Simple bi- and tricyclic inhibitors of human steroid 5alpha-reductase

A number of tricyclic thiolactams, bicyclic lactams, and bicyclic thiolactams have been prepared and evaluated in vitro as inhibitors of types 1 and 2 steroid 5alpha-reductase. The tricycles with an 8-chloro substituent in the C-ring are nM (IC50) inhibitors of type 1 steroid 5alpha-reductase (SR). In all the cases studied, lactams are more potent than the corresponding thiolactams. Activity against type 2 SR is greatly enhanced by a styryl (or azo) substituent on the aryl ring of the tri- and bicycles and also a related tricyclic aryl acid.     

Shared role for differentially methylated domains of imprinted genes

For most imprinted genes, a difference in expression between the maternal and paternal alleles is associated with a corresponding difference in DNA methylation that is localized to a differentially methylated domain (DMD). Removal of a gene's DMD leads to a loss of imprinting. These observations suggest that DMDs have a determinative role in genomic imprinting. To examine this possibility, we introduced sequences from the DMDs of the imprinted Igf2r, H19, and Snrpn genes into a nonimprinted derivative of the normally imprinted RSVIgmyc transgene, created by excising its own DMD. Hybrid transgenes with sequences from the Igf2r DMD2 were consistently imprinted, with the maternal allele being more methylated than the paternal allele. Only the repeated sequences within DMD2 were required for imprinting these transgenes. Hybrid transgenes containing H19 and Snrpn DMD sequences and ones containing sequences from the long terminal repeat of a murine intracisternal A particle retrotransposon were not imprinted. The Igf2r hybrid transgenes are comprised entirely of mouse genomic DNA and behave as endogenous imprinted genes in inbred wild-type and mutant mouse strains. These types of hybrid transgenes can be used to elucidate the functions of DMD sequences in genomic imprinting.     

Functional classes of bronchial mucosa genes that are differentially expressed in asthma

Background  

Asthma pathogenesis and susceptibility involves a complex interplay between genetic and environmental factors. Their interaction modulates the airway inflammation and remodelling processes that are present even in mild asthma and governs the appearance and severity of symptoms of airway hyperresponsiveness. While asthma is felt to develop as the result of interaction among many different genes and signalling pathways, only a few genes have been linked to an increased risk of developing this condition.     

Analysis of the steroid binding domain of rat steroid 5alpha-reductase (isozyme-1): the steroid D-ring binding domain of 5alpha-reductase.

We have previously shown that the photoactive 4-azasteroid, [1,2 3H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5alpha-androst an-17beta-carboxamide is an effective probe of rat steroid 5alpha-reductase (isozyme-1) (5alphaR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5alphaR-1 activity were ultraviolet (UV)-photolyzed with [3H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5alphaR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55-56 min. Rechromatography of this fraction using a modified gradient (elution 54-55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15-18 of the 5alphaR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an approximately 12-fold increase in the Km for testosterone, whereas the Km for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.     

Differences in steroid 5alpha-reductase iso-enzymes expression between normal and pathological human prostate tissue.

We studied the expression level and cell-specific expression patterns of 5alpha-reductase (5alpha-R) types 1 and 2 iso-enzymes in human hyperplastic and malignant prostate tissue by semi-quantitative RT-PCR and in situ hybridisation analyses. In situ hybridisation established that 5alpha-R1 mRNA is preferentially expressed by epithelial cells and little expressed by stromal cells whereas 5alpha-R2 mRNA is expressed by both epithelium and stroma. Semi-quantitative RT-PCR has been performed on total RNA from different zones of normal prostate, BPH tissues and liver. We found that 5alpha-R1 and 5alpha-R2 mRNAs expression was near the same in all zones of normal prostate. In BPH tissue, 5alpha-R1 and 5alpha-R2 mRNAs expression was slightly but significantly increased, when it was compared to the levels recorded for normal prostate. In cancer samples, 5alpha-R1 mRNA expression was higher than in normal and hyperplastic prostate but the level of 5alpha-R2 mRNA was not statistically different from that observed in the different zones of normal prostate. In liver, 5alpha-R2 mRNA level was similar to that measured in BPH but 5alpha-R1 mRNA expression was ten times higher. The increase observed in 5alpha-R isoenzymes expression in BPH tissue could play an important role in the pathogenesis and/or maintenance of the disease.     

