Identification of antigenic proteins from Echinostoma caproni (Trematoda) recognized by mouse immunoglobulins M, A and G using an immunoproteomic approach

Antigenic proteins of Echinostoma caproni (Trematoda) against mouse IgM, IgA, IgG, IgG1 and IgG2a were investigated by immunoproteomics. Excretory/secretory products (ESP) of E. caproni separated by two-dimensional (2D) gel electrophoresis were transferred to nitrocellulose membranes and probed with the different mouse immunoglobulin classes. A total of four proteins (enolase, 70 kDa heat-shock protein (HSP-70), actin and aldolase) were accurately identified. Enolase was recognized in eight different spots of which seven of them were detected in the expected molecular weight and were recognized by IgA, IgG or IgG and IgG1. Another spot identified as enolase at 72 kDa was only recognized by IgM. Digestion with N-glycosidase F of the 72 kDa band rendered a polypeptide with an apparent molecular weight similar to that expected for enolase recognized by Western immunoblotting using anti-enolase antibodies. This suggests that glycosylated forms of enolase may be involved in the early thymus-independent responses against E. caproni. Early IgM responses were also generated by actin and the HSP-70 which suggests that these proteins are exposed early to the host and may be of importance in the parasite establishment. The IgA responses also appear to be mediated by the HSP-70 and aldolase which could be related with the close contact of these proteins with the host mucosal surface after secretion.     

Phylogeny of protein-folding trajectories reveals a unique pathway to native structure

To scrutinize how a protein folds at atomic resolution, we performed 200 molecular dynamics simulations (each of 50 ns) of the miniprotein Trp-cage on the computational grid. Within the trajectories, 58 folding and 31 unfolding events were identified and subjected to extensive comparison and classification. Based on an analogy with biological sequences, the folding and unfolding trajectories (arrays of sequential snapshots of structures) were aligned by dynamic programming allowing gaps. A phylogenetic tree derived from the alignments revealed four distinct groups of the trajectories, characterized by the Trp side-chain motions and the main-chain motions. It was found that only one group attained the native structure and that the other three led to pseudonative structures having the correct main-chain trace but different nonnative Trp side-chain rotamers, indicating that those four folded structures were each attained through a unique folding pathway.     

Metabolic basis for the differential susceptibility of Gram-positive pathogens to fatty acid synthesis inhibitors

The rationale for the pursuit of bacterial type 2 fatty acid synthesis (FASII) as a target for antibacterial drug discovery in Gram-positive organisms is being debated vigorously based on their ability to incorporate extracellular fatty acids. The regulation of FASII by extracellular fatty acids was examined in Staphylococcus aureus and Streptococcus pneumoniae, representing two important groups of pathogens. Both bacteria use the same enzymatic tool kit for the conversion of extracellular fatty acids to acyl-acyl carrier protein, elongation, and incorporation into phospholipids. Exogenous fatty acids completely replace the endogenous fatty acids in S. pneumoniae but support only 50% of phospholipid synthesis in S. aureus. Fatty acids overcame FASII inhibition in S. pneumoniae but not in S. aureus. Extracellular fatty acids strongly suppress malonyl-CoA levels in S. pneumoniae but not in S. aureus, showing a feedback regulatory system in S. pneumoniae that is absent in S. aureus. Fatty acids overcame either a biochemical or a genetic block at acetyl-CoA carboxylase (ACC) in S. aureus, confirming that regulation at the ACC step is the key difference between these two species. Bacteria that possess a stringent biochemical feedback inhibition of ACC and malonyl-CoA formation triggered by environmental fatty acids are able to circumvent FASII inhibition. However, if exogenous fatty acids do not suppress malonyl-CoA formation, FASII inhibitors remain effective in the presence of fatty acid supplements.     

