Are both distinct epithelial and stromal cells molecular analysis from phyllodes tumors versus fibroadenoma components affected in breast fibroepithelial progression?

PURPOSE: To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell. METHODS: Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression. RESULTS: All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples. CONCLUSIONS: Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions.

Are both distinct epithelial and stromal cells molecular analysis from phyllodes tumors versus fibroadenoma components affected in breast fibroepithelial progression?

Purpose: To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell. Methods: Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression. Results: All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples. Conclusions: Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions.

Exome sequencing and targeted gene panels: a simulated comparison of diagnostic yield using data from 158 patients with rare diseases.

Next-generation sequencing (NGS) has altered clinical genetic testing by widening the access to molecular diagnosis of genetically determined rare diseases. However, physicians may face difficulties selecting the best diagnostic approach. Our goal is to estimate the rate of possible molecular diagnoses missed by different targeted gene panels using data from a cohort of patients with rare genetic diseases diagnosed with exome sequencing (ES). For this purpose, we simulated a comparison between different targeted gene panels and ES: the list of genes harboring clinically relevant variants from 158 patients was used to estimate the theoretical rate of diagnoses missed by NGS panels from 53 different NGS panels from eight different laboratories. Panels presented a mean rate of missed diagnoses of 64% (range 14%-100%) compared to ES, representing an average predicted sensitivity of 36%. Metabolic abnormalities represented the group with highest mean of missed diagnoses (86%), while seizure represented the group with lowest mean (46%). Focused gene panels are restricted in covering select sets of genes implicated in specific diseases and they may miss molecular diagnoses of rare diseases compared to ES. However, their role in genetic diagnosis remains important especially for well-known genetic diseases with established genetic locus heterogeneity.

Mutational Portrait of Lung Adenocarcinoma in Brazilian Patients: Past, Present, and Future of Molecular Profiling in the Clinic.

Objectives: Approximately 60% of lung adenocarcinomas (LAs) carry mutations that can guide treatment with tyrosine-kinase inhibitors (TKI) and other targeted therapies. Data on activating mutations in EGFR and other tyrosine-kinase receptor (TKR) genes in highly admixed populations, such as that of Brazil, are scarce. In this study, we comprehensively analyzed the actionable alteration profile of LA in Brazilian patients. Materials and Methods: EGFR driver mutation data were collected from a large Brazilian LA cohort covering an 8-year period of molecular testing in a single institution. Tests were performed using three distinct methods, and demographic and histopathological data were analyzed. For a subset of patients, driver mutations in KRAS, NRAS, and BRAF and gene fusions involving TKR genes (before TKI treatment) and EGFR T790M (after TKI treatment) were assessed. Results: EGFR mutations were detected in 25% of 1,316 LAs evaluated, with exon 19 deletions and exon 21 L858R TKI sensitizing mutations representing 72.5% of all mutations. Mutation rates were higher in women and non-smokers (p < 0.001). Next-generation sequencing was very sensitive, with a lower rate of inconclusive results compared with Sanger sequencing and pyrosequencing. EGFR/RAS/BRAF hotspot gene panels were applied in 495 LA cases and detected oncogenic mutations in 51.3% of samples, most frequently in EGFR (22.4%) and KRAS (26.9%). In subgroups of 36 and 35 patients, gene fusions were detected in 11.1% of tumors and EGFR T790M resistance mutations were detected in 59% of plasma samples from patients previously treated with TKI, respectively. Conclusion: This report provides the first comprehensive actionable alteration portrait of LA in Brazil. The high rate of actionable alterations in EGFR and other driver genes in LA reinforces the need to incorporate TKI guided by molecular diagnostics into clinical routines for patients in both public and private healthcare systems.

A comparison between SOLiD 5500XLand Ion Torrent PGM-derived miRNA expression profiles in two breast cell lines.

