Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein

Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) μg μL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.     

Production of panose from maltose by intact cells of Aureobasidium pullulans

Panose, a major component of isomalto-oligosaccharides, was selectively produced from maltose using transglucosylation reaction catalyzed by intact cells of Aureobasidium pullulans. When 50 %(w/v) maltose was used as a substrate, the maximum concentration of panose accumulated in the final reaction mixture was about 50 %(w/w) after 120 hr reaction at 55 °C.     

An Imported Case of Echinococcosis of the Liver in a Korean Who Traveled to Western and Central Europe

Echinococcus granulosus, an intestinal tapeworm of dogs and other canids, infects humans in its larval stage and causes human echinococcosis or hydatid disease. In the Republic of Korea, 31 parasite-proven human echinococcosis cases have been reported, most of which were imported from the Middle East. We recently examined a 61-year-old Korean man who had a large cystic mass in his liver. ELISA was negative for tissue parasitic infections, including echinococcosis, cysticercosis, paragonimiasis, and sparganosis. The patient underwent surgery to remove the cyst, and the resected cyst was processed histopathologically for microscopic examinations. In sectioned cyst tissue, necrotizing protoscolices with disintegrated hooklets of E. granulosus were found. In some areas, only freed, fragmented hooklets were detected. The patient had traveled to western and central Europe in 1996, and had no other history of overseas travel. We report our patient as a hepatic echinococcosis case which was probably imported from Europe.     

An optical multi-sensing system for detection of cardiovascular toxicity

A mini-microscope-based system for multisite detection of cardiovascular toxicity was developed. The mini-microscope consisted of an image sensor and lens module extracted from an inexpensive webcam. The flipped lens module enabled cells to be magnified and monitored during testing. The portability and compactness of this system enables short-term and potential long-term experimentation inside a conventional incubator. The toxicity test results demonstrated that the normalized beating rates of cardiac muscle cells selected from multiple regions increased over time when treated with 100 nM isoprenaline. The presented system could be a promising cost-effective cell-based testing tool for discovering and screening drugs.     

Acoustic Characteristics of Stridor in Multiple System Atrophy

Nocturnal stridor is a breathing disorder prevalent in patients with multiple system atrophy (MSA). An improved understanding of this breathing disorder is essential since nocturnal stridor carries a poor prognosis (an increased risk of sudden death). In this study, we aimed to classify types of stridor by sound analysis and to reveal their clinical significance. Patients who met the criteria for probable MSA and had undergone polysomnography (PSG) were recruited. Patients were then assessed clinically with sleep questionnaires, including the Pittsburgh Sleep Quality Index, and the Hoehn and Yahr scale. Nocturnal stridor and snoring were analyzed with the Multi-Dimensional Voice Program. Nocturnal stridor was recorded in 22 patients and snoring in 18 patients using the PSG. Waveforms of stridors were classified into rhythmic or semirhythmic after analysis of the oscillogram. Formants and harmonics were observed in both types of stridor, but not in snoring. Of the 22 patients diagnosed with stridor during the present study, fifteen have subsequently died, with the time to death after the PSG study being 1.9 ± 1.4 years (range 0.8 to 5.0 years). The rhythmic waveform group presented higher scores on the Hoehn and Yahr scale and the survival outcome of this group was lower compared to the semirhythmic waveform group (p = 0.030, p = 0.014). In the Kaplan Meier’s survival curve, the outcome of patients with rhythmic waveform was significantly less favorable than the outcome of patients with semirhythmic waveform (log-rank test, p < 0.001). Stridor in MSA can be classified into rhythmic and semirhythmic types and the rhythmic component signifies a poorer outcome.     

Effector cell expression of NK1.1, a murine natural killer cell-specific molecule, and ability of mice to reject bone marrow allografts

The rejection of Hh-1 incompatible bone marrow cells in irradiated mice is mediated by NK cells and is genetically regulated. We tested the role of the NK-specific gene, NK1.1, in regulating the rejection of allogeneic bone marrow cell grafts. NK1.1+ mice, that are known to display strong resistance against Hh-1 incompatible grafts, were crossed to H-2/Hh-1 identical NK1.1-, poor responder mice, and the progeny were backcrossed to the poor responder parent. The segregating mice were individually typed for their expression of NK1.1 and the ability to resist Hh-1 incompatible bone marrow cells (BMC). A strong correlation was noted between expression of NK1.1 and rejection of H-2d/Hh-1d BMC. Our results support the idea that NK1.1 is one of the genes responsible for strong resistance to Hh-1d (determinant 2) but not for Hh-1j (determinant 3) BMC grafts. We suggest that the NK1.1 molecule functions as an accessory molecule in the cellular interactions involving the recognition of Hh-1 determinants.     

Sustained release of an anti-glaucoma drug: demonstration of efficacy of a liposomal formulation in the rabbit eye

Topical medication remains the first line treatment of glaucoma; however, sustained ocular drug delivery via topical administration is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Currently, daily topical administration for lowering the intra-ocular pressure (IOP), has many limitations, such as poor patient compliance and ocular allergy from repeated drug administration. Poor compliance leads to suboptimal control of IOP and disease progression with eventual blindness. The delivery of drugs in a sustained manner could provide the patient with a more attractive alternative by providing optimal therapeutic dosing, with minimal local toxicity and inconvenience. To investigate this, we incorporated latanoprost into LUVs (large unilamellar vesicles) derived from the liposome of DPPC (di-palmitoyl-phosphatidyl-choline) by the film hydration technique. Relatively high amounts of drug could be incorporated into this vesicle, and the drug resides predominantly in the bilayer. Vesicle stability monitored by size measurement and DSC (differential scanning calorimetry) analysis showed that formulations with a drug/lipid mole ratio of about 10% have good physical stability during storage and release. This formulation demonstrated sustained release of latanoprost in vitro, and then tested for efficacy in 23 rabbits. Subconjunctival injection and topical eye drop administration of the latanoprost/liposomal formulation were compared with conventional daily administration of latanoprost eye drops. The IOP lowering effect with a single subconjunctival injection was shown to be sustained for up to 50 days, and the extent of IOP lowering was comparable to daily eye drop administration. Toxicity and localized inflammation were not observed in any treatment groups. We believe that this is the first demonstration, in vivo, of sustained delivery to the anterior segment of the eye that is safe and efficacious for 50 days.