Interrogating health-related public databases from a food toxicology perspective: computational analysis of scoring data.

Over the last 15 years, an expanding number of databases with information on noxious effects of substances on mammalian organisms and the environment have been made available on the Internet. This set of databases is a key source of information for risk assessment within several areas of toxicology. Here we present features and relationships across a relatively wide set of publicly accessible databases broadly within toxicology, in part by clustering multi-score representations of such repositories, to support risk assessment within food toxicology. For this purpose 36 databases were each scrutinized, using 18 test substances from six different categories as probes. Results have been analyzed by means of various uni- and multi-variate statistical operations. The former included a special index devised to afford context-specific rating of databases across a highly heterogeneous data matrix, whereas the latter involved cluster analysis, enabling the identification of database assemblies with overall shared characteristics. One database - HSDB - was outstanding due to rich and qualified information for most test substances, but an appreciable fraction of the interrogated repositories showed good to decent scoring. Among the six chosen substance groups, Food contact materials had the most comprehensive toxicological information, followed by the Pesticides category.

Genetically modified plants for non-food or non-feed purposes: straightforward screening for their appearance in food and feed.

Genetically modified (GM) plants aimed at producing food/feed are part of regular agriculture in many areas of the World. Commodity plants have also found application as bioreactors, designated non-food/non-feed GM (NFGM) plants, thereby making raw material for further refinement to industrial, diagnostic or pharmaceutical preparations. Many among them may pose health challenge to consumers or livestock animals, if occurring in food/feed. NFGM plants are typically released into the environment, but are grown under special oversight and any among several containment practices, none of which provide full protection against accidental dispersal. Adventitious admixture with food or feed can occur either through distributional mismanagement or as a consequence of gene flow to plant relatives. To facilitate NFGM surveillance we propose a new mandatory tagging of essentially all such plants, prior to cultivation or marketing in the European Union. The suggested tag--Plant-Made Industrial or Pharmaceutical Products Tag (PMIP-T)--is envisaged to occur as a transgenic silent DNA identifier in host plants and designed to enable technically simple identification and characterisation of any NFGM. Implementation of PMIP-T would permit inexpensive, reliable and high-throughput screening for NFGM specifically. The paper outlines key NFGM prospects and challenges as well as the PMIP-T concept.

Computational detection of allergenic proteins attains a new level of accuracy with in silico variable-length peptide extraction and machine learning.

The placing of novel or new-in-the-context proteins on the market, appearing in genetically modified foods, certain bio-pharmaceuticals and some household products leads to human exposure to proteins that may elicit allergic responses. Accurate methods to detect allergens are therefore necessary to ensure consumer/patient safety. We demonstrate that it is possible to reach a new level of accuracy in computational detection of allergenic proteins by presenting a novel detector, Detection based on Filtered Length-adjusted Allergen Peptides (DFLAP). The DFLAP algorithm extracts variable length allergen sequence fragments and employs modern machine learning techniques in the form of a support vector machine. In particular, this new detector shows hitherto unmatched specificity when challenged to the Swiss-Prot repository without appreciable loss of sensitivity. DFLAP is also the first reported detector that successfully discriminates between allergens and non-allergens occurring in protein families known to hold both categories. Allergenicity assessment for specific protein sequences of interest using DFLAP is possible via ulfh@slv.se.

Assessment of the safety of foods derived from genetically modified (GM) crops.

This paper provides guidance on how to assess the safety of foods derived from genetically modified crops (GM crops); it summarises conclusions and recommendations of Working Group 1 of the ENTRANSFOOD project. The paper provides an approach for adapting the test strategy to the characteristics of the modified crop and the introduced trait, and assessing potential unintended effects from the genetic modification. The proposed approach to safety assessment starts with the comparison of the new GM crop with a traditional counterpart that is generally accepted as safe based on a history of human food use (the concept of substantial equivalence). This case-focused approach ensures that foods derived from GM crops that have passed this extensive test-regime are as safe and nutritious as currently consumed plant-derived foods. The approach is suitable for current and future GM crops with more complex modifications. First, the paper reviews test methods developed for the risk assessment of chemicals, including food additives and pesticides, discussing which of these methods are suitable for the assessment of recombinant proteins and whole foods. Second, the paper presents a systematic approach to combine test methods for the safety assessment of foods derived from a specific GM crop. Third, the paper provides an overview on developments in this area that may prove of use in the safety assessment of GM crops, and recommendations for research priorities. It is concluded that the combination of existing test methods provides a sound test-regime to assess the safety of GM crops. Advances in our understanding of molecular biology, biochemistry, and nutrition may in future allow further improvement of test methods that will over time render the safety assessment of foods even more effective and informative.

