Design,synthesis and evaluation of γ-turn mimetics as LSD1-selective inhibitors

Lysine-specific demethylase 1 (LSD1) is an attractive molecular target for cancer therapy. We have previously reported potent LSD1-selective inhibitors (i.e., NCD18, NCD38, and their analogs) consisting of trans-2-phenylcyclopropylamine (PCPA) or trans-2-arylcyclopropylamine (ACPA) and a lysine moiety that could form a γ-turn structure in the active site of LSD1. Herein we report the design, synthesis and evaluation of γ-turn mimetic compounds for further improvement of LSD1 inhibitory activity and anticancer activity. Among a series of γ-turn mimetic compounds synthesized by a Mitsunobu-reaction-based amination strategy, we identified 1n as a potent and selective LSD1 inhibitor. Compound 1n induced cell cycle arrest and apoptosis through histone methylation in human lung cancer cells. The γ-turn mimetics approach should offer new insights into drug design for LSD1-selective inhibitors.     

A cylindrical thermistor probe for experiments on suppositories in rabbits

A cylindrical thermistor probe with a rubber disk stopper, which is beneficial for inserting a suppository into the rectum of rabbits without removal of the probe from the rectum, was developed. It is possible to measure body temperature continuously by using this probe in an experiment to assess the effects of a suppository on the body temperature of rabbits. After insertion of a suppository, leakage of the melted suppository from the rectum was not observed. No differences between this thermistor probe and an ordinary thermistor probe in the ability of the probe to detect the febrile response of rabbits injected with a bacterial pyrogen were observed. From these results, it could be concluded that this newly improved cylindrical thermistor probe is suitable for studying the effect of suppositories on the body temperature of rabbits.     

Quinoline-based, glucose-pendant fluorescent zinc probes

Quinoline-based tetradentate ligands with glucose pendants, N,N'-bis[2-(β-d-glucopyranosyloxy)ethyl]-N,N'-bis[(6-methoxyquinolin-2-yl)methyl]ethylenediamine (N,N'-6-MeOBQBGEN) and its N,N-counterpart, N,N-6-MeOBQBGEN, have been prepared, and their fluorescence-spectral changes upon Zn binding were investigated. Upon excitation at 336?nm, N,N'-6-MeOBQBGEN showed weak fluorescence (?≈ 0.016) in HEPES buffer (HEPES 50?mM, KCl 100?mM, pH?7.5). In the presence of Zn, N,N'-6-MeOBQBGEN exhibited a significant increase in fluorescence (?=0.096) at 414?nm. The fluorescence enhancement is specific for Zn and Cd (I(Cd) /I(Zn) of 50% at 414?nm). On the other hand, N,N-6-MeOBQBGEN exhibited a smaller fluorescence enhancement upon Zn complexation (?=0.043, λ(ex) =334?nm, λ(em) =407?nm) compared with N,N'-6-MeOBQBGEN. Fluorescence microscopic analysis using PC-12 rat adrenal cells revealed that N,N'-6-MeOBQBGEN exhibits a 1.8-fold higher fluorescence-signal response to Zn ion concentration compared with sugar-depleted compound 2 (N,N'-bis[(6-methoxyquinolin-2-yl)methyl]ethylenediamine), due to its enhanced uptake into cells due to the targeting ability of the attached carbohydrates.     

Truncation and non-natural amino acid substitution studies on HTLV-I protease hexapeptidic inhibitors

The culprit behind adult T-cell leukemia, myelopathy/tropical paraparesis, and a plethora of inflammatory diseases is the human T-cell leukemia virus type 1 (HTLV-I). We recently unveiled a potent hexapeptidic HTLV-I protease inhibitor, KNI-10166, composed mostly of natural amino acid residues. Herein, we report the derivation of potent tetrapeptidic inhibitor KNI-10516, possessing only non-natural amino acid residues.     

C-terminal Src kinase controls development and maintenance of mouse squamous epithelia

Carboxy-terminal Src kinase (Csk) is a negative regulator of Src family kinases, which play pivotal roles in controlling cell adhesion, migration, and cancer progression. To elucidate the in vivo role of Csk in epithelial tissues, we conditionally inactivated Csk in squamous epithelia using the keratin-5 promoter/Cre-loxP system in mice. The mutant mice developed apparent defects in the skin, esophagus, and forestomach, with concomitant hyperplasia and chronic inflammation. Histology of the mutant epidermis revealed impaired cell-cell adhesion in basal cell layers. Analysis of primary keratinocytes showed that the defective cell-cell adhesion was caused by cytoskeletal remodeling via activation of the Rac1 pathway. Mutant keratinocytes also showed elevated expression of mesenchymal proteins, matrix metalloproteinases (MMPs), and the proinflammatory cytokine TNF-alpha. Inhibition of the expression of TNF-alpha and MMP9 by the anti-inflammatory reagent FK506 could cure the epidermal hyperplasia, suggesting a causal link between inflammation and epidermal hyperplasia. These observations demonstrate that the Src/Csk circuit plays crucial roles in development and maintenance of epithelia by controlling cytoskeletal organization as well as phenotypic conversion linked to inflammatory events.     

