High pressure, a tool for exploring heme protein active sites

High pressure is an interesting and suitable parameter in the study of the dynamics and stability of proteins. The effects of pressure on proteins delineates its volumic (deltaV degrees ) and energetic (deltaG degrees ) parameters. An enormous amount of effort has been invested by several laboratories in developing basic theory and high pressure techniques that allow the determination of barotropic parameters. Cytochrome P450s, one of the largest super families of heme proteins, are good models for high pressure studies. Two distinct pressure-induced spin transitions of the heme iron in the active site and a P450 to P420 inactivation process have been characterized. The obtained reaction volumes of these two processes for a series of analog-bound cytochrome P450s are compared. We have shown that both the spin volume and the inactivation volume are dependent on the substrate analogs which are known to modulate the polarity and hydration of the heme pocket. Several linear correlations were found between these reaction volumes and the physico-chemical properties of the heme protein such as the polarity-induced exposure of tyrosines, the hydration of the cytochrome CYP101 heme pocket, and the mobility and binding of the substrates indicate that they constitute the main contribution to the complex thermodynamic reaction volume parameters. This interpretation allows us to conclude that cytochrome CYP101, CYP2B4 and CYP102 possess a similar mechanism of substrate binding. Interestingly the barotropic behaviors of monomeric cytochrome P450s are quite different from those of oligomeric and hetorooligomeric cytochrome P450s. The interactions of heterooligomeric subunits influence the stability of individual cytochrome P450s and the asymmetric organization of subunits which can control and modulate the activity and the recognition with NADPH-cytochrome P450 reductase.     

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.     

The Steve Biko affair: a case study in medical ethics

Steve Biko died in detention in South Africa in 1977. Critical ethical issues are raised both by the conduct of the doctors responsible for Biko's care and by the subsequent response of the medical profession as a whole. Because those issues are relevant to all healthcare professionals everywhere, the Biko affair provides a useful case study in medical ethics. We discuss the case in this article, describing how we use it in our teaching.     

Regulation of glucose utilization and lipogenesis in adipose tissue of diabetic and fat fed animals: Effects of insulin and manganese

In order to evaluate the modulatory effects of manganese, high fat diet fed and alloxan diabetic rats were taken and the changes in the glucose oxidation, glycerol release and effects of manganese on these parameters were measured from adipose tissue. An insulin-mimetic effect of manganese was observed in the adipose tissue in the controls and an additive effect of insulin and manganese on glucose oxidation was seen when Mn²⁺ was addedin vitro. The flux of glucose through the pentose phosphate pathway and glycolysis was significantly decreased in high fat fed animals. Although thein vitro addition of Mn²⁺ was additive with insulin when¹⁴CO2 was measured from control animals, it was found neither in young diabetic animals (6–8 weeks old) nor in the old (16 weeks old). Both insulin and manganese caused an increased oxidation of carbon-1 of glucose and an increase of its incorporation into¹⁴C-lipids in the young control animals; the additive effect of insulin and manganese suggests separate site of action. This effect was decreased in fat fed animals, diabetic animals and old animals. Manganese alone was found to decrease glycerol in both the control and diabetic adipose tissue inin vitro incubations. The results of the effects of glucose oxidation, lipogenesis, and glycerol release in adipose tissue of control and diabetic animals of different ages are presented together with the effect of manganese on adipose tissue from high fat milk diet fed animals.     

Biodiesel production from waste cooking oil: 2. Economic assessment and sensitivity analysis

The economic feasibilities of four continuous processes to produce biodiesel, including both alkali- and acid-catalyzed processes, using waste cooking oil and the ‘standard’ process using virgin vegetable oil as the raw material, were assessed. Although the alkali-catalyzed process using virgin vegetable oil had the lowest fixed capital cost, the acid-catalyzed process using waste cooking oil was more economically feasible overall, providing a lower total manufacturing cost, a more attractive after-tax rate of return and a lower biodiesel break-even price. On the basis of these economic calculations, sensitivity analyses for these processes were carried out. Plant capacity and prices of feedstock oils and biodiesel were found to be the most significant factors affecting the economic viability of biodiesel manufacture.     

Cognitive abilities - the result of selective pressures on food acquisition?