Next-generation sequencing (NGS) platforms allow the analysis of hundreds of millions of molecules in a single sequencing run, revolutionizing many research areas. NGS-based microRNA studies enable expression quantification in unprecedented scale without the limitations of closed-platforms. Yet, whereas a massive amount of data produced by these platforms is available, comparisons of quantification/discovery capabilities between platforms are still lacking. Here we compare two NGS-platforms: SOLiD and PGM, by evaluating their microRNA identification/quantification capabilities using two breast-derived cell-lines. A high expression correlation (R2 > 0.9) was achieved, encompassing 97% of the miRNAs, and the few discrepancies in miRNA counts were attributable to molecules that have very low expression. Quantification divergences indicative of artefactual representation were seen for 14 miRNAs (higher in SOLiD-reads) and another 10 miRNAs more abundant in PGM-data. An inspection of these revealed an increased and statistically significant count of uracyls and uracyl-stretches for PGM-enriched miRNAs, compared to SOLiD and to the miRBase. In parallel, adenines and adenine-stretches were enriched for SOLiDderived miRNA reads. We conclude that, whereas both platforms are overall consistent and can be used interchangeably for microRNA expression studies, particular sequence features appear to be indicative of specific platform bias, and their presence in microRNAs should be considered for database-analyses.

BRCA1 deficiency is a recurrent event in early-onset triple-negative breast cancer: a comprehensive analysis of germline mutations and somatic promoter methylation.

PURPOSE: BRCA1 germline mutation is closely associated with triple-negative breast cancer. BRCA deficiency leads to impaired DNA repair and tumor development, and understanding this deficiency, in both hereditary and sporadic scenarios, is of great clinical and biological interest. Here, we investigated germline or somatic events that might lead to BRCA1 impairment in triple-negative breast cancer. We also analyzed the clinical implications associated with BRCA deficiency. METHODS: Next-generation sequencing for the BRCA1/2 genes and multiplex ligation-dependent probe amplification (MLPA) for the BRCA1 gene were performed for mutation screening. A customized bisulfite next-generation sequencing approach was used for assessing BRCA1 promoter methylation status in tumor tissue. RESULTS: A total of 131 triple-negative cases were assessed, and germline pathogenic variants were detected in 13.0% of all cases and in 26% of cases diagnosed in young women. Most germline pathogenic variants (88.2%) occurred in the BRCA1 gene. BRCA1 promoter hypermethylation was detected in 20.6% of tumors; none of these tumors were in BRCA1/2 pathogenic variant carriers. BRCA1 impairment by either germline or somatic events was significantly more frequent in young women (55% in those ≤ 40 years; 33% in those 41-50 years; 22% in those > 50 years of age) and associated with better overall and disease-free survival rates in this group of patients. CONCLUSIONS: BRCA1 deficiency was recurrent in early-onset triple-negative breast cancer in Brazilian patients and associated with improved survival. With the new treatment modalities being investigated, including poly (ADP-ribose)-polymerase (PARP) inhibitor therapy, our results suggest that a significant proportion of young women with this subtype of tumor might benefit from PARP inhibitor treatment, which warrants further investigation.

Anti-cardiolipin and anti-ß2-glycoprotein I antibodies: normal reference ranges in northwestern Italy.

Laboratory tests for anticardiolipin antibodies (aCL) and anti-ß2glycoprotein I antibodies (a-ß2GPI) face problems common to many autoantibody assays: the lack of a reference standard and the need for each laboratory to assess assay-specific cut-off values. The aims of the study were to evaluate the reference range upper limits (99th percentile) used for aCL and a-ß2GPI in the northwest of Italy and to investigate the analytical performances of these assays with the newly obtained reference ranges. We assayed aCL and a-ß2GPI in 104 serum samples from patients without a history of thrombosis, pregnancy morbidity, tumours, infections and/or autoimmune diseases (30 males and 74 non-pregnant females). We tested all the commercial assays available in our regions (i.e. Orgentec Diagnostika, Aesku Diagnostics and Inova Diagnostics ELISA; CliA Zenit-RA and EliA Phadia Laboratory Systems). A further 30 serum samples, including 10 from healthy subjects, 10 from antiphospholipid syndrome (APS) patients and 10 from septic patients were assessed to investigate the analytical performance of the obtained cut-off limits. Reference range upper limits obtained with the commercial kits differ among assays and from the values reported by the manufacturer. Moreover, normal reference ranges calculated for IgG and IgM aCL differed from the arbitrary selected laboratory classification values suggested in the guidelines of 40 GPL and MPL.

Down-regulation of ANAPC13 and CLTCL1: Early Events in the Progression of Preinvasive Ductal Carcinoma of the Breast.

