Background α-Klotho (αKl) regulates mineral metabolism such as calcium ion (Ca2+) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type αKl has been demonstrated, less is known regarding the physiological importance of soluble-type αKl (sαKl) in circulation. Objectives The aims of this study were: 1) to establish a sandwich ELISA system enabling detection of circulating serum sαKl, and 2) to determine reference values for sαKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects. Results We successively developed an ELISA to measure serum sαKl in healthy volunteers (n=142, males 66) of ages (61.1 ± 18.5 yr). The levels (mean ± SD) in these healthy control adults were as follows: total calcium (Ca; 9.46 ± 0.41 mg/dL), Pi (3.63 ± 0.51 mg/dL), Blood urea nitrogen (BUN; 15.7 ± 4.3 mg/dL), creatinine (Cre; 0.69 ± 0.14 mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)2D; 54.8 ± 17.7 pg/mL), intact parathyroid hormone (iPTH; 49.2 ± 20.6 pg/mL), calcitonin (26.0 ± 12.3 pg/mL) and intact Fibroblast growth factor (FGF23; 43.8 ± 17.6 pg/mL). Serum levels of sαKl ranged from 239 to 1266 pg/mL (mean ± SD; 562 ± 146 pg/mL) in normal adults. Although sαKl levels were not modified by gender or indices of mineral metabolism, sαKl levels were inversely related to Cre and age. However, sαKl levels in normal children (n=39, males 23, mean ± SD; 7.1 ± 4.8 years) were significantly higher (mean ± SD; 952 ± 282 pg/mL) than those in adults (mean ± SD; 562 ± 146, P<0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that sαKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum sαKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of α-klotho gene. In this patient, sαKl level was notably lower than those of age matched controls. Conclusion We established a detection system to measure human serum sαKl for the first time. Age, Ca and Pi seem to influence serum sαKl levels in a normal population. This detection system should be an excellent tool for investigating sαKl functions in mineral metabolism.

p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G 1 arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G 1 arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.

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In spite of prior blockade of the obturator nerve with 1% mepivacaine (8 ml) utilizing a nerve stimulator, violent leg jerking was evoked during transurethral electroresection of a bladder tumour approximately 1 h after the blockade in a 68-year-old man. The patient became severely hypotensive immediately following the jerking, and a large lower abdominal swelling concurrently developed. The urgent laparotomy indicated that the left obturator artery was severely injured by the resectoscope associated with the bladder perforation, causing acute massive haemorrhage. The patient recovered uneventfully after adequate surgery. Investigation of the literature suggested that both our nerve stimulation technique and anatomical approach were appropriate. It was therefore unlikely that our block resulted in failure because of an inappropriate site for deposition of the anaesthetic. However, consensus does not appear to have been obtained as to the concentration and volume of the anaesthetic necessary for prevention of the obturator nerve stimulation during the transurethral procedures. The concentration and volume of mepivacaine we used might have been too low and/or small, respectively, to profoundly block all the motor neuron fibres of the nerve. Alternatively, stimulation of the obturator nerve might occur because of the presence of some anatomical variant, such as the accessory obturator nerve or its abnormal branching. In conclusion, some uncertainty appears to exist in the effectiveness of the local anaesthetic blockade of the obturator nerve. In order to attain profound blockade of the motor neuron fibres of the obturator nerve and thereby prevent the thigh-adductor muscle contraction which can lead to life-threatening situations, we recommend, even with a nerve stimulator, to use a larger volume of a higher concentration of local anaesthetic with a longer duration in the obturator nerve block for the transurethral procedures.

Preventive use of olanzapine combined with triplet therapy gives better results than those from previously reported studies of triplet therapy.

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In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.