Breast cyst fluid heparan sulphate is distinctively N‐sulphated depending on apocrine or flattened type

Breast cyst fluid (BCF) contained in gross cists is involved with its many biomolecules in different stages of breast cystic development. Type I apocrine and type II flattened cysts are classified based on biochemical, morphological and hormonal differences, and their different patterns of growth factors and active biocompounds may require different regulation. In a previous paper, hyaluronic acid in a very low content and chondroitin sulphate/dermatan sulphate were identified and characterized in BCF. In this new study, various apocrine and flattened BCFs were analyzed for HS concentration and disaccharide pattern. Apocrine HS was found specifically constituted of N‐acetyl groups contrary to flattened HS richer in N‐sulphate disaccharides with an overall N‐acetylated/N‐sulphated ratio significantly increased in apocrine compared with flattened (13.5 vs 3.7). Related to this different structural features, the charge density significantly decreased (~?30%) in apocrine versus flattened BCFs. Finally, no significant differences were observed for HS amount (~0.9–1.3 µg ml^?1) between the two BCF types even if a greater content was determined for flattened samples. The specifically N‐sulphated sequences in flattened BCF HS can exert biologic capacity by regulating growth factors activity. On the other hand, we cannot exclude a peculiar regulation of the activity of biomolecules in apocrine BCF by HS richer in N‐acetylated disaccharides. In fact, the different patterns of growth factors and active biocompounds in the two types of cysts may require different regulation by specific sequences in the HS backbone possessing specific structural characteristics and distinctive chemical groups. Copyright © 2015 John Wiley & Sons, Ltd.     

Steroid biochemistry and categorization of breast cyst fluid: relation to breast cancer risk

Patients bearing macrocysts of the breast are at higher risk of later developing cancer. The fluid filling the cysts (breast cysts fluid, BCF) contains unusual amounts of steroid conjugates, first androgen and estrogen sulfates. Measuring BCF cations (K⁺, Na⁺) allows categorization of cysts into two major subsets (type I and type II) that are associated with a different degree and/or turnover of apocrine metaplastic cells in the lining epithelium. Type I cysts (high K⁺/ Na⁺ ratio) accumulate huge amounts of dehydroepiandrosterone sulfate, estrone sulfate, androstane-3, 17β-diol glucuronide, androsterone glucuronide and contain more testosterone and dihydrotestosterone than type II. Conversely, type II cysts (low K⁺/Na⁺ ratio) contain more progesterone and pregnenolone. A cohort study was started in 1983 at the Cancer Prevention Center, Ravenna, Italy, with the aim of evaluating the relationships between the biochemistry of BCF and the incidence of breast cancer in women with gross cystic disease (GCD) of the breast. The bimodal distribution of the cationic pattern has been confirmed from data obtained in 798 patients aspirated. The risk of cyst relapse was significantly higher among women with type I cysts or with multiple cysts at presentation. Twelve incident cases of breast cancer have been diagnosed among women whose BCF was categorized. Eleven out of 12 cases had type I or multiple cysts. The cumulative incidence of breast cancer among patients bearing type I cysts was 2.5%. We conclude that women with GCD bearing type I cysts have an increased breast cancer risk when compared with the counterpart bearing type II cysts or the general population.     

