Corepressor binding to progesterone and glucocorticoid receptors involves the activation function-1 domain and is inhibited by molybdate

Corepressors are known to interact via their receptor interaction domains (RIDs) with the ligand binding domain in the carboxyl terminal half of steroid/nuclear receptors. We now report that a portion of the activation function-1 domain of glucocorticoid receptors (GRs) and progesterone receptors (PRs), which is the major transactivation sequence, is necessary but not sufficient for corepressor [nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptor (SMRT)] RID binding to GRs and PRs in both mammalian two-hybrid and coimmunoprecipitation assays. Importantly, these two receptor sequences are functionally interchangeable in the context of GR for transactivation, corepressor binding, and corepressor modulatory activity assays. This suggests that corepressors may act in part by physically blocking portions of receptor activation function-1 domains. However, differences exist in corepressor binding to GRs and PRs. The C-terminal domain of PRs has a higher affinity for corepressor than that of GRs. The ability of some segments of the coactivator TIF2 to competitively inhibit corepressor binding to receptors is different for GRs and PRs. With each receptor, the cell-free binding of corepressors to ligand-free receptor is prevented by sodium molybdate, which is a well-known inhibitor of receptor activation to the DNA-binding state. This suggests that receptor activation precedes binding to corepressors. Collectively, these results indicate that corepressor binding to GRs and PRs involve both N- and C-terminal sequences of activated receptors but differ in ways that may contribute to the unique biological responses of each receptor in intact cells.     

Evolutionary analysis of the segment from helix 3 through helix 5 in vertebrate progesterone receptors

The interaction between helix 3 and helix 5 in the human mineralocorticoid receptor [MR], progesterone receptor [PR] and glucocorticoid receptor [GR] influences their response to steroids. For the human PR, mutations at Gly-722 on helix 3 and Met-759 on helix 5 alter responses to progesterone. We analyzed the evolution of these two sites and the rest of a 59 residue segment containing helices 3, 4 and 5 in vertebrate PRs and found that a glycine corresponding to Gly-722 on helix 3 in human PR first appears in platypus, a monotreme. In lamprey, skates, fish, amphibians and birds, cysteine is found at this position in helix 3. This suggests that the cysteine to glycine replacement in helix 3 in the PR was important in the evolution of mammals. Interestingly, our analysis of the rest of the 59 residue segment finds 100% sequence conservation in almost all mammal PRs, substantial conservation in reptile and amphibian PRs and divergence of land vertebrate PR sequences from the fish PR sequences. The differences between fish and land vertebrate PRs may be important in the evolution of different biological progestins in fish and mammalian PR, as well as differences in susceptibility to environmental chemicals that disrupt PR-mediated physiology.     

Molecular investigations of pathogenesis-related Bet v 1 homologues in Passiflora (Passifloraceae)

The major birch pollen allergen, Bet v 1, responsible for allergic reactions in many areas of the world, is homologous to a large number of pathogenesis-related proteins (PRs), identified as PR10. As part of a long-range investigation of these types of proteins and of evolution in Passiflora,DNA sequences from eight Bet v 1 homologue isoforms were obtained from five species of this genus in Brazil, and their sequences compared among themselves and with 30 others from 8 different species, classified in different taxonomic units. The objective was a first characterization of these PRs in wild passionflowers, and their use for evolutionary and applied investigations. High interspecific, but low intraspecific variability was observed, as expected from multigenic families subjected to concerted evolution. The relationships obtained both within Passiflora and between it and seven other genera probably best reflect functional similarities than evolutionary history.     

Recommendations for naming plant pathogenesis-related proteins

Pathogenesis-related proteins (abbreviated PRs) are defined as plant proteins that are induced in pathological or related situations. We propose a unifying nomenclature for PRs based on their grouping into families sharing amino acid sequences, serological relationship, and/or enzymatic or biological activity. The nomenclature classifies novel proteins identified by electrophoresis or chromatography along with those established by other workers. The previously proposed system of the five well-established families from tobacco is extended to accommodate a further six families. Families are indicated by arabic numerals and individual members are named by lower case letters in the order in which they are described. Additional rules are proposed to deal with forms containing more than a single polypeptide and as yet unclassified PRs. PR genes whose sequences are conserved but whose designations are not based on function are to be designated Ypr in accordance with the recommendations of the Commission on Plant Gene Nomenclature.     

Molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.     

Health risk assessment from dermal exposure to pesticide residues on vegetables among greengrocers in fresh market,Bangkok, Thailand

Pesticide residues (PRs) in market vegetables have been reported regularly. Greengrocers may be exposed to several sorts of PRs on vegetables through hand contact. Health risk assessment from occupational exposure to PRs on vegetables is particularly concerning. This study was conducted among 91 vegetable vendors at a large fresh market in Bangkok. Hand wipe samples were collected in the dry season to extract and analyze PRs including organophosphates (OPs), pyrethroids (PYs), and carbamates (CAs) by gas chromatography (GC-FPD/GC-μECD) and liquid chromatography (LC–MS). The results showed that all wipe samples contained OPs, PYs, and CAs, mainly chlorpyrifos (0.01–0.14 µg/hands) and cypermethrin (0.42–11.64 µg/hands). The frequently detected PRs were aldicarb (87.2%), carbofuran (69.2%), permethrin (63.7%), and profenofos (60.0%). At 99th percentile values of PR exposure, average daily dose was 2.42 × 10^?5 mg/kg/d and hazard index did not exceed the acceptable level (0.287). Glove wearing, hand washing, and work-related factors were significantly associated with PRs on hands after adjusted for gender (p-value < 0.05). Greengrocers may therefore not be at risk from PRs on vegetables and exposure via hands during their work. However, these findings suggest that proper personal hygiene practices among greengrocers should be considered to prevent them from PR exposure and potential health risks.     

鸡隐孢子虫在安徽的首次定种及感染情况的数学分析

本次调研共采集合肥地区五个大型鸡场的150个新鲜鸡粪便样品,结果在62个粪样中检出了隐孢子虫,总阳性检出率为1.3％。因五个鸡场均发现有隐孢子虫,说明该虫感染在合肥地区较为普遍。不过,各鸡场之间的阳性检出率从10.0%到83.3％高低不等。本研究同时剖检了其中四个鸡场的38只病死鸡尸,发现隐孢子虫的有11只,死鸡感染率为28.9％。通过鉴定,首次认为安徽省有火鸡孢子虫（C.Meleagridis）和贝氏隐孢子虫（C.baileyi)两个虫种。统计分析得知：隐孢子虫的阳性检出率与鸡群的日龄呈一种极显著的抛物线形相关关系;与球虫感染相比,虽然阳性检出率略低,但差异不显著,均可达到同（第）一位（原虫）感染水平;不过,二者之间不存在有直线相关关系。     

Toward a universal inhibitor of retroviral proteases: comparative analysis of the interactions of LP-130 complexed with proteases from HIV-1, FIV, and EIAV.

One of the major problems encountered in antiviral therapy against AIDS is the emergence of viral variants that exhibit drug resistance. The sequences of proteases (PRs) from related retroviruses sometimes include, at structurally equivalent positions, amino acids identical to those found in drug-resistant forms of HIV-1 PR. The statine-based inhibitor LP-130 was found to be a universal, nanomolar-range inhibitor against all tested retroviral PRs. We solved the crystal structures of LP-130 in complex with retroviral PRs from HIV-1, feline immunodeficiency virus, and equine infectious anemia virus and compared the structures to determine the differences in the interactions between the inhibitor and the active-site residues of the enzymes. This comparison shows an extraordinary similarity in the binding modes of the inhibitor molecules. The only exceptions are the different conformations of naphthylalanine side chains at the P3/P3' positions, which might be responsible for the variation in the Ki values. These findings indicate that successful inhibition of different retroviral PRs by LP-130 is achieved because this compound can be accommodated without serious conformational differences, despite the variations in the type of residues forming the active-site region. Although strong, specific interactions between the ligand and the enzyme might improve the potency of the inhibitor, the absence of such interactions seems to favor the universality of the compound. Hence, the ability of potential anti-AIDS drugs to inhibit multiple retroviral PRs might indicate their likelihood of not eliciting drug resistance. These studies may also contribute to the development of a small-animal model for preclinical testing of antiviral compounds.     

