[Application of process management in nephrology. Hemodialysis process management]. / Aplicación de la gestión por procesos en nefrología. Gestión del proceso hemodiálisis.

Total Quality Management techniques have recently been introduced into clinical practice. We describe the application of process management to hemodialysis therapy in a Spanish public hospital. The "ownership" of the hemodialysis process and its limits have been defined. We present a flowchart with all the activities involved in the process and the task description. Monitoring indicators have been selected according to the recommendations of the US Committee on the National Report on Health Care Delivery. Data sources for indicators have also been described.

Aplicación de la gestión por procesos en nefrología. Gestión del proceso hemodiálisis / Process management in nephrology. Management of the hemodialysis process

Las técnicas de gestión de calidad total han sido introducidas recientemente en la práctica clínica. Este artículo describe la aplicación de la gestión de procesos a la hemodiálisis (HD), como tratamiento renal sustitutivo de la insuficiencia renal crónica, en un hospital público español. Se ha definido el proceso con un diagrama de flujo en el que aparecen todas las actividades del mismo. Los indicadores para su monitorización han sido seleccionados siguiendo las recomendaciones del Committee on the National Report on Health Care Delivery norteamericano. Se describen los estándares y las fuentes de datos para los indicadores (AU)

The autoregulation of retinal ganglion cell number.

The development of the nervous system is dependent on a complex set of signals whose precise co-ordination ensures that the correct number of neurones are generated. This regulation is achieved through a variety of cues that influence both the generation and the maintenance of neurones during development. We show that in the chick embryo, stratified retinal ganglion cells (RGCs) are themselves responsible for providing the signals that control the number of RGCs that are generated, both by inhibiting the generation of new ganglion cells and by killing incoming migratory ganglion cells. Selective toxicological ablation of RGCs in the chick embryo resulted in the achronic generation of ganglion cells, which eventually led to the repopulation of the ganglion cell layer and a large decrease in the physiological cell death affecting postmitotic migratory neurones. Interestingly, the application of exogenous NGF reversed the effects of ganglion cell ablation on ganglion cell death. Because the only source of NGF in the retina is that produced by the stratified ganglion cells, we infer that these differentiated neurones regulate their own cell number by secreting NGF, a neurotrophin that has previously been shown to be responsible for the death of migrating ganglion cells.

Programmed cell death in the developing somites is promoted by nerve growth factor via its p75(NTR) receptor.

Neurotrophins control neuron number during development by promoting the generation and survival of neurons and by regulating programmed neuronal death. In the latter case, the cell death induced by nerve growth factor (NGF) in the developing chick retina is mediated by p75(NTR), the common neurotrophin receptor (J. M. Frade, A. Rodriguez-Tebar, and Y.-A. Barde, 1996, Nature 383, 166-168). Here we show that NGF also induces the programmed death of paraxial mesoderm cells in the developing somites. Both NGF and p75(NTR) are expressed in the somites of chick embryos at the time and the place of programmed cell death. Moreover, neutralizing the activity of endogenous NGF with a specific blocking antibody, or antagonizing NGF binding to p75(NTR) by the application of human NT-4/5, reduces the levels of apoptotic cell death in both the sclerotome and the dermamyotome by about 50 and 70%, respectively. Previous data have shown that Sonic hedgehog is necessary for the survival of differentiated somite cells. Consistent with this, Sonic hedgehog induces a decrease of NGF mRNA in somite explant cultures, thus showing the antagonistic effect of NGF and Sonic hedgehog with respect to somite cell survival. The regulation of programmed cell death by NGF/p75(NTR) in a mesoderm-derived tissue demonstrates the capacity of neurotrophins and their receptors to influence critical developmental processes both within and outside of the nervous system.

Ariadne-1: a vital Drosophila gene is required in development and defines a new conserved family of ring-finger proteins.

We report the identification and functional characterization of ariadne-1 (ari-1), a novel and vital Drosophila gene required for the correct differentiation of most cell types in the adult organism. Also, we identify a sequence-related gene, ari-2, and the corresponding mouse and human homologues of both genes. All these sequences define a new protein family by the Acid-rich, RING finger, B-box, RING finger, coiled-coil (ARBRCC) motif string. In Drosophila, ari-1 is expressed throughout development in all tissues. The mutant phenotypes are most noticeable in cells that undergo a large and rapid membrane deposition, such as rewiring neurons during metamorphosis, large tubular muscles during adult myogenesis, and photoreceptors. Occasional survivors of null alleles exhibit reduced life span, motor impairments, and short and thin bristles. Single substitutions at key cysteines in each RING finger cause lethality with no survivors and a drastic reduction of rough endoplasmic reticulum that can be observed in the photoreceptors of mosaic eyes. In yeast two-hybrid assays, the protein ARI-1 interacts with a novel ubiquitin-conjugating enzyme, UbcD10, whose sequence is also reported here. The N-terminal RING-finger motif is necessary and sufficient to mediate this interaction. Mouse and fly homologues of both ARI proteins and the Ubc can substitute for each other in the yeast two-hybrid assay, indicating that ARI represents a conserved novel mechanism in development. In addition to ARI homologues, the RBR signature is also found in the Parkinson-disease-related protein Parkin adjacent to an ubiquitin-like domain, suggesting that the study of this mechanism could be relevant for human pathology.

