Characterisation of non-specific esterase isoenzyme forms in normal and leukaemic myeloid cells

The generic term 'esterases' broadly represents enzymes that hydrolyse aliphatic or aromatic esters. and within this definition is a group of esterases that show a preference for short acyl chain esters such as a-naphthyl acetate, propionate or butyrate. These are ti often collectively referred to as 'non-specific esterases' (NSE), but a more accurate and descriptive designation is according to the substrate used. The demonstration of esterases by azo-dye techniques has found particular applications in haematological cytochemistry, and, with specific reference to normal myeloid cells, a-naphthyl acetate esterase (ANAE) 0 cytochemical reactions of moderate-strong diffuse type are typically associated with cells of monocyte/macrophage lineage. In contrast. granulocytes at all levels of morphological differentiation are cytochemically ANAE negative. Isoelectric focusing (IEF) studies of ANAE isoenzymes have further revealed the existence of two species with apparent differences in lineage affiliation. The first (ComEst) is expressed by both granulocytes and monocytes, and comprises a series of isoenzymes with isoelectric points (p1) ranging from 6.3 to 7.9, whereas a second (MonEst) species that is specifically expressed by haemopoietic cells of monocytic/macrophage origin is seen on IEF zymograms as a series of isoforms within a relatively narrow pI range of 5.5-6.2. These present studies were undertaken in order to extend what is currently known of the cellular, kinetic and molecular features of the two main myeloid esterase species. It was considered that these investigations were necessary to clarify the nature of atypical cytochemical reactions in leukaemic and dyshaemopoietic myeloid cells, to establish whether or not the two species were related or distinct enzymes, to gain further insights into their possible functional role(s), and to provide molecular details of relevance for the longer term aim of cloning MonEst protein in particular. Studies reported here of normal myeloid cells confirmed the lineage affiliation of the two main esterase species, and analysis of a large number of acute myeloid leukaemias also resolved the nature of atypical ANAE cytochemistries. Abnormally increased focal and granular reactions of myeloid blasts was shown to be due to over-expression of ComEst, and the lack of ANAE cytochemical staining in a significant proportion of monocytic leukaemias was shown to result from a failure to synthesise MonEst. As a prelude to the biochemical purification of myeloid esterases, the ComEst and MonEst species were also investigated to determine their chromatographic characteristics. This involved an evaluation of a wide range of column gels including ion-exchange, hydrophobic interaction, affinity, and gel filtration. The purification protocol resulting from these evaluations successfully permitted the purification of ComEst to a highly enriched state and MonEst to homogeneity. Subsequent molecular and kinetic analyses revealed that enzymatically active MonEst exists in its native state as an apparent trimer which, under non-reducing conditions, dissociates to inactive 63 kDa monomers. In contrast, native ComEst was shown to be a 68 kDa monomer which retained enzymatic activity following SDS treatment, and was not dissociated under reducing conditions. Lectin affinity studies confirmed that both esterase species were glycoproteins but differed in that MonEst contained oligomannosidic-type glycan(s) whilst ComEst contained a mixture of fucosylated and non-fucosylated biantennary N-acetyllactosamine-type glycan(s). Neuraminidase, a-mannosidase, a-L-fucosidase, and endoglycosidase H were shown to have no effect on the pI distribution of individual ComEst or MonEst isoforms, but endoglycosidase treatment did reduce the Mr of MonEst from 63 to 60 kDa. Enzyme kinetic studies also revealed that purified ComEst preferentially hydrolysed esters of short acyl chain length (C2 and C3) whilst MonEst hydrolysed esters of higher acyl chain length (butyrate > propionate > acetate). However, MonEst failed to hydrolyse a wide range of natural and synthetic peptidase substrates thus tending to exclude its functional role in peptide processing. Possible differences in reaction mechanisms of the two esterase species were also evaluated by examining the inhibitory effects of representative enzyme inhibitors which demonstrated that serine and histidine residues were required. for MonEst but not ComEst activity. N-terminus amino acid sequencing of purified MonEst indicated almost complete identity with human alveolar macrophage esterase. differing only in a Val-Thr substitution at position 12, and close similarities with rabbit liver carboxylesterase. In summary, substrate and inhibitor studies strongly suggested that the MonEst and ComEst species should be classified as carboxylesterases (EC 3.1.1.1) and acetylesterases (EC 3.1.1.6) respectively and that, together with distinct differences in their molecular and biochemical characteristics, it is concluded that these are unrelated myeloid enzymes which share only the ability to hydrolyse a-naphthyl acetate. Although yet to be established, the kinetic and molecular differences reported here may have fundamental relevance with respect to the biofunctional role(s) of these enzymes

Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line

Mammalian cells obtain nucleic acid precursors through the de novo synthesis of nucleotides and the salvage of exogenous nucleobases and nucleosides. The first step in the salvage pathway is transport across the plasma membrane. Several transport activities, including equilibrative and concentrative mechanisms, have been identified by their functional properties. We report here the functional cloning of a 2.6-kilobase pair human cDNA encoding the nitrobenzylmercaptopurine riboside (NBMPR)- insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human leukemia cells. Expression of this cDNA conferred an NBMPR-insensitive, sodium-independent nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characteristic of the ei transporter. The cDNA contained a single open reading frame that encoded a 456-residue protein with 11 potential membrane-spanning regions and two consensus sites for N-glycosylation in the first predicted extracellular loop. The predicted protein was 50% identical to the recently cloned human NBMPR-sensitive, equilibrative nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carboxyl-terminal portion of the ENT2 protein was nearly identical to a smaller protein in the GenBank(TM) data base (human HNP36, 326 residues) that has been identified as a growth factor- induced delayed early response gene of unknown function. Comparison of the ENT2 and HNP36 nucleotide sequences suggested that HNP36 was translated from a second start codon within the ENT2 open reading frame. Transient expression studies with the full-length ENT2 and a 5\u27-truncated construct that lacks the first start codon (predicted protein 99% identical to HNP36) demonstrated that only the full-length construct conferred uridine transport activity to the cells. These data suggest that the delayed early response gene HNP36 is a truncated form of ENT2 and that the full-length open reading frame of ENT2 is required for production of a functional plasma membrane ei transporter

Cloning, genomic organization and chromosomal localization of the gene encoding the murine sodium-dependent, purine- selective, concentrative nucleoside transporter (CNT2)

A PCR-based strategy was used to isolate a 2653 bp cDNA encoding the mouse sodium-dependent, purine nucleoside selective, concentrative nucleoside transporter (designated mCNT2). The deduced protein sequence exhibits 93 and 80% identity to the previously cloned rat and human sodium-dependent, purine nucleoside selective, nucleoside transporters, respectively. Characterization of 3H-nucleoside uptake by COS-1 cells transiently transfected with the cDNA demonstrated that it encoded a functional nucleoside transport activity with selectivity for purine nucleosides. The cDNA was used to screen a murine (strain 129SvJ/6) genomic library in pBeloBAC11 to identify a clone containing the mCNT2 gene. A PCR strategy was used to identify and sequence the intron-exon boundaries and to determine the approximate sizes of the introns. The mCNT2 gene spans approximately 13.7 kb and is encoded by 15 exons. The gene was mapped to mouse chromosome 2e3 by fluorescence in situ hybridization. (C) 2000 Elsevier Science B.V. All rights reserved

E.L., a modern-day Phineas Gage: Revisiting frontal lobe injury

BackgroundHow the prefrontal cortex (PFC) recovers its functionality following lesions remains a conundrum. Recent work has uncovered the importance of transient low-frequency oscillatory activity (LFO; < 4 Hz) for the recovery of an injured brain. We aimed to determine whether persistent cortical oscillatory dynamics contribute to brain capability to support 'normal life' following injury.MethodsIn this 9-year prospective longitudinal study (08/2012-2021), we collected data from the patient E.L., a modern-day Phineas Gage, who suffered from lesions, impacting 11% of his total brain mass, to his right PFC and supplementary motor area after his skull was transfixed by an iron rod. A systematic evaluation of clinical, electrophysiologic, brain imaging, neuropsychological and behavioural testing were used to clarify the clinical significance of relationship between LFO discharge and executive dysfunctions and compare E.L.´s disorders to that attributed to Gage (1848), a landmark in the history of neurology and neuroscience.FindingsSelective recruitment of the non-injured left hemisphere during execution of unimanual right-hand movements resulted in the emergence of robust LFO, an EEG-detected marker for disconnection of brain areas, in the damaged right hemisphere. In contrast, recruitment of the damaged right hemisphere during contralateral hand movement, resulted in the co-activation of the left hemisphere and decreased right hemisphere LFO to levels of controls enabling performance, suggesting a target for neuromodulation. Similarly, transcranial magnetic stimulation (TMS), used to create a temporary virtual-lesion over E.L.'s healthy hemisphere, disrupted the modulation of contralateral LFO, disturbing behaviour and impairing executive function tasks. In contrast to Gage, reasoning, planning, working memory, social, sexual and family behaviours eluded clinical inspection by decreasing LFO in the delta frequency range during motor and executive functioning.InterpretationOur study suggests that modulation of LFO dynamics is an important mechanism by which PFC accommodates neurological injuries, supporting the reports of Gage´s recovery, and represents an attractive target for therapeutic interventions.FundingFundação de Amparo Pesquisa Rio de Janeiro (FAPERJ), Universidade Federal do Rio de Janeiro (intramural), and Fiocruz/Ministery of Health (INOVA Fiocruz)