Native Human Cells Research Articles

Nanomaterials based on chitosan for skin regeneration: an update.

Nanomaterials based on chitosan for skin regeneration: an update.

Abstract P133: Human Induced Pluripotent Stem Cells-derived Vascular Endothelial And Smooth Muscle Cells And Fractions Of Native Human Arterioles Exhibit Robust Expression Of Marker Genes

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial and smooth muscle cells (iEC and iVSMC). The defined genetic makeup and the ease of genetic engineering in multiple cell types on an isogenic background make these cells potentially powerful tools for studying molecular mechanisms underlying vascular function and disease. While several studies have characterized iEC and iVSMC and compared them with other cultured cells, no studies have analyzed these cells in conjunction with native human cells. In this study, we characterized iEC, iVSMC, and fractions of native human arterioles by examining large panels of marker genes. iEC and iVSMC were differentiated (n=4) from a custom-made iPSC line. Differentiated cells were sorted using VE-cadherin ( CDH5 ) to obtain iEC (positive selection) and iVSMC (negative selection). Arterioles were isolated from adipose tissues obtained from 4 humans, and the endothelial and vessel remainder fractions were separated by scraping. RNA-seq was performed on all samples. We focused the analysis on 10 and 15 marker genes for EC and VSMC, respectively, that we curated from the literature. iEC exhibited robust expression of 9 of the 10 EC markers such as CD34 (FPKM, 137±58), PECAM1 (153±32), and VWF (44±7), while iVSMC had negligible expression of EC marker genes. Conversely, iVSMC exhibited robust expression of 11 of the 15 VSMC marker genes, while the expression of these genes in iEC was depleted by several fold. Eight of the 10 EC marker genes were enriched in the endothelial fraction of native arterioles by several fold compared with intact arterioles, while 12 of the 15 VSMC marker genes were substantially depleted in the endothelial fraction. EC marker genes were significantly depleted and VSMC marker genes enriched in the vessel remainder fraction. In summary, iEC and iVSMC, as well as endothelial and vessel remainder factions from native human arterioles, exhibit robust expression of large panels of EC and VSMC marker genes based on agnostic RNA-seq analysis. These novel findings indicate that iEC, iVSMC, and endothelial and vessel remainder factions of native human arterioles are relevant tools for studying molecular regulation in specific vascular cell types.

Engineering Antibodies Targeting p16 MHC-Peptide Complexes.

Senescent cells undergo a permanent cell cycle arrest and drive a host of age-related pathologies. Recent transgenic mouse models indicate that removing cells expressing the senescence marker p16Ink4a (p16) can increase median lifespan and delay the onset of many aging phenotypes. However, identifying and eliminating native human cells expressing p16 has remained a challenge. We hypothesize that senescent cells display peptides derived from p16 in major histocompatibility complex (MHC)-peptide complexes on the cell surface that could serve as targetable antigens for antibody-based biologics. Using Fab-phage display technology, we generated antibodies that bind to a p16 MHC-peptide complex from the human leukocyte antigen (HLA) allele HLA-B*35:01. When converted to single-chain Fab chimeric antigen receptor (CAR) constructs, these antibodies can recognize naturally presented p16 MHC-peptide complexes on the surface of cells and activate Jurkat cells. Furthermore, we developed antibodies against predicted p16 MHC-peptide complexes for HLA-A*02:01 that specifically recognize their respective antigen on the surface of cells. These tools establish a platform to survey the surface of senescent cells and provide a potential novel senolytic strategy.

Systematic characterization of AT1 receptor antagonists with label-free dynamic mass redistribution assays

IntroductionIn the drug discovery field, the binding affinities and binding kinetics of drug candidates are very important. Angiotensin II type 1 (AT1) receptor antagonists, e.g., candesartan, telmisartan, irbesartan, losartan and valsartan, show high affinities and long-lasting bindings to the receptor, making them preferred medications for hypertension treatment. However, the molecular binding properties of AT1 receptor antagonists are controversial. MethodsIn this work, we established a profile to study the phenotypic properties of AT1 receptor antagonists with label-free dynamic mass redistribution (DMR) assays in native human cells. With noninvasive features, DMR assay were conducted in multiple formats. Eleven antagonists were systematically evaluated with angiotensin II as an agonist probe in the Hep G2 cell line, which endogenously expresses the AT1 receptor. ResultsThe IC50 values to the AT1 receptor of individual antagonist varied with different incubation times. The antagonists showed competitive behavior with angiotensin II. Schild analysis was used to analyze the competitive behavior of the antagonist. All of the antagonist showed long-lasting possession of the AT1 receptor, except telmisartan. The systematic evaluation of the antagonists implied that 11 antagonists showed high binding affinity but distinct binding modes to AT1 receptor. DiscussionThis study demonstrated that the DMR assay has great potential for determining the pharmacological parameters of ligands. This work may serve as guidance for other receptor and ligand assays.