Statistical evaluation of local alignment features predicting allergenicity using supervised classification algorithms.

BACKGROUND: Recently, two promising alignment-based features predicting food allergenicity using the k nearest neighbor (kNN) classifier were reported. These features are the alignment score and alignment length of the best local alignment obtained in a database of known allergen sequences. METHODS: In the work reported here a much more comprehensive statistical evaluation of the potential of these features was performed, this time for the prediction of allergenicity in general. The evaluation consisted of the following four key components. (1) A new high quality database consisting of 318 carefully selected, non-redundant allergens and 1,007 sequences carefully selected to be non-allergens. (2) Three different supervised algorithms: the kNN classifier, the Bayesian linear Gaussian classifier, and the Bayesian quadratic Gaussian classifier. (3) A large set of local alignment procedures defined using the FASTA3 alignment program by means of a wide range of different parameter settings. (4) Novel performance curves, alternative to conventional receiver-operating characteristic curves, to display not only average behaviors but also statistical variations due to small data sets. RESULTS: The linear Gaussian classifier proved most useful among the tested supervised machine learning algorithms, closely followed by the quadratic Gaussian equivalent and kNN. The overall best classification results were obtained with a novel feature vector consisting of the combined alignment scores derived from local alignment procedures using different substitution matrices. CONCLUSIONS: The models reported here should be useful as a part of an integrated assessment scheme for potential protein allergenicity and for future comparisons with alternative bioinformatic approaches.

Retinoids as ligands and coactivators of protein kinase C alpha.

Whereas retinoic acids control nuclear events, a second class of retinol metabolites, that is, the hydroxylated forms exemplified by 14-hydroxy-retro-retinol (HRR), operate primarily in the cytoplasm. They function as regulatory cofactors for cell survival/cell death decisions. In accordance with these biological aspects, we demonstrate that these retinoids bound protein kinase C (PKC) alpha with nanomolar affinity and markedly enhance the activation of PKC alpha and the entire downstream MAP kinase pathway by reactive oxygen species. HRR was 10 times more efficient than retinol, and the optimum doses are 10-7 and 10-6 M, respectively. PKC alpha activation was reversed rapidly by imposition of reducing conditions. The retinoid binding site was mapped to the first cysteine-rich region in the regulatory domain, C1A, yet was distinct from the binding sites of diacylglycerol and phorbol esters. The C1B domain bound retinoids poorly. The emerging theme is that retinoids serve as redox regulators of protein kinase C.

The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors.

Vitamin A and its biologically active derivatives, the retinoids, are recognized as key regulators of vertebrate development, cell growth, and differentiation. Although nuclear receptors have held the attention since their discovery a decade ago, we report here on serine/threonine kinases as a new class of retinoid receptors. The conserved cysteine-rich domain of the NH(2)-terminal regulatory domains of cRaf-1, as well as several select domains of the mammalian protein kinase C (PKC) isoforms alpha, delta, zeta, and mu, the Drosophila and yeast PKCs, were found to bind retinol with nanomolar affinity. The biological significance was revealed in the alternate redox activation pathway of these kinases. Retinol served as a cofactor to augment the activation of both cRaf and PKC alpha by reactive oxygen, whereas the classical receptor-mediated pathway was unaffected by the presence or absence of retinol. We propose that bound retinol, owing to its electron transfer capacity, functions as a tag to enable the efficient and directed redox activation of the cRaf and PKC families of kinases.

Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing.

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.

F-actin as a functional target for retro-retinoids: a potential role in anhydroretinol-triggered cell death.

