Regulatory networks underlying mycorrhizal development delineated by genome-wide expression profiling and functional analysis of the transcription factor repertoire of the plant symbiotic fungus Laccaria bicolor.

BACKGROUND: Ectomycorrhizal (ECM) fungi develop a mutualistic symbiotic interaction with the roots of their host plants. During this process, they undergo a series of developmental transitions from the running hyphae in the rhizosphere to the coenocytic hyphae forming finger-like structures within the root apoplastic space. These transitions, which involve profound, symbiosis-associated metabolic changes, also entail a substantial transcriptome reprogramming with coordinated waves of differentially expressed genes. To date, little is known about the key transcriptional regulators driving these changes, and the aim of the present study was to delineate and functionally characterize the transcription factor (TF) repertoire of the model ECM fungus Laccaria bicolor. RESULTS: We curated the L. bicolor gene models coding for transcription factors and assessed their expression and regulation in Poplar and Douglas fir ectomycorrhizae. We identified 285 TFs, 191 of which share a significant similarity with known transcriptional regulators. Expression profiling of the corresponding transcripts identified TF-encoding fungal genes differentially expressed in the ECM root tips of both host plants. The L. bicolor core set of differentially expressed TFs consists of 12 and 22 genes that are, respectively, upregulated and downregulated in symbiotic tissues. These TFs resemble known fungal regulators involved in the control of fungal invasive growth, fungal cell wall integrity, carbon and nitrogen metabolism, invasive stress response and fruiting-body development. However, this core set of mycorrhiza-regulated TFs seems to be characteristic of L. bicolor and our data suggest that each mycorrhizal fungus has evolved its own set of ECM development regulators. A subset of the above TFs was functionally validated with the use of a heterologous, transcription activation assay in yeast, which also allowed the identification of previously unknown, transcriptionally active yet secreted polypeptides designated as Secreted Transcriptional Activator Proteins (STAPs). CONCLUSIONS: Transcriptional regulators required for ECM symbiosis development in L. bicolor have been uncovered and classified through genome-wide analysis. This study also identifies the STAPs as a new class of potential ECM effectors, highly expressed in mycorrhizae, which may be involved in the control of the symbiotic root transcriptome.

Anti-estrogenic activity of a human resveratrol metabolite.

BACKGROUND AND AIMS: Resveratrol, the most investigated dietary compound in studies aimed at linking wine consumption to human health, is an extremely minor component of this beverage and it is generally studied in vitro as the unconjugated aglycone at concentrations largely exceeding those found in the human circulatory system after dietary intake. Moreover, following intestinal absorption, trans-resveratrol and its glucoside, which are naturally present in wine and other food sources, are converted to sulphate and glucuronide metabolites. An estrogenic activity has previously been documented for resveratrol, yet nothing is known about the activity of its blood-circulating metabolic derivatives. METHODS AND RESULTS: Using a yeast two-hybrid detection system relying on the interaction between the ligand-binding domain of the human oestrogen receptors α and ß and the human coactivator Tif2, we have systematically examined the oestrogen agonist and antagonist activities of the two main resveratrol forms present in planta (trans-resveratrol and trans-resveratrol-3-O-glucoside) and of the three main metabolites found in human plasma (trans-resveratrol-3-O-sulphate, trans-resveratrol-3-O-glucuronide and trans-resveratrol-4'-O-glucuronide). Only resveratrol-3-O-sulphate was found to display a fairly strong and oestrogen receptor α-preferential antagonistic activity, which was confirmed in a human breast adenocarcinoma cell line containing a luciferase reporter gene under the control of an oestrogen-responsive promoter. CONCLUSIONS: We show, for the first time, that resveratrol-3-O-sulphate, but neither of its metabolites, is endowed with anti-estrogenic activity and how human metabolism of phenolic substances plays a pivotal role in modulating their biological effect.

Safety of pregnancy after breast cancer in young women with hormone receptor-positive disease: a systematic review and meta-analysis.