Electrolytes and Trace Elements in Human Breast Cyst Fluid

Gross cystic breast disease (GCBD) is one of the most common breast diseases, and women with apocrine (type I) cysts are at higher risk of developing breast cancer than women with flattened (type II) cysts. Type I cysts contain fluid with an electrolyte composition similar to that of intracellular fluid (Na/K ratio <3), whereas type II cysts fluid’s content resembles that of plasma (Na/K ratio >3). The electrolyte composition of breast cyst fluid (BCF) has been investigated intensively; however, there have been only a few studies in literature reporting the content of trace elements in BCF. The aim of this study was to compare the concentrations of Na, K, Ca, P, Zn, Cu, Fe, and Na/K and trace element ratios in breast cyst fluid in two subgroups of breast cysts. Sixty-three BCF were obtained by needle aspiration from premenopausal women with GCBD diagnosed by clinical, xheromammographic, and cytological studies. After separation of cells for cytological evaluation, the cyst fluid was centrifuged and supernatant stored at −80°C until the analysis. Sodium, potassium, calcium, phosphorus, and iron were measured using Roche Diagnostics commercial kits on Hitachi 747-200 autoanalyzer. Measurements of copper and zinc were performed by flame atomic absorption spectrophotometer on Shimadzu AAS 680. We found statistically significant higher K, lower Na, higher phosphorus concentrations, and lower Na/K ratios in type I cysts when compared with type II cysts’ values. Median values of Na/K ratio in type I and in type II were 0.32 and 6.2, respectively. Higher Zn, Cu, and Fe concentrations with respect to median values were noted in type I cysts; higher [Na.Cu/K.Zn], [Na.Cu/K.Fe], and [Na.Zn/K.Fe] ratios were found in type II cysts. A significant negative correlation existed between Na/K and Cu, and a significant positive correlation between Na/K and Fe in type II cysts (r = −0.660, p = 0.007; r = 0.615, p = 0.014, respectively). We can conclude that the trace elements content of BCF, in addition to electrolytes, could be useful in classifying the breast cyst. Future studies in larger series are needed to confirm these data.     

p27Kip1 as a prognostic factor in breast cancer: a systematic review and meta‐analysis

The aim of this study was to comprehensively evaluate via a meta‐analysis the association between p27 expression and clinical outcome in breast cancer patients. We conducted a meta‐analysis of 20 studies (n= 6463 patients) that evaluated the correlation between p27 expression and indicators of breast cancer clinical outcome, including overall survival (OS), disease‐free survival (DFS) and relapse‐free survival (RFS). Data pooling was performed by RevMan 4.2. A total of 60% (9 of 15) of the studies showed a significant association between p27 high expression and OS, whereas 25% (2 of 8) and 60% (3 of 5) studies demonstrated a correlation between p27 high expression and DFS and RFS, respectively. The relative risks (RRs) were 1.34 (1.26–1.42) for OS (P < 0.00001), 1.27 (1.10–1.47) for DFS (P= 0.001) and 1.49 (0.92–2.42) for RFS (P= 0.10). In lymph node‐negative breast cancer patients, the RRs for OS and RFS were 1.84 (1.30–2.59; P= 0.0005) and 1.30 (0.20–8.50; P= 0.78), respectively. In lymph node‐positive breast cancer patients, the RRs for OS and RFS were 2.99 (1.77–5.07; P < 0.0001) and 1.49 (0.80–2.77; P= 0.21), respectively. This meta‐analysis indicates that reduced p27 is an independent prognostic factor for poor overall and disease‐free cancer survival.     

Cytokines in human breast cyst fluid

Gross cystic breast disease is a common benign disorder in which palpable cysts occur in the breast and are normally treated by aspiration of the contents. The cysts are classified as either Type 1, containing a high level of potassium ions and a low level of sodium ions, or as Type 2, with low potassium and high sodium ion concentrations. Steroid sulphatase activity in MDA-MB-231 and MCF-7 cell lines is regulated by exogenous breast cyst fluid (BCF), possibly because of cytokines in the BCF. A screening method was used to determine the range of cytokines in eight BCFs, four of each type. This was an array system, which uses antibodies immobilised on a membrane to qualitatively detect 79 different cytokines or growth factors. Nine cytokines were detected well above background levels: all were found in both types of BCF, but only epidermal growth factor (EGF) was higher in Type 1. All the other factors were higher in Type 2 BCF. Two of these cytokines, IL-6 and EGF, have previously been suggested to affect steroid sulphatase expression and several (MIP-1β, IL-8, NAP-2) are known to affect MCF-7 cell chemotaxis. In addition two cytokines were measured by ELISA in 57 BCFs, and both IL-1β and IL-13 were found in BCF, with significantly higher amounts of IL-1β in Type 1 than Type 2 BCF (35.5 ± 4.4 pg/ml versus 9.9 ± 2.9 pg/ml).     

