A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes

Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC₅₀ values of 5.8 (meta isomer) and 3.0 μM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K_I of 0.46 μM. Radiolabeled forms of both analogues selectively labeled the β-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.     

Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA

Studies increasingly indicate that inflammation induced by β-amyloid (Aβ) contributes to the progression of Alzheimer’s disease (AD). How to inhibit the enhanced production of proinflammatory cytokines stimulated by Aβ is an important research subject for the treatment of AD. In this study, we investigated the inhibitory effect and the molecular mechanism of the lipoxin A₄ (LXA₄) on the production of interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) induced by β-amyloid in the cortex and hippocampus of mice, and in Aβ-stimulated BV2 cells, a mouse microglial cell line. LXA₄ down-regulated the protein expression of IL-1β and TNFα, attenuated the gene expressions of IL-1β and TNFα, inhibited the degradation of IκBα, inhibited translocation of NF-κB p65 subunit into the nucleus induced by β-amyloid in the cortex and hippocampus of mice, and in Aβ-stimulated BV2 cells, and the inhibitory effects were dose dependently elevated. Our findings suggest that LXA₄ inhibits the production of IL-1β and TNFα induced by β-amyloid in the cortex and hippocampus of mice, and in BV2 microglial cells via the NF-κB signal pathway.     

Artificial production and natural breeding of the endangered frog species Odorrana ishikawae, with special reference to fauna conservation in the laboratory

Odorrana ishikawae is listed as a class IB endangered species in the IUCN Red List and is protected by law in both Okinawa and Kagoshima Prefectures, Japan. Here, in an effort to help effectively preserve the genetic diversity of this endangered species in the laboratory, we tested a farming technique involving the artificial breeding of frogs, and also promoted natural breeding in the laboratory. Field-caught male/female pairs of the Amami and Okinawa Island populations were artificially bred using an artificial insemination method in the 2004, 2006, and 2008 breeding seasons (March to April). Although fewer than 50% of the inseminated eggs achieved metamorphosis, approximately 500, 300, and 250 offspring from the three respective trials are currently being raised in the laboratory. During the 2009 and 2010 breeding seasons, second-generation offspring were produced by the natural mating activities of the first offspring derived from the two artificial matings in 2004. The findings and the methods presented here appear to be applicable to the temporary protection of genetic diversity of local populations in which the number of individuals has decreased or the environmental conditions have worsened to levels that frogs are unable to survive by themselves.     

Evaluation of the lower protein limit in the budding yeast Saccharomyces cerevisiae using TIPI-gTOW

Background

Identifying permissible limits of intracellular parameters such as protein expression provides important information for examining robustness. In this study, we used the TEV protease-mediated induction of protein instability (TIPI) in combination with the genetic Tug-of-War (gTOW) to develop a method to measure the lower limit of protein level. We first tested the feasibility of this method using ADE2 as a marker and then analyzed some cell cycle regulators to reveal genetic interactions.

Results

Using TIPI-gTOW, we successfully constructed a strain in which GFP-^TDegFAde2 was expressed at the lower limit, just sufficient to support cellular growth under the -Ade condition by accelerating degradation by TEV protease. We also succeeded in constructing a strain in which the minimal level of GFP-^TDegFCdc20 was expressed by TIPI-gTOW. Using this strain, we studied genetic interactions between cell cycle regulators and CDC20, and the result was highly consistent with the previously identified interactions. Comparison of the experimental data with predictions of a mathematical model revealed some interactions that were not implemented into the current model.

Conclusions

TIPI-gTOW is useful for estimating changes in the lower limit of a protein under different conditions, such as different genetic backgrounds and environments. TIPI-gTOW is also useful for analyzing genetic interactions of essential genes whose deletion mutants cannot be obtained.     

Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin–AcGFP in Z discs

Nanometry is widely used in biological sciences to analyze the movement of molecules or molecular assemblies in cells and in vivo. In cardiac muscle, a change in sarcomere length (SL) by a mere ∼100 nm causes a substantial change in contractility, indicating the need for the simultaneous measurement of SL and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes at high spatial and temporal resolution. To accurately analyze the motion of individual sarcomeres with nanometer precision during excitation–contraction coupling, we applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nanoscale analysis of single sarcomere dynamics and [Ca²⁺]_i changes via the expression of AcGFP in Z discs. We found that the averaging of the lengths of sarcomeres along the myocyte, a method generally used in today’s myocardial research, caused marked underestimation of sarcomere lengthening speed because of the superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that after treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (cell-SPOCs) at partial activation with blockage of sarcoplasmic reticulum functions, and the waveform properties were indistinguishable from those obtained in electric field stimulation. The myosin activator omecamtiv mecarbil markedly enhanced Z-disc displacement during cell-SPOC. Finally, we interpreted the present experimental findings in the framework of our mathematical model of SPOCs. The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation–contraction coupling in cardiomyocytes under various settings.     

Effect of Calcium in Assay Medium on D Value of Bacillus stearothermophilus ATCC 7953 Spores

The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of D value.     

Salivary proline-rich protein polymorphisms in Chinese, Malays and Indians in Singapore

Salivary proline-rich protein (PRP) polymorphism, PRH1, PRH2, Ps, Pm (PmF), PmS and Gl, were investigated in three ethnic groups in Singapore: Chinese, Malays and Indians. The phenotype and gene frequencies were presented and comparison with other ethnic groups was made. The As protein, which was recently found in Japanese but not in Caucasians as a new allelic product of the PRH1 locus, was also observed in Chinese and Malays but not in Indians. Another allelic product (Ps4) of Ps protein polymorphism was found in Malays but not in Chinese and Indians. The results indicate the usefulness of salivary PRP polymorphism as markers in population genetic studies.     

Mechanism of action of a streptococcal preparation (OK-432) in prevention of autoimmune diabetes in NOD mice. Suppression of generation of effector cells for pancreatic B cell destruction

We have previously reported that treatment with a streptococcal preparation (OK-432), one of the biologic response modifiers, inhibits insulitis and development of autoimmune diabetes in nonobese diabetic (NOD) mice and Bio-Breeding rats used as animal models of insulin-dependent diabetes mellitus (IDDM). We studied the mechanism by which OK-432 inhibited development of IDDM in NOD mice. In female NOD mice diabetes spontaneously developed from 10 to 15 wk of age and the cumulative incidence of diabetes amounted to 74.7% at 30 wk of age. NOD mice, however, never developed diabetes over the observation period of up to 45 wk of age when they were i.p. injected with 0.1 mg of OK-432 weekly from 4 to 15 wk of age. OK-432 treatment in younger age had a stronger inhibitory effect on the development of diabetes. Diabetes was transferred to young, irradiated mice by spleen cells of nontreated, adult mice, but barely transferred by those of OK-432-treated mice. Furthermore, these spleen cells of OK-432-treated mice did not suppress transfer of diabetes by diabetic mice spleen cells. Treatment with cyclophosphamide promoted development of diabetes in nontreated NOD mice due to removal of suppressor cells. However, cyclophosphamide did not show the promotive effect in OK-432-treated mice. Taken together, these results indicate that OK-432 treatment prevented development of diabetes mainly by suppression in generation of the effector cells for pancreatic B cell destruction. The same OK-432 treatment did not suppress the immune response to exogenous AG such as xenogeneic SRBC and allogeneic tumor cells. The study suggests that BRM in the natural environment such as streptococci may suppress induction and progression of autoimmunity and be useful for the immunotherapy of human IDDM.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments for a chemokine receptor-binding domain of FROUNT,a cytoplasmic regulator of chemotaxis

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain ¹H, ¹³C, and ¹⁵N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the ¹H–¹⁵N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.