MethylAction: detecting differentially methylated regions that distinguish biological subtypes

DNA methylation differences capture substantial information about the molecular and gene-regulatory states among biological subtypes. Enrichment-based next generation sequencing methods such as MBD-isolated genome sequencing (MiGS) and MeDIP-seq are appealing for studying DNA methylation genome-wide in order to distinguish between biological subtypes. However, current analytic tools do not provide optimal features for analyzing three-group or larger study designs. MethylAction addresses this need by detecting all possible patterns of statistically significant hyper- and hypo- methylation in comparisons involving any number of groups. Crucially, significance is established at the level of differentially methylated regions (DMRs), and bootstrapping determines false discovery rates (FDRs) associated with each pattern. We demonstrate this functionality in a four-group comparison among benign prostate and three clinical subtypes of prostate cancer and show that the bootstrap FDRs are highly useful in selecting the most robust patterns of DMRs. Compared to existing tools that are limited to two-group comparisons, MethylAction detects more DMRs with strong differential methylation measurements confirmed by whole genome bisulfite sequencing and offers a better balance between precision and recall in cross-cohort comparisons. MethylAction is available as an R package at http://jeffbhasin.github.io/methylaction.     

Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled

In previous work, we identified a Saccharomyces cerevisiae glycogen synthase gene, GSY1, which codes for an 85-kDa polypeptide present in purified yeast glycogen synthase (Farkas, I., Hardy, T.A., DePaoli-Roach, A.A., and Roach, P.J. (1990) J. Biol. Chem. 265, 20879-20886). We have now cloned another gene, GSY2, which encodes a second S. cerevisiae glycogen synthase. The GSY2 sequence predicts a protein of 704 residues, molecular weight 79,963, with 80% identity to the protein encoded by GSY1. Amino acid sequences obtained from a second polypeptide of 77 kDa present in yeast glycogen synthase preparations matched those predicted by GSY2. GSY1 resides on chromosome VI, and GSY2 is located on chromosome XII. Disruption of the GSY1 gene produced a strain retaining about 85% of wild type glycogen synthase activity at stationary phase, while disruption of the GSY2 gene yielded a strain with only about 10% of wild type enzyme activity. The level of glycogen synthase activity in yeast cells disrupted for GSY1 increased in stationary phase, whereas the activity remained at a constant low level in cells disrupted for GSY2. Disruption of both genes resulted in a viable haploid that totally lacked glycogen synthase activity and was defective in glycogen deposition. In conclusion, yeast expresses two forms of glycogen synthase with activity levels that behave differently in the growth cycle. The GSY2 gene product appears to be the predominant glycogen synthase with activity linked to nutrient depletion.     

Evidence that the two sucrose synthetase genes in maize are related

Summary Evidence is presented that the sucrose synthetase coding sequence at the Shrunken locus is distantly related to the sequence encoding a second, minor sucrose synthetase present in maize endosperm. Three doubly mutant sh bz strains lacking at least part of the Sh coding sequence produce an antigenically cross-reactive protein having the same electrophoretic mobility as the Sh-encoded, 92-kD sucrose synthetase monomer, but differing in primary structure. An mRNA is present in endosperm of mutants with deletions at the Sh locus that is weakly homologous to the Sh coding sequence and encodes a 92-kD protein precipitable with antiserum to sucrose synthetase. We conclude that the genes encoding the two different proteins are related.     

Alu repeated DNAs are differentially methylated in primate germ cells.

A significant fraction of Alu repeats in human sperm DNA, previously found to be unmethylated, is nearly completely methylated in DNA from many somatic tissues. A similar fraction of unmethylated Alus is observed here in sperm DNA from rhesus monkey. However, Alus are almost completely methylated at the restriction sites tested in monkey follicular oocyte DNA. The Alu methylation patterns in mature male and female monkey germ cells are consistent with Alu methylation in human germ cell tumors. Alu sequences are hypomethylated in seminoma DNAs and more methylated in a human ovarian dysgerminoma. These results contrast with methylation patterns reported for germ cell single-copy, CpG island, satellite, and L1 sequences. The function of Alu repeats is not known, but differential methylation of Alu repeats in the male and female germ lines suggests that they may serve as markers for genomic imprinting or in maintaining differences in male and female meiosis.     