Therapy of Relapsed/Refractory Acute Lymphoblastic Leukemia Today and Tomorrow.

New diagnostics and treatments, including the use of new drugs, have advanced considerably the treatment of acute lymphoplastic leukemia (ALL) in the past few years. Monoclonal antibodies and immunoconjugates targeting antigens CD19 and CD22 show greater efficacy and more favourable toxicity profiles than standard salvage chemotherapeutic protocols. Two of these drugs - blinatumomab and inotuzumab ozogamicin - have already made their way into clinical practice. Ponatinib and other new generation tyrosine kinase inhibitors allow dose reduction of intensive cytostatic regimens in Ph-positive ALL patients and slowly start to overshadow the importance of allogeneic hematopoietic cell transplants. For the time being, their use is reserved for relapsed/refractory ALL, but they are already available as a first line therapy in clinical trials. An entirely new group of living drugs is emerging for the treatment of ALL - chimeric antigen receptor T-cells produced by genetic modification of native human cells. Chimeric antigen receptor T-cells can be looked upon as in vitro trained professional blast killers. They show an efficacy never seen before for the treatment of relapsed/refractory ALL. On the other hand, this treatment still presents significant risks, mainly due to cytokine release syndrome. Ruxolitinib, mTOR inhibitors, bortezomib, and other drugs for targeted treatment of ALL are currently being evaluated in clinical trials. The article focuses on current options and news in the field of relapsed and refractory ALL treatment. This work was created at Masaryk University as part of the project “New Approaches in Research, Diagnostics and Therapy of Hematological Malignancies VI”, number MUNI/A/1105/2018, supported by Czech Ministry of Education, Youth and Sports in 2019. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 28. 8. 2018 Accepted: 10. 1. 2019.

Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics

Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell stateinformation. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay fortransposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both invitro and invivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.

Signaling profile of a new PAR2 inhibitor with anti‐inflammatory effects

The protease‐activated receptor‐2 (PAR2) belongs to an atypical subfamily of G protein–coupled receptors (GPCR), activated by the proteolytic cleavage of their N‐terminal region by enzymes such as thrombin or trypsin. This cleavage exposes a region of the N‐terminal extracellular domain (the “tethered ligand”) to bind to the extracellular loop 2 and others domains of the PAR2. This results in the stabilization of an active conformation of the receptor. Short synthetic peptides mimicking the tethered ligand sequence are also able to activate PAR2. This subfamily of GPCR is largely involved in inflammatory responses and may therefore represent a promising therapeutic target for the treatment of immune‐mediated inflammatory diseases. It is now recognized that activated‐GPCRs can engage multiple signaling pathways and that specific ligands can selectively promote the engagement of different subsets of these pathways. We therefore characterized the functional selectivity of PAR2 modulators in the context of their potential action as anti‐inflammatory drugs. Elucidating the various signaling pathways should help directed targeting in the quest for therapeutic efficacy; and reciprocally minimize undesirable side effects. For this, we have established the exhaustive signaling signatures of PAR2 modulators using bioluminescence resonance energy transfer (BRET)‐based biosensors in heterologous and native human cell based systems. First, the repertoire of signaling pathways that can be triggered by trypsin and a PAR2 activating peptide (SLIGKV‐NH2) were established. We then identified a compound with a particularly interesting signaling profile among the different modulators tested. Indeed, we showed that compound C5 acts as a negative allosteric modulator (NAM) for the Gα13/Gαq/DAG/Ca2+/PKC signaling pathways activated by both agonists (SLIGKV‐NH2 or trypsin) whereas it has no effect on Gα12/Gαi2/GαoA/GαoB pathways activated by trypsin or SLIGKV‐NH2 and acts as a positive allosteric modulator (PAM) for βarrestin2 recruitment induced by the short peptide. Using a mouse BRET‐based signaling array, we showed that C5 presents a similar signaling profile on the mouse PAR2, establishing inter‐species translation of the results obtained with human receptor in rodent model. Finally, we have evaluated the anti‐inflammatory activity of C5 by measuring its impact on (i) cytokines secretion induced by PAR2 in HCT‐116 cells and (ii) volume of paw edema in rodent inflammatory models. Our preliminary data indicated that C5 presents anti‐inflammatory properties in vitro and in vivo. Overall, our results suggest that C5, by selectively inhibiting PAR2‐induced Gα13/Gαq/DAG/Ca2+/PKC signaling pathways, leads to the anti‐inflammatory effects observed in vitro and in vivo. PAR2 functional selectivity highlights the opportunity to design new drugs that specifically block PAR2‐activated signaling pathway in disease, without affecting beneficial PAR2 signaling in normal physiology.Support or Funding InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Holi colours contain PM10 and can induce pro-inflammatory responses.