Alterations in the gene expression profile in epithelial cells during breast ductal carcinoma (DC) progression have been shown to occur mainly between pure ductal carcinoma in situ (DCIS) to the in situ component of a lesion with coexisting invasive ductal carcinoma (DCIS-IDC) implying that the molecular program for invasion is already established in the preinvasive lesion. For assessing early molecular alterations in epithelial cells that trigger tumorigenesis and testing them as prognostic markers for breast ductal carcinoma progression, we analyzed, by reverse transcription-quantitative polymerase chain reaction, eight genes previously identified as differentially expressed between epithelial tumor cells populations captured from preinvasive lesions with distinct malignant potential, pure DCIS and the in situ component of DCIS-IDC. ANAPC13 and CLTCL1 down-regulation revealed to be early events of DC progression that anticipated the invasiveness manifestation. Further down-regulation of ANAPC13 also occurred after invasion appearance and the presence of the protein in invasive tumor samples was associated with higher rates of overall and disease-free survival in breast cancer patients. Furthermore, tumors with low levels of ANAPC13 displayed increased copy number alterations, with significant gains at 1q (1q23.1-1q32.1), 8q, and 17q (17q24.2), regions that display common imbalances in breast tumors, suggesting that down-regulation of ANAPC13 contributes to genomic instability in this disease.

Vertebrados da Estação Ecológica Serra Geral do Tocantins: faunística, biodiversidade e conservação no Cerrado brasileiro / Vertebrate fauna of Estação Ecológica Serra Geral do Tocantins: biodiversity and conservation in the Brazilian Cerrado hotspot

Inventários e estudos faunísticos detalhados sobre vertebrados são uma das fontes mais relevantes de dados para interpretações de padrões detalhados de diversidade biológica. Dados básicos e de boa qualidade sobre faunística são ainda mais urgentes em regiões pouco estudadas e sob intensa ameaça antrópica, tais como a região do Cerrado, um dos 34 hotspots globais para a conservação da biodiversidade. Apresentamos aqui uma síntese dos resultados dos inventários de vertebrados na Estação Ecológica Serra Geral do Tocantins (~716.000 ha), a segunda maior unidade de conservação em todo o Cerrado. Foram registradas 450 espécies de vertebrados na EESGT e entorno imediato, incluindo 17 espécies ameaçadas, 50 espécies endêmicas do Cerrado e 11 espécies com distribuição potencialmente restrita. Do total de espécies amostradas, 180 são novos registros para a região do Jalapão. Ao menos 12 espécies amostradas foram consideradas potenciais espécies novas, das quais quatro foram descritas recentemente, a partir do material obtido no inventário. Os resultados evidenciam que a EESGT é uma das mais importantes áreas protegidas no Brasil central, contribuindo para a persistência de espécies ameaçadas, dependentes dos últimos grandes blocos contínuos de vegetação nativa de Cerrado. Nossos resultados indicam ainda que a conservação da EESGT e suas principais subunidades é crucial para a representatividade do sistema de áreas protegidas do Cerrado, protegendo potenciais endemismos restritos que aliam alta vulnerabilidade intrínseca e valor como indicadores de padrões e processos biogeográficos formadores da rica e cada vez mais ameaçada fauna Neotropical.

Basic taxonomic and distributional data on vertebrates are one of the most useful and reliable sources of information for conservation planning. Biological data are even more relevant in rich and highly threatened regions such as the Brazilian Cerrado, one of the least studied global biodiversity hotspots. Herein we provide a summary of the results of a vertebrate survey at Estação Ecológica Serra Geral do Tocantins (~716.000 ha), the second largest protected area in the Cerrado region. We recorded 450 species in EESGT and surroundings, including 17 threatened species, 50 Cerrado endemics and 11 potential restricted-range species. Our results also added 180 new vertebrate records for the Jalapão region. At least 12 species were considered potential undescribed taxa; four of these were recently described based on specimens obtained in the present study. Our results indicate that EESGT is among the most biologically relevant protected areas in the Cerrado. Proper management will favor the persistence of threatened vertebrates dependent on the last remaining large blocks of pristine Cerrado savannas. Moreover, EESGT and its major biological subunits contribute decisively to the representativeness of the reserve system in the Cerrado, conserving presumed narrow endemics with high intrinsic vulnerability and high potential value as indicators of biogeographic processes of diversification in rich and complex Neotropical biotas.

Genotype, phenotype and hormonal levels correlation in non-classical congenital adrenal hyperplasia.