The retro-retinoids, metabolites of vitamin A (retinol), belong to a family of lipophilic signalling molecules implicated in regulation of cell growth and survival. Growth-promoting properties have been ascribed to 14-hydroxy-retro-retinol (14HRR), while anhydroretinol (AR) was discovered to act as a natural antagonist triggering growth arrest and death by apoptosis. Based on morphological studies and inhibition of apoptosis by the kinase blocker, herbimycin A, it has been suggested that retro-retinoids exhibit their function in the cytosolic compartment. F-actin emerged as a functional target for retro-retinoid action. By FACS analysis and fluorescence microscopy of phalloidin-FITC labeled cells we demonstrated that F-actin reorganization was an early event in AR-triggered apoptosis. Fluorescence images of AR-treated fibroblasts displayed short, thick, stick-like and punctate structures, and membrane ruffles at the cell periphery along with an increased diffuse staining pattern. Reversal of the AR effect by 14HRR or retinol indicates that F-actin is a common site for regulation by retro-retinoids. Inhibition of both cell death and actin depolymerisation by bcl-2 implies that cytoskeleton reorganization is downstream of bcl-2-related processes. Furthermore, stabilization of microfilaments by jasplakinolide increased the survival potential of AR treated cells, while weakening the cytoskeleton by cytochalasin B abetted apoptosis. Thus the cytoskeleton is an important way station in a communication network that decides whether a cell should live or die.

The beta-gal interferon assay: a new, precise and sensitive method.

Cells of a human glioblastoma line were stably transfected with a glial fibrillary acidic protein (GFAP) promoter sequence/lacZ reporter gene. Following this modification, they produced Escherichia coli beta-galactosidase constitutively in amounts that could be measured through their conversion of an added fluorophore into a product readily estimated by fluorimetry. Human interferons (IFN) selectively and in a dose-dependent manner reduce the formation of beta-galactosidase in this system. We have used it as the basis for a novel assay that is sensitive (4-40 pg/ml), precise, completed in 30 h, and applicable to both type I and type II human IFNs. Statistical analysis showed interassay relative standard deviations ranging from 5% to 11%, and most individual assays revealed potencies with limits of error within 85%-115%. Neither partially trypsin-digested IFN nor the other cytokines and mitogens we tested reacted in this system, except for tumor necrosis factor-alpha (TNF-alpha). The high selectivity was further shown by the loss of response to IFN in the presence of the appropriate specific anti-IFN or anti-IFN-gamma receptor antibodies.

In vitro cellular responses to cytokines and erythropoietin.

Cytokines and polypeptide hormones act through high-affinity binding to cognate transmembrane receptor molecules, expressed on target cells. The impact of such ligand molecules is conveyed to the cell nucleus by specific signal transduction mechanisms and is ultimately manifested as changes in gene expression, largely accomplished by transcription-regulatory factors. Depending on target cell maturation and receptor signalling pathways, cell-cycle progression or growth inhibition may follow from ligand/receptor interactions. We have employed cellular growth as an endpoint for potency determination of several human bioactive substances, such as interferons (IFNs), IL-2, G-CSF, GM-CSF and erythropoietin (Epo), using murine or human cell lines as indicators. The conversion of the tetrazolium salt MTT by mitochondrial reductase to blue formazan served as an endpoint in such estimations. In addition to a cellular growth suppression IFN assay, a reporter gene-modified human glioblastoma line was devised to provide an implement for high-throughput potency assessment of interferons. The bioassay systems were all designed according to the parallel line assay model and were subjected to extensive validation procedures. Both intra- and inter-assay variations were consistently within the range of immunometric counterparts; hence precision and reproducibility do not need to be compromised when using biological determination methods. Furthermore, the advantage of monitoring downstream signal transduction effects of ligand binding, particularly over immunometry, is evident since it reflects a pharmacodynamic cellular response. The assays were operating in the pM range and their sensitivity could hence compete with immunometric counterparts. When applicable, the aforementioned approaches were combined with physicochemical characterization of the respective ligands, which further enhanced the physiological relevance of the cellular readout. Accordingly, such two-part assays should provide alternatives to traditional in vivo activity determinations of biological substances.

The level of lipopolysaccharide-binding protein is significantly increased in plasma in patients with the systemic inflammatory response syndrome.