BackgroundAt Holi festivals, originally celebrated in India but more recently all over the world, people throw coloured powder (Holi powder, Holi colour, Gulal powder) at each other. Adverse health effects, i.e. skin and ocular irritations as well as respiratory problems may be the consequences. The aim of this study was to uncover some of the underlying mechanisms.MethodsWe analysed four different Holi colours regarding particle size using an Electric field cell counting system. In addition, we incubated native human cells with different Holi colours and determined their potential to induce a pro-inflammatory response by quantifying the resulting cytokine production by means of ELISA (Enzyme Linked Immunosorbent Assay) and the resulting leukocyte oxidative burst by flow cytometric analysis. Moreover, we performed the XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and Propidium iodide cytotoxicity tests and we measured the endotoxin content of the Holi colour samples by means of the Limulus Amebocyte Lysate test (LAL test).ResultsWe show here that all tested Holi colours consist to more than 40 % of particles with an aerodynamic diameter smaller than 10 μm, so called PM10 particles (PM, particulate matter). Two of the analysed Holi powders contained even more than 75 % of PM10 particles.Furthermore we demonstrate in cell culture experiments that Holi colours can induce the production of the pro-inflammatory cytokines TNF-α (Tumor necrosis factor-α), IL-6 (Interleukine-6) and IL-1β (Interleukine-1β). Three out of the four analysed colours induced a significantly higher cytokine response in human PBMCs (Peripheral Blood Mononuclear Cells) and whole blood than corn starch, which is often used as carrier substance for Holi colours. Moreover we show that corn starch and two Holi colours contain endotoxin and that certain Holi colours display concentration dependent cytotoxic effects in higher concentration. Furthermore we reveal that in principle Holi colours and corn starch are able to generate an oxidative burst in human granulocytes and monocytes. In Holi colour 1 we detected a fungal contamination.ConclusionsSome of the observed unwanted health effects of Holi colours might be explained by the high content of PM10 particles in conjunction with the possible induction of a pro-inflammatory response and an oxidative leukocyte burst.

Characterisation of neurons derived from a cortical human neural stem cell line CTX0E16.

IntroductionConditionally immortalised human neural progenitor cells (hNPCs) represent a robust source of native neural cells to investigate physiological mechanisms in both health and disease. However, in order to recognise the utility of such cells, it is critical to determine whether they retain characteristics of their tissue of origin and generate appropriate neural cell types upon differentiation. To this end, we have characterised the conditionally immortalised, cortically-derived, human NPC line, CTX0E16, investigating the molecular and cellular phenotype of differentiated neurons to determine whether they possess characteristics of cortical glutamatergic neurons.MethodsDifferentiated CTX0E16 cells were characterised by assessing expression of several neural fates markers, and examination of developing neuronal morphology. Expression of neurotransmitter receptors, signalling proteins and related proteins were assessed by q- and RT-PCR and complemented by Ca2+ imaging, electrophysiology and assessment of ERK signalling in response to neurotransmitter ligand application. Finally, differentiated neurons were assessed for their ability to form putative synapses and to respond to activity-dependent stimulation.ResultsDifferentiation of CTX0E16 hNPCs predominately resulted in the generation of neurons expressing markers of cortical and glutamatergic (excitatory) fate, and with a typical polarized neuronal morphology. Gene expression analysis confirmed an upregulation in the expression of cortical, glutamatergic and signalling proteins following differentiation. CTX0E16 neurons demonstrated Ca2+ and ERK1/2 responses following exogenous neurotransmitter application, and after 6 weeks displayed spontaneous Ca2+ transients and electrophysiological properties consistent with that of immature neurons. Differentiated CTX0E16 neurons also expressed a range of pre- and post-synaptic proteins that co-localized along distal dendrites, and moreover, displayed structural plasticity in response to modulation of neuronal activity.ConclusionsTaken together, these findings demonstrate that the CTX0E16 hNPC line is a robust source of cortical neurons, which display functional properties consistent with a glutamatergic phenotype. Thus CTX0E16 neurons can be used to study cortical cell function, and furthermore, as these neurons express a range of disease-associated genes, they represent an ideal platform with which to investigate neurodevelopmental mechanisms in native human cells in health and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0136-8) contains supplementary material, which is available to authorized users.

