C ameleons are genetically encoded fluorescent indicators for Ca 2ϩ based on GFP variants and calmodulin (CaM) (1, 2). They are chimeric proteins composed of a short-wavelength variant of GFP, CaM, a glycylglycine linker, the CaM-binding peptide of myosin light-chain kinase (M13), and a longwavelength variant of GFP. Ca 2ϩ binding to CaM initiates an intramolecular interaction between CaM and M13, which changes the chimeric protein from an extended to a more compact conformation, thereby increasing the efficiency of fluorescence resonance energy transfer (FRET) from the shorter-to the longer-wavelength variant of GFP. Yellow cameleons (YCs) have cyan and yellow fluorescent proteins (CFP and YFP) as the FRET donor and acceptor, respectively. YCs are classified into several groups based on the composition of their Ca 2ϩ -sensing domains. For example, YC2 has an intact CaM and thus shows high affinity for Ca 2ϩ . On the other hand, YC3 and YC4 are low-affinity indicators because of mutations in the Ca 2ϩ -binding loops of their CaM domains. These YCs have been made more resistant to acidification by replacing the original YFP with EYFP.1 (3). The improved YCs include YC2.1 and YC3.1. In addition, some YCs have been made to mature more quickly by using especially bright versions of YFP such as citrine (4) or Venus (5). In this way, YCs have been improved mainly by optimizing the YFP component.Despite the above-mentioned improvements, YCs still suffer from poor dynamic range. The best versions available currently, such as YC2.12 or YC3.12, exhibit at most a 120% change in the ratio of YFP͞CFP upon Ca 2ϩ binding in vitro. These YCs do not have good signal-to-noise ratios, particularly when they are targeted to organelles or submicroscopic environments, because of low levels of signal. It has been also suggested that their dynamic range is attenuated in vivo depending on the abundance of endogenous CaM and CaM-binding proteins that may interact with the sensing domains of YCs.In the present study, we have attempted to modify the acceptor to increase the dynamic range of the indicator. To achieve a Ca 2ϩ -dependent large change in the relative orientation and distance between the fluorophores of CFP and YFP, we assumed that optimization of the length and sequence of the linkers used in YCs would yield only moderate improvement. Thus, we took a more rigorous approach that used a circularly permuted GFP (cpGFP), in which the N and C portions were interchanged and reconnected by a short spacer between the original termini (6, 7). By using cpYFPs that are resistant to acidification and that mature efficiently, we attempted to vary the relative orientation of the two chromophores' transition dipoles. Materials and MethodsGene Construction. The cDNAs of the 5Ј portions of the cpVenus variants were amplified by PCR using sense primers containing a BamHI site and reverse primers containing the sequence encoding the linker (GGSGG) between the natural N and C termini. The cDNAs of their 3Ј portions were extended by PCR at the 5Ј ...

Associating temporally discontinuous elements is crucial for the formation of episodic and working memories that depend on the hippocampal-entorhinal network. However, the neural circuits subserving these associations have remained unknown. The layer III inputs of the entorhinal cortex to the hippocampus may contribute to this process. To test this hypothesis, we generated a transgenic mouse in which these inputs are specifically inhibited. The mutant mice displayed significant impairments in spatial working-memory tasks and in the encoding phase of trace fear-conditioning. These results indicate a critical role of the entorhinal cortex layer III inputs to the hippocampus in temporal association memory.

Defensins comprise a family of cationic antimicrobial peptides that are characterized by the presence of six conserved cysteine residues. We identified two novel human β-defensin (hBD) isoforms by mining the public human genomic sequences. The predicted peptides conserve the six-cysteine motif identical with hBD-4, termed hBD-5 and hBD-6. We also evaluated the characteristics of the mouse homologs of hBD-5, hBD-6, and HE2β1, termed mouse β-defensin (mBD)-12, mBD-11, and mouse EP2e (mEP2e). The mBD-12 synthetic peptide showed salt-dependent antimicrobial activity. We demonstrate the epididymis-specific expression pattern of hBD-5, hBD-6, mBD-11, mBD-12, and mEP2e. In situ hybridization revealed mBD-11, mBD-12, and mEP2e expression in the columnar epithelium of the caput epididymis, contrasting with the predominant expression of mBD-3 in the capsule or septum of the whole epididymis. In addition, the regional specificity of mBD-11, mBD-12, and mEP2e was somewhat overlapping, but not identical, in the caput epididymis, suggesting that specific regulation may work for each member of the β-defensin family. Our findings indicated that multiple β-defensin isoforms specifically and cooperatively contribute to the innate immunity of the urogenital system.