Structure-Based Identification of Natural Products as SARS-CoV-2 Mpro Antagonist from Echinacea angustifolia Using Computational Approaches

Coronavirus disease-19 (COVID-19) pandemic, caused by the novel SARS-CoV-2 virus, continues to be a global threat. The number of cases and deaths will remain escalating due to the lack of effective therapeutic agents. Several studies have established the importance of the viral main protease (M^pro) in the replication of SARS-CoV-2 which makes it an attractive target for antiviral drug development, including pharmaceutical repurposing and other medicinal chemistry approaches. Identification of natural products with considerable inhibitory potential against SARS-CoV-2 could be beneficial as a rapid and potent alternative with drug-likeness by comparison to de novo antiviral drug discovery approaches. Thereof, we carried out the structure-based screening of natural products from Echinacea-angustifolia, commonly used to prevent cold and other microbial respiratory infections, targeting SARS-CoV-2 M^pro. Four natural products namely, Echinacoside, Quercetagetin 7-glucoside, Levan N, Inulin from chicory, and 1,3-Dicaffeoylquinic acid, revealed significant docking energy (>−10 kcal/mol) in the SARS-CoV-2 M^pro catalytic pocket via substantial intermolecular contacts formation against co-crystallized ligand (<−4 kcal/mol). Furthermore, the docked poses of SARS-CoV-2 M^pro with selected natural products showed conformational stability through molecular dynamics. Exploring the end-point net binding energy exhibited substantial contribution of Coulomb and van der Waals interactions to the stability of respective docked conformations. These results advocated the natural products from Echinacea angustifolia for further experimental studies with an elevated probability to discover the potent SARS-CoV-2 M^pro antagonist with higher affinity and drug-likeness.     

Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia

Serotonin (5-HT) plays a critical role in modulating synaptic plasticity in the marine mollusc Aplysia and in the mammalian nervous system. In Aplysia sensory neurons, 5-HT can activate several signal cascades, including PKA and PKC, presumably via distinct types of G protein-coupled receptors. However, the molecular identities of these receptors have not yet been identified. We here report the cloning and functional characterization of a 5-HT receptor that is positively coupled to adenylyl cyclase in Aplysia neurons. The cloned receptor, 5-HT_apAC1, stimulates the production of cAMP in HEK293T cells and in Xenopus oocytes. Moreover, the knockdown of 5-HT_apAC1 expression by RNA interference blocked 5-HT-induced cAMP production in Aplysia sensory neurons and blocked synaptic facilitation in nondepressed or partially depressed sensory-to-motor neuron synapses. These data implicate 5-HT_apAC1 as a major modulator of learning related synaptic facilitation in the direct sensory to motor neuron pathway of the gill withdrawal reflex.     

Addition of insoluble fiber to isolation media allows for increased metabolite diversity of lab-cultivable microbes derived from zebrafish gut samples

ABSTRACT

There is a gap in measured microbial diversity when comparing genomic sequencing techniques versus cultivation from environmental samples in a laboratory setting. Standardized methods in artificial environments may not recapitulate the environmental conditions that native microbes require for optimal growth. For example, the intestinal tract houses microbes at various pH values as well as minimal oxygen and light environments. These microbes are also exposed to an atypical source of carbon: dietary fiber compacted in fecal matter. To investigate how the addition of insoluble fiber to isolation media could affect the cultivation of microbes from zebrafish intestines, an isolate library was built and analyzed using the bioinformatics pipeline IDBac. While all isolation media encouraged the growth of species from several phyla, the extent of growth was greater with the addition of fiber allowing for easier isolation. Furthermore, fiber addition altered the metabolism of the cultivated gut-derived microbes and induced the production of unique metabolites that were not produced when microbes were otherwise grown on standard isolation media. Addition of this inexpensive carbon source to the media supported the cultivation of a diverse community whose secondary metabolite production may more closely replicate their metabolite production in vivo.     