Currently, there is no way to predict with a high degree of sensitivity and specificity which patients are likely to develop systemic inflammatory response syndrome (SIRS) following systemic infection, trauma, organ rejection, or blood loss. The level of human lipopolysaccharide-binding protein (LBP) was determined in the plasma of 22 patients with a clinical diagnosis of early SIRS. Twenty-nine plasma samples from healthy volunteers were used as controls. The mean level of LBP in the plasma of healthy volunteers was 7.7 micrograms/ml (standard deviation, 6.2 micrograms/ml). Twenty-one of 22 patients (95%) with SIRS had an LBP level on admission at least 2 standard deviations above the mean LBP level for a healthy volunteer control group (range, 4.9 to 114.2 micrograms/ml; mean, 36.6 micrograms/ml; standard deviation, 22.2 micrograms/ml; P < 0.0001). The level of LBP in the plasma of the majority of patients with early SIRS is significantly increased compared to that in healthy controls. The sensitivity, specificity, and predictive value of elevated plasma LBP levels in patients with SIRS remain to be determined.

Retro-retinoids in regulated cell growth and death.

Vitamin A serves as a prohormone from which three classes of active metabolites are derived: the aldehydes, the carboxylic acids, and the retro-retinoids. Although these three classes are united under the rubric of signal transduction, they act by different molecular mechanisms: the 11-cis-retinaldehydes combine with opsin to form the universal visual pigments and the retinoic acids form ligands for transcription factors, whereas the retro-retinoids, as shown here, intersect with signal transduction at a cytoplasmic or membrane site. The retro-retinoid, anhydroretinol (AR), has long been known to act as a growth inhibitor in lymphocytes, whereas 14-hydroxy-4,14-retro-retinol (14-HRR) is required for normal lymphocyte proliferation. A mutually reversible relationship exists between these two retro-retinoids as one can reverse the effects of the other when given in pharmacological doses. The common explanation for reversible inhibition is competition for a shared receptor. We now provide evidence that when AR is given to T cells unmitigated by 14-HRR, rapid cell death can occur. The circumstances are closely related to nonclassical forms of apoptosis: within 2 h of AR administration the T cells undergo widespread morphological changes, notably surface blebbing and ballooning and, inevitably, bursting. In contrast, nuclear changes are comparatively mild, as indicated by absence of chromatin condensation and overt DNA cleavage to discrete nucleosomal fragments, although DNA nicks are readily discernible by terminal deoxynucleotidyl transferase assay. What further distinguishes the AR-induced form of apoptosis from classical ones is a lack of requirements of messenger RNA and protein synthesis, suggesting that the events leading to cell death are primarily initiated and play themselves out in the cytoplasm. This view is further reinforced by the finding that herbimycin A can prevent the onset of programmed cell death. The importance of our findings is that they strongly suggest a second messenger role for vitamin A metabolites in the cytoplasmic realm that has not been seen previously. These findings are entirely compatible with a general notion that in a cell requiring multiple coordinated signals for survival, the provision of an unbalanced signal can initiate programmed cell death. Collectively, our data also challenge the paradigm that retinoids (outside vision) solely mediate their function via the steroid/ retinoic acid receptor family of nuclear transcription factors. Instead, a mode of action in the cytoplasmic realm akin to one attributed to other small lipophilic second messenger molecules, such as diacyl glycerol or ceramide, may apply to retro-retinoids.

In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity.

The human erythroleukemia cell line TF-1 was employed for the determination of proliferative stimulation induced by recombinant human erythropoietin (rhEpo). Potencies of various intact and sugar-trimmed rhEpo preparations were estimated using the International Standard for Human r-DNA-derived Epo (87/684) as a reference for activity. The cellular response was measured in a multi-channel photometer using a colorimetric microassay, based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan, by viable cells. The linear part of the log dose-response relationship encompassed 2.5-90 pM and activity of rhEpo preparations was measured at doses between 3 and 60 pM. The assay was designed as a parallel line test, using three or four concentrations for potency determinations, which fulfills pharmacopoeial requirements for assay validity. Inter-assay relative standard deviation varied between 4.1% and 12.6% and most assays revealed potencies with limits of error within 87-113%. In order to acquire an additional means for an efficient probing of physiologically relevant features of rhEpo, a luminiscence-dependent Western detection system, based on a combined isoelectric focusing/sodium dodecyl sulphate-polyacrylamide gel electrophoresis separation, was established. As opposed to conventional electrophoresis the two dimensional approach enabled the disclosure of minor truncations in the rhEpo-attached glycan moieties using picomolar quantities of the hormone. Moreover, the separated isoforms of rhEpo were quantified by computer-assisted densitometry and compared with the 87/684 standard. Accordingly, results obtained by the cellular response were balanced against the general pattern observed and the relative amounts of separated rhEpo isomers as determined by the quantitative Western analysis. The method described should be suitable for potency assessments of pharmaceutical formulations of rhEpo.