Non-classical congenital adrenal hyperplasia (NCAH) is a morbid condition sustained by the reduced function of one of the enzymes involved in the adrenal steroid biosynthesis pathway, mainly the 21-hydroxylase. Different degrees of enzyme activity impairment determine different clinical pictures, with childhood or post-pubertal onset. The aim of this study was to evaluate the relationship between genotype, phenotype, and adrenal hormonal levels in a group of 66 patients affected by NCAH attending outpatient pediatric or endocrinological Clinics. Our findings show that age at pubarche/menarche was significantly younger, height SD score) and Δ bone age-chronological age were significantly higher in patients with a more severe enzyme activity impairment, while cutaneous androgenization and menstrual irregularities in post-pubertal girls were not related to the grading of genotype.

Correlation between genotype and hormonal levels in heterozygous mutation carriers and non-carriers of 21-hydroxylase deficiency.

Congenital adrenal hyperplasia, both in its classic (CCAH) and non-classic form (NCAH), is a morbid condition sustained by the absent or reduced function of one of the enzymes involved in cortisol biosynthesis - mainly 21 hydroxylase - associated with different levels of clinical androgenization. In a wide group of relatives of patients affected by CCAH and NCAH (no.=222) and healthy volunteers (no.=30), a clinical, hormonal and genetic evaluation was performed in order to differentiate between the condition of heterozygous mutation carrier and non-carrier of any among 21-hydroxylase gene (CYP21) mutations. This study shows that clinical presentation and basal 17α-hydroxyprogesterone (17α-OHP) are not able to differentiate between heterozygous carriers and non-carriers, whereas 17α-OHP value after ACTH bolus is significantly different between heterozygous carriers and non-carriers: p<0.001 with a cut-off value of 3 ng/ml (90% sensitivity and 74,3% specificity). Moreover, our data indicate that 17α-OHP response to ACTH may be a useful tool to select subjects for genetic analysis.

Directly observed therapy (DOT) is not the entire answer: an operational cohort analysis.

SETTING: New Jersey Medical School National Tuberculosis Center-Lattimore Clinic, a TB Clinic for an inner city population of Newark, New Jersey, USA. OBJECTIVE: Directly observed therapy (DOT) is the recommended standard of TB care. Recent reports suggest that DOT may not be any better than self-administered therapy (SAT). To quantify the impact of different levels of SAT, DOT, and active case management on outcomes of TB treatment at our location, we reviewed the outcomes of six TB patient-cohorts from Newark between 1 January 1994 and 31 December 1996. STUDY DESIGN: A retrospective cohort study of the outcomes of 343 tuberculosis patients treated during the years 1994-1996. The three treatment strategies were 1) self-administered with occasional selective directly observed therapy, 2) universal directly observed therapy alone (universal DOT), and 3) universal DOT with nurse case management (NCM). RESULTS: The first two cohorts who began treatment during the transition may have received more than one treatment strategy. However, universal DOT did not significantly improve the TB treatment completion rates of Cohort 2 over SAT therapy with selective DOT given to Cohort 1. Universal DOT with NCM, Cohorts 3, 4, 5, and 6, significantly increased the TB treatment completion rates by three to six times. A cohort-specific step-wise reduction in duration of treatment from a median of 11.6-7.5 months and an increase in completion rates from 57-81% resulted. The most desirable and optimal (shortest) duration of treatment completion coincided with the application of universal DOT combined with NCM.

[Prolonged electroencephalographic and video monitoring an experience from a military Chilean hospital]. / Monitorización electroencefalográfica mediante video prolongado. Experiencia del Hospital Militar de Santiago. 1991-1996.

BACKGROUND: Cerebral activity must be registered for prolonged periods in certain clinical situations that are not resolved with conventional electroencephalography. AIM: To report the experience with prolonged electroencephalographic and video monitoring at a Neurology Department, of a Military hospital in Santiago. PATIENTS AND METHODS: A retrospective analysis of patients referred for continuous electroencephalographic and video monitoring between 1991 and 1996. Three hundred thirty six patients, aged 3 months to 60 years old, were studied in the period and in 244, there was information about the diagnosis, treatment and evolution. RESULTS: Monitoring was performed in an outpatient basis in 84% of subjects and lasted between 2 and 7 hours. One hundred ten patients were epileptics, 77 patients had a suspicion of epilepsy, 13 patients had possible pseudoseizures and 33 patients had miscellaneous diagnoses. In 154 patients, electroencephalographs recorded during wakefulness, somnolence and spontaneous dream, were normal. Intercritical recordings with epileptic activity were obtained in 76 patients and in 30 of these, critical epileptic activity was also recorded, not always associated to clinical manifestations. Unspecific electroencephalographic alterations were recorded in 14 patients. CONCLUSIONS: Prolonged electroencephalographic and video monitoring can be useful for patients with complex neurological problems.