BACKGROUND: Despite increasing evidence on the safety of pregnancy after anticancer treatments in breast cancer survivors, many physicians and patients remain concerned about a potential risk of pregnancy specifically in the case of hormone receptor-positive breast cancer. MATERIALS AND METHODS: A systematic literature search of Medline, Embase and Cochrane library with no language or date restriction up to 31 March 2023 was carried out. To be included, articles had to be retrospective and prospective case-control and cohort studies as well as clinical trials comparing survival outcomes of premenopausal women with or without a pregnancy after prior diagnosis of hormone receptor-positive breast cancer. Disease-free survival (DFS) and overall survival (OS) were the outcomes of interest. Pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated. Study protocol is registered in PROSPERO (n. CRD42023394232). RESULTS: Out of 7796 screened studies, 8 were eligible to be included in the final analysis. A total of 3805 patients with hormone receptor-positive invasive early breast cancer were included in these studies, of whom 1285 had a pregnancy after breast cancer diagnosis. Median follow-up time ranged from 3.8 to 15.8 years and was similar in the pregnancy and non-pregnancy cohorts. In three studies (n = 987 patients) reporting on DFS, no difference was observed between patients with and those without a subsequent pregnancy (HR 0.96, 95% CI 0.75-1.24, P = 0.781). In the six studies (n = 3504 patients) reporting on OS, patients with a pregnancy after breast cancer had a statistically significant better OS than those without a pregnancy (HR 0.46, 95% CI 0.27-0.77, P < 0.05). CONCLUSIONS: This systematic review and meta-analysis of retrospective cohort studies provides updated evidence that having a pregnancy in patients with prior history of hormone receptor-positive invasive early breast cancer appears safe without detrimental effect on prognosis.

Genomic profiling of carbohydrate metabolism in the ectomycorrhizal fungus Tuber melanosporum.

• Primary carbohydrate metabolism plays a special role related to carbon/nitrogen exchange, as well as metabolic support of fruiting body development, in ectomycorrhizal macrofungi. In this study, we used information retrieved from the recently sequenced Tuber melanosporum genome, together with transcriptome analysis data and targeted validation experiments, to construct the first genome-wide catalogue of the proteins supporting carbohydrate metabolism in a plant-symbiotic ascomycete. • More than 100 genes coding for enzymes of the glycolysis, pentose phosphate, tricarboxylic acid, glyoxylate and methylcitrate pathways, glycogen, trehalose and mannitol metabolism and cell wall precursor were annotated. Transcriptional regulation of these pathways in different stages of the T. melanosporum lifecycle was investigated using whole-genome oligoarray expression data together with real-time reverse transcription-polymerase chain reaction analysis of selected genes. • The most significant results were the identification of methylcitrate cycle genes and of an acid invertase, the first enzyme of this kind to be described in a plant-symbiotic filamentous fungus. • A subset of transcripts coding for trehalose, glyoxylate and methylcitrate enzymes was up-regulated in fruiting bodies, whereas genes involved in mannitol and glycogen metabolism were preferentially expressed in mycelia and ectomycorrhizas, respectively. These data indicate a high degree of lifecycle stage specialization for particular branches of carbohydrate metabolism in T. melanosporum.

A class III transcription factor composed of RNA.

It is generally assumed that the machinery that transcribes genes is composed entirely of polypeptides. However, in vitro transcription by silkworm RNA polymerase III requires a transcription factor that is not a polypeptide. This component, TFIIIR, is distinct from the previously identified transcription components: RNA polymerase III, and the accessory factors TFIIIA, TFIIIB, TFIIIC, and TFIIID. The newly discovered TFIIIR is a macromolecule that appears to be composed of RNA. It is resistant to heat, detergent, phenol, protease, and deoxyribonuclease, but it is sensitive to alkali and ribonuclease.

Retinoic acid synthesis in the somatic cells of rat seminiferous tubules.

