Investigation of a MMP-2 Activity-Dependent Anchoring Probe for Nuclear Imaging of Cancer

Purpose

Since matrix metalloproteinase-2 (MMP-2) is an important marker of tumor malignancy, we developed an original drug design strategy, MMP-2 activity dependent anchoring probes (MDAP), for use in MMP-2 activity imaging, and evaluated the usefulness of this probe in in vitro and in vivo experiments.

Methods

We designed and synthesized MDAP₁₀₀₀, MDAP₃₀₀₀, and MDAP₅₀₀₀, which consist of 4 independent moieties: RI unit (¹¹¹In hydrophilic chelate), MMP-2 substrate unit (short peptide), anchoring unit (alkyl chain), and anchoring inhibition unit (polyethylene glycol (PEGn; where n represents the approximate molecular weight, n = 1000, 3000, and 5000). Probe cleavage was evaluated by chromatography after MMP-2 treatment. Cellular uptake of the probes was then measured. Radioactivity accumulation in tumor xenografts was evaluated after intravenous injection of the probes, and probe cleavage was evaluated in tumor homogenates.

Results

MDAP₁₀₀₀, MDAP₃₀₀₀, and MDAP₅₀₀₀ were cleaved by MMP-2 in a concentration-dependent manner. MDAP₃₀₀₀ pretreated with MMP-2 showed higher accumulation in tumor cells, and was completely blocked by additional treatment with an MMP inhibitor. MDAP₃₀₀₀ exhibited rapid blood clearance and a high tumor accumulation after intravenous injection in a rodent model. Furthermore, pharmacokinetic analysis revealed that MDAP₃₀₀₀ exhibited a considerably slow washout rate from tumors to blood. A certain fraction of cleaved MDAP₃₀₀₀ existed in tumor xenografts in vivo.

Conclusions

The results indicate the possible usefulness of our MDAP strategy for tumor imaging.     

Input integration around the dendritic branches in hippocampal dentate granule cells

Recent studies have shown that the dendrites of several neurons are not simple translators but are crucial facilitators of excitatory postsynaptic potential (EPSP) propagation and summation of synaptic inputs to compensate for inherent voltage attenuation. Granule cells (GCs)are located at the gateway for valuable information arriving at the hippocampus from the entorhinal cortex. However, the underlying mechanisms of information integration along the dendrites of GCs in the hippocampus are still unclear. In this study, we investigated the input integration around dendritic branches of GCs in the rat hippocampus. We applied differential spatiotemporal stimulations to the dendrites using a high-speed glutamate-uncaging laser. Our results showed that when two sites close to and equidistant from a branching point were simultaneously stimulated, a nonlinear summation of EPSPs was observed at the soma. In addition, nonlinear summation (facilitation) depended on the stimulus location and was significantly blocked by the application of a voltage-dependent Ca²⁺ channel antagonist. These findings suggest that the nonlinear summation of EPSPs around the dendritic branches of hippocampal GCs is a result of voltage-dependent Ca²⁺ channel activation and may play a crucial role in the integration of input information.     

New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood

Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.     

A Behavioral Mechanism of How Increases in Leg Strength Improve Old Adults’ Gait Speed

We examined a behavioral mechanism of how increases in leg strength improve healthy old adults’ gait speed. Leg press strength training improved maximal leg press load 40% (p = 0.001) and isometric strength in 5 group of leg muscles 32% (p = 0.001) in a randomly allocated intervention group of healthy old adults (age 74, n = 15) but not in no-exercise control group (age 74, n = 8). Gait speed increased similarly in the training (9.9%) and control (8.6%) groups (time main effect, p = 0.001). However, in the training group only, in line with the concept of biomechanical plasticity of aging gait, hip extensors and ankle plantarflexors became the only significant predictors of self-selected and maximal gait speed. The study provides the first behavioral evidence regarding a mechanism of how increases in leg strength improve healthy old adults’ gait speed.     

Predominant phosphatase in the ocular lens regulated by physiological concentrations of magnesium and calcium

A magnesium-dependent phosphatase with a molecular weight of about 55 kDa was found in the lens of chicken embryo, mouse, rabbit and bovine. It appears to be unique to the lens and, when activated by magnesium, accounts for the majority of the phosphatase activity in the lens.Phosphatases in the lens were separated by using high-resolution gel permeation column chromatography, and phosphatase activity was determined with p-nitrophenyl phosphate (pNPP) as a substrate. The 55-kDa phosphatase showed very low basal activity, but the activity was increased concentration-dependently by magnesium ion (Mg(2+)), and at 1 mM Mg(2+), this enzyme accounted for over 50% of the total phosphatase activity in the lens. Calcium potently inhibited the magnesium-activated phosphatase activity in a dose-dependent (IC(50); about 50 microM), uncompetitive manner. The phosphatase activity was high in the acidic pH range, with an optimum pH value of 5.5. The characteristics of the lens Mg(2+)-dependent phosphatase, such as substrate specificity and sensitivity to various phosphatase inhibitors, showed little similarity to those of any reported phosphatase. The Mg(2+) and Ca(2+) concentrations that were found to be effective in this study are similar to physiological concentrations in the lens; therefore it is likely that these ions physiologically regulate the 55-kDa phosphatase activity in the lens.     

