Apolipoprotein A-I alpha -helices 7 and 8 modulate high density lipoprotein subclass distribution.

Mice have a monodisperse high density lipoprotein (HDL) profile, whereas humans have two major subfractions designated HDL(2) and HDL(3). Human apoA-I transgenic mice exhibit a human-like HDL profile, indicating that the amino acid sequence of apoA-I is a determinant of the HDL profile. Comparison of the primary sequence of mouse and human apoA-I and the previously designated "hinge" domain of apoA-I led us to hypothesize that alpha-helices 7 and 8 (7/8) are determinants of HDL subclass distribution. The following proteins were expressed in Escherichia coli: human apoA-I, T7-hAI; mouse apoA-I, T7-mAI; chimeric human apoA-I containing murine helices 7/8 in place of human helices 7/8, T7-hAI(m7/8); and the reciprocal chimera, T7-mAI(h7/8). The recombinant proteins were examined for their association with human plasma HDL subclasses. The results demonstrated that T7-hAI bound HDL(2) and HDL(3) equally well, whereas T7-mAI bound to HDL(2) preferentially. T7-hAI(m7/8) behaved like T7-mAI, and T7-mAI(h7/8) behaved like T7-hAI. Thus, alpha-helices 7/8 are strong contributors to the pattern of HDL subclass association. Self-association, alpha-helicity, cholesterol efflux, and lecithin-cholesterol acyltransferase activity of the recombinant proteins were also assessed. Human apoA-I self-associates more and activates human lecithin-cholesterol acyltransferase better than mouse apoA-I. These differential characteristics of human and mouse apoA-I are not dependent on helices 7/8.     

Reduction of atherosclerosis by the peroxisome proliferator-activated receptor alpha agonist fenofibrate in mice

Several clinical and angiographic intervention trials have shown that fibrate treatment leads to a reduction of the coronary events associated to atherosclerosis. Fibrates are ligands for peroxisome proliferator-activated receptor alpha (PPARalpha) that modulate risk factors related to atherosclerosis by acting at both systemic and vascular levels. Here, we investigated the effect of treatment with the PPARalpha agonist fenofibrate (FF) on the development of atherosclerotic lesions in apolipoprotein (apo) E-deficient mice and human apoA-I transgenic apoE-deficient (hapoA-I Tg x apoE-deficient) mice fed a Western diet. In apoE-deficient mice, plasma lipid levels were increased by FF treatment with no alteration in the cholesterol distribution profile. FF treatment did not reduce atherosclerotic lesion surface area in the aortic sinus of 5-month-old apoE-deficient mice. By contrast, FF treatment decreased total cholesterol and esterified cholesterol contents in descending aortas of these mice, an effect that was more pronounced in older mice exhibiting more advanced lesions. Furthermore, FF treatment reduced MCP-1 mRNA levels in the descending aortas of apoE-deficient mice, whereas ABCA-1 expression levels were maintained despite a significant reduction of aortic cholesterol content. In apoE-deficient mice expressing a human apoA-I transgene, FF increased human apoA-I plasma and hepatic mRNA levels without affecting plasma lipid levels. This increase in human apoA-I expression was accompanied by a significant reduction in the lesion surface area in the aortic sinus. These data indicate that the PPARalpha agonist fenofibrate reduces atherosclerosis in these animal models of atherosclerosis.     

Chemical-modification rescue assessed by mass spectrometry demonstrates that gamma-thia-lysine yields the same activity as lysine in aldolase

