The Apolipoprotein E/CI/CII Gene Cluster and Late-Onset Alzheimer Disease

The chromosome 19 apolipoprotein E/CI/CII gene cluster was examined for evidence of linkage to a familial Alzheimer disease (FAD) locus. The family groups studied were Volga German (VG), early-onset non-VG (ENVG; mean age at onset <60 years), and late-onset families. A genetic association was observed between apolipoprotein E (ApoE) allele ε4 and FAD in late-onset families; the ε4 allele frequency was .51 in affected subjects, .37 in at-risk subjects, .11 in spouses, and .19 in unrelated controls. The differences between the ε4 frequencies in affected subjects versus controls and in at-risk subjects versus controls were highly significant (standard normal deviate [Z_SND]) = 7.37, P < 10⁻⁹; and Z_SND = 4.07, P < .00005, respectively). No association between the ε4 allele and FAD was observed in the ENVG or VG groups. A statistically significant allelic association between ε4 and AD was also observed in a group of unrelated subjects; the ε4 frequency was .26 in affected subjects, versus .19 in controls (Z_SND = 2.20, P < .03). Evidence of linkage of ApoE and ApoCII to FAD was examined by maximum-likelihood methods, using three models and assuming autosomal dominant inheritance: (1) age-dependent penetrance, (2) extremely low (1%) penetrance, and (3) age-dependent penetrance corrected for sporadic Alzheimer disease (AD). For ApoCII in late-onset families, results for close linkage were negative, and only small positive lod-score-statistic (Z) values were obtained (model 1, maximum Z [Z_max] = 0.61, recombination fraction [θ] = .30; model 2, Z_max = 0.47, θ = .20). For ApoE in late-onset kindreds, positive Z values were obtained when either allele frequencies from controls (model 1, Z_max = 2.02, θ = .15; model 2, Z_max = 3.42, θ = .05) or allele frequencies from the families (model 1, Z_max = 1.43, θ = .15; model 2, Z_max = 1.70, θ = .05) were used. When linkage disequilibrium was incorporated into the analysis, the Z values increased (model 1, Z_max = 3.17, θ = .23; model 3, Z_max = 1.85, θ = .20). For the ENVG group, results for ApoE and ApoCII were uniformly negative. Affected-pedigree-member analysis gave significant results for the late-onset kindreds, for ApoE (Z_SND = 3.003, P = .003) and ApoCII (Z_SND = 2.319, P = .016), when control allele frequencies were used but not when allele frequencies were derived from the families.     

Inhibition of growth and decreased survival of B104 rat neuroblastoma cells after exposure to hyperbaric oxygen

Summary The toxic effects of hyperbaric oxygen (HBO) on growth and survival of B104 rat neuroblastoma cells were investigated. Cells in log phase growth were incubated at 37°C with 10 atm O₂ for 1 to 4 h. After exposure to HBO, cells were monitored for their subsequent growth and survival. Two hours of exposure caused a slowing of growth, which returned to normaly by the end of the 7th d of the postexposure period. Exposures to O₂ of 3 h or longer caused a complete cessation of growth for 4 d after the exposure and very litle or no recovery after this period. Increased hydrostatic pressure for 6 h using helium as the inert gas had no effect on growth. A colony formation assay was used to quantitative the degree of cell death induced by HBO. The resulting survival curve was of the exponential type with a broad shoulder between 0 to 2.5 h of exposure to 10 atm O₂. The curve fell off sharply at 2.5 h with an exponential decrease in survival when the exposure to HBO was extended to 4 h. At 2 h about 50% of cells were killed, but at 4 h only 2% survived the treatment. These results show that the depression of the growth rate by HBO is related to the number of cells that are killed by the exposure. This system provides a model in which the molecular and cellular effects of HBO can be investigated. This work was supported by the National Institutes of Health, Bethesda, MD, Grants AM-21423 and CA-14489, and by grants from the W. W. Smith Foundation and the Philadelphia Foundation. The studies presented here are part of a dissertation submitted by G. J. Gendimenico in partial fulfillment of the requirements for the degree of Ph.D. in pharmacology from the University of Pennsylvania.     

Excitation energy transfer from tryptophan residues of peptides and intrinsic proteins to diphenylhexatriene in phospholipid vesicles and biological membranes

An efficient excitation energy transfer from tryptophan residues of intrinsic membrane proteins to an extrinsic fluorescent probe (diphenylhexatriene) has been demonstrated in rat erythrocyte ghosts. To correlate this transfer with the localization of the probe, a model system has been investigated. It consists of peptides containing lysine and tryptophan residues bound to negatively charged phosphatidylserine vesicles. Absorption and fluorescence spectroscopies were used to follow peptide binding and diphenylhexatriene incorporation. Peptide binding is accompanied by a blue shift of the tryptophan fluorescence together with an increase of the quantum yield and of the fluorescence decay time. An experimental Föster critical distance value of 4.0 nm was found for energy transfer from tryptophan residues of peptides to diphenylhexatriene which approaches the range of calculated values (3.1–3.7 nm) using a two-dimensional model. These results demonstrate that efficient energy transfer can occur from tryptophan residues of intrinsic proteins to diphenylhexatriene without any interaction between diphenylhexatriene and proteins in biological membranes.     

