Hematopoietic stem cells in research and clinical applications: The "CD34 issue"

In this paper, experimental findings concerning the kinetics of hematopoietic reconstitution are compared to corresponding clinical data. Although not clearly apparent, the transplantation practice seems to confirm the basic proposals of experimental hematology concerning hematopoietic reconstitution resulting from successive waves of repopulation stemming from different subpopulations of progenitor and stem cells. One of the "first rate" parameters in clinical transplantations in hematology; i.e. the CD34+ positive cell dose, has been discussed with respect to the functional heterogeneity and variability of cell populations endowed by expression of CD34. This parameter is useful only if the relative proportion of stem and progenitor cells in the CD34+ cell population is more or less maintained in a series of patients or donors. This proportion could vary with respect to the source, pathology, treatment, processing procedure, the graft ex vivo treatment and so on. Therefore, a universal dose of CD34+ cells cannot be defined. In addition, to avoid further confusion, the CD34+ cells should not be named "stem cells" or "progenitor cells" since these denominations only concern functionally characterized cell entities.     

Nanosecond dynamics of acetylcholinesterase near the active center gorge

To delineate the role of peptide backbone flexibility and rapid molecular motion in acetylcholinesterase catalysis and inhibitor association, we investigated the decay of fluorescence anisotropy at three sites of fluorescein conjugation to cysteine-substitution mutants of the enzyme. One cysteine was placed in a loop at the peripheral site near the rim of the active center gorge (H287C); a second was in a helical region outside of the active center gorge (T249C); a third was at the tip of a small, flexible omega loop well separated from the gorge (A262C). Mutation and fluorophore conjugation did not appreciably alter catalytic or inhibitor binding parameters of the enzyme. The results show that each site examined was associated with a high degree of segmental motion; however, the A262C and H287C sites were significantly more flexible than the T249C site. Association of the active center inhibitor, tacrine, and the peripheral site peptide inhibitor, fasciculin, had no effect on the anisotropy decay of fluorophores at positions 249 and 262. Fasciculin, but not tacrine, on the other hand, dramatically altered the decay profile of the fluorophore at the 287 position, in a manner consistent with fasciculin reducing the segmental motion of the peptide chain in this local region. The results suggest that the motions of residues near the active center gorge and across from the Cys(69)-Cys(96) omega loop are uncoupled and that ligand binding at the active center or the peripheral site does not influence acetylcholinesterase conformational dynamics globally, but induces primarily domain localized decreases in flexibility proximal to the bound ligand.     

Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties

Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast and slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme. In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency. The ring opening was catalyzed most efficiently at pH 8.0 and competitively inhibited by the reaction product, N-carbamyl-β-alanine. At lower pH values DHPases catalyzed the reverse reaction, the closing of the ring. Apparently, eukaryote DHPases are enzymatically as well as phylogenetically related to the de novo biosynthetic dihydroorotase (DHOase) enzymes. Modeling studies showed that the position of the catalytically critical amino acid residues of bacterial DHOases and eukaryote DHPases overlap. Therefore, only a few modifications might have been necessary during evolution to convert the unspecialized enzyme into anabolic and catabolic ones.     

Determinants of LV diastolic function during atrial fibrillation: beat-to-beat analysis in acute dog experiments

Left ventricular (LV) diastolic function during atrial fibrillation (AF) remains poorly understood due to the complex interaction of factors and beat-to-beat variability. The purpose of the present study was to elucidate the physiological determinants of beat-to-beat changes in LV diastolic function during AF. The RR intervals preceding a given cardiac beat were measured from the right ventricular electrogram in 12 healthy open-chest mongrel dogs during AF. Doppler echocardiography and LV pressure and volume beat-to-beat analyses were performed. The LV filling time (FT) and early diastolic mitral inflow velocity-time integral (E(vti)) were measured using the pulsed Doppler method. The LV end-diastolic volume (EDV), peak systolic LV pressure (LVP), minimum value of the first derivative of LV pressure curve (dP/dt(min)), and the time constant of LV pressure decay (tau) were evaluated with the use of a conductance catheter for 100 consecutive cardiac cycles. Beat-to-beat analysis revealed a cascade of important causal relations. LV-FT showed a significant positive linear relationship with E(vti) (r = 0.87). Importantly, there was a significant positive linear relationship between the RR interval and LV-EDV in the same cardiac beat (r = 0.53). Consequently, there was a positive linear relationship between LV-EDV and subsequent peak systolic LVP (r = 0.82). Furthermore, there were significant positive linear and negative curvilinear relationships between peak systolic LVP and dP/dt(min) (r = 0.95) and tau (r = -0.85), respectively, in the same cardiac beat. In addition, there was a significant negative curvilinear relationship between dP/dt(min) and tau (r = -0.86). We have concluded that the determinants of LV diastolic function in individual beats during AF depend strongly on the peak systolic LVP. This suggests that the major benefit of slower ventricular rate appears related to lengthening of LV filling interval, promoting subsequent higher peak systolic LVP and greater LV relaxation.     