At physiological plasma concentrations, retinoic acid (RA) cannot cross the blood-testis barrier formed by Sertoli and peritubular cells, and it is thought to be mainly synthesized in situ through the oxidation of retinol. We have thus examined the in vitro RA biosynthetic capacity of cultured Sertoli and peritubular cells isolated from the seminiferous tubules of prepubertal rats, using holo-cellular retinol binding protein (CRBP) as a substrate. Although both somatic cell types contain CRBP and retinoic acid nuclear receptors, RA synthesis was only detected with Sertoli cell subcellular fractions. Most of the RA synthesizing activity of these cells is contributed by a microsomal-cytosolic system that shares many functional similarities with a RA biosynthetic pathway originally identified in rat liver. RA synthesis is maximal at a time of postnatal life (20 days) preceding meiotic cell accumulation and remains nearly constant thereafter. The unique ability of Sertoli cell subcellular fractions to support RA formation from holoCRBP, along with the observed age-dependent modulation of this activity, indicate that Sertoli cells represent the main site of intratubular RA production and that they may play a key role in controlling RA-dependent processes within the seminiferous tubule.

Transfer RNA gene redundancy and translational selection in Saccharomyces cerevisiae.

A total of 274 transfer RNA genes, representing the entire tRNA gene set of the yeast Saccharomyces cerevisiae, has been extracted from the whole genome sequence of this organism using a dedicated search algorithm (Pol3scan). All tRNA genes were assigned to 42 classes of distinct codon specificity. Accordingly, four deviations from previously proposed rules for third position wobble pairing in yeast, three G:U and one A:I codon-anticodon pairings, were found to be required to account for the reading of 61 coding triplets. The gene copy number for individual tRNA species, which ranges from one to 16, correlates well with both the frequency of codon occurrence in a sample of 1756 distinct protein coding sequences (r = 0.82) and the previously measured intracellular content of 21 tRNA species. A close link between tRNA gene redundancy and the overall amino acid composition of yeast proteins was also observed. Regression analysis values for individual protein coding sequences proved to be effective descriptions of the translational selective pressure operating on a particular gene. A significantly stronger co-adaptation between codon choice and tRNA gene copy number was observed in highly expressed genes. These observations strongly support the notion that intracellular tRNA levels in normally growing yeast cells are mainly determined by gene copy number, which, along with codon choice, is the key parameter acted upon by translational selection.

Crystal structure of the trigonal form of human plasma retinol-binding protein at 2.5 A resolution.

The three-dimensional structures of the liganded and unliganded forms of human plasma retinol binding protein (RBP) in the trigonal crystal form have been solved at 2.5 A resolution. The final model of RBP complexed with retinol (holoRBP, space group R3, a = b = 104.0 A, c = 74.4 A) has a crystallographic R factor of 0.176 for 9652 reflections. The unliganded form, obtained through a purification procedure which included steps based on hydrophobic interaction chromatography, crystallized isomorphously with holoRBP and its structure has been refined to an R factor of 0.190 for 9614 reflections. The structure of the trigonal holo protein is quite similar to that of the orthorhombic form: the root-mean-square deviation of all the equivalent alpha-carbons in the two chains is 0.53 A. The structural comparison between the liganded and unliganded forms of RBP in the crystal did not reveal gross conformational changes. The most significant difference between the two forms of the protein is a conformational change involving residues from 34 to 37. In this region, the movements of side-chains of Leu35 and Phe36 are most noticeable. In particular, in the unliganded form the side-chain ring of the latter residue is in the place previously occupied by the alcoholic moiety of retinol. Our data are consistent with a model in which a region comprising these residues and at least part of the opening of the beta-barrel is involved in the recognition between RBP and transthyretin. In the case of the unliganded form, the central cavity, that is occupied by the vitamin in the two human crystalline holoRBPs, is filled by electron density that, at the present resolution, we interpret as solvent.

TFIIIC-independent in vitro transcription of yeast tRNA genes.

