Independent responses of Pacinian and Non-Pacinian systems with hand-transmitted vibration detected from masked thresholds

This study was designed to identify psychophysical channels responsible for the detection of hand-transmitted vibration. Perception thresholds for vibration (16, 31.5, 63 and 125 Hz sinusoidal for 600 ms) at the distal phalanx of the middle finger and the whole hand were determined with and without simultaneous masking stimuli (1/3 octave bandwidth Gaussian random vibration centered on either 16 Hz or 125 Hz for 3000 ms, varying in magnitude 0 to 30 dB above threshold). At all frequencies from 16 to 125 Hz, absolute thresholds for the hand were significantly lower than those for the finger. Changes in threshold as a function of masker level were used to estimate the thresholds of three psychophysical channels (i.e. P, NP I, and NP II channels). Increased vibrotactile sensitivity of the hand compared to the finger seems to be not entirely due to increased spatial summation via the Pacinian system (P channel); non-Pacinian system (NP I and NP II channels) also contributed to perception. Differing transmission of vibration between the hand and the finger may have also influenced the thresholds.     

Transport of dopamine and levodopa and their interaction in COS-7 cells heterologously expressing monoamine neurotransmitter transporters and in monoaminergic cell lines PC12 and SK-N-SH

The present study investigated the effects of levodopa, a precursor of dopamine (DA) therapeutically used for the treatment of Parkinson's disease, on DA transport in the two different systems, COS-7 cells heterologously expressing rat monoamine transporter cDNA and in monoaminergic cell lines PC12 and SK-N-SH. Levodopa enhanced uptake of [3H]DA and [3H]norepinephrine (NE) but not [3H]serotonin in the transfected COS-7 cells in a concentration-dependent manner. On the other hand, in PC12 and SK-N-SH cells where NET is functionally expressed, levodopa enhanced [3H]DA and [3H]NE uptake at low concentrations and inhibited the uptake at higher concentrations. The effects of levodopa on catecholamine transporters in the opposite direction suggest a different mechanism at the intra- and extracellular sites in a levodopa transport-dependent and independent manner.     

Density- and serum-dependent regulation of the Reck tumor suppressor in mouse embryo fibroblasts

Reck is a membrane-anchored glycoprotein identified as a transformation suppressor. Accumulating evidence indicates that Reck negatively regulates a wide spectrum of matrix metalloproteinases and is commonly down-regulated in a variety of malignant solid tumors. Physiological cues that regulate Reck expression, however, remained unknown. In this study, we found that Reck expression was up-regulated at high cell density, low serum, or after treatment with some kinase inhibitors, such as PP2 (Src inhibitor), LY294002 (PI3-kinase inhibitor), and PF573228 (FAK inhibitor), in mouse embryo fibroblasts. Curve fitting indicated that the levels of Reck protein and Reck mRNA are quadratic in the cell density. Other factors, including serum, extracellular matrix components (type I collagen and fibronectin), the kinase inhibitors, and some of their oncogenic targets (v-Src and PIK3CA mutants), modify the shape of the quadratic curve. Comparison of these modifications implicated Src in Reck down-regulation under sparse conditions, PI3-kinase in serum-induced Reck down-regulation, and FAK in Reck down-regulation at high cell density. Fibronectin and type I collagen down-regulated Reck, supporting the role of integrin-FAK signaling in Reck down-regulation at high cell density. Our study has revealed multiple signaling pathways impinging on Reck in cultured mouse embryo fibroblasts and sets a foundation for future studies to find effective Reck inducers of potential value in cancer therapy.     

Crystallization and preliminary X-ray investigation of non-specific complexes of a mutant ribonuclease T1 (Y45W) with 2'AMP and 2'UMP

We have succeeded in crystallizing complexes of a mutant ribonuclease T1 (Y45W) with the non-cognizable ribonucleotides 2'AMP and 2'UMP by macroscopic seeding of microcrystals of the mutant enzyme complexed with 2'GMP, which is the cognizable nucleotide inhibitor. The mutant enzyme has a tryptophan residue instead of Tyr45 of the wild-type enzyme and thus this mutation enhances the binding of ribonucleotides to the enzyme. The space group is P212121 with unit cell dimensions a = 49.40 A, b = 46.71 A, c = 41.02 A for the complex with 2'AMP and a = 48.97, b = 46.58 A, c = 40.97 A for the complex with 2'UMP, both of which are poorly isomorphous to the mother crystals. Diffraction data for the complexes with 2'AMP and 2'UMP were collected on a diffractometer at 1.7 A and 2.4 A resolution, respectively. The present studies show that crystallization of non-specific complexes of other protein-ligand systems with the dissociation constants around 10(-3) M, or even larger, could be feasible by application of the seeding technique. A comparison of the crystal structures of the complexes with that with 2'GMP may serve as a structural basis for the determination of differences between the specific and non-specific interactions of the enzyme.     

