Changes in Retinal N-Acylethanolamines and their Oxylipin Derivatives During the Development of Visual Impairment in a Mouse Model for Glaucoma

Neurons are especially susceptible to oxidative damage, which is increasingly implicated in neurodegenerative disease. Certain N‐acylethanolamines (NAEs) have been shown to protect neurons from oxidative stress. Since glaucoma may be considered a neurodegenerative disorder and the survival of retinal neurons could also be influenced by N‐acylethanolamines, our goal was to quantify changes in certain N‐acylethanolamine species and their oxylipin derivatives in the retina of a mouse model for glaucoma. We also sought to identify relationships between these and parameters of glaucoma disease development, specifically intraocular pressure, visual acuity, and contrast sensitivity. Five N‐acylethanolamine species and three NAE oxylipin derivatives were quantified in retina from young and aged DBA/2Crl mice. N‐Acylethanolamines and NAE‐oxylipins in retinal extracts were quantified against deuterated standards by isotope dilution gas chromatography–mass spectrometry. Levels (nmol/g dry weight) of N‐arachidonoylethanolamine (anandamide; NAE 20:4) were significantly (p = 0.008) decreased in aged (2.875 ± 0.6702) compared to young animals (5.175 ± 0.971). Conversely, the anandamide oxylipin, 15(S)‐HETE ethanolamide (15(S)‐HETE EA), was significantly (p = 0.042) increased in aged (0.063 ± 0.009) compared to young animals (0.039 ± 0.011). Enzymatic depletion of the anandamide pool by 15‐lipoxygenase and consequent accumulation of 15(S)‐HETE ethanolamine may contribute to decreased visual function in glaucomatous mice. Since N‐acylethanolamines effectively attenuate glaucoma pathogenesis and associated visual impairment, our data provides additional rationale and novel targets for glaucoma therapies.     

Polyaniline‐modified cellulose nanofibrils as reinforcement of a smart polyurethane

Segmented polyurethanes exhibiting shape memory properties were modified by the addition of polyaniline (PANI)‐coated cellulose nanofibrils (CNFs). The two‐phase structure of the polymer is responsible for the material's ability to ‘remember’ and autonomously recover its original shape after being deformed in response to an external thermal stimulus. PANI was grown on the surface of the CNFs via in situ polymerization. Modified nanocrystals were added to the segmented polyurethane in concentrations ranging from 0 to 15 wt%. The changes in the material properties associated with the percolation of the coated fibrils appear at higher concentrations than previously observed for non‐modified CNFs, which suggests that fibril agglomeration is occurring due to the PANI coating. The shape memory behavior of the composites is maintained at about the same level as that of the unfilled polyurethane only up to 4 wt% of fibrils. At higher concentrations, the rigidity of the nanofibrils as well as their interaction with the hard‐segment phase and the increasing difficulty of dispersing them in the polymer collaborate to produce early breakage of the specimens when stretched at temperatures above the melting point of the soft segments. Copyright © 2010 Society of Chemical Industry     

Analysis of 59 cases of non-traumatic sudden death in various neurological diseases]

Sudden death is a common complication of myocardial infarction, necrotic pancreatitis and other diseases. Physicians usually neglect the possibility of neurological disorders. 59 cases of sudden death among 314 autopsied cases with neurological diseases were analyzed. The most frequent cause of neurological sudden death was cerebrovascular disease (CVD). It was present in 53 (89.9%) cases. 48 of them had hemorrhagic CVD. 37 of these 48 cases were due to hypothalamic lesions. In 37 cases general autopsy was performed; pathological abnormalities of heart, lungs, stomach etc, were found in 28 of them. In conclusion, the size and location of the lesion correlated with the prognosis of the disease. Concomitant multiple organ damage may deteriorate the lesion. Awareness of sudden death resulting from CVD may elevate the rate of correct diagnosis.     

