Dextran enzyme imine complexes : a preliminary study : this thesis was presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

A model system involving the formation of protein-dextran complexes has been investigated with a view to improving existing methods of drug administration. Activation of the dextran was achieved by periodate oxidation to give levels of 7%, 21% and 56% activated glucose moieties. The protein-dextran complexes were investigated with the prospect of obtaining sustained release of proteins from the dextran in an unmodified form. Covalent conjugation of proteins to carbohydrate polymers is known to confer stability on the protein. The proteins in this study were bound to the dextran through imine bonds. The proteins investigated were lysozyme, trypsin, amylase, alcohol dehydrogenase and catalase. The selection covered a range of molecular weights and varying enzymatic activities. As might be predicted, the speed of complex formation was shown to be greater at the 21% level of activation compared to the 7% activation of dextran in all cases studied. Lysozyme, the smallest protein, readily formed complexes at all three levels of activation. At the 56% level the resulting complex had an extremely high MW, greater than 1MDa. The extensive binding between the dextran and lysozyme molecules resulted in a complex that was inactive and showed no signs of releasing any lysozyme, active or inactive. At the lower levels of activation, complex was formed with relative ease. Upon conjugation lysozyme exhibited only minimal activity. Release of a lysozyme-like species with normal lytic activity was observed. Precautions were taken to minimise possible autolysis in the trypsin study. Once complexed it was postulated that autolysis would be prevented or minimised. Similarly the 56% level of activation appeared to be too high to obtain a viable complex for facile trypsin release. Sustained release of a trypsin-like protein was observed with complexes at the 7% and 21% levels. SEC and SDS-PAGE, in conjunction with a positive BAPNA assay gave support to the released species being trypsin-like. While complexed to the dextran trypsin showed no signs of activity. Released trypsin-like species and unreacted trypsin showed similar tryptic maps from a synthetic peptide, the peptide was designed to show distinctive fragments. α-Amylase, twice the MW of trypsin and over three times the MW of lysozyme, formed complexes with ease at both 7% and 21% levels of activation. Conjugation to dextran did not effect the activity of α-amylase. Over time the release of an α-amylase-like species from the complex was observed. Alcohol dehydrogenase and catalase are both high MW proteins. Complex formation was observed for each protein. Subsequent experiments showed that upon release the proteins appeared to dissociate, most probably into their subunits. It is also possible that the dimers and monomers bound to the dextran. The main advantage of conjugation in this case appeared to be to confer stability on the proteins. The ADH-complex exhibited enzymatic activity. At 7% and 21% activation levels the lower MW proteins formed complexes with dextran that exhibited release of a protein species. The higher MW proteins were possibly stabilised when conjugated to dextran, but dissociated upon release. Investigations have shown that the level of activation chosen affects the extent of binding and therefore the functions of the resultant complex. Thus activation levels can be manipulated depending on the desired result. While lower dextran activation levels appeared to be more suited for smaller MW proteins, there were indications that the larger MW proteins could form beneficial complexes at higher activation levels. Results indicated that conjugation to periodate activated dextran could be extended to further proteins with the possibility of therapeutic or commercial applications

Perceptions of the natural environment in undergraduate students: influence of degree programme and learning style

 A study of over 400 first year undergraduates sought to link environmental perception with the degree programme being taken and their perceived approach to learning using a range of measures. A questionnaire was used to assess whether students had a strong feeling of closeness of association with nature; assessed their views on environmental issues and if they thought of the natural environment in a wide, ‘wholistic’ way or a narrow, compartmentalist way. Results indicated that their environmental perception varied significantly according to the type of degree being taken, with those taking natural science subjects and arts tending to perceive a greater closeness to nature, hold more pro-environmental views and perceive the natural environment in a more integrative way than students taking psychology and law degrees. Students taking pharmacy and biomedical programmes scored more highly on pro-environmental views than students taking either psychology or law but tended to perceive the environment in a more narrow, compartmentalist way. The students perceived approach to learning was not a significant controlling variable in determining their closeness to nature, to their environmental views or perception of the natural environment