scully, an essential gene of Drosophila, is homologous to mammalian mitochondrial type II L-3-hydroxyacyl-CoA dehydrogenase/amyloid-beta peptide-binding protein.

The characterization of scully, an essential gene of Drosophila with phenocritical phases at embryonic and pupal stages, shows its extensive homology with vertebrate type II L-3-hydroxyacyl-CoA dehydrogenase/ERAB. Genomic rescue demonstrates that four different lethal mutations are scu alleles, the molecular nature of which has been established. One of them, scu3127, generates a nonfunctional truncated product. scu4058 also produces a truncated protein, but it contains most of the known functional domains of the enzyme. The other two mutations, scu174 and scuS152, correspond to single amino acid changes. The expression of scully mRNA is general to many tissues including the CNS; however, it is highest in both embryonic gonadal primordia and mature ovaries and testes. Consistent with this pattern, the phenotypic analysis suggests a role for scully in germ line formation: mutant testis are reduced in size and devoid of maturing sperm, and mutant ovarioles are not able to produce viable eggs. Ultrastructural analysis of mutant spermatocytes reveals the presence of cytoplasmic lipid inclusions and scarce mitochondria. In addition, mutant photoreceptors contain morphologically aberrant mitochondria and large multilayered accumulations of membranous material. Some of these phenotypes are very similar to those present in human pathologies caused by beta-oxidation disorders.

Vitronectin is expressed in the ventral region of the neural tube and promotes the differentiation of motor neurons.

The extracellular matrix protein vitronectin and its mRNA are present in the embryonic chick notochord, floor plate and in the ventral neural tube at the time position of motor neuron generation. When added to cultures of neural tube explants of developmental stage 9, vitronectin promotes the generation of motor neurons in the absence of either notochord or exogenously added Sonic hedgehog. Conversely, the neutralisation of endogenous vitronectin with antibodies inhibits over 90% motor neuron differentiation in co-cultured neural tube/notochord explants, neural tube explants cultured in the presence of Sonic hedgehog, and in committed (stage 13) neural tube explants. Furthermore, treatment of embryos with anti-vitronectin antibodies results in a substantial and specific reduction in the number of motor neurons generated in vivo. These results demonstrate that vitronectin stimulates the differentiation of motor neurons in vitro and in vivo. Since the treatment of stage 9 neural tube explants with Sonic hedgehog resulted in induction of vitronectin mRNA expression before the expression of floor plate markers, we conclude that vitronectin may act either as a downstream effector in the signalling cascade induced by Sonic hedgehog, or as a synergistic factor that increases Shh-induced motor neuron differentiation.

Control of early cell death by BDNF in the chick retina.

The developing chick retina undergoes at least two discrete periods of programmed cell death. The earlier period coincides with the main onset of neuron birth and migration (embryonic day 5-7), whereas the latter one corresponds to the well-documented process of retinal ganglion cell death following tectal innervation (embryonic day 10-14; Rager, G. H. (1980) Adv. Anat. Embryol. Cell Biol. 63, 1-92). In the early period, apoptosis is induced by nerve growth factor (NGF) acting via its p75 receptor (Frade, J. M., Rodríguez-Tébar, A. and Barde, Y.-A. (1996) Nature 383, 166-168). Here, we show that the application of brain-derived neurotrophic factor (BDNF) to chick embryos in ovo prevented retinal cell death in the early period, whereas exogenously applied NGF and neurotrophin-3 had no such effect. The addition of BDNF to embryos resulted in about 70% increase in the number of retinal ganglion cells in both E6 and E9 retinas relative to controls. BDNF is first expressed in both the pigment epithelium and neural retina of embryonic day 4 embryos, and at the same stage of development, its TrkB receptor is expressed in the neural retina. Our data indicate that early cell death is an important process in the neurogenesis of retinal ganglion cells and is regulated by locally produced BDNF.

Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation.