Locating and capturing food are suggested as significant selection pressures for the evolution of various cognitive abilities in mammals and birds. The hypothesis is proposed that aspects of food procuring behaviour should be strongly indicative of particular cognitive abilities.Experimental data concerning higher mental abilities in mammals and birds are reviewed. These data deal with self-recognition studies, rule-learning experiments, number concept, deceptive abilities, tool-use and observational learning.A Darwinian approach reveals: (1) the adaptiveness of particular abilities for particular niches, (2) that in complex foraging environments, increases in foraging efficiencies in animals should result from the evolution of particular cognitive abilities, (3) that phenomena such as convergent mental evolution should be expected to have taken place across taxonomic groups for species exploiting similar niches, (4) that divergence in mental ability should also have taken place where related species have exploited dissimilar niches.Experimental data of higher mental abilities in animals concur with a Darwinian explanation for the distribution of these cognitive abilities and no anomalies have been found.There are, as a consequence, significant implications for the welfare of animals subject to training when training methodology gives little or no consideration to the various mental abilities of species.     

Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.     

Substrate-stereoselectivity of a high-affinity glutamate transporter cloned from the CNS of the cockroach Diploptera punctata

A cDNA encoding a Na(+)-dependent glutamate transporter has been cloned from the brain of the cockroach Diploptera punctata. The cDNA encodes a transporter protein of 481 amino acids, designated DipEAAT1, which when expressed in baculovirus infected insect cells, resulted in a 40-50 fold increase in [(3)H]L-glutamate uptake. DipEAAT1 mRNA is expressed in the brain, as is the RNA encoding TrnEAAT1, a related transporter recently isolated from the caterpillar Trichoplusia ni. The affinity of these transporters for L-glutamate and several structural analogues was compared. Both have a high affinity for L-glutamate, their presumed primary substrate, but quite different affinities for D-aspartate. TrnEAAT1 was found to be similar to other glutamate transporters in that its ability to transport [(3)H]L-glutamate into cells was inhibited strongly by D- and L- isomers of aspartate and its analogues. DipEAAT1, by contrast, was inhibited weakly by all D- isomers tested. The affinity of DipEAAT1 for [(3)H]D-aspartate was found to be an order of magnitude lower than that of TrnEAAT1, revealing an unusual stereoselectivity for aspartate substrates by the cockroach transporter. The activity of DipEAAT1 was also unaffected by the presence of Zn(++) in the bathing solution, despite the presence of a putative Zn(++)-binding motif conferring Zn(++)-sensitivity on some mammalian glutamate transporters.     

The catalytic activity of the Src family kinases is required to disrupt cadherin-dependent cell-cell contacts

Despite the importance of epithelial cell contacts in determining cell behavior, we still lack a detailed understanding of the assembly and disassembly of intercellular contacts. Here we examined the role of the catalytic activity of the Src family kinases at epithelial cell contacts in vitro. Like E- and P-cadherin, Ca(2+) treatment of normal and tumor-derived human keratinocytes resulted in c-Yes (and c-Src and Fyn), as well as their putative substrate p120(CTN), being recruited to cell-cell contacts. A tyrosine kinase inhibitor with selectivity against the Src family kinases, PD162531, and a dominant-inhibitory c-Src protein that interferes with the catalytic function of the endogenous Src kinases induced cell-cell contact and E-cadherin redistribution, even in low Ca(2+), which does not normally support stable cell-cell adhesion. Time-lapse microscopy demonstrated that Src kinase inhibition induced stabilization of transiently formed intercellular contacts in low Ca(2+). Furthermore, a combination of E- and P-cadherin-specific antibodies suppressed cell-cell contact, indicating cadherin involvement. As a consequence of contact stabilization, normal cells were unable to dissociate from an epithelial sheet formed at high density and repair a wound in vitro, although individual cells were still motile. Thus, cadherin-dependent contacts can be stabilized both by high Ca(2+) and by inhibiting Src activity in low (0.03 mM) Ca(2+) in vitro.     

Base Composition Skews, Replication Orientation, and Gene Orientation in 12 Prokaryote Genomes

Variation in GC content, GC skew and AT skew along genomic regions was examined at third codon positions in completely sequenced prokaryotes. Eight out of nine eubacteria studied show GC and AT skews that change sign at the origin of replication. The leading strand in DNA replication is G-T rich at codon position 3 in six eubacteria, but C-T rich in two Mycoplasma species. In M. genitalium the AT and GC skews are symmetrical around the origin and terminus of replication, whereas its GC content variation has been shown to have a centre of symmetry elsewhere in the genome. Borrelia burgdorferi and Treponema pallidum show extraordinary extents of base composition skew correlated with direction of DNA replication. Base composition skews measured at third codon positions probably reflect mutational biases, whereas those measured over all bases in a sequence (or at codon positions 1 and 2) can be strongly affected by protein considerations due to the tendency in some bacteria for genes to be transcribed in the same direction that they are replicated. Consequently in some species the direction of skew for total genomic DNA is opposite to that for codon position 3. Received: 2 February 1998 / Accepted: 15 June 1998