Structure-activity relationships of 9β-estrogens

The 9β isomers of estradiol-17β, estradiol-17α, estrone and 17-ethinylestradiol-17β were synthesized and compared with their 9α-counterparts in the rat uterine cytosol estrogen receptor, utero-tropic, and gonadotropin release inhibition assays. Except for 17-ethinyl-9β-estradiol-17β which was as active as its 9α isomer in the uterotropic assay, none of the 9β estrogens exhibited any biological activity which was equal to or greater than their 9α counterparts. For examples, 9β-estradiol-17β was $\text{1}\text{10}$ as active as estradiol-17β, and 9β-estrone was $\text{1}\text{4}$ as active as estrone in the uterotropic assay.     

Preformulation Studies of a Prodrug of Δ9-Tetrahydrocannabinol

Preformulation studies were performed on a hemiglutarate ester prodrug of Δ⁹-tetrahydrocannabinol (THC-HG), to facilitate the development of stable formulations by hot-melt methods. The various studies performed included solid-state thermal characterization, pKa, logP, aqueous and pH dependent solubility, pH stability and effect of moisture, temperature and oxygen on solid-state stability. A hot-melt method was utilized to fabricate THC-HG incorporated poly (ethylene oxide) (PEO) matrices and the bioadhesive properties, release profiles and post-processing stability of these matrices were assessed as a function of the polymer molecular weight. The prodrug exhibited a T _g close to 0°C, indicating its amorphous nature. Thermogravimetric analysis revealed a rapid weight loss after 170°C. The prodrug exhibited a seven-fold higher aqueous solubility as compared to the parent drug (THC). Also, the solubility of the compound increased with increasing pH, being maximum at pH 8. The prodrug exhibited a v-shaped pH-rate profile, with the degradation rate minimum between pH 3 and 4. The moisture uptake and drug degradation increased with an increase in relative humidity. Solid-state stability indicated that the prodrug was stable at −18°C but demonstrated higher degradation at 4°C, 25°C and 40°C (51.6%, 74.5% and 90.1%, respectively) at the end of 3-months. THC-HG was found to be sensitive to the presence of oxygen. The release of the active from the polymeric matrices decreased, while bioadhesion increased, with an increase in molecular weight of PEO.     

9-Cis-retinoyl-β-glucuronide is a major metabolite of 9-Cis-retinoic acid

The in vivo metabolism of 9-cis-retinoic acid (9-c-RA), an endogenous ligand of retinoid X receptors (RXRs), which can also bind to retinoic acid receptors (RARs), was examined in pregnant mice and rats following a single oral dose of 100 mg 9-cis- retinaldehyde (9-c-RAL) / kg body weight. 9-Cis-retinoyl-β-glucuronide (9-c-RAG), a metabolite not found in vivo before, was a major metabolite of 9-c-RA in mouse plasma and was also present in all mouse tissues examined as well as in rat plasma. In both species putative oxidation products of retinoic acids and high levels of retinyl esters were found. Concentrations of retinoic acid isomers and retinoyl-β-D-glucuronides in the mouse plasma greatly exceeded those of the rat plasma. The finding of high levels of 9-c-RAG underlines the importance of glucuronidation in the metabolism of retinoids.     

Short form of α9 promotes α9β1 integrin-dependent cell adhesion by modulating the function of the full-length α9 subunit

The α9β1 integrin is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin-C, and osteopontin. A 2.3-kb truncated form of α9 integrin subunit cDNA was identified by searching the Medline database. This splice variant, which we called the short form of α9 integrin (SFα9), encodes a 632-aa isoform lacking transmembrane and cytoplasmic domains, and its authentic expression was verified by PCR and Western blotting. SFα9 is expressed on the cell surface but cannot bind ligand in the absence of the full-length α9 subunit. Over-expression of SFα9 in cells expressing full-length α9 promotes α9-dependent cell adhesion. This promoting effect of SFα9 requires the authentic cytoplasmic domain of the co-expressed full-length α9 subunit. Thus, SFα9 is a novel functional modulator of α9β1 integrin by inside-out signaling.     

The effects of PGD2 and 9-deoxy-Δ9-PGD2 on colony formation of murine osteosarcoma cells

Effects of antineoplastic prostaglandins (PG), PGD₂ and 9-deoxy-Δ⁹-PGD₂, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD₂ significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 ug/ml. The IC₅₀ value was calculated to be 0.72 ug/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-Δ⁹-PGD₂ between 0.01 to 1 ug/ml, the IC₅₀ value being 0.22 ug/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 ug/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-Δ⁹-PGD₂ at a concentration of 5 ug/ml. These results suggest that PGD₂ and 9-deoxy-Δ⁹-PGD₂ are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.     

Antibacterial activity of Δ9-tetrahydrocannabinol and cannabidiol

The minimum inhibiting concentrations (MIC) of ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD) for staphylococci and streptococci in broth are in the range of 1–5 g/ml. In the same range, both compounds are also bactericidal. In media containing 4% serum or 5% blood the antibacterial activity is strongly reduced (MIC 50g/ml). Gram-negative bacteria are resistant to THC and CBD.     