To identify proteins that interact in vivo with muscle components we have used a genetic approach based on the isolation of suppressors of mutant alleles of known muscle components. We have applied this system to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. We show that the structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle.

Primary structure of Lep d I, the main Lepidoglyphus destructor allergen.

The most relevant allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been characterized. Lep d I is a monomer protein of 13273 Da. The primary structure of Lep d I was determined by N-terminal Edman degradation and partially confirmed by cDNA sequencing. Sequence polymorphism was observed at six positions, with non-conservative substitutions in three of them. No potential N-glycosylation site was revealed by peptide sequencing. The 125-residue sequence of Lep d I shows approximately 40% identity (including the six cysteines) with the overlapping regions of group II allergens from the genus Dermatophagoides, which, however, do not share common allergenic epitopes with Lep d I.

cDNA sequence analysis of the main olive allergen, Ole e I.

Olea europaea (Ole e) I-specific cDNA sequences were amplified by 3'-RACE-PCR, using specific primers based on the N-terminal sequence of the allergen, and cloned into appropriate vectors. The nucleotide sequence data obtained revealed the presence of isogenic variation in Ole e I gene(s). The molecular mass, pI, amino acid composition and sequence of the predicted polypeptides agree with data previously obtained by analysis of purified Ole e I from pollen. Furthermore, by treatment of purified Ole e I with specific glycopeptide hydrolases it has been demonstrated the presence of N-glycosylation in the allergen, and there is a unique concensus site for N-linked glycosylation at positions 111-113 of the deduced amino acid sequence. The Ole e I predicted sequence shows a significant homology with three putative proteins encoded respectively by the another-specific LAT52 gene from tomato and the pollen specific genes Zmc13 from maize and OSPSG from rice, suggesting that these proteins could have a role in one of the development processes unique to male gametophytes.

Abnormal muscle development in the heldup3 mutant of Drosophila melanogaster is caused by a splicing defect affecting selected troponin I isoforms.

The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6a1, 6a2, 6b1, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6b1, resulting in the failure to produce all exon 6b1-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor.

Troponin I is encoded in the haplolethal region of the Shaker gene complex of Drosophila.

We have analyzed one of the nine complementation groups that constitute the haplolethal (HL) region of the Shaker gene complex (ShC). Five mutations, including a dominant lethal, define this complementation group: HL I. Mutant phenotypes show abnormal embryogenesis with structural defects in the nervous system and aberrant degeneration of specific adult muscles in addition to altered action potentials. HL I encodes a family of proteins with extensive homology to invertebrate troponin I (TnI). Members of this family are brought about by two alternative and two mutually exclusive exons in conjunction with two differential polyadenylation sites. Transcription analysis indicates that some isoforms are adult specific and others are synthesized throughout development, except during early metamorphosis. Certain isoforms of Drosophila TnI are expressed in specific muscles. The specificity of mutant phenotypes suggests a functional role of particular TnI isoforms in the development and the mature activity of muscle and nervous systems.

Antibodies against Drosophila potassium channels identify membrane proteins across species.

Shaker is a complex locus (ShC) in Drosophila that encodes components of the K+ channel responsible for the IA current. We have raised antibodies against synthetic peptides of selected sequences from the Sh products. One of the antisera identifies a 71 kDa protein band in immunoblots from Drosophila neural membrane proteins. We demonstrate that this protein is encoded within the viable (V) region of the ShC since deletions and breakpoints in this part of the complex eliminate this band from the immunoblots. Certain Sh mutations abolish the production of this product while other do not seem to interfere with it. The same antiserum identifies bands of different apparent molecular weight (Mr) in membrane extracts of nervous systems of a variety of organisms including vertebrates.

Differential splicing and alternative polyadenylation generates distinct NCAM transcripts and proteins in the mouse.

The neural cell adhesion molecule (NCAM) exists in at least three different protein isoforms which are selectively expressed by different cell types and at different stages of development. They are encoded by four to five different transcripts that are derived from a single gene. Here we report the exon--intron structure of the 3' part of the mouse NCAM gene. This region contains six exons. The 5' exon is constitutively expressed in all four prominent size classes of NCAM mRNAs detected in the mouse brain. The second exon contains the poly(A) addition sites for the two smaller mRNAs of 5.2 and 2.9 kb which differ in the length of their 3' non-coding regions and seem both to encode NCAM-120. This second exon is absent in the largest 7.4 kb transcript which encodes NCAM-180; in the 6.7 kb mRNA, which appears to code for NCAM-140, the second and the fifth exon have been spliced out. This data explains how the prominent four transcripts and three protein isoforms of mouse NCAM are generated from a single gene. The alternatively spliced fifth exon is surrounded by inverted repeats potentially capable of secondary structure formation, that may sequester this exon in a loop.