Building a retinal mosaic: cell-fate decision in the fly eye

Across the animal kingdom, color discrimination is achieved by comparing the outputs of photoreceptor cells (PRs) that have different spectral sensitivities. Much remains to be understood about how the pattern of these different PRs is generated and maintained. The Drosophila eye has long provided a beautiful system for understanding various aspects of retinal-cell differentiation. Recent progress in this field is revealing that a highly ordered series of events, involving cell-cell communication, localized signaling and stochastic choices, creates a complex mosaic of PRs that is reminiscent of the human retina. Notably, several of the factors used in generating the retinal mosaic of the fruitfly have corresponding functions in vertebrates that are likely to have similar roles.     

Structures of signaling complexes of lipid receptors S1PR1 and S1PR5 reveal mechanisms of activation and drug recognition

Sphingosine-1-phosphate (S1P) is an important bioactive lipid molecule in cell membrane metabolism and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, physiological homeostasis, and pathogenic processes in various organs. S1PRs are lipid-sensing receptors and are therapeutic targets for drug development, including potential treatment of COVID-19. Herein, we present five cryo-electron microscopy structures of S1PRs bound to diverse drug agonists and the heterotrimeric Gi protein. Our structural and functional assays demonstrate the different binding modes of chemically distinct agonists of S1PRs, reveal the mechanical switch that activates these receptors, and provide a framework for understanding ligand selectivity and G protein coupling.Subject terms: Cryoelectron microscopy, Lipid signalling     

Altered gag polyprotein cleavage specificity of feline immunodeficiency virus/human immunodeficiency virus mutant proteases as demonstrated in a cell-based expression system

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.     

Identification,purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).     

Induction of long-lasting depolarization in medioventral medulla neurons by cholinergic input from the pedunculopontine nucleus.

Stimulation of the pedunculopontine nucleus (PPN) is known to induce changes in arousal and postural/locomotor states by activation of such descending targets as the caudal pons and the medioventral medulla (MED). Previously, PPN stimulation was reported to induce prolonged responses (PRs) in intracellularly recorded caudal pontine neurons in vitro. The present study used intracellular recordings in semihorizontal slices from rat brain stem (postnatal days 12-21) to determine responses in MED neurons following PPN stimulation. One-half (40/81) of MED neurons showed PRs after PPN stimulation. MED neurons with PRs had shorter duration action potential, longer duration afterhyperpolarization, and higher amplitude afterhyperpolarization than non-PR MED neurons. PR MED neurons were significantly larger (568 +/- 44 microm2) than non-PR MED neurons (387 +/- 32 microm2). The longest mean duration PRs and maximal firing rates during PRs were induced by PPN stimulation at 60 Hz compared with 10, 30, or 90 Hz. The muscarinic cholinergic agonist carbachol induced depolarization in all PR neurons tested, and the muscarinic cholinergic antagonist scopolamine reduced or blocked carbachol- and PPN stimulation-induced PRs in all MED neurons tested. These findings suggest that PPN stimulation-induced PRs may be due to activation of muscarinic receptor-sensitive channels, allowing MED neurons to respond to a transient, frequency-dependent depolarization with long-lasting stable states. PPN stimulation appears to induce PRs in large MED neurons using parameters known best to induce locomotion.     

Differential regulation of arylalkylamine N-acetyltransferase activity in chicken retinal ganglion cells by light and circadian clock

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48?h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30-60?min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects. (Author correspondence: mguido@fcq.unc.edu.ar ).     

Lack of relationship between the expression of hsp27 heat shock estrogen receptor-associated protein and estrogen receptor or progesterone receptor status in male breast carcinoma

Estrogen, through estrogen receptors (ERs), may regulate the synthesis of progesterone receptors (PRs) and of a heat shock estrogen receptor-associated protein (hsp27). In female breast carcinoma (FBC) both proteins serve as surrogate indicators for the presence of functional ERs. In addition, the expression of these proteins was related to other prognostic indicators of value in female breast tumours. Endocrine disorders, hormone therapy and altered estrogen metabolism have been associated with the development of male breast cancer (MBC), suggesting that evaluation of the expression of ER, PR and hsp27 might improve our understanding of the biology of this tumour. ER and PR status and hsp27 expression were evaluated by immunohistochemistry in 16 primary MBC patients. The interrelationships between these parameters were established and compared with the clinicopathological data on the tumours. Ten (56%) MBC patients were ER-positive, 69% were PR-positive and all samples were hsp27-positive. Our series of MBC patients showed a positive correlation between ERs and PRs, however there was a lack of correlation between hsp27 and ERs or PRs. MBCs did not exhibit any correlation between the biomarkers studied and known prognostic indicators for females (e.g. Scarff-Bloom-Richardson (SBR) or modified SBR (MSBR) grade, T stage, lymph node status). This is the first published series reporting the incidence of hsp27 in MBC. The lack of association between the expression of ERs and hsp27 found in MBC differs from the results reported for FBC, moreover the expression of ERs, PRs or hsp27 did not correlate with the clinicopathological parameters that have prognostic value in females. Although the data were obtained from a relatively small sample population, our findings suggest that MBC and FBC are biologically different tumours with respect to the expression of the studied proteins.     

Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease

Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV-1 PR), were cocrystallized with the C2-symmetric inhibitor, TL-3. The mutants of FIV PR were designed to replace residues involved in enzyme-ligand interactions by the corresponding HIV-1 PR residues at the structurally equivalent position. TL-3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3'), small differences are evident in the conformation of the Phe side chains of TL-3 at the P1 and P1' positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL-3 in HIV-1 PR and correlate with a significant improvement in the inhibition constants of TL-3 with the two mutant FIV PRs. Large differences between the HIV-1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL-3 at P4 and P4'. In HIV-1 PR:TL-3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV-1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4' positions. These findings provide a structural explanation of the observed Ki values for TL-3 with the different PRs and will further assist in the development of improved inhibitors.     

A primary response with an initial negative wave

In the anterosuperior and posterosuperior portions of the ectosylvian gyrus of dogs small foci were found in which clicks evoked responses with parameters close to those of primary responses (PRs), but with an initial negative wave. An analysis of PRs with an initial wave of different polarity was carried out by studying the reproducibility of the response during an increase in the frequency of stimulation and during the action of various drugs. PRs with an initial negative ("negative" focus) and positive (auditory area AI) waves were found to be reproduced when the frequency of stimulation was 20–30 Hz, whereas the negative phase of the classical PR disappeared when the stimulation frequency reached 10–15 Hz. The polarity of the response in these foci was unchanged after injection of a lethal dose of nembutal, but the negative phase of the classical PR disappeared during moderately deep anesthesia. Strychnine, on the other hand, considerably increased the amplitude of the negative wave in the positive-negative complex, but the initial negative potential was only very slightly and temporarily increased, and it was lost in the subsequent strychnine spike. GABA inhibited both the PR with initial negative wave and also the negative phase of the classical PR. The results suggest that PRs with different polarities of their initial wave differ in origin. The results of experiments with GABA indicate that PRs with an initial negative wave arise through excitation of apical dendrites.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 5, pp. 488–496, September–October, 1970.     

Synthetic "interface" peptides alter dimeric assembly of the HIV 1 and 2 proteases.

Retroviral proteases are obligate homodimers and play an essential role in the viral life cycle. Dissociation of dimers or prevention of their assembly may inactivate these enzymes and prevent viral maturation. A salient structural feature of these enzymes is an extended interface composed of interdigitating N- and C-terminal residues of both monomers, which form a four-stranded beta-sheet. Peptides mimicking one beta-strand (residues 95-99), or two beta-strands (residues 1-5 plus 95-99 or 95-99 plus 95-99) from the human immunodeficiency virus 1 (HIV1) interface were shown to inhibit the HIV1 and 2 proteases (PRs) with IC50's in the low micromolar range. These interface peptides show cognate enzyme preference and do not inhibit pepsin, renin, or the Rous sarcoma virus PR, indicating a degree of specificity for the HIV PRs. A tethered HIV1 PR dimer was not inhibited to the same extent as the wild-type enzymes by any of the interface peptides, suggesting that these peptides can only interact effectively with the interface of the two-subunit HIV PR. Measurements of relative dissociation constants by limit dilution of the enzyme show that the one-strand peptide causes a shift in the observed Kd for the HIV1 PR. Both one- and two-strand peptides alter the monomer/dimer equilibrium of both HIV1 and HIV2 PRs. This was shown by the reduced cross-linking of the HIV2 PR by disuccinimidyl suberate in the presence of the interface peptides. Refolding of the HIV1 and HIV2 PRs with the interface peptides shows that only the two-strand peptides prevent the assembly of active PR dimers. Although both one- and two-strand peptides seem to affect dimer dissociation, only the two-strand peptides appear to block assembly. The latter may prove to be more effective backbones for the design of inhibitors directed toward retroviral PR dimerization in vivo.