Genetic predisposition of six well‐defined polymorphisms in HMGB1/RAGE pathway to breast cancer in a large Han Chinese population

Breast cancer constitutes an enormous burden in China. A strong familial clustering of breast cancer suggests a genetic component in its carcinogenesis. To examine the genetic predisposition of high mobility group box‐1/receptor for advanced glycation end products (HMGB1/RAGE) pathway to breast cancer, we genotyped six well‐defined polymorphisms in this pathway among 524 breast cancer patients and 518 cancer‐free controls from Heilongjiang province, China. There were no deviations from Hardy–Weinberg equilibrium for all polymorphisms. In single‐locus analysis, the frequency of rs1800624 polymorphism mutant A allele in RAGE gene was significantly higher in patients than in controls (24.52% versus 19.50%, P = 0.006), with the carriers of rs1800624‐A allele being 1.51 times more likely to develop breast cancer relative to those with rs1800624‐GG genotype after adjustment (95% confidence interval or CI: 1.17–1.94, P = 0.001). In HMGB1 gene, haplotype analysis did not reveal any significance, while in RAGE gene, haplotypes C‐T‐A and C‐A‐G (alleles in order of rs1800625, rs18006024, rs2070600) were significantly associated with an increased risk of breast cancer (adjusted OR = 2.72 and 10.35; 95% CI: 1.20–6.18 and 1.58–67.80; P = 0.017 and 0.015 respectively). In further genetic score analysis, per unit and quartile increments of unfavourable alleles were significantly associated with an increased risk of breast cancer after adjustment (odds ratio or OR = 1.20 and 1.26; 95% CI: 1.09–1.32 and 1.12–1.42; P < 0.001 and <0.001 respectively). Our findings altogether demonstrate a significant association between RAGE gene rs1800624 polymorphism and breast cancer risk, and more importantly a cumulative impact of multiple risk associated polymorphisms in HMGB1/RAGE pathway on breast carcinogenesis.     

Seawater recirculation through subducting sediments sustains a deeply buried population of sulfate‐reducing bacteria

Subseafloor sulfate concentrations typically decrease with depth as this electron acceptor is consumed by respiring microorganisms. However, studies show that seawater can flow through hydraulically conductive basalt to deliver sulfate upwards into deeply buried overlying sediments. Our previous work on IODP Site C0012A (Nankai Trough, Japan) revealed that recirculation of sulfate through the subducting Philippine Sea Plate stimulated microbial activity near the sediment–basement interface (SBI). Here, we describe the microbial ecology, phylogeny, and energetic requirements of population of aero‐tolerant sulfate‐reducing bacteria in the deep subseafloor. We identified dissimilatory sulfite reductase gene (dsr) sequences 93% related to oxygen‐tolerant Desulfovibrionales species across all reaction zones while no SRB were detected in drilling fluid control samples. Pore fluid chemistry revealed low concentrations of methane (<0.25 mM), while hydrogen levels were consistent with active bacterial sulfate reduction (0.51–1.52 nM). Solid phase total organic carbon (TOC) was also considerably low in these subseafloor sediments. Our results reveal the phylogenetic diversity, potential function, and physiological tolerance of a community of sulfate‐reducing bacteria living at ~480 m below subducting seafloor.     

Quantification of the sulfates of 16α-hydroxy androgens that are possible precursors of estriol-3-sulfate in human breast cyst fluid

The concentration of 16 alpha-hydroxydehydroepiandrosterone-3-sulfate (16 alpha-OHDHAS) was determined in 29 samples of human breast cyst fluid (BCF) and in 15 of these, androst-5-ene-3 beta,16 alpha,17 beta-triol-3-sulfate (A-TriolS) was also assayed. The median value of both was about 100 ng/mL and the ranges were from 1.4 to about 1800 ng/mL. There was a significant association in the values for the two sulfates (p less than 0.05). These concentrations are consistent with a role for 16 alpha-hydroxy androgens as possible precursors for estriol-3-sulfate. The latter is highly elevated relative to other body fluids in BCF. The androgens also correlated directly with the concentrations of K+, an indicator of apocrine proliferation of breast cysts.     