Interactions of yeast valyl-tRNA synthetase with RNAs and conformational changes of the enzyme.

Different conformations have been identified for the enzyme valyl-tRNA synthetase from yeast inside its complex with one tRNA molecule by neutron scattering. One form is identical to that of the free enzyme in solution; the other form is more contracted, having a radius of gyration which is smaller by 10% and a specific volume which is smaller by 1%. The contracted conformation has been found for the complexes with tRNA^Val and tRNA^Asp in phosphate buffer (pH 6.3) provided the ionic strength is lower than about 150 mm. In higher ionic strength (up to about 500 mm) the enzyme still forms a complex with tRNA^Val but its conformation remains that of the free protein in solution. In the complex with tRNA₃^Leu, the enzyme conformation is that of the free state even at the lowest ionic strength examined (that of the phosphate buffer, 60 mm). The free enzyme is an elongated molecule of radius of gyration 40 Å (a compact protein of the same molecular weight would have a radius of gyration of 30 Å).The positioning within the complex of tRNA^Val, on the one hand, and tRNA₃^Leu, on the other, is very different. The first tRNA is intimately associated with the enzyme, lying predominantly closer to the centre of mass of the complex than the protein. In the complex with tRNA₃^Leu, the tRNA lies further away from the centre of mass of the complex than the protein.Small concentrations of tRNA^Val, tRNA^Asp, tRNA₃^Leu or Escherichia coli 5 S ribosomal RNA cause the enzyme to aggregate into dimers, trimers and higher aggregates provided the ionic strength of the buffer is below 150 mm. In higher ionic strength or for [RNA]: [enzyme] > 1 the aggregates are dissociated to yield the one-to-one RNA-enzyme complex.     

Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized.

Summary A HindIII (17.0 kb) and an EcoRl restriction fragment (6.9 kb) of Klebsiella pneumoniae nif DNA were cloned on two small amplifiable plasmids, pCM1 and pSA30 respectively. These plasmids between them carry 14 of the 15 known Klebsiella nif genes. The operon for the three structural genes for nitrogenase, nifpHDK, is carried on pSA30: four and five of the remaining six operons are on pCRA37 and pCM1 respectively. All of the nif genes were assigned to endonculease restriction fragments of DNA using the Southern blotting technique (Southern, 1975) with total DNA of nif insertion mutants and radioactive plasmid DNA which contained cloned nif DNA sequences. Their locations were consistent with the genetic map of nif genes. The estimated size of the nif gene cluster was 24 kb.     

Metabolism of inorganic sulphate in the isolated perfused rat liver. Effect of sulphate concentration on the rate of sulphation by phenol sulphotransferase

1. The metabolism of inorganic [³⁵S]sulphate (Na₂³⁵SO₄) was studied in the isolated perfused rat liver at three initial concentrations of inorganic sulphate in the perfusion medium (0, 0.65 and 1.30mm), in relation to sulphation and glucuronidation of a phenolic drug, harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole). 2. [³⁵S]Sulphate rapidly equilibrated with endogenous sulphate in the liver. It was excreted in bile and reached, at the lowest concentration in the perfusion medium, concentrations in bile that were much higher than those in the perfusion medium; at the higher sulphate concentrations, these concentrations were equal. The physiological concentration of inorganic sulphate in the liver, available for sulphation of drugs, is similar to the plasma concentration. 3. At zero initial inorganic sulphate in the perfusion medium, the rate of sulphation was very low and harmol was mainly glucuronidated. At 0.65mm-sulphate glucuronidation was much decreased and considerable sulphation took place, indicating efficient competition of conjugation by sulphation. At 1.30mm-sulphate the sulphation increased still further. 4. The results suggest that an important factor in sulphation is the relatively high K_m of synthesis of adenosine 3′-phosphate 5′-sulphatophosphate (the co-substrate of sulphation) for inorganic sulphate, which is of the order of the plasma concentration of inorganic sulphate. The steady-state adenosine 3′-phosphate 5′-sulphatophosphate concentration may determine the rate of sulphate conjugation of drugs in the rat in vivo.     

Immobilization of yeast microbodies and the properties of immobilized microbody enzymes

Summary Yeast microbodies isolated from methanol-grown cells of Kloeckera sp. No. 2201 were immobilized by two types of entrapping techniques: photocrosslinking of liquid oligomers of suitable photosensitive resins and crosslinking of albumin molecules with glutaraldehyde. The apparent activities of catalase, alcohol oxidase, and D-amino acid oxidase in the gel-entrapped microbodies were 40–50, 70–80, and ca. 50% respectively as compared with those in the free microbodies. Alcohol oxidase in the immobilized microbodies, similarly to that in free ones, oxidized methanol, ethanol, n-propanol, n-butanol, n-amyl alcohol, and benzyl alcohol. Some properties of catalase and alcohol oxidase in the microbodies immobilized by the above-mentioned techniques were studied in comparison with those of the enzymes in the free microbodies.     

Physicochemical conditions and benthic macroinvertebrates of a tertiary sewage treatment system

Hydrobiologia - Physicochemical conditions and benthic macroinvertebrates were studied in the Beaumont, Texas sewage treatment plant from August, 1973 to June, 1974. Physicochemical conditions...