Direct interaction in T-cells between thetaPKC and the tyrosine kinase p59fyn.

The protein kinase C (PKC) family has been clearly implicated in T-cell activation as have several nonreceptor protein-tyrosine kinases associated with the T-cell receptor, including p59fyn. This report demonstrates that thetaPKC and p59fyn specifically interact in vitro, in the yeast two-hybrid system, and in T-cells. Further indications of direct interaction are that p59fyn potentiates thetaPKC catalytic activity and that thetaPKC is a substrate for tyrosine phosphorylation by p59fyn. This interaction may account for the localization of thetaPKC following T-cell activation, pharmacological disruption of which results in specific cell-signaling defects. The demonstration of a physical interaction between a PKC and a protein-tyrosine kinase expands the class of PKC-anchoring proteins (receptors for activated C kinases (RACKs)) and demonstrates a direct connection between these two major T-cell-signaling pathways.

Synthesis and evaluation of 11beta-substituted 21-chloro/iodo-(17alpha,20E/Z)-19-norpregna-1,3,5(10),20-te traene-3, 17beta-diols: high-affinity ligands for the estrogen receptor.

We have synthesized six new estrogens substituted at the 11beta-position with a methoxy or vinyl group and at the 17alpha-position with an (E)- or (Z)-chloro/iodovinyl moiety. The products were obtained in good overall yields from the corresponding tri-n-butylstannylvinyl intermediates using the electrophilic halodestannylation methodology. The six new ligands were compared to the 11beta-unsubstituted chloro/iodovinyl derivatives and the 11beta-methoxy (E)- and (Z)-iodovinyl estrogens to evaluate the effects of 11beta-substitution and 20E/Z-stereochemistry. While all the compounds exhibited high affinity for the estrogen receptor, the 20Z-isomers demonstrated higher affinity than the corresponding 20E-isomers. In addition, the presence of the lipophilic 11beta-substituent was favored over either no substituent or a polar (methoxy) group. Within each isomeric series, the presence of the 21-halo substituent had different effects. For the 20E-series, the 21-chloro products had a higher affinity than the 21-iodo analogue, whereas for the 20Z-series the effect was reversed. These results provide additional insights into the interaction of substituted estradiols with the hormone binding domain of the estrogen receptor.

Novel high-affinity steroidal estrogenic ligands: synthesis and receptor binding of 11 beta-vinyl-17 alpha-E/Z-phenylselenovinyl estradiols.

Previous studies from our laboratory demonstrated separately the tolerance of the estrogen receptor for the 17 alpha-phenylselenovinyl substituent and the enhancement of affinity imparted by the 11 beta-vinyl moiety. Our recent publication suggested that the two groups could be combined within a single structure and retain high affinity for the estrogen receptor. As a result, we have prepared in good overall yields the E- and Z-isomers of 11 beta-vinyl-17 alpha-phenylselenovinyl estradiol. Evaluation of the new steroids with receptor isolated from lamb cytosol indicated that both isomers are poorer ligands than estradiol at 4 degrees C, but both are better than estradiols. at 25 degrees C. This behavior had not been observed for the 11 beta-unsubstituted 17 alpha-E/Z phenylselenovinyl estradiols. Of particular interest was the observation that, unlike previous isomer pairs, the E-isomer possessed a greater affinity than the Z-isomer. The results suggest that relatively small changes in structure may impart significant differences in the interactions with the receptor and provide the basis for further ligand design.

Mycobacterium tuberculosis miniepidemic in a church gospel choir.

BACKGROUND: Conventional contact investigation and molecular fingerprinting of Mycobacterium tuberculosis isolates in tuberculosis (TB) outbreaks have facilitated recognition as well as application of public health control activities. Singing in a choir as an activity that promotes TB transmission has been occasionally recognized. Such avocational transmission in a middle class community can occur with attendant difficulties encountered in contact investigation. METHODS: Five cases of TB (one index case; three secondary cases; one unassociated case) were identified among members of a famous church gospel choir in Newark, NJ. DNA fingerprinting and susceptibility testing were done on all retrieved strains. Of 306 choir members who had tuberculin tests, 19% were reactors. The presumed outbreak site was investigated. RESULTS: Four of the five patients were tenors, and one was an alto. Tenors were approximately twice as likely to be tuberculin reactors than subjects with other vocal ranges combined (relative risk, 2.04; 95% confidence interval, 1.17 to 3.56). An air ventilation outlet was directly in front of the tenor section. Some limited extra-church activity between choir members may have contributed to transmission. CONCLUSION: Conventional contact investigation must be supplemented by newer techniques, such as DNA fingerprinting, in identifying possible outbreak transmission. Singing, location of a ventilation outlet, and exposure time may have contributed to TB transmission in this outbreak. Transmission need not only be in congregate settings among well-defined socioeconomic groups but may occur unexpectedly in middle class communities.