Identification of a novel cell death-inducing domain reveals that fungal amyloid-controlled programmed cell death is related to necroptosis

Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a β-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid-controlled fungal programmed cell death and mammalian necroptosis.Programmed cell death (PCD) plays a central role in response to nonself and host defenses in animals, plants, fungi, and bacteria (1–4). Altruistic cell suicide hinders pathogen replication and promotes survival in multicellular organisms, and provides benefits at the population level in unicellular microbes. Questions surrounding the classification, evolutionary origin, and level of transkingdom conservation of PCD modalities remain open and often polemic (5). Classification systems of PCD types are based on cytological or molecular markers that can differ between phyla, making it difficult to merge cell death processes into unifying categories (5).In filamentous fungi, a form of PCD termed heterokaryon incompatibility occurs in response to conspecific nonself (3). Cell death by incompatibility takes place following fusion of cells from genetically distinct individuals and is controlled by het genes. One of the best-characterized incompatibility systems involves a cell death-inducing protein termed HET-S, which is controlled by amyloid templating. HET-S of Podospora anserina exhibit an N-terminal cell death execution HeLo domain and a C-terminal regulatory prion forming domain (PFD) that adopts a specific β-solenoid amyloid fold, characterized by the stacking of two pseudorepeats of an elementary amyloid motif (6–8). When the HET-S PFD is converted to the β-solenoid fold, the HeLo domain undergoes refolding, and an N-terminal transmembrane helix (TMH) is exposed, causing HET-S to behave as a pore-forming toxin (9). On transconformation, HET-S relocates from the cytoplasm to the cell membrane, where it exerts toxicity (10).Conversion of the HET-S PFD region can be achieved by two means. The first described mechanism occurs in the context of HET-S/[Het-s] incompatibility (9). [Het-s] is a variant form of HET-S that has lost the pore-forming activity and can stably propagate as a prion. [Het-s] and HET-S strains are incompatible, because [Het-s] acts as a seed converting the PFD of HET-S, thereby leading to activation of the HeLo cell death-inducing domain. A second, recently described mode of activation involves a Nod-like receptor protein (NLR), termed NWD2, encoded by the gene adjacent to het-S in the genome (11, 12). The NWD2 NLR displays a 21-aa N-terminal region homologous to the HET-s amyloid motif. On binding of a specific ligand to NWD2, these N-terminal extensions adopt an amyloid fold and convert HET-S to the toxic pore-forming state.The NWD2/HET-S system is one of several examples in which activation of cell death and host defense cascades relies on prion-like and/or amyloid polymerization mechanisms (13, 14). Activation of ASC (apoptosis-associated speck-like protein with a caspase recruitment domain) by the NLRP3 human NLR depends on a prion-like polymerization of the PYRIN domain and the HET-s-prion motif is able to substitute for the PYRIN signaling domain in NLRP3-dependent ASC activation (13). Another example is the mammalian RIP1/RIP3 kinase complex controlling necroptosis, assembled via short amyloid motifs termed RHIM (15). One downstream effector of the RIP1/RIP3 complex in necroptosis is the mixed lineage kinase domain-like protein (MLKL), which is a target of RIP3 (16). In fungi, we have identified several other gene clusters analogous to Nwd2/het-S in which an NLR and a putative cell death effector protein are encoded by adjacent genes and in which the N-terminal extension of the NLR protein is homologous to the C-terminal end of the effector (12, 17). One such proposed prion motif, termed PP, was found to be associated with HeLo domain proteins, lipases, and regulatory NLRs (12). Herein we characterize a protein identified in Chaetomium globosum bearing this motif, termed HELLP.     

From the Cover: Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode of plant odor reception and defense