Direct binding of RalA to PKCη and its crucial role in morphological change during keratinocyte differentiation

During differentiation, keratinocytes undergo a dramatic shape change from small and round to large and flat, in addition to production of proteins necessary for the formation of epidermis. It has been shown that protein kinase C (PKC) η is crucial for keratinocyte differentiation. However, its role in this process has yet to be fully elucidated. Here, we show that catalytic activity is not necessary for enlarged and flattened morphology of human keratinocytes induced by overexpression of PKCη, although it is important for gene expression of the marker proteins. In addition, we identify the small G protein RalA as a binding partner of PKCη, which binds to the C1 domain, an indispensable region for the morphological change. The binding led activation of RalA and actin depolymerization associated with keratinocyte differentiation. siRNA techniques proved that RalA is involved in not only the keratinocyte differentiation induced by PKCη overexpression but also normal keratinocyte differentiation induced by calcium and cholesterol sulfate. These results provide a new insight into the molecular mechanism of cytoskeletal regulation leading to drastic change of cell shape.     

Microbiology of nitrogen cycle in animal manure compost

Composting is the major technology in the treatment of animal manure and is a source of nitrous oxide, a greenhouse gas. Although the microbiological processes of both nitrification and denitrification are involved in composting, the key players in these pathways have not been well identified. Recent molecular microbiological methodologies have revealed the presence of dominant Bacillus species in the degradation of organic material or betaproteobacterial ammonia‐oxidizing bacteria on nitrification on the surface, and have also revealed the mechanism of nitrous oxide emission in this complicated process to some extent. Some bacteria, archaea or fungi still would be considered potential key players, and the contribution of some pathways, such as nitrifier denitrification or heterotrophic nitrification, might be involved in composting. This review article discusses these potential microbial players in nitrification–denitrification within the composting pile and highlights the relevant unknowns through recent activities that focus on the nitrogen cycle within the animal manure composting process.     

Neuropathic Pain in Rats with a Partial Sciatic Nerve Ligation Is Alleviated by Intravenous Injection of Monoclonal Antibody to High Mobility Group Box-1

High mobility group box-1 (HMGB1) is associated with the pathogenesis of inflammatory diseases. A previous study reported that intravenous injection of anti-HMGB1 monoclonal antibody significantly attenuated brain edema in a rat model of stroke, possibly by attenuating glial activation. Peripheral nerve injury leads to increased activity of glia in the spinal cord dorsal horn. Thus, it is possible that the anti-HMGB1 antibody could also be efficacious in attenuating peripheral nerve injury-induced pain. Following partial sciatic nerve ligation (PSNL), rats were treated with either anti-HMGB1 or control IgG. Intravenous treatment with anti-HMGB1 monoclonal antibody (2 mg/kg) significantly ameliorated PSNL-induced hind paw tactile hypersensitivity at 7, 14 and 21 days, but not 3 days, after ligation, whereas control IgG had no effect on tactile hypersensitivity. The expression of HMGB1 protein in the spinal dorsal horn was significantly increased 7, 14 and 21 days after PSNL; the efficacy of the anti-HMGB1 antibody is likely related to the presence of HMGB1 protein. Also, the injury-induced translocation of HMGB1 from the nucleus to the cytosol occurred mainly in dorsal horn neurons and not in astrocytes and microglia, indicating a neuronal source of HMGB1. Markers of astrocyte (glial fibrillary acidic protein (GFAP)), microglia (ionized calcium binding adaptor molecule 1 (Iba1)) and spinal neuron (cFos) activity were greatly increased in the ipsilateral dorsal horn side compared to the sham-operated side 21 days after PSNL. Anti-HMGB1 monoclonal antibody treatment significantly decreased the injury-induced expression of cFos and Iba1, but not GFAP. The results demonstrate that nerve injury evokes the synthesis and release of HMGB1 from spinal neurons, facilitating the activity of both microglia and neurons, which in turn leads to symptoms of neuropathic pain. Thus, the targeting of HMGB1 could be a useful therapeutic strategy in the treatment of chronic pain.