Effect of modified oligopeptides from the beetle Allomyrina dichotoma on Escherichia coli infection in mice

The novel peptides based on a putative active site of defensin, an anti-bacterial peptide from the beetle Allomyrina dichotoma, were synthesized. These synthetic oligopeptides exhibited strong anti-bacterial activity in vitro, even against antibiotic-resistant pathogenic bacteria. Then, anti-bacterial activity of two newly synthesized peptides, RLYLRIGRR-NH(2) (peptide A) and RLRLRIGRR-NH(2) (peptide B) was also examined by macroscopic and histopathologic assessment during the course of infection in mice inoculated with antibiotic-resistant pathogenic Escherichia coli (E. coli) in vivo. Peptide B decreased the mortality of mice inoculated with antibiotic-resistant pathogenic E. coli. The results of macroscopic and histopathologic examinations revealed that peptide B could protect the mice from infection. In contrast, peptide A failed to protect mice from infection with antibiotic-resistant pathogenic E. coli. Also, modified peptides A and B produced no toxicity or side effects in mice. These results suggest that peptide B is useful for developing novel antibiotics against antibiotic-resistant pathogenic bacteria.     

In vitro characterization of the inhibitory effects of ketoconazole on metabolic activities of cytochrome P-450 in canine hepatic microsomes

OBJECTIVE: To evaluate the inhibitory potency of ketoconazole (KTZ) on the metabolic activities of isozymes of cytochrome P-450 (CYP) in dogs. ANIMALS: 4 healthy 1-year-old male Beagles. PROCEDURE: Hepatic microsomes were harvested from 4 dogs after euthanasia. To investigate the effects of KTZ on CYP metabolic activities, 7-ethoxyresorufin, tolbutamide, bufuralol, and midazolam hydrochloride were used as specific substrates for CYP1A1/2, CYP2C21, CYP2D15, and CYP3A12, respectively. The concentrations of metabolites formed by CYP were measured by high-performance liquid chromatography, except for the resorufin concentrations that were measured by a fluorometric method. The reaction velocity-substrate concentration data were analyzed to obtain kinetic variables, including maximum reaction velocity, Michaelis-Menten constant, and inhibitory constant (Ki). RESULTS: KTZ competitively inhibited 7-ethoxyresorufin O-deethylation and midazolam 4-hydroxylation; it noncompetitively inhibited tolbutamide methylhydroxylation. Bufuralol 1'-hydroxylation was inhibited slightly by KTZ. The mean Ki values of KTZ were 10.6+/-6.0, 170+/-2.5, and 0.180+/-0.131 microM for 7-ethoxyresorufin O-deethylation, tolbutamide methylhydroxylation, and midazolam 4-hydroxylation, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs, KTZ at a therapeutic dose may change the pharmacokinetics of CYP3A12 substrates as a result of inhibition of their biotransformation. Furthermore, no influence of KTZ on the pharmacokinetics of CYP1A1/2, CYP2C21, and CYP2D15 substrates are likely. In clinical practice, adverse drug effects may develop when KTZ is administered concomitantly with a drug that is primarily metabolized by CYP3A12.     

Hexachlorophene and cuprizone induce the spongy change of the developing rat brain by different mechanisms: the role of 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)

The goal of this research was to identify mechanisms responsible for the spongy change induced in rats after repeated hexachlorophene (HCP) or cuprizone (CPZ) dosing. Rats were dosed with 35 mg/kg HCP for 5 days followed by drug withdrawal for 7 days suffered spongy changes to the white matter of the cerebrum, cerebellum, medulla oblongata, and spinal cord that were accompanied by degeneration of oligodendroglia. The severity of both lesions increased prominently on day 5; however, the spongy change decreased and degeneration of oligodendroglia reversed on day 12 (7 days after dosing ceased). On day 12, cerebral cortex oligodendroglia were stained strongly by anti-CNPase. Other rats were fed for 8 days with powdered chow containing 1% (w/w) CPZ, which was then withdrawn for 16 days. The rats exhibited the spongy change in the white matter of the cerebrum and cerebellum as well as oligodendroglial cell death from day 3. The severity of both lesions increased prominently on day 8. Cerebral cortex oligodendroglia were stained strongly by anti-CNPase on days 3 to 8 and decreased to the control levels by day 24 (16 days after dosing ceased). Electron microscopy revealed that oligodendroglia frequently displayed apoptotic morphology. These findings suggest that CNPase expression was induced in the course of restoration following HCP-induced insults to oligodendroglia and the myelin sheath, and in the course of demyelination by CPZ-induced damage to oligodendroglia. However, the role of CNPase on both courses is unclear.     