Plants receive volatile compounds emitted by neighboring plants that are infested by herbivores, and consequently the receiver plants begin to defend against forthcoming herbivory. However, to date, how plants receive volatiles and, consequently, how they fortify their defenses, is largely unknown. In this study, we found that undamaged tomato plants exposed to volatiles emitted by conspecifics infested with common cutworms (exposed plants) became more defensive against the larvae than those exposed to volatiles from uninfested conspecifics (control plants) in a constant airflow system under laboratory conditions. Comprehensive metabolite analyses showed that only the amount of (Z)-3-hexenylvicianoside (HexVic) was higher in exposed than control plants. This compound negatively affected the performance of common cutworms when added to an artificial diet. The aglycon of HexVic, (Z)-3-hexenol, was obtained from neighboring infested plants via the air. The amount of jasmonates (JAs) was not higher in exposed plants, and HexVic biosynthesis was independent of JA signaling. The use of (Z)-3-hexenol from neighboring damaged conspecifics for HexVic biosynthesis in exposed plants was also observed in an experimental field, indicating that (Z)-3-hexenol intake occurred even under fluctuating environmental conditions. Specific use of airborne (Z)-3-hexenol to form HexVic in undamaged tomato plants reveals a previously unidentified mechanism of plant defense.In response to herbivory, plants emit specific blends of volatiles (1). When undamaged plants are exposed to volatiles from neighboring herbivore-infested plants, they begin to defend against the impending infestation of herbivores (2, 3). This so-called “plant–plant signaling” has been reported in several plant species (4). For example, a study on the expression profiles of defense-related genes when Arabidopsis was exposed to several volatiles, including green leaf volatiles and a monoterpene, showed that the manner of induction varied with the gene monitored or the volatile used, suggesting that the plant responses were specific to the individual volatile compound (5). Kost and Heil (6) reported that the secretion of extrafloral nectar (an alternative food for carnivores) in undamaged lima bean plants was enhanced by volatiles from infested conspecific plants; this reaction was specific to (Z)-3-hexenyl acetate. Recently, Kikuta et al. (7) showed that wound-induced volatile organic compounds from Chrysanthemum cinerariaefolium induced the biosynthesis of pyrethrins in volatile-exposed neighboring plants. In this plant–plant signaling system, a blend of five compounds at specific concentrations was essential for the pyrethrin biosynthesis in receiver plants.These previous studies on plant–plant signaling raise questions about how different airborne volatiles are received by undamaged neighboring plants. Tamogami et al. (8) reported that airborne (E)-nerolidol was metabolized by Achyranthes bidentata plants into (E)-4,8-dimethyl-1,3,7-nonatriene. However, the mechanisms involved in the reception of airborne (E)-nerolidol in plants remained unclear. To date, only the receptor for ethylene, ETR1, a typical histidine kinase involved in a two-component regulatory system, has been identified (9, 10); no information exists on receptors for other volatile compounds in plants. In this study, we conducted comprehensive analyses of metabolic changes in intact tomato plants (Solanum lycopersicum) exposed to volatiles emitted from conspecifics infested with common cutworm (CCW; Spodoptera litura) and also conducted bioassays and biochemical analyses. We report that (Z)-3-hexenol emitted from herbivore-infested tomato plants is used by undamaged plants to form a glycoside with defensive function against CCW.     

A 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase forms a stable complex with the catalytic subunit leading to markedly altered enzyme activity.

In an effort to clarify effects of specific protein-protein interactions on the properties of the dodecameric enzyme aspartate transcarbamoylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), we initiated studies of a simpler complex containing an intact catalytic trimer and three copies of a fragment from the regulatory chain. The partial regulatory chain was expressed as a soluble 9-kDa zinc-binding polypeptide comprising 11 amino acids encoded by the polylinker of pUC18 fused to the amino terminus of residues 84-153 of the regulatory chain; this polypeptide includes the zinc domain detected in crystallographic studies of the holoenzyme. In contrast to intact regulatory chains, the zinc-binding polypeptide is monomeric in solution because it lacks the second domain responsible for dimer formation and assembly of the dodecameric holoenzyme. The isolated 9-kDa protein forms a tight, zinc-dependent complex with catalytic trimer, as shown by the large shift in electrophoretic mobility of the trimer in nondenaturing polyacrylamide gels. Enzyme assays of the complex showed a hyperbolic dependence of initial velocity on aspartate concentration with Vmax and Km for aspartate approximately 50% lower than the values for free catalytic subunit. A mutant catalytic subunit containing the Lys-164----Glu substitution exhibited a striking increase in enzyme activity at low aspartate concentrations upon interaction with the zinc domain because of a large reduction in Km upon complex formation. These changes in functional properties indicate that the complex of the zinc domain and catalytic trimer is an analog of the high-affinity R ("relaxed") state of aspartate transcarbamoylase, as proposed previously for a transiently formed assembly intermediate composed of one catalytic and three regulatory subunits. Conformational changes at the active sites, resulting from binding the zinc-containing polypeptide chains, were detected by difference spectroscopy with trinitrophenylated catalytic trimers. Isolation of the zinc domain of aspartate transcarbamoylase provides a model protein for study of oligomer assembly, communication between dissimilar polypeptides, and metal-binding motifs in proteins.     