Evidence for a Ustilago maydis steroid 5alpha-reductase by functional expression in Arabidopsis det2-1 mutants

We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction with its host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5alpha-steroid reductases. Udh1 differs from those of known 5alpha-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulum localization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants, which display a dwarf phenotype due to a mutation in the 5alpha-steroid reductase encoding DET2 gene. det2-1 mutant plants expressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-type Columbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosette leaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reduced light conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth in det2-1 plants. Our results demonstrate the existence of a 5alpha-steroid reductase encoding gene in fungi and suggest a common ancestor between fungal, plant, and mammalian proteins.     

The mechanism of rat epididymal 4-ene steroid 5 alpha-reductase

Epididymal nuclear 4-ene steroid 5 alpha-reductase catalyses the bisubstrate reaction between testosterone and NADPH to produce 5 alpha-dihydrotestosterone (DHT) and NADP+. Previous studies from this laboratory have demonstrated that the 4-ene steroid 5 alpha-reductase reaction proceeds through the direct transfer of protons from NADPH to testosterone, and that while the product DHT does not affect 4-ene steroid 5 alpha-reductase activity, NADP+ is a potent inhibitor of this enzyme. In the present studies we have investigated the mechanism of 4-ene steroid 5 alpha-reductase with respect to the binding of the substrates, testosterone and NADPH. Kinetic analyses revealed that testosterone does not alter the Kmapp for NADPH, and that NADPH does not alter the Kmapp for testosterone. These findings excluded the possibility that the mechanism of 4-ene steroid 5 alpha-reductase is of the ping-pong variety, and that the sequential addition of both substrates is required before any products are released. The lack of change in Kmapp, observed for either substrate, further suggests that both testosterone and NADPH are able to bind to the free enzyme, negating the possibility that substrate addition occurs in an ordered manner. Indeed the kinetic profiles are entirely consistent with the mechanism of 4-ene steroid 5 alpha-reductase being a rapid equilibrium random sequential process in which the binding of the first substrate has no affect on the binding of the second. Mean values for the dissociation constants, Ktestosterone and KNADPH, were 200 nmol/l and 50 nmol/l, respectively. These findings, coupled with those from earlier studies, suggest that the mechanism of epididymal nuclear 4-ene steroid 5 alpha-reductase is a rapid equilibrium random bireactant process, with the possible dead-end complex: testosterone-4-ene steroid 5 alpha-reductase-NADP+.     

Densely methylated sequences that are preferentially localized at telomere-proximal regions of human chromosomes

We have constructed a library of densely methylated DNA sequences from human blood DNA by selecting fragments with a high affinity for a methyl-CpG binding domain (MBD) column. PCR analysis of the library confirmed the presence of known densely methylated CpG island sequences. Analysis of random clones, however, showed that the library was dominated by sequences whose G+C content and CpG frequency were intermediate between those of bulk genomic DNA and bona fide CpG islands. When human chromosomes were probed with the library by fluorescent in situ hybridisation (FISH), the predominant sites of labelling were at terminal regions of many chromosomes, approximately corresponding to T-bands. Analysis of the methylation status of random clones indicated that all were heavily methylated at CpGs in blood DNA, but many were under-methylated in sperm DNA. Lack of methylation in germ cells may reduce CpG depletion at some sub-terminal sequences and result in a high density of methyl-CpG when these regions become methylated in somatic cells.     

Solubilization of human prostatic 5 alpha-reductase

A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.     

Screening for differentially methylated genes among human colorectal cancer tissues and normal mucosa by microarray chip

High density DNA methylation microarrays were used to study the differences of gene methylation level in six pairs of colorectal cancer (CRC) and adjacent normal mucosa. We analyzed the profile of methylated genes by NimbleGen Microarray and the biologic functions by NIH-NAVID. In addition, preliminary validation studies were done in six pairs of samples by MSP (methylation-specific PCR). A total of 4,644 genes had a difference in methylation levels. Among them 2,296 were hypermethylated (log2ratio > 1), 2,348 genes were hypomethylated (log2ratio < ?1), in which 293 hypermethylated and 313 hypomethylated genes were unmapped according to the NIH-NAVID. All these genes were randomly distributed on all the chromosomes. However, chromosome 1 contained the most of the hypermethylated genes (232 genes), followed by chromosome 19 (149 genes), chromosome 11 (144 genes), chromosome 2 (141 genes), chromosomes 3 (127 genes). Through the analysis of the statistics, There were 2 hypermethylated/3 hypomethylated genes involved in six pairs of samples simultaneously, followed by 10/14 in five samples, 34/37 in four samples, 101/113 in three samples, 341/377 in two samples, 1,808/1,804 in one sample. According to gene ontology analysis, some physiological processes play important roles in the cell division and the development of tumor, such as apoptosis, DNA repair, immune, cell cycle, cell cycle checkpoint, cell adhesion and invasion etc. Through Preliminary validation, there were two genes (St3gal6, Opcml) in thirty top-ranking genes shown hypermethylated in six pairs of CRC and adjacent normal mucosa. Conclusions High density DNA methylation microarrays is an effective method for screening aberrantly methylated genes in CRC. The methylated genes should be further studied for diagnostic or prognostic markers for CRC.     

