Androgens in women

The role of androgen treatment in women remains controversial. The proposed “Female Androgen Insufficiency Syndrome” (Fertility and Sterility, April 2002) describes a number of non-specific symptoms including unexplained fatigue, decreased well being/dysphoric mood and/or blunted motivation and diminished sexual function. An estimated 40% of women experience sexual dysfunction, highlighting the need for ongoing research into this field in order to fully define the possible contribution of androgen insufficiency. The increasing availability of products, such as dehydroepiandrosterone (DHEA) supplements also points to the need for controlled studies to assess the safety of these and other preparations.

Measurement of androgens in women requires sensitive assays with the ability to detect low levels and a narrow range with precision. Normal ranges of androgens for women of reproductive and post-reproductive age remain poorly defined. Debate exists as per importance of measurement of free versus total testosterone, with the ‘free androgen index’ offering an alternative method of assessment of testosterone availability.

Testosterone treatment is being developed for women in the form of transdermal patches, gels or cream, with percutaneous implants in common usage in some countries. Recent research has highlighted alternative means of administration, such as oral inhalation or buccal lozenge. DHEA is widely available in some countries. Research to date has demonstrated improvements in libido and sexual function, mood and well being. Evidence points to other potential benefits of androgen treatment, including preservation of bone mass, a possible protective role in breast cancer and beneficial effects on cognition.

Adverse effects of androgen treatment in women are dose-dependent and include virilisation, mood disturbance and acne. These are uncommon if appropriate doses are administered and highlight the need for treatment to be closely monitored clinically and biochemically. Beneficial effects of testosterone treatment in post-menopausal women with lowered androgen levels have been well documented, and preliminary evidence suggests a role for treatment in pre-menopausal women with symptoms and lowered testosterone levels.     

Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia

OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic anion uptake transporter and has been shown to be a higher affinity bilirubin uptake transporter than OATP1B3. Using human embryonic kidney (HEK 293) cells stably transfected with OATP1B1, we have studied the effects of indinavir, saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function. These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support the hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important mechanism in drug-drug and drug-endogenous substance interactions.     

Functional domains and temperature-sensitive mutations in SPE-9, an EGF repeat-containing protein required for fertility in Caenorhabditis elegans

The spe-9 gene is required for fertility in Caenorhabditis elegans and encodes a sperm transmembrane protein with an extracellular domain (ECD) that contains 10 epidermal growth factor (EGF) repeats. Deletion analysis reveals that the EGF repeats and the transmembrane domain are required for fertilization. In contrast, the cytoplasmic region of SPE-9 is not essential for fertilization. Individual point mutations in all 10 EGF motifs uncover a differential sensitivity of these sequences to alteration. Some EGF repeats cannot tolerate mutation leading to a complete lack of fertility. Other EGF repeats can be mutated to create animals with temperature-sensitive (ts) fertility phenotypes. All ts mutations were generated by changing either conserved cysteine or glycine residues in the EGF motifs. For two endogenous ts alleles of spe-9, loss of function at nonpermissive temperatures is not due to protein mislocalization or degradation. Additionally, the proper localization of SPE-9 in sperm is not altered in a genetically interacting fertility mutant (spe-13) or a mutant that affects sperm vesicle-plasma membrane fusion (fer-1). Like the EGF repeats in the Notch/LIN-12/GLP-1 receptors and their ligands, the EGF repeats in SPE-9 may carry out different functions. Because EGF motifs are found in many proteins in different species, similar experimental strategies could be used to generate useful temperature-sensitive mutations in other EGF motif-containing molecules.     

Cytotoxicity of the E(2)-isoprostane 15-E(2t)-IsoP on oligodendrocyte progenitors

