The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono-and diglyceride specific lipase

The extracellular lipase from Penicillium camembertii has uniquesubstrate specificity restricted to mono- and diglycerides.The enzyme is a member of a homologous family of lipases fromfilamentous fungi. Four of these proteins, from the fungi Rhizomucormiehei, Humicola lanuginosa, Rhizopus delemar and P.camembertii,have had their structures elucidated by X-ray crystallography.In spite of pronounced sequence similarities the enzymes exhibitsignificant differences. For example, the thermo-stability ofthe P.camembertii lipase is considerably lower than that ofthe H.lanuginosa enzyme. Since only the P.camembertii enzymelacks the characteristic long disulfide bridge, correspondingto Cys22–Cys268 in the H.lanuginosa lipase, we have engineeredthis disulfide into the former enzyme in the hope of obtaininga significantly more stable fold. The properties of the doublemutant (Y22C and G269C) were assessed by a variety of biophysicaltechniques. The extra disulfide link was found to increase themelting temperature of the protein from 51 to 63°C. However,no difference is observed under reducing conditions, indicatingan intrinsic instability of the new disulfide. The optimal temperaturefor catalytic activity decreased by 10°C and the optimumpH was shifted by 0.7 units to more acidic.     

Coherent diffusion of isotopic impurities in solid helium

A theory of coherent diffusion (impuriton propagation) of dilute isotopic impurities in solid He is described. An expression for the mean square distance R ² (t) traveled by the impurity is derived. For short times pure band propagation occurs and so R ² (t) grows as t ². For times longer than a characteristic impurity-phonon scattering time this time dependence becomes linear, and a diffusion constant can be defined. This characteristic scattering time is estimated to be ~7 × 10^–5 T ^–9 sec (T in K) for a ³He impurity in ⁴He at V _m = 21 cm³/mole. The corresponding diffusion constant is consistent with that measured in the NMR experiment of Allen and Richards if the ³He-⁴He tunneling frequency J ₃₄ is taken as 7 × 10⁵ sec^–1.     

Activation and adherence of lyophilized human platelets on canine vessel strips in the Baumgartner perfusion chamber

The Baumgartner perfusion technique was used to test the ability of rehydrated lyophilized human platelets to adhere to the thrombogenic surface of a denuded arterial vessel segment and to undergo platelet activation under conditions of high shear. Twenty preparations of washed platelets were stabilized by 1-hour or 2-hour exposure to paraformaldehyde before freeze-drying in a Virtis 600 lyophilizer. The response of these fixed-dried preparations was compared with that of non-fixed platelets in fresh citrated whole blood. The outcome of each perfusion experiment was quantified in photomicrographs by morphometric estimation of the percent area of the vessel segment covered by adherent platelets after immunofluorescent staining with monoclonal antibodies to glycoprotein Ib (CD42) or glycoprotein IIbIIIa (CD41a). Evidence of activation in nonadherent platelets was examined by flow cytometry for CD62 and GP53 expression. In addition, thromboxane B2 was measured by radioimmunoassay as an index of platelet responsiveness to activation conditions during perfusion. The percent vessel coverage observed with lyophilized platelets in recombined whole blood was somewhat less than that of platelets in fresh whole blood (39% vs 73%, respectively). In other perfusion experiments performed with non-denuded vessel segments, the percent coverage was reduced by half or more for both types of platelet preparation. Scanning electron microscopy confirmed that the lyophilized platelets did not adhere to areas of intact endothelium. Furthermore, the lyophilized platelets showed a small-but-significant rise in CD62 or CD63 activation antigen expression and generated thromboxane B2 at about one third the rate of fresh platelets in these perfusion experiments. The thromboxane generation during perfusion was inhibited in fresh or lyophilized platelet preparations by pretreatment with indomethacin or PGE-1. We interpret these data as evidence of the ability of our lyophiilized platelet preparations to respond at least partially to physiologic stimuli and to adhere to appropriate thrombogenic sites to support hemostasis.     