Amino acid sequence, post-translational modifications, binding and labelling of porcine odorant-binding protein.

An odorant-binding protein, migrating in SDS-PAGE with an apparent molecular weight of 22 kDa and an isoelectric point of 4.2, has been purified from pig nasal mucosa. Its complete amino acid sequence was determined by a combination of mass spectrometry and Edman degradation procedures. The protein consists of a single polypeptide chain of 157 amino acids, presenting at the N-terminus a pyroglutamic acid residue. The two cysteine residues, occurring in the primary structure at positions 63 and 155, are involved in an intramolecular disulphide bridge. Sequence comparison with other lipocalins revealed a good similarity with bovine odorant-binding protein, the only member of this class which does not contain disulphide bonds and of which the three-dimensional structure recently has been resolved. Nine out of the 1 6 residues lining the binding pocket in bovine OBP are conserved in the porcine protein, suggesting structural similarities in this region of the molecule. The synthesis of a fluorescent photoaffinity labelling agent and of two tin-containing thymol analogues is also described. These compounds together with other ligands were able to bind the protein as revealed by competitive binding experiments.

Stereochemical probes for the estrogen receptor: synthesis and receptor binding of (17 alpha,20E/Z)-21-phenyl-19-norpregna-1,3,5(10), 20-tetraene-3,17 beta-diols.

Previous studies from our laboratory using 17 alpha-E- and 17 alpha-Z-halovinyl and phenylthiovinyl estradiols demonstrated a marked preference for the Z stereochemistry and a significant steric tolerance for the Z-vinyl substituent. To further explore the extent of that stereochemical preference and steric tolerance we have prepared stereoselectively the 17 alpha-E- and 17 alpha-Z-phenylvinyl estradiols (E- and Z-styrylestradiols). The results, in addition to demonstrating a facile preparation of the target compounds, supported the previously observed stereochemical and steric effects. The relative binding affinities for the Z isomer were 3-4 fold greater than the E isomer at both 4 degrees C and 25 degrees C, and only one-half to one-fourth those of estradiol under similar conditions. The developing model for ligand-accessible space within the estrogen receptor suggests that Z-phenylvinyl estradiols may provide interesting and useful probes for mapping the receptor.

Synthesis and estrogen receptor binding of (17 alpha, 20E)- and (17 alpha, 20Z)-21-phenylthio- and 21-phenylseleno-19-norpregna-1,3,5(10),20-tetraene-3,17 beta-diols.

Previous studies from our laboratory using 17 alpha-E- and 17 alpha-Z-halovinyl estradiols demonstrated a marked enhancement of receptor binding by the Z-isomers. This suggested tolerance at the 17 alpha-position was not previously observed by investigations using 16 alpha and 17 alpha-substituted estradiols. Because of the synthetic access provided by vinyl tin chemistry, we prepared the 17 alpha-E and Z-phenylthiovinyl and phenylselenovinyl estradiols and compared their binding characteristics to those of the previously reported 16 alpha/17 alpha-phenylseleno and methylseleno estradiols. The results, in addition to demonstrating a facile preparation of the target compounds, indicated that significant receptor affinity was retained by these compounds (relative binding affinity = 24.5-117). The highest affinity was demonstrated by the 17 alpha-Z-phenylthiovinyl estradiol 5a, which, by molecular modeling, exhibited a significantly different molecular conformation from the corresponding 17 alpha-E-phenylthiovinyl isomer or the 17 alpha-phenyl-thioethynyl analog. The current series possessed better binding characteristics than the phenylseleno and methylseleno estradiols but somewhat poorer binding than the 17 alpha-E/Z-halovinyl series. The observations suggest that some steric limitations exist in a portion of the 17 alpha-region, and that the region is better accessed by compounds possessing Z-vinyl stereochemistry.