Oxidant stress plays a significant role in the pathogenesis of periventricular leukomalacia (PVL). Isoprostanes (IsoPs) are bioactive products of lipid peroxidation abundantly generated during hypoxic-ischemic injuries. Because loss of oligodendrocytes (OLs) occurs early in PVL, we hypothesized that IsoPs could induce progenitor OL death. 15-E(2t)-IsoP but not 15-F(2t)-IsoP elicited a concentration-dependent death of progenitor OLs by oncosis and not by apoptosis, but exerted minimal effects on mature OLs. 15-E(2t)-IsoP-induced cytotoxicity could not be explained by its conversion into cyclopentenones, because PGA(2) was hardly cytotoxic. On the other hand, thromboxane A(2) (TxA(2)) synthase inhibitor CGS12970 and cyclooxygenase inhibitor ibuprofen attenuated 15-E(2t)-IsoP-induced cytotoxicity. Susceptibility of progenitor OLs was independent of TxA(2) receptor (TP) expression, which was far less in progenitor than in mature OLs. However, TxA(2) synthase was detected in precursor but not in mature OLs, and TxA(2) mimetic U46619 induced hydroperoxides generation and progenitor OL death. The glutathione synthesis enhancer N-acetylcysteine prevented 15-E(2t)-IsoP-induced progenitor cell death. Depletion of glutathione in mature OLs with buthionine sulfoximine rendered them susceptible to cytotoxicity of 15-E(2t)-IsoP. These novel data implicate 15-E(2t)-IsoP as a product of oxidative stress that may contribute in the genesis of PVL.     

Particulate phase cigarette smoke increases MnSOD, NQO1, and CINC-1 in rat lungs

Loss of antioxidant/oxidant homeostasis perpetuates inflammation in the lungs and may contribute to the development of COPD and lung cancer. Cigarette smoke (CS) is a primary source of airway oxidative stress and recruits inflammatory cells into smokers' lungs. However, whether these consequences are attributable to a specific or the collective fraction of CS is unknown. We investigated whether the particulate or the gas phase of CS would alter expression of the antioxidant enzymes MnSOD and NQO1 or CINC-1. Sprague Dawley rats were exposed to sham (n = 10) or the particulate phase (PP; n = 10) or gas phase (n = 10) of a Kentucky reference cigarette (1R4F) for 2 h/d for 28 d, after which animals were sacrificed and the lower left lobe of the lung was removed. Immunoblots for SOD and NQO1 revealed that lungs exposed to PP had higher MnSOD/actin and NQO1/actin ratios than either sham-or gas phase-treated animals. In contrast, CuZnSOD remained unchanged. In PP-exposed animals, CINC-1 was 3-fold higher than in sham-exposed animals. The increases in MnSOD and NQO1 protein were associated with increases in total SOD, NQO1, and MPO activities. These data provide evidence that the PP of CS alters oxidant/antioxidant homeostasis in the lungs and participates in the pathogenesis of CS-induced lung diseases such as COPD and cancer.     

Isomer-specific contractile effects of a series of synthetic f2-isoprostanes on retinal and cerebral microvasculature

F2-isoprostanes (F2-IsoP's) are biologically active prostanoids formed by free radical-mediated peroxidation of arachidonic acid. Four different F2-IsoP regioisomers (5-, 8-, 12-, and 15-series), each comprising eight racemic diastereomers, total 64 compounds. Information regarding the biological activity of IsoP's is largely limited to 15-F2t-IsoP (8-iso-PGF2alpha). We recently demonstrated that 15-F2t-IsoP and its metabolite, 2,3-dinor-5,6-dihydro-15-F2t-IsoP, evoked vasoconstriction and TXA2 generation in retina and brain microvasculature. We have now examined and compared the biological activities of a series of recently synthesized new 5-, 12-, and 15-series F2-IsoP isomers in pig retinal and brain microvasculature. We hereby show that other 15-series F2-IsoP isomers, 15-epi-15-F2t-IsoP, ent-15-F2t-IsoP, and ent-15-epi-15-F2t-IsoP, are also potent vasoconstrictors. The 12-series isomers tested, 12-F2t-IsoP and 12-epi-12-F2t-IsoP, also caused marked vasoconstriction. Of the 5-series isomers tested, 5-F2t-IsoP and 5-epi-5-F2t-IsoP possessed no vasomotor properties, whereas ent-5-F2t-IsoP caused modest vasoconstriction. The vasoconstriction of ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP was abolished by removal of the endothelium, by TXA2 synthase and receptor inhibitor (CGS12970, L670,596), and by receptor-mediated Ca2+ channel blockade (SK & F96365); correspondingly, these isomers increased TXB2 formation by activating Ca2+ influx (detected with fura 2-AM) through non-voltage-dependent receptor-mediated Ca2+ entry (SK & F96365 sensitive) in endothelial cells. In conclusion, as seen with 15-F2t-IsoP, ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP constricted both retinal and brain microvessels by inducing endothelium-dependent TXA2 synthesis. These new findings broaden the scope of our understanding regarding the potential involvement of F2-IsoP's as mediators of oxidant injury.     