Purification, cloning, and bacterial expression of retinol dehydratase from Spodoptera frugiperda.

Anhydroretinol and 14-hydroxy-4,14-retro-retinol, retro-retinoids endogenous to both mammals and insects, act as agonist and antagonist, respectively, in controlling proliferation in lymphoblasts and other retinol-dependent cells. We describe here the identification, purification, cloning, and bacterial expression of the enzyme retinol dehydratase, which converts retinol to anhydroretinol in Spodoptera frugiperda. Retinol dehydratase has nanomolar affinity for its substrate and is, therefore, the first enzyme characterized able to utilize free retinol at physiological intracellular concentrations. The enzyme shows sequence homology to the sulfotransferases and requires 3'-phosphoadenosine 5'-phosphosulfate for activity.

13,14-Dihydroxy-retinol, a new bioactive retinol metabolite.

Deprivation of vitamin A (retinol) leads to reduced potential of B cell proliferation and nearly complete block of T cell activation in vitro. Retinol, which is thought to function as a pro-hormone, is enzymatically converted into intracellular messenger molecules. Thus, 14-hydroxy-retro-retinol (14-HRR) is an intracellular messenger molecule linked to activation and growth regulation of lymphocytes; whereas, anhydroretinol, another natural retro-retinoid, is an antagonist of 14-HRR effects. In this article, we describe the isolation, structure determination, synthesis, and biological properties of a new intracellular retinol derivative, 13,14-dihydroxy-retinol (DHR), which also supports the viability of retinol-deprived lymphocytes. DHR is found in numerous cell lines representing a large cross-section of tissues and animals from insects to mammals. In T lymphocytes the production of DHR and 14-HRR is up-regulated by phorbol ester. DHR is converted to 14-HRR by mild acid treatment, but not by cells; therefore DHR is not a biosynthetic intermediate in the conversion of retinol to 14-HRR. DHR is a distinct end point of retinol metabolism. Although it is linked to cell proliferation, its biological role remains to be determined.

In vitro bioassay with enhanced sensitivity for human granulocyte colony-stimulating factor.

A method for the determination of human granulocyte colony-stimulating factor (hG-CSF) activity, based on stimulation of cellular proliferation, was developed using a subclone of the murine myeloid leukemia cell line NFS-60, with an improved sensitivity for hG-CSF, as indicator. The optimal range for quantitative analysis of hG-CSF was about 4-60 pg ml-1. The stimulatory effect was measured by a colorimetric microassay: the optical density of formazan, which is produced by viable cells from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), was obtained by reading plates in a multi-channel photometer. The assay was designed as a five-dose parallel line test, employing three or four doses for potency determinations, which fulfil pharmacopoeial requirements for assay validity. Inter-assay relative standard deviation (RSD) varied between 5.2 and 12.0%. Most assay experiments revealed potencies within limits of error of 90-110% and the mean index of precision value was 0.057. The recently developed yeast cell-derived International Standard (88/502) served as a reference for activity of rhG-CSF. Specificity of the assay was demonstrated by absence of response upon exposure to a panel of biomolecules, including recombinant human interleukin-3, and by the suppression of growth stimulation in the presence of neutralizing anti hG-CSF antibodies. Potency readings of unglycosylated rhG-CSF were dependent on pH of assay medium with higher relative activities observed at pH 6.6 than at 7.4. Moreover, SDS-PAGE analysis of the carbohydrate-deficient preparation, following incubation at physiological pH, revealed several high molecular weight rhG-CSF bands and decreased monomeric form. The method described was found suitable for potency assessments of pharmaceutical formulations of hG-CSF.

