Abnormalities of human sex chromosomes

Summary The cytogenetic study of a pair of identical, mentally-retarded twins with the chromosome complement 48,XXXY is reported, along with extensive clinical and endocrinological studies of one twin.The genetic and clinical features of 30 reported 48,XXXY individuals were summarized and compared to those of 47,XXY and 49,XXXXY individuals. For 47,XXY the mean maternal age clearly is increased; for 48,XXXY it appears definitely but only slightly increased; and for 49,XXXXY it may not be increased at all. Developmental defects, similar in type, appear to be progressively more marked when an additional 1, 2, or 3 X chromosomes are added to the normal male chromosome complement. 47,XXY individuals may be either normal in intelligence or mentally retarded, whereas severe mental retardation has been present in all those with the complements 48,XXXY and 49,XXXXY.The interesting suggestion of increased twining associated with poly-X male complements is noted.

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Zusammenfassung Die cytogenetische Untersuchung eines Paares eineiiger, geistig zurückgebliebener Zwillinge mit dem Chromosomenstatus 48,XXXY wird dargestellt; bei dem einen Paarling konnten außerdem ausgedehnte klinische und endokrinologische Studien durchgeführt werden.Außerdem wurden die genetischen und klinischen Merkmale der 30 bekannten Fälle mit 48,XXXY dargestellt und mit denen von Patienten mit 47,XXY und mit 49,XXXXY verglichen. Bei Fällen mit 47,XXY ist das mütterliche Alter deutlich erhöht; bei 48,XXXY ist es eindeutig, aber nur leicht erhöht; es sieht so aus, als ob es für 49,XXXXY überhaupt nicht erhöht wäre. Defekte der Entwicklung, die dem Typ nach ähnlich sind, scheinen dem Ausmaß nach desto mehr ausgeprägt zu sein, je mehr X-Chromosomen zusätzlich bei dem normalen männlichen Chromosomensatz vorhanden sind. 47,XXY Individuen können entweder schwachsinnig sein oder eine normale Intelligenz haben; dagegen zeigten alle Fälle mit 48,XXXY und 49,XXXXY einen schweren Schwachsinn.Es wird die interessante Frage aufgeworfen, ob die Zwillingshäufigkeit bei Poly X-Männern erhöht ist.

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Research supported by grants HD 04134, HL 09011, RR-47, AM-11105, and TIAM 53950-11 from the National Institutes of Health.     

Benzodiazepine-Induced Hyperphagia: Development and Assessment of a 3D Pharmacophore By Computational Methods

Abstract

Benzodiazepine receptor (BDZR) ligands are structurally diverse compounds that bind to specific binding sites on GABAA receptors and allosterically modulate the effect of GABA on chloride ion flux. The binding of BDZR ligands to this receptor system results in activity at multiple behavioral endpoints, including anxiolytic, sedative, anticonvulsant, and hyperphagic effects. In the work presented here, a computational procedure developed in our laboratory has been used to obtain a 3D pharmacophore for ligand recognition of the GABAA/BDZRS initiating the hyperphagic response. To accomplish this goal, 17 structurally diverse compounds, previously assessed in our laboratory for activity at the hyperphagic endpoint, were used. The result is a four-component 3D pharmacophore. It consists of two proton acceptor atoms, the centroid of an aromatic ring and the centroid of a hydrophobic moiety in a common geometric arrangement in all compounds with activity at this endpoint. This 3D pharmacophore was then assessed and successfully validated using three different tests. First, two BDZR ligands, which were included as negative controls in the set of seventeen compounds used for the pharmacophore development, did not fit the pharmacophore. Second, some benzodiazepine ligands known to have activity at the hyperphagia endpoint, but not included in the pharmacophore development, were used as positive controls and were found to fit the pharmacophore. Finally, using the 3D pharmacophore developed in the present work to search 3D databases, over 50 classical benzodiazepines were found. Among them, were benzodiazepine ligands known to have an effect at the hyperphagic endpoint. In addition, the novel compounds also found in this search are promising therapeutic agents that could beneficially affect feeding behavior.     

Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.     