Improved capacity of a monkey-tropic HIV-1 derivative to replicate in cynomolgus monkeys with minimal modifications

Human immunodeficiency virus type 1 (HIV-1) hardly replicates in Old World monkeys. Recently, a mutant HIV-1 clone, NL-DT5R, in which a small part of gag and the entire vif gene are replaced with SIVmac239-derived ones, was shown to be able to replicate in pigtail monkeys but not in rhesus monkeys (RM). In the present study, we found that a modified monkey-tropic HIV-1 (HIV-1mt), MN4-5S, acquired the ability to replicate efficiently in cynomolgus monkeys as compared with the NL-DT5R, while neither NL-DT5R nor MN4-5S replicated in RM cells. These results suggest that multiple determinants may be involved in the restriction of HIV-1 replication in macaques, depending on the species of macaques. The new HIV-1mt clone will be useful for studying molecular mechanisms by which anti-viral host factors regulate HIV-1 replication in macaques.     

Analysis of the effectiveness of control measures against Schistosoma mekongi using an intra- and inter-village model in Champasak Province, Lao PDR

Schistosomiasis mekongi is prevalent in the Khong district of Lao PDR, made up of one big island, Khong, and numerous small islands in the Mekong River. Schistosoma mekongi is spread by Neotricula aperta as the intermediate host along the Mekong River. Therefore, even if an epidemic of S. mekongi were stamped out in a certain village, infection may recur if the source of infection is a village located in the upper reaches of the Mekong River. The purpose of this study was to construct a mathematical model for the transmission of S. mekongi among villages from the upper to lower Mekong River to estimate the effect of control measures against it. The chief characteristic of the present model is competence in dealing with the spread of infection among villages through the Mekong River in consideration of the reduction in longevity of cercariae and miracidia and their diffusion in the river. The model also takes into account seasonal fluctuation in the water level of the Mekong River, which affects human behavior in terms of water contact. The results of simulations indicated that the prevalence of schistosomiasis mekongi would be suppressed to a low level for a long time in a village further downstream when universal mass treatment is performed in villages further upstream simultaneously.     

High-dose green tea polyphenols induce nephrotoxicity in dextran sulfate sodium-induced colitis mice by down-regulation of antioxidant enzymes and heat-shock protein expressions

Previously, we reported that oral feeding of 1% green tea polyphenols (GTPs) aggravated the dextran sulfate sodium (DSS)-induced colitis in mice. In the present study, we assessed the toxicity of 1% GTPs in several organs from normal and DSS-exposed mice. Sixty-two male ICR mice were initially divided into four groups. Non-treated group (group 1, n = 15) was given standard diet and water, GTPs (group 2, n = 15) received 1% GTPs in diet and water, DSS (group 3, n = 15) received diet and 5% DSS in water, and GTPs + DSS group (group 4, n = 17) received 1% GTPs in diet and 5% DSS in water. We found that group 4 significantly increased (P < 0.05) kidney weight, the levels of serum creatinine and thiobarbituric acid-reactive substances in both kidney and liver, as compared with those in group 3. The mRNA expression levels of antioxidant enzymes and heat-shock proteins (HSPs) in group 4 were lower than those of group 3. For instance, heme oxygenase-1 (HO-1), HSP27, and 90 mRNA in the kidney of group 4 were dramatically down-regulated as compared with those of group 3. Furthermore, 1% GTPs diet decreased the expression of HO-1, NAD(P)H:quinone oxidoreductase 1 (NQO1) and HSP90 in kidney and liver of non-treated mice. Taken together, our results indicate that high-dose GTPs diet disrupts kidney functions through the reduction of antioxidant enzymes and heat-shock protein expressions in not only colitis but also non-treated ICR mice.     

Comparative studies of genome-wide maps of nucleosomes between deletion mutants of elp3 and hos2 genes of Saccharomyces cerevisiae

In order to elucidate the influence of histone acetylation upon nucleosomal DNA length and nucleosome position, we compared nucleosome maps of the following three yeast strains; strain BY4741 (control), the elp3 (one of histone acetyltransferase genes) deletion mutant, and the hos2 (one of histone deactylase genes) deletion mutant of Saccharomyces cerevisiae. We sequenced mononucleosomal DNA fragments after treatment with micrococcal nuclease. After mapping the DNA fragments to the genome, we identified the nucleosome positions. We showed that the distributions of the nucleosomal DNA lengths of the control and the hos2 disruptant were similar. On the other hand, the distribution of the nucleosomal DNA lengths of the elp3 disruptant shifted toward shorter than that of the control. It strongly suggests that inhibition of Elp3-induced histone acetylation causes the nucleosomal DNA length reduction. Next, we compared the profiles of nucleosome mapping numbers in gene promoter regions between the control and the disruptant. We detected 24 genes with low conservation level of nucleosome positions in promoters between the control and the elp3 disruptant as well as between the control and the hos2 disruptant. It indicates that both Elp3-induced acetylation and Hos2-induced deacetylation influence the nucleosome positions in the promoters of those 24 genes. Interestingly, in 19 of the 24 genes, the profiles of nucleosome mapping numbers were similar between the two disruptants.