The role of active site residues in fructose 1,6-bisphosphate aldolase is investigated by chemical-modification rescue. An active-site mutation, K107C, is constructed in a background where the four solvent-accessible cysteine residues are converted to alanine. The resulting mutant, tetK107C, when reacted with bromoethylamine (BrEA), shows a 40-fold increase in activity (to 80% that of wild type). Determination of the sites and their degree of modification using electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) is developed, allowing correlation of activity after chemical modification rescue to the degree of modification. The stoichiometry of the reaction is 2.5 aminoethylations per subunit, as measured by ESI-FTMS. Protein modification with a double-labeled mix (1:1) of natural abundance isotope (d(0)-BrEA) and 2-bromoethyl-1,1,2,2-d4-amine hydrobromide (d(4)-BrEA), followed by dialysis and trypsin digestion, shows aminoethylated peptides as "twin peptides" separated by four mass units in ESI-FTMS analysis. Using this detection procedure under nondenaturing (native) conditions, C107 is aminoethylated, whereas the four buried thiols remain unlabeled. Aminoethylation of other residues is observed, and correlates with those peptides containing histidine, methionine, and/or the amino terminus. Quantification of the aminoethylation reaction is achieved by labeling with nondeuterated d(0)-BrEA under denaturing conditions following double labeling under native conditions. In addition to complete labeling all five thiols, the intensity of the d(0)-BrEA peak for C107 containing peptides increases, and the change in the d(0)/d(4) ratio between native and denaturing conditions shows 82 +/- 4.5% aminoethylation at C107. This correlation of modification with the recovered activity, indicates that gamma-thia-lysine replaces lysine in the catalytic mechanism. Kinetic constants measured for the rescued K107C mutant enzyme with the substrates fructose 1-phosphate and fructose 1,6-bisphosphate are consistent with the role of the positively charged lysine binding to the C6-phosphate. ESI-FTMS, combined with this double-labeling procedure, allows precise identification of sites and measurement of degree of protein modification.     

Parents' age at birth of their offspring and child survival

This study presents some new results on parental age as a risk factor for child survival. The study is based on individual registration forms for live births and infant deaths collected in Hungary from 1984 to 1988. Logistic regression models have been fitted for early neonatal and neonatal mortality on the one hand, and post-neonatal mortality on the other hand. Children of older males and females have significantly higher early neonatal and neonatal mortality rates compared to those of younger males and females. The impact of age of both parents remains, however, slighter than that of other biological characteristics such as previous number of fetal deaths, induced abortions, or live births. The authors discuss possible biological explanations.     

Use of bioconversion for the preparation of [4-14C]-labeled 7 alpha- and 7 beta-hydroxylated derivatives of dehydroepiandrosterone and epiandrosterone

The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-dehydroepiandrosterone were prepared with use of the yeast-expressed human cytochrome p4507B1. Epiandrosterone (EPIA), 5 alpha-androstane-3beta,17 beta-diol, and 5 alpha-androstane-3,17-dione were obtained after incubation of [4-14C]-5 alpha-dihydrotestosterone with Escherichia coli-expressed (3beta,17 beta)-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-EPIA produced were prepared after incubation with mycelium of Rhizopus nigricans. Each labeled steroid was purified by chromatography and identified by crystallization to constant specific activity after isotopic dilution with each authentic steroid carrier. Production yields and radio-purity measurements allowed the use of such procedures for the preparation of the described radio-steroids for studies of metabolism and mode of action.     

Engineering of phytase for improved activity at low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

Viral and bacterial serology of free-ranging Pacific walrus

Serum or heparinized plasma samples were obtained between 1994 and 1996 from 20 male and 20 female adult free-ranging Pacific walrus (Odobenus rosmarus divergens) from St. Lawrence Island and Round Island, Alaska. Samples were screened for antibodies to some potentially pathogenic bacteria and viruses. No sample had detectable antibody to Brucella spp. Three of 40 (8%) had low antibody titers to Leptospira interrogans serovars. Phocine distemper virus antibodies were not detected. Serologic responses to one or more caliciviruses (San Miguel sea lion virus 12 or vesicular exanthema of swine serotypes E54, F55, G55, 1934B) were detected in 18% (seven of 40) walrus. Antibodies to one or more subtypes of influenza A virus (H10, N2, N3, N5, N6, N7) were detected in 21% (eight of 38). Periodic screening of free-ranging populations for exposure to infectious diseases has become an important component of bio-monitoring programs to facilitate understanding and detecting trends in marine mammal populations.     

Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.     

Casein utilization by Streptococcus thermophilus results in a diauxic growth in milk

In milk, Streptococcus thermophilus displays two distinct exponential growth phases, separated by a nonexponential one, during which proteinase synthesis was initiated. During the second exponential phase, utilization of caseins as the source of amino acids resulted in a decrease in growth rate, presumably caused by a limiting peptide transport activity.