Kinetic properties of native and mutagenized isoforms of mitochondrial alcohol dehydrogenase III purified from Kluyveromyces lactis

By computer modelling and protein engineering we have investigated changes in two amino acid residues located in the coenzyme pocket of the yeast Kluyveromyces lactis mitochondrial alcohol dehydrogenase III. These two residues, Gly 225 and Ala 274, were hypothesized to be involved in the enzyme discrimination between NAD(H) and NADP(H). Upon changing Gly 225 to Ala we produced an enzyme (mutant G225A) showing very little difference from the wild-type. On the contrary, change at position 274 of Phe instead of Ala (mutant A274F) caused a significant increase of K(m) values for NAD(P) and for NADPH and even a more marked decrease in catalytic activity. The k(cat)/K(m) rates for NADP(H) were also decreased in this mutant. Enzymes with the double changes at 225 and 274 (mutant G225A-A274F) showed, apart the substantial low K(m) value for NADPH and its high catalytic efficiency, kinetic parameters relative to coenzymes which were not additive over the single substitutions. Surprisingly, enzymes with changes at the two positions reduced efficiently acetaldehyde, displaying a K(m) value 10-fold lower and a catalytic efficiency sevenfold higher with respect to parent or singularly mutated enzymes. None of the engineered enzymes would convert formaldehyde, glutaraldehyde or aromatic aldehydes but all enzymes reduced propionaldehyde and butyraldehyde at relative reaction rates approximately half of that exhibited by acetaldehyde. Interestingly only mutant A274F was able to oxidize methanol almost as well as ethanol. In addition, this mutant was capable to convert secondary and cyclic alcohols, at a rate not detected in the other isoforms. These results are in general agreement with the prediction that increasing the size of amino acids in the proximity of the coenzyme pocket would hamper the accommodation of NADP but discord the increased affinity for NADPH as well as for alcoholic or aldehydic substrates with high steric hindrance.     

Immunolocalization of sperm protein 17 in human testis and ejaculated spermatozoa.

Sperm protein 17 (Sp17) is a highly conserved mammalian protein whose primary function is still poorly understood. Immunohistochemistry (IHC) in the human testis reveals the presence of Sp17 in some spermatocytes and abundantly in spermatids. All spermatogonia, Sertoli cells, and Leydig cells appear to be immunonegative for Sp17, whereas some interstitial cells are immunopositive. IHC recognized two distinct populations (immunopositive or not for Sp17) in the ejaculated spermatozoa. Although it will be necessary to clarify why some ejaculated spermatozoa do not contain Sp17, its distribution suggests that this protein may be associated with some phases of germinal cell differentiation.     

In an elite cross of maize a major quantitative trait locus controls one-fourth of the genetic variation for grain yield

Quantitative trait loci (QTLs) for grain yield, dry matter content and test weight were identified in an F₂ segregating population derived from a single cross between two elite maize lines (B73 and A7) and testcrossed to two genetically divergent in breds. Most of the QTLs inferred were consistent across locations, indicating that the expression of the genes influencing the traits under investigation was largely independent of the environment. By using two different tester lines we found that QTLs exhibited by one tester may not necessarily be detected with the second one. Only loci with larger effects were consistent across testers, suggesting that interaction with tester alleles may contribute to the identification of QTLs in a specific fashion. Analysis across both testers revealed four significant QTLs for grain yield that explained more than 35% of the phenotypic variation and showed an overall phenotypic effect of more than 2t/ha. The major QTL for grain yield, located in the proximity of the Nucleolus Organiser Region, accounted for 24.5% of the phenotypic variation for grain yield and showed an average effect of allele substitution of approximately 1 t/ha. Marker-assisted introgression of the superior A7 allele at this locus in the B73 genetic background will not differ from qualitative trait introgression and will eventually lead to new lines having superior testcross performance.     

Ultrastructural localization of acetylcholinesterase in Axolotl brain capillaries

The ultrastructural distribution of cholinesterases on the brain capillaries of Axolotl has been studied. The Axolotl brain contains branching, Anastomosing capillary network and capillary loops. The presence of acetylcholinesterases is seen on the basal lamina and in the spaces between the endothelial cells and the pericytes of both types of vessels. The role of this enzyme in the blood-brain barrier is discussed.     

Apolipoprotein AIMilano. Disulfide-linked dimers increase high density lipoprotein stability and hinder particle interconversion in carrier plasma

The in vitro metabolism of high density lipoproteins (HDL) in carriers of the apolipoprotein AIMilano (apoAIM) mutant was investigated during incubation of whole plasma and isolated lipoprotein fractions. A reduced cholesterol esterification (16.5 versus 25.0% for controls) and a decreased exchange of lipids between HDL and lower density lipoproteins was observed during incubation (6 h at 37 degrees C) of AIM plasma. Control HDL3 were converted to larger, faster-floating HDL particles, whereas only a fraction of AIM HDL3 followed the same pathway. Incubations were also carried out by mixing HDL3 from controls and AIM carriers with a lipoprotein-depleted plasma fraction in the presence of triglyceride-rich particles isolated from Intralipid. AIM HDL3 again showed a reduced capacity for lipid exchange; some HDL3 particles followed a "normal" conversion to faster-floating, larger HDL, whereas the small AIM HDL3 were not modified, indicating that AIM HDL3 are a mixture of metabolically functional and nonfunctional particles. Following transformation of the apoAIM homo- and heterodimers into their normal counterparts, i.e. monomeric apoAI and -AII, by reduction and carboxamidomethylation of AIM HDL3, the modified HDL3 behave like control HDL3 during incubation with lipoprotein-depleted plasma and triglyceride-rich particles. The presence of AIM dimers is most likely responsible for the increased HDL3 stability in the AIM carriers, indicating that apolipoprotein composition plays a major role in HDL particle interconversion.     