Single mutations of residues outside the active center of the xylanase Xys1 Delta from Streptomyces halstedii JM8 affect its activity

Mutagenesis of the xylanase Xys1 of Streptomyces halstedii JM8 has been done by error prone PCR. Mutants with modified hydrolytic activity were isolated, the recombinant variant proteins purified and the catalytic activities of each one determined and compared with the wild type enzyme. Two of the isolated single point mutants, m1 (G133D) and m8 (N148D), showed 22-25% increase in specific activity towards xylan compared to wild type xylanase. Two other mutants, m5a (D175A) and m7 (T160A), showed a significant reduction in specific activity of 40-50% with respect to the wild type enzyme. These residues are mainly located in the beta alpha-loops of the xylanase, the region showing the main structural divergences within family 10 of xylanases. This study shows the usefulness of random mutagenesis to point out some key residues not directly involved in the active center, but in which mutation produces subtle structural rearrangements affecting the enzymatic function.     

Evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in environmental samples

Microarrays fabricated with oligonucleotides longer than 40 bp have been introduced for monitoring whole genome expression but have not been evaluated with environmental samples. To determine the potential of this type of microarray for environmental studies, a 50-mer oligonucleotide microarray was constructed using 763 genes involved in nitrogen cycling: nitrite reductase (nirS and nirK), ammonia monooxygenase (amoA), nitrogenase (nifH), methane monooxygenase (pmoA), and sulfite reductase (dsrAB) from public databases and our own sequence collections. The comparison of the sequences from pure cultures indicated that the developed microarrays could provide species-level resolution for analyzing microorganisms involved in nitrification, denitrification, nitrogen fixation, methane oxidation, and sulfite reduction. Sensitivity tests suggested that the 50-mer oligonucleotide arrays could detect dominant populations in the environments, although sensitivity still needs to be improved. A significant quantitative relationship was also obtained with a mixture of DNAs from eight different bacteria. These results suggest that the 50-mer oligonucleotide array can be used as a specific and quantitative parallel tool for the detection of microbial populations in environmental samples.     

Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain

Synaptotagmin I has two tandem Ca(2+)-binding C(2) domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C(2)B domain, one of them in the cation-free form at 1.50 A resolution, two in the Ca(2+)-bound form at 1.04 A (two bound Ca(2+) ions) and 1.65 A (three bound Ca(2+) ions) resolution and one in the Sr(2+)-bound form at 1.18 A (one bound Sr(2+) ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca(2+) ions (sites 1 and 2). At higher Ca(2+) concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca(2+) coordination site (site 3). Divalent cation binding sites 1 and 2 in the C(2)B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca(2+) sites in the crystal structures, while it is coordinated to Ca(2+) in site 2 in the NMR structure. In the presence of Sr(2+), which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.     

Infection of Calomys callosus (Rodentia Cricetidae) with strains of different Trypanosoma cruzi biodemes: pathogenicity, histotropism, and fibrosis induction

The influence of different Trypanosoma cruzi biodemes on the evolution of the infection and on the histopathological lesions of the heart and skeletal muscles, during the experimental infection of Calomys callosus, was investigated. Three groups of C. callosus were infected, respectively, with parasite strains representative of three different Biodemes: Type I (Y strain), Type II (21 SF strain), and Type III (Colombian strain). For each group, normal C. callosus were also used as controls. Marked differences have been detected in the responses of C. callosus to the infection with the three strains in this model. The strains Types I and II (Y and 21 SF) determined moderate lesions, mostly in the myocardium, with low parasitism, a rapid course, and total regression of the lesions by the 60th day of infection. Differently, Type III strain (Colombian), was more pathogenic for C. callosus and induced necrotic-inflammatory lesions in skeletal muscles and myocardium, in correspondence to intracellular parasitism. Proliferation of fibroblasts and amorphous matrix deposits, followed by interstitial fibrosis were present. Progressive regression of the inflammatory changes and collagen deposits occurred spontaneously. The progression and regression of both inflammation and fibrosis induced by the Colombian strain were further submitted to quantitative evaluation by morphometry. Results of the morphometric studies presented good correlation with the histopathological findings. The results confirm the importance of the different biodemes in the determination of tissue lesions and the peculiarities of response of C. callosus to infection with T. cruzi.