Analysis of cDNA clones that code for the transmembrane forms of the mouse neural cell adhesion molecule (NCAM) and are generated by alternative RNA splicing.

The neural cell adhesion molecule (NCAM) exists in at least three different isoforms. In the mouse, NCAM proteins with apparent Mr's of 180,000, 140,000 and 120,000 have been distinguished. These are encoded by 4 to 5 different transcripts. Here we report the full amino acid sequence of an isoform which most likely represents NCAM-140. The N-terminal extracellular portion of the 829-residue polypeptide appears to be identical to all three NCAM proteins. The Mr of 91,276 is considerably smaller than the estimate based on SDS-gel electrophoresis. The 147 C-terminal residues are distinct from NCAM-120 and contain the putative transmembrane and cytoplasmic domains. The transcript encoding NCAM-140 contains almost 3.2 kb non-coding sequence with a canonical polyadenylation signal. While the 5' sequences of NCAM-140 hybridize with all NCAM mRNAs, the 3' probes recognize only the two larger transcripts of 7.4 and 6.7 kb. From S1 nuclease protection analyses and hybridization studies of several NCAM cDNA clones with genomic NCAM sequences one can conclude that the different NCAM transcripts are generated by alternative splicing. In addition to the two alternative splice sites in the sequence encoding the extracellular domains, a third one can be predicted approximately 320 nt downstream of the start of the NCAM-140-specific sequence portion. This finding is in agreement with the existence of an extra exon in the chicken NCAM-180. Comparison between mouse and chicken NCAM amino acid sequences revealed the highest homology in the second and fifth Ig-like domains and in the cytoplasmic parts suggesting that these regions serve highly conserved functions.

Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli.

This communication deals with the location of penicillin-binding proteins in the cell envelope of Escherichia coli. For this purpose, bacterial cells have been broken by various procedures and their envelopes have been fractioned. To do so, inner (cytoplasmic) and outer membranes were separated by isopycnic centrifugation in sucrose gradients. Some separation methods (Osborn et al., J. Biol. Chem. 247:3962-3972, 1972; J. Smit, Y. Kamio, and H. Nikaido, J. Bacteriol. 124:942-958, 1975) revealed that penicillin-binding proteins are not exclusively located in the inner membrane. They are also found in the outer membrane (A. Rodríguez-Tébar, J. A. Barbas, and D. Vásquez, J. Bacteriol. 161:243-248, 1985). Under the milder conditions for cell rupture used in this work, an intermembrane fraction, sedimenting between the inner and outer membrane, can be recovered from the gradients. This fraction has a high content of both penicillin-binding proteins and phospholipase B activity and may correspond to the intermembrane adhesion sites (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-769, 1982). We postulate that this intermembrane fraction is a labile structure that contains a high amount of all penicillin-binding proteins which are usually found in both the inner and outer membranes when the adhesion sites are destroyed by the cell breakage and fractionation procedures.

An unusual linkage between polysaccharides and some major proteins from the outer membrane of Escherichia coli.

A small population of OmpA, a major protein from the outer membrane of Escherichia coli, was found covalently associated with either lipopolysaccharide or O-antigen polysaccharide. Radioactive oligosaccharide was elicited linked to OmpA after treatment of the membranes with periodate that hydrolyzed large sugars. Association of saccharides to OmpA could be enhanced by treatment of the outer membrane with NaBH4.

Final steps of the maturation of Omp F, a major protein from the outer membrane of Escherichia coli.

Pulse-labelling experiments with E. coli cells allowed us to follow the incorporation of de novo proteins into the outer membrane of the cell envelope. Labelled membrane samples containing increasingly different levels of newly synthesized Omp F protein were subjected to chemical cross-linking with a bifunctional cleavable reagent in order to investigate the process of trimer formation of the protein. From the results obtained, we conclude that the formation of functional Omp F trimers is substantially delayed to, and can be distinguished from, the incorporation of Omp F monomers to the outer membrane.

Location of some proteins involved in peptidoglycan synthesis and cell division in the inner and outer membranes of Escherichia coli.

Inner and outer membranes of Escherichia coli were separated by isopycnic centrifugation in sucrose gradients and analyzed for the presence of penicillin-binding proteins. All penicillin-binding proteins--except penicillin-binding protein 3, which is found almost exclusively in the cytoplasmic membrane and is involved in septum formation--are also found in gradient fractions corresponding to the outer membrane. Our results support the hypothesis that approximately half of the total amount of penicillin-binding proteins may be sacculus-located proteins linked to the outer membrane, probably through peptidoglycan bridges.