Utilizing induced pluripotent stem cells (iPSCs) to understand the actions of estrogens in human neurons

This article is part of a Special Issue “Estradiol and Cognition”.Over recent years tremendous progress has been made towards understanding the molecular and cellular mechanism by which estrogens exert enhancing effects on cognition, and how they act as a neuroprotective or neurotrophic agent in disease. Currently, much of this work has been carried out in animal models with only a limited number of studies using native human tissue or cells. Recent advances in stem cell technology now make it possible to reprogram somatic cells from humans into induced pluripotent stem cells (iPSCs), which can subsequently be differentiated into neurons of specific lineages. Importantly, the reprogramming of cells allows for the generation of iPSCs that retain the genetic “makeup” of the donor. Therefore, it is possible to generate iPSC-derived neurons from patients diagnosed with specific diseases, that harbor the complex genetic background associated with the disorder. Here, we review the iPSC technology and how it's currently being used to model neural development and neurological diseases. Furthermore, we explore whether this cellular system could be used to understand the role of estrogens in human neurons, and present preliminary data in support of this. We further suggest that the use of iPSC technology offers a novel system to not only further understand estrogens' effects in human cells, but also to investigate the mechanism by which estrogens are beneficial in disease. Developing a greater understanding of these mechanisms in native human cells will also aid in the development of safer and more effective estrogen-based therapeutics.

Computational investigation of drug action on human-induced stem cell-derived cardiomyocytes.

We compare experimental and computational results for the actions of the cardioactive drugs Lidocaine, Verapamil, Veratridine, and Bay K 8644 on a tissue monolayer consisting of mainly fibroblasts and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSc-CM). The choice of the computational models is justified and literature data is collected to model drug action as accurately as possible. The focus of this work is to evaluate the validity and capability of existing models for native human cells with respect to the simulation of pharmaceutical treatment of monolayers and hiPSc-CM. From the comparison of experimental and computational results, we derive suggestions for model improvements which are intended to computationally support the interpretation of experimental results obtained for hiPSc-CM.

Profiling of histamine H4 receptor agonists in native human monocytes

Since the identification of the histamine H₄ receptor, several ligands activating this receptor have been described and more compounds are in development. These ligands are well characterized in pharmacological assays, including radioligand competition binding studies, GTPγS and GTPase assays. In most cases, these experiments are performed in transfected cell lines, expressing unnaturally high levels of target receptors and G-protein signalling components. In this study we investigated the specific properties of H₄ receptor ligands in native cells. Histamine and five different H₄ receptor agonists - 4-methylhistamine, UR-PI376, clobenpropit, VUF8430 and ST-1006 - were characterized in freshly isolated human monocytes. The ligands (10 nM-10 μM) were tested as inhibitors of IL-12p70 secretion from human monocytes and the effects of the H₂ receptor antagonist ranitidine and the H₄ receptor antagonist JNJ7777120 on their action was investigated. Histamine and all the tested agonists reduced IL-12p70 secretion into monocyte supernatants by 40-70%. The potencies varied with pEC50 values ranging from 5.7 to 6.9, depending on the agonist used. All potencies were lower than those determined in the original investigations of the compounds. Pretreatment of monocytes with H₂ or H₄ receptor antagonists showed that some H₄ receptor ligands also had low activity at the H₂ receptor. Our study demonstrates discrepancies between the potencies obtained from assays in transfected cell lines and assays in native human cells, indicating the importance of evaluating H₄ receptor ligands in native cells.

