A doubly responsive probe for the detection of Cys4-tagged proteins.

Recombinant proteins bearing a tag are crucial tools for assessing protein location or function. Small tags such as Cys4 tag (tetracysteine; Cys-Cys-X-X-Cys-Cys) are less likely disrupt protein function in the living cell than green fluorescent protein. Herein we report the first example of the design and synthesis of a dual fluorescence and hyperpolarized (129)Xe NMR-based sensor of Cys4-tagged proteins. This sensor becomes fluorescent when bound to such Cys4-tagged peptides, and the (129)Xe NMR spectrum exhibits a specific signal, characteristic of the biosensor-peptide association.

Comedolytic effect of topically applied active vitamin D3 analogue on pseudocomedones in the rhino mouse.

BACKGROUND: Retinoids and active vitamin D(3) analogues regulate the proliferation and differentiation of keratinocytes and are effective in the treatment of psoriasis. Retinoids are known to be effective against acne vulgaris through comedolysis. However, the comedolytic effect of active vitamin D(3) analogues has not been reported. OBJECTIVES: To investigate whether maxacalcitol, one of the active vitamin D(3) analogues, has a comedolytic effect by using spontaneously comedogenic rhino mice. METHODS: Rhino mice were treated topically with tretinoin and maxacalcitol once daily for 2 and 4 weeks, respectively. The dermal side of the epidermal sheet was observed to determine the size of the utricle. Haematoxylin and eosin-stained vertical sections were used to measure utricle diameter and density and to evaluate histological changes. RESULTS: Maxacalcitol (25 microg g(-1)) and tretinoin (0.1%) significantly decreased the size and the diameter of the utricle after 1 week of treatment. However, maxacalcitol did not affect the density while tretinoin did have an effect. Histopathologically, maxacalcitol and tretinoin markedly induced epidermal hyperplasia accompanied by a minor accumulation of inflammatory cells in the dermis, with and without hypercornification, respectively. CONCLUSIONS: These results indicate that maxacalcitol has a prominent effect on comedolysis and that its mechanism of action may be different from that of retinoids.

Regulation of hydroxyindole-O-methyltransferase gene expression in Japanese quail (Coturnix coturnix japonica).

Hydroxyindole-O-methyltrasferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In this study, the expression of the HIOMT gene in Japanese quail was investigated with respect to tissue distribution and the effects of light and vitamin A deficiency. HIOMT mRNA in the pineal gland and eye had a clear daily rhythm with peak values in daytime. The testis also contained a detectable amount of HIOMT mRNA, which did not display a rhythmic change over a 24-h period. When birds were rendered vitamin A deficient through feeding with a vitamin A-free diet, the daily rhythm of the HIOMT gene almost disappeared in both the pineal gland and eye due to increases in the nighttime values. Our previous observations and these results suggest that vitamin A and a photo-signal are required to maintain the rhythmic expression of the HIOMT gene as well as the arylalkylamine N-acetyltransferase gene.

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): I. Rhythmic pattern and effect of light.

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] is the rate-limiting enzyme in melatonin synthesis, and its activity exhibits a diurnal rhythm similar to that of the melatonin content in the pineal gland and retina of Japanese quail. Studies were conducted to characterize the Japanese quail AANAT cDNA, and to evaluate the expression of AANAT mRNA in the pineal gland, the retina, and other peripheral tissues. The nucleic acid sequence of a 400 bp cDNA clone obtained by RT-PCR manifested 78 and 95% homology compared to the rat and chicken AANAT cDNA, respectively, while the deduced amino acid sequence homology was 82 and 99%, respectively. AANAT mRNA content in a single pineal gland or an aliquot of eye lysate was measured by a micro-lysate protection assay. The expression of AANAT mRNA in the pineal gland and the retina exhibited circadian rhythm with peak levels at night. AANAT mRNA was also detected in the testis, but did not display a rhythmic change over a 24 hr period. AANAT mRNA was not detected in other tissues studied. Darkness during the day did not increase the pineal AANAT mRNA levels. However, unexpected light-exposure for 2 hr just after lights-off blocked the increase in AANAT mRNA, and at midnight remarkably decreased AANAT mRNA by 50%.

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): II. Effect of vitamin A deficiency.

Levels of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] mRNA, AANAT activity, and melatonin display a rhythmic pattern in both the pineal gland and the retina. It has been shown that vitamin A is required to maintain the rhythm of melatonin synthesis in the pineal gland of Japanese quail. To understand the mechanism underlying the direct relationship among these factors, we developed an assay system sensitive enough to determine AANAT mRNA, AANAT activity, and melatonin content from a single pineal gland of Japanese quail. Positive direct relationships were found among these three parameters. We next deprived Japanese quail of vitamin A by feeding them a vitamin A-free diet supplemented with retinoic acid, and examined the effects of vitamin A deficiency on the expression of AANAT mRNA in the pineal gland and the retina. Vitamin A deficiency reduced both the expression of AANAT mRNA and melatonin content in the pineal gland. Retinal AANAT mRNA rhythm disappeared in vitamin A-deficient quails. Moreover, the responsiveness of the pineal gland and the retina to light was reduced by vitamin A deficiency when compared with the control group.

Bone-resorbing activities of 24-epi-1 alpha-hydroxyvitamin D2 and 24-epi-1 alpha,25-dihydroxyvitamin D2.

Bone-resorbing activities of 24-epi-1 alpha-hydroxyvitamin D2 [24-epi-1 alpha(OH)D2], 24-epi-1 alpha,25-dihydroxyvitamin D2 [24-epi-1,25(OH)2D2], and 1 alpha,24S,25-trihydroxyvitamin D2 [1,24S,25(OH)3D2], which might be a metabolite of 24-epi-1,25(OH)2D2, were investigated. In an in vitro bone resorption test, the activity of 24-epi-1 alpha(OH)D2 was similar to that of 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3] at 10(-9) M-10(-6) M. The activity of 24-epi-1,25(OH)2D2 was weaker than that of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] at 10(-11) M-10(-8) M. On the other hand, the activity of 1,24S,25(OH)3D2 was similar to that of 24-epi-1,25(OH)2D2 at 10(-11) M-10(-9) M. In the formation assay of osteoclast-like cells, the activity of 24-epi-1 alpha(OH)D2 was weaker than that of 1 alpha(OH)D3 at 10(-7) M. The activity of 24-epi-1,25(OH)2D2 was almost similar to that of 1,25(OH)2D3 at 10(-11) M-10(-7) M. The activity of 1,24S,25(OH)3D2 was significantly weaker than that of 24-epi-1,25(OH)2D2 at 10(-11) M-10(-9) M. In the two experiments, the potencies of 24-epi-1,25(OH)2D2 were about 100 times higher than those of 24-epi-1 alpha(OH)D2. In an in vivo/in vitro bone resorption test, the activity of 24-epi-1 alpha(OH)D2 was almost similar to those of 1 alpha(OH)D3 and 1,25(OH)2D3 and higher than those of 24-epi-1,25(OH)2D2 and 1,24S,25(OH)3D2. 24-epi-1 alpha-(OH)D2 and 1 alpha(OH)D3 were longer lasting than 24-epi-1,25(OH)2D2 and 1,25(OH)2D3 in this experiment.(ABSTRACT TRUNCATED AT 250 WORDS)