Detection of extrathoracic metastases by positron emission tomography in lung cancer

BACKGROUND: Accurate staging of non-small cell lung cancer is essential for treatment planning. We evaluated in a prospective study the role of whole-body 2-[18F]fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) in mediastinal nodal staging with a positive predictive value of 96%. The study was continued to further evaluate the value of whole-body FDG PET in detecting unexpected extrathoracic metastases (ETMs) in patients qualifying for surgical treatment by conventional staging. METHODS: One hundred patients underwent clinical evaluation, chest and upper abdominal computed tomography scan, mediastinoscopy (lymph nodes greater than 1 cm on computed tomography), and routine laboratory tests. In 94 patients with stage IIIa or less and 6 with suspected N3 a whole-body FDG PET was performed. If clinical signs of ETMs were present additional diagnostic methods were applied. All findings in the FDG PET were confirmed histologically or radiologically. RESULTS: Unexpected ETMs were detected in 13 (14%) of 94 patients (stage IIIa or less) at 14 sites. In addition 6 of 94 patients were restaged up to N3 after PET. The suspected N3 disease (stage IIIb) on computed tomography was confirmed by PET in all 6 patients. There was no false positive finding of ETM. Weight loss was correlated with the occurrence of ETM: more than 5 kg, 5 of 13 patients (38%); more than 10 kg, 4 of 6 patients (67%). Pathologic laboratory findings were not predictive for ETM. CONCLUSIONS: Whole-body FDG PET improves detection of ETMs in patients with non-small cell lung cancer otherwise elegible for operation. In 14% of patients (stage IIIa or less), ETMs were detected, and in total, 20% of the patients were understaged.     

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone 1(-1) and 7 g fusion glucagon 1(-1). The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e., high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content.     

Flow cytometric quantitation of yeast a novel technique for use in animal model work and in vitro immunologic assays

Animal models of fungal and other infectious diseases often require that the number of organisms in tissue be quantified, traditionally by grinding organs, plating them on agar and counting colony forming units (CFU). This method is labor intensive, slow as some fungi require two weeks of culture and limited in reliability by poor plating efficiency. To circumvent these problems, we developed a flow cytometric method to quantify yeast. In vitro cultured Blastomyces dermatitidis, Cryptococcus neoformans, Candida albicans and Histoplasma capsulatum yeast were labelled with specific monoclonal or polyclonal antibodies to stain surface determinants or with Calcofluor to stain cell-wall chitin. A defined number of fluorescently labelled beads were added prior to acquisition by flow cytometry as a reference standard for quantitation. Beads were readily distinguished from yeast by forward scatter, side scatter and intensity of fluorescence. Cultured yeast were enumerated by both standard CFU determination and flow cytometry in a range of 10(2) to 10(7) cells. Only flow cytometry enabled discrimination of live and dead yeast by using appropriate fluorescent dyes. The flow cytometric method was applied to murine models of histoplasmosis and blastomycosis to quantify the burden of fungi in the lungs of infected mice. Labelling yeast with Calcofluor alone resulted in unacceptably high levels of nonspecific binding to mouse cell debris. In contrast, labelling H. capsulatum with a rabbit polyclonal antiserum and B. dermatitidis with a monoclonal antibody to the surface protein WI-1 permitted accurate quantitation. We conclude that this flow cytometry technique is rapid, efficient and reliable for quantifying the burden of infection in animal models of fungal disease. The technique also should lend itself to performing cytotoxicity assays that require discrimination of live and dead fungi, or phagocytosis assays that require discrimination of intracellular and extracellular organisms.     

Vitamin E supplementation attenuates myointimal proliferation of the abdominal aorta after balloon injury in diet-induced hypercholesterolemic rabbits

