Molecular cloning and characterization of neutral ceramidase homologue from the red flour beetle, Tribolium castaneum.

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C(6), C(12), C(16), C(18:1), and C(24:1)-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe(2+). These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.

Role of Drosophila alkaline ceramidase (Dacer) in Drosophila development and longevity.

Ceramidases catalyze the hydrolysis of ceramides to generate sphingosine (SPH) and fatty acids, and ceramide metabolism is implicated in various biological responses in Drosophila melanogaster. Here we report the cloning, biochemical characterization, and functional analysis of a Drosophila alkaline ceramidase (Dacer). Dacer, a membrane-bound protein of 284 amino acids, shares homology with yeast and mammalian alkaline ceramidases. Overexpression of Dacer in High Five insect cells increases ceramidase activity in the alkaline pH range, indicating that Dacer is a bona fide alkaline ceramidase. Dacer mRNA is highly expressed in the midgut and at the pupal stage. An inactivation of Dacer by insertional mutagenesis increases the levels of ceramides in both Drosophila pupae and adult flies. Dacer inactivation increases Drosophila pre-adult development time, lifespan, and anti-oxidative stress capacity. Collectively, these results suggest that Dacer plays an important role in the Drosophila development and longevity by controlling the metabolism of ceramides.

Punicic acid from Trichosanthes kirilowii seed oil is rapidly metabolized to conjugated linoleic acid in rats.

The incorporation and metabolism of orally administered punicic acid (PA), one isomer of conjugated linolenic acid (CLNA), in rat tissues and plasma were studied over a 24-hour period. The punic acid was derived from Trichosanthes kirilowii Maxim seed oil, a unique PA-containing material, and identified and analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry. The results show that PA was incorporated and metabolized to 9c,11t-conjugated linoleic acid (CLA) in rat plasma, liver, kidney, heart, brain, and adipose tissue. The level of PA and CLA in liver and plasma was higher than in brain, heart, kidney, and adipose tissue, and the lowest accumulation occurred in the brain. The observation that PA can be converted into 9c,11t-CLA has gained increased importance since it has been demonstrated that 9c,11t-CLA exerts many biological activities. Therefore natural resources containing CLNA, especially edible T. kirilowii seed, could be a potential dietary source of CLA, following PA metabolism. PA is expected to be used as a functional food and nutraceutical.

[Human aqueous humor levels of levofloxacin 0.5%, gatifloxacin 0.3% and levofloxacin 0.3% ophthalmic solution after topical dosing].

OBJECTIVE: To compare the aqueous humor concentration of levofloxacin 0.5%, gatifloxacin 0.3% and levofloxacin 0.3% ophthalmic solution after topical dosing in human eyes. METHODS: A randomized, double-blind and parallel study was conducted. Senile cataract patients (150 eyes in 150 cases) receiving phacoemulsification in eye center affiliated second hospital of Zhejiang university were randomized into three groups: levofloxacin 0.5%, 50 eyes;gatifloxacin 0.3%, 50 eyes and levofloxacin 0.3%, 50 eyes. Each group was randomized into five sub-groups, with 10 eyes in each sub-group. Each group received either levofloxacin 0.5% or topical gatifloxacin 0.3% or levofloxacin 0.3% preoperatively. One drop of antibiotic was administered every 15 minutes for 4 doses. More than 100 microl aqueous samples were obtained from different sub-groups during surgery, 15.0, 30.0, 60.0, 120.0 and 180.0 min after the last drop, respectively. Then 100 microl aqueous samples were put into test tube by sample injector accurately. Concentration of the antimicrobial agents was determined by the HPLC (high pressure liquid chromatography). Variance analysis was performed to detect differences among the antibiotic treatment groups. The differences between two groups were assumed by t-test. RESULTS: Humor concentration of levofloxacin 0.5% (1.61 +/- 0.48), (2.41 +/- 0.80), (2.93 +/- 0.50), (2.56 +/- 0.63), (1.87 +/- 0.88) mg/L was significantly higher than that of gatifloxacin 0.3% (0.70 +/- 0.18), (1.29 +/- 0.54), (1.59 +/- 0.67), (1.41 +/- 0.50), (1.13 +/- 0.28) mg/L and levofloxacin 0.3% (0.55 +/- 0.39), (1.15 +/- 0.42), (1.38 +/- 0.49), (1.02 +/- 0.33), (0.55 +/- 0.31) mg/L at the same period after the last drop (F = 23.64, 12.82, 21.13, 25.00, 12.22;P < 0.05). CONCLUSION: Topically applied levofloxacin 0.5% had the higher aqueous humor drug level than gatifloxacin 0.3% and levofloxacin 0.3%.

[Investigation on release model of insoluble drug in hydroxypropyl methycellulose matrix tablets].

OBJECTIVE: To investigate the release model of insoluble drug carbamazepine (CBM) based on HPMC matrix tablets. METHODS: CBM release profile from matrices and HPMC erosion rate were determined. RESULT: The mathematical model by matrix erosion rate and drug release from HPMC K15M were established for the fractional HPMC and CBM released as M(P(t))/M(P(infinity))=-[0.8095ln((t))+1.2775]Meq((-0.0622t-0.305)) and M(d(t))/M(d(infinity))=-[0.1891t-0.1294]Meq(-0.9326). In comparison with the data of HPMC K4M matrix erosion and CBM release from HPMC K4M matrices, theoretical value agreed well with experimental data. CONCLUSION: The two mathematical models can be satisfactorily applied to insoluble drug release, which is governed by matrix erosion.