Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance

The selectivity of interaction between bovine spinal cord myelin basic protein (MBP) and eight different spin-labeled lipid species in complexes with dimyristoylphosphatidylglycerol (DMPG) and between spin-labeled phosphatidylglycerol and spin-labeled phosphatidylcholine in complexes of MBP with various mixtures of DMPG and dimyristoylphosphatidylcholine (DMPC) has been studied by electron spin resonance (ESR) spectroscopy. In DMPC/DMPG mixtures, the protein binding gradually decreased with increasing mole fraction of DMPC in a nonlinear fashion. The lipid-protein binding assays indicated a preferential binding of the protein to phosphatidylglycerol relative to phosphatidylcholine without complete phase separation of the two lipids. The outer hyperfine splittings (2Amax) of both phosphatidylglycerol and phosphatidylcholine labeled at C-5 of the sn-2 chain (5-PGSL and 5-PCSL, respectively) were monitored in the lipid-protein complexes as a function of the mole fraction of DMPC. The increases in the value of Amax induced on binding of the protein were larger for 5-PGSL than for 5-PCSL, up to 0.25 mole fraction of DMPC. Beyond this mole fraction the spectral perturbations induced by the protein were similar for both lipid labels. The ESR spectra of phosphatidylglycerol and phosphatidylcholine labeled at C-12 of the sn-2 chain were two component in nature, indicating indicating a direct interaction of the protein with the lipid chains, at mole fractions of DMPC up to 0.25. Quantitation of the motionally restricted spin-label population by spectral subtraction again indicated a preferential interaction of the protein with phosphatidylglycerol relative to phosphatidylcholine. Up to DMPC mode fractions of 0.25, the microenvironment of the protein was enriched in DMPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spin-label ESR studies on the interaction of bovine spinal cord myelin basic protein with dimyristoylphosphatidylglycerol dispersions

Electron spin resonance (ESR) spectroscopy and chemical binding assays were used to study the interaction of bovine spinal cord myelin basic protein (MBP) with dimyristoylphosphatidylglycerol (DMPG) membranes. Increasing binding of MBP to DMPG bilayers resulted in an increasing motional restriction of PG spin-labeled at the C-5 atom position in the acyl chain, up to a maximum degree of association of 1 MBP molecule per 36 lipid molecules. ESR spectra of PG spin-labels labeled at other positions in the sn-2 chain showed a similar motional restriction, while still preserving the chain flexibility gradient characteristic of fluid lipid bilayers. In addition, labels at the C-12 and C-14 atom positions gave two-component spectra, suggesting a partial hydrophobic penetration of the MBP into the bilayer. Spectral subtractions were used to quantitate the membrane penetration in terms of the stoichiometry of the lipid-protein complexes. Approximately 50% of the spin-labeled lipid chains were directly affected at saturation protein binding. The salt and pH dependence of the ESR spectra and of the protein binding demonstrated that electrostatic interaction of the basic residues of the MBP with the PG headgroups is necessary for an effective association of the MBP with phospholipid bilayers. Binding of the protein, and concomitant perturbation of the lipid chain mobility, was reduced as the ionic strength increased, until at salt concentrations above 1 M NaCl the protein was no longer bound. The binding and ESR spectral perturbation also decreased as the protein charge was reduced by pH titration to above the pI of the protein at approximately pH 10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fermentation parameters of Peptostreptococcus productus on gaseous substrates (CO,H2/CO2)

Intrinsic growth and substrate uptake parameters were obtained for Peptostreptococcus productus, strain U-1, using carbon monoxide as the limiting substrate. A modified Monod model with substrate inhibition was used for modeling. In addition, a product yield of 0.25 mol acetate/mol CO and a cell yield of 0.034 g cells/g CO were obtained. While CO was found to be the primary substrate, P. productus is able to produce acetate from CO₂ and H₂, although this substrate could not sustain growth. Yeast extract was found to also be a growth substrate. A yield of 0.017 g cell/g yeast extract and a product yield of 0.14 g acetate/g yeast extract were obtained. In the presence of acetate, the maximum specific CO uptake rate was increased by 40% compared to the maximum without acetate present. Cell replication was inhibited at acetate concentrations of 30 g/l. Methionine was found to be an essential nutrient for growth and CO uptake by P. productus. A minimum amount of a complex medium such as yeast extract (0.01%) is, however, required.     

