Electroanalytical properties of a novel biosensor modified with zirconium alcoxide porous gels for the detection of acetaminophen

The development of composite electrodes for biosensors construction based on HRP and zirconium alcoxide film for acetaminophen detection and finally, acetaminophen determination in pharmaceutical products is described. The enzyme immobilization is performed by retention in a polyetilenimine and zirconium alcoxide porous gel film, technique that offers a good entrapping and in the mean times a "protective" environment for the biocomponent. The operation principle of the biosensor is based on monitoring the amperometric signal generated by reduction at the electrode surface of the enzymatically generated quinoneimine from acetaminophen. The resulting biosensor shows a linear response towards acetaminophen with a linear range of 1.96 x 10(-5)M and 2.55 x 10(-4)M and a limit of detection of 1.17 x 10(-7)M. The proposed biosensor shows long term stability and good reproducibility.     

The in situ up‐regulation of chondrocyte interleukin‐1–converting enzyme and interleukin‐18 levels in experimental osteoarthritis is mediated by nitric oxide

Objective

To investigate in situ the relationship between 2 key mediators implicated in osteoarthritic (OA) cartilage: nitric oxide (NO) and interleukin‐1–converting enzyme (ICE). Interleukin‐18 (IL‐18) was also studied and served as reference for the effects of ICE.

Methods

An OA model was created in dogs by sectioning (stab wound) the anterior cruciate ligament of the right stifle joint. Three experimental groups were studied: unoperated untreated dogs, operated untreated dogs (OA), and OA dogs treated with oral N‐iminoethyl‐L ‐lysine (L‐NIL), a specific inhibitor of inducible nitric oxide synthase (iNOS) (10 mg/kg twice a day starting immediately after surgery). At 12 weeks after surgery, cartilage from the femoral condyles and tibial plateaus were processed for immunohistochemistry for ICE, IL‐18, and protease inhibitor 9 (PI‐9), a natural inhibitor of ICE, followed by morphometric analysis. Cartilage specimens from the femoral condyles of untreated OA dogs were dissected and incubated with specific inhibitors of different signaling pathways likely to be involved in the OA process: SB 202190 (10 μM; a p38 mitogen‐activated protein kinase [MAPK] inhibitor), PD 98059 (100 μM; a MAPK kinase 1/2 [MEK‐1/2] inhibitor), NS‐398 (10 ng/ml; a specific cyclooxygenase 2 [COX‐2] inhibitor), and L‐NIL (50 μM).

Results

Both ICE and IL‐18 were present in situ in the canine cartilage, with a significant increase in the level of these 2 proteins in OA cartilage. In contrast, the level of PI‐9 was lower in OA than in normal cartilage (difference not statistically significant). Compared with untreated OA cartilage, oral treatment with L‐NIL significantly decreased ICE and IL‐18 levels in cartilage from the femoral condyles and tibial plateaus, to values similar to those in normal dogs. L‐NIL also increased the PI‐9 level in normal dogs compared with OA dogs, reaching statistical significance for femoral condyle cartilage. Interestingly, in vitro experiments demonstrated significant inhibition of ICE levels by p38, MEK‐1/2, and COX‐2 inhibitors, but not by the iNOS inhibitor.

Conclusion

This study demonstrated that in situ in OA cartilage, the stimulation of chondrocytes by NO is at least partly responsible for the up‐regulation of ICE and IL‐18 synthesis while decreasing the level of the ICE inhibitor PI‐9. The ICE level is controlled by the activation of at least 2 MAPK pathways, p38 and MEK‐1/2. Interestingly, it appears that ICE synthesis is not regulated by the endogenous production of NO. These data highlight the role played by iNOS in regulating the synthesis of major catabolic factors involved in OA cartilage degradation.

     

Putative virulence factors identified in Vibrio vulnificus strains isolated from oysters and seawater in Mexico

The presence of Vibrio vulnificus was analyzed in oyster and estuarine water samples from Mexico by PCR amplification of the vvhA gene and some putative virulence factors were tested. Samples were collected from 12 different sampling points over a one-year period; 31% samples were positive for V. vulnificus and all isolates were identified as biotype 1. All strains were cytotoxic and proteolytic, 98% showed adherence to epithelial cells, 91.4% were DNase-positive, 77.6% were mucinase-positive, 97.8% were lecithinase-positive and 79.8% were lipase positive. Regarding colony morphology, 51% strains were opaque, 20% were translucid, 28% were both opaque and translucid, and 80.8% showed a capsule. This is the first report on the isolation of V. vulnificus strains from environmental samples in Mexico, which may pose a health risk for local fisherman and seafood consumers.     