Regulation of glycosaminoglycan biogenesis is critical for sensitive‐period‐dependent vocal ontogeny

In the brain, the extracellular matrix (ECM) plays a central role during neural development and thus modulates critical‐period regulated behavioral ontogeny. The major components of the ECM are glycosaminoglycans (GAGs) including chondroitin sulfate (CS). However, the specific roles of GAGs in behavioral development are largely unknown. It has been shown that xylosides affect the biological functions of GAGs through modulating GAG biosynthesis. Particularly, xylosides affect GAG biosynthesis through priming of GAG chains (priming activity), competing with endogenous core proteins that carry GAG initiation sites (decoy activity), or both. Using birdsong as our model, we investigated, for the first time, how xyloside‐mediated modulation of GAG biogenesis affects song development. Xylosides infused into motor cortex of juvenile birds alter song development by specifically affecting ontogeny of the stereotyped sequence rather than the acoustic structure of syllables. Further analyses reveal that observed changes can be attributed to the priming activity rather than the decoy activity of xylosides. Collectively, these results suggest that regulation of GAG biogenesis through chemical biology approaches may allow promising therapeutic interventions of critical‐period‐dependent central nervous system plasticity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1401–1412, 2017     

Glycosaminoglycan and DNA Binding Induced Intra‐ and Intermolecular Exciton Coupling of the bis‐4‐Aminoquinoline Surfen

Despite the diverse biological activities of the glycosaminoglycan (GAG) antagonist surfen, the molecular details of its interaction with biomacromolecules remain poorly understood. Therefore, heparin and DNA binding properties of surfen were studied by circular dichroism (CD) and UV absorption spectroscopy methods. High‐affinity (K_a ~ 10⁷ M^‐1) association of surfen to the chiral heparin chain gives rise to a characteristic biphasic CD pattern due to the conformational twist of the aminoquinoline moieties around the central urea bridge. At higher drug loading, intermolecular stacking of surfen molecules alters the induced CD profile and also provokes strong UV hypochromism. In contrast to the right‐handed heparin template, binding of surfen to the left‐helicity chondroitin sulfate chains produces inverted CD pattern. Large UV hypochromism as well as polyphasic induced ellipticity bands indicate that surfen intercalates between the base pairs of calf‐thymus DNA. Extensive CD spectroscopic changes observed at higher drug binding ratios refer to cooperative binding interactions between the intercalated drug molecules. The inherent conformational flexibility of surfen demonstrated here for the first time is important in its binding to distinct macromolecular targets and should be considered for rational drug design of novel GAG antagonists. Chirality 27:605–612, 2015. © 2015 Wiley Periodicals, Inc.     

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4⁺, CD8⁺ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3⁺ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.     

Comprehensive analysis of autophagy‐related prognostic genes in breast cancer

Accumulating evidence revealed that autophagy played vital roles in breast cancer (BC) progression. Thus, the aim of this study was to investigate the prognostic value of autophagy‐related genes (ARGs) and develop a ARG‐based model to evaluate 5‐year overall survival (OS) in BC patients. We acquired ARG expression profiling in a large BC cohort (N = 1007) from The Cancer Genome Atlas (TCGA) database. The correlation between ARGs and OS was confirmed by the LASSO and Cox regression analyses. A predictive model was established based on independent prognostic variables. Thus, time‐dependent receiver operating curve (ROC), calibration plot, decision curve and subgroup analysis were conducted to determine the predictive performance of ARG‐based model. Four ARGs (ATG4A, IFNG, NRG1 and SERPINA1) were identified using the LASSO and multivariate Cox regression analyses. A ARG‐based model was constructed based on the four ARGs and two clinicopathological risk factors (age and TNM stage), dividing patients into high‐risk and low‐risk groups. The 5‐year OS of patients in the low‐risk group was higher than that in the high‐risk group (P < 0.0001). Time‐dependent ROC at 5 years indicated that the four ARG–based tool had better prognostic accuracy than TNM stage in the training cohort (AUC: 0.731 vs 0.640, P < 0.01) and validation cohort (AUC: 0.804 vs 0.671, P < 0.01). The mutation frequencies of the four ARGs (ATG4A, IFNG, NRG1 and SERPINA1) were 0.9%, 2.8%, 8% and 1.3%, respectively. We built and verified a novel four ARG–based nomogram, a credible approach to predict 5‐year OS in BC, which can assist oncologists in determining effective therapeutic strategies.     

