Synthesis of carbamate-type caged derivatives of a novel glutamate transporter blocker

L-threo-beta-Benzyloxyaspartate (L-TBOA) and (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (L-TFB-TBOA) are potent nontransportable blockers for glutamate transporters. We synthesized a carbamate-type coumarin derivative of L-TBOA 3a as a caged blocker and compared 3a with the corresponding ester-type analogs 1. The carbamate 3a was less sensitive to photolysis than the ester 1 but was more stable in the aqueous solution. The [6,7-bis(carboxymethoxy)-coumarin-4-yl]methylcarbonyl (BCMCMC) group exhibited good results both in photoreactivity and stability. Therefore, we examined photolysis of N-BCMCMC-TBOA 3b and N-BCMCMC-TFB-TBOA 4, which immediately released blockers to show glutamate uptake inhibition.     

Syntheses of optically pure beta-hydroxyaspartate derivatives as glutamate transporter blockers

DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a blocker and the transporters, we synthesized the optically pure isomers (L- and D-TBOA) and its erythro-isomers. L-TBOA is the most potent blocker for the human excitatory amino acid transporters (EAAT1-3), while D-TBOA revealed a difference in the pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives) of L-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers.     

A protecting group for carboxylic acids that can be photolyzed by visible light

We report on a photolabile protecting (caging) group that is new for carboxylic acids. Unlike previously used caging groups for carboxylic acids, it can be photolyzed rapidly and efficiently in the visible wavelength region. The caging group 7-N,N-diethyl aminocoumarin (DECM) was used to cage the gamma-carboxyl group of glutamic acid, which is also a neurotransmitter. The caged compound has a major absorption band with a maximum at 390 nm (epsilon(390) = 13651 M(-)(1) cm(-)(1)). Experiments are performed at 400 nm (epsilon(400) = 12232 M(-)(1) cm(-)(1)) and longer wavelengths. DECM-caged glutamate is water soluble and stable at pH 7.4 and 22 degrees C. It photolyzes rapidly in aqueous solution to release glutamic acid within 3 micros with a quantum yield of 0.11 +/- 0.008 in the visible region. In whole-cell current-recording experiments, using HEK-293 cells expressing glutamate receptors and visible light for photolysis, DECM-caged glutamate and its photolytic byproducts were found to be biologically inert. Neurotransmitter receptors that are activated by various carboxyl-group-containing compounds play a central role in signal transmission between approximately 10(12) neurons of the nervous system. Caged neurotransmitters have become an essential tool in transient kinetic investigations of the mechanism of action of neurotransmitter receptors. Previously uncaging the compounds suitable for transient kinetic investigations required ultraviolet light and expensive lasers, and, therefore, special precautions. The availability of caged neurotransmitters suitable for transient kinetic investigations that can be photolyzed by visible light allows the use of simple-to-use, readily available inexpensive light sources, thereby opening up this important field to an increasing number of investigators.     

Effects of threo-β-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5)

D,L-threo-beta-Benzyloxyaspartate (D,L-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.     

Factors affecting the uncaging efficiency of 500 nm light-activatable BODIPY caging group

Photoremovable protective groups, or caging groups, enable us to regulate the activities of bioactive molecules in living cells upon photoirradiation. Nevertheless, requirement of UV light for activating caging group is a significant limitation due to its cell toxicity and its poor tissue penetration. Our group previously reported a 500?nm light-activatable caging group based on BODIPY scaffold, however, its uncaging efficiency was lower than those of conventional caging groups. Here we show that the uncaging quantum yield (QY) of BODIPY caging group depends upon the driving force of photo-induced electron transfer (PeT). We also found that the uncaging QY increased in less polar solvents. We applied these findings to develop BODIPY-caged capsaicin, which is well localized to low-polarity intracellular compartments, as a tool to stimulate TRPV1 in live cells in response to blue-green light.     