Depletion of pre-beta-high density lipoprotein by human chymase impairs ATP-binding cassette transporter A1- but not scavenger receptor class B type I-mediated lipid efflux to high density lipoprotein

The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cellular unesterified cholesterol and phospholipid to lipid-poor apolipoprotein A-I. Chymase, a protease secreted by mast cells, selectively cleaves pre-beta-migrating particles from high density lipoprotein (HDL)(3) and reduces the efflux of cholesterol from macrophages. To evaluate whether this effect is the result of reduction of ABCA1-dependent or -independent pathways of cholesterol efflux, in this study we examined the efflux of cholesterol to preparations of chymase-treated HDL(3) in two types of cell: 1) in J774 murine macrophages endogenously expressing low levels of scavenger receptor class B, type I (SR-BI), and high levels of ABCA1 upon treatment with cAMP; and 2) in Fu5AH rat hepatoma cells endogenously expressing high levels of the SR-BI and low levels of ABCA1. Treatment of HDL(3) with the human chymase resulted in rapid depletion of pre-beta-HDL and a concomitant decrease in the efflux of cholesterol and phospholipid (2-fold and 3-fold, respectively) from the ABCA1-expressing J774 cells. In contrast, efflux of free cholesterol from Fu5AH to chymase-treated and to untreated HDL(3) was similar. Incubation of HDL(3) with phospholipid transfer protein led to an increase in pre-beta-HDL contents as well as in ABCA1-mediated cholesterol efflux. A decreased cholesterol efflux to untreated HDL(3) but not to chymase-treated HDL(3) was observed in ABCA1-expressing J774 with probucol, an inhibitor of cholesterol efflux to lipid-poor apoA-I. Similar results were obtained using brefeldin and gliburide, two inhibitors of ABCA1-mediated efflux. These results indicate that chymase treatment of HDL(3) specifically impairs the ABCA1-dependent pathway without influencing either aqueous or SR-BI-facilitated diffusion and that this effect is caused by depletion of lipid-poor pre-beta-migrating particles in HDL(3). Our results are compatible with the view that HDL(3) promotes ABCA1-mediated lipid efflux entirely through its lipid-poor fraction with pre-beta mobility.     

Geographical determinants and environmental implications of livestock production intensification in Asia

Under growing and urbanizing demand, livestock production is rapidly evolving in South, East and South-east Asia, with both an increase of production and a shift to intensive production systems. These changes infer impacts on the environment, on public health and on rural development. Environmental impacts are mainly associated with a mismanagement of animal excreta, leading to pollution of surface water, ground water and soils by nutrients, organic matter, and heavy metals. In the framework of the Livestock Environment and Development Initiative, this research aims at assessing, on a regional scale, the impacts of livestock production on nutrient fluxes. Phosphate (P(2)O(5)) mass balances were chosen as an indicator and were calculated on the basis of spatially modelled livestock densities, estimated excretion values and crop uptake. The results show a strong West--East gradient regarding the distribution of monogastrics, with clear concentration in densely populated areas and around urban centres. P(2)O(5) overloads are estimated on 23.6% of the study area's agricultural land, mainly located in eastern China, the Ganges basin and around urban centres such as Bangkok, Ho Chi Minh City and Manila. On average, livestock manure is estimated to account for 39.4% of the agricultural P(2)O(5) supply (the remaining share being supplied by chemical fertilisers). Livestock is the dominant agricultural source of P(2)O(5) around urban centres and in livestock specialised areas (southern and north-eastern China), while chemical fertilisers are dominant in crop (rice) intensive areas.     

Phenotype and genotype of Candida albicans strains isolated from pregnant women with recurrent vaginitis

Fourteen out of 80 pregnant women receiving prenatal care presented signs and symptoms of recurrent vaginal candidiasis. Candida albicans strains were isolated from 12 patients (85.7%), and these were submitted to morphotyping (morphological characteristics of the colony), antifungal typing (pattern of sensitivity to amphotericin B, 5-fluorcytosine, myconazole, ketoconazole and fluconazole) and genotyping (electrophoretic migration of DNA fragments digested with EcoRI and HinfI). Alteration of morphotype and antifungal type was observed in 50% of the patients, but the genotype of the strains isolated from the same patients at different times was identical in all subjects. The predominant morphotypes presented continuous fringes and the basic changes observed among antifungal types was the emergence of strains resistant to myconazole, which was the drug used for the treatment of the first episode of vaginitis. We conclude that recurrent vaginal candidiasis is caused by the persistence of a single yeast genotype that undergoes morphological and behavioral changes in the presence of antifungal agents due to the selective pressure to which it is submitted. This revised version was published online in August 2006 with corrections to the Cover Date.