The most peculiar transcriptional property of eukaryotic tRNA genes, as well as of other genes served by RNA polymerase III, is their complete dependence on the intragenic interaction platform provided by transcription factor IIIC (TFIIIC) for the productive assembly of the TBP-containing initiation factor TFIIIB. The sole exception, in yeast, is the U6 RNA gene, which is able to exploit a TATAAATA element, 30 bp upstream of the transcription start site, for the TFIIIC-independent assembly of TFIIIB. To find out whether this extragenic core promoter organization and autonomous TFIIIB assembly capacity are unique features of the U6 gene or also apply to other genes transcribed by RNA polymerase III, we scanned the 5'-flanking regions (up to position -100) of the entire tRNA gene set of Saccharomyces cerevisiae searching for U6-like TATA motifs. Four tRNA genes harboring such a sequence motif around position -30 were identified and found to be transcribed in vitro by a minimal system only composed of TFIIIB and RNA polymerase III. In this system, start site selection is not at all affected by the absence of TFIIIC, which, when added, significantly stimulates transcription by determining an increase in the number, rather than in the efficiency of utilization, of productive initiation complexes. A specific TBP-TATA element interaction is absolutely required for TFIIIC-independent transcription, but the nearby sequence context also contributes to the efficiency of autonomous TFIIIB assembly. The existence of a TFIIIB assembly pathway leading to the faithful transcription of natural eukaryotic tRNA genes in the absence of TFIIIC provides novel insights into the functional flexibility of the eukaryotic tRNA gene transcription machinery and on its evolution from an ancestral RNA polymerase III system relying on upstream, TATA- centered control elements.

Crystallization of human plasma apo-retinol-binding protein.

Crystals of three forms of human plasma apo-retinol-binding protein have been obtained using the procedure described for the holoprotein. The apoprotein was prepared by a novel method, which uses hydrophobic interaction and immobilized dye chromatography. The three forms were separated by fast protein liquid chromatography. All of the crystals are isomorphous and diffract to 2.5 A resolution. These crystals will be useful for studies of the mechanism of binding of retinol to its carrier using X-ray diffraction techniques.

Crystallization and preliminary X-ray data of human plasma retinol-binding protein.

Crystals of human plasma retinol-binding protein have been obtained from 4.5 M-NaCl buffered at pH 6.8 with 20 mM-cacodylate. The crystals are trigonal with space group R3 and unit cell dimensions, referred to the hexagonal system, a = b = 104.2 A and c = 74.5 A. The crystals diffract to a resolution of 2.0 A.

Deformed erythrocytes in muscular dystrophies.

Because of previous reports of a possible correlation between echinocytogenesis and muscular dystrophies, we investigated the time-dependent development of echinocytes in the blood of normal subjects, patients, and healthy carries of Duchenne dystrophy. There was a quantitatively significant increase of echinocytes in patients and carriers.

Carrier detection in Duchenne muscular dystropy.

We investigated the number of echinocytes, the serum hemopexin level, and spectrin band II phosphorylation in the blood of normal subjects, patients, and carriers of Duchenne dystrophy. The patients and carriers exhibited quantitatively significant differences with respect to controls.

The isolation by self forming gradients of Percoll of plasma membrane enriched fractions from bovine retinal pigment epithelium.

A method is described for the preparation of plasma membrane enriched fractions from bovine retinal pigment epithelium (RPE) by means of differential centrifugation followed by the use of self forming gradients of Percoll. A detailed analysis of the distribution of organelle specific markers (nuclei, mitochondria, lysosomes, endoplasmic reticulum, cytosol) in the different fractions is presented. Comparison of 125I-wheat germ agglutinin (WGA) binding with more conventional plasma membrane enzyme markers demonstrates that also in RPE radiolabeled lectin is a specific and extremely sensitive marker to follow quantitatively the distribution of outer cell membranes. Results of 125I-WGA displacement experiments indicate that plasma membranes are mostly (90%) composed of right side out vesicles or sheets. On the basis of 125I-WGA radioactivity the overall recovery of plasma membranes was about 10% and purification over 15 fold. NADH cytochrome c reductase activity, which is shown to be a specific marker for endoplasmic reticulum in retinal pigment epithelium, has been utilized to evaluate microsomal contamination of the plasma membrane preparation.

Fearful expressions enhance recognition memory: electrophysiological evidence.

Facial expressions play a key role in affective and social behavior. However, the temporal dynamics of the brain responses to emotional faces remain still unclear, in particular an open question is at what stage of face processing expressions might influence encoding and recognition memory. To try and answer this question we recorded the event-related potentials (ERPs) elicited in an old/new recognition task. A novel aspect of the present design was that whereas faces were presented during the study phase with either a happy, fearful or neutral expression, they were always neutral during the memory retrieval task. The ERP results showed three main findings: An enhanced early fronto-central positivity for faces encoded as fearful, both during the study and the retrieval phase. During encoding subsequent memory (Dm effect) was influenced by emotion. At retrieval the early components P100 and N170 were modulated by the emotional expression of the face at the encoding phase. Finally, the later ERP components related to recognition memory were modulated by the previously encoded facial expressions. Overall, these results suggest that face recognition is modulated by top-down influences from brain areas associated with emotional memory, enhancing encoding and retrieval in particular for fearful emotional expressions.