Nucleobase‐caged peptide nucleic acids: PNA/PNA duplex destabilization and light‐triggered PNA/PNA recognition

The 2‐(o‐nitrophenyl)‐propyl (NPP) group is used as caging group to mask the nucleobases adenine and cytosine in N‐(2‐aminoethyl)glycine peptide nucleic acids (aeg‐PNA). The adeninyl and cytosinyl nucleo amino acid building blocks Fmoc‐a^NPP‐aeg‐OH and Fmoc‐c^NPP‐aeg‐OH were synthesized and incorporated into PNA sequences by Fmoc solid phase synthesis relying on high stability of the NPP nucleobase protecting group toward Fmoc‐cleavage, coupling, capping, and resin cleavage conditions. Removal of the nucleobase caging group was achieved by UV‐LED irradiation at 365 nm. The nucleobase caging groups provided sterical crowding effecting the Watson–Crick base pairing, and thereby, the PNA double strand stabilities. Duplex formation can completely be suppressed for complementary PNA containing caging groups in both strands. PNA/PNA recognition can be completely restored by UV light‐triggered release of the photolabile protecting group. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Coumarin-caged glycine that can be photolyzed within 3 microseconds by visible light

The synthesis and characterization of a new photolabile precursor of glycine (coumarin-caged glycine) are reported. The new compound is suitable for rapid chemical kinetic investigations of the membrane-bound neurotransmitter receptor activated by glycine. Unlike previously used caging groups for glycine, this precursor can be photolyzed rapidly and efficiently in the visible wavelength region. This allows the use of a relatively inexpensive light source. The alpha-carboxyl group of glycine was covalently coupled to the 7-(diethylamino)coumarin (DECM) caging group. The caged compound has a major absorption band with a maximum at 390 nm (epsilon390 = 13,900 M-1 cm-1). Photolysis was performed at wavelengths of >or=400 nm (epsilon400 = 12,400 M-1 cm-1). Under physiological conditions, DECM-caged glycine is water soluble and stable. In the visible wavelength region, it photolyzes rapidly to release glycine with a half-life of approximately 2.5 micrometers and a quantum yield of 0.12 +/- 0.01. The experimental results demonstrated that neither DECM-caged glycine nor its byproduct inhibits or activates human alpha1 glycine receptors expressed on the surface of HEK 293 cells.     

Photocaged variants of the MunI and PvuII restriction enzymes

Regulation of proteins by light is a new and promising strategy for the external control of biological processes. In this study, we demonstrate the ability to regulate the catalytic activity of the MunI and PvuII restriction endonucleases with light. We used two different approaches to attach a photoremovable caging compound, 2-nitrobenzyl bromide (NBB), to functionally important regions of the two enzymes. First, we covalently attached a caging molecule at the dimer interface of MunI to generate an inactive monomer. Second, we attached NBB at the DNA binding site of the single-chain variant of PvuII (scPvuII) to prevent binding and cleavage of the DNA substrate. Upon removal of the caging group by UV irradiation, nearly 50% of the catalytic activity of MunI and 80% of the catalytic activity of PvuII could be restored.     

光敏掩蔽基团在化学生物学中的应用

光敏掩蔽基团技术是通过运用一种由光子控制的光敏化合物,在光子激发后,被该化合物掩蔽处于惰性状态的生物活性分子重新被激活、释放以调节生理功能的化学生物学研究方法,它对生命活动的控制具有实时、原位、精确、快速的优势。该文综述了不同光敏基团的结构与功能,包括硝基苄基类、香豆素类、喹啉类、吲哚类、硝基二苯并呋喃类等,它们通过掩蔽神经递质、钙离子、蛋白质、缩氨酸、核苷酸、遗传物质等重要的生理活性物质来高选择性地调控不同的生物学过程。     

Caged RNA: photo-control of a ribozyme reaction.

We report here the first photo-chemical control of a ribozyme reaction by the site-specific modification of the 2'-hydroxyl nucleophile in the hammerhead system with a caging functionality. Rapid laser photolysis of the O-(2-nitrobenzyl) caging group initiates an efficient and accurate hammerhead-catalyzed cleavage of substrate RNA under native conditions. RNAs in which reactive functionalities or recognition elements are caged in this manner will be useful tools to probe RNA reactivity and dynamics.     

