The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b₅ reductase (CYB5R) and cytochrome b₅ (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Daily nurse requirements planning based on simulation of patient flows

Nurses account for approximately 50 % of total hospital budgets and their allocation to medical units and shifts can significantly affect the quality of care provided to patients. The adoption of flexible shift schedules and the assessment of actual nursing time can enable sensible resource planning, balancing the quality of care with efficiency in resource use. Starting from the concept that nurse requirements are triggered by patient needs, which are stochastic in nature both for clinical activities and their duration, this paper proposes an innovative Nurse Requirement Planning model grounded on the concept of the clinical pathway (the “standard” sequence of diagnostic, therapeutic and care activities a patient with certain pathology should undertake over time) with its inner routing probability and patient dependence on nurses, which can be correlated to the time needed to perform nursing tasks. In merging and modelling these two aspects, the method summarizes the best features of acuity-quality and timed-task/activity techniques, well known although not usually applied for reasons of demands on clinicians’ time. Instead, in this paper, for each shift of the day, hospital management is enabled to choose the optimal number of nurses to meet actual requirements according to a desired service level and personnel saturation by means of a tool that simulates the patient flow in a medical unit based on automatic data retrieval from hospital databases. The validation and verification of the proposal were undertaken in a stroke unit.     

A QTL on distal Chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors

C57BL/6J-c^2J (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J × BALB/c)F₁× c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c. When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases. Received: 14 December 1999 / Accepted: 18 February 2000     

Genetic and physiological analysis of Rht8 in bread wheat: an alternative source of semi-dwarfism with a reduced sensitivity to brassinosteroids

Over the next decade, wheat grain production must increase to meet the demand of a fast growing human population. One strategy to meet this challenge is to raise wheat productivity by optimizing plant stature. The Reduced height 8 (Rht8) semi-dwarfing gene is one of the few, together with the Green Revolution genes, to reduce stature of wheat (Triticum aestivum L.), and improve lodging resistance, without compromising grain yield. Rht8 is widely used in dry environments such as Mediterranean countries where it increases plant adaptability. With recent climate change, its use could become increasingly important even in more northern latitudes. In the present study, the characterization of Rht8 was furthered. Morphological analyses show that the semi-dwarf phenotype of Rht8 lines is due to shorter internodal segments along the wheat culm, achieved through reduced cell elongation. Physiological experiments show that the reduced cell elongation is not due to defective gibberellin biosynthesis or signalling, but possibly to a reduced sensitivity to brassinosteroids. Using a fine-resolution mapping approach and screening 3104 F(2) individuals of a newly developed mapping population, the Rht8 genetic interval was reduced from 20.5 cM to 1.29 cM. Comparative genomics with model genomes confined the Rht8 syntenic intervals to 3.3 Mb of the short arm of rice chromosome 4, and to 2 Mb of Brachypodium distachyon chromosome 5. The very high resolution potential of the plant material generated is crucial for the eventual cloning of Rht8.     

Genomic dissection of pod shattering in common bean: mutations at non‐orthologous loci at the basis of convergent phenotypic evolution under domestication of leguminous species

The complete or partial loss of shattering ability occurred independently during the domestication of several crops. Therefore, the study of this trait can provide an understanding of the link between phenotypic and molecular convergent evolution. The genetic dissection of ‘pod shattering’ in Phaseolus vulgaris is achieved here using a population of introgression lines and next‐generation sequencing techniques. The ‘occurrence’ of the indehiscent phenotype (indehiscent versus dehiscent) depends on a major locus on chromosome 5. Furthermore, at least two additional genes are associated with the ‘level’ of shattering (number of shattering pods per plant: low versus high) and the ‘mode’ of shattering (non‐twisting versus twisting pods), with all of these loci contributing to the phenotype by epistatic interactions. Comparative mapping indicates that the major gene identified on common bean chromosome 5 corresponds to one of the four quantitative trait loci for pod shattering in Vigna unguiculata. None of the loci identified comprised genes that are homologs of the known shattering genes in Glycine max. Therefore, although convergent domestication can be determined by mutations at orthologous loci, this was only partially true for P. vulgaris and V. unguiculata, which are two phylogenetically closely related crop species, and this was not the case for the more distant P. vulgaris and G. max. Conversely, comparative mapping suggests that the convergent evolution of the indehiscent phenotype arose through mutations in different genes from the same underlying gene networks that are involved in secondary cell‐wall biosynthesis and lignin deposition patterning at the pod level.     

New semidominant mutations that affect mouse development

Dominantly acting mutations that produce visible phenotypes are frequently recovered, either during routine maintenance of colonies or from mutagenesis experiments. We have studied 12 dominant mouse mutations that cause a tail dysmorphology, a coat spotting phenotype, or a combination of these. The majority of these mutations act in a semidominant manner with the homozygous state associated with embryonic lethality and a visible phenotype at or before midgestation. The homozygous phenotypes include axis truncation and neural crest cell defects, as may be expected from the heterozygous phenotypes. The majority of mutations, however, also produced other phenotypes that include neural tube closure defects and aberrant heart looping. In one coat spotting mutant the homozygous condition is lethal before neural crest cell production commences. The mutated genes often function in processes additional to those alluded to by the heterozygous phenotype.     

Stage-specific requirement of a mitogen-activated protein kinase by Trypanosoma brucei

In cycling between the mammalian host and the tsetse fly vector, African trypanosomes undergo adaptive differentiation steps that are coupled to growth control. The signaling pathways underlying these cellular processes are largely unknown. Mitogen-activated protein kinases (MAPKs) are known mediators of growth and differentiation in other eukaryotic organisms. To establish the function of a MAPK homologue, TbMAPK2, in T. brucei, a null mutant was constructed. Bloodstream forms of a deltamapk2/deltamapk2 clone were able to grow normally and exhibited no detectable phenotype. When these cells were triggered to differentiate in vitro, however, they developed to the procyclic (fly midgut) form with delayed kinetics and subsequently underwent cell cycle arrest. Introduction of an ectopic copy of the TbMAPK2 gene into the null mutant restored its ability to differentiate and to divide. In contrast, a TbMAPK2 mutant, in which the T190 and Y192 residues of the activating phosphorylation site were replaced by A and F, was unable to restore the growth and differentiation phenotypes. Analysis of the DNA content and the nucleus/kinetoplast configuration of individual cells showed that the null mutant was arrested in all phases of the cell cycle and that 25-30% of the cells had failed to segregate their nucleus and kinetoplast correctly. This implies that cell cycle progression by the procyclic form depends on a constitutive stimulus exerted by the signaling cascade operating through TbMAPK2.