The effects of vitamin E supplementation in a dose of 450 mg/1000 g chow on the myointimal proliferation of the abdominal aorta after balloon injury were studied in 4 groups of rabbits (24 each). The animals were fed regular diet, regular diet plus vitamin E, 1% cholesterol-enriched diet, and 1% cholesterol-enriched diet plus vitamin E. Each animal underwent a balloon injury of the abdominal aorta and left common iliac artery after 2 weeks of feeding. The animals remained on their respective diets thereafter. In 8 balloon-injured and 8 sham-operated animals of each group, the abdominal aortas were harvested 3 days after the procedure for the analysis of prostacyclin and thromboxane A2 synthesis, thiobarbituric acid reactive substances (TBARS) levels, enzyme activities of glutathione reductase (GR) and glutathione peroxidase (GP) as well as reduced (GSH) and oxidized (GSSG) glutathione levels, 3H-thymidine uptake, cholesterol as well as vitamin E contents. In the other 8 balloon-injured rabbits of each group, the tissue was harvested 3 weeks later for the morphometric study. In dependent of high cholesterol feeding, the vitamin E-treated rabbits had lower aortic production of thromboxane B2, higher 6-keto-PGF1 alpha and higher 6-keto-PGF1 alpha/thromboxane B2 ratios in both procedures. The aortic TBARS levels of the rabbits treated high cholesterol alone were significantly higher than the other three groups in both procedures. Balloon injury had a trend to increase TBARS levels and had significantly higher 3H-thymidine uptake (each p < 0.001) than sham operation in each group. Vitamin E supplement to high cholesterol diet or regular chow reduced aortic TBARS levels (p < 0.005 and 0.01, respectively) and 3H-thymidine uptake (p < 0.05 and 0.01, respectively), as well as attenuated myointimal proliferation of the abdominal aorta and left common iliac artery after balloon injury; but only supplement to high cholesterol diet reached statistical significances (both p < 0.05 compared to rabbits fed high cholesterol alone). These results suggest that vitamin E supplement changes prostanoid metabolism to a favorable pattern and reduces lipid peroxidation of the abdominal aortic wall, thus attenuates myointimal proliferation after balloon injury; these presentations are particularly obvious in diet-induced hypercholesterolemic rabbits.     

Small-dose hyperbaric versus plain bupivacaine during spinal anesthesia for cesarean section

In a double-blind, randomized trial, 98 parturients undergoing cesarean section received either hyperbaric or plain bupivacaine 6.6 mg combined with sufentanil 3.3 microg as part of a combined spinal-epidural procedure. To prevent hypotension, 1000 mL of lactated Ringer's solution, 500 mL of hydroxyethyl starch 6%, and ephedrine 5 mg were administered i.v. The height of the block was equal in both groups, but more patients in the plain group had blocks that were either too high or too low (P < 0.01). The number of patients requiring epidural supplementation was equal in both groups. Strict criteria were used to treat hypotension. The overall incidence of systolic blood pressure (<90 mm Hg) was 13%, whereas it was more pronounced in the plain group (21% vs 6% in the hyperbaric group, P < 0.05), which required more ephedrine (P < 0.05) and in which a greater incidence of nausea was noticed (P < 0.05). We conclude that the use of a small dose of intrathecal bupivacaine combined with sufentanil plus our described preloading regimen resulted in a lower incidence of hypotension. Further, we conclude that the use of hyperbaric bupivacaine in this manner provides a more reliable block and a lower incidence of hypotension than plain bupivacaine. Implications: A small dose of hyperbaric bupivacaine 0.5% combined with sufentanil used intrathecally during cesarean section offered a more reliable cephalad spread of the spinal block than the glucose-free combination, which was reflected in a lower incidence of hypotension and nausea.     

Changes in brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia, spinal cord, and gracile nuclei following cut or crush injuries

In the present study, we evaluated changes in brain-derived neurotrophic factor (BDNF) immunoreactivity in the rat lumbar (L) 5 dorsal root ganglion (DRG) and areas where afferents from the DRG terminate, the L5 spinal cord and gracile nuclei, following unilateral sciatic nerve transection or crush. From 3 days to 4 weeks following cut or crush injury, the percentage of medium and large BDNF-immunoreactive neurons in the ipsilateral DRG increased significantly compared with those on the contralateral side. Following cut injury, there was no significant change in the percentage of small BDNF-immunoreactive neurons in the ipsilateral DRG; however, the intensity of immunoreactivity of these cells decreased. Following crush injury, however, both the percentage and intensity of small BDNF-immunoreactive neurons in the ipsilateral DRG significantly increased. Following cut injury, the expression of BDNF-immunoreactive axonal fibers decreased markedly in the ipsilateral superficial laminae of the L5 spinal cord and increased significantly in the ipsilateral deeper laminae of the spinal cord and gracile nuclei. Crush injury induced a marked increase in the expression of BDNF-immunoreactive axonal fibers in the superficial laminae of the spinal cord and gracile nuclei. These differences in BDNF response in the DRG and spinal cord after cut or crush injuries may reflect differences in trophic support to the injured DRG neurons and altered neuronal activity in the spinal cord and gracile nuclei following different types of peripheral nerve injury.