Evidence that cytosine residues within 5'-CCTGG-3' pentanucleotides can be methylated in human DNA independently of the methylating system that modifies 5'-CG-3' dinucleotides

In contrast to the complex sequence specificities of the prokaryotic DNA methylating systems, the mammalian machinery identified thus far methylates cytosine residues within the context of a 5'-CG-3' dinucleotide. To explore the possibility that cytosine residues that do not precede guanine may be independently methylated in mammalian DNA, we have examined a region of the human myogenic gene, Myf-3, which is not targeted by the methylating system that methylates 5'-CG-3' dinucleotides. Our investigations have revealed cytosine methylation within the 5'-CCTGG-3' pentanucleotides specified by the 0.8-kb Myf-3 probe. We have also found that in DNA from neoplastic cells, in which 5'-CG-3' dinucleotides within Myf-3 become abnormally hypermethylated, cytosine residues within 5'-CCTGG-3' pentanucleotides are not methylated. Moreover, methylation of 5'-CCTGG-3' pentanucleotides was not detected within the closely related Myf-4 gene, which is normally 5'-CG-3' hypermethylated. These findings indicate the existence of a system that methylates 5'-CCTGG-3' pentanucleotides independently of the system that methylates cytosine residues within 5'-CG-3' dinucleotides. It is possible that the 5'-CCTGG-3' methylating system influences the fate of foreign integrated DNA.     

3,3-diphenylpentane skeleton as a steroid skeleton substitute: novel inhibitors of human 5alpha-reductase 1

We designed and synthesized novel type 1 5alpha-reductase inhibitors by using 3,3-diphenylpentane skeleton as a substitute for the usual steroid skeleton. 4-(3-(4-(N-Methylacetamido)phenyl)pentan-3-yl)phenyl dibenzylcarbamate (11k) is a competitive 5alpha-reductase inhibitor with the IC(50) value of 0.84 microM.     

Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate

In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled.     

Evidence that two forms of choline acetyltransferase are differentially expressed in subclasses of enteric neurons

Cholinergic neurons have been revealed in the enteric nervous system by functional and biochemical studies but not by antibodies that provide excellent localisation of the synthesising enzyme, choline acetyltransferase (ChAT), in the central nervous system. In order to determine whether a newly described peripheral form of ChAT (pChAT) is a ChAT enzyme of enteric neurons, we have compared pChAT distribution with that of the common form of ChAT, cChAT, by quantitative analysis of the co-localisation of pChAT and cChAT with other neurochemical markers in enteric neurons of the guinea-pig ileum. We found classes of neuron with strong pChAT immunoreactivity (IR) and others with strong cChAT-IR. In myenteric ganglia, strong pChAT-IR was in calbindin-positive intrinsic primary afferent neurons (IPANs), whereas cChAT-IR of these neurons was weak. Calretinin neurons were immunoreactive for cChAT, but not pChAT. Only 4% of nitric oxide synthase (NOS) neurons (possibly interneurons) were pChAT-immunoreactive, similar to observations with cChAT. NOS-immunoreactive inhibitory motor neurons stained with neither cChAT nor pChAT antisera. In the submucosal ganglia, pChAT-IR was strongly expressed in IPANs (identified by cytoplasmic staining for the neuronal nuclear marker, NeuN) and in neuropeptide Y (NPY)-immunoreactive secretomotor neurons, but not in calretinin-immunoreactive neurons. cChAT-IR occurred weakly in submucosal IPANs and also labelled NPY- and calretinin-immunoreactive neurons. Submucosal vasoactive-intestinal-peptide-immunoreactive neurons (non-cholinergic secretomotor neurons) were not reactive for either form of ChAT.