Genetic and enzymatic analyses of metalloprotease (aureolysin) from Staphylococcus aureus isolated from domestic animals.

The gene (aur) encoding the metalloprotease (aureolysin) of Staphylococcus aureus from domestic animals was analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing. The aur gene was detected in all 74 isolates from cows, pigs and chickens by PCR amplification and was classified into types I and II by PCR-RFLP patterns. The type II aur gene was contained in 36 (94.7%) of 38 protease-positive isolates as judged by skim milk agar plate culture, while type I was contained in 28 (77.8%) of 36 protease-negative isolates. The deduced amino acid sequences of aureolysins from type I and II isolates were almost identical with those of the published data. Subsequently, the two aureolysins were purified from the culture supernatants of type I and II isolates. The molecular weights of purified type I and II aureolysins were both estimated at 34kDa by SDS-polyacrylamide gel electrophoresis. These aureolysins had maximum proteolytic activity at 30-50 degrees C and pH 7.0-8.0. Their activity was inhibited by metal- and zinc-specific inhibitors, such as EDTA, EGTA and 1,10-phenanthroline. Specific activity (activity/protein) of type II aureolysin was two times higher than that of type I. These results indicated that the aur gene is highly conserved with two allelic forms (types I and II) among bovine, porcine and avian isolates of S. aureus.     

Bacterial Pleuritis with Thickened Mesothelial Hyperplasia in a Young Beagle Dog

A five-month-old male beagle dog suddenly became moribund. Bloody fluid accumulated in the thoracic and abdominal cavities, and soft yellow flecks were floating in the thoracic fluid. The mediastinum and pericardium became dark reddish with villous thickening. Other parietal and pulmonary pleurae were rough, and the organs adhered to each other. Histologically, most mediastinal pleura formed papillary projections covered by a single layer of mesothelial cells. Many macrophages and neutrophils infiltrated the submesothelial connective tissue. At the mediastinum adjacent to the pericardium, cuboidal mesothelial cells proliferated solidly and formed a thick surface stratum. The flecks consisted of gram-negative filamentous or small bacillary (coccoid) bacteria. In the right posterior lobe of the lung, neutrophilic infiltration and a large encapsulated abscess including a bacterial colony were present. We diagnosed this case as “bacterial pleuritis with thickened mesothelial hyperplasia”. The cause of the pleuritis might be a chronic pleural infection spread via the lung abscess.     

Quantitative loop-mediated isothermal amplification method for the detection of <Emphasis Type="Italic">Vibrio nigripulchritudo</Emphasis> in shrimp

Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60 min at 63°C. Quantitative analysis was then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification of the initial DNA concentration with a sensitivity of 10² copy numbers equivalent to 2.3 colony forming units/ml or 0.3 fg/μl. The LAMP assay was able to specifically detect two representative strains of V. nigripulchritudo, whereas other Vibrio and non-Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time (T _t) versus the log of bacterial number. A high correlation coefficient (R ² = 0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.     

Growth comparison of Asian Nile and red tilapia strains in controlled and uncontrolled farm conditions

Growth of several genetically improved Nile tilapia (GIFT or Genetically Improved Farmed Tilapia, FaST or Freshwater Aquaculture Center Selected Tilapia, SEAFDEC-selected) and domesticated red tilapia (BFS or Binangonan Freshwater Station, NIFI-red or National Inland Fisheries Institute red, HL or Hacienda Luisita) stocks were compared in controlled (tank) and uncontrolled farm conditions (lake-based cages) with unselected NIFI or Chitralada Nile tilapia as control. Specific growth rate differed significantly (P = 0.009) in tank-reared Nile tilapia stocks where GIFT grew best at 1.358%/day followed by FaST (1.307%/day), control stock NIFI (1.257%/day) and SEAFDEC-selected (1.202%/day). Genetic effect explained 84.4% of the variance in growth of Nile tilapia in tanks. Although Nile tilapia growth in cages followed the same trend where GIFT grew best at 1.570%/day, no significant stock differences (P = 0.479) were noted. Meanwhile, red tilapia reared in either tanks or cages showed no significant stock differences in terms of growth. However, survival of the red tilapia stocks in cages differed significantly with HL having the highest percentage survival at 93.3%. The different growth responses of the Nile tilapia stocks especially under controlled (tank) farm conditions were largely due to genetic factors (stock differences).Under uncontrolled farm conditions, environmental factors were generally observed to have also affected the survival and to some extent, the growth of Asian Nile and red tilapia stocks.