Steroid antagonism of the ‘hypertensinogenic’ activity of 9α-fluoroprednisolone

We have previously reported that adrenocortical steroids raise blood pressure by a ‘hypertensinogenic’ mechanism of action which is not simply related to their classical ‘mineralocorticoid’ or ‘glucocorticoid’ actions. This study presents evidence for specific antagonism of this ‘hypertensinogenic’ activity. The effects of separate IV infusions of prednisolone (P) 100 mg/d and 9α-fluoro-prednisolone (9αF-P) 0.6 mg/d on mean arterial pressure (MAP), plasma [K], plasma [glucose] and urinary NA excretion (UNaV) after 2 days were studied in sheep. In the same group of sheep which received P alone for 2 days, 9αF-P was given for a further 2 days while continuing the P infusion (P + 9αF-P). P alone had no effect on MAP or plasma [K] or U_NaV but increased plasma [glucose], effects which are characteristic of ‘glucocorticoid’ activity. 9αF-P alone increased MAP by 14 mmHg (P<0.001) and reduced plasma [K] and U_NaV but had no effect on plasma [glucose]. Thus 9αF-P exhibited both ‘hypertensinogenic’ and ‘mineralocorticoid’ activity. In the sheep which received the combined P + 9αF-P infusion, the increase in MAP normally produced by 9αF-P was blocked. Although pretreatment with P blocked the pressor effect of 9αF-P, it did not alter the ‘mineralocorticoid’ effects, namely hypokalaemia and urinary Na retention, produced when 9αF-P was infused alone. These results provide further evidence for our concept of a ‘hypertensinogenic’ class of steroid activity and are the first demonstration of specific antagonism of steroid induced hypertension.     

The effects of Δ9-tetrahydrocannabinol Δ9-THC) on the regional concentrations of spermidine in the rat brain

The effects of intravenous administration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC, 2 mg/kg, i.v.) on the regional brain spermidine concentrations of the rat were examined. Thirty minutes after vehicle treatment, the spermidine concentrations were: for the medulla oblongata/pons, 68.2 ± 7.7 μg/g; the hypothalamus, 67.7 ± 2.6 μg/g; the midbrain, 59.1 ± 4.4 μg/g; the cerebellum, 47.3 ± 5.9 μg/g and for the cortex, 13.8 ± 0.8 μg/g. Thirty minutes after Δ⁹-THC, these concentrations were reduced in the midbrain (47.0 ± 8.0% of control, P < 0.0001) and cortex (69.4 ± 7.4% of control, P < 0.009). The spermidine concentrations were not significantly altered in the medulla oblongata/pons (86.5 ± 13.3%, P > 0.36), hypothalamus (107.2 ± 11.8, P > 0.36) or cerebellum (89.0 ± 14.4%, P < 0.48). These results suggest that spermidine within the midbrain and cortex may be involved in the expression of some of the actions of Δ⁹-THC.     

Discovery of 2,3′-diindolylmethanes as a novel class of PCSK9 modulators

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of low density lipoprotein receptor (LDLR). Anti-PCSK9 agents have been approved for the treatment of hypercholesterolemia. We recently discovered a series of small-molecule PCSK9 modulators that contains a relatively small pharmacophore of 2,3′-diindolylmethane with molecular weights around only 250. These molecules can significantly lower the amount of PCSK9 protein in a cell-based phenotypic assay. Our SAR studies yielded compound 16 with a IC₅₀-value of 200 nM. No obvious cytotoxicity was observed at concentrations below 50 µM.     

What are the genomic drivers of the rapid evolution of PRDM9?

Mammalian Prdm9 has been proposed to be a key determinant of the positioning of chromosome double-strand breaks during meiosis, a contributor to speciation processes, and the most rapidly evolving gene in human, and other animal, genomes. Prdm9 genes often exhibit substantial variation in their numbers of encoded zinc fingers (ZFs), not only between closely related species but also among individuals of a species. The near-identity of these ZF sequences appears to render them very unstable in copy number. The rare sequence differences, however, cluster within ZF sites that determine the DNA-binding specificity of PRDM9, and these substitutions are frequently positively selected. Here, possible drivers of the rapid evolution of Prdm9 are discussed, including selection for efficient pairing of homologous chromosomes or for recombination of deleterious linked alleles, and selection against depletion of recombination hotspots or against disease-associated genome rearrangement.     

Immunohistochemical localization of thymosin β9 in bovine tissues

Summary Using an indirect fluorescent antibody technique with frozen sections, the localization of thymosin ₉ was investigated for the first time in bovine thymus, spleen, lung, muscle and liver. The antibodies used have been raised against the N-terminal fragment 1–14 of thymosin ₉ in order to minimize the cross-reactivity with thymosin ₄ which was found to be also present in bovine tissues. The specific antibodies against thymosin ₉ raised in our laboratory allowed us to localize this peptide in presence of the highly homologous and always accompanying thymosin ₄ in different tissues. Although thymosin ₉ was first isolated from calf thymus, it could be also detected in other bovine organs. The highest density of positive immunoreaction was found to be in spleen sections. In the muscle tissue a pronounced fluorescence intensity was present in the region of the sarcolemn.     

Preparation of 9-β-D-Arabinofuranosylguanine (araG)

Abstract

A convenient practical synthesis of araG is described.     

Purification of Cas9—RNA complexes by ultrafiltration

The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.