Diversity decoupled from sulfur isotope fractionation in a sulfate‐reducing microbial community

The extent of fractionation of sulfur isotopes by sulfate‐reducing microbes is dictated by genomic and environmental factors. A greater understanding of species‐specific fractionations may better inform interpretation of sulfur isotopes preserved in the rock record. To examine whether gene diversity influences net isotopic fractionation in situ, we assessed environmental chemistry, sulfate reduction rates, diversity of putative sulfur‐metabolizing organisms by 16S rRNA and dissimilatory sulfite reductase (dsrB) gene amplicon sequencing, and net fractionation of sulfur isotopes along a sediment transect of a hypersaline Arctic spring. In situ sulfate reduction rates yielded minimum cell‐specific sulfate reduction rates < 0.3 × 10^?15 moles cell^?1 day^?1. Neither 16S rRNA nor dsrB diversity indices correlated with relatively constant (38‰–45‰) net isotope fractionation (ε³⁴S_{sulfide‐sulfate}). Measured ε³⁴S values could be reproduced in a mechanistic fractionation model if 1%–2% of the microbial community (10%–60% of Deltaproteobacteria) were engaged in sulfate respiration, indicating heterogeneous respiratory activity within sulfate‐reducing populations. This model indicated enzymatic kinetic diversity of Apr was more likely to correlate with sulfur fractionation than DsrB. We propose that, above a threshold Shannon diversity value of 0.8 for dsrB, the influence of the specific composition of the microbial community responsible for generating an isotope signal is overprinted by the control exerted by environmental variables on microbial physiology.     

PA‐MSHA inhibits proliferation and induces apoptosis through the up‐regulation and activation of caspases in the human breast cancer cell lines

To investigate the effects of PA‐MSHA (Pseudomonas aeruginosa‐mannose sensitive hemagglutinin) on inhibiting proliferation of breast cancer cell lines and to explore its mechanisms of action in human breast cancer cells. MCF‐10A, MCF‐7, MDA‐MB‐468, and MDA‐MB‐231HM cells were treated with PA‐MSHA or PA (Heat‐killed P. aeruginosa) at different concentrations and different times. Changes of cell super‐microstructure were observed by transmission electron microscopy. Cell cycle distribution and apoptosis induced by PA‐MSHA were measured by flow cytometry (FCM) with PI staining, ANNEXIN V‐FITC staining and Hoechst33258 staining under fluorescence microscopy. Western blot was used to evaluate the expression level of apoptosis‐related molecules. A time‐dependent and concentration‐dependent cytotoxic effect of PA‐MSHA was observed in MDA‐MB‐468 and MDA‐MB‐231HM cells but not in MCF‐10A or MCF‐7 cells. The advent of PA‐MSHA changed cell morphology, that is to say, increases in autophagosomes, and vacuoles in the cytoplasm could also be observed. FCM with PI staining, ANNEXIN V‐FITC and Hoechst33258 staining showed that the different concentrations of PA‐MSHA could all induce the apoptosis and G₀–G₁ cell cycle arrest of breast cancer cells. Cleaved caspase 3, 8, 9, and Fas protein expression levels were strongly associated with an increase in apoptosis of the breast cancer cells. There was a direct relationship with increased concentrations of PA‐MSHA but not of PA. Completely different from PA, PA‐MSHA may impart antiproliferative effects against breast cancer cells by inducing apoptosis mediated by at least a death receptor‐related cell apoptosis signal pathway, and affecting the cell cycle regulation machinery. J. Cell. Biochem. 108: 195–206, 2009. © 2009 Wiley‐Liss, Inc.     

A New Heterotrophic Dinoflagellate from the North‐eastern Pacific,Protoperidinium fukuyoi: Cyst–Theca Relationship,Phylogeny, Distribution and Ecology

The cyst–theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown‐coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst‐defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North‐east Pacific, its distribution reflecting optimal summer sea‐surface temperatures of ~14–18 °C and salinities of 22–34 psu.     