SPL7013 Gel (VivaGel?) Retains Potent HIV-1 and HSV-2 Inhibitory Activity following Vaginal Administration in Humans

SPL7013 Gel (VivaGel^®) is a microbicide in development for prevention of HIV and HSV. This clinical study assessed retention and duration of antiviral activity following vaginal administration of 3% SPL7013 Gel in healthy women. Participants received 5 single doses of product with ≥5 days between doses. A cervicovaginal fluid (CVF) sample was collected using a SoftCup™ pre-dose, and immediately, or 1, 3, 12 or 24 h post-dose. HIV-1 and HSV-2 antiviral activities of CVF samples were determined in cell culture assays. Antiviral activity in the presence of seminal plasma was also tested. Mass and concentration of SPL7013 in CVF samples was determined. Safety was assessed by reporting of adverse events. Statistical analysis was performed using the Wilcoxon signed-rank test with Bonferroni adjustment; p≤0.003 was significant. Eleven participants completed the study. Inhibition of HIV-1 and HSV-2 by pre-dose CVF samples was negligible. CVF samples obtained immediately after dosing almost completely inhibited (median, interquartile range) HIV-1 [96% (95,97)] and HSV-2 [86% (85,94)], and activity was maintained in all women at 3 h (HIV-1 [96% (95,98), p = 0.9]; HSV-2 [94% (91,97), p = 0.005]). At 24 h, >90% of initial HIV-1 and HSV-2 inhibition was maintained in 6/11 women. SPL7013 was recovered in CVF samples obtained at baseline (46% of 105 mg dose). At 3 and 24 h, 22 mg and 4 mg SPL7013, respectively, were recovered. More than 70% inhibition of HIV-1 and HSV-2 was observed if there was >0.5 mg SPL7013 in CVF samples. High levels of antiviral activity were retained in the presence of seminal plasma. VivaGel was well tolerated with no signs or symptoms of vaginal, vulvar or cervical irritation reported. Potent antiviral activity was observed against HIV-1 and HSV-2 immediately following vaginal administration of VivaGel, with activity maintained for at least 3 h post-dose. The data provide evidence of antiviral activity in a clinical setting, and suggest VivaGel could be administered up to 3 h before coitus.

Trial Registration

The study is registered at ClinicalTrials.gov under identifier: {"type":"clinical-trial","attrs":{"text":"NCT00740584","term_id":"NCT00740584"}}NCT00740584     

Attenuation of multi-targeted proliferation-linked signaling by 3,3'-diindolylmethane (DIM): from bench to clinic

Emerging evidence provide credible support in favor of the potential role of bioactive products derived from ingesting cruciferous vegetables such as broccoli, brussel sprouts, cauliflower and cabbage. Among many compounds, 3,3'-diindolylmethane (DIM) is generated in the acidic environment of the stomach following dimerization of indole-3-carbinol (I3C) monomers present in these classes of vegetables. Both I3C and DIM have been investigated for their use in preventing, inhibiting, and reversing the progression of cancer - as a chemopreventive agent. In this review, we summarize an updated, wide-ranging pleiotropic anti-tumor and biological effects elicited by DIM against tumor cells. It is unfeasible to point one single target as basis of cellular target of action of DIM. We emphasize key cellular and molecular events that are effectively modulated in the direction of inducing apoptosis and suppressing cell proliferation. Collectively, DIM orchestrates signaling through Ah receptor, NF-κB/Wnt/Akt/mTOR pathways impinging on cell cycle arrest, modulation of key cytochrome P450 enzymes, altering angiogenesis, invasion, metastasis and epigenetic behavior of cancer cells. The ability of DIM to selectively induce tumor cells to undergo apoptosis has been observed in preclinical models, and thus it has been speculated in improving the therapeutic efficacy of other anticancer agents that have diverse molecular targets. Consequently, DIM has moved through preclinical development into Phase I clinical trials, thereby suggesting that DIM could be a promising and novel agent either alone or as an adjunct to conventional therapeutics such as chemo-radio and targeted therapies. An important development has been the availability of DIM formulation with superior bioavailability for humans. Therefore, DIM appears to be a promising chemopreventive agent or chemo-radio-sensitizer for the prevention of tumor recurrence and/or for the treatment of human malignancies.     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.     

L-type Ca2+ channels in Ca2+ channelopathies

Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.