The entorhinal cortex provides the major cortical input to the hippocampus, and both structures have been implicated in memory processes. The dynamics of neuronal circuits in the entorhinal-hippocampal system were studied in slices by optical imaging with high spatial and temporal resolution. Reverberation of neural activity was detected in the entorhinal cortex and was more prominent when the inhibition due to gamma-aminobutyric acid was slightly suppressed. Neural activity was transferred in a frequency-dependent way from the entorhinal cortex to the hippocampus. The entorhinal neuronal circuit could contribute to memory processes by holding information and selectively gating the entry of information into the hippocampus.

Formation of neurofibrillary tangles (NFTs) is a common neuropathological feature found in several neurodegenerative diseases, including Alzheimer's disease. We have developed a transgenic (Tg) mouse expressing mutant human tau (V337M), derived from frontotemporal dementia parkinsonism-17. V337M Tg mice revealed tau aggregations in the hippocampus, which fulfills the histological criteria for NFTs in human neurodegenerative diseases. Concurrent with the accumulation of RNA and phosphorylated tau, neurons exhibited morphological characteristics of degenerating neurons, which include a loss of microtubules, accumulation of ribosomes, plasma and nuclear membrane ruffling, and swelling of the Golgi network. Thus, mutant tau induces neuronal degeneration associated with the accumulation of RNA and phosphorylated tau. The functional consequences of this neuronal degeneration was evidenced by the reduction of hippocampal neural activity and behavioral abnormality in Tg mice.

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SummaryPrenatal exposure to valproic acid (VPA), an established antiepileptic drug, has been reported to impair postnatal cognitive function in children born to VPA-treated epileptic mothers. However, how these defects arise and how they can be overcome remain unknown. Using mice, we found that comparable postnatal cognitive functional impairment is very likely correlated to the untimely enhancement of embryonic neurogenesis, which led to depletion of the neural precursor cell pool and consequently a decreased level of adult neurogenesis in the hippocampus. Moreover, hippocampal neurons in the offspring of VPA-treated mice showed abnormal morphology and activity. Surprisingly, these impairments could be ameliorated by voluntary running. Our study suggests that although prenatal exposure to antiepileptic drugs such as VPA may have detrimental effects that persist until adulthood, these effects may be offset by a simple physical activity such as running.

Aim : We evaluated the efficacy of an α 1a/d blocker, naftopidil, on storage symptoms in patients with benign prostatic hyperplasia (BPH), using frequency/volume charts (FVC). Methods : A total of 81 patients with BPH (52-91 years, mean age 69.0 years) were studied. The inclusion criteria were: (i) one or more episode(s) of urinary urgency/day; (ii) a score of eight or more points on the International Prostate Symptom Score (I-PSS); and (iii) three or more points in any of the scores for three items (frequency, nocturia, and urgency) of the I-PSS. The patients received 50-75 mg/day of naftopidil for 6 weeks. All the patients were examined for 2-day FVC before and after the administration of naftopidil. I-PSS, quality of life index, and uroflowmetry were also evaluated. Results : Total I-PSS decreased from 19.1 to 10.5 points ( P < 0.0001), with significant improvement of both storage and voiding symptom scores ( P < 0.0001, both). The score for urgency decreased from 3.1 to 1.4 ( P < 0.0001). Daytime and night-time frequency decreased from 9.3 to 8.0 ( P < 0.0001) and from 2.7 to 2.0 ( P = 0.0009), respectively. Mean volume/void increased from 174.0 to 188.6 mL ( P = 0.0453). Nocturia decreased from 3.2 to 2.3 ( P < 0.0001) in 40 patients who suffered from nocturia two times or more. Notably, significant improvement of nocturia was observed in the patients both with and without nocturnal polyuria ( P = 0.0006 and 0.0135, respectively). Conclusion : The α 1a/d blocker naftopidil improves not only voiding symptoms but also storage symptoms, and is effective for nocturia in patients with BPH regardless of the existence of nocturnal polyuria.