Inhibition and oscillatory activity in human motor cortex

Using transcranial magnetic stimulation (TMS) important information can be obtained about the function of motor cortical circuitry during performance of voluntary movements by conscious human subjects. In particular, pairs of TMS pulses can probe inhibitory pathways projecting onto corticospinal neurones, which themselves project to motoneurones innervating hand muscles. This allows investigation of inhibitory circuitry involved in the performance of specific motor tasks, such as the precision grip. Previous studies have shown that pronounced synchronous oscillatory activity within the hand motor system is present at both cortical and muscular level when subjects maintain steady grasp of an object in a precision grip. The origin of this synchronous activity is unknown. However modelling studies have suggested that inhibitory pathways are likely to play an important role in the generation of cortical oscillations, and therefore TMS was used in this Thesis to investigate the origin of synchrony present during the precision grip task. In the first study, parameters of the paired-pulse test used to measure intracortical inhibition were examined. It was found that by modifying the intensities of the stimuli, and the interval between the paired-pulses, different phases of inhibition could be measured. This enabled specific use of TMS to investigate inhibitory pathways. Both single and paired-pulse TMS were then delivered to the motor cortex of subjects performing a precision grip task. It was found that low intensity TMS could reset the phase of muscle oscillatory activity, consistent with corticospinal neurones being part of the circuitry that generates the oscillatory rhythm. When, in the paired-pulse test, a low intensity stimulus was followed a few milliseconds later with a larger TMS stimulus, in the paired-pulse test, strong intracortical inhibition could be measured. This suggested that inhibitory interneurones activated by low intensity TMS could play an important role in the rhythm-generating network. An additional study looked at the importance of cutaneous receptor feedback on synchrony, by studying the effects of local anaesthesia of the index finger and thumb. Whereas low intensity TMS was shown to enhance synchronous activity between muscle pairs, suppression of cutaneous feedback from the digits reduced it. Results in this Thesis suggest that inhibitory interneurones within the motor cortex are important in the generation of synchronous activity within the hand motor system. This synchrony is also under the influence of cutaneous afferent input

Eric Hoffer : implications for a social theory of communication

Thesis (M.A.)--University of Kansas, Speech and Drama, 1971

Relaxin: a new cardiovascular hormone in humans? Comparative potency and mechanisms of action

INTRODUCTION: The focus of this MD thesis has been relaxin, a member of the insulin family, which is a protein composed of two disulphide linked chains of approximately 6000 Daltons. Relaxin has been traditionally recognised as a hormone of parturition, though more recently it has been postulated that relaxin may be involved in cardiovascular regulation. We used concentrations similar to those found in the plasma in physiological (non-pregnant, pregnancy) and pathophysiological (chronic heart failure) states. Firstly, we characterised the effects of relaxin in small human resistance arteries ex vivo using wire myography obtained from gluteal biopsies taken from patients with coronary heart disease (CHD) and normal left ventricular systolic function. We also studied the same effects in larger calibre arteries (internal mammary) and veins (saphenous) using standard organ bath techniques. The effect of relaxin in veins has not previously been described. Internal mammary arteries and saphenous veins were obtained from patients undergoing coronary artery bypass surgery. Small pulmonary arteries were obtained from patients undergoing thoracotomy for bronchial carcinoma. In addition, we wished to determine if a transcardiac or transpulmonary gradient of relaxin could be measured to suggest either pulmonary or cardiac secretion or clearance of the hormone. Relaxin secretion in heart failure has previously been described. Lastly, we wished to determine whether an increased relaxin plasma concentration in patients with chronic heart failure (CHF), is of prognostic importance. METHODS AND RESULTS i)comparative potency of relaxin compared to other vasodilators: Small resistance arteries were obtained from biopsies taken from patients with CHD. Each set of vessels was preconstricted with noradrenaline. Thereafter, cumulative concentration response (relaxation) curves (CRCs) were constructed with known vasodilators 25 atrial natriuretic peptide (ANP), epoprostenol, substance P and relaxin (n=8). Relaxin was found to be a more potent vasodilator than ANP and equipotent to epoprostenol. ii) mechanism of vasorelaxation: CRCs to relaxin (as above) were constructed to identify the importance of the endothelium – following the removal of the endothelium by the established method of intraluminal rubbing with a human hair. We found that relaxin is endothelium dependent. iii) interaction of relaxin with nitric oxide and other possible mechanisms of vasodilation and importance of ACE inhibitor treatment: We identified the importance of the effect of ACE inhibitor treatment on the action of relaxin in human resistance arteries. Relaxin’s vasodilatory action was significantly reduced in those patients on ACE inhibitors (n=28) compared with those patients not on ACE inhibitors (n=30). In patients treated with an ACE inhibitor, we found that manipulation of prostanoids is important. Indomethacin, (a cyclooxygenase inhibitor) (n=8) blocked relaxin’s vasodilatory action. Manipulation of the cAMP second messenger system, with milrinone, (a cAMP phosphodiesterase inhibitor) (n=6) is also important as relaxin’s vasodilatory action was enhanced. Manipulation of cyclic GMP second messenger system is also important. ODQ, (a guanylate cyclase inhibitor) (n=10) reduced relaxin’s action while zaprinast, (a cGMP phosphodiesterase inhibitor) (n=7) enhanced relaxin’s action. Manipulation of nitric oxide with L-NAME (n=8) and L-NOARG (n=10), nitric oxide synthase (NOS) inhibitors and EDHF with apamin and charybdotoxin (potassium channel blockers) (n=7) had a curious effect causing the opposite action to that expected, by enhancing relaxin’s vasodilatory action. In patients not treated with an ACE inhibitor, we found that manipulation of nitric oxide with L-NAME (n=8) and LNOARG (n=8), is important, as both reduced relaxin’s vasodilatory action. Manipulating the cGMP second messenger system with ODQ (n=8) greatly reduced relaxin’s action. but zaprinast (n=9) did not. Manipulation of EDHF with apamin and charybdotoxin (n=8) had no effect on relaxin’s action. Manipulation of prostanoids with indomethacin (n=10) reduced relaxin’s action but manipulation of cAMP with milrinone (n=8), had no effect. 26 iv)relaxin and small human pulmonary arteries: We determined, using wire myography, that relaxin is not a vasodilator of small pulmonary resistance arteries (n=5). v)relaxin and large calibre vessels: We determined, using the organ bath technique, that relaxin is not a vasodilator of larger calibre arteries i.e. internal mammary arteries removed from patients during coronary artery bypass surgery (n=5).Relaxin is not a venodilator studying saphenous veins removed from patients during coronary artery bypass surgery (n=5). vi)transmyocardial and transpulmonary gradient of relaxin: Plasma relaxin concentrations were measured using a validated assay. Samples were taken from patients undergoing CABG surgery, from the aorta, coronary sinus, pulmonary artery and pulmonary vein. We found that in 20 patients with normal left ventricular function that there was no transpulmonary gradient but there was a transcardiac gradient suggesting net cardiac extraction of relaxin. vii)prognostic value of relaxin in patients with chronic heart failure: Relaxin was compared with N-terminal pro brain natriuretic peptide to determine whether relaxin is of prognostic importance. Plasma concentrations of the hormones were measured in 87 patients admitted with CHF. These patients were followed up for a year during which time hospitalisations due to CHF and death were recorded. While NT-proBNP was found to be a powerful and independent predictor of outcome in these patients, relaxin was not. CONCLUSION. In addition to its established role in pregnancy, relaxin has many other actions. In particular, its antihypertensive, antithrombotic and vasodilatory properties suggest that relaxin may have a central role in cardiovascular regulation

