Determination of boswellic acids in brain and plasma by high-performance liquid chromatography/tandem mass spectrometry

Peritumoral edema, one of the major causes for neurological disorders in brain tumor patients, is mainly treated with steroids, which unfortunately have significant side effects and interfere with the efficacy of chemotherapy. Boswellic acids, the main active ingredients of Boswellia serrata, are antiinflammatory agents, inhibiting 5-lipoxygenase, the key enzyme of leukotriene biosynthesis and one of the pathophysiological mechanisms of peritumoral edema. Based on positive results in clinical trials and animal studies, B. serrata resin dry extract was designated an orphan drug by the European Commission for the treatment of peritumoral edema resulting from brain tumors. Thus boswellic acids may be alternative drugs to corticosteroids. However, the question of the availability of boswellic acids in brain has not been addressed until now. Accordingly, a highly sensitive LC/MS method has been developed for the simultaneous determination of KBA and AKBA, the most potent boswellic acids, in plasma and brain. This method involves matrix-assisted liquid-liquid extraction on Extrelut NT followed by separation on reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using atmospheric pressure chemical ionization. Excellent linearity was obtained for the entire calibration range from 5 to 1500 ng/mL KBA and AKBA in plasma and 5 to 1000 ng/mL KBA and AKBA in brain. Validation assays of the lower limit of quantification as well as for the intra- and interday precision and accuracy met the international acceptance criteria for bioanalytical method validation. Moreover, the interchangeability of calibration curves generated in pork and rat brain homogenates could be demonstrated. Using the developed analytical method, KBA and AKBA could be detected for the first time in brain up to a concentration of 99 and 95 ng/g of brain, respectively, 3 h after the single oral administration of 240 mg/kg of dry B. serrata resin extract to Wistar rats. The developed method represents an appropriate tool to further study the time-dependent distribution of KBA and AKBA in plasma and brain as well as the absolute brain concentration after multiple doses and contributes thus to the optimization of the dosage regimen and to a better understanding of the therapeutic effects of B. serrata.     

The chilling of carcasses

Biochemical processes and structural changes that occur in muscle during the first 24h postmortem play a great role in the ultimate quality and palatability of meat and are influenced by the chilling processes that carcasses are subjected to after slaughter. For beef and lamb, employing chilling parameters that minimize cold shortening is of greatest importance and can be best addressed by ensuring that muscle temperatures are not below 10°C before pH reaches 6.2. For pork, because of the impact of high muscle temperatures and low pH on the development of pale, soft, and exudative (PSE) pork, a more rapid chilling process is needed to reduce PSE with the recommended internal muscle temperature of 10°C at 12h and 2-4°C at 24h. Spray chilling, a system whereby chilled water is applied to carcasses during the early part of postmortem cooling, is used to control carcass shrinkage and to improve chilling rates through evaporative cooling. Delayed chilling can be used to reduce or prevent the negative effects of cold shortening; however, production constraints in high-volume facilities and food safety concerns make this method less useful in commercial settings. Electrical stimulation and alternative carcass suspension programs offer processors the opportunity to negate most or all of the effects of cold shortening while still using traditional chilling systems. Rapid or blast chilling can be an effective method to reduce the incidence of PSE in pork but extreme chilling systems may cause quality problems because of the differential between the cold temperatures on the outside of the carcass compared to the warm muscle temperatures within the carcass (i.e., muscles that are darker in color externally and lighter in color internally).     

Imaging proliferation in vivo with [F-18]FLT and positron emission tomography

Positron emission tomography (PET) is now regularly used in the diagnosis and staging of cancer. These uses and its ability to monitor treatment response would be aided by the development of imaging agents that can be used to measure tissue and tumor proliferation. We have developed and tested [F-18]FLT (3'-deoxy-3'-fluorothymidine); it is resistant to degradation, is retained in proliferating tissues by the action of thymidine kinase 1 (TK), and produces high-contrast images of normal marrow and tumors in canine and human subjects.     

Circulating dopamine-beta-hydroxylase (DbetaH) and catecholamines in a paediatric phaeochromocytoma

Nasal airways resistance was measured in ten patients with allergic rhinitis during intranasal application of an extract of grass pollen. Pretreatment with placebo did not inhibit the increase in nasal airways resistance, whereas ICI 74,917 administered from a pressurized aerosol gave almost complete protection. ICI 74,917 was well tolerated and no evidence was obtained of local hyposensitization during the period of the study.     