Functional selectivity of G-protein-coupled receptors: From recombinant systems to native human cells

In the mid 1990s, it was assumed that a two-state model, postulating an inactive (R) state and an active (R*) state provides the molecular basis for GPCR activation. However, it became clear that this model could not accommodate many experimental observations. Accordingly, the two-state model was superseded by a multi-state model according to which any given ligand stabilizes a unique receptor conformation with distinct capabilities of activating down-stream G-proteins and β-arrestin. Much of this research was conducted with the β2-adrenoceptor in recombinant systems. At the molecular level, there is now no doubt anymore that ligand-specific receptor conformations, also referred to as functional selectivity, exist. This concept holds great potential for drug discovery in terms of developing drugs with higher selectivity for specific cells and/or cell functions and fewer side effects. A major challenge is the analysis for functional selectivity in native cells. Here, I discuss our current knowledge on functional selectivity of three representative GPCRs, the β2-adrenoceptor and the histamine H2- and H4-receptors, in recombinant systems and native human cells. Studies with human neutrophils and eosinophils support the concept of functional selectivity. A major strategy for the analysis of functional selectivity in native cells is to generate complete concentration/response curves with a large set of structurally diverse ligands for multiple parameters. Next, correlations of potencies and efficacies are analyzed, and deviations of the correlations from linearity are indicative for functional selectivity. Additionally, pharmacological inhibitors are used to dissect cell functions from each other.

Dissociations in the effects of β2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

In neutrophils, activation of the β2-adrenergic receptor (β2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3′,5′-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2 •−) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2 •− formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2 •− production than in stimulating cAMP accumulation. (−)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2 •− production. Moreover, (−)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2 •− assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2 •− production after β2AR-stimulation although cAMP-increasing substances inhibited O2 •− production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2 •− production in a cAMP-independent manner.

Expression of sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes

The SERCA family includes 3 genes ( SERCA1–3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early ( ETF) and Late ( LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg 334, Arg 396 and Arg 638 were directly assigned to the ETF and ii) Arg 198 was assigned as the only tryptic site to the LTF. Arg 671, Lys 712/Lys 713 and Lys 728 were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl 2, EGTA or AlF 4 − strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b–3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg 1002 as an additional tryptic site in SERCA3b–3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.

Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes

We describe the use of modified fluorescent-labeled oligonucleotide probes in the sequence-specific detection of messenger RNAs in live human cells. To make this detection possible, we developed a previously undescribed probe design that combines earlier quenched autoligation chemistry with a previously undescribed fluorescence resonance energy transfer (FRET) strategy to lower background signals. The probe pairs consisted of a nucleophilic 3'-phosphorothioate probe carrying a Cy5 FRET acceptor, and an electrophilic probe containing the combination of a 5' end electrophile/quencher and a fluorescein FRET donor. Probes were introduced to HL-60 cells by use of the streptolysin O pore-forming peptide. Signals from three different messenger RNAs, as well as 28S ribosomal RNA, could be detected and quantitated by flow cytometry. Probes targeted to ribosomal sequences and beta-actin mRNA also could be detected over background by confocal fluorescence microscopy. Varying the target site and probe backbone chemistry were found to have large effects on signal. The data suggest that quenched autoligating probes may be of general utility as biological tools in following localization, transcription, and processing of eukaryotic cellular messages and may have applications in diagnostic or prognostic analysis of disease-related RNAs in human tissues.

Human Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (acat1) Sequences Located in Two Different Chromosomes (7 and 1) Are Required to Produce a Novel ACAT1 Isoenzyme with Additional Sequence at the N Terminus

A rare form of human ACAT1 mRNA, containing the optional long 5'-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7. To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5'-untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1(ORF). The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.

Release of T-antigen, a carcinoma marker from native human cells, by endo-α- N-acetylgalactosaminidase of Alcaligenes SP

The endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. could release T-antigen (Ga1 beta 1----3GalNAc), a carcinoma-associated marker from asialo glycophorin and human cells. The released T-antigen from human asialo erythrocytes was determined by thin layer chromatography and gas liquid chromatography. The released T-antigen from human gastric carcinoma cell Kato III was identified by high performance liquid chromatographies with a reverse-phase column and a size fractionation column. Released T-antigen could be analyzed quantitatively by a sensitive method including pyridylamino derivatization and following high performance liquid chromatography. This suggests that the enzyme is useful for detection and determination of T-antigen from cells.