Comparison of femoral blood gases and muscle near-infrared spectroscopy at exercise onset in humans

We hypothesized that near-infrared spectroscopy (NIRS) measures of hemoglobin and/or myoglobin O2 saturation (IR-SO2) in the vascular bed of exercising muscle would parallel changes in femoral venous O2 saturation (SfvO2) at the onset of leg-kicking exercise in humans. Six healthy subjects performed transitions from rest to 48 +/- 3 (SE)-W two-legged kicking exercise while breathing 14, 21, or 70% inspired O2. IR-SO2 was measured over the vastus lateralis muscle continuously during all tests, and femoral venous and radial artery blood samples were drawn simultaneously during rest and during 5 min of exercise. In all gas-breathing conditions, there was a rapid decrease in both IR-SO2 and SfvO2 at the onset of moderate-intensity leg-kicking exercise. Although SfvO2 remained at low levels throughout exercise, IR-SO2 increased significantly after the first minute of exercise in both normoxia and hyperoxia. Contrary to the hypothesis, these data show that NIRS does not provide a reliable estimate of hemoglobin and/or O2 saturation as reflected by direct femoral vein sampling.     

Abnormalities of retinal metabolism in diabetes or experimental galactosemia. VI. Comparison of retinal and cerebral cortex metabolism, and effects of antioxidant therapy

Metabolic abnormalities observed in retina and in cerebral cortex were compared in diabetic rats and experimentally galactosemic rats. Diabetes or experimental galactosemia of 2 months duration significantly increased oxidative stress in retina, as shown by elevation of retinal thiobarbituric acid reactive substances (TBARS) and subnormal activities of antioxidant defense enzymes, but had no such effect in the cerebral cortex. Activities of sodium potassium adenosine triphosphatase [(Na,K)-ATPase] and calcium ATPase became subnormal in retina as well as in cerebral cortex. In contrast, protein kinase C (PKC) activity was elevated in retina but not in cerebral cortex in the same hyperglycemic rats. Dietary supplementation with an antioxidant mixture (containing ascorbic acid, Trolox, alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, and selenium) prevented the diabetes-induced and galactosemia-induced elevation of retinal oxidative stress, the elevation of retinal PKC activity and the decrease of retinal ATPases. In cerebral cortex, administration of the antioxidant diet also prevented the diabetes-induced decreases in (Na,K)-ATPase and calcium ATPases, but had no effect on TBARS and activities of PKC and antioxidant-defense enzymes. The results indicate that retina and cerebral cortex differ distinctly in their response to elevation of tissue hexose, and that cerebral cortex is more resistant than retina to diabetes-induced oxidative stress. The greater resistance to oxidative stress in cerebral cortex, as compared to retina, is consistent with the resistance of cerebral cortex to microvascular disease in diabetes, and with a hypothesis that oxidative stress contributes to microvascular disease in diabetes. Dietary supplementation with these antioxidants offers a means to inhibit multiple hyperglycemia-induced retinal metabolic abnormalities.     

Metaphors in the talk of "depressed" women in psychotherapy.

Variations in how we describe, explain, and treat "depression" and in who is labeled "depressed" attest to the significance of the social-historical contexts in which our understandings and practices are based. A prevalent, modern-day (Western) conception of "depression" is as a gendered (primarily female), devalued condition characterized by a discourse of the deficient self. In an effort to learn more about the particular forms of this discourse, the metaphors used by 10 depressed women (aged 28–62 yrs) to talk to their psychotherapists about their "selves" in relation to their perceived difficulties were studied. Two broad cultural imperatives were constructed from these metaphors: (1) Don't be too mothering and (2) Don't be too child-like. At the core of these 2 imperatives were messages about the importance of autonomy, for both oneself and others. This valuing of autonomy can be clearly located at the heart of 20th-century Western ideals. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

An experimental stationary quadrotor with variable DOF

Unmanned air vehicles (UAV) and especially quadrotors have drawn great attention in recent years because of their maneuverability, ease of design and control. Most of the works concentrate mostly on control; yet, design and communication are also some sub-topics. In this research, a stationary, four rotor air vehicle with limited/controlled degree of freedom is constructed so that various control algorithms and their changing effects with varying vehicle dynamics can be studied on the ground for safety purposes. Ascending/descending, pitch/yaw/roll motions can be limited/controlled by setting the vehicle’s degree of freedom mechanically, resulting better net observability of the control algorithms on the vehicle’s dynamic performance. Design, in terms of mechanics, mechatronics and software is presented and the usability of the system is shown. Parallel self tuning fuzzy PD + PD control is applied to the system for preliminary studies and results are discussed. Inspite of the sensor noise, satisfactory pitch/roll/yaw control is achieved.