluation of mouse and hamster fetuses at term

The morphology of otoliths in CD-1 mouse and Syrian hamster fetuses exposed to the fungicide dinocap were evaluated at the end of gestation. Pregnant mice were dosed by gavage with 0, 10, 15, 30, or 60 mg/kg/day dinocap in corn oil on days 7-16 of gestation. Pregnant hamsters were dosed by the same route with 0, 50, 100, or 200 mg/kg/day on days 7-14 of gestation. At the end of gestation (day 18 in mice, day 15 in hamsters) dams were killed and all fetuses were removed and fixed overnight in 70% ethanol. Fetal heads were then removed, left in 70% ethanol for at least 3 days, and then dehydrated in a graded ethanol series and cleared with methyl salicylate. Otoliths were examined by darkfield microscopy, and each otolith was scored for morphological completeness on a scale of 0 to 3. Otolith development was complete by day 18 of gestation in control mouse fetuses. Otolith development was complete in many, but not all, of the hamster fetuses by day 15 of gestation. In the mouse, dinocap exposure inhibited fetal otolith formation in a dose-related manner, with a significant effect on total otolith score occurring at 10 mg/kg/day and above. Dinocap affected otolith formation in the hamster only at 100 mg/kg/day (200 mg/kg/day was embryolethal), concomitant with severe maternotoxicity and fetotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.     

Variation in the ribosomal RNA genes among individuals of Vicia faba

Summary Length heterogeneity in the ribosomal repeat of Vicia faba is due to the presence of variable numbers of a 325 bp subrepetitive element within the nontranscribed spacer region. The distribution of size classes among 88 individuals within a population was investigated by blot-hybridization. We find that individual plants can exhibit more than 20 size classes and that hybridization patterns are highly diverse from individual to individual, more so than for any species so far investigated. In contrast, no such differences are observed in patterns for different tissues from a single plant or from parental to F₁ generation. Some changes were observed in the F₂ generation. We conclude that unequal recombination can give rise to the diversity that we observe for the V. faba rDNA repeats.     

High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.

The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.