Activation of the human NPY gene during neuroblastoma cell differentiation: induced transcriptional activities of AP-1 and AP-2.

During functional neuronal differentiation of human SH-SY5Y neuroblastoma cells, induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), the mRNA expression of c-fos and c-jun displayed a synchronous and biphasic type of induction for both mRNAs, with an early transient (30 to 120 min) and a later (> 8 h) more persistent increase. This was coupled to increased in vitro DNA binding activity of cFos/cJun AP-1 heterodimers in SH-SY5Y nuclear extracts using the electrophoretic mobility shift assay. Functional AP-1 activity was demonstrated in differentiating SH-SY5Y cells by transient transfection assays using a TPA-responsive reporter plasmid. The second expression phase of these protooncogenes was paralleled by a sustained induction of neuronal differentiation markers, as exemplified by growth-associated protein 43 and neuropeptide tyrosine (NPY) mRNAs. DNA-protein interaction between an evolutionarily conserved region (-73 to -45) of the human NPY promoter, containing potential binding sites for AP-1, AP-2, and Sp1, and nuclear extracts prepared from untreated and TPA-treated SH-SY5Y cells revealed one complex (CI) that was unaffected and three complexes (CII to CIV) that were induced by TPA treatment. Competition for DNA binding using AP-1, AP-2, and Sp1 consensus sequences and an anti-cJun antibody, respectively, revealed cooperative interactions between AP-1, AP-2, and Sp1 transcription factors and the NPY promoter. In addition, TPA-mediated induction of AP-2 DNA binding activity to the NPY promoter was not dependent on increased AP-2 mRNA expression. This high degree of complexity presumably involved in NPY gene expression during neuronal differentiation of SH-SY5Y cells suggests productive cooperative interactions between multiple transcription factors.

Intracellular signaling activity of synthetic (14R)-, (14S)-, and (14RS)-14-hydroxy-4,14-retro-retinol.

14-Hydroxy-4,14-retro-retinol (14-HRR), first isolated from cultures of lymphoblastoid 5/2 and HeLa cells and characterized by NMR, UV, and CD, is a metabolite of retinol which promotes growth of B lymphocytes in culture and activation of T lymphocytes by antigen receptor-mediated signals. It is also produced by various tested cell lines: fibroblasts, leukemia, and Drosophila cells. 14-HRR is the first bioactive retro-retinoid to be discovered and, after retinal and retinoic acid, is the third intracellular messenger molecule derived from retinol. Physical properties and intracellular signaling activities of synthetic (14R)-HRR, (14S)-HRR, and racemic 14-HRR are described. CD spectra indicate that natural 14-HRR isolated previously was a mixture of enantiomers. B-cell survival and T-cell activation assays performed in the optimal range of (7-1.6) x 10(-7) M surprisingly showed that all 14-HRR compounds exhibit similar activity, with the 14R enantiomer exhibiting slightly higher activity in comparison to the 14S enantiomer. However, because of the semiquantitative nature of the assays, the conclusion as to which enantiomer is more active and which is the true ligand for the target receptor must await characterization of this protein.

Growth control or terminal differentiation: endogenous production and differential activities of vitamin A metabolites in HL-60 cells.

Vitamin A (retinol) is a prohormone that exerts its pleiotropic biological effects after conversion into multiple metabolites. In this report we describe the identification of three endogenous, retinolderived effector molecules, 14-hydroxy-retro-retinol (14-HRR), anhydroretinol (AR), and retinoic acid (RA) and a putative storage form of retinol, retinylesters (RE) in the human promyelocytic leukemia cell line HL-60. Exogenous application of the retinol metabolites in retinol-depleted serum-free cultures of HL-60 allowed the identification of unique cellular functions for each metabolite: 14-HRR is a growth factor for HL-60. AR is a functional antagonist of 14-HRR with growth-inhibiting activity, and RA is a potent inducer of granulocyte differentiation accompanied by growth arrest. Finally, intracellular RE serves as storage form allowing continuous production of 14-HRR when no external retinol is available.