Selection at the wobble position of codons read by the same tRNA in Saccharomyces cerevisiae.

The transfer RNA gene complement of Saccharomyces cerevisiae was utilized for a whole-genome analysis of the deviation from a neutral usage of pyrimidine-ending cognate codons, that is, codons read by a single tRNA species having either inosine or guanosine as the first anticodon base. Mutational pressure at the wobble position was estimated from the base composition of the noncoding portion of the yeast genome. The selective pressure for translational efficiency was inferred from the degree of codon adaptation to tRNA gene redundancy and from mRNA abundance data derived from yeast transcriptome analysis. Amino acid conservation in orthologous comparisons with wholly sequenced microbial genomes was used to estimate translational accuracy requirements. A close correspondence was observed between the usage of wobble position pyrimidines and the frequency predicted by mutational bias. However, in the case of four cognate pairs (Gly: ggu/ggc; Asn: aau/aac; Phe: uuu/uuc; Tyr: uau/ uac) all read by guanosine-starting anticodons, we found evidence for a strong selective pressure driven by translational efficiency. Only for the glycine pair, wobble pyrimidine choice also appears to fulfill a translational accuracy requirement. Wobble pyrimidine selection is strictly related to the number of hydrogen bonds formed by alternative cognate codons: whenever a different number of hydrogen bonds can be formed at the wobble position, there is selection against six- or nine-hydrogen-bonded codon-anticodon pairs. Our results indicate that an intrinsic codon preference, critically dependent on the stability of codon-anticodon interaction and mainly reflecting selection for the optimization of translational efficiency, is built into the translational apparatus.

Vitamin A uptake from retinol-binding protein in a cell-free system from pigment epithelial cells of bovine retina. Retinol transfer from plasma retinol-binding protein to cytoplasmic retinol-binding protein with retinyl-ester formation as the intermediate step.

We have investigated the steps by which retinol, released from plasma retinol-binding protein (RBP), enters the cells and is accumulated for the most part as a retinyl-ester, only a small fraction of it being present as a complex with cytoplasmic retinol-binding protein (CRBP). For this purpose, we have developed a cell-free system composed of plasma membrane-enriched fractions from bovine retinal pigment epithelium which selectively incorporates exogenous vitamin A when presented as a retinol-RBP complex. Upon incubation in the presence of [3H]retinol-RBP, isolated plasma membrane fractions take up and esterify retinol. A 4-fold reduction of total vitamin A incorporation is observed in conditions which specifically inhibit retinyl-ester formation, thus indicating that the two processes of retinol uptake and esterification are functionally coupled. Evidence is presented that retinol bound to a plasma membrane receptor sharing functional and structural similarities with CRBP is the actual substrate for esterification. Vitamin A accumulation seems to require retinol esterification to allow the recycling of a limited number of free, plasma membrane-associated, retinol receptors. Mobilization of retinol stored as a membrane-bound retinyl-ester is mediated by a membrane-associated hydrolase activity selectively controlled by the level of apo-CRBP which acts as a carrier for the released retinol. Up to 90% of membrane-bound vitamin A is released upon incubation in the presence of apo-CRBP (11 microM) with concomitant formation of retinol-CRBP. The overall process, in which retinol never needs to leave its binding proteins, allows the accumulation of vitamin A in the form of a membrane-bound retinyl-ester and its regulated mobilization as a retinol-CRBP complex.

Serum antiproteases and respiratory diseases of the horse.

The serum antiprotease (AAT) levels are reported in healthy horses and horses with respiratory diseases. Of the methods used, only the STIC test seemed to give useful results; this test showed variations in horses with respiratory diseases, especially in horses with acute alveolar pulmonary emphysema.

Selective inactivation of two components of the multiprotein transcription factor TFIIIB in cycloheximide growth-arrested yeast cells.

Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.