In vivo selective inhibition of mitogen-activated protein kinase kinase 1/2 in rabbit experimental osteoarthritis is associated with a reduction in the development of structural changes

OBJECTIVE: The primary aim of this study was to investigate, using an experimental rabbit model of osteoarthritis (OA), the effect of a selective mitogen-activated protein kinase kinase 1/2 (MEK-1/2) inhibitor, PD 198306, on the development of structural changes. Additional aims were to assess the effects of the inhibitor on levels of phosphorylated extracellular signal-regulated kinase 1/2 (phospho-ERK-1/2) and matrix metalloproteinase 1 (MMP-1; collagenase 1) in OA chondrocytes. METHODS: After surgical sectioning of the anterior cruciate ligament of the right knee joint, rabbits with OA were separated into 3 experimental groups: oral treatment with placebo or with PD 198306 at a therapeutic concentration of 10 mg/kg/day or 30 mg/kg/day. Each treatment started immediately after surgery. The animals were killed 8 weeks after surgery. Macroscopic and histologic studies were performed on the cartilage and synovial membrane. The levels of phospho-ERK-1/2 and MMP-1 in OA cartilage chondrocytes were evaluated by immunohistochemistry. Normal, untreated rabbits were used as controls. RESULTS: OA rabbits treated with the highest dosage of MEK-1/2 inhibitor showed decreases in the surface area (size) of cartilage macroscopic lesions (P < 0.002) and in osteophyte width on the lateral condyles (P = 0.05). Histologically, the severity of synovial inflammation (villous hyperplasia) was also reduced (P < 0.02). In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in the superficial layer stained positive for phospho-ERK-1/2 and MMP-1 compared with normal controls. Rabbits treated with the highest dosage of PD 198306 demonstrated a significant and dose-dependent reduction in the level of phospho-ERK-1/2 and a lower level of MMP-1. CONCLUSION: This study demonstrates that, in vivo, PD 198306, a selective inhibitor of MEK-1/2, can partially decrease the development of some of the structural changes in experimental OA. This effect was associated with a reduction in the level of phospho-ERK-1/2 in OA chondrocytes, which probably explains the action of the drug.     

Microcoil embolisation for ablation of septal hypertrophy in hypertrophic obstructive cardiomyopathy

BACKGROUND AND AIM: Percutaneous transcatheter occlusion with ethanol injection of septal arteries is an efficient treatment procedure of hypertrophic obstructive cardiomyopathy (HOCM). The aim of our study was to evaluate the feasibility and efficiency of septal artery embolisation with microcoils. METHODS: Microcoils were delivered through the guide-wire lumen of a 2mm-diameter coaxial balloon positioned inside the target vessel as distally as possible. One or more 0.018"-straight microcoils (Hilal straight coils, Cook, USA) were used for each target vessel until complete flow obstruction was achieved. The intraventricular pressure gradient was measured before, during and after the procedure. Septal branch occlusion was finally documented by coronary angiography. RESULTS: We treated 7 patients (5 males; mean age: 48 +/- 10 years). All patients were symptomatic (NYHA class III or IV). The target vessels were successfully occluded in all patients without complications. Moderate pain was recorded during and after the procedure and the CK level increased five- to ten-fold. The pressure gradient diminished during the procedure from 72 +/- 21 mmHg to 30 +/- 15 mmHg. The number of coils delivered ranged from 3 to 7 per patient. The embolised septal branches included 1 vessel in 5 patients, 2 vessels in 1 patient and 3 vessels in 1 case. After the procedure, the pressure gradient, evaluated by transthoracic echocardiography, was 34 +/- 16 mmHg and 42 +/- 12 mmH at 3 month-follow-up. Clinical improvement was recorded in all patients after the procedure (NYHA class I or II). Temporary pacing was necessary in 3 patients during and immediately after the procedure but no patient needed permanent pacing. CONCLUSIONS: Microcoil embolisation is an efficient and safe approach for transcatheter ablation of septal hypertrophy in HOCM. This technique induced myocardial necrosis without the toxic effects of alcohol, reducing the risk of complications such as permanent pacemaker implantation or ethanol flow to other myocardial regions.