Environmental Enrichment Alters Splenic Immune Cell Composition and Enhances Secondary Influenza Vaccine Responses in Mice

Chronic stress has deleterious effects on immune function, which can lead to adverse health outcomes. However, studies investigating the impact of stress reduction interventions on immunity in clinical research have yielded divergent results, potentially stemming from differences in study design and genetic heterogeneity, among other clinical research challenges. To test the hypothesis that reducing glucocorticoid levels enhances certain immune functions, we administered influenza vaccine once (prime) or twice (boost) to mice housed in either standard control caging or environmental enrichment (EE) caging. We have shown that this approach reduces mouse corticosterone production. Compared with controls, EE mice had significantly lower levels of fecal corticosterone metabolites (FCMs) and increased splenic B and T lymphocyte numbers. Corticosterone levels were negatively associated with the numbers of CD19⁺ (r² = 0.43, p = 0.0017), CD4⁺ (r² = 0.28, p = 0.0154) and CD8⁺ cells (r² = 0.20, p = 0.0503). Vaccinated mice showed nonsignificant differences in immunoglobulin G (IgG) titer between caging groups, although EE mice tended to exhibit larger increases in titer from prime to boost than controls; the interaction between the caging group (control versus EE) and vaccine group (prime versus boost) showed a strong statistical trend (cage-group*vaccine-group, F = 4.27, p = 0.0555), suggesting that there may be distinct effects of EE caging on primary versus secondary IgG vaccine responses. Vaccine-stimulated splenocytes from boosted EE mice had a significantly greater frequency of interleukin 5 (IL-5)-secreting cells than boosted controls (mean difference 7.7, IL-5 spot-forming units/10⁶ splenocytes, 95% confidence interval 0.24–135.1, p = 0.0493) and showed a greater increase in the frequency of IL-5–secreting cells from prime to boost. Our results suggest that corticosterone reduction via EE caging was associated with enhanced secondary vaccine responses, but had little effect on primary responses in mice. These findings help identify differences in primary and secondary vaccine responses in relationship to stress mediators that may be relevant in clinical studies.     

An oxidatively releasable caging group that senses lipid peroxidation

A new radical-sensitive caging technique releases a caged molecule under lipid peroxidation conditions. In a competitive oxidation study with a model lipid, methyl linoleate, the oxidatively releasable xanthenyl caging groups is found to be 1.93 ± 0.55 times more reactive than the lipid model compound.     

Evaluation of individually ventilated cage systems for laboratory rodents: occupational health aspects

New ventilated caging systems for laboratory animals were compared with conventional caging regarding allergen distribution, ergonomic suitability, cage environment and animal welfare. This paper presents occupational health evaluations. Mice were placed in individually ventilated cage (IVC) systems, a ventilated cabinet, and in cages on open shelves (conventional husbandry). The IVC systems were studied at negative and positive airflow. Aeroallergens were sampled on filters (n = 204, including controls) in undisturbed rooms and during cage changing. Concentrations of mouse urinary allergen (Mus m 1) in filter eluates were measured using sandwich ELISA. An ergonomic evaluation was performed with measurement of traction forces. Staff exposure during cage changing was high in all systems, range 116-4430 ng Mus m 1/m3. In undisturbed animal rooms, allergen levels were orders of magnitude higher when using conventional caging compared with ventilated systems; P < 0.001. At positive pressure both IVCs leaked allergen (median Mus m 1 concentration was < 0.08 ng/m3 at negative, but 6.5 ng/m3 (IVC1) and 0.8 ng/m3 (IVC2S) at positive pressure). The IVC systems had ergonomic disadvantages compared with the conventional husbandry and the ventilated cabinet, for instance with cages in unsuitable working heights. Ventilated husbandry solutions reduce levels of airborne allergen substantially at negative pressure, but are ergonomically less suitable. To prevent allergen exposure during cage changing, we propose that this procedure should be performed under ventilated conditions. Producers and users must cooperate in optimizing animal caging systems for both animals and staff.     

Inhibition of non-vesicular glutamate release by group III metabotropic glutamate receptors in the nucleus accumbens