Conversion of aquaporin 6 from an anion channel to a water-selective channel by a single amino acid substitution

Aquaporin (AQP) 6 belongs to the aquaporin water channel family. Unlike other aquaporins, AQP6 functions not as a water channel but as an anion-selective channel. Single-channel analyses have shown AQP6 to flicker rapidly between closed and open status. The atomic structure of AQP1 and amino acid sequence alignments of the mammalian aquaporins reveal two well conserved glycine residues: Gly-57 in transmembrane helix (TM) 2 and Gly-173 in TM5 reside at the contact point where the two helices cross in human AQP1. Uniquely, all known mammalian orthologs of AQP6 have an asparagine residue (Asn-60) at the position corresponding to Gly-57. Here we show that a single residue substitution (N60G in rat AQP6) totally eliminates the anion permeability of AQP6 when expressed in Xenopus oocytes, but the N60G oocytes exhibit significantly higher osmotic water permeability under basal conditions. Replacement of the glycine at this site in AQP0, AQP1, and AQP2 blocked expression of the mutants at the oocyte plasma membrane. We propose that the asparagine residue at the contact point between TM2 and TM5 in AQP6 may function as a teeter board needed for rapid structural oscillations during anion permeation.     

Identification of strategies used to cope with chronic pain in older persons receiving primary care from a Veterans Affairs Medical Center

OBJECTIVES: To identify the strategies used by older persons to cope with chronic noncancer pain, determine the perceived effectiveness of the strategies, and ascertain factors associated with their use. DESIGN: Cross-sectional telephone survey. SETTING: Primary care practice located at a Veterans Affairs Medical Center in New England. PARTICIPANTS: Two hundred forty-five patients (aged 65-90) with chronic pain. MEASUREMENTS: Qualitative methods were used to ascertain participants' coping strategies, and their effectiveness was determined using a five-category response scale (1=not at all effective to 5=extremely effective). In multivariate analyses, associations between participants' demographic, medical, psychosocial, and pain characteristics and prevalent coping strategies were assessed. RESULTS: Participants had a mean age+/-standard deviation of 75+/-5.1; 84% were male. Overall, 240 (98%) participants had employed at least one coping strategy in the previous month; the mean number used per participant was 2.8+/-1.4. Prevalent coping strategies included analgesic medications (used by 187/240=78% participants), exercise (35%), cognitive methods (37%), religious activities (21%), and activity restriction (20%). The proportion of participants who used a given strategy and rated it quite a bit or extremely effective exceeded 50% for only five of the 15 identified strategies. Women were more likely than men to use cognitive coping methods (odds ratio (OR)=3.2, 95% confidence interval (CI)=1.5-6.8) and religious activities (OR=2.6, 95% CI=1.2-5.7). Participants with chronic pain due to a musculoskeletal cause were more likely to use analgesic medications than those with pain due to all other causes (OR=3.2, 95% CI=1.6-6.4), whereas those with trauma-related pain were less likely to use exercise than those with pain due to all other causes (OR=0.2, 95% CI=0.1-0.7). CONCLUSION: Older primary care patients use a broad variety of coping strategies to cope with chronic pain. Studies are needed to confirm these findings in other older populations and to characterize the longitudinal effects of the coping strategies. Given the finding that the perceived effectiveness of most coping strategies was modest, efforts to increase their effectiveness in older persons are indicated.     