Missense mutations in the regulatory domain of PKC gamma: a new mechanism for dominant nonepisodic cerebellar ataxia

We report a nonepisodic autosomal dominant (AD) spinocerebellar ataxia (SCA) not caused by a nucleotide repeat expansion that is, to our knowledge, the first such SCA. The AD SCAs currently comprise a group of > or =16 genetically distinct neurodegenerative conditions, all characterized by progressive incoordination of gait and limbs and by speech and eye-movement disturbances. Six of the nine SCAs for which the genes are known result from CAG expansions that encode polyglutamine tracts. Noncoding CAG, CTG, and ATTCT expansions are responsible for three other SCAs. Approximately 30% of families with SCA do not have linkage to the known loci. We recently mapped the locus for an AD SCA in a family (AT08) to chromosome 19q13.4-qter. A particularly compelling candidate gene, PRKCG, encodes protein kinase C gamma (PKC gamma), a member of a family of serine/threonine kinases. The entire coding region of PRKCG was sequenced in an affected member of family AT08 and in a group of 39 unrelated patients with ataxia not attributable to trinucleotide expansions. Three different nonconservative missense mutations in highly conserved residues in C1, the cysteine-rich region of the protein, were found in family AT08, another familial case, and a sporadic case. The mutations cosegregated with disease in both families. Structural modeling predicts that two of these amino acid substitutions would severely abrogate the zinc-binding or phorbol ester-binding capabilities of the protein. Immunohistochemical studies on cerebellar tissue from an affected member of family AT08 demonstrated reduced staining for both PKC gamma and ataxin 1 in Purkinje cells, whereas staining for calbindin was preserved. These results strongly support a new mechanism for neuronal cell dysfunction and death in hereditary ataxias and suggest that there may be a common pathway for PKC gamma-related and polyglutamine-related neurodegeneration.     

Catabolism of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyptila

The present study describes the distribution and properties of enzymes of the catabolic pathway of pyrimidine nucleotides in Riftia pachyptila, a tubeworm living around deep-sea hydrothermal vents and known to be involved in a highly specialized symbiotic association with a bacterium. The catabolic enzymes, 5'-nucleotidase, uridine phosphorylase, and uracil reductase, are present in all tissues of the worm, whereas none of these enzymatic activities were found in the symbiotic bacteria. The 5'-nucleotidase activity was particularly high in the trophosome, the symbiont-harboring tissue. These results suggest that the production of nucleosides in the trophosome may represent an alternative source of carbon and nitrogen for R. pachyptila, because these nucleosides can be delivered to other parts of the worm. This process would complement the source of carbon and nitrogen from organic metabolites provided by the bacterial assimilatory pathways. The localization of the enzymes participating in catabolism, 5'-nucleotidase and uridine phosphorylase, and of the enzymes involved in the biosynthesis of pyrimidine nucleotides, aspartate transcarbamylase and dihydroorotase, shows a non-homogeneous distribution of these enzymes in the trophosome. The catabolic enzymes 5'-nucleotidase and uridine phosphorylase activities increase from the center of the trophosome to its periphery. In contrast, the anabolic enzymes aspartate transcarbamylase and dihydroorotase activities decrease from the center toward the periphery of the trophosome. We propose a general scheme of anatomical and physiological organization of the metabolic pathways of the pyrimidine nucleotides in R. pachyptila and its bacterial endosymbiont.     

The role of photophosphorylation in SO2 and SO 3 2- inhibition of photosynthesis in isolated chloroplasts

Sulphur dioxide inhibits noncyclic photophosphorylation in isolated envelope-free chloroplasts. This inhibition was shown to be reversible and competitive with phosphate, with an inhibitor constant of K_i=0.8mM. The same inhibition characteristics were observed when phosphoglycerate (PGA)- or ribulose-1,5-bisphosphate (RuBP)-dependent oxygen evolution was examined in a reconstituted chloroplast system in the presence of SO ₃ ^2- . Using an ATP-regenerating system (phosphocreatine-creatine kinase), it was demonstrated that the inhibition of PGA-dependent oxygen evolution is solely the result of inhibited photophosphorylation. It is concluded that at low SO₂ and SO ₃ ^2- concentrations the inhibition of photophosphorylation is responsible for the inhibition of photosynthetic oxygen evolution.Abbreviations Chl chlorophyll - PGA D-3-phosphoglyceric acid trisodium salt - Pi inorganic phosphate - RuBP D-ribulose-1,5-bisphosphoric acid tetrasodium salt     

Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant k_on for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction. © 1998 John Wiley & Sons, Inc. Biopoly 46: 465–474, 1998