Previous in vitro studies have shown that group III metabotropic glutamate receptors (mGluRs) regulate synaptic glutamate release. The present study used microdialysis to characterize this regulation in vivo in rat nucleus accumbens. Reverse dialysis of the group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (L-AP4) decreased, whereas the antagonist (R,S)-alpha-methylserine-O-phosphate (MSOP) increased the extracellular level of glutamate. The decrease by L-AP4 or the increase by MSOP was antagonized by co-administration of MSOP or L-AP4, respectively. Activation of mGluR4a by (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid or mGluR6 by 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid had no effect on extracellular glutamate. (R,S)-4-Phosphonophenylglycine (PPG), another group III agonist with high affinity for mGluR4/6/8, reduced extracellular glutamate only at high concentrations capable of binding to mGluR7. The increase in extracellular glutamate by MSOP was tetrodotoxin-independent, and resistant to both the L-type and N-type Ca2+ channel blockers. L-AP4 failed to block 30 mm K+-induced vesicular glutamate release. Blockade of glutamate uptake by d,l-threo-beta-benzyloxyaspartate caused a Ca2+-independent elevation in extracellular glutamate that was reversed by L-AP4. Finally, (S)-4-carboxyphenylglycine, an inhibitor of cystine-glutamate antiporters, attenuated the L-AP4-induced reduction in extracellular glutamate. Together, these data indicate that group III mGluRs regulate in vivo extracellular glutamate in the nucleus accumbens by inhibiting non-vesicular glutamate release.     

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.     

Nectar depletion and its implications for honeyeaters in heathland near Sydney

Several researchers have attempted to calculate whether depression of nectar resources by Australian honeyeaters is likely to limit their densities. Such calculations can be misleading, however, and do not directly test whether birds depress nectar availability. I monitored changes in nectar availability during the 8–9 months that honeyeaters bred in heathland near Sydney, and caged inflorescences to test whether nectar availability was being depressed by birds. There were pronounced seasonal changes in nectar availability in each of 2 years, and caging substantially increased the amounts of nectar in inflorescences during months when nectar production was low. The effects of caging must have resulted from exclusion of honeyeaters, as: (i) open-ended cage controls showed that the effects of caging resulted from exclusion of foragers, not from artifacts of caging; (ii) day-only and night-only caging showed that nectar was depleted only during the day: and (iii) observations showed that cages did not exclude any diurnal foragers other than honeyeaters. Resident honeyeaters spent more time foraging during months when nectar was scarce, implying that the rates at which they could obtain nectar were affected by changes in nectar availability. It is therefore possible that the depletion of nectar by honeyeaters could have limited their densities. However. I argue that such limitation could only be inferred safely if nectar-supplementation experiments showed survival and/or reproduction to be limited by nectar availability.     

A renewed look at laboratory rodent housing and management

Since its publication in 1996, the Guide for the Care and Use of Laboratory Animals (National Research Council, Washington DC, National Academy Press) has become a primary source of information for institutional animal care and use committees (IACUCs) and research facility managers. In the ensuing years, recommendations relating to laboratory animal care have evolved in response to new scientific information and use of new technology such as ventilated caging. In this article, recent publications are examined to determine the potential impact of new scientific evidence on current practices for the housing and care of laboratory rodents. The discussion points out recent advances in technology and new knowledge of the conditions for the housing of various laboratory rodents, including cage space, single versus group housing, ventilated caging systems, thermoregulation, bedding materials, and enrichment. This new information is provided to aid IACUCs and facility managers in making decisions regarding the housing and care of laboratory rodents.     

The calm mouse: an animal model of stress reduction

Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a "calm mouse model" with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.     

Urologic syndrome associated with wire caging in AKR mice

Individually housed male AKR/NCrlBR mice used in a chronic inhalation experiment were noted to develop a severe obstructive genitourinary condition. The mouse urologic syndrome (MUS) had one or more of the following features: bladder distension; peripreputial urine staining, alopecia, and edema; paraphimosis; urethral blockage; ulcerative balanophosthitis; hydronephrosis; pyelonephritis; rectal prolapse; and perineal ulcerative dermatitis. MUS was less severe and less prevalent in similarly housed B6C3F1/CrlBR and NIH-Swiss mice used in the same experiment. Epidemiologic evidence within the animal facility restricted the syndrome to the inhalation toxicology area. The effects of intermittent water deprivation as well as wire caging on the development of MUS were studied because these conditions were only utilized in the inhalation facility. Male AKR/NCrlBR mice, caged individually in suspended wire caging or kept isolated in polystyrene shoebox style cages containing wire floorwalk bottoms, all developed MUS within 16 weeks. Mice which were housed directly on hardwood bedding in identical plastic caging remained free of the syndrome, as did castrated males which were kept in suspended wire cages. Water deprivation was not found to be a major contributing factor to the development of the condition, but was found to augment its severity. We concluded that although MUS is probably multifactorial in etiology, housing susceptible animals on wire bottom caging may exacerbate the incidence and severity of the condition in certain strains of male mice.