Identification of the Human Papillomavirus Genotypes,According to the Human Immunodeficiency Virus Status in a Cohort of Women from Maputo,Mozambique

Background: Human papillomavirus (HPV) infection is now a well-established cause of cervical cancer and other anogenital cancers. An association between human immunodeficiency virus (HIV) infection and higher HPV incidence and prevalence are commonly reported. This study was conducted to demonstrate HPV prevalence, genotypes and its characteristics, according to the HIV status in women from Maputo in Mozambique. Methods: A total of 233 participants with ages ranging from fourteen to forty-five were included. Cervical samples were collected, DNA extracted, and HPV genotyping was performed using the HPV Direct Flow CHIP Kit. Results: In total, 177 HIV-negative and 56 HIV-positive women were included in the analysis. The overall HPV prevalence was 63% and was significantly higher among HIV-positive women (79% versus 58% among HIV-negative women; p = 0.005). The prevalence of multiple HPV type infections was 32%. High-risk HPV types 52, 68, 35, 18 and 16 were the most frequent. A higher proportion of HIV-positive women had multiple HPV types compared with HIV-negative women. Conclusions: This study demonstrated a high prevalence of HPV in the study cohort. HIV-positive women were identified as having the highest HPV prevalence and infection with multiple HPV types across all ages. High-risk genotypes were the most commonly found.     

Spontaneous T-cell proliferation in the non-obese diabetic mouse to a peptide from the unique class II MHC molecule, I-Ag7, which is also protective against the development of autoimmune diabetes

Aims/hypothesis. Major histocompatibility complex class II molecules present antigenic peptides to T-cells and have an important role in T-cell thymic education. The mechanism by which major histocompatibility complex alleles confer a high genetic risk for autoimmune diabetes is not known. One hypothesis is that during positive thymic selection, the peripheral T-cell repertoire is modelled by major histocompatibility complex-restricted presentation of self major histocompatibility complex molecule-derived peptides, some of which mimic tissue autoantigens. The sequence similarity between a known T-cell epitope of glutamic acid decarboxylase-65, 509:VPPSLRTLED and the non-obese diabetic mouse class II major histocompatibility complex molecule I-A^g7 86:VPTSLRRLEQ is consistent with this. Methods. We measured spontaneous proliferation of peripheral T-cells from non-obese diabetic mice and other, non-diabetes-prone strains, to the I-A^{g7 86–101} and glutamic acid decarboxylase-65^509–524 peptides, binding of these peptides to intact I-A^g7 and assessed the effect of tolerance induction on diabetes development, by injecting young non-obese diabetic mice with high doses of peptide. Results. T-cells from non-obese diabetic, but not other mice strains, spontaneously proliferate to the I-A^{g7 86–101} and glutamic acid decarboxylase-65^509–524 peptides, but not control peptides. Both test peptides bind I-A^g7. Tolerance induction prolongs diabetes-free survival in non-obese diabetic mice when either the I-A^{g7 86–101} or glutamic acid decarboxylase-65^509–524 peptide, but not control peptide, is used. Conclusion/interpretation. A peptide from the unique class II major histocompatibility complex, diabetes-susceptibility molecule, I-A^g7, presented by I-A^g7 is a target of T-cell responses in diabetes-prone non- obese diabetic mice and tolerance induction against the peptide offers appreciable protection against the development of diabetes. [Diabetologia (1999) 42: 560–565] Received: 18 September 1998 and in final revised form: 22 December 1998     

Treatment of myelodysplastic syndromes with retinoic acid and 1 alpha-hydroxy-vitamin D3 in combination with low-dose ara-C is not superior to ara-C alone. Results from a randomized study. The Scandinavian Myelodysplasia Group (SMG)

63 evaluable patients with myelodysplastic syndromes (MDS) and 15 with acute myelogenous leukemia (AML) were randomized between low-dose ara-C (arm A) and low dose ara-C in combination with 13-cis-retinoic acid (13-CRA) and 1 alpha-hydroxy-vitamin D3 (1 alpha D3) (arm B). 69 patients were evaluable and 18 (26.1%) responded to therapy. The addition of 13-CRA and 1 alpha D3 had no positive influence on survival of the patients, remission rates or duration of remissions. 12/27 patients in arm A and 6/29 patients in arm B progressed from MDS to AML during the course of the study (p = 0.0527). Arm B gave significantly more side-effects than arm A (p = 0.005). Therapeutic effects of 13-CRA and 1 alpha D3 on MDS is not supported by this study. However, an inhibiting effect on AML development in some MDS subgroups cannot be excluded.