Characterization of the three-dimensional solution structure of human profilin: 1H, 13C, and 15N NMR assignments and global folding pattern

Human profilin is a 15-kDa protein that plays a major role in the signaling pathway leading to cytoskeletal rearrangement. Essentially complete assignment of the 1H, 13C, and 15N resonances of human profilin have been made by analysis of multidimensional, double- and triple-resonance nuclear magnetic resonance (NMR) experiments. The deviation of the 13C alpha and 13C beta chemical shifts from their respective random coil values were analyzed and correlate well with the secondary structure determined from the NMR data. Twenty structures of human profilin were refined in the program X-PLOR using a total of 1186 experimentally derived conformational restraints. The structures converged to a root mean squared distance deviation of 1.5 A for the backbone atoms. The resultant conformational ensemble indicates that human profilin is an alpha/beta protein comprised of a seven-stranded, antiparallel beta-sheet and three helices. The secondary structure elements for human profilin are quite similar to those found in Acanthamoeba profilin I [Archer, S. J., Vinson, V. K., Pollard, T. D., & Torchia, D. A. (1993), Biochemistry 32, 6680-6687], suggesting that the three-dimensional structure of Acanthamoeba profilin I should be analogous to that determined here for human profilin. The structure determination of human profilin has facilitated the sequence alignment of lower eukaryotic and human profilins and provides a framework upon which the various functionalities of profilin can be explored. At least one element of the actin-binding region of human profilin is an alpha-helix. Two mechanisms by which phosphatidylinositol 4,5-bisphosphate can interfere with actin-binding by human profilin are proposed.     

Specific and quantitative immunoprecipitation of tropomyosin and other cytoskeletal proteins by magnetic separation

Immunoprecipitation is a powerful technique for purifying many proteins for which specific antibodies exist. Magnetic separation has recently been demonstrated to be effective in the immunoprecipitation of cell-surface proteins. We have used magnetic separation with anti-immunoglobulin or protein A bound to magnetic particles to immunoprecipitate labeled muscle tropomyosin and several other cytoskeletal proteins for which specific antibodies exist. We have not found it necessary to bind antigen-specific antibody to the magnetic particles, increasing the versatility of the technique. The quantitative recovery of tropomyosin from muscle cultures using magnetic separation is superior to Staph A (protein A-positive Staphylococcus aureus cells). The specificity of magnetic separation also compares favorably with Staph A for immunoprecipitation of muscle tropomyosin. Fibroblast tropomyosin, vimentin (from muscle and osteoblast) and myosin heavy chain are other cytoskeletal proteins that are easily recovered with magnetic separation. Magnetic separation, therefore, appears to be a valuable technique for the immunoprecipitation of cytoskeletal proteins from various cell types.     

Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the increased vascular permeability and angiogenesis associated with many malignant tumors. In addition, VPF/VEGF is strongly expressed by epidermal keratinocytes in wound healing and psoriasis, disorders that are also characterized by increased microvascular permeability and angiogenesis. In this study, we investigated the expression of VPF/VEGF in three bullous diseases with subepidermal blister formation that are characterized by hyperpermeable dermal microvessels and pronounced papillary dermal edema. The expression of VPF/VEGF mRNA was strongly up-regulated in the lesional epidermis of bullous pemphigoid (n = 3), erythema multiforme (n = 3), and dermatitis herpetiformis (n = 4) as detected by in situ hybridization. Epidermal labeling was particularly intense over blisters, but strong expression was also noted in areas of the epidermis adjacent to dermal inflammatory infiltrates at a distance from blisters. Moreover, the VPF/VEGF receptors, flt-1 and KDR, were up-regulated in endothelial cells in superficial dermal microvessels. High levels of VPF/VEGF (138-238 pM) were detected in blister fluids obtained from five patients with bullous pemphigoid. Addition of blister fluid to human dermal microvascular endothelial cells exerted a dose-dependent mitogenic effect that was suppressed after depletion of VPF/VEGF by immunoadsorption. These findings strongly suggest that VPF/VEGF plays an important role in the induction of increased microvascular permeability in bullous diseases, leading to papillary edema and fibrin deposition and contributing to the bulla formation characteristic of these disorders.     

Evaluation and treatment of pain: aspects for nerve injury and rehabilitation

Pain, or at least discomfort in the form of hypersensitivity, is always a sequela to nerve injury and nerve repair. A carefully planned pain evaluation and treatment program has been shown to result in a decrease in pain and improved function. For a hand therapist, the commitment to evaluation and management of pain is a personal as well as a professional dedication. Patients may greatly vary in their responses to injury, symptomatology, and treatment interventions. Although the hand therapist can successfully intercede with many patients, it is often advantageous to involve a clinical psychologist. Psychosocial and psychophysiologic data can be imperative in evaluating the possibilities of patient outcome with regard to pain, evaluation, and treatment. Psychophysiologic information is likely to increase the hand therapist's evaluation and treatment potency. A psychological assessment and/or intervention is appropriate at several points of treatment--for example, after an appropriate interval of healing time in which an injury should resolve itself, after a major invasive procedure such as surgery, or when a condition is chronic and all other avenues of treatment have been unsuccessful.