Expression of recombinant anti‐breast cancer immunotherapeutic monoclonal antibody in baculovirus–insect cell system

The anti‐breast cancer monoclonal antibody (mAb) BR55 was expressed in the baculovirus–insect cell expression system, which is advantageous because of its high production capacity, cell culture flexibility and glycosylation capability. The baculovirus–insect cell expression system was successfully established for production of mAb BR55 and mAb BR55 fused with the KDEL (Lys–Asp–Glu–Leu) endoplasmic reticulum (ER) retention signal (mAb BR55K). The heavy chain (HC) and light chain (LC) genes of mAb BR55 were cloned under the control of the polyhedrin (P_PH) and P₁₀ promoters, respectively, in the pFastBacDual vector. The antibody gene‐expression cassettes carrying both the HC and LC genes were transferred into a bacmid in Escherichia coli (DH10Bac). The bacmid carrying the expression cassettes was transfected into Sf9 insect cells to generate baculovirus expressing mAb BR55 and BR55K. Western blot analysis confirmed the expression of mAb BR55 and BR55K in baculovirus‐infected insect cells. Cell direct enzyme linked immunosorbent assay (ELISA) showed that both mAbs from insect cell lysates or cell culture medium bound to MCF‐7 human breast cancer cells. Both mAb BR55 and BR55K were successfully purified using a Protein A affinity column. Collectively, these results suggest that the anti‐breast cancer mAb BR55 can be expressed, properly assembled and purified from the baculovirus expression system, which can serve as an alternative system for antibody production.     

Allopregnanolone, pregnenolone sulfate, and epitestosterone in breast cyst fluid

The risk of breast cancer is 2 to 5 times higher in patients suffering from gross cystic disease. Breast cysts are categorized into two groups (type I and type II) according to the concentration of electrolytes in the cyst fluid. The two types also differ with respect to accumulation of steroids and steroidogenic enzyme activity. In type I cysts a higher risk of breast carcinoma could be expected. Here, we studied a possible relationship between the type of cyst and levels of epitestosterone (an endogenous antiandrogen), allopregnanolone (a product of 5alpha-reductase activity), and pregnenolone-sulfate (an activator of N-methyl-D-asparate receptors). We have found five times higher levels of epitestosterone in BCF in comparison with the circulation. Allopregnanolone levels were similar to those in plasma of women in the luteal phase of the menstrual cycle. Pregnenolone-sulfate levels in BCF were about two orders of magnitude higher when compared with the circulation. No differences were found in concentrations of the steroids studied between the types of cysts.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Characterization of a Deep‐Branching Heterolobosean,Pharyngomonas turkanaensis n. sp., Isolated from a Non‐Hypersaline Habitat,and Ultrastructural Comparison of Cysts and Amoebae Among Pharyngomonas Strains

An unusual heterolobosean amoeba, isolate LO, was isolated recently from a sample with a salinity of ~4‰, from Lake Turkana in East Africa. 18S rDNA phylogenies confirm that isolate LO branches among halophilic amoeboflagellates assigned to Pharyngomonas. We examined the ultrastructure of the amoeba and cyst stages of isolate LO, as well as the amoebae and cysts of Pharyngomonas kirbyi (isolates AS12B and SD1A). The amoebae of all three isolates lacked discrete dictyosomes and had discoidal/flattened mitochondrial cristae, but the mitochondria were not enrobed by rough endoplasmic reticulum. The cysts of all three isolates showed a thick, bipartite cyst wall, and lacked cyst pores. The cysts of isolate LO were distinct in that the ectocyst was very loose‐fitting, and could contain “crypts”. No flagellate form of isolate LO has been observed to date, and a salinity‐for‐growth experiment showed that isolate LO can grow at 15–100‰ salinity, indicating that it is halotolerant. By contrast, other studied Pharyngomonas isolates are amoeboflagellates and true halophiles. Therefore, we propose isolate LO as a new species, Pharyngomonas turkanaensis n. sp. It is possible that P. turkanaensis descended from halophilic ancestors, and represents a secondary reestablishment of a physiology adapted for moderate salinity.