The Transcriptional Effects of Photobiomodulation in an In Vitro Model of Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of blindness. The pathophysiology of DR is complicated, involving inflammation, oxidative stress, retinal vascular proliferation, and vascular degeneration. Symptomatically, the growth and subsequent rupture of vessels within the frame of view leads to the development of vision loss and eventual blindness. Prior to the development of symptoms, oxidative stress involved in DR leads to the activation of the transcription factor, nuclear factor-kB (NF-kB), resulting in the excess production of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1), proteins involved in vascular development and immune dysregulation, respectively. The most common therapeutic approach for DR utilizes anti-VEGF agents to reduce vascular proliferation. These treatments are expensive, invasive, frequently ineffective, and have numerous adverse effects, such as retinal detachment, infection, and inflammation inside the eye. A non-invasive alternative therapy is clearly needed. Photobiomodulation (PBM) using far-red to near infrared (NIR) light has been shown to reduce oxidative stress and inflammation in vitro and in vivo and is an ideal candidate for an alternative therapy. Indeed, PBM slows the progression of DR in animal models via attenuation of oxidative stress and by reducing the relative level of ICAM-1. We hypothesize that PBM will reduce the activity of NF-kB and reduce the production of VEGF and ICAM-1 in an in vitro model of DR. To test this hypothesis, we used an in vitro model system of cultured retinal Müller glial cells grown in normal (5 mM) or high (25 mM) glucose conditions for either 3 or 6 days to simulate normoglycemia and hyperglycemia. Cultures were treated with 670 nm light emitting diode (LED) (180 seconds at 25 mW/cm2; 4.5 J/cm2) or no light (sham) for 3 or 5 days. NF-kB activity and ICAM-1 concentrations were significantly increased under high glucose conditions, as measured by a dual luciferase assay or western blot, respectively. Treatment with 670 nm LED significantly reduced NF-kB activity of high glucose culture cells to values comparable to transcriptional activity measured under normoglycemic condition and decreased the level of ICAM-1. VEGF concentrations were not affected by high glucose or PBM. These data are in partial support of our central hypothesis that in an in vitro model of DR, 670 nm light will reduce activation of NF-kB, and reduce the synthesis of ICAM-1 and VEGF. The lack of an observable effect of hyperglycemia or PBM on VEGF concentrations indicates that the stimulation of VEGF secretion requires the activation of additional signaling pathways not induced by high glucose alone

The Effectiveness of Australian Medical Portals: Are They Meeting the Health Consumers’ Needs?

The move to using portals to distribute medical information is supported by Australian Governments and government agencies. The recent success of ‘telemedicine’ is promising for patients and governments alike as it could provide quality care and convenience for patients and reduces the burden on the health budget for governments. The Australian Government is taking a proactive role in developing medical portals to encourage the general use of the web for the dissemination of medical information (NHIMAC, 2000). Government portals such as HealthInsite (Australian) and BetterHealth (Australian Victorian Government) encourage users to access the sites (NHIMAC, 2000).. Despite the support by governments, usability tests examining portal effectiveness indicate that many portals are not effective for users. This paper presents the results of usability testing conducted on current Australian medical portals and discusses the portals’ effectiveness from the users’ perspective. The paper also discusses current technology that could improve medical portals’ effectiveness thereby better serving the needs of the health consumer