Studies on the Dutch rabbit for pyrogen test

A study was conducted on Dutch rabbits to examine their applicability for the pyrogen test in comparison with the Japanese white rabbit which, conventionally, has been mainly used for the test in Japan. The following results were obtained. Adult Dutch rabbits, so small as to weight only about 60% of average adult weight of the Japanese white, showed a food consumption as low as 40% of that in the latter. None in the Dutch rabbit group suffered dislocation of the hip-joint during restraint on a neck stock while it was frequently encountered among Japanese white rabbits. The body temperature during restraint was unstable in infantile Dutch rabbits (7-8 weeks old)(, but stable in the young (14 weeks old) and in the adult (20-47 weeks old). Adult Dutch rabbits exhibited greater febrile responses to intravenously injected bacterial pyrogen (LPS) than infantile and young Dutch rabbits and adult Japanese white rabbits. A good linear regression was observed between the dose of LPS (0.01-0.1 microgram/kg, iv) and the febrile response in adult Dutch rabbits as well as in adult Japanese white rabbits. From these data, it is concluded that the Dutch rabbit has an advantage over the Japanese white for the pyrogen test in respect of febrile responsiveness and cost of rearing.     

Application of attenuated Lactococcus garvieae strain lacking a virulence-associated capsule on its cell surface as a live vaccine in yellowtail Seriola quinqueradiata Temminck and Schlegel

An attenuated Lactococcus garvieae strain lacking a virulence‐associated capsule on its cell surface was evaluated for application as a live vaccine. The attenuated strain (MS93003A) was obtained from the parent strain (MS93003V), which produced a well‐developed capsule, by culturing on an agar medium supplemented with 2,3,5‐triphenyltetrazolium chloride. When live cells of L. garvieae (MS93003A) or formalin‐killed cells (MS93003A) were used as an injectable vaccine, protection against virulent L. garvieae (MS93003V) was conferred on Seriola quinqueradiata. The MS93003A cells did not recover their virulence even after in vivo passages in fish. MS93003A live cells also conferred long‐lasting protective immunity to S. quinqueradiata against virulent L. garvieae infection.     

Divergence of Eurosta solidaginis in response to host plant variation and natural enemies

We tested the hypothesis that forest and prairie populations of the gall-inducing fly, Eurosta solidaginis, have diverged in response to variation in selection by its host plant Solidago altissima, and its natural enemies. A reciprocal cross infection design experiment demonstrated that fly populations from the prairie and forest biomes had higher survival on local biome plants compared to foreign biome host plants. Flies from each biome also had an oviposition preference for their local plants. Each fly population induced galls of the size and shape found in their local biome on host plants from both biomes indicating a genetic basis to the differences in gall morphology. Solidago altissima from the prairie and forest biomes retained significant morphological differences in the common garden indicating that they are genetically differentiated, possibly at the subspecies level. The populations are partially reproductively isolated as a result of a combination of prezygotic isolation due to host-associated assortative mating, and postzygotic isolation due to low hybrid survival. We conclude that E. solidaginis is undergoing diversifying selection to adapt to differences between prairie and forest habitats.     

Improved conception in timed-artificial insemination using a progesterone-releasing intravaginal device and Ovsynch protocol in postpartum suckled Japanese Black beef cows

The primary objective was to determine the effect of supplemental progesterone, administered via an intravaginal device (CIDR), on conception rates to timed-artificial insemination (timed-AI) in postpartum suckled Japanese Black beef cows treated with the Ovsynch protocol. A secondary objective was to compare the effects of treatments on plasma concentrations of progesterone and estradiol. Cows in the control group (Ovsynch, n=38) received a standard Ovsynch protocol (100 microg GnRH analogue on Day 0, 500 microg PGF2alpha analogue on Day 7, and 100 microg GnRH analogue on Day 9), with AI on Day 10, approximately 20 h after the second GnRH treatment. Cows in the treatment group (Ovsynch+CIDR; n=40) received a standard Ovsynch protocol plus a CIDR for 7 days (starting on Day 0). Plasma progesterone concentrations were determined on Days 0, 1, 7, 9, 10, and 17 and plasma estradiol-17beta concentrations were determined on Days 7, 9, 10, and 17. The odds ratio for likelihood of conception was 3.29 times greater (P=0.02) in the Ovsynch+CIDR group compared to Ovsynch group. The conception rate was greater (P=0.03) in the Ovsynch+CIDR group than in the Ovsynch group (72.5% versus 47.7%). Insertion of a CIDR device significantly increased plasma progesterone concentrations only on Days 1 and 7 (P<0.001 and P=0.05, respectively), but had no significant effect on plasma estradiol-17beta concentrations. Including a CIDR with the Ovsynch protocol significantly improved conception rates in postpartum suckled Japanese Black beef cows.