XX sex reversal in the American cocker spaniel dog: phenotypic expression and inheritance

Summary This study was conducted to define the range of phenotypic expression and mode of inheritance of XX sex reversal in the cocker spaniel dog. Breeding experiments produced F1, F1BC, and F2 generations in which 29 XX true hermaphrodites and 3 XX males were defined by chromosome constitution, serial histologic sections of the gonads, and examination of the internal and external genitalia. In XX true hermaphrodites, the most common combination of gonads was bilateral ovotestes, followed by ovotestis and ovary, then ovotestis and testis. The amount of testicular tissue in the two gonads was closely correlated within each true hermaphrodite. The distribution of testicular tissue within ovotestes of true hermaphrodites was consistent with the hypothesis that testicular differentiation is initiated in the center of the gonad and spreads outward. XX males had bilateral aspermatogenic testes and the internal ducts and external genitalia were more masculinized than in true hermaphrodites. Results of breeding experiments are consistent with autosomal recessive inheritance, the affected phenotype being expressed only in dogs with an XX chromosome constitution. The phenotypic expression and mode of inheritance of this disorder is compared to XX sex reversal in humans and other animals.     

Effect of haemin on enzyme activity and cytotoxin production by Bacteriodes gingivalis W50

Abstract Bacteriodes gingivalis W50 was grown in a chemostat at pH 7.5 under haemin-limited and haemin excess conditions in order to provide cells with a known high or low inefectivity and virulence for mice, respectively. The activities of enzymes and formation of cytotoxic fermentation products by these cells were compared. No significant differences in chymotrypsin activity or in the ability to degrade hyaluronic acid were found; neither were there any significant differences in the production of butyrate, propionate or succinate. At pH 7.5, trypsin activity was 3.5-fold higher in cells grown under haemin excess conditions whereas collagenolytic activity was nearly 3-fold higher in haemin-limited cells. Although collagenolytic activity may be important in tissue damage, a high ratio of trypsin to collagen breakdown activities was associated with virulent cells grown under an excess of haemin.     

A flexible peptide spacer increases the efficacy of holoricin anti-T cell immunotoxins

Immunotoxins, constructed by chemically cross-linking an antibody and protein toxin, do not possess the high efficacy of the native toxin. Decreases in toxicity are due in part to the steric constraints imposed on the two macromolecules, which result in both decreased antibody binding and toxin function. In examining the structural features that influence efficacy in holotoxin-antibody conjugates, it was found that the incorporation of a 29-residue polypeptide, derived from the insulin B chain between the antibody and ricin moiety, resulted in an increase in both potency and efficacy. In a murine model system, potency of the peptide spacer conjugate was increased nearly 10-fold; however, when examined by the procedure used to purge bone marrow, the peptide spacer conjugate was not demonstrably more toxic to nontarget cells than the nonspacer conjugate. Thus, in addition to increases of efficacy and potency, this novel immunotoxin demonstrated increased specificity by approximately 10-fold. To test the general utility of peptide spacer inclusion, a T101-ricin conjugate was constructed with the peptide spacer. It yielded a protein synthesis inhibition rate of -0.6 log/h on MOLT-3 cells, greater than 10-fold more efficacious than a previously constructed nonspacer T101-ricin conjugate examined under similar conditions.     

PHYLOGENY OF THE SUBFAMILIES OF THE FAMILY BRACONIDAE (HYMENOPTERA: ICHNEUMONOIDEA): A REASSESSMENT

Abstract— The recently published phylogeny of Braconidae by Quicke and van Achterberg is reassessed. Character-state definitions and character polarities are evaluated, and more rigorous methods are suggested. Our results indicate that there are many more parsimonious solutions to their data set, the consensus of which differs substantially from their results. Based on our reassessment, little can be said about the relationships among braconid subfamilies. Consensus trees show the cyclostomes as a largely unresolved basal grade. The two other major lineages which have been proposed, the helconoids and microgastroids, are somewhat better resolved, but not consistently so. Relationships among the helconoids vary considerably depending on the parameters used for parsimony analysis.     

New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.